ORS Basic Science Tips

In 2022, the ORS lost a giant, Dr. Fred Nelson MD. Fred was a member of the ORS for almost 50 years. Through his clinical perspective, Fred’s myriad contributions to the society have advanced both basic and translational research. He was a longstanding member of the Scientific Communications Committee, and he pioneered the Basic Science Tips series. Here Fred showcased the research of more than 500 projects to the greater ORS community. He focused on basic science with a potential to make an eventual impact in the clinic. As such, the Scientific Communications Committee will carry forward the Basic Science Tips in hopes of honoring the legacy of Fred. We aim to produce 1-2 tips per month and to expand their reach through translation into non-English languages. While we can never replicate Fred’s insight or wit, we hope to continue to highlight the important basic science research that ORS members produce.

Currently, ORS Basic Science Tips are written by ORS Scientific Communications Committee members Mia Huang and Hugo Giambini, PhD.

Collagen XII is a Critical Regulator of Supraspinatus Tendon Mechanics and Collagen Fiber Realignment Across Sex

Collagen XII is a Critical Regulator of Supraspinatus Tendon Mechanics and Collagen Fiber Realignment Across Sex
https://www.ors.org/transactions/2023/1992.pdf

Collagen XII is a Fibril-Associated Collagen that regulates collagen fibril assembly and is primarily expressed throughout tendon growth and development. Mutations in the Col12a1 gene result in myopathic Ehlers-Danlos syndrome, a connective tissue disorder leading to distal joint hypermobility and contracture. The role of collagen XII on the supraspinatus (SSP) tendon, which experiences a complex region-specific loading environment within the rotator cuff of the shoulder, remains unknown. Researchers hypothesized that tendon-targeted deficiency and knockout of collagen XII would result in decreased SSP tendon whole-tissue and regional elastic mechanics, whole-tissue viscoelasticity, and regional collagen fiber realignment across sex.

SSP tendons from male and female, day 60 tendon-targeted collagen XII heterozygous (HET) mice (ScxCre;Col12a1f/wt), knockout (KO) mice (ScxCre;Col12a1f/f), and wild-type (WT) control mice (Cre- littermates) were used to evaluate stress relaxation at various % strains followed by ramp-to-failure. Throughout the ramp-to-failure, dynamic collagen fiber realignment was quantified using cross-polarization imaging. Images were also used to calculate regional moduli (insertion and midsubstance).

Female KO tendons exhibited a trending decrease in cross-sectional area relative to WT. Linear stiffness was significantly decreased in KO mice across sex and between female HET and KO mice. Insertion modulus was significantly reduced in HET and KO tendons across sex whereas midsubstance modulus was significantly reduced in male KO tendons and female HET and KO tendons. Percent relaxation was significantly increased in KO tendons across sex at all strain levels. Dynamic modulus was significantly decreased in male KO tendons and in female HET and KO tendons, while phase shift was significantly increased in KO tendons across sex across at all strain levels and frequencies. There was a reduction in collagen fiber realignment in HET and KO tendons across region and sex.

This study showed that tendon-targeted collagen XII knockout results in striking reductions in supraspinatus tendon mechanical properties and dynamic collagen fiber realignment across sex. Further, reductions in these properties in the HET tendons highlight the allele-dependency of collagen XII on tendon biomechanics. These outcomes elucidate the critical role of collagen XII in regulating male and female supraspinatus tendon regional and whole-tissue mechanics and dynamic structural response to load within the complex loading environment of the rotator cuff of the shoulder. A better understanding of the effects of collagen XII can be used to evaluate potential treatments modalities for myopathic Ehlers-Danlos syndrome.

Figure caption: Differences in collagen fiber realignment distribution and elastic mechanical properties for male and female WT, HET, and KO supraspinatus tendon insertion and midsubstance regions. Decreased normalized circular variance is indicative of increased collagen fiber realignment. Data as mean ± standard deviation (—p≤0.1, **p≤0.01, ***p≤0.001).

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Inflammatory Bone Loss Induced by SARS-CoV-2 Infection

Inflammatory Bone Loss Induced by SARS-CoV-2 Infection
https://www.ors.org/transactions/2023/441.pdf

Severe acute COVID-19 may be complicated by both pulmonary and extrapulmonary manifestations, such as anosmia, ageusia, diarrhoea, lymphopenia, and multi-organ dysfunction syndrome. However, clinical manifestations in the long-term post-acute sequelae of SARS-CoV-2 infection associated to pathological changes in the skeletal system remain largely unknown. In this work, we characterized the effects of SARS-CoV-2 infection on bone metabolism and its underlying mechanism during the acute and post-recovery phases in our established golden Syrian hamster model which closely mimics human infection.

6–10-week-old male or female golden Syrian hamsters (Mesocricetus auratus) were intranasally treated with 105 PFU of SARS-CoV-2 (i.e., Delta variant, strain HKU-001a) in 50 μL of PBS under intraperitoneal ketamine (200 mg/kg) and xylazine (10 mg/kg) anesthesia at 0 dpi, and then their bone tissues were collected serially after the infection. Mock-infected animals were treated with 50 μL of PBS. Their blood, bone, and lung tissues were collected at sacrifice at 4, 30, and/or 60 days post-infection (dpi) for μCT, virological, and histopathological analyses.

In general, SARS-CoV-2 caused significant multifocal loss of bone trabeculae in the long bones and lumbar vertebrae of all infected hamsters. The most prominent disease manifestations were seen at about 4 dpi and the hamsters generally recovered at about 7 to 10 dpi. Infection resulted in progressive loss of bone trabeculae at the distal metaphysis of femurs from the acute phase (4 dpi) to the post-recovery phase (30 dpi) and the chronic phase (60 dpi) of infection. On D60 and post recovery of SARS-CoV-2 infection, bone density was not restored. Instead, a gradual decrease of bone volume fraction was detected. Compared with mock-infected hamsters, a significantly higher number of tartrate-resistant acid phosphatase positive (TRAP+) osteoclasts were found in the trabeculae at the distal metaphysis of the femur, the proximal metaphysis of the tibia, and the lumbar vertebrae of infected hamsters. Moreover, bone loss was associated with SARS-CoV-2-induced cytokine dysregulation.

This study suggested that SARS-CoV-2 induces pathological bone resorption through a pro-inflammatory cascade instead of direct infection in the skeletal tissue. The findings of this study highlight the need for optimizing clinical protocols for monitoring long-term complications of COVID-19 and finding novel treatment strategies for SARS-CoV-2-induced inflammatory osteopenia/osteoporosis.

Figure caption: SARS-CoV-2 infection induces continuous inflammatory bone loss during both the acute and post-recovery phases of COVID-19. The 3D reconstructed micro-CT scans reveal that the bone trabecula (white arrow) density in the femur of SARS-CoV-2-infected hamsters (Right) is significantly lower than that of the Mock control (Left).

(Photo copyright: https://www.hku.hk/press/press-releases/detail/24565.html)

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Turkey Model to Study Acute Compartment Syndrome

Acute Compartment Syndrome (ACS) is a common and severe condition following extremity trauma. In animal models, the most common method to induce ACS is to infuse a solution such as saline or an albumin suspension directly into the muscle compartment. Small animal models are technically challenging in terms of ACS induction and clinical examination. On the other hand, Turkeys have several advantages compared to other species, including (1) large leg muscles with similar anatomy and structure as human legs, (2) amenable to clinical ultrasound and functional evaluations, (3) thin skin with little fat in the subcutaneous tissue and compartment. In this study, researchers demonstrate the feasibility of using a turkey model for the evaluation of ACS.

Thirty-two (32) one-year-old Bourbon Red Heritage turkeys were used (Figure 1A). Groups 1 & 2 had the inter compartment pressure (ICP) maintained at 50 mmHg for 2 and 6 hours, during which ultrasound shear wave elastography (SWE) and ICP measurements were measured every half hour simultaneously. Groups 3 & 4 had a fasciotomy performed after 2 and 6 hours, respectively, of ICP at 50 mmHg. The left or right leg, selected randomly, was prepared for ACS creation. The tibialis cranialis (TC) muscles, which corresponds to the tibialis anterior muscle in humans, were identified. SWE was implemented to evaluate muscle elasticity at the following six testing points: 0 (before infusion), 10, 20, 30, 40, and 50 mmHg. Muscle elasticity was measured daily in the first week after the procedure, every two days in the second week, and then weekly until sacrifice. The relation between ICP and SWE was investigated. TC isometric tetanic muscle force (ITF) was also measured. The contraction force of the TC muscle was measured under stimulating the peroneal nerve at 10V, duration 0.4s and delay 2ms with preload at 100N.

Muscle elasticity outcomes showed significant and high correlations with the actual ICPs (Figure 1B). Elasticity measures were able to describe changes in the order of 10 mmHg with ICP, demonstrating its important utility as an alternative to the invasive ICP measurements currently used in clinical practice as a diagnostic method for ACS. Additionally, this study shows the feasibility of using a turkey model as an inexpensive bipedal large ACS model (Figure 1C & D).

Reference:

Acute Compartment Syndrome Model Using Turkey Tibialis Cranialis Muscle
Ichiro Tsukamoto, Naoya Iida, Farbod Yousefi, Elameen Adam, Omar Selim, Gogyin Zhao,Tomoyuki Kuroiwa, Rou Wan, Aida Sarcon, Mohammed Abozaid, Ramona Reisdorf, Chunfeng Zhao.
Mayo Clinic Orthopedic Biomechanics Laboratory, Rochester, MN
ORS 2023 Annual Meeting Paper No. 1066

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Thank you to Mingyue Fan, Shuyan Han, Franziska Breulmann, Luca Ambrosio, and Elena Della Bella for translating this Basic Science Tip.

How hypoxic is the initial phase of fracture healing?

Oxygen is critical for cell survival, proliferation, and differentiation. Different cell types and tissues have different oxygen levels and different oxygen needs. For example, while the spleen exhibits high oxygen levels ~8-10% pO₂ at atmospheric pressure, the bone marrow environment is relatively hypoxic (1%-6% pO₂). When a bone is broken, this can dramatically change the amount of oxygen in this environment. The initial phase of fracture healing has long been thought to be characterized by severe lack of oxygen due to the rupture of blood vessels, but the intracellular oxygen levels in the bone marrow and fracture gap are unknown. In this study, researchers sought to understand how intracellular oxygen levels vary between cells in the bone marrow and how cellular oxygen levels change during fracture repair.

In cancer research, nitroimidazoles (such as EF5) have been widely used to examine and grade the hypoxic environment in tumor tissue. EF5 is a nitroimidazole that is selectively reduced by nitroreductase enzymes under hypoxic conditions, resulting in the formation of EF5 adducts in proportion to intracellular hypoxia. These adducts can then be visualized with fluorophore-coupled antibody. Researchers established EF5 staining as a precise method to investigate the tissue oxygenation based on the abundance of hypoxic cells in the bone marrow and initial phase of fracture healing.

First, the researchers performed in vitro studies in which they cultured mouse bone marrow under defined oxygen concentrations (10%, 2%, 0.5%, 0.1% pO₂) and found that the frequency of EF5-positive cells correlated positively both with environmental hypoxia and with HIF-target gene expression. This establishes EF5 as a reliable marker of cellular hypoxia: less oxygen produces greater EF5 signal.

Second, the researchers analyzed in vivo cellular oxygen levels in the bone marrow by systemic injection of EF5 in mice. The results were consistent with measurements of 4% pO₂ in murine bone marrow and revealed an approximately equal distribution of EF5-positive and EF5-negative cells, indicating heterogeneity of intracellular hypoxia under these physiologic conditions.

Finally, the researchers performed EF5 analysis of intracellular oxygen in the hematoma of the fracture gap at 3 days post-fracture (dpf). The frequency of EF5-positive cells at 3 dpf was significantly lower than the EF5-positive frequency in either the adjacent bone marrow of the same limb or bone marrow of the un-injured contralateral bone marrow. These findings suggest that the initial fracture healing is not hypoxic as the field has long assumed.

Together, the researchers have established the EF5 method as a robust approach to analyze intracellular oxygen in bone marrow and fracture repair. The results show heterogeneity of intracellular oxygen levels in native bone marrow and overturn long-standing assumptions about hypoxia in the early stages of fracture repair.

Reference:

Heterogeneity of Cellular Hypoxia in Murine Bone Marrow
Annemarie Lang, Cameron Koch, Joel D. Boerckel. Departments of Orthopaedic Surgery and Bioengineering, University of Pennsylvania, Philadelphia, PA, Department of of Radiation Oncology, University of Pennsylvania, Philadelphia, PA
ORS 2023 Annual Meeting Paper No. 482

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Thank you to Mingyue Fan, Shuyan Han, Franziska Breulmann, Luca Ambrosio, and Elena Della Bella for translating this Basic Science Tip.

Age-related stem cell dysfunction with aging

Weakening of the skeleton with age increases the susceptibility to bone-related injuries and results in impaired regeneration of bone tissue thereafter. Previous studies in mice suggest that aging is associated with declined Skeletal Stem and Progenitor Cells (SSPCs) number and function. However, findings have been inconsistent and the underlying mechanisms of this detrimental change are not well understood. Bone mineral density decreases with age, and thus it is widely thought that SSPC osteogenic potential is also reduced. Unexpectedly, while aged SSPCs exhibit a reduced self-renewal capacity compared to young SSPCs, they produce significantly more bone matrix in vitro. Therefore, this study hypothesized that aged SSPCs prematurely differentiate into bone, eventually leading to a loss in number and function of SSPCs in vivo with age.

Bone marrow stromal cells from young (8-16 wk.) and middle-aged (52-82 wk.) wild type (C57BL/6) mice and human Bone Marrow Aspirate Concentrate (BMAC) from the iliac crest of consented subjects was collected and plated individually for CFU-F and other functional assays.

Researchers observed increased osteogenic differentiation of aged murine and human derived SSPCs compared to young SSPCs in vitro. Aged SSPCs expressed osteogenic and adipogenic genes, and exhibited Alkaline Phosphatase enzymatic activity, an early marker of differentiation. Furthermore, aged SSPCs showed reduced self-renewal capacity overtime as they efficiently differentiated into mature bone forming osteoblasts. A more differentiated progenitor, the pre-BCSP (Bone, cartilage, stromal progenitor) was more abundant, proliferative, and osteo-primed in aged mice. While young and aged skeletal stem cells comparably differentiated into bone, aged non-SSPCs cells presented increased mineralized matrix deposition compared to young cells. Finally, RNA-seq showed several components of key osteogenic signaling pathways (Wnt and BMP) to be upregulated during aging.

Some takeaways from this study include: While it is thought that bone formation declines with age due to a loss of the osteogenic capacity of SSPCs, the authors showed that aged SSPCs, specifically pre-BCSPs, exhibited higher levels of osteogenic differentiation in vitro compared to their young counterparts. Importantly, aged pre-BCSPs seemed more osteo-primed & differentiated more rapidly than young pre-BCSPs, suggesting that the premature differentiation of aged SSPCs drives the decline in SSPC number with age in vivo. Finally, these outcomes pinpoint the pre-BCSP as a cellular target of interest & the BMP & WNT signaling pathways as molecular targets for therapeutic interventions to rescue SSPC function to rejuvenate the aged skeleton.

Reference:

Investigating the cellular mechanism of age–related stem cell dysfunction
Malissa Ramsukh, Sophie Morgani, Kevin Leclerc, Lindsey Remark, Margaux Sambon, Philipp Leucht. NYU Robert I. Grossman School of Medicine – Department of Orthopaedic Surgery, New York, NY, USA, NYU Robert I. Grossman School of Medicine – Department of Cell Biology, New York, NY, USA. ORS 2023 Annual Meeting Paper No. 2053

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Thank you to Baixing Chen, Sonja Häckel, Franziska Breulmann, and Elena Della Bella for translating this Basic Science Tip.

Conversion of white fat to brown/beige fat improves rotator cuff muscles quality

Muscle atrophy, fibrosis and fatty infiltration (FI) are commonly seen in rotator cuff tears (RCT). Thus, improving muscle quality is critical after RCT repair to improve clinical outcomes. Besides thermogenic function, brown/beige fat (BAT) also serves as an endocrine organ, secreting batokines (brown adipokines) to promote muscle growth. PRDM16 can determine brown fat fate and stimulate its development, and knockdown of PRDM16 ablates the genetic program of brown fat. PRDM16 expression is highly enriched in BAT, and it activates a robust brown fat phenotype when expressed in white fat cell progenitors, fibro-adipogenic progenitor (FAP) cells. Thus, we investigated the role of PRDM16 in regulating muscle function after massive tendon tears. We hypothesized that overexpression of PRDM16 would improve muscle function and ameliorate fibrosis and FI.

Transgenic mice over-expressing PRDM16 and wild type (C57BL/6J) mice underwent unilateral supraspinatus (SS) tendon transection and suprascapular nerve transection (TTDN). Post-injury evaluations included forelimb function at 6 weeks post the TTDN injury, PRDM16 expression in adipose tissue, muscle fibrosis and fat infiltration, and muscle fiber type.

There was an increased expression of PRDM16 protein in both white and brown fat in PRDM16-overexpression mice compared to wild type (WT) mice. PRDM16 significantly improved forelimb function with longer brake, stance and stride time, larger stride length and paw area in mice after RCT. PRDM16 overexpression did not improve fibrosis, however, it significantly reduced fatty infiltration area (%) after injury. Compared to WT mice, PRDM16 overexpression significantly increased MHC-IIx fiber percentage in supraspinatus muscle after TTDN.

Some takeaways from this study include that overexpression of PRDM16 can improve forelimb function, and result in significantly less fatty infiltration and increased MHC-IIx fiber type in muscle after RCT. While the functional role of MHC-IIx fiber type in rotator cuff muscle metabolism and function remains unknown, and further studies are needed to explore the relationship between BAT and muscle fiber type after rotator cuff injury, promoting BAT activity seems to be beneficial in improving rotator cuff muscle quality and shoulder function after RCT.

Reference:

He Zhang, Agustin Diaz, Mengyao Liu, Yajing Hu, Hubert Kim, Brian T. Feeley, Xuhui Liu. Department of Physical Education, Central South University, Hunan, China, San Francisco Veterans Affair Health Care System, Department of Orthopaedic Surgery, University of California, San Francisco.
Overexpression of PRDM16 Improves Muscle Function after Rotator Cuff Tears. ORS 2022 Annual Meeting Paper No. 343.

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Thank you to Lin Han, Elena Della Bella, Luca Ambrosio, Sonja Häckel, and Franziska Breulmann for translating this Basic Science Tip.

What can we learn from evolutionary adaptations of bat’s shoulder for developing new approaches to treat glenohumeral joint instability and rotator cuff pathology?

In humans, the glenohumeral joint enables the greatest mobility in the human body, at the expense of its stability. The bony anatomy between the proximal humerus and glenoid cavity of the scapula has minimal constraints, thus humans are able to perform a wide range of overhead motions. Stability in the joint is largely provided by soft tissues such as the rotator cuff and the labrum. Unfortunately, repetitive overhead motion leads to rotator cuff injuries that affect millions of people in the US each year. Bats perform repetitive overhead motion in order to achieve flight. Their survival depends on performing these motions across a relatively long lifespan without injury. In this study, investigators compared bat and mouse shoulder anatomy to understand the functional advantages of the shoulders in bats and hypothesized that the features of the proximal humerus and the glenoid cavity of the scapula in bats have evolved to provide better stability of the glenohumeral joint during repetitive overhead motion and to offload stress from the supraspinatus tendon.

Shoulders harvested from 12-week-old mice and C. perspicillata bats were fixed in three positions: full shoulder extension, intermediate shoulder extension, and full shoulder flexion. Gait and flight analyses were used to ensure position consistency by identifying the angle between the scapular spine and the humerus. Anatomical landmarks and measurements were identified and calculated from microCT images.

Scapular index (the ratio of scapula width to length) and infraspinatus index were both significantly larger in bats than in mice, while the supraspinatus index did not differ. Supraspinatus-acromion clearance (vertical distance between the insertion of the supraspinatus on the humerus and the bottom face of the acromion) increased when moving from full shoulder flexion to extension, and this trend was maintained for both. However, the supraspinatus outlet area (the area posterior to the coracoid, inferior to the acromion, and within the arch of the acromion) was significantly higher in bats compared to mice. Glenoid measurements showed that the glenoid was retroverted in bats and anteverted in mice.

Some takeaways from this study are: 1) there are functional adaptations in the shoulder anatomy of bats that contribute to increased stability of the glenohumeral joint; 2) the conserved supraspinatus index and enlarged infraspinatus index suggests that the bat has adapted to relieve stress on the supraspinatus tendon, and the infraspinatus tendon might have a role in stabilizing the glenohumeral joint; 3) the bat’s supraspinatus outlet area was significantly larger than that of mice, providing more space for the supraspinatus to pass under the coracoacromial arch, which might prevent shoulder impingement or entrapment of the supraspinatus, a pathology commonly seen in humans; 4) the glenoid is anteverted in mice and retroverted in bats, analogous to the retroverted glenoids reported in high-level overhead-throwing athletes (this is an adaptation that enables more mobility while preserving joint stability by limiting capsular tension); 5) these adaptations in bats contribute to increased stability of the glenohumeral joint and provide insight into novel surgical treatments for glenohumeral joint instability or prevention of rotator cuff tears.

Reference:

Iden Kurtaliaj, Jennifer Kunes, K. Michael Rowley, Lynn Ann Forrester, Guy M. Genin, Sharon M. Swartz, Stavros Thomopoulos. Columbia University, New York, NY; Brown University, Providence, RI. Washington University, St. Louis, MO;. ORS 2022 Annual Meeting Paper No. 1940

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Thank you to Baixing Chen for translating, and Shuyang Han for proofreading the Chinese translation, to Sonja Häckel for translating and Sebastian Wangler for revising the German translation, and to Luca Ambrosio for translating and Elena Della Bella for revising the Italian translation

Patient sex affects Knee Osteoarthritis Outcomes after ACL Reconstruction

ACL reconstruction (ACLR) is commonly performed to restore knee function and decrease the risk of posttraumatic osteoarthritis (PTOA) after ACL injury. Female sex is an established risk factor for ACL injury, although its impact on ACLR outcomes remains unclear. Given the minimal differences found from their previous study between low and high initial graft tensioning protocol conditions at 3 and 7 years, and that most of the PTOA outcomes were worse among the ACLR subjects relative to the uninjured controls, the authors followed these patients through 12 years to evaluate the extent of arthrosis following ACLR and to determine how initial graft tension may interact with patient sex to influence long-term arthrosis.

Patients with isolated unilateral ACL injuries underwent ACLR with either a bone-patellar tendon-bone or a four-stranded hamstring autograft using a low or high initial graft tension protocol. Control subjects were matched by age, sex, race, and activity level. Patient-reported and imaging outcomes related to PTOA (e.g., OARSI and WORMS difference scores) were evaluated at 10 to 12 years post-surgery. 31%, 36%, and 42% of low-tension, high-tension, and control participants, respectively, were lost to follow up at this time point.

Female patients had significantly worse Tegner scores than male patients, indicating that female patients maintain a lower activity level relative to males at 12 years post-ACLR. Contrastingly, males scored significantly worse than females in OARSI difference score and tended to score worse than females in WORMS difference score, although this finding was not significant. Both tension groups scored significantly worse than the control group in most patient-reported outcomes such as KOOS subscales, and no significant differences were observed between tension groups. Both tension cohorts scored significantly worse than the control group for the WORMS difference score and the surgical knee had significantly worse scores than the contralateral knee in both cohorts. The low-tension group exhibited these findings for the OARSI difference score, suggesting that PTOA may only be progressing in the surgical knees treated with a low initial graft tension, which departs from the WORMS and KOOS findings.

There are several take away points from the current study work: First, the female sex may be a risk factor for worse Tegner outcomes at 12 years post-ACLR. Second, low and high initial graft tensions are equivalent in terms of patient-reported and OA imaging outcomes, and arthrosis seems to progress relative to the controls regardless of initial graft tension assignment. Third, males scored worse than females in OARSI difference score and, although not significant, tended to score worse than females in WORMS difference score. Limitations of this study include loss to follow-up and the higher rate of loss to follow up in the control group relative to the tension groups.

Reference:

Costa, M. Q., Badger, G. J., Chrostek, C. A., Carvalho, O. D., Faiola, S. L., Fadale, P. D., Hulstyn, M. J., Gil, H. C., Shalvoy, R. M., & Fleming, B. C. (2022). Effects of Initial Graft Tension and Patient Sex on Knee Osteoarthritis Outcomes After ACL Reconstruction: A Randomized Controlled Clinical Trial With 10- to 12-Year Follow-up. The American journal of sports medicine, 50(13), 3510–3521.

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Thank you to Lin Han, Elena Della Bella, Luca Ambrosio, Sonja Häckel, and Sebastian Wangler for translating this Basic Science Tip.

Patient-specific Musculoskeletal Modeling Can Predict Back Muscle Activation During Dynamic Activities

Musculoskeletal (MSK) modeling of the spine can be used to study a variety of spine conditions including spine deformity and vertebral fractures. An open source full-body thoracolumbar musculoskeletal model includes patient-specific characteristics such as spinal curvature and muscle morphometry. The model has been shown to predict spine loading and muscle activity during static tasks, but has not yet been evaluated during dynamic tasks. Therefore, the authors compared the predicted back muscle activation patterns from subject-specific models with the recorded in-lab electromyography (EMG) during a dynamic box lifting task. Reflective markers used for 3D kinematic assessments and wireless electromyography sensors were placed at body segments and vertebral levels on healthy volunteers (mean age = 65). Participants performed a static standing calibration pose, four different maximal voluntary contractions (MVC) of the trunk (flexion, extension, and L/R lateral bending) while seated, and lifted and lowered a box of 10% body mass. MSK models for each participant were scaled based on the static calibration pose, total body mass, and the trunk imaging data, and used to predict muscle activity during the lifting task. To quantify the temporal similarity between measured EMG and model muscle activities, maximum absolute normalized cross-correlation (MANCC) coefficients were calculated. All of the back muscles had strong MANCC coefficients, with median values ranging from 0.83 to 0.90. Moreover, assessing model muscle activation at the nominal location of the EMG sensor produced similar, or in some case better (p-values < 0.003) MANCC coefficients than assessing it one vertebral level below or above the nominal location.

Some of the takeaways from this study are: 1) thoracolumbar spine MSK models can accurately predict the activation trend of back muscles during dynamic lifting; 2) activation trends at the nominal physical location of the sensor better matched measurements, providing a secondary confirmation of accurate modeling; 3) future applications of the model include providing estimates of musculoskeletal loading during dynamic activities to evaluate the risk of injuries, effects of spine conditions, and/or effects of treatments.

Reference:

M Mehdi Alemi, Jacob J. Banks, Andrew C. Lynch, Brett T. Allaire, Mary L. Bouxsein, and Dennis E. Anderson, Center for Advanced Orthopaedic Studies, Beth Israel Deaconess Medical Center, Boston, MA, A thoracolumbar spine musculoskeletal model predicts back muscle activation patterns during a dynamic lifting activity. ORS 2022 Annual Meeting Paper No. 1990

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Thank you to Shuyang Han, Elena Della Bella, Luca Ambrosio, and Sonja Häckel for translating this Basic Science Tip.

What is Required in the First 7 Days Post Fracture for Normal Healing?

Atrophic nonunions are a type of failed fracture healing that occurs when osteochondral callus fails to form. A key feature of fracture healing is proliferation of periosteal cells in the first 2 weeks, which appear to contribute to callus formation. To address the role of proliferation, the authors had previously investigated fracture healing in Col1-tk mice that express a herpes simplex virus (HSV)-TK ‘suicide gene’ downstream of the 3.6Col1a1 promoter, a promoter active in periosteal osteoblast lineage cells. They showed that proliferating TK-positive (tk+) cells died when dosed with ganciclovir (GCV) twice daily for 14 days starting at the time of fracture , without affecting non-dividing cells. In a new study, the authors investigated how changing the dosing duration (3, 7, or 14 days) of GCV would affect fracture healing. The right femur of mice was fractured at the mid-diaphysis and stabilized with a stainless-steel intramedullary rod. The mice were radiographed weekly until euthanasia (day 21) to track healing progression.

Radiograph analysis shows the WT control groups developed mineralized callus at similar rates where, by day 21 post fracture, 90-100% of femurs had a fully bridged callus. The tk+ mice receiving GCV for 3 days showed delayed callus formation compared to the WT group, but fully bridged calluses by day 21. tk+ mice receiving GCV for 7 and 14 days showed delayed callus formation, and by day 21 only 57% and 25%, respectively, had a fully bridged callus. microCT showed tk+ mice had significantly smaller total callus volume than WT mice for each dosing group, with a trend for progressively smaller callus with longer GCV dosing. Histology at day 21 showed that all WT mice and tk+ mice receiving GCV for 3 days had fully bridged bony callus with little cartilage. In contrast, calluses from tk+ mice dosed with GCV for 7 days were smaller with cartilage often present, while tk+ mice dosed for 14 days displayed blunted mineralized callus formation, with a large fraction of cartilage or fibrous tissue present, indicating impaired fracture healing.

There are several take away points from the current study work: First, blocking osteoblast proliferation for 14 days post fracture can lead to radiographically and histologically impaired healing, confirming previous work. Second, 7 days of GCV dosing in tk+ mice lead to impaired fracture healing when assessed 21 days after fracture, indicating that the first 7 days is a critical period for osteoblast proliferation. Third, osteoblast proliferation in the first 3 days after fracture contributes to early healing, but it is not a requirement for eventual healing. Proliferation of osteoblast lineage cells in the first 7 days post fracture seems to be required for normal healing in mice. Future work should determine if healing in the 7-days of GCV dosing group is merely delayed or leads to persistent nonunion.

Reference:

Andre F. Coello Hernandez, Jennifer A. McKenzie, Katherine R. Hixon, Anna N. Miller, Matthew J. Silva, Dept. of Orthopaedic Surgery, Dept. of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO; Proliferation of Osteoblast Lineage Cells in the First 7 Days Post Fracture Is Required for Normal Healing in Mice. ORS 2022 Annual Meeting Paper No. 1356

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Thank you to Baixing Chen, Elena Della Bella and Valentina Basoli for translating this Basic Science Tip.

Etiology and Mechanobiology of Osteoporosis Using a Multicellular Bone Mimetic Model

Bone tissue composition is altered with changes in the cell mechanical environment and in their biological responses to mechanical loads during osteoporosis. There is a link between estrogen withdrawal, osteocyte differentiation, and osteoclastogenic signaling. Osteoblasts and osteocytes undergoing estrogen withdrawal alter mineralization under mechanical stimulation and produce pro-osteoclastogenic paracrine factors. To incorporate osteoclast activities, an advanced 3D model was developed to understand the role of osteocyte and osteoblast signaling and mechanobiology in governing osteoclast resorption during osteoporosis. In this study, researchers aimed to incorporate a 3D multicellular niche within a suitable matrix, and apply exogenous mechanical loading to the constructs to investigate osteoclastogenesis under estrogen withdrawal conditions to simulate postmenopausal osteoporosis.

Osteocytes (OCY454) and osteoblasts (MC3T3) were pre-treated with 17β-Estradiol for 14 days before hydrogel encapsulation and cultured in osteogenic media in continued estrogen supplementation or no further estrogen supplementation (estrogen withdrawal*). Osteoclast precursors (RAW264.7) were encapsulated and added directly on top of the mineralized constructs and allowed to differentiate for 14 days. Oscillatory flow (~1.2 mL/min at 1 Hz) and compression (~0.5% strain at 1 Hz) were applied to the constructs, with a control group cultured in a standard static medium. Osteoclast activity was determined by analyzing the constructs at day 3, 7 and 14.

TRAP activity measured via-histology peaked 7 days after the addition of RAW cells, for both continued estrogen (E) and estrogen withdrawal (EW) groups. Mechanically stimulated constructs had significantly greater semi-quantitative CTSK fluorescence intensity 7 days after addition of RAW cells, when compared to day 3, but this decreased by day 14. Furthermore, estrogen deficient (EW) constructs increased expression of OSCAR and CTSK genes at days 7 and 14, respectively, compared to those under continuous estrogen (E). Calcium content increased from day 3 to day 14 under all conditions. Mechanically stimulated and estrogen deficient constructs (EW) illustrated a significant down-regulation of BSP2 at day 14, and a significant reduction in ALP activity and calcium content at day 7, compared to continuous estrogen groups.

Some takeaways from this study include: 1) in-vitro 3D multicellular and mechanically mimetic models can successfully simulate postmenapausal osteoporosis and recapitulate osteoclastogenesis; 2) while mineralisation is impaired in the current multicellular construct with mechanical loading, this is in contrast to the significant increase in mineralization under estrogen deficiency observed in the author’s previous studies. Potential causes for these contradictory outcomes might be related to factors produced by the osteoclasts which may have inhibited mineralised matrix formation. The reduction in osteoblast gene expression (BSP2) might also explain these changes in mineralization. 3) Further studies are needed to uncover the relative contributions of osteoclasts, osteoblasts and osteocytes to bone loss and mineralization under estrogen withdrawal and mechanical stimulation.

Reference:

Syeda M. Naqvi, Laoise M. McNamara, Mechanobiology and Medical Device Research Group, Biomedical Engineering, National University of Ireland Galway, Ireland; Osteoclast Activation and Differentiation in a Multicellular Bone Mimetic Model to Study the Etiology and Mechanobiology of Osteoporosis. ORS 2022 Annual Meeting Paper No. 1369

Essential Need for an Osteoporotic Animal Model for the Evaluation of Pain Mechanisms and Biomarkers

Osteoporosis affects 1 in 3 women and 1 in 5 men over the age of 50 leading to an increased risk of fragility fractures. Osteoporotic and fragility fracture-associated pain has been poorly studied, therefore, in this study, researchers aimed to study skeletal pain in postmenopausal osteoporosis, hypothesizing that the pain associated with osteoporosis and fractures could be linked to the altered bone remodelling but also to the dramatic changes in oestrogen levels affecting nociceptive pathways and changes in nerve markers in the serum and dorsal root ganglia (DRGs).

To mimic postmenopausal bone loss, bilateral ovariectomy (OVX) or Sham as control were performed in both young adults (10 weeks) and aged (30 weeks) C57/Bl mice with age matched non-operated controls. Nociception was assessed weekly from baseline (before OVX) to end-point using evoked and naturalistic behaviors. Evoked behaviors included hot (50°C)/cold(15°C) plates to assess thermal hyperalgesia/allodynia, while Von Frey was employed to measure mechanical allodynia. Naturalistic behaviors included burrowing and nesting to assess the mouse state and well-being. Brain-derived neurotrophic factor (BDNF), neurofilament light (NF-L), and estradiol levels, as well as gene expression profiles of pain markers in DRGs were investigated.

Mechanical allodynia was significantly decreased in old mice in both Sham and OVX when compared to unoperated controls in weeks four and five post – surgery, but while the sham group recovered this sensitivity six weeks post-surgery, the OVX group did not. Thermal hyperalgesia and naturalistic behaviors did not change in young or old mice from pre-OVX baseline to 6 weeks post-surgery in both sham and OVX mice and were comparable to the ones of unoperated controls. While the levels of estradiol were decreased in OVX mice compared to Sham, this was not statistically significant. Similarly, very few changes in the circulating amounts of nerve markers were observed. Levels of NF-L decreased in young mice 6 weeks post-OVX when compared to Sham, while BDNF levels of old mice showed a 2.6-fold decrease in OVX compared to Sham 3 weeks post-surgery. Finally, the levels of nerve marker gene expression in DRGs, expressed as fold change of OVX compared to Sham, only indicate low fold changes from OVX to Sham for both the transient receptor potential cation channel subfamily V member 1 (TRPV1) (2 fold change) and tropomyosin receptor kinase A (TrkA) (2.5 fold-change).

There are several take away points from this work: First, an increased response to mechanical and thermal stimulation that detects hyperalgesia and allodynia was not observed after OVX, indicating limited evidence of pain in this model and suggesting that the bone loss induced by OVX may not drive pain; second, the peripheral circulating levels of estradiol were high and variable after OVX, suggesting that a more sensitive method for this measurement should be implemented or that OVX may not be the best suitable model to assess the effect of estrogen deficiency on pain; third, the levels of serum nerve markers after OVX, while not significantly different, were lower with OVX than in Sham; finally, there were increments observed in some gene expression for peripheral nociceptive neuromodulators but not others. Overall, this data suggests that this model does not reflect the hormonal conditions of postmenopausal osteoporosis despite the bone loss observed. Further studies are required to analyze the contribution of bone loss and estrogen depletion to skeletal pain in osteoporosis.

Reference:

Andreea Radulescu, Xiang Li, Amy Fisher, Chantal Chenu. Investigating pain mechanisms and biomarkers in a mouse model of osteoporosis. ORS 2022 Annual Meeting Paper 1302

Intraoperative Measurements of Gracilis Muscle Properties

Surgical procedures can alter the length and force produced by muscles. However, direct measurement of muscle properties is very rare and other methods, such as computer or biomechanical models, are rarely validated using in-vivo data from humans. To address this limitation, in this study, researchers aimed to measure structure and function of the gracilis muscle during an in-vivo functioning gracilis muscle transfer from the thigh to restore elbow flexion.

Gracilis free functioning muscle was harvested from 9 patients and gracilis muscle-tendon unit (MTU) length, passive sarcomere length, and passive and active muscle tetanic tension were measured at four anatomical/joint positions. Active tension was obtained by stimulating the anterior branch of the obturator nerve at an intensity corresponding to 50% of compound muscle action potential (CMAP) determined from twitch stimulation. The muscle’s physiological cross-sectional area (PCSA) was used to calculate passive and active muscle stress. Active sarcomere length was estimated for each measured active tension using the idealized human muscle force-length relationship which assumes that maximum tension occurs at an optimal sarcomere length of 2.7µm.

The researchers observed that the greatest sarcomere shortening was observed at the shortest MTU length and lowest passive stress. The magnitude of shortening decreased with increasing MTU length. Importantly, the predicted operating range of the active sarcomeres covered the ascending and descending portions of the stress vs. sarcomere length curve.

There are several take away points from this work: First, investigators state that the measured changes in sarcomere length cannot be accounted for using any of the lumped parameter models commonly implemented in the biomechanical literature using the actual measured muscle dimensions and sarcomere length; second, increased shortening with decreased passive tension and MTU length suggests that there is significant compliance within the gracilis MTU. If such compliance is not accounted for during surgery, it is likely that a muscle would be significantly under tension and skewed to very short sarcomere length during activation. This work will provide actual numbers for the strength and length of human muscle for the first time. Thus, we will not have to extrapolate from animal studies to “guess” how much force (stress) human muscles generate.

Reference:

Benjamin I. Binder-Markey, Lomas S. Persad, Alexander Y. Shin, Kenton R. Kaufman, and Richard L. Lieber Intraoperative Experimental Measurement and Biomechanical Prediction of Gracilis Muscle Properties ORS 2022 Annual Meeting Paper No.164

Fred Nelson’s Basic Science Tips

For over six years, ORS Member Fred Nelson, MD, has circulated a Basic Science Tip of the Week to a large and growing email list. In total, he has completed 342 weekly tips directed at orthopaedic residents and staff.

These tips serve several roles:

Highlight terms and concepts that may appear on resident examinations (OITE).
Give a glimpse into future developments that may come into clinical practice in the near future.
Highlight a mechanical or biochemical development that affects current basic science applications in medicine and surgery.
Share surprising developments such as the discovery of mechanisms of action for age old traditional herbal medicine and ancient practices.

Dr. Nelson’s “Tip of the Week” were regularly featured in ORS Connect, the society’s e-newsletter.

Etiology and Mechanobiology of Osteoporosis Using a Multicellular Bone Mimetic Model

Reference:

Essential Need for an Osteoporotic Animal Model for the Evaluation of Pain Mechanisms and Biomarkers

Reference:

Andreea Radulescu, Xiang Li, Amy Fisher, Chantal Chenu. Investigating pain mechanisms and biomarkers in a mouse model of osteoporosis. ORS 2022 Annual Meeting Paper 1302

Intraoperative Measurements of Gracilis Muscle Properties

Reference:

ORS Basic Science Tips: Honoring a Legacy

The ORS recently lost a giant, Dr. Fred Nelson MD. Fred was a member of the ORS for almost 50 years. Through his clinical perspective, Fred’s myriad contributions to the society have advanced both basic and translational research. He was a longstanding member of the Scientific Communications Committee, and he pioneered the Basic Science Tips series. Here Fred showcased the research of more than 500 projects to the greater ORS community. He focused on basic science with a potential to make an eventual impact in the clinic. As such, the Scientific Communications Committee will carry forward the Basic Science Tips in hopes of honoring the legacy of Fred. We aim to produce 1-2 tips per month and to expand their reach through translation into non-English languages. While we can never replicate Fred’s insight or wit, we hope to continue to highlight the important basic science research that ORS members produce.

Are you interested in translating the Basic Science Tips into non-English languages? Please take a moment to complete this form. Thank you!

From the Back of the Rack, Winners and Losers: Episode #1

The Scientific Communications Committee of Orthopaedic Research Society made a request for a review of some old tips to see which sparkled with time, and which fizzled. A major advance on our understanding in poor outcomes in hip preservation surgeries such arthroscopy and osteotomies hinges on of the degree hip articular cartilage changes at the time of surgery. This, in part, is dependent on age and severity of deformity. In a 2009 tip with no stated reference noted that deformities of the femoral head and acetabular complex would affect forces on articular cartilage. At the time it appeared that the range of “measured” normal forces on weight bearing joints was somewhere between 2 and 5 MPa. At the time investigators used MRI and other technology to find that the forces in OA knees was significantly elevated and that it was above 3.2 MPa. It was estimated that the force on the articular cartilage toward the edge of the femoral head and acetabular was 5 MPa instead of 2 MPa. Bottom line: alterations in force concentration, intensity, and frequency play a prime role in the initiation and continuation of cartilage loss regardless of biochemical interventions.

Fast forward to 2021. Attempts at joint preservation with hip arthroscopic procedures show that articular changes seen in femoro-acetabular impingement (FAI) resulting from cam deformities do not respond as well in older individuals who have more advanced articular changes. With corrective surgery such as femoral head neck chondro-osteoplasty and other techniques may give some “playing time” to the individual. Intervention later in the process is not as rewarding. So, what are the mechanics? A good analogy for a cam deformity is a scoop of ice cream on top of a cone. In a normal hip the diameter of the scoop of ice cream is circular and wider than the cone. In FAI the scoop is more egg shaped and does not overlap the edge of the cone. The result is an increased alpha angle which is the angle made by the intersection of a line from the center line of the femoral neck to the central part of the femoral head, to a line from the central femoral head to a point where the contour of the femoral head-neck junction exceeds the radius of the femoral head. Independent of neck shaft angle an angle greater than 55^O is considered at risk of cam impingement.

Not as well studied is the effect of the femoral neck to the femoral shaft. In this current research the author examined the differences in stress characteristics between symptomatic and asymptomatic patients with cam deformities and individuals with normal hips. This investigation studied the differences in acetabular cartilage stresses subchondral bone stresses and the effects of high and low femoral neck-shaft angles on these stresses.

Three groups of 2 persons (symptomatic cam, asymptomatic cam, control without cam deformity) Each group had one with the highest femoral neck-shaft angle and one with the lowest.

	Neck shaft angle high	Neck shaft angle low
Symptomatic cam	14.1 MPa 125	15.8 MPa 119
Asymptomatic cam	10.9 MPa 134	13.0 MPa 123
No cam	6.4 MPa 132	6.5 MPa 124

Overall, the symptomatic and asymptomatic participants with low femoral neck-shaft angles had the highest cartilage and subchondral bone stresses in their respective subgroups. The control group also showed no differences between cartilage and subchondral bone stresses. If you want to know more about the overall scope of the work studying the cam femoro-acetabular impingement see Kappa Delta Award paper from 2018.

References:

Ng KCG, Mantovani G, Lamontagne M, Labrosse MR, Beaulé PE. Cam FAI and Smaller Neck Angles Increase Subchondral Bone Stresses During Squatting: A Finite Element Analysis. Clin Orthop Relat Res. 2019 May;477(5):1053-1063. doi: 10.1097/CORR.0000000000000528.PMID: 30516652

Unravelling the hip pistol grip/cam deformity: Origins to joint degeneration.

Beaulé PE, Grammatopoulos G, Speirs A, Geoffrey Ng KC, Carsen S, Frei H, Melkus G, Rakhra K, Lamontagne M.J Orthop Res. 2018 Dec;36(12):3125-3135.

Minimally Clinically Important Difference: How Minimal, How Important, and How Calculated?

As the Patient Reported Outcome Measurement Information System (PROMIS) has become more prominent in outcomes measures there has been no consensus for minimally clinically important difference (MCID) calculations. Varied MCID calculation methods have led to a range of possible values making comparison of studies difficult. PROMIS is a National Institute of Health driven question system that measures global health with physical, mental, and social domains normalized to a United States population. Each question is dependent on the answer to the previous question allowing for a process that is shorter than the typical patient reported outcome measures in use. PROMIS also crosses the musculoskeletal subspecialties and typically can be used by most patients, albeit will some require assistance. In clinical studies a p value of < 0.05 does not mean that the patient has noticed a significant difference in their condition. The original design of MCID was to determine the level of improvement that was worth the cost of a particular treatment. The MCID is useful for examining whether individual patients improve enough to reach the threshold for improvement along with quantifying the proportion of patients from a given sample who attain sufficient improvement, and to compare proportions across procedures, settings, or diagnoses.

In a current concepts article authors review 2 basic categories of MCID calculations (anchor-based and distribution-based) and the effects of sample size on the application of any given statistic¹. Anchor-based MCID calculations are made based on a patient response to an external outcome measure (anchor) relevant to the patient experience under study. The MCID can be calculated with a mean change method or receiver operator characteristic curve (ROC). The limits used to determine “meaningful change” is based on research personnel and thus may lead to post hoc revision. A credibility instrument has been developed to evaluate anchor-based MCIDs. In contrast to Anchor-based MCID, Distribution-based MCID calculations rely on cohort-specific statistical parameters. Sample size influences the significance threshold and, therefore, the MCID value. There is a floor and ceiling impact on these calculations. Sample size influences the significance threshold and, therefore, the MCID value. the calculated MCID value in the cohort can vary based on the statical method chosen for the distribution-based MCID calculation. With the insurgence of PROMIS distribution-based MCID calculation has become a preferred method. The calculated MCID value may be statistically sound, but it does not necessarily ensure correlation with clinical importance, which is the impetus behind MCID calculation at the outset. MCID variability with PROMIS scores includes problems with MCID thresholds leading to patient exclusion due to some patients not being able to improve pre-operative scores. The assigned percentage of improvement determined to be clinically relevant will impact the percentage of patients that achieve that score. The precision of the follow-up (range of days post procedure for follow-up) has key significance to the number of patients reaching selected MCID. In this Current Concepts Review the authors present a list of suggested questions before implementing clinical research reporting MCIDs.

Reference

Aditya V KarhadeAV, Christopher M Bono CM, Schwab JH, Tobert Minimum Clinically Important Difference: A Metric That Matters in the Age of Patient-Reported Outcomes. J Bone Joint Surg Am. 2021 Dec 15;103(24):2331-2337. doi: 10.2106/JBJS.21.00773. PMID: 34665785

Detecting Early Cartilage Losses In Vivo: Is There a Consensus?

Disease modification in osteoarthritis is likely to be more successful in the early course of the disease. An analogy is that the structure and content of articular cartilage is pretty much defined by late teenage. Like a care tire tread, the object is to keep it intact through 6 or more decades. Unlike a care tire tread cartilage is not entirely passive. There are hard to define homeostatic mechanisms that will help maintain cartilage during the decades. The tensile properties of type II collagen are responsible for the restraint of proteoglycan swelling pressure leading to the viscoelastic properties of articular cartilage. After the third decade these tensile properties decrease. In the earlier literature it was not clear if this was a change in quantity or structure of collagen^1,2. The earliest matrix signs of early osteoarthritis is loss of proteoglycan content (swelling pressure) and collagen integrity. Dedicated MRI technologies have been offered to detect early proteoglycan loss and changes in collagen.

For proteoglycan content an intravenous or intra-articular negatively charged gadolinium agent is injected to enhance areas of glycosaminoglycan (GAG) depletion. In healthy cartilage, the negatively charged contrast agent is repulsed by negatively charged GAGs. When the GAG structure is disrupted, the contrast agent is allowed to diffuse into the cartilage shortening the T1 relaxation time of the lesion. Another technique for proteoglycan content is T1rho (T1ρ). T1ρ is the time constant of spin-lattice relaxation in rotating frame, which characterizes relaxation of magnetization under influence of a radiofrequency (RF) field. Damage to the collagen network results in an increase of water mobility. T2 relaxation times are sensitive to tissue anisotropy and water mobility and are therefore thought to reflect integrity of the collagen network. Ultrashort echo time (UTE) T2* mapping is based on the same principle, but is also influenced by the local field inhomogeneity caused by differences in magnetic susceptibility among tissues. T2* relaxation times are sensitive to the tissue integrity and organization, specifically in densely organized tissues characterized by the short T2 relaxation times.

A systematic review of literature was taken from Embase Classic and PubMed³. Relative to T2 imaging article using polarized light microscopy were used to correlate to collagen integrity. 1471 articles were trimmed to 21 for review. The pooled correlation between dGEMRIC and proteoglycan concentration was r = 0.59 (95% CI: 0.41 to 0.73). The pooled random effects model showed a significant correlation of T1p relaxation times and proteoglycan concentration, r =0.54 (95% CI: 0.73 to 0.29. However, Considerable heterogeneity was found between studies. No correlation between T2 relaxation time and collagen concentration was found (r = 0.02). A heterogeneous set of correlations between T2 relaxation times and PLM were identified, including strong correlations to anisotropy. In their conclusion the authors note that “the relation between T2 relaxation time and cartilage properties can be attributed to the relation with collagen organization, rather than concentration.”

In most instances the onset of loss of articular cartilage homeostasis is silent since there is no nociceptive response. The imaging is expensive and is hardly a screening tool. However, in at risk populations this could play a role in early intervention research.

References:

Akizuki S, Mow VC, Müller F, Pita JC, Howell DS, Manicourt DH. Tensile properties of human knee joint cartilage: I. Influence of ionic conditions, weight bearing, and fibrillation on the tensile modulus. J Orthop Res. 1986;4(4):379-92. doi: 10.1002/jor.1100040401.PMID: 3783297
Akizuki S, Mow VC, Muller F, Pita JC, Howell DS. Tensile properties of human knee joint cartilage. II. Correlations between weight bearing and tissue pathology and the kinetics of swelling.J Orthop Res. 1987;5(2):173-86. doi: 10.1002/jor.1100050204.PMID: 3572588
EmanuelKS, Kellner LJ, Peters MJM, Haartmans MJJ, Hooijmans MT, Emans The relation between the biochemical composition of knee articular cartilage and quantitative MRI: a systematic review and meta-analysis. Osteoarthritis Cartilage. 2021 Nov 23;S1063-4584(21)00976-6. doi: 10.1016/j.joca.2021.10.016. Online ahead of print. PMID: 34826570

Note: After repeated efforts the corresponding author was not respondent to requests for accuracy.

Hypercoagulable States: Historic Road Maps to Avoidance

Venous thromboembolism is a great concern in lower limb trauma albeit the upper limb can have similar sequelae. Virchow’s trail of endothelial injury, stasis, and hypercoagulability was first described in 1856¹ and remains a focus in the assessment and prevention of VTE. Avascular necrosis of the femoral head is sometimes seen after a displaced sub capital hip fracture. “Spontaneous” avascular necrosis is often associated with steroid use and alcohol abuse. Over the past three decades the hunt for clotting disorders has led to the identification of many genetic disorders. The question could be, does our current state of assessment and intervention to prevent VTE have an impact on prevention of AVN?

Unfortunately, VTE in lower limb injuries is a come as you are party. The population tends to be younger and may not have time to get a history that may reflect increased risk. Those historical (acquired) factors include obesity, cancer, pregnancy, hormonal therapy, and antiphospholipid syndrome². Antiphospholipid syndrome is associated with an autoimmune disorder with spontaneous pregnancy loss, and systemic lupus erythematosus. Inherited disorders include factor V Leiden (more resistant to degradation by protein C leading to increased thrombus formation), protein C deficiency mutation, antithrombin III deficiency, prothrombin gene mutation, hyperhomocysteinemia, and Sticky Platelet Syndrome (platelets tend to aggregate with increased stress and epinephrine expression.

Identification of a VTE propensity in trauma requires a thorough history and physical assessment. Routine testing is not cost effective. Considerations should include a family history of VTEs and the presence of risk factors for acquired HCDs, including obesity, pregnancy or hormonal therapy, history of malignancy, and other conditions. On suspicion, a hematologic consult is warranted to obtain best testing and recommendations.

Sickle cell disease, sudden barometric decompression, and lipid storage disease are some of many conditions known to be associated with the development of avascular necrosis (AVN). Relative to VTE, AVN of the femoral head following glucocorticoid (GC) use is an interesting corollary³. High doses of dexamethasone in rats inhibit fibrinolytic activity by decreasing tissue plasminogen activator (t-PA) activity and increasing plasma plasminogen activator inhibitor-1 (PAI-1) antigen levels. PAI-1 plays a role in fibrinolysis by forming complexes with t-PA. Plasma fibrinogen and lipoprotein (a) (Lp(a)) are also abnormalities found in GC-induced or idiopathic ON. Others have shown that there was no association between thrombophilia with ON and suggested that GC-induced regional endothelial dysfunction was a more likely reason. Endothelial cell damage seen on electron microscopy has been found in steroid treated rabbits. In addition, GCs can induce endothelial cell apoptosis promoting thrombus formation and by two mechanisms. Apoptotic bodies can induce endothelial dysfunction. Also, apoptotic endothelial cells can stimulate adhesion of platelets to endothelial cells and activate platelets which would later lead to thrombus formation. Increased fat deposition may lead to passive congestion and increased intraosseous pressure that becomes associated with a hypercoagulable state. These and other mechanisms are reminiscent of Virchow’s triad.

Both clinician and researcher should keep in mind the roles of endothelial injury, stasis, and hypercoagulability in VTE and AVN.

References

Virchow, R. (1856). “Thrombose und Embolie. Gefässentzündung und septische Infektion”. Gesammelte Abhandlungen zur wissenschaftlichen Medicin(in German). Frankfurt am Main: Von Meidinger & Sohn. pp. 219–732.Matzdorff AC, Bell WR (1998). Thrombosis and embolie (1846-1856). Canton, Massachusetts: Science History Publications. ISBN 0-88135-113-X.
RomanoD, Boyle M, Isla AE, Teasdall RJ, Srinath A, Aneja Hypercoagulable Disorders in Orthopaedics: Etiology, Considerations, and Management. JBJS Rev. 2021 Oct 12;9(10). doi: 10.2106/JBJS.RVW.21.00079. PMID: 34637409
ZhangQ, Jin L V , Jin Role of coagulopathy in glucocorticoid-induced osteonecrosis of the femoral head. J Int Med Res. 2018 Jun;46(6):2141-2148. doi: 10.1177/0300060517700299. Epub 2017 May 1. PMID: 28459353

Knee Osteoarthritis: The Great Communicator

Knee osteoarthritis can be a devastating blow to the quality of life on otherwise very active individuals. Thousands of papers show investigations determine a cytokine, intracellular signaling protein, cell surface or nuclear receptor, non-coding RNA, or other pathway transmitter wherein the clinical significance is reported as “a potential target for therapeutic intervention”. However, what part of the knee, as an organ, is impacted? A glimpse into the future may involve non-coding RNAs and dividing out the components of the knee.

Non-coding RNAs can be considered to be a part of the epigenetic changes affecting cellular activity¹. The precise mechanism of action is dependent on the class of non-coding RNA. In part, these are short non-coding RNAs such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), or circular RNAs (circRNAs). It has been demonstrated that non-coding RNAs play a major role in joint development. On the other end of the spectrum are the 100s of non-coding RNAs in osteoarthritis (OA). Albeit biopsies may not be ethically appropriate, circulating and synovial fluid levels do reflect changes such as in rheumatoid arthritis (RA). To date, no consistent profile of non-coding RNAs has been identified and validated in biofluids across OA or RA studies. There are differences across studies in the joints characterized, the profiling platforms used, the biofluids profiled, and how the patient groups are defined, all of which make it difficult to directly compare findings.

Data suggest that non-coding RNAs can have both beneficial and detrimental effects on the joints. Cellular processes include inflammation, apoptosis, extracellular matrix dysregulation, chondrocyte differentiation, oxidative stress, and autophagy. This entails signaling pathways such as transforming growth factor-β, fibroblast growth factor (FGF), Wnt–β-catenin, Hedgehog, and other mediators including the transcription factors FOXM1, SOX5, SOX6 and SOX9, and estrogen receptor-α. A variety of signaling molecules, including non-coding RNAs, can induce and regulate joint inflammation through cytokines such as IL-1β, TNF and IL-6 which are often used as inflammatory markers. Specific microRNAs have been shown to have a positive effect.

There is potential of non-coding RNAs as possible therapeutics, whether systemic or intra-articular. Strategies for non-coding RNA modulation continue to emerge. For example, transplantation of cartilage pellets derived from human BMSCs that overexpress beneficial miRNAs (such as miR-27b) inhibited hypertrophic chondrocyte differentiation during cartilage defect repair. Non coding RNA can be tissue specific.

The search for biological interventions often relies on the outcome of biochemical findings in synovial fluid, articular cartilage, and synovium. However, the knee is an organ with highly varied relationships between cartilage, bone, menisci, synovium, infrapatellar fat pad, cruciate ligaments (intra-articular structures) and nearby muscle. Albeit individual biologic products from any given tissue may give insight into important degenerative factors, the varied communications between tissues of the organ may be a more critical factor. Primary total joint replacement surgery in patients without RA are a good resource to discover how the tissues communicate at the RNA and microRNA level in OA². After saving a portion for histology, remaining portions of bone, cartilage, and menisci can be homogenized by taking frozen samples (-80^OC) and pulverizing with mortar and pestle. Synovium, fat pad, anterior cruciate ligament, and vastus medialus muscle can be thawed from -80^OC and individual samples could be homogenized. RNA extraction then enables gene analysis to follow.

Will the future of therapeutic intervention rely more on the active organ tissue in any given individual and how that communicates to other tissues in the same organ? Film at 11*

References:

Ali SA, Peffers MJ, Ormseth MJ, Jurisica I, Kapoor The non-coding RNA interactome in joint health and disease. Nat Rev Rheumatol. 2021 Nov;17(11):692-705. doi: 10.1038/s41584-021-00687-y. Epub 2021 Sep 29. PMID: 34588660
Wilson T, Kaur N, Davis J, Ali Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint. J Vis Exp. 2021 Jul 22;(173). doi: 10.3791/62718. PMID: 34369924

* In the early days of television evening news relied on film motion photography. Since there was occasionally not enough time to develop the film before the evening news the anchor would indicate that the film would be available for the 11 PM news by saying at the end of the story, “Film at 11”.

Yes Virginia, There is Science in Ethics

Three years ago, a major journal reviewed a paper discussing an analysis of hidden ethical problems that occur with artificial intelligence (AI) and how those very issues may alter the scientific applications of AI. This article was aimed at AI uses in radiographs, other musculoskeletal applications, and health insurance data as well how the four medical ethics principles define the problem. The reviewing editors were concerned with the lack of “science” in AI and medical ethics and directly rejected the article. The paper was subsequently published in “Ethics in Biology, Engineering and Medicine.” The work was also presented at the Tenth International Conference on Ethics in Biology, Engineering, and Medicine. The combination of science with ethics is such an exciting merge. Science is the compilation of confirmed knowledge and the quest to discover new knowledge. Yet discovery and application can be impinged upon by ethical considerations in study design, data collection, conflicts of interest, inherent assumptions of AI data analysis, learned preexisting biases and the AI amplification of the impact of these factors. AI is emerging as a dominating force in future data use, machine learning and medical decision making. In short ethical considerations in biology, engineering and medicine are gaining their own “science” and the implications are wide spread. The following are some bullet points from over 70 presentations at the referenced meeting.

The morphing of “conflict of interest” into “competing interest.” The beauty of this term is that the researcher or author can list anything that might influence their work wherein they may not perceive this as a conflict of interest. Examples include demands to publish, conflicting teaching loads, and grant specific requirements, amongst many others. This leaves to the reader and editors their view as to whether this would be a true conflict of interest. The aim is a better understanding that naming the conflict is not the same as understanding the influence.

Tolerance for risk, for the practitioner/scientist and for the patient influences decision making process in selecting laboratory studies or surgical procedures. This entails some sole searching in that advice can be textured on the avoidance or acceptance of risk by the practitioner. The choice may leave the patient out of the loop given the practitioner/scientist avoidance or acceptance of risk.

Cross cultural ethics can impinge on international trials. These can present huge ethical problems. Amongst these is the language barrier, belief systems of the subjects, laws of the land and lifetime experiences that may make the options for study participation unintelligible.

Spiritual belief systems are often overlooked in care for patients. Even. an informed consent could be at issue if birth control is part of a study in many religious settings. Use of human tissue can equally be a religious conflict from the get go. The door is not as difficult to open as may appear. The call from the presenter of this paper is the availability of guidance. The absence of this consideration may be an ethical barrier to care.

The definition of dedicated scientist. This includes curiosity and diligence. One survey of scientists named the top ten attributes of a dedicated ethic scientist: duty to society, beneficence, conflict of interest, informed consent, integrity, non-discrimination, non-exploitation, privacy and confidentiality, professional competency, and professional discipline. At the top of another list was honesty.

Artificial Intelligence (AI) is expanding in use. In musculoskeletal medicine that includes diagnostics, virtual reality, and insurance use for hidden or covert rationing of care. This exposes both practitioner and patient to many risks that are hidden in this complex technology. The tool is for our use and to make life better. But we can clearly argue that it has altered human behavior, so the tool is no longer used in ways envisioned. The artifact has forever changed the way we relate to each other and human relations in general. Case in point twitter, face book and even the way google decides what search results to show you are based on prior human behavior with the tool but the actions change what we think of by not showing what others were less interested in. It amplifies some behaviors and extinguishes others – probably never its intent.

There are some thyroid cancers that by virtue of resident time, size, and histologic appearance can be “observed” in contrast to treated with surgery or chemotherapy. The selection of these cases is now defined but the patient must be informed so they can interface with the treatment decision to be shared by them. Here we see how surgical decision making may be impinged upon by the ethical application of mixed data. The risk of malpractice and avoidance of that risk is clearly beneficence for the surgeon. That risk avoidance may affect the potential benefice for the patient by picking a watching and waiting strategy. That strategy may increase the medical malpractice risks for the surgeon. This is most evident in the very high C-section rate in the USA as compared to the rest of the world because the first reason for many medical malpractice cases against OB-GYN surgeons in our country is for delaying a C-section when needed.

Preprint articles are published online prior to peer review. This can introduce advances prior to publication but can lead to misinformation, such as the reports on COVID and hydroxychloroquine. Steps to prevent this include being watermarked as non-peer reviewed, depending on server noting no evaluation of COI or ethics, a link to peer reviewed publication once complete, along with per reviewed literature show prior preprint including a box in the peer review article to show differences, and if never meet peer review. These preprints can equally help and hurt scientific progress and the ethics of their use is a needed ongoing discussion.

Other discussions included ethics in residency training and the conflict between benefice for the patients of only the best surgeon, the teacher, in the room doing the work and the benefits to society by training new surgeons. This is particularly when the student surgeons are learning techniques in order to become the expert surgeons in the future.

Clearly ethics impinge upon every aspect of scientific research particular in the intersection of engineering, biology, and medicine. Developing the science of ethics within scientific research, scientific design, and the practical implication of AI applications to human conditions and treatments is a necessity. This is especially true as the implications of the newer technologies expand with our growing knowledge.

Tracking Down Your Prosthetic Past

With increasing demand for prosthetic joint replacement over the past 2 decades the number of revision surgeries is also increasing. Prosthetic design had expanded to a point that recognition of a prosthetic brand can be extremely difficult. Of revision 10% of the patients have no record of the device used. Waiting until surgery can be very problematic, especially if only one of two components needs replaced. Given the wide use of artificial intelligence (AI) in radiology it is no surprise that investigators are trying to effect it use in prosthetic identification.

For those not involved in artificial intelligence (AI) the concept can be difficult. One working definition of AI/ machine learning is a set of algorithms that give the machine the ability to learn the existing patterns in a given dataset without being explicitly programmed to do so. Machine learning can be divided into two main types: conventional and deep learning. Conventional machine learning methods are mainly concerned with analyzing tabulated datasets. Deep learning methods mainly focus on analyzing imaging datasets. Both of these machine learning methods learn from labeled historical data to predict solutions to questions for the new data presented to the method.

Investigators at Massachusetts General Hospital and Harvard Medical School have developed deep learning models for the first time to detect a failed hip implant design before the revision surgery from radiographic images. They used 252 post-operative radiographic images of three known total hip replacement implant designs to develop an AI model that can automatically identify their type ¹. They were able to properly identify the specific device on 25 new radiographs to the AI model with perfect accuracy. In a subsequent work the same group used 402 radiographs and compared the performance of AI with three board-certified orthopaedic surgeons. They show that the AI model achieved on-par performance with the expert orthopaedic surgeons while being significantly faster [2].

Using training and validation data sets from 1715 patients and 1766 AP radiographs 18 different femoral components after 1000 training epochs by the deep-learning system, the system discriminated the 18 implant models with an area under the receiver-operating characteristic curve of 0.999, accuracy of 99.6%, sensitivity of 94.3%, and specificity of 99.8% in the external-testing data set of 206 AP radiographs³.

In a current article under review the processing time for the identification is 0.2 seconds. From these works it is clear that AI could play a significant role in implant identification for patients without records of an original implant waiting revision.

Unfortunately, the authors have been non respondent to multiple requests to review this synopsis. The overall impact is correct but there may be some errors in the details.

References

BorjaliA, Chen AF, Muratoglu OK, Morid MA, Varadarajan Detecting total hip replacement prosthesis design on plain radiographs using deep convolutional neural network. J Orthop Res. 2020 Jul;38(7):1465-1471. doi: 10.1002/jor.24617. Epub 2020 Feb 11. PMID: 31997411
Borjali, A. F. Chen, H. Bedair, C. Melnic, O. K. Muratoglu, M. A. Morid, and K. M. Varadarajan, “Comparing the performance of deep convolutional neural network with orthopaedic surgeons on identification of total hip prosthesis design from plain radiographs,” Med Phys mp.14705. Jan. 2021
Karnuta JM, Haeberle HS, Luu BC, Roth AL, Molloy RM, Nystrom LM, et al. Artificial Intelligence to Identify Arthroplasty Implants From Radiographs of the Hip. J Arthroplasty. 2021 Jul;36(7S):S290-294.

Another Way of Detecting Periprosthetic Infection: A Mass Spectroscopic Effort

Synovial fluid proteins reflect the response of the joint to its environment, internal challenges, and homeostasis. The capacity to protein clusters and all proteins would provide a comprehensive analysis of joint status to detect periprosthetic joint infection (PJI). Mass spectroscopy (MS) offers such an opportunity. In preliminary work MS detected PJI myeloid nuclear differentiation antigen (MNDA), polymorphonuclear leukocyte serine protease 3 (PRTN3), and lactoferrin (LTF) as promising synovial markers for PJI detection. These three measures were confirmed by individual enzyme link immunoassay (Elisa). To confirm these preliminary results investigators explored the use of parallel reaction monitoring to verify and quantitatively detect specific biomarkers seen in MS generated data. Parallel reaction monitoring (PRM) is an ion monitoring technique based on high-resolution and high-precision mass spectrometry. The principle of this technique is comparable to SRM/MRM, but it is more convenient in assay development for absolute quantification of proteins and peptides. SRM monitors only a single fixed mass window, while MRM scans rapidly over multiple (very narrow) mass windows and thus acquires traces of multiple fragment ion masses in parallel. Thus, MRM is the application of SRM to multiple product ions from one or more precursor ions.

Of 159 synovial aspirations over a 16-month period of time 117 had full data for comparison and the modified Musculoskeletal Infection Society (MSIS) criteria was used to confirm infection. After sample thawing and protein sample preparation, LC-MS/MS analyses were performed. To further quantitively verify the reliability of the selected biomarkers with parallel reaction monitoring cluster analysis and receiver operating characteristic (ROC) curves were used to evaluate the results. These were quantitatively confirmed with parallel reaction monitoring in another patient group who underwent aspiration between August 2018 and January 2019.

Cluster analysis of the differentially expressed proteins showed that the PJI and non-PJI cohorts could be clearly divided into 2 groups. However, 5 patients with PJI and 2 without PJI were mistakenly allocated to the wrong group. MNDA and PRTN3 were further verified as differentially expressed proteins. However, LTF was missed because of the low frequency of identification by MS. Combining results for 50 samples in their previous study to those of the present study a total of 167 samples were analyzed with the random forest algorithm and out-of-bag estimates That showed that PRTN3 and MNDA are the 2 most important proteins to distinguish PJI from those without PJI.

Overall, MS adds a new powerful tool for the diagnosis of PJI that may well lead to a foundation for the clinical application of MS for synovial fluid evaluation in suspected PJI.

Unfortunately, the authors have been non respondent to multiple requests to review this synopsis. The overall impact is correct but there may be some errors in the details.

References

LiR, Song L, Quan Q, Liu M-W, Chai W, Lu Q, Li X, Qin J, Chen J-Y. Detecting Periprosthetic Joint Infection by Using Mass Spectrometry. J Bone Joint Surg Am. 2021 Oct 20;103(20):1917-1926. doi: 10.2106/JBJS.20.01944. PMID: 34097653

Blood and the Meniscus: the Good, the Bad, and the Devastating

Recurrent hemophilic hemorrhage in the knee is a notorious cause of devastating degenerative osteoarthritis. The role of the meniscus in force distribution on articular cartilage is well known. For this reason, repair of peripheral tears of the meniscus in younger individuals is commonly done as an arthroscopic surgery. There is typically little intraarticular bleeding with this procedure. A small blood clot is often used to encourage vascular ingrowth from the periphery. Loss of meniscal function can be brought about by degeneration of the meniscus or changes in is stiffness¹. Investigators were concerned about the negative effects of hemarthrosis, and the effects of blood products sometimes used to improve knee function. They hypothesized that blood will enhance meniscal catabolism in a dose dependent fashion².

Porcine menisci were obtained at an abattoir and 4 mm full thickness explants were taken from the centerline of the medial meniscus (anterior to posterior) and placed in culture for 72 hours with one of the following blood conditions in the media: 0%, 2.5%, 5%. 10%, 20%, and 50% (n= 8). After washing with PBS all explants were placed in meniscal culture media for an additional 72 hours. There were no differences in DNA content or cell metabolism related to blood content in the first 72 hours. Total matrix metalloprotease activity was most pronounced at 10%and the release of sulfated glycosaminoglycans was elevated at 20%.

The presence of varied concentration affects cell catabolism over a 72-hour period. Undefined parameters included concurrent articular cartilage damage and duration and frequency of hemarthrosis. This research has some buried bed side possibilities. For intraarticular fractures articular aspiration, irrigation and/or analgesia would reduce or put off long term consequences? If so, the search for initial post traumatic interventions would have another arrow in the quiver. Another question is whether some individuals are more prone to meniscal damage with hemarthrosis only to progress to subsequent articular cartilage degeneration.

References

Seitz AM, Osthaus F, Ignatius A, Dürselen L. Degeneration alters first the biomechanical properties of human menisci before affecting the tibial cartilage. ORS 2020 Annual Meeting Paper No.0687
Betsch K , Lyons LP , Weinberg JB, Jocelyn Wittstein J , McNulty AL Blood Induces Catabolism of Meniscus Tissue. ORS 2021 Annual Meeting Paper No. 1296

Organ Engineering: When Packaging is as Important as Content, Illuminating

Human induced pluripotent stem cells (hiPSCs) offer tremendous promise for studying human embryogenesis and organ tissue engineering, while avoiding the ethical concerns associated with human embryonic stem cells. Normal embryogenesis, initially involving the formation of an epiblast structure consisting of pluripotent stem cells containing a lumen or cavity, is thought to be affected by physical forces and cell polarity along the developmental tract but the role of the surrounding microenvironment in regulating this process has been unclear. Human-pluripotent-stem-cell-based models of embryonic development have been designed involving culture of the stem cells in 3D matrices. For example, these human pluripotent stem cells self-organize to form lumens when cultured in reconstituted basement membrane (rBM)-based matrices. However, these matrices are difficult to manipulate and the stem cells rapidly lose their pluripotency. In synthetic matrices, it has been found that matrix stiffness, degradability, cell–matrix adhesion ligand type, and ligand density impact intestinal stem cell organoid formation and budding morphogenesis. Recently, it has been found that matrices viscoelasticity plays a major role in regulating various biological processes¹. Viscoelastic materials exhibit viscous dissipation of mechanical energy, and stress relaxation in response to a deformation. In a recent paper, viscoelastic alginate hydrogels with independently tunable stress relaxation (viscoelasticity), stiffness, and arginine–glycine– aspartate (RGD) ligand density in the gel were used to study hiPSC morphogenesis in 3D culture. Fast stress relaxation and high arginine–glycine–aspartate (RGD) ligand density resulted in larger lumens as well as higher rate of lumen formation in the hiPSCs.

Alginate hydrogels were formed by varying the molecular weight of the alginate used to form the gels (280 kDa slow relaxing, 70 kDa medium relaxing and 28 kDa fast relaxing) and ionic crosslinking density while holding the polymer concentration constant². 3 different RGD densities (0, 150 × 10−6 , and 1500 × 10−6 M) were tested. hiPSCs were encapsulated in the gels and their morphogenesis was monitored for 7. In a nutshell, slow stress relaxation and low ligand densities triggered apoptosis whereas high ligand density boosted viability, proliferation, and pluripotency. Further, fast stress relaxation combined with high ligand density promoted lumen formation in hiPSC clusters. On the other hand, hydrogel stiffness did not influence hiPSCs morphogenesis.

Apical markers were found to be distributed selectively at the apical surface suggesting that hiPSCs in lumen-containing clusters are polarized along the apical–basal axis. Lumen formation is accompanied by cytoplasmic translocation of nuclear yes-associated protein (YAP) and is regulated by actomyosin contractility. YAP mediates mechanotransductive response to shear stress, cell shape, and matrix elasticity and critical for hiPSC self-renewal. YAP is initially localized in the nucleus. As the lumens form over time, YAP is translocated to the cytoplasm, where it is necessarily inactive. Reduction of actomyosin contractility reduces translocation of YAP from nucleus to cytoplasm. Actomyosin contractility and actin polymerization, as well as Rac1 activity, play an important role in mediating lumen formation. Nuclear morphology of hiPSC clusters in fast relaxing high weight was similar to the human epiblast cells for nuclear area, perimeter, and circularity. Over-all, matrix mechanics need to be tuned to provide high ligand density and exhibit fast stress relaxation to obtain morphologically similar hiPSC clusters.

These results indicate that manipulating matrix viscoelasticity will play a major role in regulating stem cell morphogenesis and enhance engineered biomaterials manipulated to build organoids.

Reference

Chaudhuri O, Cooper-White J, Janmey PA, Mooney DJ, Shenoy VB.Effects of extracellular matrix viscoelasticity on cellular behaviour. 2020 Aug;584(7822):535-546. doi: 10.1038/s41586-020-2612-2. Epub 2020 Aug 26.PMID: 32848221.
IndanaD, Agarwal P, Bhutani N, Chaudhuri Viscoelasticity and Adhesion Signaling in Biomaterials Control Human Pluripotent Stem Cell Morphogenesis in 3D Culture. Adv Mater. 2021 Sep 9;e2101966. doi: 10.1002/adma.202101966. Online ahead of print. PMID: 34499389

Lungs and Bone Connected? Take a Deep Breath

Chronic obstructive pulmonary disease is often a consequence of smoking. The obstruction causes the alveoli’s elastic behavior to deteriorate, resulting in emphysema. The link between COPD, osteoporosis and intervertebral disc (IVD) on the other hand, is often overlooked. This oversight also contributes to undiagnosed osteoporosis. Deficiency and mutation of α-1 antitrypsin (AAT) protein, encoded by serpinA1, occur in 5-10% of the population, promotes elastin degradation and increases the risk of developing early emphysema. In a recently developed mouse knockout of serpinA1a-e that develops emphysema, we hypothesize that deletion of serpinA1a-e induces bone loss and IVD degeneration.

CRISPR/Cas9-generated 18-week-old male serpinA1a-e global KO mice were compared to age-matched C57BL6/J mice (n=5-6/group). In the lumbar region, L1-3 was designated for histology, L3-5 IVDs and bones were designated for qPCR analysis and L6 vertebrae was imaged by µ-CT to determine vertebral bone structure. IVD morphology and histological score was determined from Safranin-O/Fast green images. For the trabecular analysis, the growth plate was used as a landmark and trabecular bone analysis consisted of the next 30 consecutive slices. Groups were compared by a student t-test and statistical significance level was set at p<0.05.

Compared to controls, deletion of serpinA1a-e reduced Tb.BV/TV by 34% because of trabecular thinning despite greater trabecular number (Fig. 1A, A’). Further, deletion of serpinA1a-e decreased collagen1a1 gene expression, osteoblast number/bone surface and AAT1-positive osteocytes (serpinA1a) in bone. On the other hand, deletion of serpinA1a-e increased the number of TRAP+ osteoclasts (/bone surface). In the IVD, deletion of serpinA1a-e increased IVD degeneration score by ~300% by catabolism of the annulus fibrosus. IVD degeneration by deletion of serpinA1a-e was corroborated by a 2-fold downregulation of notochordal marker KRT19. Lastly, deletion of serpinA1 promoted large cavities in the bony endplate between the IVD and the growth plate.

These data demonstrate that deletion of serpinA1a-e induces bone loss and intervertebral disc degeneration. The bone loss is attributed to trabecular thinning and may be due to increased resorption and reduced bone formation. In the adjacent IVD, deletion of serpinA1a-e may have induced IVD degeneration in the annulus fibrosus by allowing degradation of elastin. Further studies will be required to determine the mechanism(s) underlying the degradation of the IVD by AAT-deficiency.

Pursuing the changes seen in this model will likely lead to a better understanding of bone and IVD changes from AAT-deficiency and inform the link between AAT, osteoporosis and IVD degeneration.

Reference

Bhadouria N, Holguin N. SerpinA1 systemic deletion models the bone loss and intervertebral disc degeneration from genetic emphysema. ORS 2021 Annual Meeting Paper No. 1159; [email protected], [email protected]

Fig. 1: (A) µCT image of L6 vertebrae of control and serpinA1a-e KO mice. (A’) Deletion of serpinA1a-e decreases trabecular bone volume fraction (Tb.BV/TV). CON-Control mice, BV/TV – bone volume/ tissue volume, *p<0.05 (CON vs serpinA1a-e global KO), Scale bar: 1 mm

How Can You Misalign a Knee Replacement, Let’s Count the Ways

Trying to achieve a perfect alignment for knee prosthetic replacement is essential to longer lasting wear of the components. Correcting varus or valgus alignment to the best physiologic position is difficult but essential. However, there are other alignment parameters that can play a very important role on long term wear. To study this, investigators used nine component alignment parameters for volumetric wear in TKR simulations of both force and displacement. They used International Organization for Standardization (ISO) preclinical testing standards for a validated TKR finite element analysis (FEA) wear model combined with a Latin Hypercube Sampling (LHS) design of experiments (DOE) approach. Since this is all computer driven they use the term in silico. A surrogate model was generated for both control regimes using stepwise multiple linear regression. Each DOE considered nine component alignment parameters: 1) internal/external (IE) rotation of the femoral component, 2) IE rotation of the tibial component, 3) femoral component varus/valgus (VV) angle, 4) tibial insert anterior/posterior (AP) position, 5) tibial component rotation in the flexion/extension (FE) axis (tibial slope), 6) location of the femoral center of rotation (CoR) in both the AP and 7) superior/inferior (SI) directions, 8) initial femoral FE angle, and 9) location of the tibial IE axis in the medial/lateral (ML) direction. Alignment changes were created by rotation or translation of the femoral or tibial component, translating the femoral rotation reference point, or translating soft tissue constraint reference points for tibial IE rotation and AP translation in force control. The range of each parameter was chosen to best satisfy the following criteria, in order of priority: 1) values reported in patients, 2) values used in previous studies on alignment, 3) reliability of convergence of the FEA model.

Both force and displacement control models show that rotational mismatch in the transverse plane dominates malalignment related wear. This matches a previous mechanical knee simulator study. For tibial slope the displacement control model predicted decreasing wear when moving away from neutral, but for force control, the trend was increasing wear with increasing posterior slope. Force-control allows the knee to find a new kinematic pattern, as would be the case in vivo.

There are several take away points from this elaborate work. Primary is the significance of rotational alignment and the other is the force control model that may capture the importance mechanical forces. Additionally tibial internal external rotation range was greater than femoral.

Reference

Mell SP, Wimmer MA, Jacobs JJ, Lundberg HJ. Optimal surgical component alignment minimizes TKR wear – An in silico study with nine alignment parameters. ORS 2021 Annual Meeting Paper No. 0041.

Intervertebral Disc Disease: Delivering the Right Message

One of the mechanisms of epigenetics is DNA methylation which has the ability to change gene expression without changing the underlying DNA sequence. Lifestyle factors, such as diet and physical activity may have a strong influence on epigenetic regulation that can last over an extended time. Endurance exercise has been found to have sex dependent effects on epigenetics. Investigators hypothesized that with exercise there would be changes in the intervertebral disk epigenetics. They used a wild type mouse compared to a SPARC-null low back pain model that has associated early degenerative disc changes. SPARC is secreted protein acidic and rich in cysteine.

8-month-old male and female SPARC-null and age-matched control (WT C57BL/6) mice (n=108) were assigned to exercise (n=56) or sedentary (n=52) groups. After 6 months of intervention, voluntary physical activity was quantified using a voluntary running test with a wheel that counted revolutions for 1 hour in a separate cage for each mouse. Locomotor capacity and radiating pain were determined along with epigenetic regulatory gene mRNA expression and global DNA methylation (5-mC: 5-methylcytosine) in IVDs were assessed.

Impact of disc degeneration on epigenetic regulatory genes in the IVDs

Lumbar IVDs from WT sedentary and SPARC-null sedentary mice presented similar global DNA methylation (%5-mC) and mRNA expression of epigenetic regulatory genes in both sexes.

Sensitivity of the epigenetic regulatory machinery to therapeutic environmental change

Exercise reduced LBP-related behaviors and the mRNA expression of Mecp2 in SPARC-null mice. Exercise significantly decreased global DNA methylation in both WT and SPARC-null mice.

Sex-specific effects of exercise on epigenetic regulatory machinery.

mRNA expression analysis revealed that exercise resulted in sex-specific changes in genes related to epigenetic regulation.

Albeit there is no difference between the epigenetics in the wild type and SPARC model changes in biologic response to disc disease can be made by and an epigenetic effector such as exercise. In humans, exercise does improve core strength which is known to improve back pain. However, in mice this does not appear to be a likely factor. Improved function appears to be related to epigenetic changes beneficial to disc function.

Intervertebral discs from exercising mice had reduced mRNA expression of Dnmt3a, Mecp2, and Tet1 in males and Dnmt3b and Mecp2 in females, whereas it increased the expression of Mbd2a in females. Dnmt3b is DNA Methyltransferase 3 Alpha, Mecp2 binds methylated CpGs which is a chromatin-associated protein that can both activate and repress transcription and is related to chronic pain. Tet1 is one of many methylcytosine dioxygenases which play a role in epigenetic translation. In the SPARC-null mice, exercise significantly reduced the mRNA expression of Mecp2 in both sexes. The authors hypothesized that a decrease in Mecp2 expression commits the alteration of global DNA methylation via DNMT-mediated maintenance of DNA methylation.

This research highlights the epigenetic impact of exercise and its effect on chronic LBP. Targeting epigenetic alterations with lifestyle change could have therapeutic impact on IVDs as well as other conditions.

Reference

Kawarai Y, Jang SH, Lee S, Millecamps M, Kang HM , Gregoire S, Suzuki-Narita M, Ohtori S , Stone L. Exercise Attenuates Low Back Pain and Alters Epigenetic Regulation in Intervertebral Discs in a Mouse Model. ORS 2021 Annual Meeting Paper No. 0057

Pain Intensity: Where are the Magnifying Glasses?

Pain intensity varies substantially for a given pathophysiology/noxious stimulus. A smiling patient with a stated pain score 10/10 is a red flag. Unhelpful thoughts (misconceptions) about pain have been measured with the use of questionnaires such as the Negative Pain Thoughts Questionnaire, the Tampa Scale for Kinesiophobia, and the Pain Catastrophizing Questionnaire. Examples of common misconceptions are “hurt equals harm,” “new symptoms are a new pathology,” and “I will not be able to accommodate these symptoms.”

In a study that used cluster analysis to identify mental health phenotypes that might respond to specific health strategies, 2 questionnaires measured unhelpful thoughts regarding pain including fear of painful movement, and the Negative Pain Thoughts Questionnaire. Heightened concerns regarding illness (symptoms of hypochondriasis) were measured using the 5-question version of the Short Health Anxiety Inventory. The 2-question version of the Patient Health Questionnaire and the 2-question version of the General Anxiety Disorder questionnaire were used to measure symptoms of depression and anxiety (distress)^1,2. Higher scores on these instruments indicate greater unhelpful thoughts or distress.

Upper-extremity-specific activity tolerance was measured with use of the Patient-Reported Outcomes Measurement Information System Physical Function Upper Extremity Computer Adaptive Test, pain intensity was measured with use of an 11-point ordinal measure of pain intensity (numerical rating scale), and pain self-efficacy was measured using a 2-question measure.

Using a clustering algorithm, 4 mental health phenotypes were identified in the study population:

Low misconceptions, low distress (77 patients)
Notable misconceptions (36 patients)
Notable symptoms of depression and notable misconceptions (19 patients)
Notable symptoms of anxiety and depression, and notable misconceptions (5 patients)

Patients with low misconceptions and low distress had significantly greater activity tolerance and pain self-efficacy than those with notable symptoms of depression and notable symptoms of anxiety. Patients with notable misconceptions and those with notable symptoms of depression had higher pain self-efficacy than those with notable anxiety, depression, and misconceptions. There was no correlation between phenotype and socioeconomic status or condition type (traumatic conditions, discrete nontraumatic conditions, or nonspecific diagnoses). In a similar fashion there was no correlation to demographics, However, the population was only 137.

Identifying and addressing unhelpful thoughts and distress may help patients interpret their symptoms in the healthiest way contributing to greater alleviation of symptoms and improved capability. There is some debate about the primacy of mind (thoughts/distress) or brain (central nervous system overactivity). There is also debate about regional pathophysiological differences that may be useful targets for treatment, particularly among people seeking care for daily pain as an illness^3,4. While the debates continue and experimental evidence is developed in attempt to resolve the debate, we can move forward with the consistent evidence that modifiable factors such as misconceptions and symptoms of worry and despair are strongly associated with variation in pain intensity, and develop health strategies that incorporate psychological and communications expertise.

Reference

MinerH, Rijk L, Thomas J, David Ring D, Reichel LM, FatehiMental-Health Phenotypes and Patient-Reported Outcomes in Upper-Extremity Illness. J Bone Joint Surg Am. 2021 Aug 4;103(15):1411-1416. doi: 10.2106/JBJS.20.01945. PMID: 34357891
https://orthobuzz.jbjs.org/tag/psychosocial/
Anne-Marie Malfait, MD, PhD, Rachel E. Miller, Richard J. Miller. Basic Mechanisms of Pain in Q4 Osteoarthritis Experimental Observations and New Perspectives Rheum Dis Clin N Am – (2021)
Malfait AM, Miller RE, Block JA. Targeting neurotrophic factors: Novel approaches to musculoskeletal pain. Pharmacol Ther. 2020 Jul;211:107553. doi: 10.1016/j.pharmthera.2020.107553. Epub 2020 Apr 18.PMID: 32311372

A Genome-engineered Bioartificial Implant: In-gene-yes

Antirheumatic biologic drugs have systemic effects due to higher levels of anticytokine agents that do not react to cytokine response. To develop an adjustable delivery system, investigators used CRISPR-Cas9 genome engineering to create a self-regulating synthetic gene circuit in induced pluripotent stem cells(iPSCs). iPSCs were edited to insert either the gene for IL-1Ra (Il1rn) or luciferase (Luc) at the Ccl2 locus, creating an inflammation-sensitive gene circuit that transcribes Il1rn or Luc in response to inflammatory activation of Ccl2 (referred as Ccl2-IL1Ra or Ccl2-Luc cells). The K/BxN murine model of arthritis has an IL-1–dependent spontaneous arthritis in the F1 generation that is observed starting from 3 to 4 weeks and progressing to advanced disease at 7 to 8 weeks of age. Comparing Ccl2-Luc cells (control) to Ccl2- IL1Ra cell (active) intraperitoneal cell injections had some remote effect on this model but that dissipated over a short time.

To maintain the cells in vivo for prolonged periods of time, tissue-engineered bioartificial implants were created using Ccl2–IL-1Ra and Ccl2-Luc iPSCs seeded onto three-dimensional woven poly(e-caprolactone) scaffolds. These cells were differentiated over 21 days into chondrocyte-like cells that produced a proteoglycan-rich matrix. The cells rapidly sensed and responded to IL-1a stimulation by producing IL-1Ra. A second set of cells were engineered to produce soluble TNF receptor 1 (sTNFR1). Implants behaved similarly in vitro. The cell-secreted anticytokine biologics suppressed the response to IL-1a or TNF-a stimulation, reducing mRNA levels for proinflammatory mediators Il6 and Ccl2, and for matrix metalloproteinases, Mmp9 and Mmp13 as compared to the control Ccl2-Luc construct.

In summary, these constructs remain responsive to levels of IL-1 and TNF-a over a period of 29 days. In a similar fashion, over a shorter period of time active mice had less swelling, better function/pain scores/ and less inflammation on histology. Most remarkable was the complete elimination of bone erosion.

K/BxN F1 mice have a marked inflammatory environment and the finding that these implants survived and functioned appropriately over 5 weeks in vivo is remarkable. With further optimization these constructs could reduce the need for frequent injections of biologic drugs in patients with RA.

Reference

Choi Y-K, Collins KH, Springer LE, Pferdehirt L, Ross AK, Wu C-L, Moutos FT, Harasymowicz NS, Brunger JM, Pham CTN, Guilak F. A genome-engineered bioartificial implant for autoregulated anticytokine drug delivery. Sci Adv. 2021 Sep 3;7(36):eabj1414. doi: 10.1126/sciadv.abj1414. Epub 2021 Sep 1. PMID: 34516920

Bone Metastases: Giving it Your Best Shot

The treatment of metastatic bone cancer has several options, all with drawbacks including local recurrence. Radiotherapy can deliver good pain relief, but surgery is often required to reinforce weakened bone. The effect of bone‐modifying agents is influenced by the bone response to a specific cancer. Similarly molecular targeted drugs are aimed only at specific cancers. Another potential targeted treatment would be the delivery of a concentrated agent into a metastatic lesion wherein the delivered drug remains within the target and the vehicle is not toxic.

Researchers designed a delivery vehicle of gelatin hydrogel microspheres (GMs) with injectable diameters ranging from 100 to 180 μm in a double deionized water swollen state. Cisplatin can be added to these hydrogels and is released in therapeutic levels only at the site of the tumor. Different levels of cross linking were tested in vitro to derive the most effective release sequence. To test this in vivo, a 29-gauge needle was used to deliver 10⁶ human breast cancer cells to the proximal tibial epiphysis of BALB/c nude mice aged 5 weeks.

After 4 weeks mice were assigned to the following four groups of 10 mice: group S had local administration of GM‐PBS; group P had intraperitoneal injection of free cisplatin solution (2 mg/kg); group F had local administration of free cisplatin solution (2 mg/kg); and group G had local administration of GM‐cisplatin (2 mg/kg, calculated for cisplatin). Intraosseous injections were performed using a syringe with a 26G needle. 5 mice of each group were sacrificed at 2 or 4 weeks. Extraosseous tumor volume was measured by an in vivo imaging system using 465 nm excitation and 520 nm absorption. Side effects were measured in weight loss and nephrotoxicity measured at different time points.

The apoptotic response to injection of free cisplatin and GM cisplatin with degradation was similar. At two- and four-weeks tumor radiant efficiency was greatest in the GM-PBS group, significantly less in the local and systemic cisplatin groups and less by an order of magnitude in the focal GM-cisplatin group. Bone volume fraction was greatest at 2 and 4 weeks in the GM-cisplatin mice. By histology tumor amount was decreased and apoptosis (TUNEL) increased in the GM-cisplatin mice. There was a significant increase of BUN and non-significant increase of creatinine in the intraperitoneal cisplatin group. There was an early significant weight lost in the intraperitoneal cisplatin group but by day 28 the groups were similar.

The success of this method relied on the elimination of harmful products such as glutaraldehyde in the gel production. Apoptosis is the mechanism of action for cisplatin and the gradual release in sufficient concentrations extended the benefit locally without harmful side effects. Expansion of this research to bedside will require evaluation of effectiveness with a variety of tumors and refinement of the intraosseous delivery system.

Reference

Kanda Y, Kakutani K, Yurube T, Zhang Z, Miyazaki S, Kakiuchi Y, Takeoka Y, Tsujimoto R, Miyazaki K, Kawamoto T, Takada T, Hoshino Y, Tabata Y, Kuroda R.J. A novel topical treatment for bone metastases using a gelatin hydrogel incorporating cisplatin as a sustained release system. Orthop Res. 2021 Mar;39(3):525-535. doi: 10.1002/jor.24874. Epub 2020 Oct 29.PMID: 33030789

A Systems Based Approach to Disease Modification in OA: The Key Might be How You Look at It

Disease modification for osteoarthritis remains elusive. One problem is that treatment is often sought when damage is already evident and opportunities to prevent tissue failure may have passed. Putting out barn fires is best done before the floor has collapsed. Anterior cruciate ligament patients do not typically have significant articular cartilage damage wherein early MRIs often reveal “bone bruises” that reflect a region of high articular cartilage impact. Not all OA develops from sudden impact. To help identify a means of defining early OA a “Systems Based Approach to Developing Osteoarthritis Prevention Strategies” has been proposed¹. This approach considers biology, mechanics, and structure.

A UTE pulse sequence detects T2 signal much sooner after radio frequency excitation than standard T2 imaging. This allows visualization and measurement of T2 signal from tissues with fast T2 decay such as menisci and deep articular cartilage which appear as black signal voids on standard T2 MRI. This is particularly true for articular cartilage which varies widely by region and depth. The use of ultrashort echo times to assess T2* relaxation is abbreviated UTE-T2*.

Potential bed side applications include detecting early OA and assessing interventions that may modify the long-term course of this disease. Using ACL injured younger patients UTE-T2* was tracked in uninjured versus recently injured patients 2 years after anatomic ACL reconstruction¹. There were significant UTE-T2* changes in deep articular cartilage as well as the meniscus. These early elevations were reversed in about half of patients by 2 years after anatomic repair. Looking at the mechanical part of the systems-based approach UTE-T2* correlated to knee adduction moment and knee center of rotation which is altered in ACL injury and with varied partial correction after ACLR. Patient related outcomes likewise correlated to UTE-T2* 2 years after ACLR. Biomarkers of collagen cleavage seen in cartilage degradation (C1,2C) and of aggrecan synthesis in cartilage (CS846) are elevated in early radiographically apparent OA. Following a 30-minute walk 2 years after anterior cruciate ligament repair these same makers were found to correlate with UTE-T2*. All these studies highlight the concept of a “Systems Based Approach to Developing Osteoarthritis Prevention Strategies”.

Unrelated to this research, detecting the long-term effect of high (basketball) versus low impact (swimming) sports on early OA is another area where UTE-T2* could prove valuable². To date these studies have revealed no differences but may be too early in life and/or too short in time to reflect a longer-term difference.

References

ChuCR, Williams AA, Erhart-Hledik JC, MR, Qian Y, Andriacchi Visualizing pre-osteoarthritis: Integrating MRI UTE-T2* with mechanics and biology to combat osteoarthritis-The 2019 Elizabeth Winston Lanier Kappa Delta Award. J Orthop Res. 2021 Aug;39(8):1585-1595. doi: 10.1002/jor.25045. Epub 2021 Apr 29. PMID: 33788306
Argentieri EC, Gold G, Majumdar S, Potter HG, Koff MF. Comparison of Meniscal T2* Metrics in Elite Basketball Players and Swimmers. ORS 2021 Annual Meeting Paper No. 1219. https://www.ors.org/Transactions/67/1219.pdf

Next Generation Sequencing: A Better Way to Track Down the Unusual Suspects

For over a century the gold standard for determining the causative agent of infection has been bacterial or fungal culture. There are number culture conditions and techniques which can lead to variation of results between laboratories. Culture techniques are impacted by fastidious organisms, time for results, and difficulty in obtaining appropriate specimens.

The use of DNA bacterial signatures has been under study for over a decade. In a more recent study, the advantage of the PCR based method has high sensitivity regardless of cell viability, fastidious growth, or previous antibiotic treatment¹. However, new systems such as the Sanger sequencing have become available. The critical difference between Sanger sequencing and next generation sequencing (NGS) is sequencing volume. While the Sanger method only sequences a single DNA fragment at a time, NGS is massively parallel, sequencing millions of fragments simultaneously per run. This high-throughput process translates into sequencing hundreds to thousands of genes at one time. NGS also offers greater discovery power to detect novel or rare variants with deep sequencing.

There are multiple approaches to apply NGS in the clinical setting but at present, amplicon sequencing has seen the most widespread adoption in the clinical laboratory because the cost per base has continued to fall and data are available in a clinically actionable time frame. Amplicon sequencing uses primers designed to amplify by PCR specific regions of highly conserved genes, typically the 16S rRNA gene and the ribosomal internal transcribed spacer which are the universal genetic signatures for bacteria and fungi respectively. The resulting PCR products (amplicons) are then sequenced to determine from which species that particular amplicon came. The process of reliably assigning taxonomy to the millions of sequence reads output by the sequencer is dependent on an accurate database of sequences with known taxonomy. Public sequence repositories are vast but contain many duplicate and poorly annotated sequences thus, diagnostic testing is should be limited to the use of curated databases.

The beauty if these systems is the short time it takes to achieve the results, the relatively low cost ($100.00), and more sensitive and specific relative to culture methods. NGS has detected signal in 9 of 11 culture negative periprosthetic joint infections. NGS can find signals from synovial fluid sample of PJI in both culture negative and positive samples. NGS had also proved effective for more fastidious bacterial such as C. acnes (formerly Propionibacterium acnes) and for detecting polymicrobial infections that would otherwise be missed.

References

Moshirabadi A, Razi M, Arasteh P, Sarzaeem MM, Ghaffari S, Aminiafshar S, Hosseinian Khosroshahy K, Sheikholeslami FM. Polymerase Chain Reaction Assay Using the Restriction Fragment Length Polymorphism Technique in the Detection of Prosthetic Joint Infections: A Multi-Centered Study. J Arthroplasty. 2019 Feb;34(2):359-364. doi: 10.1016/j.arth.2018.10.017. Epub 2018 Oct 25. PMID: 30471785
https://www.aaos.org/aaosnow/2021/jul/research/research02/

A Broad Sweep with a Useful Finish in Pediatric Musculoskeletal Infections

Musculoskeletal infections (MSKI) are common in the pediatric population, accounting for nearly 1 in every 10 consultations to pediatric orthopaedic providers at a tertiary care centers. Pediatric MSKI is a major cause of morbidity in the pediatric population due to increased risk of adverse outcomes. Rapid treatment is required to avoid these adverse outcomes. Therefore, clinicians must have an arsenal of tools that allow for efficient diagnosis, determination of the location of the infection and its severity. Currently, physicians use physical exam, imaging and measurement of the infection induced acute phase response (APR) to determine the appropriate antibiotics and surgery that will eradicate the infection.

In caring for patients with pediatric MSKI, C-reactive protein (CRP) is the most measured infection provoked APR biomarker. CRP is an acute-phase hepatic protein that increases up to 1,000-fold following interleukin-6 secretion by macrophages and T cells. It is a member if the pentraxin family of proteins and increase with injury, infection, and tissue death. Its clinical utility is well established for prognosticating the presence of an infection and its severity. However, there has been no studies establishing the dynamics of CRP in the near-term assessment that indicate the success or failure of infection treatment. Investigators wished to determine if comparing CRP levels prior to surgical intervention to early post treatment levels would be predictive of treatment success or need for escalation of care.

Of 639 patients admitted for evaluation for MSKI over a six-year period. After eliminating known infections such as osteomyelitis authors reviewed 135 who underwent surgical intervention and had both preoperative CRP values and postoperative CRP values at ~48 hours following surgical intervention. Normal was 3.0 mg/L. These patients were divided into four groups: Group 1 presurgical CRP <90 mg/L with subsequent decrease following surgery (n = 46), Group 2 presurgical CRP <90 mg/L with subsequent increase following surgery (n = 24), Group 3 presurgical CRP >90 mg/L with subsequent decrease following surgery (n = 48), Group 4 presurgical CRP >90 mg/L with subsequent increase following (n = 17). In group 1, only 2% of patients needed escalation of care, the median LOS was 3.8 days, and 6.5% of patients experienced adverse medical and musculoskeletal outcomes. Alternatively, 20.8% of patients in Group 2 necessitated escalation of care, resulting in a median LOS of 4.9 days, and an adverse outcome rate of 12.5%. In group 3, 33.3% of patients required escalation of care, the LOS was 6.8 days, with an adverse outcome rate of 35.4%. Finally, Group 4 fared worse with 70.6% of patients requiring escalation of care, leading to a median LOS of 8.8 day, and an adverse outcome rate of 41.2%. Additionally, there was a significant variance of those with disseminated disease (p=0.001) and methicillin resistant staphylococcus (MRSA) occurring in Groups 3 and 4. Likewise, there were differences in length of stay, need for escalation of care, and complications.

The implication from this work indicates that serial assessment of CRP has clinical utility in determining the adequacy of surgical and medical treatment afflicted with MSKI. Together, pre- and post-operative CRP measures can be used as a guide to help assure appropriate care and reduce adverse outcomes that can alter lifetime function.

References

HuangZY, Huang Q, Wang LY, Lei YT, Xu H, Shen B, Xing Pei Normal trajectory of Interleukin-6 and C-reactive protein in the perioperative period of total knee arthroplasty under an enhanced recovery after surgery scenario. BMC Musculoskelet Disord. 2020 Apr 21;21(1):264. doi: 10.1186/s12891-020-03283-5. PMID: 32316949
Stephanie N. Moore-Lotridge SN, Daryoush JR, Wollenman CC, Gibian JT, Johnson SR, Thomsen IP, Schoenecker JD. Can CRP Predict the Need to Escalate Care After Initial Debridement for Musculoskeletal Infection? ORS 2021 Annual Meeting Paper No. 0052

Hip Capsular Stability: Reining in the Edges

In total hip arthroplasty (THA) reconstruction of the capsular ligaments has been found to be essential in prevention of post-operative hip dislocation. This is particularly true for the posterolateral approach. In joint preservation surgery and THA loss of capsular restraint may lead to joint edge loading to articular cartilage or prosthetic wear. To study this, the in-situ stabilizing contributions generated in the native hip ligaments were quantified for force contribution of the capsule at extreme range of motion ROM and to examine their ability to prevent edge loading.

Six male and three female cadaver hips were tested for the rotational constraint contributions of the lateral and medial iliofemoral (anterior acetabular rim directed inferiorly to femur), ischiofemoral (posterior ischium directed medially to femur), and pubofemoral ligaments (anterior acetabular directed medially and inferiorly to femur). Internally and externally rotated positions in abduction, neutral and adduction at four flexion angles (EXT, F0, F30, F60, F90, FLX) were tested for a total of 36 positions. The cadaver data set was used to account for ligament attachments and directions to generate a force vector response for each of the 36 positions.

Using the torque contribution data from the cadaveric study and ligament moment arm prediction, the functioning ligament forces at 5 Nm of torque were evaluated. The force vectors generated were 3 dimensional relative to joint center and expressed in pelvic coordinates relative to acetabular articular surfaces.

Particularly in adduction and internal rotation when in extension the medial and lateral iliofemoral ligaments had the highest net force contribution. In flexion 90 degrees the highest contribution to restraint at adduction and internal rotation, a position common to THA dislocations, was the ischiofemoral ligament. The iliofemoral and ischiofemoral ligaments are important contributors for maintaining femoral head centralization in the extremes of motion.

This research highlights the significance of the need for meticulous capsular repair regardless of approach in total joint arthroplasty. Future research will be required to evaluate the relative role of these capsular ligaments in response to varying joint morphologies and mechanics as seen in pathologies such as femoralacetabular impingement and hip dysplasia. Additionally, more research will be required to determine how these roles are changed with the degradative process in the hip joint as an organ and how does that direct capsular repair in advanced stages of the disease that leads to THA.

Reference

Karunaseelan KJ, Muirhead-Allwood SK, van Arkel RJ, Jeffers JRT. The Capsular Ligaments Stabilise the Hip Joint at Extreme Ranges of Motion. ORS 2021 Annual Meeting Paper No. 0266

Post Traumatic Osteoarthritis: Salvaging Joints in That Golden Hour

During the Vietnam war the concept of the “golden hour” in trauma was born. Changes in field intervention and helicopter transport from field to aid station rose the initial survival rate to 99%. The concept of the golden hour is controversial¹. However, the long term mental and physical symptoms remained. In the past two decades there has been an equal focus on the “golden hour” in traumatic joint OA. Terminology is a bit of a barrier. Post traumatic OA can include repetitive trauma. An example of repetitive is a sudden burst of activity such as marine corps training. Another would be an anterior cruciate ligament injurie in the knee. Unlike knee PTOA, greater than 90% of ankle OA is post-traumatic². Evidence suggests the magnitude of the articular trauma at the time of initial injury dictates development of ankle PTOA. Severe ankle sprain is the #1 sports and military non-combat injury and is highly associated with end-stage OA. For the purposes of this research, PTOA is a result of a sudden impact event leading to immediate articular cartilage response. It is known that mitochondrial response to impact injury is immediate and results in chondrocyte apoptosis or death which may, in turn, boost a joint wide cytokine storm.

Twelve 2 – 5-year-old horses had an arthroscopically delivered cartilage injuries done by a tamp at three sites of both tali³. This models more closely ankle impact injury and described elsewhere⁴. 6 had immediate injection with SS-31 and 6 with saline³. Synovial fluid was collected at initial surgery before injury and then 1, 2, 4, and 6 weeks after the procedure. SS-31 is a synthetic tetrapeptide elamipretide that improves mitochondrial function by unknown mechanisms. After sacrifice at 6 weeks RNA was extracted from synovium and osteochondral samples,

Synovial fluid CPII (protein marker of type II collagen synthesis) and PGE2 (marker of inflammation) were elevated in control at 1 and 2 weeks compared to treated with lower elevations. Neither the collagen cleavage product C2C or GAG differed significantly at all time points between the two groups. Prostaglandin E synthase was increased in injured versus not injured cartilage and in synovium at 6 weeks compared to synovium taken at arthroscopy. Cardiolipin synthase 1 (an inner mitochondrial membrane phospholipid essential for mitochondrial function) and heme oxygenase 1 (an antioxidant), and runt-related transcription factor 2 (increased in cartilage degradation) expression was decreased in the synovium of treated joints.

Translating this into human trials would be difficult since the degree of injury is hard to calibrate in severe ankle sprains. However, it would be this type of population that might present an opportunity to determine short- and long-term modification of human PTOA with SS-31 and similar agents⁴.

References

Tallon The “golden hour” paradigm. Acad Emerg Med. 2002 Jul;9(7):760; author reply 760. doi: 10.1111/j.1553-2712.2002.tb02159.x.
DelcoML, Kennedy JG, Bonassar LJ, Fortier Post-traumatic osteoarthritis of the ankle: A distinct clinical entity requiring new research approaches. J Orthop Res. 2017 Mar;35(3):440-453. doi: 10.1002/jor.23462. Epub 2016 Nov 8. PMID: 27764893
Delco ML, Seewald LA , Cercone M, Wells KV, Fortier LA. Mitoprotective Therapy Reduces Inflammation and Prevents Cartilage Turnover in an In Vivo Model of Posttraumatic Osteoarthritis. ORS 2021 Annual Meeting Paper No. 0065.
Delco ML, GoodaleM, Talts JF, Pownder SL, MF, Miller AD, Nixon B, Bonassar LJ, Lundgren-Åkerlund E, Fortier Integrin α10β1-Selected Mesenchymal Stem Cells Mitigate the Progression of Osteoarthritis in an Equine Talar Impact. Am J Sports Med. 2020 Mar;48(3):612-623. doi: 10.1177/0363546519899087. Epub 2020 Jan 31. PMID: 32004077

Knee Osteoarthritis: When is it Not a Drain?

Post traumatic osteoarthritis (PTOA) is marked by a single trauma that can be associated with both an immediate and on long-term effects on cartilage homeostasis. Most interventions target the biochemical response with other biochemicals. Chondrocytes are mechanosensitive and may respond to specific intensity and frequency of forces to improve both venous and lymphatic circulation which in turn may improve trafficking of cytokines and cleaved extracellular matrix products. Investigators hypothesized that appropriate levels of exercise could reduce inflammation, decrease degradations, improve function, and reduce pain and that these would be associate with changes in vascular and lymphatic clearance.

There were four groups (N=6-8) of adult 3 month old (equivalent to ~13 year old human) male rats: medial collateral ligament section (MCL = sham) or medial meniscal transection (MMT = injured) rats were divided into exercise versus no exercise. By the time PTOA developed (21 days post-surgery in meniscal transection group) the exercise groups had treadmill exercise 5 days a week for three weeks. The end point was 6 weeks after surgery. Gait was assessed at the 6 week end point. Micro CT was used to compare cartilage loss and osteophyte size in the medial joint. The latter assessment was subjected to partial least squares discriminant analysis (PLSDA) to characterize whole joint morphology, summarized by a clustering of the measurements by the first latent variable (LV1). Morphological variables included mineralized osteophyte volume, prevalence of exposed bone, cartilage surface roughness, lesion volume, and cartilage attenuation.

MMT animals showed significant joint damage compared to sham controls, as indicated by an increased LV1 score. Exercise significantly improved joint morphology in the MMT animals, as indicated by a decrease LV1 score, with significant improvements in cartilage surface roughness, proteoglycan content, and osteophyte size; yet these remained elevated compared to the sham controls. While there were no significant differences in individual spatiotemporal parameters of gait, MMT animals had a significantly higher LV1 gait score than sham animals, shower an altered gait pattern of wider hind step widths, decreased duty factor of the involved limb, and decrease spatial symmetry. Exercise corrected this gait dysfunction, returning the LV1 score to sham levels.

In a separate experiment on non-surgical (naive) rats of the same age, near-infrared imaging was used to evaluate the effect of exercise on the clearance of intra-articular injections of NIR dye by venous (carboxylate modified IRDye800CW = red dye) and lymphatic (IRDye680RD with 40 kDa polyethylene glycol = 40 kDa PEG-NIR) mechanisms. Immediately after injection, 4 rats were treadmill exercised at 10 m/min for 30 minutes while 4 other rats remained within their cages. NIR images were collected at multiple time points up to 28 hours. Exercise increased lymphatic, but not venous, clearance of the knee joint.

Moderate exercise is a vital clinical intervention to control OA pain and improve joint function, regardless of the cause of the disease. However, the mechanism of this therapy remains elusive. The effect of exercise on human PTOA would be difficult to assess since there is a wide spectrum of injury and disease types, as well as complex clinical histories, genetic variability, etc. This preclinical study lays the groundwork to explore potential therapeutic mechanisms of exercise and could provide clinical opportunities for targeted enhancement of intervention. A practical approach would be to assess the effect of exercise on lymphatic clearance in different stages of the disease.

Reference:

Kaiser CJ, Bernard F, Pucha K, Raval S, Fulton T, Anderson S, Dixon B, Willett N. Mild Exercise Alleviates Post-Traumatic Osteoarthritis in Part by Expediting Lymphatic Joint. ORS 2021 Annual Meeting Paper No. 0066

Electron Microscopy for Bone Infections: Cutting Edge No Matter How You Slice It

Acute osteomyelitis is typically an infection seeding bone marrow due to systemic infection. However, in its chronic form cortical bone is often involved, leading to cell and bone death and eventual sequestration of the infected portion of bone. With a half-life of 25 years, osteocytes have endocrine function, provide calcium homeostasis, and have other feedback roles. On average they are 5 µm wide and 15 µm long. Osteocytes have 40 to 60 lacuna-canalicular extensions measuring 100-600 nm in width and a cell-to-distance of 20-30 µm. Determining the process of events in chronic bone infection is important to understand specific tissues at risk, why some very nearby components are not involved, and give a window into therapeutic interventions. In chronic infection involving cortical bone there is invasion of bacteria into osteocyte lacuna-canalicular networks (OLCN). The most common bacterial species is staphylococcus aureus with convert to rod shape in OLCN. Bone is very hard to process for transmission electron microscopy (TEM) as decalcification is required. Serial section electron microscopy (SSEM) involves a continuous collection of ultra‐thin sections onto a reel of Kapton tape using an automated tape‐collecting ultramicrotome. Each section is imaged sequentially using a scanning electron microscope (SEM) fitted with a backscatter electron (BSE) detector to produce an image “stack”. In soft tissues 25‐nm thin sections can be cut and imaged at a pixel size of 20–30 nm producing a stack of more than 33,000 images to reconstructing a 1 mm³ image. Investigators modified the technology for OLCN definition that would help determine why S. aureus infection of bone is incurable, and why is the efficacy of antibiotic‐loaded bone cement so limited.

Archived samples were used wherein a staphylococcal impregnated pin was inserted into the tibial canal and left if place for 14 days before sacrifice. Bone was specifically fixed, demineralized, pin pulled, and 5 µm thick paraffin sections were used to determine areas of interest. Immunogold labeling was used on 60 nm sections to determine the presence or absence of S. aureus and S. agalactiae. Once a 0.5 µm region of interest was the tissue was embedded in epoxy and 60 nm sections obtained with an ATUMtome system and collected on Kapton tape mounted on silicon wafer discs that were post-stained with uranyl acetate and lead citrate, and carbon coated. Using SEM it is possible to directly stack images take from the tape sections. A total of 264 60 nm sections were constructed into a 3D 15.83 µm X 40 µm X 45 µm block.

Both staphylococcal species are seen in diabetic foot conditions. The sequential images were aligned and segmented to allow for 3D rendering of the ROI. Using this technology in a 3D fashion this is the first reported hard tissue application to confirm the presence of each species and to detect the presence of unaffected adjacent canaliculi next to infected canaliculi.

Using this novel technique the authors hypothesize that viable osteocytes in some of the osteocyte‐lacuno canalicular networks respond to and can resist infection. Future research will be required to explain the paradox of adjacent uninfected canaliculi in life‐long deep bone infection in patients with chronic osteomyelitis.

Reference

Bentley KLdeM, Galloway CA, Muthukrishnan G, Echternacht SR, Masters EA, Zeiter S, Schwarz EM, Leckenby JI. Emerging electron microscopy and 3D methodologies to interrogate Staphylococcus aureus osteomyelitis in murine models. J Orthop Res. 2021 Feb;39(2):376-388. doi: 10.1002/jor.24968. Epub 2021 Jan 13. PMID: 33377538

Speeding Up Fracture Healing: Special Delivery

The first phase for healing after a fracture is the inflammatory phase release of prostaglandins and bradykinin within minutes. After several days this leads to the induction of new vasculature enhanced by a hypoxic environment that follows injury. Unfortunately, injury severity or concurrent disease such as type I diabetes can retard this early process. With the development of the vasculature in the first days to several weeks after a fracture oxygen supply improves. With the interplay of perivascular cells and mesenchymal stem cells vascular endothelial growth factor, secreted protein acidic and rich in cysteine (SPARC), platelet derived growth factor BB (PGFBB) and thrombin play roles in enhancing vasculature. The use of these angiogenic factors can enhance fracture healing. However, when delivered therapeutically in systemic doses sufficient to maintain therapeutic levels this can be toxic to other tissues and encourage vasculature formation outside of the site of trauma. A way to systemically deliver targeted fracture healing with these agents, or their memetics VEGF(QK), SPARC (SPARC 113), PDGF-BB(PBA2-1c ), and TP508(Thrombin), is to attach them to an acidic oligopeptide bone fracture-targeting drug delivery system that localizes attached drugs to the exposed hydroxyapatite at the site of bone fracture¹.

These conjugates were tested on endothelial cells to assess their activity. Once confirmed, the conjugates were tested in an Einhorn midshaft-stabilized femur fracture model in 12-week-old healthy Swiss Webster mice and CD1-IGS mice along with a beta cell cytotoxin streptozotocin (STZ) induced type 1 diabetes mice². Following fracture and fixation 10 mice per group were dosed daily for three weeks with one of the 4 conjugates. CT and 4 point bending testing was done at the end of the 3 weeks.

The targeted conjugation did not appear to interfere with receptor binding for any of the 4 agents. The best performing targeted angiogenic factor was the VEGF memetic QK which is a 15-amino acid synthetic peptide that binds both receptors VEGFR-1 and -2. There was a 180% increase in BV/TV of the fracture callus relative to saline control and a 242% increase in max load relative to the saline treated control. The induced diabetic mice also had a 62% increase in BV/TV, and 102% increase in max load. Liver, kidney, and ipsilateral femurs showed no adverse effects.

This methodology for targeted angiogenic delivery has the additional advantage of exploring dose and time variations based on what is known on normal healing levels. The other opportunity is investigating compounds other than angiogenic factors as therapeutics for bone fracture repair.

Reference

https://www.sciencedirect.com/science/article/abs/pii/S0168365920305630?via%3Dihub
Nielsen JJ, Low SA, Mbachu E, Chen C, Trigg L, Nordyke K, Tremby M, Low PS. Improved Bone Fracture Repair through Targeted Delivery of Angiogenic Agents. ORS 2021 Annual Meeting Paper No. 0596.

How Ageing Affects Achilles Tendons: When Push Comes to Shove

Achilles tendon injuries and disorders occur through a spectrum of ages and demands. Although macroscale mechanical properties decline with age less is know about the multiscale structural properties. To investigate this, researchers used rats aged 1, 8, 19, and 28 youth to advanced age on multiscale mechanical and structural properties of Achilles tendons and to identify the relationship between hierarchies.

Tissue Harvest and High Frequency Ultrasound Imaging (HFUS):

After sacrifice tendons were harvested for cross sectional area and echogenicity, a measure of collagen fiber linearity. With age, cross sectional area increased, and echogenicity decreased at 8 months but no significant difference comparing 8, 19, and 28 months to each other.

Multiscale Mechanics

Microscale: Nanoindentation with a flat tip devise measured storage (G’) and loss (G”) moduli, and tan δ. Tan δ expresses the out-of-phase time relationship between an impact force and the resultant force that is transmitted to the supporting body. A smaller tan δ indicates greater elasticity. Younger tendons had lower G’ and G’’ but higher Tan δ suggesting higher viscosity in younger tendons. Macroscale: Embedded legs were used for tensile testing. Younger tendons had lower toe region and linear modulus, faster stress relaxation, a higher |E*|, and a lower tan δ. On regression modeling macrostructural properties strongly predicted microstructural properties.

Alginate culture: Achilles tendon cells were expanded for one week and then placed in alginate gels of varied RGD induced cross linking and calcium concentration to tune varied hydrogel G’ and tan δ. Staining was done for F actin, staining for shape, and Edu staining (DNA synthesis) for proliferation. Modulus and viscoelasticity affected the nuclear aspect ratio (nAR) of encapsulated cells regardless of age. 1-month-old rats had increased modulus and tan δ leading to increased nAR. However, 28-month-old rats cells had increased modulus and tan δ with concomitant decreased nAR. Cell proliferation increased in stiffer gels from 28-month-old animals, but not 1-month-old animals.

Wherein tuning viscoelastic properties of hydrogels has been shown to modulate behavior of mesenchymal stem cells this study shows that tendon cells also respond to substrate stiffness and viscoelasticity and that this response is age dependent at the cellular level. Relative to age, future research may investigate protein expression based on the mechanical and the wide variation that is apparent within each age group. Another question would be if that is predictive of failure.

Reference

Freedman BR, Knecht RS, Tinguely Y, Mooney DJ. Aging and Viscoelasticity Affect Multiscale Tendon Properties and Tendon Derived Cell Behavior. ORS 2021 Annual Meeting Paper No. 0240.

Healing Osteochondritis Dissecans Lesions: Time to Get Ossified

Juvenile osteochondritis dissecans of the knee (JOCD) usually occurs in children between the ages of 8 and 18. Typically, these are thumbnail sized lesions created as a result of ischemic necrosis of epiphyseal (subarticular) cartilage and subsequent failure of endochondral ossification. With time, the necrotic epiphyseal cartilage, also called the progeny lesion, may either heal by becoming a part of parent subchondral bone via the process of endochondral ossification, or progress to clefting and eventual creation of an osteochondral fragment. It is ossification of the progeny lesion, as well as the interface between the parent bone and the progeny lesion, that is critical to spontaneous resolution. A recently introduced JOCD staging system based on short echo time imaging follows the natural history of JOCD and divides lesions into five stages with I representing early lesion, IV healed lesion and V loose body¹. There are various MRI classification systems for the evaluation of lesion instability that can assist in the clinical decision for surgical intervention versus conservative, non-operative treatment². Unfortunately, conservative treatment of stable lesions fails in up to 50% of patients, and while predictors such as age, lesion size, and MRI features seem to be associated with treatment success, it is not yet possible to reliably predict which stable lesions will heal without surgery and which will later require surgery. Clinical MR imaging typically uses long echo times (TE>20 milliseconds) and therefore cannot reliably distinguish between osseous, fibrous and cartilaginous tissues found within JOCD lesions. T2* mapping uses shorter echo times that also capture signals from trabecular bone marrow and permit the calculation of the T2* relaxation time constant, which is correlated with tissue microstructure. T2* mapping has previously been used to evaluate microstructure and composition of cartilage and subchondral bone. Investigators used T2* mapping to evaluate changes in the composition of JOCD lesions (i.e., progeny lesion, interface, and parent bone) in the distal femur of patients with JOCD (≤ 18 years) at different disease stages.

25 subjects (16 males; average age, 12.1 years) with 34 JOCD-affected knees and 13 lesion-free knees were evaluated and all lesions were graded Stage I to V according to the JOCD staging system¹. On standard imaging 11 of 34 lesions were deemed as unstable and treated surgically. With an acquisition time of 7 minutes, six echo times from 2.6 to 17.4 milliseconds were obtained. Median T2* values and volumes were measured in four 3D regions on multiple slices: within the progeny lesion; in the parent bone adjacent to lesion; in the interface situated between the progeny lesion and the parent bone; and in the control bone on the opposite condyle (normal region).

Only 5 of the 34 lesions were asymptomatic. There were 5 stage I, 13 stage II, 9 stage III, and 7 stage IV lesions. There was a significant correlation between JOCD stage and T2* values in the progeny lesion and interface. There was no correlation between T2* and age in any region. The progeny lesion and interface showed significantly higher T2* at stages I and II compared to stage IV. When compared to T2* in control bone, T2* of progeny lesion was significantly higher at stages I, II and III but significantly lower at stage IV. However, there was no significant difference between control bone T2* and stage IV interface or parent bone. Although there were significant positive correlations between patient age and volume of the progeny lesion, interface and parent bone, none were found between volume and JOCD stage.

T2* mapping is a quantitative method that can differentiate between osseous, fibrous and cartilaginous tissues within lesion and thus provides a quantitative measurement of degree of ossification in progeny lesion and interface regions. T2* is a potential imaging biomarker for the evaluation of healing progress by capturing the natural history of disease progression from its epiphyseal cartilage origin through all stages of subsequent osteogenesis as it is known from fracture healing. Lower T2* in recently healed progeny lesions (stage IV) suggests that the repair process results in osseous tissue of a different composition than native trabecular bone. This is in keeping with the knowledge from fracture healing that it will take an extended period of time in the final phase of osseous remodeling to re-establish normal trabecular architecture and bone marrow. T2* mapping may allow quantitative follow-up of JOCD to differentiate gradually healing lesions from those that do not show progression in ossification and might be at risk of becoming unstable, however future validation and follow-up studies are needed.

References

Ellermann J, Johnson CP, Wang L, et al. 2017. Insights into the Epiphyseal Cartilage Origin and Subsequent Osseous Manifestation of Juvenile Osteochondritis Dissecans with a Modified Clinical MR Imaging Protocol: A Pilot Study. Radiology 282:798-806.
Kijowski R, Blankenbaker DG, Shinki K, et al. 2008. Juvenile versus adult osteochondritis dissecans of the knee: appropriate MR imaging criteria for instability. Radiology 248:571-578.
Zbyn S, Santiago C, Johnson CP, Ludwig KD, Zhang L, Marette S, Tompkins MA, Nelson BJ, Takahashi T, Metzger GJ, Carlson CS, Ellermann JM. Quantitative MRI Evaluation of Lesion and Parent Bone in Patients with Juvenile Osteochondritis Dissecans (JOCD) of the Knee – T2* Mapping and Volumetric Study at 3T. ORS 2021 Annual Meeting Paper No. 0049.

Growing Back Toe Tips: Follow the Red Brick Road

Scientists have always been interested in the regenerative properties of planaria that can completely regenerate half transverse and longitudinal half sections. Amphibians have critters that can regrow a limb or tail after transection. Hence, all the control that goes into embryology goes into that recovery. It has also been recognized that in some mammals very distal amputation of the digit tip can regenerate completely. It turns out that osteoblasts can express limb-related developmental transcription factors, including Prrx1 and Hoxa13/Hoxd13. Investigators hypothesized that osteoprogenitors from the terminal phalanx (P3) bone directly contribute to skeletal elongation and spatial patterning during regeneration.

Osx-Cre^ERT2;Ai9 inducible reporter mice were used to trace osteoblast lineage cells, where Cre+ mice had osterix-positive (Osx+) cells pulse-labeled with a tdTomato (tdT) reporter prior to amputation. Five daily doses of tamoxifen were given to 7 to 8-week old mice to induce Cre recombination. After a three-week clearance period, the mice, along with vehicle and wild type controls had distal terminal phalangeal amputations where 25% of the bone length was resected. MicroCT scans were taken at 0 and 10, 14, 21, 28, 56 days after amputation (DPA). 5-ethynyl-2′- deoxyuridine (EdU) to track cell proliferation and calcein to track mineralization was added to drinking water 5 days and injected 2 days before sacrifice, respectively, at 10, 14, 21 and 56 DPA. Because of its high affinity for DNA, 4’,6-diamidino-2-phenylindole (DAPI) is used for counting cells, measuring apoptosis, sorting cells based on DNA content, and as a nuclear segmentation tool in high-content imaging analysis. The % signal of cells co-labeled with DAPI and tdT and cells labeled with DAPI and EdU were determined using confocal microscopy in four tissue regions of interest: periosteum, endosteum, marrow, and blastema (corresponding to the progenitor cells distal to the amputated bone). In a separate experiment 3.6Col1a1-tk (TK) mice were used, alongside wild type littermates. TK mice can have proliferating osteoblasts expressing 3.6Col1a1 ablated with twice daily administration of ganciclovir (GCV). This allowed for a separate investigation of histologic differences in regeneration in the absence of proliferating osteoblasts. For the above experiments there was a control group with no amputation.

Following amputation, Cre+ mice exhibited bony remodeling followed by osteogenesis, reaching pre-amputated length by 56 DPA. Tamoxifen-mediated recombination showed that in unamputated digits, 60% of periosteal cells and 40% of endosteal cells were osteoblast lineage, compared to less than 5% of marrow cells. This proportion increased during blastema formation before dropping to a baseline level at 21 DPA. In contrast to the minimal EdU staining seen in unamputated digits, approximately 40% of cells in all regions were EdU+ at 10 DPA, decreasing to baseline at 21 DPA. In the blastema, 40% of cells were osteoblast lineage at 14 DPA. EdU and calcein labeling revealed a proximal to distal wave of proliferation, followed by mineralization that concluded by 56 DPA. GCV-mediated ablation of proliferating osteoblasts in TK mice led to decreased P3 bone length by 21 DPA, which persisted to later time points. However, outgrowth and patterning of the surrounding connective tissue, skin, and nail resembled wild type digits at 21 DPA, suggesting that other mesenchymal cell types, such as fibroblasts, marrow stromal cells, and perivascular cells, may play a role in digit morphogenesis.

There are several take home thoughts. Removal of these osteoblast lineage cells in more proximal amputation leads to failure of regeneration. Osx+ cells exclusively formed the regenerate bone, but also appeared in the dermis, indicating that they may not be lineage-restricted. scRNA-seq data suggests that activated osteoblast lineage cells express digit-specific Hox genes. Future work will determine the function of Hox genes in osteoblast lineage cells during regeneration.

Reference

Qu F, Lenz K, Krekeler G, Radinsky S, Harris T, Silva M, Guilak F. Osteoblast Lineage Cells Play a Critical Role in Murine Digit Tip Regeneration. ORS 2021 Annual Meeting Paper No. 0007.

Getting the Meniscus Root Attached: Bridge Over Troubled Waters

The mental image of a tendon enthesis is one of parallel orientation of collagen fibers in the transition from soft tissue to bone often by a bridge of fibrocartilage. Meniscal roots are a strange enthesis in that the collagen orientation in the attachment meniscus to bone are more unaligned whereas throughout the rest of the meniscus the collagen fibers parallel to the capsular wall. In whole meniscal allograft transplants the results are better with bone-to-bone attachment in contrast to meniscal soft tissue attachment to bone. In creating tissue engineered implants native fiber orientation would be an advantage, especially for maintaining the physiologic hoop stress configuration of a normal meniscus. It is known that hyper physiological levels of glucose (4500 mg/L) and TGF-β1 (5 ng/ml) lead to a compact and unaligned collagen fiber network. Lower concentrations of glucose (500 mg/L) and TGF-β1 (0.5 ng/ml) lead to aligned collagen fiber constructs. A custom bioreactor has been developed to generate a biochemical gradient at the soft tissue-bone interface with different types of media individually applied to either boney or soft tissue portions of the engineered entheses.

Using trabecular cores were taken from 1-3 day old bovine femurs. The space between 1 cm pegs was embedded with a collagen solution with 1-3 day old bovine fibrochondrocytes. These were then clamped in a fashion to mimic native boundary conditions. Technically, there were three regions, bone only/bone+collagen/collagen only. High glucose/TGF- β 1 media was applied to bone only region while low glucose/TGF- β 1 media was applied to collagen only region (Bioreactor group). For bone+collagen region (interface region) where a clamp was placed, we believe that there would be a gradient of glucose/TGF- β 1 (high glucose/TGF- β 1 at the interface close to bone plug and lower glucose/TGF- β 1 at the interface farther down to the soft region). This was done in a bioreactor environment in place for 30 days. As control groups, constructs were cultured only with either high glucose/TGF-β1 or low glucose/TGF-β1 media. At day 30 specimens were cut in half for strain analysis at 20% and picrosirius staining for collagen orientation.

All constructs kept their shape at 30 days. The high glucose dose has a denser construct with radially oriented fibers. The low glucose group had longitudinal orientation and the bioreactor group had longitudinally aligned collagen fibers in the soft tissue zone and intermediate collagen formation at the interface. The authors note that the bioreactor group showed a mixed collagen fiber structure like a native enthesis. The intermediate feature of collagen was observed at the interface where the high and low media likely meet. Overall, for a dense collagen fiber structure, glucose would play a role while for an aligned collagen fiber structure, TGF-b1 might play an important role.

Overall, the strain analysis showed that the high and low group had a peak strain near to the interface while the bioreactor group farther away from the interface which is similar to native tissue. This might be due to the gradient in fiber organization. Further research will be required to include different biochemical stimuli, longer culture time, and the additional of mechanical force to enhance a more physiologic construct.

Reference

Kim J, Babmatee RR, Boys AJ, Estroff LA, Bonassar LJ. Gradients in Media Recapitulate Native Collagen Fiber Organization within Tissue Engineered Enthesis. ORS 2021 Annual Meeting Paper No. 0352

Nickel-Free High-Nitrogen Austenitic Steel: A New Metal on the Block

Cobalt-chromium-molybdenum alloy (CoCrMo-alloy) has been a mainstay for joint prosthetics for years. Metal on metal prosthetics may have wear debris problems due to edge loading and other mechanical issues. Also, trunnion head interface is another source of corrosive wear debris generation. The durability of CoCrMo-alloy has been matched by some ceramics such as Al₂O₃. A new generation of austenitic high-nitrogen steels (AHNS) do not contain alloying elements such as nickel and cobalt known to have biocompatibility issues.

Disks made of low carbon (LC) CoCrMo medical alloy and nickel-free high-nitrogen FeCrMnMoN steel were compared to Al₂O₃ceramic on a custom-made ball-on-flat tribometer. Testing was at constant load (37 N, pmax = 66 MPa after running-in) for 86400 cycles. Four simulated synovial fluids were prepared: Dulbecco’s phosphate buffered saline solution; newborn calf serum; hyaluronic acid (sodium hyaluronate) powder (HA); and 35 wt% hydrogen peroxide ultrapure analytical reagent (H₂O₂) to simulate inflammation. Wear was quantified through measurements of wear scar volume (OrthoLux, Redlux). Sample surfaces were characterized by open circuit potential (OCP) measurements along with scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). OCP measures when the passive film is removed and how quickly the surface repassivates.

Absent H₂O₂ there was no statistical difference in wear scar volume between LC-CoCrMo and FeCrMnMoN. Unlike FeCrMnMoN the wear of LC-CoCrMo increased with increasing H₂O₂. The OCP pre-wear values between LC-CoCrMo and FeCrMnMoN were not statistically different in tests w/o H₂O₂ and with 10 mM H₂O₂ but they were more electropositive for FeCrMnMoN than for LC-CoCrMo at 3 mM H₂O₂ (p = .031). During wear, the OCP drop of the two metals was similar except for being significantly higher for LC-CoCrMo than for FeCrMnMoN in tests with 10 mM H₂O₂ (p < .0005).

The authors note that it appears that nitrogen is the essential alloying element of AHNS. Other research has demonstrated that in combination with sufficient amounts of chromium and molybdenum nitrogen improves corrosion resistance. A long-term question will be the effects on trunnion corrosion wear in femoral components.

Reference

Nickel-Free High-Nitrogen Austenitic Steel – A replacement for CoCrMo-Alloy to Battle Tribocorrosion in Healthy and Inflammatory Simulated Synovial Fluids? Simona Radice 1 , Alfons Fischer 1 , Markus A. Wimmer. ORS 2021 Annual Meeting Paper No. 0040.

Another Point of Care Test to Rule Out Prosthetic Joint Infection: Coming to an Operating Theatre Near You

Laboratory tests used in periprosthetic joint infection are generally positive for infection when values are elevated. Conversely, it is more difficult to have that is very accurate for ruling out infection. Defensins are a family of microbicidal, and cytotoxic peptides seen in plants and animals. They are abundant in the granules of neutrophils. An alpha defensive has been found to have a high sensitivity to periprosthetic infection as an enzyme linked immunoassay (ELISA)¹. The Elisa has a very high sensitivity and specificity. The Synovasure Alpha Defensin Test is a lateral flow device that requires 0.5ml fluid and 15-minute processing at a cost of $500.00². In 195 cases the overall sensitivity of the Synovasure Alpha Defensin Test was 92.1% (95% confidence interval [CI], 83.6% to 97.1%), the specificity was 100% (95% CI, 97.0% to 100%), the positive predictive value was 100% (95% CI, 94.9% to 100%), and the negative predictive value was 95.2% (95% CI, 89.9% to 98.2%). The overall accuracy of the Synovasure test was 96.9% (95% CI, 93.4% to 98.9%). However, based on a meta-analysis of 640 laboratory-based alpha defensin test and 119 lateral flow the lateral-flow test performed worse, with sensitivity of 77.4% compared with 95.3% and specificity of 91.3% compared with 96.5%. This makes the study a less efficient rule out test³.

Calprotectin is a complex of the mammalian proteins S100A8 and S100A9. This complex chelates manganese and zinc forming a compound with antibiotic properties and, like defensin, is found to make up to 50% of neutrophil granules. It has very strong antimicrobial properties. A calprotectin lateral-flow POC test was compared to a calprotectin ELISA for PJI diagnosis in patients who underwent revision TKA using the 2013 Musculoskeletal Infection Society (MSIS) criteria to define septic and aseptic cases⁴.

Based on MSIS criteria, 53 revision TKAs were septic and 70 were aseptic. With ≥50-mg/ mL cutoff calprotectin POC performance showed a sensitivity of 98.1%, a specificity of 95.7%, a positive predictive value of 94.5%, a negative predictive value of 98.5%, and an AUC of 0.969. With ≥14-mg/mL cutoff the sensitivity was 98.1%, the specificity was 87.1%, the positive predictive value was 85.2%, the negative predictive value was 98.4%, and the AUC was 0.926. This study was all done on preserved specimens assayed in a lab doing lateral flow and Elisa side by side using unblinded technicians. However, the promise is sufficient to pursue the calprotectin lateral flow test as a multicenter study.

References

Deirmengian C, Kardos K, Kilmartin P, Cameron A, Schiller K, Parvizi J. Combined Measurement of Synovial Fluid α-Defensin and C-Reactive Protein Levels: Highly Accurate for Diagnosing Periprosthetic Joint Infection. J Bone Joint Surg Am, 2014 Sep 03;96(17):1439-1445.
Frangiamore SJ, Gajewski ND, Saleh A, Farias-Kovac M, Barsoum WK, Higuera CA. α-Defensin Accuracy to Diagnose Periprosthetic Joint Infection-Best Available Test? J Arthroplasty. 2016 Feb;31(2):456-60. doi: 10.1016/j.arth.2015.09.035. Epub 2015 Sep 28. PMID: 26545577
Suen K, Keeka M, Ailabouni R, Tran P. Synovasure ‘quick test’ is not as accurate as the laboratory-based a-defensin immunoassay: a systematic review and metaanalysis. Bone Joint J. 2018 Jan;100-B(1):66-72.
Warren J, Anis HK, Bowers K, Pannu T, Villa J, Klika AK, Colon-Franco J, Piuzzi NS, Higuera CA. Diagnostic Utility of a Novel Point-of-Care Test of Calprotectin for Periprosthetic Joint Infection After Total Knee Arthroplasty: A Prospective Cohort Study. J Bone Joint Surg Am. 2021 Jun 2;103(11):1009-1015. doi: 10.2106/JBJS.20.01089. PMID: 34038396

Hip-Spine Syndrome: Is it All in the Timing?

Pain patterns due to lumbar spinal stenosis and hip osteoarthritis are often similar. Osteoarthritic hip pain is associated with buttock, groin and thigh pain that increases with ambulation and recedes with rest. In a similar fashion spinal stenosis results in buttocks pain that progresses distally in the thigh with walking and recedes with rest. Patients with hip osteoarthritis may have alterations in gait that can be associated with low back pain. If a person has both spinal stenosis and hip osteoarthritis it can be difficult to determine which condition should be approached with surgery first. The combination of hip and spine origin of leg pain could be termed “hip-spine syndrome.”

To get some insight into surgical order investigators used the Swedish Hip Arthroplasty Register (SHAR) and the Swedish Spine Register (Swespine) to review the outcomes of individuals who had both lumbar spinal stenosis surgery (LSSS) and total hip replacement (THR). Between 2002 and 2012 found 1921 patients who had primary LSSS and THR. On review of records only 509 who had patient related outcome scores and met the surgical criteria of primary cares. Of these only 255 had both surgeries within 2 years. 84 had THR prior to LSSS and 171 had LSSS prior to THR.

The mean time from LSSS to THR was 312 days (SD 165) whereas from THR to LSSS was 460 days (SD 160). Decompression and or fusion at one versus two levels was similar for the two groups. Those with LSSS prior to THR reported significantly better outcomes 1 year after the second surgery. Overall, patients with hip-spine syndrome fair worse than those with only one of the two disorders. Ultimately, the decision on which order to do the two surgeries rests with the clinical judgement of the surgeons involved. It will take well designed prospective studies to determine which subjective and objective findings would dictate the order of procedures. For now, when it comes to a toss-up, spinal decompression comes first.

Reference

Eneqvist T, Bülow E, Nemes S, Brisby H, Fritzell P, Rolfson O. Does the order of total hip replacement and lumbar spinal stenosis surgery influence patient-reported outcomes: An observational register study. J Orthop Res. 2021 May;39(5):998-1006. doi: 10.1002/jor.24813. Epub 2020 Oct 12. PMID: 32710668

Using Portable Ultrasound in Ankle Fracture Assessment: Not Quite a Tricorder?

A supination external rotation injury of the ankle typically occurs when the foot in planted on the ground, rolls down and out (supination) and the leg is rotating inward. The resulting injury can be mild (stage 1) with a tear of the anterior inferior tibiofibular ligament, (stage 2) stage 1 combined with fracture of the fibula, (stage 3) stage 2 unstable with tear posterior tibiofibular ligament (stage 3), severe unstable (stage 4) includes Stage 3 and results in separation of the talus from the medial malleolus. Stage 4 is divided into 2 sages: Stage IVa had a superficial deltoid ligament tear and Stage IVb also has a tear of the deep deltoid ligament. Instability increases with each of these stages. The instability of stage 3 and 4 can result in ankle osteoarthritis. Surgeons rely of stress radiographs of the ankle to determine instability that would require surgical fixation. This be done by taking a radiograph with the patient laying on the affected side resulting external stress created by gravity. Alternatively, the radiograph may be taken with 45N force of the foot pushing lateral or the patient standing turning inward. Fortunately for the patient, with comparison of the two sides the gravity stress views are sufficient.

Serial sectioning of ligaments and fracture of the fibular investigators could replicate each stage and by radiograph or handheld ultrasound determine the medial malleolar to medial talar side separation, Typically the two sides are compared radiographic images wherein, based on the literature, an increase of separation greater 2 mm is an indication for surgical stabilization. As the ligaments were being incised it was not until stage 3 (complete separation of the distal anterior and posterior tibiofibular interosseus ligament) can cause significant surgical ankle separation.

The authors found that there was a moderate to strong correlation of ultrasound findings to radiology images. The strongest correlation was in the gravitational stress test (kindest to the patient). As a guide to surgical decision making the surgical team involved in this study is using the handheld ultrasound devise as a guide in the decision-making process. Based on recent correspondence they feel that “Although, most people still use the stress radiograph to evaluate the instability as most orthopaedic physicians are not familiar with the ultrasound.”

Reference

Saengsin J, Sato G, Sornsakrin P, Lubberts B, Waryasz G, Guss D, DiGiovanni CW. A Comparison of Portable Ultrasonography and the Fluoroscopy for Evaluating Medial Ankle Instability – A Cadaveric Study. ORS 2021 Annual Meeting Paper No. 0834

Recreating an Enthesis, Bridging the Gap

Most rotator cuff repairs are done on individuals over 40. The healing that would occur with a teenager would likely be better than that of an older individual albeit return to function in older individuals can be gratifying^1,2. Other series show fewer gratifying results in older individuals. Part of the problem is the failure for healing tissues to recreate the process seen in growth and development. There is a mouse model MRL/MpJ (MRL) with enhanced healing of many tissues including patellar tendon³. One of the hall marks is healing without the usual concomitant fibrosis. Investigators contrasted this mouse line with C57Bl6 (B6) mice with the hypothesis that the repair site of a surgically induced rotator cuff tear would more nearly approximate a normal complex insertional site⁴.

16-week-old MRL and B6 mice had surgical transection of the supraspinatus tendon with repair through a drill hole. Sacrifices were at 2 and 6 weeks with pre-sacrifice gait analysis compared to presurgical gait. Histology was performed for insertional bone quality and organization along with the amount of type III collagen, a surrogate for fibrosis.

At 2 weeks the injured MRL limb exhibited decreased paw area from baseline relative to B6 (p=0.01), There was increased paw area on the corresponding hind limb. However, by 6 weeks these deficits were recovered, with MRL trending to prefer the injured paw relative to baseline and trending to increased stride length on the injured side. At 6 weeks there was increased recovery of insertion site zonal architecture in the MRL mice compared to B6. Also, at 6 weeks MRL had more organized bone formation, with interdigitation from the insertion site. B6 had fibrous bone and disorganization at 6 weeks. There was a statistical trend of increased collagen III in the B6 mice at two weeks (p=0.1) and increased procollagen I cell percentage in the MRL at 6 weeks (p=0.06).

The question remains as to differences in collagen alignment, crosslinking and fibrillar size, as well as mineralization at the tendon-to-bone transition site that may enhance the integrity of the repair site. Likewise, the authors are investigating proteoglycan at the insertion site. That would be an index or more normal transition of tendon to bone. If this can be established, the question will remain as to which cellular signals are related to the altered synthesis.

Reference

AzzamMG, Dugas JR, Andrews JR, Goldstein SR , Emblom BA, Cain Jr EL Rotator Cuff Repair in Adolescent Athletes. Am J Sports Med. 2018 Apr;46(5):1084-1090. doi: 10.1177/0363546517752919. Epub 2018 Feb 13. PMID: 29438628
Bhatia S, Greenspoon JA, Horan MP, Warth RJ, Millett Two-Year Outcomes After Arthroscopic Rotator Cuff Repair in Recreational Athletes Older Than 70 Years. Am J Sports Med. 2015 Jul;43(7):1737-42. doi: 10.1177/0363546515577623. Epub 2015 Apr 1. PMID: 25834140
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3806350/
Chatterjee M, Acosta A, Srisha Gaur S, Venkatesh S, Taub PJ, Andarawis-Puri N. MRL/MpJ Exhibit Restored Function and Insertion Site Structure in Supraspinatus Tendon after Surgical Repair. ORS 2021 Annual Meeting Paper No. 1484.

Osteocyte MMP-13 Cross Talk in Osteoarthritis, When is Too Little Too Much?

Name a cell that is a systemic regulator, has a huge surface area, and can communicate like a nerve. The answer: what is an osteocyte? Osteocytes, many of which have a half life of 25 years, affect bone and calcium interchange via their perilacunar/canaliculi extensions. Perilacunar/canaliculi remodeling (PLR) is vital to calcium homeostasis and bone turnover. Osteocytes secrete metalloproteases (MMP) and cathepsin K (CatK). The lacunar-canalicular network (LCN) can be studied by silver staining and diminished area is a key hallmark in decrease PLR activity. With decreased MMP-13 there is deceased PLR. Since subchondral bone is known to play an important role in osteoarthritis, but the mechanisms by which bone supports cartilage health remain unclear. To determine if osteocyte expression of the enzymes required for perilacunar/canaliculi bone absorption support cartilage health, osteocytic MMP-13 and cathepsin K were studied in human and mouse models of osteoarthritis.

Human osteoarthritis versus normal cartilage and subchondral bone
Normal medial and lateral tibial plateau osteoarticular samples from cadavers were compared to similar specimens retrieved during joint replacement. Compared to the lateral side, the medial side showed relatively more articular cartilage changes in both the OA and control samples, but to a lesser degree in controls. Decreased osteocyte lacunocanalicular area correlated to the degree of OA change.. There was also a concurrent decrease in osteocytic MMP-13 and CatK in medial versus lateral tibial bone. That was statistically significant in OA but not in the cadaver sections.

Targeted ablation of MMP-13 in mouse osteocytes: effects on bone
Using an DMP1 promoter, Cre recombinase was expressed in osteocytes but not in cartilage. This made it possible to create a mouse in which MMP13 was selectively deleted in osteocytes (MMP-13^ocy-/-). Decreased MMP-13 expression in osteocytes, and intact MMP-13 expression in chondrocytes, was confirmed. Comparing MMMP-13^ocy-/- to controls, canalicular length was decreased in all areas (medial, lateral, anterior, and posterior areas). There was a non-significant reduction in lacunar area. There was a small but statistically significant decrease in collagen orientation. The only significant mechanical difference was decreased stiffness seen in MMMP-13^ocy-/- compared to controls.

Targeted ablation of MMP-13 in mouse osteocytes: effect on articular cartilage in normal and destabilized knees
4-month-old MMP-13^ocy-/- mice had histology differences with surface fibrillation, proteoglycan decrease, and significant increase in OARSI and Mankin scores. Comparing control to MMP-13^ocy-/- joints, destabilization of the medial ligament resulted in the same degree of articular cartilage change. This indicates that severe injury overshadows the effect of osteocytic MMP-13 deficiency in bone. By targeting osteocyte MMP-13 without any influence on cartilage MMP-13, the isolated effect of osteocyte-only ablation implies a cross talk between bone and cartilage that is not dependent on mechanical alterations. Considering the joint as an organ, this is an amazing example of the communication of bone to cartilage. The authors suggested mechanisms of osteocyte crosstalk with cartilage, such as normal osteocyte MMP-13 supporting growth factors. Wherein injury may have a more severe and immediate effect on PLR, the gradual effects of aging wherein PLR is gradually lost due to decrease MMP-13 production appears to be important to chondrocyte heath.

Reference

Mazur CM, Woo JJ, Yee CS, Fields AJ, Acevedo C, Bailey KN, Kaya S, Fowler TW, Lotz JC, Dang A, Kuo AC, Vail TP, Alliston T. Osteocyte dysfunction promotes osteoarthritis through MMP13-dependent suppression of subchondral bone homeostasis. Bone Res. 2019 Nov 5;7:34. doi: 10.1038/s41413-019-0070-y. eCollection 2019. PMID: 31700695

Tracking Down the Mystery of Development and Aging, One Cell at a Time

Ones of several single-cell RNA sequencing (scRNA-seq) approaches takes dissociated cells and passes them though a microfluidics system so that each cell is attached to a single gel microbead that is coated with oligos to do the RNA sequencing. The trick is that each bead has a genetic barcode so that you can do the RNA-seq reaction all at once, but based on the oligo barcode sequence, you can tell which cell it came from. In this manner, you can measure quantitatively several thousand genes on several thousand (typically around 10,000) single cells at once resulting in a huge amount of data per sample. However, when this is done you lose all the spatial information because the cells must be isolated/dissociated. To determine the locus of the cell in the original tissue RNA fluorescence in situ hybridization (FISH) allows for the visualization of single cells with a specific mRNA present. There are sufficient mRNA copies within the cell to make the fluorescence visible. As an example, this technology has been used to identify the cellular trajectories and lineage specification during limb bud development¹. New techniques are now being developed to allow for spatial transcriptomic analysis of cells without digestion of the tissue, preserving the spatial distribution of the cells and further allowing to study cell-cell interaction networks in details.

By crossbreeding mice with a gene such as COL1 with a reporter gene such as rose26 (tomato) it is possible to trace specific mesenchymal stem cells with osteoblast or other lineages. Using other gene markers it is possible to detect subpopulations of mesenchymal stem cells. With that background investigators employed large scale single cell transcriptome analysis to detect a unique cell type that expresses adipocyte markers but contains no lipid droplets². These appear in the pericyte and stromal cell network throughout the marrow cavity. This creates and maintains the vascular structure within bone marrow and suppresses bone formation within that space. This population is a newly identified component of marrow adipose tissue and has been labeled marrow adipogenic lineage precursors (MALPs).

Next generation sequencing is a term applied to new methodologies for quantitative RNA-sequencing, which have now been adapted for single cell omics. In other words, what is the gene expression of each individual cell rather than the average gene expression of all the cells pooled together. This can be achieved by scRNA-seq, Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), and Cytometry by Time-Of-Flight (CyTOF). scRNA-seq is described above. ATAC-seq complements scRNA-seq by finding open and closed chromatin across the genome in specific cell types. If performed on singles cells ATAC-seq can be used to analyze the epigenome of skeletal cell types at the single cell levels. CyTOF is a mass cytometry that can target multiple single cell proteomics. Isotope antibodies make it possible to detect 40 to 120 epitopes in the same cell. An article that summarizes these technologies was the product of a workshop on “Single Cell Omics” at the 2020 annual meeting of the Orthopedic Research Society. This contains more in depth methodologic information³.

Similar to the human genome project, there is an international effort to create a human cell atlas that will cover all stages of development and aging⁴. An analogy I will use is that of a stamp album. First are the stamps from a beginner’s album reflecting the texture of the various countries. Then, there are specialty albums devoted to a single country, region, or commonwealth. Finally, a complete collection reflects the perspective of a century. Stamps have changed reflecting local political and other historical movements. For the cell atlas the first phase uses massively parallel sequencing based on single-cell and/or single-nuclear RNA-sequencing to initiate an initial roadmap. The second, deeper phase uses specialized lower throughput methods to provide detailed maps for a small number of individuals and cells. The third phase will explore patient-to-patient variation encompassing age, sex, ethnicity, and geographical variation. By bringing this worldwide to musculoskeletal clinicians and scientists the network will foster collaboration both in intellect and resources.

References

KellyNH, Huynh NPT, Guilak Single cell RNA-sequencing reveals cellular heterogeneity and trajectories of lineage specification during murine embryonic limb development. Matrix Biol. 2020 Jul;89:1-10. doi: 10.1016/j.matbio.2019.12.004. Epub 2019 Dec 24.PMID: 31874220
Zhong L 1, YaoL, Qin L, et al. Single cell transcriptomics identifies a unique adipose lineage cell population that regulates bone marrow environment. Elife. 2020 Apr 14;9:e54695. doi: 10.7554/eLife.54695. PMID: 32286228
Rai MF, Wu CL, Capellini TD, Guilak F, Dicks AR, Muthuirulan P, Grandi F, Bhutani N, Westendorf JJ. Single Cell Omics for Musculoskeletal Research. Curr Osteoporos Rep. 2021 Apr;19(2):131-140. doi: 10.1007/s11914-021-00662-2. Epub 2021 Feb 9.PMID: 33559841
BaldwinMJ, Cribbs AP, Guilak F, Snelling Mapping the musculoskeletal system one cell at a time. Nat Rev Rheumatol. 2021 May;17(5):247-248. doi: 10.1038/s41584-021-00600-7. PMID: 33712803

Improving Osteoarthritis Function: Time to Prime your Engines

The effect of age on muscle strength and energy capacity can increase difficulty in ambulation with hip osteoarthritis. 30 women with moderately symptomatic hip OA were studied for walking energetics and hip abductor strength¹. Investigators measured the UCLA activity score, quantified activity frequency and intensity using accelerometers worn for seven days, used a portable oxygen exchange system to measure energy used during walking at preferred speeds. A dynamometer measured hip abductor strength. Preferred walking speed was contrasted to total energy capacity as assessed using a six-minute walk test. Greater energy used during walking was associated with lower self-reported physical activity, more sedentary time, and less light activity time. Albeit there was no associate with hip abductor strength and walking energy the two together predicted variance in self-reported physical activity.

What is the clinical usefulness of this information? It is known that increased physical function can reduce pain. Albeit OA is associated with metabolic syndrome (obesity, dyslipidemia, diabetes, and hypertension) decreased activity may also have a negative impact on cardiovascular health. While fatigue can arise from several mental and physical sources, fatigability refers to the process of becoming more fatigued with activity. Fatigability is a subjective measure that can affect the level of physical activity. On the other hand, aerobic capacity is an objective measure. If there is a relationship between fatigability and aerobic capacity, interventions to improve mobility might be possible.

A select group of 36 women with isolated hip OA were studied². All took the hip disability and osteoarthritis outcome score (HOOS) which measures pain, physical function, and quality of life. Rather than using a maximum walking speed for 10 minutes on a treadmill the individual was asked to periodically indicate hand signals whether to increase or decrease their walking speed. Aerobic capacity (VO2max) was measured by calculation from a 6-minute treadmill walk at the patients’ maximum speed determined by their adjustment of speed during the six minutes. Physical activity was measured by UCLA activity score 0 (dependent on others) – 10 (high level sports) and an accelerometer-based activity monitor to calculate number of steps per day over 7 days. There was a significant correlation between fatigability scores and UCLA scores, but fatigability did not have statistically significant association with the quantity or intensity of accelerometer‐based activity. Aerobic capacity was significantly associated with UCLA scores and the percent sedentary time. Lower aerobic capacity was related to increased fatigability scores. Together aerobic capacity and fatigability predicted UCLA scores and aerobic capacity was the strong of the two.

From these two studies it appears that aerobic capacity is a reasonable treatment target. This is particularly true in that aerobic capacity can be improved in a non weight bearing manner, opening this intervention to OA individuals with both hip and knee OA. Future studies prospectively isolating aerobic capacity enhancement will further isolate the role of aerobic capacity in OA management.

References

Foucher KC, Huang C-H, Aydemir Walking energetics and abductor strength are associated with physical activity in older women with hip osteoarthritis. Gait Posture. 2021 Feb 3;85:151-156. doi: 10.1016/j.gaitpost.2021.01.022. Online ahead of print.
FoucherKC, Aydemir B, Huang C-H, Horras M, Chmell Aerobic capacity and fatigability are associated with activity levels in women with hip osteoarthritis. J Orthop Res. 2020 Sep 12;10.1002/jor.24856. doi: 10.1002/jor.24856. Online ahead of print.

The Trunnion Music is Sour, Strike Out the Band

On average, hip replacement surgery is more successful when the center of rotation for the femoral head is located close to the original hip anatomy, the hip abductor muscles are at a physiologic lateral point, and leg length is preserved. To achieve this, most femoral components are modular, involving a femoral head that is impacted on to the femoral stem via the femoral neck or trunnion. The trunnion takes the form of a truncated cone, with a slightly smaller diameter at the tip as compared to the base. The ideal fit of the femoral head on the trunnion is free of any motion. However, some modular junctions develop fretting corrosion, which is caused by micromotion and results in metallic breakdown. This fretting corrosion and subsequent debris release into the surrounding joint space can lead to a painful adverse local tissue reaction within the capsule, requiring surgical interventions and implant revision. This phenomenon is linked in particular to implants with CoCrMo alloy femoral heads. To determine if any aspect of the alloy itself was a risk factor for corrosion and debris release, investigators retrieved 108 wrought CoCrMo alloy femoral heads with severe fretting and corrosion from the Biocompatibility and Implant Pathology repository, constituting 1.1% of the 1002 wrought CoCrMo femoral heads collected from revision surgeries between 06/2004-06/2019. Implants were revised with multiple diagnoses, including aseptic loosening, infection, adverse local tissue reactions, pain, and more.

Molds were made of the femoral head-trunnion interface and scanned with a white light confocal sensor (RedLux) to create detailed maps of the taper junction surface. From these maps, volumetric material loss could be calculated, acting as an estimate for debris released to the tissue. The femoral heads were sectioned, and samples were processed to visualize different aspects of alloy microstructure, namely average grain size, carbon content, and microstructural banding. These samples were assessed using light and scanning electron microscopy. Microstructural characteristics of the alloy were then correlated with volumetric material loss.

Material loss could not be computed in 30% of the cases because there was no undamaged surface from which a calculation could be made. Of the remaining 70%, the average material loss was 0.9 mm³ (0.0 mm³– 20.7 mm³). Mean grain size of all the samples was 4.99±1.72µm and there was no correlation to material loss. Low carbon content was seen in 92% of heads, but no statistically significant correlation was found between carbon content and material loss. However, microstructural banding was observed in 81% of the heads, and those with this feature had 5x more material loss than those without.

Alloy banding was the only microstructural characteristic explored here that showed a correlation with volumetric material loss. Banding reflects a slight inhomogeneity in implant alloy processing and seems to be linked to more severe forms of chemically dominated corrosion secondary to fretting, also known as column damage. The authors conclude that “Optimization of the alloy microstructure, especially with respect to chemical homogeneity, can remove preferential corrosion sites, reducing material loss and subsequent ALTR in THA patients.” More information about this topic and further findings from this work can be found in a soon to be published manuscript in CORR, “Are Damage Modes Related to Microstructure and Material Loss in Severely Damaged CoCrMo Femoral Heads?”

Reference

McCarthy SM, Queiroz Neto M, Je M, Hall DJ, Jacobs JJ, Pourzal R. Effects of Alloy Microstructure on Material Loss in Severely Damaged CoCrMo THA Femoral Head Tapers. Trans ORS 46: 0383, 2021.

Meniscal Physiology: How Much is too Much and When do Things Go Bad?

The lateral meniscus takes over 50% of the load of the lateral compartment and the medial meniscus takes 50% [Ref]. In addition, the lateral meniscus absorbs relatively more rotation forces than the medial meniscus. It would be anticipated that meniscal changes would be more likely to occur in elite basketball players who perform cutting maneuvers in contrast to elite swimmers. A non-invasive method to assess meniscal status is ultra-short echo (UTE) MRI. UTE sequences can capture signal from highly ordered collagen structures, such as menisci, which normally display as structures with limited signal intensity on standard-of-care imaging sequences. UTE sequences also permit the calculation of the T2* relaxation time constant. T2* values have been correlated with tissue microstructure. In particular, collagen disruption and disorganization is associated with longer relaxation times indicating the presence of degeneration.

In a longitudinal study designed to run at time 0, 12 months, and 30 months, 16 elite college basketball player and 14 elite swimmers had UTE-MRI imaging performed. At each time point, a three-dimensional, Cones UTE sequence was acquired for evaluation of meniscal T2* metrics (TEs: 5 echoes between 0.03-24 ms, TR: 188 ms, slice thickness: 3 mm, field of view: 16 cm, acquisition matrix: 256x256mm, RBW: ±83.3kHz, Flip-Angle: 16°). All menisci were manually segmented using MeVisLab software, and T2* values were calculated using a mono-exponential fit. There were significant differences of meniscal T2* values between the lateral to medial compartments. However, there was no differences between elite swimmers and basketball players.

The authors note that the lateral meniscus has been found to have a thicker zone of uncalcified fibrocartilage and increased calcified tissue at the anterior and posterior horns compared to the medial meniscus and this may account for differences in T2* metrics. The authors note that the absence of differences between swimmers and basketball players may require larger numbers and the assessment of meniscal subregions. Another consideration for this very young group of athletes is that they may, at this time in their lives, be within their “envelope of function” for the menisci.

Reference

Seedhom BB, Hargreaves DJ Transmission of the load in the knee joint with special reference to the role of the menisci: part II: experimental results, discussion and conclusions. Engineering in Medicine, 1979. 1979;8:220-228.
Chen B, Zhao Y, Cheng, Ma Y, EY, Kavanaugh^A, Liu S, Du Three-dimensional ultrashort echo time cones (3D UTE-Cones) magnetic resonance imaging of entheses and tendons. doi: Epub 2018 Jan 5. 10.1016/j.mri.2017.12.034. PMID: 29309823.
Argentieri EC, Gold G, Majumdar S, Potter HG, Koff MF. Comparison of Meniscal T2* Metrics in Elite Basketball Players and Swimmers. ORS 2021 Annual Meeting Paper No. 1219.
Dye SF. The knee as a biologic transmission with an envelope of function: a theory. Clin Orthop Relat Res. 1996 Apr;(325):10-8. PMID: 8998861

Cell-secreted Extracellular Matrix in MSC Spheroids: Filling the Gap

Delayed and non-unions of fractures remain major clinical problems. The use of bone autographs is the gold standard but involves surgical procedures that can cause donor site morbidity. Mesenchymal stem cells are an enticing choice. However, their implantation faces many limitations such as loss of cell viability, low cell replication, and poor matrix production.

Researchers performed an experiment to assess if the use of mesenchymal stem cell-derived extracellular matrix (ECM) would enhance cellular activity, promote in vitro osteogenesis, and bone formation in a surgically induced bone defect. Mesenchymal stem cell-derived ECM was produced in vitro, lyophilized, and added to MSC spheroids.

In vitro spheroid production with loaded 0, 1 µg, 2.5 µg, 5µg, and 7.5 µg ECM.

The ECM modulated MSC adhesion and mechano-sensing after 48 hours when aggregated with up to 7.5 µg. Picrosirius red and Alcian blue staining for collagen and GAG increased with each increment of ECM. Immunostaining of after 3 days showed increased yes-associated protein-1 (YAP) expression and nuclear translocation, which is associated with cell proliferation and differentiation. Expression genes for integrin α2 and β1, N-cadherin, and yes-associated protein were all increased at 3 days accounting for mechano-sensing and proliferation.

21-day in vitro cell production

Compared to 5 µg type I collagen loaded spheroids, spheroids loaded with 5 mg of ECM had increase collagen, GAG, and DNA content at 21 days with a highly mineralized matrix.

4-week and 12-week fracture gap bone formation

ECM-loaded spheroids were entrapped in alginate hydrogels modified with RGD adhesive ligands on the alginate backbone. These were placed in a 6 mm segmental defect created in the right femur of skeletally mature immunodeficient rats. Alginate gels containing non-loaded spheroids or spheroids with identical masses of free ECM were used as controls. At 4 weeks after implantation the ECM-loaded spheroids group showed enhanced defect bridging. There were areas of hypertrophic cartilage formation adjacent to the implanted ECM-loaded spheroids seen on Safranin-O staining. This suggested bone healing through endochondral ossification. At 12 weeks defects treated with ECM-loaded spheroids showed mature cortical bone with an internal bone marrow. ECM-loaded spheroids resulted in a union rate over 60% and higher bone volume based on micro CT analysis. The control groups did not achieve union in any animals.

Reference

Gonzalez-Fernandez T, Tenorio AJ , Saiz AM Jr, Leach K. Incorporation of Cell-secreted Extracellular Matrix into MSC Spheroids Promotes Endochondral Ossification for Repair of Segmental Bone Defects. ORS 2021 Annual Meeting Paper No. 0314

Neurogenic Therapies for Osteoarthritic Knee Pain: A Road Paved in Uncertainties

Pain protects the organism from potential harm. In the case of joints the congenital absence of pain results in joint destruction. Once the stimulus passes the pain will usually cease. However, the musculoskeletal system is particularly prone to have active pain messages in the absence of significant disease. Degenerative disease of the spine and knees is a leading cause of pain and disability. In many instances the apparent level of disease is based on physical exam and imaging. This long-lasting nerve excitatory state may be due to effects at the peripheral nerve ending or more central in the spinal cord, thalamus, or cerebral cortex. Both patients and surgeons are frustrated with 20% of total joint replacement surgeries that result in continued pain. Albeit some of these poor results might be due to reasons aside from the nervous systems, many patients retain hypersensitivity to stimuli such as direct pressure that would not normally produce pain. Both for central and peripheral pain generators there are many neurotrophic factors under study for potential intervention. Neurotrophins include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT)-3, and neurotrophin-4 (NT)-4. The GDNF family of ligands include (GDFLs): Glial cell line-derived neurotrophic factor (GDNF), neurturin, persephin, and artemin (also known as neublastin and enovin). NGF has received the most clinical attention.

Initial phase 2 osteoarthritis (OA) clinical trials of an injectable antibody against NGF produced remarkable pain relief. However, a major side effect was rapidly progressive OA (RPOA). To understand current NGF targeting treatment strategies it is important to further define the role of NGF in nociception and inflammatory pain; summarize the literature on NGF in joint pain and experimental models of OA; and review the clinical development of strategies to block NGF.

The role of NGF in nociception and inflammatory pain

NGF is known to be pro-algesic when injected systemically or when directly injected into the joint. On this stimulation there is an increase in nerve ingrowth into synovium. Several studies have shown that the presence of inflammatory factors will upregulate NGF. Trk is tyrosine receptor kinase and TrkA is key in nociception highly selective for NGF. The NGF-TrkA complex can have an effect on the peripheral nerves and dorsal root ganglion with changes in neurotransmitters such as substance P and calcitonin gene-related peptide. Increased NGF has been found in the channels that cross subchondral bone and penetrate cartilage in OA.

NGF in bone pain and experimental models of OA

NGF can also be detected in OA synovial fluid. Increased NGF has been found in the channels that cross subchondral bone and penetrate cartilage in OA, and in synovium- and NGF expression is associated with symptomioatic knee OA.

Current clinical development of strategies to block NGF

The initial anti-NFG studies of 2010 were resumed in 2015 with lower doses. In 2017 the FDA granted Fast Track designation to tanezumab for the treatment of chronic pain in patients with OA and chronic lower back pain. Studies with the lower doses reflect pain relief superior to placebo and NSAIDS but not as significant as the higher doses. Albeit the progression to rapid joint destruction was not as great there was increased progression to TKA tanezumab 2.5 mg (5.2%) and 5 mg (8.0%) compare to NSAIDs (3.2%) and placebo. Because of the problem with anti-NGF therapies some work has started with TrkA small molecule antagonists.

Other pain mediators

BDNF has been shown to be increased centrally as well in peripheral nerve in chronic pain and fibromyalgia. There are drugs being developed to target BDNF-TrkB. The role of NT-3 in peripheral pain is under study. The GDFLs have been investigated for potential treatment in Parkinson’s disease. These ligands are also involved in diabetic nerve pain and bone pain. There several early trials involving GDFLs.

Thus far

To date, NGF antagonists have been the only product to achieve phase 3 studies and be submitted to the FDA for clinical use. Lower dosage was the trade off to avoid serious neurogenic side effects. However, there is significant decreased pain relief. These are no disease modifying effects and long-term usage may delay the time to joint replacement.

References:

Anne-Marie Malfait, MD, PhD, Rachel E. Miller, Richard J. Miller. Basic Mechanisms of Pain in Q4 Osteoarthritis Experimental Observations and New Perspectives Rheum Dis Clin N Am – (2021)
Malfait AM, Miller RE, Block JA. Targeting neurotrophic factors: Novel approaches to musculoskeletal pain. Pharmacol Ther. 2020 Jul;211:107553. doi: 10.1016/j.pharmthera.2020.107553. Epub 2020 Apr 18.PMID: 32311372

Estimating Tibial Fracture Healing: A Whole New Twist

The assessment of progression of fracture healing in tibial shaft fractures has always been difficult. The radiographic union scale for tibial fractures (RUST) was developed as a semiquantitative method based on fracture healing for each of four cortexes. The potential ranges of scores is 4 to 12 (each cortex 1-4). RUST has been accepted and applied to the femur as well. However, its use in earlier phases of healing is limited. To this end a virtual mechanical testing based on low dose computed tomography had been developed. Low dose CT scans are reconstructed in 3 dimensions (3D). In a simulation environment, these 3D models can be subjected to virtual mechanical tests such as torsion loading. In preclinical validation studies, virtual mechanical testing was shown to be a reliable surrogate for postmortem physical biomechanical testing¹. In clinical translation, this method was used to assess virtual torsional rigidity (VTR) in the fractured limb relative to that in the intact bone². This would allow for virtual testing of mechanical integrity prior to the time of eventual fracture healing based on painless ambulation healing across 3 cortices.

The authors hypothesized that midshaft tibial fracture patients with comorbidities would have lower virtual mechanical integrity and long times to clinical union in nailed tibial shaft fractures². Scans were done on 27 closed and low-grade open fractures 12 weeks after injury. 20 had no comorbidities and 7 had comorbidities (cigarette smoking, alcoholism, drug abuse, hepatitis C, diabetes, ischemic heart disease). Only two patients had multiple risk factors. All fractures eventually healed. The time to healing was based on healing across at least three cortices and ambulation without pain. The comorbidity group had lower VTR scores compared to the no comorbidity group. VTR significantly correlated with time to union but this was a moderate effect and the two lowest VTRs were non comorbidity patients. Despite the low numbers the effect size was large. Based on this study it is likely that CT and finite element base virtual torsion analysis could be a valuable tool in identifying patients at risk of fracture delayed union or nonunion.

References

Schwarzenberg P, Klein K, Ferguson SJ, von Rechenberg B, Darwiche S, Dailey HL. Virtual Mechanical Tests Out-Perform Morphometric Measures for Assessment of Mechanical Stability of Fracture Healing in Vivo. J Orthop Res. 2021 Apr;39(4):719-726. doi: 1002/jor.24866
Dailey HL, Schwarzenberg P, Daly CJ, Boran SAM, Maher MM, Harty Virtual Mechanical Testing Based on Low-Dose Computed Tomography Scans for Tibial Fracture: A Pilot Study of Prediction of Time to Union and Comparison with Subjective Outcomes Scoring. J Bone Joint Surg Am. 2019 Jul 3;101(13):1193-1202. doi: 10.2106/JBJS.18.01139.
Schwarzenberg P, McCarthy A , Harty JA , Dailey HL. Clinical Application of Virtual Mechanical Testing Measures Slow Fracture Healing in Patients with Comorbidities. ORS 2021 Annual Meeting Paper No. 0788

Intervertebral Disc Inflammation: A CRISPR View of Treatment

The origin of back pain is often hard to determine. It is known that inflammation is often associated with intervertebral disc disease. This inflammation is associated with cytokine pathways leading to matrix degradation including collagen and proteoglycans. Local anti-inflammatory delivery can be done with injectable nanoparticles but with time the effects would be lost. However, investigators have demonstrated that CRISPR aimed at cellular epigenic mechanism could modulate focal tissue activity using a lentil viral vector. A major inflammatory cytokine in degenerative intervertebral discs is tumor necrosis factor. A major receptor for this pathway is tumor necrosis factor receptor-1 (TNFR-1). The epigenome target is the methylation of the H3K9 histone at the transcription start site located in the promoter region of the TNFR1 gene. The lentil virus delivery system restricts the effect to the disc.

Researchers used adult rats (Sprague-Dawley 3-6 months) for luciferase expression (n-5), cell cytokine expression (n=5), and long-term degeneration (n-6).

Luciferase Versus PBS control

7 days after injection of the lentil virus vector into the IVD animals were sacrificed with direct imaging of the IVD segment. Disc tissue was extracted along with tissues from heart, lung, liver, kidney, spleen, and a non-injected IVD and soaked with luciferin followed by imaging with an IVIS system. The result was direct florescence noted in only the injected tail segment. Only the inject IVD cells showed luminescence. This demonstrates the focal isolation of the lentil viral injection.

Cell expression TNFR-1

21 Days after IVD injection of PBS or lentil viral injection of CRISPR for TNFR-1 or a non-target CRISPER (NT) rats were sacrificed for immunohistology assessment of cell expression TNFR-1. The cell expression for TNFR-1 was ½ of the expression for PBS and NT injected discs.

IVD degeneration

3 months after IVD injection of PBS or lentil viral injection of CRISPER for TNFR-1 or a non-target CRISPER (NT) rats were sacrificed for histology evaluation using a 10 point degeneration For scoring, stained IVD sections were evaluated using a previously reported method (Evashwick-Rogler et al 2018, JOR Spine 2018:1:e1014). The combined degeneration score (0-10) consisted of five categories, (1) AF, (2) AF/NP border, (3) NP cellularity, (4) matrix of NP, and (5) endplate. Each section received a score of 0-2, with 0 normal morphology and 2 severe degeneration. For TNFR-1 CRISPER the score was closer to the naïve state compared to PBS and NT.

This project confirms the potential of lentil viral focal CRISPER epigenetic injection to deliver an anti-inflammatory treatment with longer term effects.

References

Stover JD, Weston J, Trone M, Zeidan M, Lawrence B, Bowles RD. In Vivo CRISPR Receptor Modulation for Treating Disc Degeneration. ORS 2021 Annual Meeting Paper No. 0059

Rotator Cuff Repair, Bridging the Gap

A problem with rotator cuff tendon repair is that we have not been able to replicate the same process seen in entheses development in early life¹. By age 75 half of the population has a rotator cuff tear. Tears that occur earlier in life tend to retract leaving a gap that may be difficult to close. Added to this is fatty degermation of the affected muscles. Despite this, surgical technology has led to attempts to close and/or augment these gaps in 50- to 60-year-old patients. However, the manipulation of cytokines and breakdown products in wound repair could lead to the reproduction of developmental anatomy in adult insertion repairs.

To replicate the problem of a retracted tear investigators used adult New Zealand rabbits with a 5 mm gap created in the distal infraspinatus tendon. There were 4 groups: 1) no repair, 2) direct repair, 3) repair with electrochemically aligned collagen scaffold (ELAC), and 4) repair with ELAC and mesenchymal stem cells (MSC). ELAC is a methodology to form highly oriented electrochemically aligned collagen threads with mechanical properties close to those of natural tendon. A steel suture was used in the repairs to determine degree of retraction. After 6 months whole joints were excised on both sides. After microCT shoulders were allotted to biomechanical testing or histology with trichrome and picrosirius staining for collagen and immunohistochemistry for type I and III collagen, and tenomodulin.

All sutured tendons remained intact with no suture pull out. However, there was retraction of the direct repair group. Maximum load testing revealed all repair groups superior to no repair. The ELAC + MSCs was marginally superior to direct repair. There was considerable loss of the original ELAC collagen I fiber content but a significant increase in de novo type I collagen, particularly for ELAC + MSCs. Collagen fibers alignment was similar between native tendon and ELAC+MSCs (p = 0.17). ELAC alone differed significantly from native tendon fiber alignment (p = 0.02). Scarce tenomodulin was seen in direct repair whereas ELAC alone was greater but inferior to ELAC + MSCs.

Translating this into chronic retracted tears will be problematic but this interface appears to be a strong step to a solution for so many active individuals who would just like to be able to put a one-gallon jug of milk into the refrigerator or reaching across to attach a seat belt.

Reference

Nelson, F. On the Horizon from the ORS: Recreating Normal Tendon and Ligament Attachments After Injury J Am Acad Orthop Surg.2008 May 16(5) 237-239
McClellan P , Ina JG , Knapik D , Valente A , Wen Y, Wu C, James Anderson J, Gillespie RJ, Ozan Akkus O. Bulk Tendon Regeneration in Electrochemically Aligned Collagen Scaffolds in a Rabbit Rotator Cuff Model. ORS 2021 Annual Meeting Paper No. 0104.rabbits

Cam Deformity and Developmental Dysplasia: Getting the Position Right

The common denominator for increase articular cartilage (AC) wear in cam deformity leading to femoroacetabular impingement (FAI) and development dysplasia of the hip (DDH) are increased forces on AC. The degree of deformity is often defined with AP and lateral radiographs to assess femoral and acetabular shapes relative to “normal” ranges.

24 cadaver hips had CT to measure femoral head alpha angles, head-neck offset, neck angles, version. The acetabulum was measured for lateral center-edge angle, inclination, version. The spinopelvic features of pelvic incidence was also measured. 12 hips had characteristic FAI findings and 12 under-coverage of DDH. He specimens were then denuded down to the bone and capsule for 6 degrees of freedom robotic testing and connected to a universal force-moment sensor. Anatomic landmarks were digitized. internalexternal rotations to 5 Nm in each position.

Ixtension (toe-off), Neutral 0° (standing), Flexion 30° (heel strike), Flexion 60° (mid-flexion), Flexion 90° (sitting); and performed hip internalexternal rotations to 5 Nm in each position.

The FAI group had axial alpha angle = 63 ± 6°, radial alpha angle = 74 ± 3°, and femoral head-neck offset = 4 ± 2 mm). The DDH group had acetabular undercoverage (lateral center-edge angle = 18 ± 2°, inclination = 13 ± 3°). The FAI group also indicated larger pelvic incidence angles (54 ± 11°) compared to the DDH group (42 ± 7°). Femoral neck and version angles together predicted ROM in Neutral 0° (R2 = 60%) and Flexion 30° (R 2 = 58%); whereas pelvic incidence and femoral neck-shaft angle predicted ROM in Flexion 60° (R2 = 77%). In Flexion 90°, pelvic incidence and radial alpha angle together predicted ROM (R2 = 81%), where pelvic incidence alone accounted for 63% of this variance.

Pelvic incidence was a stronger predictor of hip ROM (R2 = 63%, deep hip flexion) than the characteristic femoral and acetabular parameters of FAI and DDH¹. It is not surprising that many athletic injuries occur in more squat positions such as climbing and ice hockey such as goalies who are more flexed while repeating internal rotation activities².

References

Ng KCG, Bankes MJK, Grammatopoulos G, Jeffers JRT, Cobb JP. Effects Of Pelvic Incidence On Hip Impingement And Dysplasia Mobility. ORS 2021 Annual Meeting Paper No. 0079
Whiteside D, Deneweth JM, Bedi A, Zernicke RF, Goulet GC. Femoroacetabular Impingement in Elite Ice Hockey Goaltenders: Etiological Implications of On-Ice Hip Mechanics. Am J Sports Med. 2015 Jul;43(7):1689-97. doi: 10.1177/0363546515578251. Epub 2015 Apr 15. PMID: 25878118

Osteoarthritic Cartilage Preservation: Delay of Game, Still First Down

Epidermal growth factor receptors (EGFR) are found on articular chondrocytes and are particularly important for homeostasis of chondrocytes that occupy the superficial layer of cartilage. Rosa-diphtheria toxin receptor (DTR) is a receptor for bacterial diphtheria toxin. DTR has been found to be nearly identical with human full-length heparin binding EGF-like growth factor (HBEGF). Genetically enhanced EGFR activity can be achieved by adopting the HBGEF gene. To study the impact of EGFR there were five experiments. For appropriate mouse models they bred Col2-Cre mice or Aggrecan-CreER mice with Rosa-DTR mice to generate Col2-Cre DTR (HBEGF Over^Col2) and Aggrecan-CreER DTR (HBEGF Over^AgcER) mice. The medial meniscotibial ligament was sectioned to create an osteoarthritis (OA) model. A method that creates and enduring structure to signal EGFR was created and then tested on the OA model.

Examining articular cartilage phenotype in HBEGF Over^Col2 and HBEGF Over^AgcER mice.

The growth plate thickness was greatest in the 1-month-old over the 5-month-old HBEGF Over^Col2mice with the proliferative zone showing the greatest increase. Calcified articular cartilage, uncalcified articular cartilage, and total articular cartilage thickness were increased in overexpressing mice at 1 and 5 months.

Delineating the cellular and molecular changes in articular cartilage

In 5-month-old HBEGF Over^Col2 mice there was higher articular chondrocyte staining for Ki67 (cell proliferation), TUNEL (apoptosis), and PRG4 (superficial zone protein important in lubrication). In dissociated cell cultures from 5-month-old mice HBEGF Over^Col2 had higher stain levels of 5-Ethynyl-2′-deoxyuridine (DNA synthesis) and colony forming units from fibroblasts.

Characterizing the responses to meniscal destabilization

Relative to wild type mice with sham surgery the HBEGF Over^AgcER with meniscal destabilization had a similar lack of histology evidence of OA development. Gefinitib eliminated the protective effect of Over^AgcER mice.

Synthesizing and characterizing TGFa-NPs

Poly(ethylene glycol)-polycaprolactone (PEG-PCL), poly[L-lysine-block-poly(ε-caprolactone)] (PLL-PCL), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-5000] (DSPE-PEG5K-N₃) were assembled in a fashion to create an appropriate nanoparticle (NP) size and surface charge (zeta potential) that would provide the improved cartilage penetration and the increased joint retention. The diameter is 20-40 nm and retrains stability of size for 7 days in water and 24 hours in bovine serum. TGF-α was attached to this construct via copper-free click chemistry.

Studying the effect of intra-articular injection of TGFa-NPs on attenuating OA progression

By the addition of rhodamine it was found that bovine articular cartilage samples retained TGFa-NPs best and the pentation into bovine articular cartilage took 6 days to reach maximum appearance. Meniscus destabilized mice who had intraarticular TGFa-NPs had lower Mankin scores approaching shame mice, uncalcified cartilage thickness approaching sham mice, and less synovitis approaching sham mice.

Reference

Wei Y, Luo L, Gui T, Yu F, Yan L, Yao L, Zhong L, Yu W, Han B, Patel JM, Liu JF, Beier F, Levin LS, Nelson C, Shao Z, Han L, Mauck RL, Tsourkas A, Ahn J, Cheng Z, Qin L. Targeting cartilage EGFR pathway for osteoarthritis treatment. Sci Transl Med. 2021 Jan 13;13(576):eabb3946. doi: 10.1126/scitranslmed.abb3946. PMID: 33441426

Trimming the Fat in Osteoarthritis

The knee is an organ comprised of cartilage, fibrocartilage, bone, synovial lining, ligaments, capsule, and adipose tissue, including the large anterior infrapatellar pat pad (IFP). The role of inflammatory cells and cytokines from the synovium has been well studied. The IFP is also known to be rich in stem cells as well inflammatory cells such as macrophages and leukocytes. Because Hartley guinea pigs naturally develop knee osteoarthritis (OA) that is similar to human disease, they were used as a model for determining the role of the IFP in OA progression¹.

Ten 3-month-old guinea pigs had a unilateral IFP excision from one knee, with the opposite knee serving as a sham surgery control. Notably, Hartley guinea pigs develop OA after three months; thus, this treatment was meant to be preemptive. Gait analyses was collected prior to surgery and then monthly for the duration of the study. Animals were harvested at 7 months of age, when moderate OA was expected to be present. Microcomputed tomography (microCT) and histopathology were performed on all 20 limbs. The microCT was scored using a clinically-relevant scoring system. Histology scoring was based on Mankin criteria modified to consider species-specific features of OA.

Stride length was not statistically different for either hindlimb throughout the study. Limbs receiving resection of the IFP had a decreased/improved microCT score (1.048 compared to 6.500) compared to contralateral intact knees (p<0.0001). The mean modified Mankin score of knees with IFP removal was 2.556 versus 12.56 in naïve knees (p<0.0004). In limbs with IFP resection, this area was replaced with fibrous connective tissue. Ongoing work continues to determine the contribution of the IFP to joint homeostasis and OA, as well as the potential benefit of the connective tissue in place of adipose.

Resection of the fat pad is commonly done in humans receiving reconstructive knee surgery, with no known reports of adverse effects beyond decreased range of motion due to local fibrosis². Based on a review of the contribution of the IFP to knee pain, there are cytokines and other mechanisms whereby synovial tissue and adipose tissue have a combined effect on pain and, in all probability, OA progression.

Reference

Afzali MF, Radakovich LB, Pixler ZC, Campbell MA, Sanford JL, Marolf AJ, Donahue T, Santangelo, Kelly S. Early removal of the infrapatellar fat pad beneficially alters the pathogenesis of primary osteoarthritis in the Hartley guinea pig ORS 2020 Annual Meeting Paper No.0166
Belluzzi E, Stocco E, Pozzuoli A, Granzotto M, Porzionato A, Vettor R, De Caro R, Ruggieri P, Ramonda R, Rossato M, Favero M, Macchi V. Contribution of Infrapatellar Fat Pad and Synovial Membrane to Knee Osteoarthritis Pain. Biomed Res Int. 2019 Mar 31;2019:6390182. doi: 10.1155/2019/6390182. eCollection 2019.PMID: 31049352

It Takes a Village to Raise a Child

Clinicians have seen, in the absence of obvious injury, severe degenerative rotator cuff disease with minimal articular cartilage change. Alternatively, severe degenerative joint disease may occur in the presence of an intact rotator cuff. It is known that aged tissues are more likely to respond to stress with an inflammatory cascade. Investigators hypothesized that explants from aged mice would demonstrate increased inflammatory cytokine production leading to more rapid cell death and matrix degeneration in an initially normal appearing tendon¹.

Bone-tendon-muscle (BTM) tissue explants, as well as isolated bone, tendon, and muscle tissues, were taken from 4-month (young) and 24-month (old) mice. Spent culture media after 24 hours for each group was tested for production of catabolic proteins (IL-1β, IL-6, IL-10, TNF-α, INF-γ, KC/GRO) and anabolic proteins. Tendons in BTM explants were also harvested for analysis of gene expression for collagen 1, scleraxis, tenomodulin, MMP-13, p16^INK, p19^ARF, p53, MMP-3, IL-1β, TNF-α, and IL-6 along with confocal assessment of cell viability.

Cell viability was reduced to 10% by 5 day with a slightly less loss in older mice. Except for elevated IL-1β in aged mice, there were no significant differences in cytokines present in BTM culture media. However, isolated tendon cultures showed significant reductions in inflammatory cytokine production (IFN-γ, IL-10, KC/GRO, IL-6), while significant increases were found in aged muscle (IFN-γ, IL-10, KC/GRO, IL-1β, IL-6) and bone (IFN-γ, IL-10, IL-6). The authors noted that the combined cytokine crosstalk between the three tissue in a complete explant likely explains the difference between BTM and individual tissue cytokine responses. They also demonstrated that increased gene expression does not necessarily lead to increased protein production, particularly in aged tissues.

In an unrelated rat experiment it was found that the reaction of subchondral bone to tendon change is a rat model resulted in subchondral bone changes².

With these results there may be a more global view to rotator cuff/glenoid joint pain and dysfunction. The glenoid joint is complex and like the knee, it is a very complex organ. That organ, aside from the blood vessels and nerves, includes muscle, tendon, bone (both insertional and subchondral), cartilage, fibrocartilage, and synovium. As the authors clearly state, there is cross talk between these components. But in what direction? The mechanical state of the shoulder, variations in anatomy, activity/injury history, and other environmental factors all lead to the variations seen clinically. These authors have pressed on to highlight the importance of three integral tissues in this complex process. This is an important introduction to the consideration of all parts of the puzzle.

References

Connizzo BK, Grodzinsky AJ. Tendon inflammatory responses are altered with age in an in vitro rotator cuff model of secondary joint damage. ORS 2020 Annual Meeting Paper No.0397.
Parks AN, McFaline-Figueroa J, Coogan A, Poe-Yamagata E, Guldberg RE, Platt MO, Temenoff JS. Supraspinatus tendon overuse results in degenerative changes to tendon insertion region and adjacent humeral cartilage in a rat model. J Orthop Res. 2016 Dec 21. doi: 10.1002/jor.23496. [Epub ahead of print] PMID: 28001327

Reaching for the Scars

Despite advances in tissue engineering the ability to get tendons to heal to bone or tendon to tendon does not completely recreate the original embryology and mature construct¹. In order to better understand improved tendon healing a study was developed to investigate the association between expression of healing biomarkers and patient reported outcome after Achilles tendon rupture (ATR)². Competitive level athletes are very often selected for surgical versus non surgical repair.

At the time of Achilles tendon repair a 1-2 mm tendon biopsy was taken and frozen. These were later homogenized, RNA extracted, and cDNA synthesized to detect mRNA expression of FGF, FN, COL I, COL III and MMP-9. GAPDH was used for normalization. Portions were set aside for immunohistochemistry staining for FGF, FN, COL I, COL III and MMP-9. The mRNA levels were contrasted to ATRS, a measure of Achilles tendon function and pain, in this case at one year.

There was a positive correlation between FGF mRNA expression and ATRS 1 year after rupture. N = 12. Likewise, there was a positive correlation between COL III expression and 1-year ATRS1. On immunohistochemistry the alignment of collagen fibers was disorganized relative to the more parallel pattern of the more proximal sample. In their discussion the authors suggest that Higher FGF expression at both mRNA and protein level may activate stem cells in the injured tendon, leading to increased production of tendon-related genes and improved healing outcome. They suggest the same is true for COL III to predict tendon strength at one year.

For high level athletes would this or other analyses at the time of injury predict outcome or return to sport? Also, does this suggest potential tissue engineering strategies?

Figure 1. a) FGF immunoreactivity in injured human tendon and b) Correlation between

FGF gene expression and patient reported outcome measure as total ATRS from patient

suffering with ATR (*p < 0.05 by Spearman’s rank correlation coefficients).

References

Nelson, F. Recreating Normal Tendon and Ligament Attachments After Injury. J Am Acad Orthop Surg 2008;16:237- 240.
FGF and COL III expression effects long term patient outcome after Achilles Tendon Rupture. Chen J, Ahmed AS , Ackermann P. ORS 2020 Annual Meeting Paper No.0712.

Chronic Ankle Sprains: Time to Shape Up

Most people who experience a lateral ankle sprain can anticipate uneventful recovery without a propensity for recurrent sprain. If chronic ankle instability (CAI) occurs and physical therapy fails to resolve the problem the options are ankle supports or reconstruction surgery. Unfortunately, surgery does not always result in a fully functional ankle for sports and other activities such as hiking on uneven surfaces.

To the knowledge of the authors no one has investigated variations in the structure of the talus and calcaneus that can lead to CAI¹. 26 patients with unilateral CAI and 26 normal individuals had CT of both ankles. Both the calcaneus and talus were segmented for additional manipulation. Shape modes can be used to capture shape variations that other techniques cannot achieve because the shape modes are independent from each other. These manipulations allowed for group comparisons of shapes.

The CAI patients generally had flatter talar joint surfaces and a flattened calcaneal ground‐contact surface. The CAI patients seem to be exhibiting less congruent subtalar joints as well as a flattened calcaneal ground‐ contact surface. Variations in the medial and lateral calcaneal tuberosities can alter the loading moment, which is formed by a pair of the ground reaction force and joint reaction force. The authors speculate that with a lateral sift of the ground reaction force the patient may try to minimize this inversion movement to avoid losing balance. The resulting compensating reaction may lead to a recurrent lateral ankle sprain. In individuals with CAI there is a decrease in the inclination angle of the talar neck relative to the body. Also, a distal extension of the lateral tuberosity is more likely, which with or without decreased talar neck inclination angle can cause the subtalar joint to be more vertically oriented. An analogy would be the difference between stacking rectangular blocks side to side versus on edge.

The use of this imaging technology could help lead to a better understanding of several activity related injuries.

Reference

Tümer N, Vuurberg G, Blankevoort L, Kerkhoffs GMJ, Tuijthof GJM, Zadpoor AA. Typical Shape Differences in the Subtalar Joint Bones Between Subjects with Chronic Ankle Instability and Controls. J Orthop Res. 2019 Sep;37(9):1892-1902. doi: 10.1002/jor.24336. Epub 2019 May 26. PMID: 31042001

Thrombosis: Pulling Our Risk Out of the Fire

Lower limb fractures that require fixation are associated with a high risk for thromboembolic disease. This can occur despite prophylactic intervention. Increasing blood viscosity is known to play a role in thrombus formation. Thrombelastography is able to measure viscosity with maximal amplitude (mA), a measure of clot strength, > 65mm being predictive of in-hospital VTE. Thrombelastography, inflammatory cytokines, and plasminogen activator factor were assessed from blood samples obtained 1 hour pre and postoperative, daily for five days, and at 2 and 6 weeks.

Twelve femur fractures aged 24-66 years underwent serial TEG and systemic inflammatory marker analysis. Hypercoagulability peaked at two weeks, median admission mA = 59.8mm (57.3mm-61.6mm) and 71.0mm (70.3mm-74.3mm) at 2-weeks (p=0.002). IL-6 was elevated on admission 45.6 (25.4-72.1) and returned to normal by 2-weeks 2.8 (1.8-6.8); p = 0.02). CRP reach a wide range of peaks at 24, 48, and 72 hours with closer to normal values at 2 weeks and normal at six weeks. The patient who presented with the highest mA on admission experienced a PE and remained hypercoagulable until 6-week follow-up. PAI-1 had a strong correlation with mA at 2-weeks (r = 0.80).

TEG appears to be a valuable point-of-care to tool to evaluate the risk for thromboembolism.

So, what can TEG do for other conditions? We are in the midst of A COVID-19 pandemic. It is recognized that prime feature for those who succumb is a “cytokine storm”. The cytokine features of this storm include IL-1, Il-6 and the other usual suspects². 24 COVID-19 ICU patients has TEG, CRP, and clotting studies performed. TEG ranged 58.0-92.0 (avg 79.1). CRP ranged 3.9-34.2 (avg 16.1). Serum D-dimer is often used to detect clot formation. Tis was elevated in all patients. Most platelet counts were within normal range.

Wherein the prime feature is death following femoral fracture fixation is ARDS and DIVC for this is different. There is something very similar here in the research on TEG and venous or arterial thrombosis. There is an opportunity to seek out those out risk and pull their fat out of the fire.

References

You D, Cantle P, Korley R, Lee A, McDonald B , Skeith L, Soo A, Schneider PS. Individual Hypercoagulability and Inflammatory Response Following Surgical Fixation of Femur Fractures ORS 2020 Annual Meeting Paper No.0061.
https://www.sciencedirect.com/science/article/pii/S2666379120300215
Panigada M, Bottino N, Tagliabue P, Grasselli G, Novembrino C, Chantarangkul V, Pesenti A, Peyvandi F, Tripodi A.J Thromb Haemost. Hypercoagulability of COVID-19 patients in intensive care unit: A report of thromboelastography findings and other parameters of hemostasis. 2020 Jul;18(7):1738-1742. doi: 10.1111/jth.14850. Epub 2020 Jun 24. PMID: 32302438

Exosomes: They Can Grow on You

Purified exosome products (PEP) are now commercially available. Exosomes are a type of extracellular vesicles that can be derived from a variety of cells, including stem cells. Exosomes are produced in the endosomal compartment of most eukaryotic cells. An endosome defined by intraluminal vesicles that bud inward into the endosomal lumen. If the multivesicular body fuses with the cell surface, these intraluminal vesicles are released as exosomes. Exosomes can also be extracted from plasma. Platelet rich plasma has been applied to a variety of musculoskeletal conditions.

To test the effects of exosomes on tendon healing investigators used commercially prepared exosome lyophilized powders. This preparation is not specific to a given cell source and is prepared from plasma donation that have expired but are still sterile. This exosome powder can then be expanded in plasma for study.

Characterization of PEP

Using NanoSite analysis the major peak particle size was 100 nanometers. Transmission electron microscopy demonstrated spherical particles approximately 100 nm in size. There are approximately 2.5 X 10¹¹ particles per ml. Enzyme‐linked immunosorbent assay kits analysis demonstrated CD63, seen on the surface of exosomes, and acetylcholinesterase (AChE) seen in the interior of exosomes.

Tenocyte response

On confocal microscopy red dye markers show that the particles are taken up by the cytoplasm. For increasing cell growth the optimum concentration of PEP leveled off at 5%. The media optimum dose was 5% PEP and 10% fetal bovine serum (FBS). Cell migration was greater in PEP compared to media alone and the increased confluency was the same for FBS and FBS plus 5% PEP. On the first day qRT-PCR (timed response for mRNA expression) for (SCX (scleraxis), COL1A, COL3A1 (type I and III collagen), TNMD (tenomodulin), and MKX (Mohawk) was the same for media alone, PEP, FBS, and FBS+PEP. However, at three days those markers as well as DCN (decorin) were significantly increased in the PEP groups. PEP combined with FBS increased TNMD and DCN expression. Collagen expression was increased over DMEM similarly. Compared to PEP or FBS alone PEP and FBS together was significantly greater. The combination of FBS and PP significantly decreased apoptosis. However, the increased apoptosis with exposure to dexamethasone was significantly reduced with FBS and PEP compared to FBS alone.

In summary, this research demonstrates PEP enhancing cell proliferation, increasing total collagen deposition, maintaining tenogenic phenotype and migration capability, as well as attenuating dexamethasone‐induced cell apoptosis. Future research may show longer term tissue engineering and tendon injury recovery effects. The question remains, would tenocyte or tenoblast specific exosomes be more effective?

Reference

Qi J, Liu Q, Reisdorf RL, Boroumand S, Behfar A, Moran SL, Amadio PC, Gingery A, Zhao C. Characterization of a purified exosome product and its effects on canine flexor tenocyte biology. J Orthop Res. 2020 Aug;38(8):1845-1855. doi: 10.1002/jor.24587. Epub 2020 Jan 24.J Orthop Res. 2020. PMID: 31930553

Nanoparticles Going on a Scavenger Hunt

Half of the content of most intra-articular injections are eliminated from the joint between 30 minutes and several days following injection. The rate of this elimination has an inverse relationship to molecular size. Nanoparticle attachment has been used to improve the joint resident time of a variety of potential joint reserving agents. There are three components to the resident time of the therapeutic agent within the joint: the rate of elimination of the nanoparticle, breakdown of the nanoparticle, and rate of dissociation of the therapeutic molecule from the nanoparticle. A fourth consideration is cellular uptake by targeted cells wherein there is pharmacologic activity without cellular toxicity.

It is known that nanoparticle (NP) associated with manganese dioxide (MnO₂) are H₂O₂ scavengers. These nanoparticles are rapidly internalized by chondrocytes and bone marrow macrophages. The PEG stabilized MnO2 NPs had a hydrodynamic diameter of 15 nm, TEM size of 8 nm and had a cationic surface charge. The NPs effectively scavenged H2O2, with 5mg/mL MnO2 NPs neutralizing 55% of 100uM H2O2 in PBS. Chondrocytes in monolayer showed uptake of MnO2 NPs without cytotoxicity.

Chondrocytes in media

Chondrocytes were exposed to standard control media, media containing IL-1β, IL-1β with NPs, and NPs alone. Compared to IL-1β conditions wherein there was an upregulation of both MMP1 and MMP13 by 125.8-fold and 70.5-fold, NPs maintained these genes at baseline conditions. COL2 was upregulated 2.2 fold with IL-1β indicating a possible initial anabolic response to the inflammation but was not upregulated with combined NP and IL-1β. IL-1β upregulated antioxidants glutathione peroxidase 10.2 fold and manganese superoxide dismutase 43.4 fold but treatment with NPs and IL-1β maintained these genes at control conditions. A number of other antioxidants had similar findings.

Bone marrow derived macrophages in media

Lipopolysaccharide stimulation of TNF-α in rat bone marrow derived macrophages was reduced in a dose dependent fashion by the NPs. An anti-inflammatory cytokine IL-10 was minimally elevated in a dose dependent manner but it did not reach statistical significance.

This nanoparticle approach is based on the persisting intracellular presence of the nanoparticle. For intra-articular application it will be required to answer a number of questions. How long will the application be effective? Is there any toxicity with a more prolonged presence? How will this NP work with an already stimulated and possibly dedifferentiated chondrocyte? Film at 11.

References

Kumar S, Adjei I, Brown S, Sharma B. Chondroprotective Mechanisms of Manganese Dioxide Nanoparticles for Intra-Articular Therapies ORS 2020 Annual Meeting Paper No.0639
KumarS, Adjei IM, Brown SB, Liseth O, Sharma Manganese Dioxide Nanoparticles Protect Cartilage From Inflammation-Induced Oxidative Stress. Biomaterials. 2019 Dec;224:119467. doi: 10.1016/j.biomaterials.2019.119467. Epub 2019 Sep 11.

A Novel Way to Lick Your Wounds

With increasing incidence of obesity diabetic foot ulcers and leg ulcers are becoming more common. Both decreased protective sensation and abnormal vascular supply play a role. Once an ulcer has occurred there are many wound dressings used to help in ulcer healing. Sericin is a high cysteine content protein made by silkworms seen in the cocoons. It is incorporated in skin products as well as a variety of wound dressing products for use in chronic wound conditions such as venous stasis ulcers in hopes of improved vasodilatation. 4-hexaresorcinol (4HR) found in some commercial skin products and is associated with the elaboration of VEGF-A and VEGF-C. To study the effects and mechanisms of these two agents human umbilical vein endothelial cell (HUVEC) cultures and an induced diabetic mouse model were used. The mouse model was created by a diabetic inducing agent STZ followed by a deep burn on the back of 20 Wistar rats.

human umbilical vein endothelial cell (HUVEC) cultures

HUVECs were growth to 70% confluency and treated with varied dose of 4HR. Lysed cells were evaluated with western blot for TGF-β1. In similar culture TGF-β1 was added in various doses to measure western blot for VEGF-a and VEGF-C. To secure the role of TGF-β1 was assessed by adding 4HR after incubation with siRNA for TGF-β and VEGF-A and VEGF-C. A similar experiment was done with A83-01, an inhibitor of ALK5. ALK5 block SMADS initiated by TGF-β1.

4HR increased VEGF-A, VEGF-C, and TGF-β1 expression. TGF-β1 also increased VEGF-A and VEGF-C expression.
TGF-β1 small interfering RNA (siRNA) inhibited VEGF-A and VEGF-C expression normally induced by 4HR application. ALK5 inhibitor, A8301, had a similar effect.

Diabetic Rat skin burn model

After diabetic induction thermal back skin burn were created on a 2cm X 2cm area of skin with hair removed. There were three groups: ointment by itself, ointment with sericin (a known inflammatory macrophage stimulator) and ointment with 4HR (a known M2 macrophage stimulator). Ointment was applied every three days and heat photographs were take 1, 2, and 3 weeks after burn. Skin samples were thin sliced for confocal microscopy and other samples used for immunohistology for TNF-α, TGF-β1, VEGF-A, and apoptosis-inducing factor (AIF). Tissue was also save for extraction and western blot analysis for TGF-β1, VEGF-A, VEGF-C, TNF-α, hypoxia inducible factor-1α (HIF-1α), AIF, and β-actin.

4HR and sericin wound sizes were small that control ointment.
On confocal microscopy only 4HR showed significant increase in vascularity but sericin had vasodilatation.
Skin thickness on H and E stain was greater in 4HR.
With western blot TGF-β1 and apoptosis inducing factor (AIF) were higher in the 4HR group. TNF-α and HIF-1α was higher in the sericin group.
Immunohistochemistry was also in accordance with those of tissue Western blot. TGF-β1 and AIF were highly expressed in 4HR administered groups and TNF-α in the sericin group.
Wound temperature was highest in the sericin group followed by 4HR and ointment alone.

The take home for this is that 4HR-induced angiogenic factors (VEGFs) are controlled by TGF-β1 expression and subsequent activation of SMADs/VEGFs signaling. The impact on M2 macrophages has major implication for eventual application to human ulcer conditions.

References

Kim D-W, Jo Y-Y, Garagiola U, Choi J-Y, Kang Y-J, Oh J-H, Kim S-G. Increased Level of Vascular Endothelial Growth Factors by 4-hexylresorcinol Is Mediated by Transforming Growth Factor-β1 and Accelerates Capillary Regeneration in the Burns in Diabetic Animals. Int J Mol Sci. 2020 May 14;21(10):E3473. doi: 10.3390/ijms21103473. PMID: 32423083

Bioprinting: Hot off the Spinning Press

The number of papers found in PubMed relative to bone tissue engineering jumped from nearly 100 in 1993, to 1000 in 2005 and about 3700 per year for the past three years. Investigators have developed a bone forming constructs that have a relative time released expression of vascular endothelial growth factor (VEGF) followed by bone morphogenic protein (BMP)¹. Applied to challenging defects the methodology continues under investigation. The problem is made very complex by the nature of the construct (scaffold), cytokine dose over time, and the sequencing of cytokines. A recent approach is the development of bioprinting and the capacity to incorporate more than one material in the bioprinting process.

To expand on the potential of the bioprinting investigators hypothesized that they could create a bioprinting spatial gradient of VEGF that would accelerate vascularization of an implant which would also have a slowly releasing low doses of BMP-2 from defined regions of the implant. This combination would enhance bone healing with minimal ectopic bone. Bioinks that included RGD and methylcellulose were printed within networks of PCL filaments to generate 3 experimental groups; 1. isolated VEGF Gradient, 2. BMP-2 gradient, and 3. a composite (VEGF+BMP-2) with the osteoinductive bioink in the periphery with the vascular bioink in the center.

In the in vitro analyses addition of laponite to the bioink allowed for a slower release of BMP with subsequent osteogenesis in the BMP alone arm. nHA provided for a spatial gradient of VEGF with in vivo even vascularization throughout the construct. In vivo the composite implant had increases in vessel volume and diameter within the defect site. There was also a larger number of mature vessels were also observed in the BMP-2 gradient and composite groups. Albeit at 12 weeks more new bone formation was seen in all three experimental groups safranin O staining revealed that after 12 weeks there was still new bone developing via endochondral ossification in the BMP-2 gradient and composite groups. There was little ectopic bone formed. µCT showed that the composite group had the most consistent bone regeneration and new bone formation was as early as 8 weeks post-implantation.

The take home is that the technology of this construct provides a spatial time release of VEGF and BMP that leads to greater vascularity and enchondral ossification without excess amounts of cytokines that could lead to heterotopic bone. This was a 5 mm defect in a rat model. This should lead to larger constructs and bioprinting techniques that can be applied clinically.

References

Kempen DH, Yaszemski MJ, Heijink A, et al: Non-invasive monitoring of BMP-2 retention and bone formation in composites for bone tissue engineering using SPECT/CT and scintillation probes. J Control Release 2009;134:169176.
Freeman FE, Pitacco P. Nulty J, Browe DC, Shin J-Y, Alsberg E, Kelly DJ. 3D Bioprinting Spatio-Temporally Defined Patterns of Growth Factors to Tightly Control Tissue Regeneration. ORS 2020 Annual Meeting Paper No.0155.

How to Stop Pushing Mesenchymal Progenitor Cells off a Cliff

Human studies show that chronic inflammation. as seen in diabetes and rheumatoid arthritis, impacts both fracture healing and mesenchymal progenitor cells. Serum transfer-induced RA mice (K/BxN) develop fracture nonunion partially due to elevated expression of nuclear factor kappa B (NF-κB) activation. Experimentally induced increased expression of NF-κB in mice leads to decreased progenitor cell differentiation into osteoblast and chondrocyte, fibrotic tissue formation, and fracture non-union. It is not clear what is downstream from NFκB that leads to this phenomenon.

Animal Studies have found that DNA methyltransferase 3b (Dnmt3b) is highly expressed in fracture callus during fracture repair. Of the Dnmt family only Dnmt3b is responsive to cytokines in MPCs, indicating an essential role of Dnmt3b in fracture healing. Inflammation decreases Dnmt3b expression in MPCs which is a likely a downstream mechanism for fracture nonunion. Inflammatory signals inhibit Dnmt3b in an NF-κB-dependent manner. In line with these findings, Dnmt3b gain-of-function (GOF) in MPCs shows a protective effect from inflammation in vitro.

Rbpjκ, also known as CBF1, is the human homolog for the Drosophila gene Suppressor of Hairless. This promoter region classically increases Notch signaling which inhibits MPC chondrogenic and osteogenic differentiation. Rbpjκ may be a downstream target of Dnmt3b in MPCs. This pathway has a negative impact on MPC differentiation into chondrogenic and osteogenic cells. Investigators have shown that the MPC differentiation defect observed in Dnmt3b loss-of-function (LOF) cells is due to upregulation of Rbpjκ. Dnmt3b physically binds to the CpG island (a CG repeating segment) in Rbpjκ promoter resulting in DNA methylation reduction in the CpG island. This leads to the reduction of the Notch signaling that suppresses chondrogenic and osteogenic function.

So how can we promote better fracture healing in patients with inflammatory disease. An existing medication is DAPT (dual antiplatelet therapy). Experimentally DAPT has been found to restore fracture healing under K/BxN induced fracture nonunion conditions in mice. The question is whether this can translate into human applications for fracture healing in inflammatory disease,

Reference

Ying J, Xu T, Wang C, O’Keefe R, Abu-Amer Y, Shen J. Epigenetic Regulation of Bone Regeneration in Inflammation Disease. ORS 2020 Annual Meeting Paper No.0196

Ying J, Xu T, Wang C, Jin H, Tong P, Guan J, Abu-Amer Y, O’Keefe R, Shen J. Dnmt3b ablation impairs fracture repair through upregulation of Notch pathway. JCI Insight, 2020. PMID: 32051335.

Osteoblasts: Always Looking for a Nest

Cells get their signals in a variety of ways: chemical, physical, or mechanical. Generation of biochemical signals via ion channels and receptors often depend on mechanical stimuli such as loading, electromagnetic fields, and surface stiffness. But it may be a surprise that osteoblasts would seek favored structural environments.

One of the fields of research and clinical application is nanotechnology. Here we are looking for dimensions of 100 nm (1000 angstrom) or less.

Working down the scale from macro to micro to nano investigators have been adding nanostructures to both microsmooth and microrough implants to improve the early healing and long-term osseointegration of implants. The superposition of nanostructures onto microrough surfaces synergistically enhanced osteoblast differentiation and local factors important for bone formation compared to unmodiﬁed microrough controls. These results suggest that osteoblasts are very sensitive to the hierarchical structure of the surface they attached to for the production of new bone.

After lipids, water and proteins adsorb onto a fresh implant surface, their conformation influences subsequent cell behavior. Attachment of platelets is associated with fibrin production and is dependent on surface properties. Neutrophils, macrophages, and multipotent progenitor cells follow based on surface-dependent attachment and signaling. It is fascinating to note that osteoclasts leave behind a nanostructure environment during the formation of the resorption pit that includes collagen tufts and fibers (100 nm). From a micro standpoint, grit blasted versus smooth dental implants and pedicle screws have been evaluated with CT imaging and histology, showing that osseointegration is improved on the textured surfaces. Indeed, pull out strength is far stronger in the grit blasted group. In vitro experiments using titanium substrates with a complex topography characterized by 30 µm diameter craters superimposed with irregular pits and peaks approximately 3 µm in diameter to mimic the osteoclast resorption pit, elicited the most differentiated osteoblast phenotype. Experiments show have shown both improved pull out strength and positive cytokine activity for osteoblasts with increased nanoroughness. In vitro experiments also show that osteoblasts respond with more anabolic activity on microtextured surfaces that are nanorough compared to nanosmooth. Likewise, MSC differentiation into osteoblasts is greater on the nano-rough surface.

So, what is the best surface for osteointegration to metallic surfaces? Through eons of evolution, nature has provided osteoclast pit surfaces that drive osteoblasts wild. The problem is how science with all of the 3D printing technology can mimic the surface similar to osteoclast pit. This could allow a surface to trick a cell into thinking a new implant is actually native bone? Dr. Boyan and her lab has demonstrated that when a surface mimics the roughness of the natural bone surface, multipotent progenitor cells sense it and differentiate into osteoblasts. Engineering this type of surface is essential to tailoring the implant to the specific patient.

Reference

Gittens RA, Olivares-Navarrete R, Schwartz Z, Boyan BD. Implant osseointegration and the role of microroughness and nanostructures: lessons for spine implants. Acta Biomater. 2014 Aug;10(8):3363-71. doi: 10.1016/j.actbio.2014.03.037. Epub 2014 Apr 8. Review. PMID: 24721613

Glucocorticoids: When is too Much, too Much?

It is rare that a short, tapered glucocorticoids (GC) exposure results in femoral head avascular necrosis also known as osteonecrosis of the femoral head. Longer term relative higher dose corticosteroid however have a varied risk of that complication. Early detection of ONFH, especially before symptoms occur, may improve the effectiveness of interventions. Magnetic resonance imaging (MRI) is the most effect tool for early detection but too expensive for routine surveillance of long term steroid use. Based on previous literature there are a number of serum biomarkers that mat be useful in detecting ONFH. Given that disturbances in bone and lipid metabolism, inflammation and cell apoptosis, coagulopathies, and hematologic condition that impact local circulation the list of serum biomarker suspects includes type I collagen crosslinked C-telopeptide (β-CTX), amino terminal telopeptide of procollagen type I (PINP), osteocalcin (OCN), apolipoprotein (apo)-A1, apo-B, high density lipoprotein cholesterol (HDL-C), and low density lipoprotein cholesterol (LDL-C), tartrate resistant acid phosphatase (TRAP), sclerostin (SOST), adiponectin (ADPN), leptin (LEP), and interleukin 33 (IL-33).

Patients who required a dose of ≥30mg/d prednisone equivalent dose for ≥3 weeks or GC pulse therapy for ≥3 days were enrolled. Hematologic studies and serum studies for the above marker were collected antecedent to GC treatment and then, along with, MRIs of both hips at 1, 3, 12, and 24 months. A nested case control study was created by taking 7 patients who developed ONFH between 1 and 3 months and comparing them to 20 non ONFH patients wherein there was no significant differences between age, gender, and disease type in the two groups. The cumulative dose of GC was not significantly different between the two groups (p=0.082; Table 1).

The β-CTX levels were significantly lower in GC-induced ONFH patients both before (p<0.001) and after (p<0.05) short term GC therapy. Among the patients with decreased PINP after GC therapy the mean reduction in PINP was significantly greater in the osteonecrosis group than in the control group (by 60.7±9.0% vs. 41.1±5.6%; p<0.05). There were no linear correlations or rank correlations between the cumulative dose of GC and any of the 13 biomarkers.

Overall Lower β-CTX and PINP are promising biomarkers for the early diagnosis of GC induced ONFH. Large scale prospective studies will help confirm these findings and possibly give cut off values for ordering MRIs.

Reference

Wang XY, Hua BX, Jiang C, Yuan HF, Zhu L, Fan WS, Ji ZF, Wang Z, Yan ZQ. Serum Biomarkers Related to Glucocorticoid-Induced Osteonecrosis of the Femoral Head: A Prospective Nested Case-Control Study. J Orthop Res. 2019 Nov;37(11):2348-2357. doi: 10.1002/jor.24400. Epub 2019 Jul 22.

Bone Tissue Engineering: Like Your Favorite Beverage - What and How Much is in the Mix

In initial fracture healing one of the earliest steps is increased vascularity. Vascular endothelial growth factor (VEGF) is expressed early and bone morphogenic proteins soon follow. VEGF is known to stimulate osteoblast differentiation, vascularization and mineralization during skeletal development. Critical to any bone tissue engineered construct is a vascular system. 3D printing on a micro scale has made possible a more precise construct.

To this end investigators created a gelatin-nanohydroxyapatite (gel-nHA) 3D bioprinted scaffold with an interconnected pore network seeded with human musculoskeletal stem cells (hMSCs) subjected to osteogenic differentiation for two weeks. They then added lentiviral-GFP transfected human umbilical vein endothelial cells (HUVECs) to form a capillary-like network within the scaffold pores over the next two weeks. Bioprinting required crosslinking the gel-nHA with genipen in a sacrificial gel, pluronic acid, that gave temporary support for the printing and was later dissolved away. Pore size was 0.92 ± 0.14 mm, a lateral pore size of 0.5 ± 0.15 mm, and a porosity of approximately 60%. With water added there was no significant swelling after a leveling off period and the compressive modulus dropped from 36.4 ± 9.6 kPa to 5.1 ± 2.3 kPa. After UV sterilization, nine constructs were placed in culture with hMSC seeded at 10⁵ hMSCs/scaffolds for 7 days in growth medium (GM) and then osteogenic medium (OM) for another 7 days. They were then divided into three groups: 1. HUVECs added in 1:1 OM and endothelial growth media (EM); 2. no added HUVECs and 1:1 OM:EM; and 3. HUVECs added and 1:1 GM:EM.

All samples resulted in engineered bone matrix. The two conditions with HUVECs formed tubular structures within the bone constructs, with the assembly of a complex capillary-like network visible by fluorescence microscopy in the live tissue and in histology. CD31 immunostaining confirmed significant vascular lumen formation. Quantitative real-time PCR was used to quantify osteogenic differentiation and endothelial response. Alkaline phosphatase and runt-related transcription factor 2 upregulation confirmed early osteogenic commitment of hMSCs. Even with OM removed under HUVECs support there was clear osteogenesis.

The future of this novel construct will rely on the ability of native vasculature to grow into and anastomose with a larger construct designed to fill defects.

Reference

Chiesa I, De Maria C, Lapomarda A, Fortunato GM, Montemurro F, Di Gesu R, Tuan RS, Vozzi G, Gottardi R. Endothelial cells support osteogenesis in an in vitro vascularized bone model developed by 3D bioprinting. Biofabrication. 2020 Jan 10. doi: 10.1088/1758-5090/ab6a1d. [Epub ahead of print] PMID: 31929117

Stiff Joints: Rounding Up the Usual Suspects

One of the features of osteoarthritis (OA) symptomology and complications following knee replacement surgery is fibrosis. Various members of the transforming growth factor beta (TGF-β) family are essential for normal development and upkeep of joint components such as the articular cartilage and synovium. However, an abnormal increase in the activity of TGF-β members can help drive chondrocyte hypertrophy, ossification, and fibrosis seen in OA. In particular, TGF-β1 is one of the main regulators of matrix deposition in response to traumatic, acute, or chronic injuries such as OA. Many cell types produce TGF-β1, but a key source in the synovium are CD163+ cells, often associated with alternatively polarized, M2 macrophages. Previous studies have documented a higher incidence of fibrotic conditions such as scleroderma, keloid scar formation, and chronic kidney disease in black individuals. Retrospective analyses of prospectively collected patient-reported outcomes at the orthopaedic clinic of Louisiana State University Health Sciences Center in New Orleans (LSUHSC-NO) show significant disparities in the pre-operative severity of knee OA pain and symptoms (i.e. stiffness) by race based on widely used, validated patient-reported outcomes (PROs) questionnaires. Black patients consistently report worse knee OA problems than white patients, regardless of disease stage and after controlling for BMI, age, sex, insurance type, access to healthcare resources, and socioeconomic status. To evaluate biological factors that may help explain these racial disparities, the LSUHSC-NO orthopaedic research team developed a repository to bank joint tissues and cell lines, synovial fluid (SF), and blood from consented knee replacement candidates. The team hypothesizes that molecular pathways, specifically those causing black patients to be at higher risk of fibrotic conditions, may help identify the underlying drivers of OA-related racial disparities.

Knee OA patients who underwent total knee arthroplasty were selected for analyses of PROs by means of the Knee Injury and Osteoarthritis Outcome Scores (KOOS) (n=247); a subset of black (n=20) and white (n=20) patients, balanced by sex and PROs, were selected for more in-depth analyses. Function was assessed by PROs mainly adjusted for age, sex, and BMI. Formalin-fixed, paraffin-embedded femoral condyle and synovium were serially sectioned and the first set of sections stained with Safranin O and H&E, respectively, for assessment of OA severity of the articular surface and synovitis stage using brightfield microscopy and validated histopathology scoring methods. Synovial fibrosis was measured from a second set of slides stained by picrosirius (PS) technique. PS epifluorescence was captured by confocal microscopy and quantified by dividing the total pixels corresponding to the fluorescence emitted by collagen fibers, irrespective of type and age of deposition, over the total area of the tissue section. TGF-β1 and CD163 were co-detected by indirect immunofluorescence of a third set of serial sections and measured similarly to PS. PROs were compared by race and adjusted by analysis of covariance; fibrosis, TGF-β1, and CD163 levels were compared by Student’s t-test and expressed as mean±standard deviation, where p<0.05 was deemed significant.

Analysis of 247 knee OA patients showed that black patients reported worse PROs compared to white patients, in the symptoms (e.g., stiffness, swelling) (p=0.002), pain (p=0.0003), and activities of daily life (p=0.0006) subscales of the KOOS. Among the 40 patients selected for in-depth histopathology, there was no difference by race in histopathology scores for OA severity. However, black patients, particularly those with low synovitis, manifested a statistically significant 31% (p=0.0002) increase in synovial fibrosis. Correspondingly, black patients manifested a 40% increase in TGF-β1 expression in the synovial subintima relative to white patients (p<0.0001). Notably, there were no significant differences between racial subgroups in the number of CD163+ cells in the synovium, thus ruling out a dysfunction for sources of TGF-β1 with that phenotype and suggesting that further research must be conducted on the release and activation of TGF-β1 and its canonical, receptor-mediated cascade that targets collagen transcription during OA.

The research described provides evidence that black patients are at higher risk of a more severe synovial fibrosis phenotype in a TGF-β1-dependent manner and this process may influence differences in OA symptom severity reported by the racial subgroups. Knowledge gained from this research should yield a more comprehensive view of each patient’s disease state and inform individualized treatment strategies.

Although there is no literature linking synovial fibrosis to similarities in genetics or development of other fibrotic conditions, we must note that a TGF-β receptor-activated, Smad-mediated pathway modulates the transcription of collagen for deposition into the extracellular matrix in various fibrotic diseases by indirect (e.g. pro-inflammatory) or direct (e.g. genetic polymorphisms) influences. For example, there are multiple genetic alterations known to cause enough dysfunction in this cascade to drive keloid formation², a condition that is predominant in black individuals. Although black patients are at higher risk of developing various conditions of exacerbated fibrosis such as keloid formation and scleroderma, this study is one of the first to demonstrate a similar trend of increased scarring within the joint capsule during OA relative to race. Therefore, after adjusting for co-variates and considering co-morbidities, OA-induced synovial fibrosis warrants more in-depth cellular (e.g. myofibroblast content), pro-fibrotic protein and transcriptome profiling upstream and downstream of TGF-β1 in low- and high-risk patient populations to find the influencing stimuli or genetic alterations that may fuel such increases in the severity of synovial fibrosis.

References

Hodgeson SM, Jordan KE, O’Brien SY, Leonardi C, Dasa V, Marrero L. Race-Dependent Synovial Fibrosis May Impact Outcomes in Patients Undergoing Total Knee Arthroplasty. ORS 2019 Annual Meeting Paper No. 0133
Glass DA 2nd. Current Understanding of the Genetic Causes of Keloid Formation. J Investig Dermatol Symp Proc. 2017 Oct;18(2):S50-S53. doi: 10.1016/j.jisp.2016.10.024. Review. PMID: 28941494

Figure 1. Sections of synovium from OA patients can be stained by the picrosirius method. The dye specifically binds to collagen fibers and can be detected by either polarized or epifluorescence microscopy (shown) and quantified over total tissue area to highlight the severity of synovial fibrosis. (A) White patient with low fibrosis and (B) black patient with high fibrosis had equivalent histopathology scores for OA severity at the articular surface and synovitis grades measured using validated methods.

Fatty Infiltration: Turning Back the Muscular Clock

Chronic and unrepaired acute rotator cuff (RC) tears are associated with muscle atrophy and fatty infiltration (FI). When a repair is done, the muscle usually does not recover. Animal studies suggest that muscle resident fibro/adipogenic progenitors (FAPs) are a cellular source of RC muscle recovery in FI. FAPs can also generate brown/beige adipose tissue (BAT). A study was designed to see if Amibegron, a beta3 adrenergic receptor agonist, can induce FAP BAT differentiation leading to improved RC muscle rehabilitation and shoulder function after delayed RC tear repair in a mouse model.

Five PDGFRa-GFP reporter mice to track FAP cells, ten UCP1-knockout (KO) mice and ten colony control C57BL/6J mice at 3 months old underwent unilateral supraspinatus tendon transection followed by 6 weeks delayed repair surgery (TT+DR). A similar grouping was used for sham repair surgery approach without tendon sectioning. At time of repair or sham surgery all mice received 10mg/kg Amibegron (5 mice) or 5% DMSO vehicle (5 mice) administered intraperitoneally each day for 6 weeks starting on the day of surgery. At 6 weeks gait analysis was followed by sacrifice, muscle weight and histology for UCP1 along with red O staining for muscle fat.

In the wild type delayed repair group there was significant muscle atrophy, FI and upregulated UCP1 expression (muscle weight loss: -20.58% ± 3.04%, vs -1.97 ± 1.23% in sham, p < 0.0001; fat fraction area: 8.64% ± 3.41%, vs 0.77% ± 0.49% in sham, p = 0.0009; and UCP1 fraction area: 2.74% ± 1.35%, vs 0.04% ± 0.03% in sham, p = 0.0021). In wild type mice,10mg/kg Amibegron significantly reduced muscle atrophy, FI and improved shoulder function in the delayed repair group (muscle weight loss: -10.31% ± 7.84%, vs -19.62 ± 3.33% in vehicle, p = 0.0403; fat fraction area: 5.37% ± 2.30%, vs 9.34% ± 1.51% in vehicle, p = 0.0120). Amibegron significantly improved shoulder function, as evidenced by increased stride length and paw area in wildtype mice after RC repair. However, Amibegron did not improve muscle atrophy, FI, or gait in the delayed repair group of UCP1 KO mice.

This study demonstrated that beta3 adrenergic receptor agonist can significantly improve muscle quality and shoulder function after delayed RC repair. In the future Amibegron might be used in RC tear patients before/after tendon repair to improve clinical outcome.

Reference

Wang Z, Liu X, Liu M, Lee C, Kim H, Feeley BT. Beta-3 Adrenergic Receptor Agonist Improves Rotator Cuff Muscle Rehabilitation and Shoulder Function after Delayed Tendon Repair in Mice. ORS 2019 Annual Meeting Paper No. 0039

Fragility Fractures: You Have Some Skin in this Game

Bone mineral density is one factor used to predict the risk for fragility fractures. Another method to assess fracture risk is the FRAX score, which has been used by many “bone health” centers. The FRAX score includes The Fracture Risk Assessment Tool that combines the history of a fracture with age, sex, race, height, weight, and social habits such as smoking and drinking, to determine the risk of major facture in the next 10 years. FRAX scores have been compared to the use of BMD alone¹. Hopefully this will capture the elements of bone as a composite².

Recognizing that type I collagen in skin might reflect bone quality, investigators studied ultrasound echogenicity of the skin as an index of collagen quality that might correlate with collagen maturity in bone. At the time of instrumented, posterior lumbar fusion, bone biopsies of the iliac crest and pedicles were obtained. Preoperative and day of surgery dermal layer thickness and echogenicity of the lower third of the dermis (reticular dermis) were calculated and averaged. Echogenicity is an index of collagen integrity and may be reflective of accumulated cross-links. Aged or impaired collagen in the bone tissue results in increased cross-linking. To eliminate factors of race and anti-osteoporotic medications, skin and bone parameters were compared in Caucasian patients without anti-osteoporotic treatment only. Bone collagen cross-links were measured by Fourier-transform infrared spectroscopy (FTIR).

The mean age was 60.6 ±11.7. Among 19 men, no correlation between US measurements and bone collagen cross-links could be observed. However, among 24 women, dermal layer thickness had a significantly negative correlation with iliac crest trabecular bone collagen cross-links (r=-0.51, p=0.01), iliac crest cortical bone cross-links (r=-0.50, p=0.02) and pedicle cross-links (r=-0.59, p=0.01). Also, echogenicity of the lower dermal layer showed significant positive correlation with the presence of collagen cross-links in trabecular bone of the vertebra (r=0.59, p=0.01). Adjusting for age, the US measurements compared to collagen cross-links were weakest in the cortical bone of the iliac crest and more significant at the other anatomic locations. In both genders, US measurements showed no correlation with BMD.

Of note, similar factors may determine both dermal layer and bone quality such as specific changes in type I collagen, changes in compositional structure, or both. In the future, the use of skin ultrasound may become important in our understanding of fragility fractures risk and bone quality.

Reference

McCloskey EV, Odén A, Harvey NC, Leslie WD, Hans D, Johansson H, Barkmann R, Boutroy S, Brown J, Chapurlat R, Elders PJ, Fujita Y, Glüer CC, Goltzman D, Iki M, Karlsson M, Kindmark A, Kotowicz M, Kurumatani N, Kwok T, Lamy O, Leung J, Lippuner K, Ljunggren Ö, Lorentzon M, Mellström D, Merlijn T, Oei L, Ohlsson C, Pasco JA, Rivadeneira F, Rosengren B, Sornay-Rendu E, Szulc P, Tamaki J, Kanis JA. A meta-analysis of trabecular bone score in fracture risk prediction and its relationship to FRAX. J Bone Miner Res. 2015 Oct 24. doi: 10.1002/jbmr.2734. [Epub ahead of print]
Malluche HH, Porter DS, Mawad H, Monier-Faugere M-C, Pienkowski D. Low-Energy Fractures without Low T-Scores Characteristic of Osteoporosis: A Possible Bone Matrix Disorder. J Bone Joint Surg Am, 2013 Oct 02;95(19):e139 1-6. doi: 10.2106/JBJS.L.01281
Salzmann SN, Okano I, Rentenberger C, Winter F, Miller CO, Schadler P, Sax OC, Miller TT, Shue J, Boskey AL, Sama1 AA, Cammisa FP, Girardi FP, Hughes AP. Ultrasound Measurement of the Skin as a Potential Marker of Bone Quality: A Prospective Pilot Study of Patients undergoing Lumbar Spinal Fusion. J Orthop Res 2019:1–8. doi:10.1002/jor.24438.

Exercise: Keeping it Fluid

The benefits of exercise for joint health as well as for disease modification is difficult to assess. Long term studies require intensive imaging or analyses to destroy the joint. However, synovial fluid analyses might prove to be useful to monitor the effects of exercise on the whole joint including cartilage, bone, fat, menisci, and the synovium itself. To test this, investigators analyzed synovial fluid (SF) from exercised and non-exercised control mice¹.

14 mice were housed in similar cages beginning from 17:00 throughout the night. 8 had voluntary running wheels wherein the 8 averaged 10.8 ± 3.9 km of running overnight ending at 07:30. All 14 were euthanized between 08:00-09:00. Metabolites extracted from synovial fluid were analyzed by high performance liquid chromatography and mass spectrometry. Statistical analyses were performed to identify mechanosensitive metabolites and pathways using MetaboAnalyst and MATLAB. These included Kolmogorov-Smirnov (KS) tests, volcano plot analysis, hierarchical cluster analysis (HCA), partial least squares-discriminant analysis (PLS-DA), and correlation coefficient analysis. Detected metabolite features were matched to known metabolite identities using METLIN, and enriched pathways were determined using mummichog.

It was found that there are distinct mechanosensitive metabolites including coenzyme A derivatives, prostaglandin derivatives, phospholipid species, tryptophan, methionine, vitamin D3, fatty acids, and thiocholesterol. Enrichment analysis identified exercise linked pathways including amino acid metabolism, inflammatory pathways, citrulline-nitric oxide cycle, catecholamine biosynthesis, ubiquinol biosynthesis, and phospholipid metabolism.

Following acute exercise, enhanced amino acid metabolism may supply chondrocytes with necessary nutrients to maintain a healthy matrix. Specific exercise may help maintain appropriate composition and concentration of SF phospholipids for joint lubrication. This might vary in health and disease. Acute exercise may enhance the production of antioxidants such as ubiquinol preventing oxidative stress-induced damage in OA.

These tools could help guide individuals in their specific exercise programs that fall within their “envelope of function”² whether they are disease free or are early in the degenerative process.

Reference

Carlson AK, Rawle RA, Bothner B, Lopes EBP, Griffin TM, June RK. In Vivo Mechanotransduction: Acute Exercise-Induced Metabolic Changes in Mouse Synovial Fluid. ORS 2019 Annual Meeting Paper No. 0114
Dye SF. The Knee as a Biologic Transmission with an Envelope of Function: A Theory. Clin Orrthop Rel Res, 1996, 323, 10-18

Chipping Away at OA

Research on interventions in human osteoarthritis rarely reaches clinical application due to its complex etiologies (trauma, aging, deformity, contribution of inflammation); multiple tissues affected in the organ called a joint (cartilage, synovium, bone, ligaments, fat pad, and menisci); and the difficulty of translation of animal studies into a human platform. The other problem with animal studies is that effective intervention should occur earlier in the disease. To deal with these problems investigators from multiple institutions engineered a stem cell-based 3D human micro-joint chip (mJoint), which is physiologically analogous to the native joint, and capable of modeling pathogenesis of joint diseases to evaluate disease modifying medication development.

3D printing created bioreactors with multiple flows. P5 mesenchymal stem cells were encapsulated in photo-crosslinked 15% methacrylated gelatin and then differentiated to synovium, cartilage, bone, and fat pad tissues. Cartilage and bone tissues were engineered to be in contact in a biphasic scaffold. After 28 days of differentiation, these tissues were assembled in the interconnected chambers of the “mJoint” bioreactor (Fig. 1). In top flows were tissue-specific media for maintaining the tissue phenotypes, and the bottom flows passed simulated “synovial fluid” (SSF) and were shared by fat pad, synovium, and the cartilage that adjoined bone (macrophages not used in the initial project). This multi-tissue construct is called the “mJoint”. After culturing the tissues in the “mJoint” chip for 4 weeks, the proinflammatory cytokine IL-1β was added to the synovium-specific medium at 10 ng/mL to simulate OA, while other tissues were not directly challenged. Real-time polymerase chain reaction, histology, and enzyme-linked immunosorbent assay were used to assess the function of all four tissues.

Under standard media conditions specific tissue phenotypes were maintained for 4 weeks. Calcium deposition in bone, glycosaminoglycan formation in cartilage, new and old collagen in synovium, and fat droplet formation in fat pad tissue were all documented. After IL-1β stimulation, MMP1, MMP13, TNF-α and IL-6 were all upregulated in the synovial tissue. Because of tissue crosstalk, the generation of degenerative arthritis in “mJoint” chip was reflected in decreased expression of aggrecan and collagen type II and increased levels of MMP13 and aggrecanase-1 by the cartilage tissue.

This was the first prototype of the “mJoint”, which included osteochondral unit, fat pad and synovium. Adding a macrophage compartment into the system will serve as a generator of proinflammatory cytokines. Eventually, the “mJoint” can be used to evaluate the effectiveness of therapeutic interventions on human tissues.

Acknowledgements

Supported by the National Institutes of Health (UG3TR002136).

Reference

Li Z, Lin Z, Lopez MR, O’Donnell B, Li X, Ian J. Moran IJ, Alexander PG, Goodman SB, Bunnell BA, Lin H, Tuan RS. Organ-on-a-chip System for the Modeling of Synovial Joint Pathologies. ORS 2019 Annual Meeting Paper No. 0148

Fig. 1 The “mJoint” chip. (A) The 3D model designed using SolidWorks. (B) A “mJoint” bioreactor fabricated by 3D printing. (C) A schematic showing the co-culture of engineered tissues – fat pad, synovium, and osteochondral complex from left to right – in the “mJoint” bioreactor with different types of media.

Microbiome: Picking Your Bacterial Friends Wisely

Together, bacteria and archaea make up the human microbiome. Albeit one thinks of the gut in terms of biome the biome involves the entire human body including the oral cavity and open wounds. Shifts in the makeup of these biomes can have an impact on a variety of conditions including tissue composition and open wounds. Two recent ORS abstracts highlight this.

Humans live in symbiosis with millions of bacteria on their skin, a human microbiome, since this enveloping organ is exceptionally resistant to microbial invasion. However, in the setting of diabetes mellitus, a disease process accompanied by neuropathy, vasculopathy (large and small vessel) and leukocyte dysfunction, any breach of skin integrity, especially in and around the foot, is an immunocompromised district in which bacterial contamination can rapidly progress to a deep infection with limb-jeopardizing and life-threatening consequences.

Some patients with infected diabetic foot ulcers (DFU) respond quite favorably to local wound care and antibiotics, avoiding amputation. Yet, other patients go through months of serial surgical debridements and prolonged parenteral antibiotic administration, only to have a lower extremity amputation which may have been inevitable regardless of clinical management. Might the microbacterial communities present in these wounds be predictive of ultimate outcome so as to mitigate the time, physical/emotional complications and expense of failed treatment?

In a prospective study, patients had their DFU assessed using bacterial cultures and 16S rRNA sequencing for microbiome analysis prior to the initiation of antibiotics and at 4 and 8 weeks after the initiation of treatment. All patients received six weeks of parenteral antibiotics and daily wet to dry dressing changes. Those DFUs that healed at 12 weeks had a significantly greater pre-antibiotic abundance of Actinomycetales (p = 0.04), Bacillales (p < 0.01) and Staphylococcaecae (p = 0.02) with significantly lower levels of Streptococcaecae (p = 0.02) compared to non-healing DFUs. Increasing levels of Staphylococcaecae at 8 weeks was a bad prognostic sign for DFU healing.

More research is needed to control for variables such as the severity and control of hyperglycemia, associated multi-organ morbidity, obesity, adequacy of arterial inflow and venous outflow, nutritional status, size, and the depth and location of the DFU. However, this is a first step, an isolated study of the microbiome as it relates to DFU healing, which will hopefully stimulate additional investigation.”

The administration of antibiotics to affect gut flora in animal models could be analogous to human differences caused by multiple rounds of oral antibiotics throughout life. To study modifications to the gut microbiota generated by chronic oral antibiotics, 7 groups of 4 week old mice were given antibiotics as individual treatments or as an antibiotic cocktail with sweetener, until skeletal maturity at 16 weeks of age². To test the effects of sweetener alone, a group that was received only sweetener was added. Femora were then harvested for analysis. Changes in gut microbiota resulted in decreased cross sectional area, moment of inertia, section modulus and whole bone strength, compared to untreated animals. While all of the oral antibiotics were associated with impaired bone geometry, only neomycin was associated with impaired bone tissue quality. Animals receiving sweetener alone had bone geometry similar to that of untreated animals but tissue strength was increased. Because sweetener alone was found to increase bone strength the addition of sweetener will influence the results of antibiotics. To clarify this, next generation bacterial genome sequencing will be required to isolate the organisms and/or mechanistic pathways linking oral neomycin to impaired bone quality.

References

Brodell JD Jr, MacDonald A, Oh I, Smith BL, Schwarz EM, Daiss J. Microbiome Analysis for Assessment of Treatment Response and Salvage Prognosis in Infected Diabetic Foot Ulcers. ORS 2019 Annual Meeting Poster No. 1196
Vasquez-Bolanos LS, Luna M, Hernandez CJ. Selective Removal of Components of the Gut Microbiome has Differential Effects on Bone Strength. ORS 2019 Annual Meeting Poster No. 0705.

Footnote: In the 2019 ORS symposium “The Microbiome and Musculoskeletal Disease” the following topics highlighted. Christopher Hernandez “What is the Microbiome and How is it Relevant to Musculoskeletal Disease?” Julia Charles “Overview of an Emerging Field” and Michael Zuscik “Role of the Dysbiotic Gut Microbiome in Arthritis: Implications for Development of Novel DMOADS”

Meniscal vs Synovial Cell Origin in Meniscal Repair: And the winner is...

Animal models for meniscal regeneration and repair had used an ultrapurified low endotoxin alginate (UPAL) gel wherein the origin of the cells that seed the gel is not clear. The embryological source of the meniscus is concurrent with the development of the synovium which becomes a distinct layer from the capsule and bone. To clarify the origin of the reparative cells in meniscal defects a freeze-thaw method was used to destroy surrounding meniscal and/or synovial cells that could populate the defect.

32 mature Japanese rabbits had a 2 mm hole punch into the anterior horn of the medial meniscus. The UPAL get was applied CaCl2 gel applied after application. 8 rabbits were in each of four groups.

No surrounding meniscal or synovial freeze thaw
Only surrounding meniscal freeze thaw
Only surrounding synovial freeze thaw
Both surrounding meniscal and synovial freeze thaw

The successful freeze-thaw killing of surrounding meniscal and/or synovial cells in the targeted tissues was confirmed by tissue using Hoechst and propidium iodide stain. Hematoxylin and eosin, Safranin-O, and Toluidine-blue staining quantified the regenerated tissue within the defects.

There was no significant changes in all groups at 1 week. However, at six weeks the defects in groups A and B were completely filled with thick fibrocartilage-like tissues and reparative tissues contained large round or ovoid cells rich in cytoplasm. The matrix around these cells was positively stained by safranin-O and toluidine blue. The synovial cell deprived groups C and D had histologic findings similar to week one.

This study more strongly implicates synovial cells as the chief contributor to meniscal injury repair. This has significant impact on future meniscal tissue engineering studies.

Reference

Kim WY, Onodera T, Kondo F, Terkawi MA, Homan K, Hishimura R, Iwasaki N. Which contributes to meniscal regeneration, synovium or meniscus? In vivo rabbit model study with Ultrapurified Low Endotoxin Alginate (UPAL) gel and freeze-thaw method. ORS 2019 Annual Meeting Paper No. 0154

Beyond the Genome Genie

There is a multiplicity of factors, in some instances acting on the same set of genes, that can contribute to degradative pathways in both injury and ageing articular cartilage. It is for this reason that many “targets” for therapeutic interventions that interfere with specific degradative pathways fail to come to clinical fruition. However, with epigenetics that tact may be taking on an effort to maintain homeostasis of the existing cartilage.

The term epigenetics comes from the words “beyond genetics”. Early on, the term referred to CpG methylation modification of DNA or posttranslational modifications (PTMs) of histone proteins that package DNA into chromatin. Added to this genetic dance are modulations of mRNA translation and degradation by microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and other noncoding RNA species. All of these can play a role throughout growth, development, and homeostasis. The fundamental molecular mechanisms by which cell type-specific patterns in gene expression are established and retained during successive mitotic divisions require epigenetic control.

Repair by CpG Methylation

There are three distinct DNA methyltransferases (DMNT) that act differently. DNMT3B is a case in point that is required for chondrocyte maturation and endochondral bone. Many developmental programs are reactivated in response to injury such as fracture repair. In homeostasis DNMT3B, but not DNMT3A, is expressed in healthy articular cartilage but decreases with age and following injury along with altered DNA methylation in osteoarthritis (OA) patients. There is a demethylation process by ten-eleven translocation (TET) proteins further complicating this picture. The relative importance of TET1, TET2, and TET3 in contributing to these 5-hmC changes in OA remains to be determined.

Repair by Histone Writers, Erasers, and Readers

Post-translational modifications of the histone proteins in the nucleus can have a huge impact on genetic control. Histones can be acetylated by a number of histone/lysine acetyltransferases. In human cells, histone deacetylation is catalyzed by 18 histone lysine deacetylases (HDAC/KDAC). Overall, the protein acetyl writers (lysine acetyl transferases), erasers (lysine deacetylases) and readers (bromodomain containing acetyl-lysine binding proteins) play roles in OA. Another pathway, lysine and arginine methyltransferases, can also lead to promotion or inhibition of specific genes. There are myriad varieties of these methyltransferases and deficiencies in their expression are associated with a number of abnormal musculoskeletal phenotypes.

Epigenetics and orthopaedic oncology

Mutant histones are present in most giant cell tumors and chondrosarcomas. There are differences in DNA methylation seen in Ewing sarcomas and chondrosarcomas. Epigenetic alterations may be as prevalent in musculoskeletal tumors as in soft tissue cancer types.

The Future

One limitation of possible epigenetic pharmacotherapies is that epigenetic regulators may act broadly in multiple cell types. Studies with epigenetic inhibitors in cancer patients have revealed a remarkable short term tolerance for these drugs in patients, and there is a concern regarding the unpredictable long term side effects of systemic use. It is possible that local administration of these drugs may be useful for treatment of orthopedic conditions and that research will identify new epigenomic targets that can reset cell function and slow tissue degeneration. For now, rubbing the magic lamp to recover cartilage normalcy may be “beyond the genie” at present, but the new frontier beyond the genome may define epigenetic drugs that could be used to prime the musculoskeletal healing process.

Reference

van Wijnen AJ, Westendorf JJ. Epigenetics as a New Frontier in Orthopedic Regenerative Medicine and Oncology. J Orthop Res. 2019 Apr 12. doi: 10.1002/jor.24305. [Epub ahead of print]

Muscle Loss: It’s Time to Muscle In

Significant, non-recoverable muscle loss is seen in both the civilian and military sectors. Investigators developed a scaffold-less method to tissue-engineer skeletal muscle units (SMUs) to treat volumetric muscle loss (VML) in a sheep model. They also developed an engineered neural conduit (ENC) to bridge the SMU and damaged nerve. ENC was constructed by using sheep bone marrow stromal cells (BMSCs) induced to a tendon lineage and upon monolayer formation, rolled into a cylinder around a silicone tube forming a hollow tube construct 3 mm in diameter and 6.5 cm in length. The ENC can then be stored at -80C until surgical use. The silicone tubing is removed prior to use. For SMU fabrication, sheep muscle satellite cells were seeded and allowed to form a confluent monolayer of myotubes. Subsequently, minutien pins were used to create a 3-D cylindrical construct with a length of 12-14 cm and a diameter of 6 to 8 mm. Two to three SMUs were sutured together, just prior to implantation to repair a 30% VML. The construct is attached to the native muscle with sutures at the tendon/bone ends and a second suture is used along the length of the SMU to hold it in place so it does not dislodge and move from the defect site. A subset of constructs were used to measure contractile properties following 3D formation.

SMUs had an average isometric tetanic force of 694 ± 828 µN prior to use for VML repair. Light microscopy showed that the 3D SMU construct consisted of highly aligned and dense myotube network and highly aligned collagen. The SMU technology was successful in repairing a 30% VML in sixty sheep.

Reference

Novakova SS, Nutter GP, Larkin LM. In Vitro Assessment of Scaffoldless Tissue-Engineered Skeletal Muscle for Volumetric Muscle Loss Repair in Sheep. ORS 2019 Annual Meeting Poster No. 1165

Getting to the Root of the Pain

Lumbar nerve root pain is a major source of controversy for best treatments. Nerve compression with pain and motor loss is typically seen as a surgical problem. However, there is a large percentage of patients with positive evidence of nerve irritation based on exam but falling short of obvious motor or sensory loss despite MRI and/or EMG findings. Epidural and focal nerve injections are a popular resource wherein the outcome is not entirely predictable. Systemic medications have both short and long term undesirable effects. Actual nerve root swelling can be seen on some MRIs. In response to some disc herniations there are abundant inflammatory cell responses with infiltration of a variety of leukocytes, such as macrophages.

Fullerene C₆₀ and a chemical derivative, fullerol C₆₀-OH, have been found to have a positive effect on lumbar radiculopathy. cFLFLF is a ligand for formyl peptide receptor (FPR-1) targeting inflammatory cells near radiculopathy. With this in hand investigators developed a nanomedicine Fluorescence labeled Targeted C₆₀ derivative, “FT-C₆₀”. The modular design of FT-C₆₀ includes functionalized C₆₀ as the therapeutic moiety, polyethylene glycol (PEG) and lysine (K) as linker and spacer, and cFLFLF peptide as targeting moiety, and a near infrared fluorescence dye to ease detection for in vitro and in vivo validations.

In vitro evaluation on anti-inflammatory, targeting properties, and cytotoxicity of FT-C₆₀ were studied in macrophages (Raw 264.7) with and without lipopolysaccharide (LPS) stimulation. FT-C₆₀ demonstrated preferential binding towards LPS stimulated macrophages compared to non-LPS control. FT-C₆₀ significantly attenuated LPS-induced pro-inflammatory based on inhibition of mRNA expression of IL-6, TNF-α, IL-1, and COX-II.

In vivo evaluation involved the use of a lumbar radiculopathy mouse model (L4-5 disc punctured by needle from left anterior) to assess pain alleviation effect and targeting property of FT-C₆₀. There were three punctured groups: no treatment, systemic FT-C₆₀, and local C₆₀. Ipsilateral and contralateral von Frey tests were performed to measure hyperalgesia. Single intravenous injection of FT-C₆₀ diminished mechanical hyperalgesia in the mouse model of radiculopathy for up to POD 12.

Ex vivo Organs and spines were harvested at POD 7 and POD 14 for ex vivo IVIS fluorescence imaging and histology analysis. Near infrared fluorescence imaging of mouse spine clearly depicted targeting property of FT-C₆₀ towards inflammatory infiltration of injured disc at POD 14. At POD 7, there was both degenerative disc and massive inflammatory cell infiltration present in the anterior ipsilateral (left) side near exposed, untreated nerve root whereas systemic FT-C₆₀ dramatically reduced calculated tissue volume of inflammatory infiltration.

This promising data warrants further exploration and characterization of FT-C₆₀class for future translational studies. If effective, this would have tremendous impact when radicular involvement in spine related pain is not clear.

Reference

Xiao L, Li T, Zhang Y, Dai J, Shen FH, Jin L, Dorn HC, Li X. Developing a Targeted Nanomedicine to Alleviate Lumbar Radiculopathy via Systemic Delivery. ORS 2019 Annual Meeting Paper No. 0168

Enhancing Fracture Healing: A Bony Fountain of Youth

Repairing larger bone defects continues to be a challenge. Although bone morphogenetic protein 2 (BMP-2) has been used for several decades to enhance bone healing the application requires super-physiologic amounts. One of the consequences is an initial osteoclastic drive and other soft tissue effects that can lead to serious complications. Cervical spine surgery is an example where BMP-2 has been used to encourage fusion but where there are instances of initial bone absorption and collapse as well of soft tissue swelling associate with respiratory interference. As a potential alternative, c-Jun N-terminal kinase 3 (JNK3) was recently identified as mechanically-activated intra-cellular modulator of the superior osteogenic potential of mesenchymal stromal/stem cells (MSCs) derived from children (C-MSC) in comparison to those stem cells from adults (A-MSC). Investigators have now designed a therapeutic molecule to facilitate JNK3 activation in adult MSCs in an attempt to reproduce the function seen in youth.

A JNK3 activator (JNK3*) molecule was designed by complexing a 25 amino acid peptide with in house fabricated nanoparticles of hydroxyapatite (nHA). Following selection of the optimal JNK3 activator dose, the nanomedicine was incorporated into a 3D highly porous collagen-HA scaffolds based delivery platform (JNK3* scaffold). The effects on adult MSCs were then assessed by evaluating its ability to take-up JNK3* nanoparticles and any resultant osteogenic commitment by means of PCR, alkaline phosphatase (ALP) activity and calcium deposition. A rat calvarial defect was then used to test JNK3* scaffold, JNK3* free scaffold, and defect alone.

There was no toxic effect on C-MSCs and A-MSCs cultured with 0-5 µM JNK3*. In a test of cell uptake of JNK3* there was a concentration-dependent increase on the capacity of stem cells to take up the nanoparticles. Cells treated with JNK3* with no nHA carrier were unable to take-up the molecule. Also, the JNK3* nanomedicine increased the osteogenic capacity of A-MSCs as demonstrated by significantly higher levels of ALP activity compared with un-treated C-MSCs while the addition of a JNK3 inhibitor (SR-3576) blocked the pro-osteogenic action of the JNK3*. There was significant upregulation of ALP, PGF and JNK3 for JNK3* scaffold compared to cells cultured on the JNK3*free scaffold. A JNK3-dependent increase in ALP activity and mineralization capacity of A-MSCs cultured on the JNK3* scaffold was also demonstrated. Adult rats treated with the cell-free JNK3*scaffold for two weeks showed the infiltration of a larger amount of blood vessels, host stem cells and mineralized tissue in samples treated with JNK3* scaffolds when compared with JNK3*free scaffolds and empty defects. More importantly, the host stem cells that colonized JNK3* scaffold in vivo showed significant upregulation of genes associated with pro angiogenic and pro osteogenic pathways such as vascular endothelial growth factor (VEGF), metalloproteinase 9 (MMP9), runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP). Bone healing occurred as early as 28 days post-implantation with a significantly higher bone volume fraction (BV/TV) observed in rats treated with JNK3* scaffold (25.06 %) compared to rats treated with JNK3*free scaffolds (6.04%) and empty defects (2.58%).

This cell-free, off-the-shelf, mechanobiology-informed therapeutic delivery platform recapitulated children’s bone healing capacity in adults by facilitating JNK3 activation.

Reference

Gonzalez-Vazquez A, Raftery R, Chen G, Murray DJ, O’Brien FJ. Mechanobiology-informed Tissue Engineering: Developing scaffold-based therapies for adult bone repair by harnessing age-altered JNK3 activation in stem cells. ORS 2019 Annual Meeting Paper No. 0141.

Measuring Vertebral Stiffness and Displacements Using Tomosynthesis: A Weighty Solution for Thin Bones

To date, the study of stress/strain relationships in bone required ex vivo measures. These often require removal of the soft tissues around bones and joint. This could lead to calculations that would not occur in a living subject. Current assessment of osteoporotic fracture risk based on BMD is useful¹, but ultimately relies on surrogates for biomechanical properties. One of the consequences of osteoporosis is vertebral fractures that occur with minimal force such as open a stuck window or picking up a heavy suitcase. Computerized tomography can be used to study vertebral trabecular architecture in of evaluating bone deformity living subject but is not capable under loading conditions in a human being. Digital tomosynthesis based digital volume correlation technique (DTS-DVC) is a technique that allows for live bone deformation measurements. In DTS the x-ray source and detector are moved together through an arc of 60^O during exposure rapidly producing sharply focused planes. These 2-dimensional slice images have a high in-plane resolution of 150-300 µm with about one-fifth of the exposure of a computed tomography examination.

Investigators used DTS-DVC to measure T12 and L1 in five patients with T-score: -1.8 to -4.3 and no vertebral fracture, twice in supine (“non-loaded”), once in standing position (“loaded”), and once in standing position while holding a 4kg weight (milk jug) in each hand². DVC analysis was performed for the T12 and L1 levels. An additional 61 year old patient with an osteoporotic T11 fracture was also imaged, once in non-loaded and once in the standing position. For this patient, the fractured T11 and the adjacent intact T10 were analyzed. Calculations took into account the person’s weight and weight above the level studied.

10 repeat scans of top endplate displacements were -0.0068 mm and 0.0322 mm respectively corresponding to a total stiffness error of 604 N/mm. The mean in vivo top endplate displacement achieved by standing was 17-32 times the mean error for the T12 and L1 levels (Figure 1) resulting in stiffness values ranging 1284 to 7766 N/mm. Holding milk jugs resulted in a qualitative and statistically demonstrable increase in displacements, but no demonstrable change in stiffness (Figure 2). The displacement of the fractured T11 vertebra under loads from standing was 3.1 times that of the adjacent intact T10, and the T11 had a stiffness that was 38.6% of the intact T10 (2468 vs 6388 N/mm) (Figure 3).

One step further. Bone is a composite and BMD may not reflect the true mechanical quality of bone. DTS-DVC would be insensitive and only seek the mechanical quality of bone. The potential in both clinical medicine and research is evident.

References

Siris ES, Genant HK, Laster AJ, Chen P, Misurski DA, Krege JH. Enhanced prediction of fracture risk combining vertebral fracture status and BMD. Osteoporos Int. 2007 Jun;18(6):761-70. Epub 2007 Jan 24. PMID: 17245546
Oravec D, Soni P, Zauel R, Rao S, Flynn MJ, Yeni YN. Clinical measurement of vertebral stiffness and displacements using tomosynthesis based digital volume correlation. ORS 2019 Annual Meeting Poster No. 2252
Malluche HH, Porter DS, Mawad H, Monier-Faugere M-C, Pienkowski D. Low-Energy Fractures without Low T-Scores Characteristic of Osteoporosis: A Possible Bone Matrix Disorder. J Bone Joint Surg Am, 2013 Oct 02;95(19):e139 1-6. doi: 10.2106/
JBJS.L.01281

Repairing Rotator Cuffs: Calling in the Troops

To some extent bone healing after a fracture replicates embryology. The enchondral callus of a fracture that is not rigidly fixed goes through many of the steps of bone growth and development. Unfortunately, tears of the supraspinatus do not respond according and surgical repair does not replicate the initial biology of a growing tendon attachment¹. Similarly, repair of rotator cuff tendons does not typically reproduce the native fibrocartilage insertion site.

It is known that there are intrinsic progenitor cells resident in tendon, and these progenitor cells are regulated by a specialized population of endothelial cells that produce various signaling molecules. Tendon derived cells can improve supraspinatus tendon (SST) repair heling. Because there are not a large number of cells from tendon tissues investigators obtain a greater population from muscle to use those endothelial cells in an effort to enhance tendon repair healing. They euthanized six C57BL/6 mice for muscle muscle-derived activated endothelial cells (mECs) harvest and transfected these with adenoviral E4ORF1 and labeled with green fluorescent protein (GFP). Then one hundred fourteen C57BL/6 mice underwent unilateral SST detachment and repair with implantation of 100,000 mECs in a fibrin glue (FG) carrier vehicle (study group) or FG alone (control group) at the tendon-to-bone interface. Three mice each were euthanized at 3 and 7 days after the surgery to check cell viability and localization using fluorescent microscopy. The remaining animals were sacrificed at 7, 14, and 28 days.

Biomechanical testing: The failure force in the study group was significantly higher than that of the control groups at day #14 (2.87 vs 1.86 N, p=0.0098) but there was no statistical difference in failure force at day #28 (3.73 vs 3.65 N).

Cell tracking: There were GFP positive viable mECs at the repair site of SST at day #3 in the study group (please see figure). At day #7, the GFP positive cells were obviously decreased.

Histomorphologcial evaluations: There was improved continuity of the repaired SST and the bone tissue in the mECs group at day #14. At day #28, the picrosirius red staining revealed better organized collagen fibers in the mECs group.

Quantitative analysis with µCT: There were no significant differences between the study and control groups at day #28 in both trabecular and cortical bone analysis.

Comprehensive RNA analysis: Preliminary analysis suggests that integrin binding sialoprotein and endothelin 1 play important roles at day #7.

This study demonstrated that activated endothelial cells derived from muscle tissue can improve healing of the tendon to bone tissue. Activated muscle-derived endothelial cells may be a promising cell source for the future treatment of soft tissue injuries.

References

Nelson, F. On the Horizon from the ORS: Recreating Normal Tendon and Ligament Attachments After Injury J Am Acad Orthop Surg.2008 May 16(5) 237-239
Wada S, Lebaschi A, Nakagawa Y, Carballo C, Wang D, Nemirov D, Album Z, Croen B, Chen T, Ying L, Deng X-H, Mendias C, Rodeo S. Muscle-Derived Activated Endothelial Cells Improve Tendon-To-Bone Healing in a Murine Model of Rotator Cuff Repair: Comprehensive Gene Expression Analysis using RNA Sequencing. ORS 2019 Annual Meeting Paper No. 0010

Detecting Early Aging in Articular Cartilage: Depends on How You Slice It

With the exception of prolonged weight loss there are no disease modifying interventions for osteoarthritis. For this reason, an increasing number of potentially disease modifying pharmaceutical and surgical early interventions for OA have been proposed. Radiographic imaging is not useful since more advance changes are required to reveal detectable radiographic change. MRI has improved the capacity to follow specific articular cartilage component change. T1ρ (rho) imaging measures the relative amount of proteoglycan based on water content. The time of T2 signal decay can measure collagen integrity by looking at a series of signal decays over time. Delayed g enhanced magnetic resonance imaging of cartilage (dGEMRIC) in the most validated indirect measure of cartilage glycosaminoglycan content and thus detects the earliest signs of OA.

Femoral acetabular impingement (FAI) is a common precursor to human hip OA. When FAI is detected on radiographs individuals can be followed for the early development of OA. However, effective use of MRI data requires isolation of articular cartilage from neighboring structures, thus segmented. Segmentation in hips is very difficult due to thinner cartilage compared to the knee and due to the more extreme curvature of the hip. Segmentation done by hand can take several hours making large studies impractical. The same factors also made semi-automated segmentation problematic.

Investigators designed a semi-automated MRI segmentation method for the hip to be fast, accurate, reproducible, and clinically viable. Radiographs were oriented to specific pelvic land marks to detect FAI. Cam morphology was defined as an alpha angle (reflects flattened superior lateral curviature of femoral head) greater than 60˚ on anteroposterior radiographs and pincer morphology deﬁned as a center-edge angle (vertical line from center of femoral head and line from center to acetabular margin) greater than 39^O on AP and lateral radiographs.

An academic musculoskeletal clinician manually segmented sagittal dGEMRIC images developing regions of interest (ROI) clockwise around the center of the femoral head at 30˚ intervals excluding the chondrolabral junction. The process on average is 60 to 90 minutes. The first step of the semi-automated segmentation method is a fully automated hierarchical partitioning of the data volume generated by a software bespoke segmentation approach applied recursively. The second step is a user selectin of regions of interest using a bespoke segmentation. Combined, the steps take 5 to 10 minutes. The selection of the anatomical structure of interest takes a newly trained musculoskeletal radiology operator on average 5 min per hip.

Accuracy and Dice similarity coefﬁcient (DSC) for the comparison of semi-automated and manual segmentations was 0.9886 and 0.8803, respectively. Intra-observer and inter-observer reproducibility of the semi-automated segmentation method gave an accuracy of 0.9997 and 0.9991, and DSC of 0.9726 and 0.9354, respectively.

Reference

Fernquest S, Park D, Marcan M, Palmer A, Voiculescu I, Glyn-Jones S. Segmentation of hip cartilage in compositional magnetic resonance imaging: A fast, accurate, reproducible, and clinically viable semi-automated methodology. J Orthop Res. 2018 Feb 22. doi: 10.1002/jor.23881. [Epub ahead of print] PMID: 29469172

Initial Implant Fixation: If Push Comes to Shove

There is a large array of orthopaedic implants that require secure initial fixation for the proper initial and long-term function of the implant. These include spinal ﬁxators, bone fracture ﬁxation plates, tendon repair anchors, ligament reconstruction ﬁxation screws, external fixation devices, chondral repair implants, dental implants, and total joint replacement implants. With the exception of total joint replacement implants and suture anchors most entail the use of screws. The long-term risk is screw loosening. Even though there are surface modifications for joint prostheses their initial pull out strength is 1/10^th that of a screw.

Additive manufacturing (AM) technology, more colloquially known as 3D printing, enables intricate parts to be manufactured from orthopaedic-grade metal alloys. The objective of this reported research was to use the design freedoms of additive manufacturing to develop an implant fixation surface that anchors in bone attaining closer to screw-level pull-out strength from a push-ﬁt device.

3 pegs, similar is size for knee implant fixation, we used for pull out testing after insertion in sets of 4. One set was solid Ti pegs with a rough surface similar to contemporary cementless orthopaedic implants (please see figure). Another set was porous (mesh) structure pegs similar to porous implants. The third was the investigational barbed ﬁxation pegs with dedicated ﬁxation features – a geometry that could only be manufactured using AM technology. The AM fixation device is a porous lattice structure with barbs that allow for push in but resisting extraction. An ACL interference screw of equivalent size was also tested as a control. A synthetic bone model with little inter specimen variability was used for the pull-out test. Four frozen distal femoral condyles from subjects aged 38-57 were also used.

Overall the barbed ﬁxation technology inverted traditional push-in/pull-out ﬁxation mechanics, such that its pull out strength was equivalent to a press-ﬁt pegs’ push in, and its push in equivalent to a press-ﬁt pegs’ pull out. Although the interference screw vastly outperformed solid and porous peg designs, the optimum barbed ﬁxation surface achieved screw-level ﬁxation. The trends for the cadaveric tests replicated those seen in the synthetic bone tests. The push in was less for the barbed construct while pull out was greater.

The barbed ﬁxation is anchored under intact trabeculae. Removal of the implant requires localized cancellous bone fracture which is similar to screw ﬁxation. However, screws may be removed with torque rather than pull out. The new barbed fixation technology could be applied to porous implants to improve their initial stability, encouraging implant bone ingrowth and secure long-term implant fixation. It offers scope to design smaller implant fixation features whilst ensuring secure initial fixation, thus enabling minimally invasive implants that preserve bone stock. It also creates the possibility for cementless prostheses that do not require impaction, avoiding the risk of iatrogenic intraoperative fractures.

Reference

van Arkel RJ, Ghouse S, Milner PE, Jeffers JRT. Additive manufactured push-fit implant fixation with screw-strength pull out. J Orthop Res. 2018 May;36(5):1508-1518. doi: 10.1002/jor.23771. Epub 2017 Nov 22.

Cartilage Repair: Making it Stick

Limited cartilage defects have a host of operative solutions. Cell based therapies such as autologous chondrocyte transplantation and microfracture appear to have reasonable bone adhesion but the cartilage to cartilage interface has been a major mimesis. In fact, there is significant die back of the normal cartilage at the graft interface articular cartilage wall. Albeit osteochondral allografts and autografts have good articular cartilage maintenance and bone fixation, graft wall association with the host remains problematic.

FGF18 (sprifermin) is predominately expressed in early development with properties that may promote regeneration in adult tissues. FGF18 is currently under investigation as a treatment for knee OA. An intraarticular injection of FGF18 in a rat meniscal tear OA model resulted in a dose-dependent increase in cartilage formation. A recent clinical trial examined changes in tibiofemoral cartilage thickness in OA patients following intraarticular injection of recombinant human FGF18 (sprifermin). Investigators hypothesized that sprifermin would enhance chondrocyte proliferation, matrix biosynthesis, and cartilage–cartilage integration and strength

6 mm cartilage cylinders were harvested from the trochlear grove of juvenile bovine knees. After overnight explant culture a central 3 mm defect was created in the center of each explant, removed, and replaced. These constructs were cultured in control or sprifermin-containing medium (weekly 24-h exposure of 100 ng/ml sprifermin) for 4 weeks.

In biosynthesis studies

sprifermin-treated samples had an integration strength of 93 versus 59kPa for control samples (p<0.0001)
GAG content was higher in both the core and annulus in sprifermin samples compared to the controls.
there was significant increased collagen in sprifermin samples, seen only in the core.
solid volume was increased in both annulus and core for sprifermin treated samples.

From micro CT all samples had wall gaps but sprifermin-treated explants had a signiﬁcantly lower gap volume/defect. Electron microscopy demonstrated that sprifermin-treated samples had a smaller gap between inner and outer cartilage. Safranin O/Fast Green staining revealed no qualitative differences with some samples from both groups having reduced staining at the cartilage–cartilage interface. Similarly TUNEL assay demonstrated a region of apoptotic cells at the marginal tissues in samples from both groups but sprifermin-treated samples had signiﬁcantly more total cells/mm2, and more non-apoptotic cells/mm2.

The role of sprifermin in cartilage transplant and tissue engineered cartilage remains to be clarified.

Reference

Sennett ML, Meloni GR, Farran AJE, Guehring H, Mauck RL, Dodge GR. Sprifermin treatment enhances cartilage integration in an in vitro repair model. J Orthop Res. 2018 Oct;36(10):2648-2656. doi: 10.1002/jor.24048. Epub 2018 Jul 26.

Osteoporosis: Gone with the WNT

There are a number of bone-building therapeutics for patients with severe osteoporosis and high fracture risk. Bisphosphonates have proved to be effective in blocking bone absorption but carry the potential risk of bone fragility. Teriparide is a bone-forming agent of 1-34 amino acid replication of parathyroid hormone agent that requires daily injections. Denosumab is a fully human monoclonal antibody that, similar to naturally occurring osteoprotegerin which inhibits RANKL by blocking the receptor and is now available in the treatment of bone absorption particularly from tumors. It requires an injection every two to four weeks.

The WNT signaling pathway is a key regulator of bone metabolism, particularly bone formation. Osteocytes are the most abundant bone cells and direct bone remodeling by a number of pathways. WNT signaling has a positive impact on osteocyte driven bone formation. Recent investigators have studied components of the WNT signaling pathways as potential therapeutic targets in animal models. However, only inhibitors of sclerostin have been tested in phase three clinical studies. Sclerostin inhibits the canonical WNT signaling pathway interfering with WNT ligands which bind to LRP5 or LRP6 and Frizzled co-receptors (FZD co-receptors) on the cell surface. These receptors transduce a signal that results in stabilization and accumulation of intracellular β-catenin which translocates to the nucleus binding transcription factors that trigger transcription genes in osteoblasts.

There are a number of sclerostin antibody products including blosozumab and romosozumab. However in phase three human studies there has been concern for adverse cardiovascular and cerebrovascular events. Relationship to vascular calcification has not been proved.

There are other WNT related avenues with therapeutic potential.

There is a mouse strain with low bone mass owing to deficiency in osteoblast number that lack the WNT co-receptor LRP5. Lithium chloride restored bone volume to that of wild-type mice by stimulating bone formation without an effect on bone resorption. Lithium inhibits glycogen synthase kinase-3β preventing phosphorylation of β-catenin increasing its stabilization, which induces its nuclear translocation. In a large epidemiological studying bipolar patients taking lithium it has been found that these have denser bone.
Protein kinase C-binding protein NELL1 promotes bone formation by binding to β1 integrin which regulates the activity of runt-related transcription factor 2 (RUNX2). RUNX2 stimulates the canonical WNT signaling. In ovariectomized mice systemic administration of recombinant human NELL1 increases bone formation and bone mass and decreases the number of osteoclasts. There are other studies targeting NELL1
Sirtuins are a family of stress-responsive proteins that are regulators of several processes related to ageing, cancer, metabolism and inflammation. SIRT1 and SIRT2 both bind to β-catenin, but SIRT1 activates the WNT pathway, whereas SIRT2 inhibits it. Treatment of mice with a SIRT1 activator decreased sclerostin expression and increased cortical periosteal mineralizing surfaces and serum procollagen type 1 amino-terminal (P1NP). Administration of another SIRT1 activator to aged male mice increased bone mass and when given to ovariectomized mice immediately after ovariectomy partially reversed bone loss.

Reference

Appelman-Dijkstra NM, Papapoulos SE. Clinical advantages and disadvantages of anabolic bone therapies targeting the WNT pathway. Nat Rev Endocrinol. 2018 Oct;14(10):605-623. doi: 10.1038/s41574-018-0087-0. PMID: 30181608

Back Pain, Joint Causes

A common procedure for back pain is facet joint injection. Based on literature, facet joints share about 20% to 30% load distribution in the spine with the rest shared by the intervertebral disc (IVD). Due to unbalanced loads in a scoliotic spine, the facet load share may be increased. Osteoarthritis is present in 15% to 45% of the facet joints in low back pain patients. Investigators hypothesized that degenerative changes, degradative cytokines, and degradative proteases are elevated in facet joints of younger patient with scoliosis¹. During scoliosis corrective surgery they studied facet joints from 20 patients age 11-19. For comparison 6 non scoliotic facets were obtained from tissue donors between 17-60 years old, mean average 34.33. Articular cartilage explants, histology that included immunohistochemistry, and articular cartilage protein extraction was assessed with western blotting for protein analyses.

Histology

Safranin-O fast green staining show a decreased and variable proteoglycan content in contrast to the donated facets. Based on OARSI grade the OA phenotype was increased. Immunohistochemistry staining with Ki-67 revealed increased cell proliferation in the scoliotic facets. Cell density was also increased. Immunohistochemistry also showed increased MMP-3, MMP-13, and IL-1 in the scoliotic facets.

Protein extracts

Pooled extracts from 10 apex facets contrasted to a non-scoliotic facet revealed fragmentation of decorin and chondroadherin. Biglycan fragments were present in all samples despite degree of curvature but a 22kDa fragment appeared only in curves > 70^o.

The authors suggest that, similar to patients with age related OA, the premature joint degeneration seen in scoliotic patients is likely to contribute to the pain perceived in some individuals.

Another group of investigators evaluated the role of facet joints in torsion mechanics of healthy and degenerated human lumbar discs with different compressive preloads (300-1200 N) with and without facet joints². They found that disc torsion mechanics are likely resistant to compositional changes during early degeneration. But with a degenerative decrease in annulus fibrosis glycosaminoglycan (AF GAG) content there was decreased torsional stiffness from the healthiest disc to the most degenerated disc based on AF GAG content (85% decrease in the most degenerated disc). The decrease in torsional stiffness increases disc compliance resulting in a greater proportion of hysteresis energy being absorbed by the facet joints.

Given the significance of facet joints as a pain generator in back and neck pain these studies direct clinicians to a heightened appreciation of how these joints may be influenced by age and acquired spinal deformity.

Reference

Bisson DG, Lama P, Abduljabbar F, Rosenzweig DH, Saran N, Ouellet JA, Haglund L Facet joint degeneration in adolescent idiopathic scoliosis JOR Spine. 2018;1:e1016.
Bezci SE, Eleswarapu A, Klineberg EO, O’Connell GD. Contribution of facet joints, axial compression, and composition to human lumbar disc torsion mechanics. J Orthop Res. 2018 Feb 12. doi: 10.1002/jor.23870. [Epub ahead of print]

Lactation Bone Loss: A Second Chance

It is well recognized that the calcium demand of lactation may lead to bone loss. This would be particularly true for multiple pregnancies with breast feeding. However, the incidence of fragility fractures in later life does not appear to be dramatically higher for lactating mothers. Investigators may have come up with a reason.

Microstructural Analysis

Starting at age 3 months, 9 reproductive rats underwent 3 cycles of pregnancy and lactation, with a 6-week post-weaning recovery period between each cycle. At 12 months these rats, along with 12 virgin rats of the same age, had ovariectomy (OVX). All 9 reproductive and 8 virgin rats had µCT of the proximal tibia just prior to surgery and then 4, 8, and 12 weeks after surgery. A trabecular volume of interest (VOI) was identified for pre-OVX µCT scans. Each was subjected to individual trabecular segmentation (ITS), to isolate individual trabecular elements. The extent of bone loss and changes in connectivity were tracked for each trabecula, and the baseline characteristics associated with connectivity deterioration were identified. Whole bone stiffness was estimated by finite element analysis.

At 12 weeks post-OVX, the virgin rats had decreases of 76% bone volume fraction (BV/TV), 87% connectivity density (Conn.D), 52% trabecular number (Tb.N), and 22% whole-bone stiffness. There was no change in trabecular thickness (Tb.Th,). Albeit the reproductive rats has a 53% BV/TV decrease after OVX, there were no changes in Conn.D, Tb.N, Tb.Th, or whole-bone stiffness. Lending credence to the concern about bone loss with lactation, prior to OVX reproductive rats had 49%, 77%, and 50% lower BV/TV, Conn.D, and Tb.N, respectively, than virgins. Reproductive rats had 13-17% greater Tb.Th, as well as 22%, 10-13%, and 7-12% greater polar moment of inertia (pMOI), cortical area (Ct.Area), and cortical thickness (Ct.Th) than virgins throughout the study. By 12 weeks post-OVX, baseline differences in trabecular microstructure between reproductive and virgin rats were eliminated. Whole bone stiffness, cortical thickness, and trabecular thickness were greater for the reproductive rats.

Cell activities

17 rats (9 reproductive, 8 virgin) were euthanized at 4 weeks post-OVX. Proximal tibiae were embedded and longitudinal sections were stained with Goldner’s Trichrome. The numbers and surfaces of osteoblasts and osteoclasts (N.Ob/BS, N.Oc/BS, Ob.S/BS, Oc.S/BS) were quantified within the secondary trabecular bone. Histomorphometry indicated that virgin and reproductive rats had highly similar osteoblast and osteoclast numbers and surfaces at 4 weeks post-OVX. µCT -based trabecular dynamic analysis showed that virgin rats underwent a 125-179% greater rate of rod disconnection and plate perforation after OVX than the reproductive group. Results from the study confirm the long-lasting effects of reproduction on maternal bone as prior to OVX (surgical menopause), and suggest that the reproduction-associated changes in maternal bone phenotype, especially trabecular thickness, may explain the reduced OVX bone loss.

Overall, lactating mothers might be losers in the beginning but winners after menopause. Human cadaver studies taking benefit of lactation history might tell the story.

Reference

de Bakker C, Leavitt L, Zhao H, Li Y, Krickus C, Huang M, Tseng W-J, Liu XS. Microarchitectural Adaptations in Rat Maternal Bone Induced by Pregnancy and Lactation Exert Protective Effects against Future Estrogen Deficiency. ORS Annual Meeting Paper No.0256, 2018

Musculodystrophy, Cleaving a Way to a Cure

The lack of a muscle protein, dystrophin, is generally accepted as a cause of Duchenne Muscular Dystrophy (DMD). Beyond the muscle fragility is the effect of the dystrophin mutation reflected as a muscle stem cell disease that results in muscle progenitor cell (MPC) depletion/dysfunction in DMD progression.

The term CRISPR/Cas9 stands or clustered regularly interspersed short palindromic repeats (CRISPR) and the CRISPER associated protein 9 (Cas9). CRISPR is an adaptive immune system for bacteria. On infection by a virus (phage) the bacteria’s Cas genes are activated. Cas gene products cut viral DNA sequences sites called protospacers and then enter those sequences into the bacterial DNA. The viral sequences are identified by a protospacer adjacent motif (PAM) which is rarely seen in the host genome. Hence, replication of this sequence will not adversely affect the host. In the event of a second phage attack CAS genes are activated and CRISPR RNA (ccRNA) recognizing the phage sequence is generated. These associate with CAS nucleases to cleave both DNA strands of the invader. The type II CRISPR family uses ccrRNA and an additional tracrRNA to target specific DNA sequences. These have been combined to create a single guide RNA (gRNA) to direct sequence-specific Cas9 double-stranded DNA cleavage and present a simple, programmable RNA method that has been used for genome targeting and genome editing in eukaryotes. With DNA manipulation the accuracy of this system has been markedly enhanced to avoid unwanted mutations¹.

Investigators used in vitro methods to study recovery of MPC functionality (or intrinsic properties)². They also used twelve-week-old mice with transplantation of 2 million dystrophin deficient MPCs cells (non-treated and CRISPR dystrophin treated) injected into the gastrocnemius muscle.

MPC functional recovery

Gene expression analysis revealed that mRNA levels in dystrophin-restored MPCs were increased for multiple myogenic markers. Myogenic differentiation potential was also increased. The chondrogenic and osteogenic potentials of dystrophin-restored MPCs was increase if cells were incubated in chondrogenic and osteogenic differentiation media. Also, restoration of dystrophin in dystrophic MPCs improved hypoxic stress resistance and promoted cellular proliferation.

Cell transplantation

Modified MPCs ex vivo to restore dystrophin expression were placed in the muscles of mdx mice. GA muscles were harvested and frozen sections were evaluated for engraftment using beta-galactosidase and antidystrophin antibody staining. While no dystrophin expression was detected in control-injected muscles (-CRISPR), muscles that had been transplanted with CRISPR/Cas9-corrected cells (+CRISPR) generated engrafted areas with dystrophin-positive myofibers.

References

Gibson GJ, Yang M. What rheumatologists need to know about CRISPR/Cas9. Nat Rev Rheumatol. 2017 Apr;13(4):205-216. doi: 10.1038/nrrheum.2017.6. Epub 2017 Feb 9.
Matre PR, Wu J, Mu X, Lu A, Deepak R, Darabi R, Huard J. Dystrophin Restoration by CRISPR/Cas9-Mediated Gene Editing Improves Properties of Muscle Progenitor Cells. ORS Annual Meeting Paper No.0262, 2018

Preventing Heterotopic Ossification: No Bones About It

Heterotopic ossification (HO), also called fibrocytis ossificans, can be associated with blunt muscle injury in younger athletes. It can also occur as a complication of total hip replacement surgery. During the past two decades HO had been a serious problem for service members who receive high-energy penetrating injuries in blast-related amputations. The prophylaxis for HO that is typically offered includes radiation and anti-inflammatories. However, in the military theater logistics and the traumatic condition of the patients dictates against those. Thus far, no safe and effective alternative prophylaxis has been identiﬁed for that patient population.

Palovarotene is a highly selective retinoic acid receptor gamma (RAR-γ) agonist. It is being investigated as a potential orphan drug treatment for fibrodysplasia ossificans progressive, a rare genetic disorder. It has also been shown to reduce bone formation in traumatic models of HO. Given the safety of palovarotene in a mass trauma population a study was designed to determine the effects of Palovarotene on inﬂammation, progenitor cell proliferation, and gene expression using a blast-related amputation rodent model. 5 non-treated and 5 treated animals were sacrificed at post-operative day 1, 3, 7, 14, and 21. There were 12 naïve controls. Palovarotene was given orally every other day for 14 days. The ability of Raman spectroscopy to detect early HO before radiographic changes occur was also tested.

Cytokine expression

Palovarotene dampened the systemic inﬂammatory response including the cytokines IL-6 (p<0.01), TNF-a (p<0.001), and IFN-g (p<0.03) as well as the local inﬂammatory response via a 76% reduction in the inﬁltration of connective tissue progenitor cells at post-operative day (POD)-7 (p<0.03).

Ossification progenitor cells

Palovarotene decreased osteogenic connective tissue progenitor (CTP-O) colonies by as much as 98% both in vitro (p<0.04) and in vivo (p<0.01). Palovarotene signiﬁcantly decreased expression of osteo- and chondrogenic genes by POD-7, including BMP4 (p<0.02).

Raman spectroscopic detection of markers of early HO formation

Raman spectroscopy of affected soft tissue specimens detected differences between the two groups by POD-1 (p<0.001). Palovarotene inhibited traumatic HO formation through multiple inter-related mechanisms including anti-inﬂammatory, anti-proliferative, and gene expression modulation. Raman spectroscopy was able to detect markers of early HO formation before it becomes radiographically evident. During amputation debridement this could facilitate earlier diagnosis and treatment.

Refernce

Wheatley BM, Cilwa KE, Dey D, Qureshi AT, Seavey JG, Tomasino AM, Sanders EM, Wesley Bova W, Boehm CA, Iwamoto M, Potter BK, Forsberg JA, Muschler GF, Davis TA. Palovarotene Inhibits Connective Tissue Progenitor Cell Proliferation in a Rat Model of Combat-Related Heterotopic Ossiﬁcation. J Orthop Res. 2018 Apr;36(4):1135-1144. doi: 10.1002/jor.23747. Epub 2017 Nov 16. PMID: 28960501

A Crystal Ball to Predict Periprosthetic Osteolysis

Following joint replacement surgery, patients can develop asymptomatic peri-prosthetic osteolysis, which can lead to substantial bone loss prior to diagnosis. Revision surgery to treat osteolysis may be more challenging due to the extent of bone loss. Investigators set out to identify urinary biomarkers that differentiate total hip replacement patients who develop osteolysis from patients who do not.

Samples from 24-h urine collections prior to surgery and annually thereafter were saved in 26 patients. 16 developed osteolysis an average of 7.5 years after surgery. 10 did not have osteolysis over the 19 year follow-up period. At the first radiographic sign of osteolysis marker analysis was done on samples from the preoperative, first post-operative, and for the 6 years preceding diagnosis. Patients in the osteolysis and non-osteolysis groups were matched according to time post-surgery. They did not differ in the male to female ratio or age at surgery. Seven biomarkers were measured: free deoxypyridinoline (DPD), cross-linked N-telopeptides (NTX), interleukin-6 (IL-6), interleukin-8 (IL-8), osteoprotegerin (OPG), α-crosslaps (α-CTX), and β-crosslaps (β-CTX). Type I collagen cross-linked N-telopeptide (NTx) and deoxypyridinoline (DPD) levels in human urine are indicators of bone resorption. As bone ages the C-terminal telopeptide, another bone resorption marker, converts as the alpha form of aspartic acid present in CTx converts to the beta form (beta-CTx). Interleukin-6 (IL-6), interleukin-8 (IL-8) are markers of inflammation. Osteoprotegerin attaches to receptor activator of nuclear factor κB (RANK) and thus protects bone by inhibiting osteoclastic bone resorption.

DPD demonstrated the highest ability to predict osteolysis in Receiver Operating Characteristic (ROC) analyses at 6 years prior to osteolysis diagnosis with an area under the curve (AUC) 0.844. A panel of α-CTX combined with IL-6 was able to identify at-risk patients with an AUC of 0.941 or greater at all post-operative time points. The AUC was 1.000 pre-operatively. Markers for collagen degradation and inflammation are potential non-invasive biomarkers to identify patients at risk for peri-implant osteolysis long before the emergence of radiographic signs. The high accuracy of the pre-operative biomarker levels identify patient-specific factors leading to early osteolysis and the need to follow these patients more routinely.

Reference

Ross RD, Deng Y, Fang R, Frisch NB, Jacobs JJ, Sumner DR. Discovery of biomarkers to identify peri-implant osteolysis before radiographic diagnosis. J Orthop Res. 2018 Jun 5. doi: 10.1002/jor.24044. [Epub ahead of print]. PMID: 29873110

Signal Minded About Cortical Bone Quality

Magnetic resonance imaging (MRI) prepares the magnetization then acquires its signal after a short time delay, which is called the echo time that is typically 5-20ms. This finite echo time makes the tissue that has short T2 relaxation to appear dark/black (hence invisible!) in MR images. Short-T2 tissue includes mineralized tissue (e.g., cortical bone) and tissue with well-organized/dense collagen fibers (deep articular cartilage, tendon and ligament). One recent effort in MRI technology is to develop methods that has very short echo time (≤1ms), hence enabling the visualization of invisible tissues¹.

In one investigation both 2D and 3D methods of MRI-UTE-T2* were used on 58 subjects to detect changes in deep articular cartilage within two years of an anterior cruciate rupture and repair². 2D and 3D imaging correlated for changes in the medial femoral condyle compared to uninjured controls but on 3D imaging showed a difference in the medial tibial plateau relative to controls. Significance of the results varied in different regions of the tissue analyzed. The elevated UTE-T2* values in regions of deep tibiofemoral cartilage may be early indications of cartilage at risk for accelerated OA development.

Another investigation used UTE-MRI for cortical bone. Investigators sought a balance between imaging speed and quantification errors using different imaging trajectories, thus facilitating the clinical use of quantitative UTE-MRI for cortical bone imaging. Bovine cortical bone samples in formalin were scanned using 3 techniques designed to measure the magnetization transfer ratio (MTR) and MT modeling of water and collagen proton fractions and exchange rates³. There was excellent bi-component T2* decay observed in cortical bone with information on short (bound water) and long (pore water) T2*s (0.27 vs 2.34 ms) and relative fractions (75% vs 25%). Excellent MT modeling was achieved with macromolecular proton fraction of ~61%. Collectively, these studies show that UTE-MRI may facilitate clinical determination of deep cartilage² and cortical bone³ quality quantitatively.

References

Du J, Takahashi AM, Chung CB. Ultrashort TE spectroscopic imaging (UTESI): application to the imaging of short T2 relaxation tissues in the musculoskeletal system. J Magn Reson Imaging. 2009 Feb;29(2):412-21. doi: 10.1002/jmri.21465.
Williams A, Titchenal MR, Guha a, Chu CR.MRI UTE-T2* Shows High Incidence of Cartilage Subsurface Matrix Changes 2 Years After ACL Reconstruction. ORS 2018 Annual Meeting Paper No.0173.
Zhao W, Ma Y, Jerban S, Carl M, Chang EY, Du J. Quantitative ultrashort echo time (UTE) magnetic resonance imaging of cortical bone: speed vs. accuracy. ORS 2018 Annual Meeting Poster No.1557.

Obesity and Osteoarthritis Inflammation: A Gut Reaction

Fifteen years ago counseling obese patients with knee osteoarthritis would have focused on the impact of force on articular cartilage. Metabolic syndrome (MS) is the combination of diabetes, hypertension, dyslipidemia, and obesity. The impact of MS on the cardiovascular system has been recognized for over a decade. More recently the impact of MS on knee OA has been studied to the extent that some feel that OA is a component of MS. The impact of obesity in the alteration of gut flora has recently gained attention.

Gut microbiome dysbiosis is associated with increased systemic inflammation. One hallmark of this is macrophage migration to the synovium and accelerated knee OA. Oligofructose is a non-digestible prebiotic fiber that can restore the microbial community profile of an obese individual to that of a lean gut.

Investigators created an obese mouse model of trauma induce OA to test hypotheses that a proinflammatory shift in the gut microbiome causes more severe OA and that reversal of that shift with oral oligofructose would mitigate the effects of obesity on OA progression. They had a control weight gain population over twelve weeks to demonstrate that oligofructose does not prevent obesity in mice consuming a high-fat diet. Also, oligofructose reversed the effect of obesity on the gut microbiome and the colon transcriptome.

Oligofructose-supplemented obese mice have reduced systemic inflammation. There were four cytokines which are significantly increased in obese mice that were also significantly reduced in the oligofructose-supplemented group. These were the mouse homolog of IL-8, MIP-1B, M-CSF, and Tnf. This is matched by the reduction of obesity-associated macrophage migration to the synovium.

In a meniscal destabilization model oligofructose-supplemented obese mice were completely rescued from the deleterious effect of obesity. Combined with the above findings the protective effects of oligofructose in the OA of obesity are associated with the suppression of the MCP-1/Tnf inflammatory profile.

Reference

Schott EM, Farnsworth CW, Grier A, Lillis JA, Soniwala S, Dadourian GH, Bell RD, Doolittle ML, Villani DA, Awad H, Ketz JP, Kamal F, Ackert-Bicknell C, Ashton JM, Gill SR, Mooney RA, Zuscik MJ. Targeting the gut microbiome to treat the osteoarthritis of obesity. JCI Insight. 2018 Apr 19;3(8). pii: 95997. doi: 10.1172/jci.insight.95997. [Epub ahead of print] PMID: 29669931, PMCID: PMC5931133

Micro Managing Bone Defects

The gold standard for augmentation of bone defects is graft autograph taken at time of surgery. However, donor site complications detract from use of larger quantities of bone. The use of bone graft substitutes is often disappointing and graft failures can occur. Likewise, growth factor augmentation may have its complications and failures because of timing of release. Non-viral gene delivery vectors such as polyethylenimine, chitosan or nano-hydroxyapatite have been used to give controlled expression and release of growth factors such as bone morphogenetic protein-2 (BMP-2) or vascular endothelial growth factor (VEGF) from plasmid DNA (pDNA) at a defect site by transfecting host mesenchymal stem cells (MSCs).

Investigators aimed to create a gene activated scaffold with a novel Star-Shaped Poly-L-Lysine Polypeptides (star-PLLs). Star-PLLs are non-viral gene delivery vectors incorporated into collagen scaffolds. Due to their unimolecular, modifiable design, they have the ability to complex nucleic acids with a high cargo loading capability. Multiple Star-PLL-pDNA nanoparticles were studied. An optimal starPLL-scaffold combination to induce osteogenesis (star-PLL-03-pBMP2/pVEGF collagen-HA scaffold) was identified and implanted into an 7mm critical sized cranial defect in a Wistar rat model.

In vitro the osteogenic capability of the pBMP2 & pVEGF gene activated scaffold compared to a non-gene activated scaffold was greater (p<0.001). In vivo, microCT analysis at four weeks revealed that the pBMP2 and pVEGF dual loaded star-PLL-gene activated collagen-HA scaffold increased new bone volume by 14.1±3.1% within the defect compared to an empty defect (0.27±0.24%) or gene free collagen-HA scaffold (2.2±0.9%). Histology revealed consistent and extensive deposition of highly mineralized osteoid in the gene activated scaffold group which was not observed in the scaffold alone group.

Continued research on this delivery system may result in a cell free, “off the shelf”, gene activated scaffold for the healing of critical sized bone defects.

Reference

Walsh D, Raftery R, Castano IM, Murphy R, Heise A, Cryan S-A,O’Brien F. Gene Activated Scaffolds Incorporating Star Shaped Polypeptides Accelerate Bone Tissue Regeneration in Vitro and in Vivo. Trans ORS 2018 Paper 0264

Sound Advice for Tendon Scars

There is significant variation in scar formation following tendon injury and repair. To study scar formation in animal models mechanical, biochemical, and cellular assessment requires destruction of the tendon in each of the stages of healing. Investigators have developed an ultrasound (US)-based metric to quantify scar tissue volume (STV), allowing for a non-invasive in vivo characterization of tendon healing wherein image segmentation STV strongly correlates with traditional end-point metrics of gliding function.

A flexor (FDL) was transected and repaired in 12 week old S100a haploid (better healers) and wild type mice. At 7, 14, 20, and 28 days following flexor digitorum profundus tendon repair a 70-MHz transducer probe was used to obtain 105 frames of B-mode images in the sagittal plane were taken with 0.04mm steps to capture the entire width of the tendon (please see figure). For each stage of healing images were compared to histology at 14 and 28 days. Scar formation was quantified by measuring metatarsophalangeal (MTP) joint angle. Lower gliding resistance and higher MTP flexion angle indicate decreased scar and better gliding function.

SCV correlated positively with decreased flexion and increased gliding resistance. Traditional endpoint analyses of S100a4GFP/+ repairs demonstrate a significant 36% increase in MTP flexion angle, and a 43% decrease in gliding resistance, relative to WT, consistent with decreased scar formation. Consistent with these data, STV was significantly decreased in S100a4GFP/+ repairs (1.003 ± 0.08mm3), relative to WT at day 14 (0.8 ± 0.04mm3, p=0.03). Segmented US assessment of STV correlated with histology-based segmentation approaches.

Based on these findings there may be an increasing role for the use of US to asses therapeutic strategies aimed at scar reduction in tendon repair.

Reference

Ackerman LE, Loiselle AE. Development of Longitudinal, Non-invasive Ultrasonography to Assess Scar Formation During Flexor Tendon Healing. Trans ORS 2018 Paper 0306

Is it Ready? You Don’t Have to Stick a Fork In It

Cartilage tissue engineering constructs are evaluated based on molecular content and mechanical properties that change depending on the length of culture and tissue source. The assessment requires tissue destruction. To assess cartilage matrix development over time multiple constructs must be made. Non-destructive near infrared (NIR) spectral data has been used to evaluate engineered cartilage grown with bovine chondrocytes. This investigation hypothesized that NIR spectroscopic data along with multivariate partial least square (PLS) analysis techniques could be used to predict the compositional and biomechanical properties of engineered cartilage.

6 months old Yorkshire porcine stifle joint cartilage was digested, centrifuged. The pellets were dispersed and encapsulated at 60 million/mL in 1% methacrylated hyaluronic acid (MeHA) hydrogels polymerized by UV exposure. 4 mm biopsy punches created chondrocyte laden hydrogel constructs and cultured in cytokine supplemented glass-bottomed six-well cell culture plates. NIR spectral data for sulfated glycosaminoglycan (sGAG) content and total collagen as well as mechanical properties were collected from constructs at days 0, 14, 28, and 42. For correlation, biochemical and mechanical testing was performed on constructs at 2, 4 and 6 weeks. Porcine data were compared to previously published bovine data.

There are differences between species as to the change in rate of synthesis (first derivative) for collagen and sGAG. PLS analysis of NIR spectral data predicted biochemical composition and dynamic moduli of porcine constructs with 11.6%, 15.4% and 9.25% error for the calibration and validation of the dynamic modulus, sGAG content and collagen content, respectively. The predicted parameters had significant (p<0.0001) correlations with experimental values for dynamic modulus (r= 0.84), sGAG content (r=0.71) and collagen content (r=0.93).

This non-destructive method to determine the composition and mechanical properties of cartilage could allow researchers and clinicians to monitor tissue engineered constructs in real time to determine the optimal time for implantation without sacrificing samples.

Reference

Kandel1 S, Querido W, Falcon JM, Yousefi F, Reiners DJ, Kim M, Mauck RL, Pleshko N. Near Infrared Spectroscopy Predicts the Compositional and Biomechanical Properties of Porcine Engineered Cartilage. Trans ORS 2018 Paper 0306

Stem Cell Extracellular Vesicles: It’s All in the Message

The importance of perivascular cells (pericytes) in their role as stem cells has drawn exponential attention over the past decade. Their role in forming new tissue as well as paracrine signaling is becoming far better understood¹.

Researchers have investigated the role of extracellular vesicles (EVs) secreted by human bone marrow derived MSC (BMMSC) in human OA cartilage repair. EVs are small membrane-enclosed particles released by cells. TNF-alpha-stimulated OA chondrocyte monolayer cultures were treated with BMMSC-EVs and pro-inflammatory gene expression was measured by qRT-PCR after 48 h². When co-cultured with OA chondrocytes, BMMSC-EVs abrogated the TNF-alpha-mediated upregulation of COX2 and pro-inflammatory interleukins and inhibited TNF-alpha-induced collagenase activity. Addition of BMMSC-EVs to untreated cultures of chondrocytes isolated from OA patients stimulated production of proteoglycans and type II collagen by these cells.

In a corollary project 2^nd passaged OA chondrocytes were cultured in fibrin glue and monolayer supplemented with MSC-derived EVs with or without the presence of TNF-α³. Dimethyl-methylene blue (DMMB) was used to measure the content of glycosaminoglycan (GAG, DMMB normalized for DNA) and histological analyses of paraffin sections of the fibrin constructs after 4 weeks. MSC-derived conditioned medium (CM) and EV-depleted CM were used as controls. Cultures of OA chondrocytes treated with MSC-EV contained more GAGs and type II collagen.

Proliferation was determined by ethynyldeoxyuridine (EdU) incorporation. Once Edu is incorporated by the cell it can be conjugated with fluorescent azides. Proliferation of OA chondrocytes was increased by MSC-EV

Inflammation was determined by gene expression of COX-2 and interleukins 1alpha, 1beta, 6, 8 and 17, NFkB nuclear translocation and phosphorylation of IkB kinase. MSC-derived EVs decreased TNF-α –induced gene expression of COX2 and interleukins 1alpha, 1beta, 6, 8 and 17. In addition, a decreased nuclear translocation of NFkB-p65 subunit and phosphorylated p65 and decreased phosphorylation of IkB kinase were observed.

Given different methods of extracting EVs, variation with tissue source, age of donor, and other factors clinical applications will require biochemical monitoring for consistency of chemical content and dose before consistent results can be appreciated.

References

Caplan AI. New MSC: MSCs as pericytes are Sentinels and gatekeepers. J Orthop Res. 2017 Jun;35(6):1151-1159. doi: 10.1002/jor.23560. Epub 2017 Apr 11. PMID: 28294393
Vonk LA, van Dooremalen SFJ, Liv N, Klumperman J, Coffer PJ, Saris DBF, Lorenowicz MJ. Mesenchymal Stromal/stem Cell-derived Extracellular Vesicles Promote Human Cartilage Regeneration In Vitro. Theranostics. 2018 Jan 1;8(4):906-920. doi: 10.7150/thno.20746. eCollection 2018. PMID: 29463990
Vonk LA,Coffer PJ,Saris DBF, Lorenowicz MJ. MSC-Derived Extracellular Vesicles as New Treatment for Osteoarthritis ORS 2018 0586

Chemokine Signaling: Rounding up Some of the Unusual Suspects

CC chemokines are chemokines that have two cysteine-cysteine pairs that attach to each other in a fold of the protein near the amino terminus. CCL indicates the roles of these proteins as ligands which are associated with chemokine activity. This group of molecules immediately makes one think of inflammatory processes where macrophages with CCL receptors (CCLR) are involved. Case in point is monocyte chemoattractant protein-1 (MCP-1 or CCL2). That axis is typically associated with degradation.

To identify mechanisms of cartilage healing in juveniles, investigators focused on chemokines for their effects on the migration of stem cells and progenitor cells wherein a cartilage laceration injury would heal. They identified a CCL21/CCR7 axis as a candidate related to juvenile cartilage repair. Full-thickness lacerations were done on 3-week old (juvenile), and 8-week old (adult) CCR7-/- and WT mice. At 4 and 8 weeks postoperatively, histological evaluation was performed. Distribution of CCL21/CCR7 expression during articular cartilage repair was confirmed by immunohistochemical staining. The CCR7-/- juvenile mice did not repair the cut and had degeneration at the cut margin. CCR7-/- and wild type adults had similar degenerative changes. CCL21 and CCR7 were detected at the injury site in juvenile mice but CCL21 and CCR7 were not detected at the injury site in adults.

In a separate experiment they made osteochondral defects in 15 week old rabbits and filled them with gel or gel with CCL21. A third group had no fill. At 16 weeks postoperatively, the reparative tissues in CCL21 group were predominantly hyaline-like cartilage with histological scores significantly better than those of other groups. Macroscopic score was 2.7 ± 0.95 in defect alone group, 4.0 ± 1.49 in the vehicle group, 6.1 ± 0.99 in the CCL21 group. Histological scores likewise progressed from 4.3 ± 2.75 defect, 11.4 ± 2.76 gel vehicle, and 19.1 ± 4.25 CCL21. There was no difference in subchondral bone volumes.

CCL21 possibly recruited chondrogenic progenitor cells from bone marrow resulting in the enhancement of cartilage repair in the rabbit. Further CCL21/CCR7 axis may constitute part of the molecular control of juvenile cartilage repair. This raises the possibility that the modulation of CCL21 can improve cartilage repair in the adults.

Reference

Joutoku Z, Onodera T, Momma D, Matsuoka M, Baba R, Hontani K, Matsubara S, Homan K, Hishimura R, Iwasaki N CCL21/CCR7 axis regulating juvenile cartilage repair can enhance cartilage healing in adult. Trans Orthop Res Sco. 2018. Paper 0064

Fractures, Macrophages, and Osteoblasts: Connecting the Dots

Polarization is not simply macrophage activation but exclusive selection of a differentiation path among several possible options. The term polarization has been borrowed from immunology, which due to the diversity and volatility of macrophage phenotypes may not be quite appropriate¹. Polarization to M1 macrophages is associated with inflammatory factors which produce distinct pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IL-23. On the other hand M2 polarization leads to activated healing macrophages with suppressed production of pro-inflammatory cytokines. Hence it seems logical that the path to new bone formation in fracture healing would be more related to M2 macrophages.

Within minutes of a fracture, the inflammatory phase of fracture healing starts. Shortly after both macrophages and mesenchymal stem cells (MSCs) arrive in the surrounding tissue. Investigators wished to define if M1 macrophages and MSCs worked in concert to promote bone healing². They co-cultured undifferentiated M0, pro-inflammatory M1, and anti-inflammatory M2 macrophages with primary murine bone marrow derived MSCs in vitro to determine the cross-talk between polarized macrophages and MSCs and the effects on osteogenesis.

After 4 weeks of co-culture, MSCs grown with macrophages had enhanced bone mineralization compared to MSCs grown alone. Of note, M1 macrophages had the highest level of bone formation after 4 weeks of culture. M1 cells are very important in the early stages of fracture healing, as the M1 chemokines call in cells to mop up the dead cellular debris and begin healing. Also, if M1 cells were converted to M2 before 72 hours, optimal bone formation does not occur.

The variables may be:

Prostaglandin E2 (PGE2) secretion occurs early in osteogenesis.
Treatment with celecoxib, a cyclooxygenase-2 (COX-2) selective inhibitor, significantly reduces bone mineralization in all co-cultures but most dramatically in the M1-MSC co-culture.
The presence of macrophages reduced the secretion of osteoprotegerin (OPG), the decoy RANKL receptors suggesting that macrophages may indirectly modulate osteoclast activity in addition to enhancing bone formation.

It appears that an initial pro-inflammatory phase modulated by M1 macrophages promotes osteogenesis in MSCs via the COX-2-PGE2 pathway. In this case, the interactions between macrophages and bone marrow MSCs provide opportunities to optimize bone healing and other regenerative processes via modulation of the inflammatory response and MSC phenotype change. This is in contrast to collaborative MSC signaling that affects surrounding cells³. It is interesting to note that over a decade ago it was found that inhibiting TNFα early in fracture healing ended up with poor healing⁴.

Reference

Konttinen YT, Pajarinen J, Takakubo Y, Gallo J, Nich C, Takagi M, Goodman SB. J Long Term Eff Med Implants. 2014;24(4):267-81. Macrophage polarization and activation in response to implant debris: influence by “particle disease” and “ion disease”. PMID: 25747030
Lu LY, Loi F, Nathan K, Lin TH, Pajarinen J, Gibon E, Nabeshima A, Cordova L, Jämsen E, Yao Z, Goodman SB. Pro-inflammatory M1 macrophages promote Osteogenesis by mesenchymal stem cells via the COX-2-prostaglandin E2 pathway. J Orthop Res. 2017 Nov;35(11):2378-2385. doi: 10.1002/jor.23553. Epub 2017 Mar 13. PMID: 28248001
Caplan AI. Mesenchymal Stem Cells: Time to Change the Name! Stem Cells Transl Med. 2017 Jun;6(6):1445-1451. doi: 10.1002/sctm.17-0051. Epub 2017 Apr 28. PMID: 28452204
Gerstenfeld LC, Cho TJ, Kon T, Aizawa T, Cruceta J, Graves BD, Einhorn TA. Impaired intramembranous bone formation during bone repair in the absence of tumor necrosis factor-alpha signaling. Cells Tissues Organs. 2001;169(3):285-94. PMID: 11455125

Bone Density Assessment: Breaking It Down

The clinical assessment of fracture healing can be a costly and possibly inaccurate process. While computerized 3D imaging has improved the reliability of healing assessment, current imaging assesses only mineral aspects of a process that involves many tissues in the evolution of repair that varies with age and location. There is evidence that ultrasound can detect early callus formation, but is limited to superficial assessment and results are highly dependent on operator expertise. Radiographic assessment remains the most commonly used technique, but does not take into account cartilage and non-mineralized osteoid that may be sufficient to allow weight bearing.

Investigators designed a two-part study to validate the use of impedance spectroscopy to monitor fracture healing, with stages of healing confirmed through histological evidence. Impedance spectroscopy measures impedance with varying voltages and frequencies, encompassing resistive and capacitive components due to the ionic environment and cell membranes, respectively. Impedance magnitude (|Z|) reﬂects the magnitude of the combined components, of which tissue conductivity is often a dominant term. The phase angle (θ), provides information about how resistive or capacitive the measurement is (0˚ is fully resistive, -90˚ is fully capacitive).

Cadaver bone study

A comminuted fracture was created in a thawed cadaveric leg using a chisel, and fragments were fixed with pins and external fixation. Cartilage, trabecular bone, and a mixture of the two were inserted into the fracture gaps. Measurements were taken with two stainless steel pins as electrodes 2.7cm apart.

Cartilage exhibited smaller |Z| than trabecular bone across all measured frequencies (p<0.002), as well as more negative θ from 20Hz to 1kHz (p<0.05) and less negative θ from 50kHz to 500kHz (p<0.002). The heterogeneous mixture of cartilage and trabecular bone resulted in measurements that fell approximately halfway between the individual tissues. A metal plate across the fractures did not affect the measurements, with no effect from screws if electrodes are placed far from the screws.

Mouse fracture model study

Adult wild-type C57BL/6 mice had un-stabilized mid tibia fractures, with calluses dissected and isolated at 4, 8, 14, and 21 days, reflecting vascular, cartilage, mixed and trabecular bone healing phases. Calluses were measured with impedance spectroscopy and tissue composition was quantified with histomorphometry.

Impedance magnitude had a positive relationship with % trabecular bone and a negative relationship with % cartilage. The opposite relationships were found when comparing phase angle to these phases.

The future

In open procedures the development of micro- and nanofabrication implants that feature wireless capabilities have potential use for early detection of bone healing and fracture nonunion.

Reference

Lin MC, Yang F, Herfat ST, Bahney CS, Marmor M, Maharbiz MM. New opportunities for fracture healing detection: Impedance spectroscopy measurements correlate to tissue composition in fractures. J Orthop Res. 2017 Dec;35(12):2620-2629. doi: 10.1002/jor.23570. Epub 2017 Jun 9. PMID: 28383765

Tendon to Bone Healing: All in Good Time My Little Pretty, All in Good Time

Following injury and repair, reducing the time of tendon to bone healing helps avoid cartilage deterioration and muscle atrophy that may be hard to recover. It normally requires less time for bone to bone to heal in contrast to tendon to bone. The region of tendon healing ideally results in reformation of the original tendon, fibrocartilage, and bone. Very often, for many reasons, remodeling falls short of that goal.

Low intensity pulsed ultrasound (LIPUS) has been studied in fracture healing, both animal and clinical studies. It has also been studied in animals for effect on tendon to bone healing from the inflammatory phase to remodeling. It has been found that there may be increased cell permeability in the first week wherein the LIPUS would be adverse. Hence investigators hypothesized that LIPUS starting at week two would accelerate new bone formation and remodeling.

The adult rabbit model entailed a distal 1/3 patellectomy including stripping of fibrocartilage with reattachment of the tendon to bone. In the rabbits, starting at the second week thaey used LIPUS until sacrifice at 4, 8, and 16 weeks. LIPUS was adapted to a 1.5MHz frequency, 1:4 duty cycle and 30mW/cm2 spatial and temporal average incident intensity. Daily treatment times were 20 minutes. There were 18 controls.

The qRT-PCR showed that LIPUS group has significantly lower TNF-α and IL-1 at weeks 4 and 8 and less IL-6 at week 4. Both IL-10 and TGF-β were significantly increased at weeks 4,8, and 16. Histologically, the LIPUS group showed more advanced remodeling of the lamellar bone and marrow cavity than the control group. The area and length of the new bone in the LIPUS group were signiﬁcantly greater than the control group at postoperative weeks 8 and 16. More fibrocartilage interface to tendon appeared at weeks 8 and 16 in the LIPUS group. SR-mCT demonstrated more advanced new bone formation and remodeling in the LIPUS group and biomechanical test results showed failure load, ultimate strength and energy at failure signiﬁcantly higher compared to the control group.

This model is not the same as the more difficult issue of rotator cuff repair where the tissue damage and biological environment is not the same as surgically induced tendon to bone repair. However, the time has come to look at the role of LIPUS and other physical modalities in enhancing surgical repair.

Reference

Lu H, Liu F, Chen H, Chen C, Qu J, Xu D, Zhang T, Zhou J, Hu J. The effect of low-intensity pulsed ultrasound on bone-tendon junction healing: Initiating after inflammation stage. J Orthop Res. 2016 Oct;34(10):1697-1706. doi: 10.1002/jor.23180. Epub 2016 Feb 18. PMID: 26833973

Tendons in 3D: A New Twist

The role of cellular and matrix architecture in by region and depth in articular cartilage and by composite structure and function in bone. These regional differences also vary when they are investigated in different joints and bones. The biomechanical, and functional characteristics of the Achilles tendon are closely related to its composition and microstructure. Type I collagen is the predominant component of tendons and is mainly responsible for the tissue’s function. Although elastin has been found in varying proportions in other connective tissues, previous studies report that tendons contain very small quantities of elastin. It is believed that elastin plays a role in the in the initial elongation of the tendon as seen on a stress/strain curve referred to as the toe region. However, the morphology and the microstructural relationship among the elastic ﬁbres, collagen, and cells in tendon tissue have not been well examined.

Investigators hypothesize the elastic ﬁbres have a unique role in mechanical function and microstructural arrangement in Achilles tendons. Using cryosectioning the sections were stained with the nucleic acid-selective ﬂuorescent dye Acridine Orange (AO) for imaging the nucleus of tenocytes, and the ﬂuorescent dye SRB was used to label the elastin. Similarly, a laser which was tuneable was set at 890nm to acquire the SHG signals from the collagen. The elastin and cells ﬂuorescent signals were collected by photomultiplier tubes at 565–600 and 590–680nm, respectively. The SHG signals were directly collected by a secondary detector at 445nm for transmitted lights. With 3D imaging processing and 2D and fast fourier transform and alignment analysis matrix and cellular relationships could be studied in detail.

Strong association of orientations between the elastin and tenocytes within the longitudinal collagen ﬁbril framework was found that remained when the collagen ﬁbrils become oblique and spiral. This anatomical structural knowledge may enrich the theory of mechanical and biological information transduction in tendon tissue to develop future imaging techniques investigating tendon pathology. Future studies can focus on the quantitative evaluation of elastic ﬁbre meshwork in relation to tenocytes and collagen matrix as they change in aged tendons and different pathological conditions.

Reference

Pang X, Wu JP, Allison GT, Xu J, Rubenson J, Zheng MH, Lloyd DG, Gardiner B, Wang A, Kirk TB. Three dimensional microstructural network of elastin, collagen, and cells in Achilles tendons. J Orthop Res. 2017 Jun;35(6):1203-1214. doi: 10.1002/jor.23240. Epub 2017 May 2.

Treating Legg-Calvé-Perthes Disease: Adds a Whole New Meaning to Support Group

One of the holy grails of orthopaedics is finding a way to preserve femoral head shape during recovery from osteonecrosis. Whether in a pediatric or older adult population treatment options are often met with femoral head collapse, bone loss, and deformity. The problem in Legg Calvé Perthes disease is that bone absorption is not coupled with new bone formation. Creeping substitution is not the mechanism of repair. Hence, efforts to decrease absorption and increase new bone formation would be potentially useful in preventing femoral head deformity. Using an immature pig model of surgically imposed osteonecrosis systemically administered ibandronate decreased bone absorption and femoral head deformity¹. In subsequent research the osteoclast number per bone surface was significantly lower in a bone morphogenic protein plus ibandronate (BMP+IB) group compared with a non-weight bearing alone group. Calcein labeling showed significantly higher bone formation in the BMP and BMP+IB groups than in the NWB group (p < 0.05)².

For the next step the same investigators used the same drugs in an immature pig osteonecrosis model to determine the effects of BMP-2 and IB on the mineral content and nanoindentation properties of the bone following ONFH. There were five groups: normal control, untreated, IB, BMP, and BMP + IB (n = 5/group). Using backscattered electron imaging and Raman spectroscopy both BMP and BMP + IB groups showed calcium content in the trabecular bone to be similar to the normal group, while the IB and no-treatment groups showed a significant increase in the calcium content compared to the normal group. The carbonate content relative to phosphate was significantly increased in the IB and BMP + IB groups (p < 0.01) compared to the normal group. No significant difference was found between the BMP and the normal group. The nanoindentation modulus of the bone in the IB group was significantly increased compared to the normal group (p < 0.05). No significant differences were observed between the BMP and BMP + IB groups compared to the normal group. The nanoindentation hardness measurements in the IB group were also significantly increased compared to the BMP and BMP + IB groups (p < 0.05).

In summary, necrotic femoral heads treated with BMP or BMP + IB had bone material properties comparable to normal bone whereas the bone in the IB group retained the increased mineral content and the nanoindentation hardness found in the necrotic bone. Hence, BMP or BMP + IB better restores the normal mineral content and nanomechanical properties after ONFH than IB treatment alone. Question: will this biological support prove effective in humans?

References.

Kim HKW, Pathophysiology and New Strategies for the Treatment of Legg-Calvé-Perthes Disease. J Bone Joint Surg Am. 2012;94:659-69
Kim HK, Aruwajoye O, Du J, Kamiya N. Local administration of bone morphogenetic protein-2 and bisphosphonate during non-weight-bearing treatment of ischemic osteonecrosis of the femoral head: an experimental investigation in immature pigs. J Bone Joint Surg Am. 2014 Sep 17;96(18):1515-24. doi: 10.2106/JBJS.M.01361. PMID: 25232075
Aruwajoye OO,Aswath PB, Kim KW. Material properties of bone in the femoral head treated with ibandronate and BMP-2 following ischemic osteonecrosis. J Orthop Res. 2017 Jul;35(7):1453-1460. doi: 10.1002/jor.23402. Epub 2016 Sep 22.

Viscosupplementation in Obese Advanced Osteoarthritis Patients: Think Twice Before You Shoot

Viscosupplementation (VS) became very popular at the end of the 1990’s. It appeared to be an alternative to surgery or at least would delay joint replacement. However, several metanalyses led the Academy of Orthopaedic Surgeons to recommend against VS as a treatment for osteoarthritis. VS is still controversial. A problem is the absence of well-identiﬁed patient specific factors that may influence response.

Investigators used the results of a controlled, multicenter, double-blind, randomized, non-inferiority trial of 166 patients comparing 3 weekly IA injections of HA (HAnox-M or BioHA) for symptomatic tibiofemoral OA¹. In the process they wanted to identify clinical (demographic, anthropometric, knee effusion, patient global assessment, and WOMAC score) and radiological (OARSI grade, patello-femoral involvement) factors associated with lack of response. VS response was deﬁned according to OMERACT–OARSI response criteria at month 6. Predictors of response were investigated in univariate then in multivariate analysis.

The baseline characteristics and treatment effectiveness were similar between the 2 HA groups and thus that data was pooled. This was an older group with a mean age 65.2. 60.8% were women and 44.0% had severe TF space narrowing. At 6 months, 113 patients (68.1%) were responders. Multivariate analysis revealed obesity (BMI > 30kg/m2) and OARSI grade 3 were signiﬁcantly associated with VS failure, p value 0.001 and 0.008, respectively. The combination of obesity and severe TF space narrowing signiﬁcantly increased the risk of VS failure. Baseline pain intensity and functional impairment were not associated with VS response.

The authors used the terms “clinical and radiological factors associated with lack of relevant response”. Others might have applied the term phenotype. One way or the other this article highlights the careful thought in prospectively recording that should be applied to musculoskeletal research. However, caution must be taken in that the use of “phenotypes” must stand on the shoulders of validation².

References

Eymard F, Chevalier X, Conrozier T. Obesity and radiological severity are associated with viscosupplementation failure in patients with knee osteoarthritis. J Orthop Res. 2017 Oct;35(10):2269-2274. doi: 10.1002/jor.23529. Epub 2017 Feb 9. PMID: 28128473
Deveza LA, Melo L, Yamato TP, Mills K, Ravi V, Hunter DJ. Knee osteoarthritis phenotypes and their relevance for outcomes: a systematic review. Osteoarthritis 2017 Aug 25. pii: S1063-4584(17)31156-1. doi: 10.1016/j.joca.2017.08.009. [Epub ahead of print]. PMID: 28847624

Mesenchymal Stem Cells: It’s All in a Name

Years ago it was recognized that fracture callus was formed by cells derived from the surrounding muscle. It has been found that, regardless of the tissue (muscle fat, etc.) the origin of stem cells is pericytes¹. The first phase of any healing process is inflammation. Inflammation is inherently associated with the development of new vasculature. Research on this and other processes led to the discovery of Human Mesenchymal Stem Cells, hMSCs, first named as such over 25 years ago. It was found that these cells could differentiate into a number of different mesodermal phenotypes in cell culture. The capacity to form skeletal tissue in vitro encouraged the use of hMSCs for the fabrication of tissue engineered skeletal repair tissue with subsequent transplantation to in vivo sites. Based on markers CD 146 and smooth muscle actin additional research has shown that most, if not all, MSCs are derived from perivascular cells otherwise known as pericytes.

Beyond the role of providing stem cells for musculoskeletal repair a complete re-evaluation of the role and functions of MSCs in the body has been found. As a case in point, the skeleton is a preferred organ for cancer dissemination for various tumor malignancies. To date, most efforts to understand skeletal metastasis have investigated the invasive and digestive capability of disseminated tumor cells (DTCs). Studies have focused on cytokines such as tumor necrosis factor alpha (TNF-α). TNF-α upregulates degradative enzymes such as metalloprotease 13 leading to a breakdown of vascular barriers. Recent investigations have determined that role of MSCs extends beyond that function. As an example these cells may act as a gatekeeper for metastasis of melanoma into bone. In this fascinating work it was seen that pericytes would “grab” meleanoma cells into the marrow cavity. The platelet derived growth factor PDGF-BB plays a role in the process. Knock out mice for PPDGF-BB are free of melanoma metastaces¹.

In research where allogenic MSCs are mixed with autologous chondrones and placed in chondral defects in the human knee it was determined that no allogenic DNA remained at one year. Investigators hypothesized that the role of the MSCs was to communicate to the chondrones messages associated with the tilling of the defects with chondrocytes².

It has been suggested that the name of MSCs be changes to Medicinal Signaling Cells to more accurately reflect the fact that these cells seek sites of injury or disease and secrete immunomodulatory and trophic are more like therapeutic drugs. It is, indeed, the patient’s own site-specific and tissue-specific resident stem cells that construct the new tissue as stimulated by the bioactive factors secreted by the exogenously supplied MSCs³. It should be noted that there are currently almost 800 clinical trials listed using the MSCs as listed on clinicaltrials.gov.

References

Caplan AI. New MSC: MSCs as pericytes are Sentinels and gatekeepers. J Orthop Res. 2017 Jun;35(6):1151-1159. doi: 10.1002/jor.23560. Epub 2017 Apr 11. PMID: 28294393
de Windt TS, Vonk LA, Saris DBF. Response to: Mesenchymal Stem Cells: Time to Change the Name! Stem Cells Transl Med. 2017 Jul 11. doi: 10.1002/sctm.17-0120. [Epub ahead of print]. PMID: 28696033
Caplan AI. Mesenchymal Stem Cells: Time to Change the Name! Stem Cells Transl Med. 2017 Jun;6(6):1445-1451. doi: 10.1002/sctm.17-0051. Epub 2017 Apr 28. PMID: 28452204

Is There a “Genei” in the Bottle?

Rheumatoid arthritis (RA) and other autoimmune diseases are characterized by transient disease flares that are mediated by increased levels of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF). Current drug therapies for RA include a variety of biologic protein drugs (e.g., adalimumab and etanercept) that are effective in about 50% of patients but have to be delivered continuously at relatively high doses. This study applied tools of genome engineering and synthetic biology to modify stem cells such that they deliver a TNF in an autoregulated manner as the basis of a cellular “vaccine” for inflammatory diseases such as RA.

Terms:

Genome engineering refers to the use of molecular tools to modify DNA in a controlled manner.

Induced pluripotent stem cells (iPSCs) are a form of stem cell that possess pluripotent capabilities similar to those of embryonic, but can be created from a variety of adult cells by the expression of 4 specific transcription factors (Oct4, Sox2, Klf4, and Myc).

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that has been implicated in a number of autoimmune diseases. A number of TNF inhibitors are currently available and form the major class of biologic drug therapies for arthritis.

The chemokine CC ligand 2 (Ccl2) gene is also known as macrophage chemoattractant protein-1 gene (Mcp-1). The gene product regulates trafficking of monocytes/macrophage, basophile, and T lymphocytes by stimulating the ligand 2 receptor. This gene is upregulated by both TNF-α and IL-1.

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR Associated Protein 9 (Cas9). The CRISPR/Cas9 system uses a based bacterial anti phage immune system to cut and paste specific genes into specific sites of DNA, and has been adapted for genome editing of non-bacterial cells. As a case in point genes that encode IL-1 and TNF-α antagonists can be inserted at the Ccl2 locus to confer cytokine-activated and feedback-controlled expression of biologic therapies.

The research:

Cell monolayer

In response to TNF, stem cells rapidly express Ccl2. The CRISPR/Cas9 system was used at the Ccl2 promoter to insert a DNA segment that can transcribe soluble TNF receptor-1 (sTNR1). In this manner, cells will automatically produce the inhibitor of TNF in response to extracellular levels of TNF.

Wild type and Ccl2-sTNFR1 cells were then transfected with a viral NF-κβ reporter luminescent gene. With TNF-α stimulation after 24 hours NF-κβ was upregulated in both WT and Ccl2-sTNFR1 cells. However, at 48 and 72 hours there was a sharp decrease in Ccl2-sTNFR1 cells. This confirms that the Ccl2-sTNFR1 construct results in a feedback mechanism. A second confirmation of feedback was achieved by measures of sTNR1 showing declines at lower does of TNF- α and after 48 hours with a higher dose. NF-κβ fell only slight over 72 hours after wild type stimulation with TNF-α but fell dramatically in the Ccl2-sTNFR1cells. The expression of IL-6 had a similar decrease of upregulation relative wo WT.

Stem cell engineered cartilage

WT, Ccl2-luc, Ccl2-Il1ra, and Ccl2-sTNFR-1 cells were engineered into cartilage constructs. On stimulation with IL-1 or TNF- α the WT, Ccl2-luc cartilage underwent degradative changes with upregulation of metalloproteases and aggrecanases. On the other hand the Ccl2-Il1ra, and Ccl2-sTNFR-1 cartilage responded with a reduced inflammatory response.

Conclusion

Given the role of inflammation in RA and osteoarthritis and the dysregulated responses to pro-inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α) a local therapy absent of systemic effects would be dramatic. Using the CRISPR/Cas9 genome-engineering system antagonizing the IL-1- or TNF-α-mediated inflammation in an autoregulated, feedback-controlled manner is a potential solution. Also, this opens innovative possibilities for safer and more effective therapeutic approaches for a wide variety of diseases.

Reference

Brunger JM, Zutshi A, Willard VP, Gersbach CA, Guilak F. Genome Engineering of Stem Cells for Autonomously Regulated, Closed-Loop Delivery of Biologic Drugs. Stem Cell Reports. 2017 May 9;8(5):1202-1213. doi: 10.1016/j.stemcr.2017.03.022. Epub 2017 Apr 27.

Video available at: https://youtu.be/BqgQHGWJdMg

Tenascin Function in Rotator Cuff Tears: A Snippet

By age 80 the incidence of rotator cuff tears is 50%. Trauma, overuse, subacromial bony impingement, and cigarette smoking are known to be associated with tears. Although many rotator cuff tears are associated with injury, tears in the absence of trauma are common, particularly in older individuals. Evidence for heritable predisposition to rotator cuff tears (RCTs) is growing.

The TNC gene encodes tenascin. Tenascin is a glycoprotein responsible for some cell signaling, embryological development, and matrix protein associations. TNC is a modular ECM glycoprotein composed of a series of epidermal growth factor like repeats, ﬁbronectin type III-like repeats and a C-terminal ﬁbrinogen-like globular domain. Its expression is upregulated in wound healing and with inflammation subsequent to mechanical and degradative stress.

To investigate the role of TNC gene expression in rotator cuff tears 59 unrelated Caucasian individuals with surgically diagnosed full thickness RCTs (cases) were compared to 32 elderly Caucasian controls with intact rotator cuffs. Both were screened for differences in candidate genes: TNC, Col5A1, TIMP-1, MMP-1, MMP-2, MMP-3, MMP-9, and MMP-13. A ﬁrst cohort (59 cases; 32 controls) was genotyped. Using the Sequenom MassARRAY iPLEX 30 of 142 single nucleotide polymorphisms (SNPs) were tested for differences in proportions between cases and controls.

A second, matched cohort (96 patients; 44 controls) was also genotyped for the same 30 SNPs, but with the KASPTM genotyping technology. These two cohorts were combined to find six TNC SNPs that were signiﬁcantly associated with RCT. A missense SNP in exon10 might be of biological signiﬁcance because it varies the amino acid sequence close to the TNC-FNIII5 domain. The FNIII5 domain binds multiple growth factors and co-ligates with integrins during tendon healing.

It remains to be seen if these and other SNPs are associated with less favorable results in rotator cuff tendon repair and progression.

Reference

Kluger R, Burgstaller J, Vogl C, Brem G, Skultety M, Mueller S. Candidate gene approach identifies six SNPs in tenascin-C (TNC) associated with degenerative rotator cuff tears. J Orthop Res. 2017 Apr;35(4):894-901. doi: 10.1002/jor.23321. Epub 2016 Jun 19.

Where Are Your Weight Bearing Joints?

I am faced with a number of published journal articles wherein the phrases “weight-bearing”, “non-weight-bearing”, “non-load bearing” are applied to segregate upper limb joint forces (supposedly less) from those of the lower limbs. Normal force per area is often measured in Pascals (grams per Meter². In lower limb joints (ankle, hip, and knee) it is pretty well known that force concentration is 2 to 5 megapascals (MPa) with a wider range for some activities. 2 MPa (just under 300#/in2) is roughly 10 times the pressure in a car tire. As a point of comparison, normal atmospheric pressure at sea level is 100 kilopascals. So what about the upper limb?

Over a decade ago investigators recognized that despite the fact that investigators have explored static joint contact stresses estimated in vitro in many joints and in a number of species, although only rarely in vivo. Reviewing the literature on contact forces in both upper and lower limb joints they found that, despite a number of widely varying techniques (and spatial resolutions) to measure these contact stresses, reported ranges of static peak normal stresses were relatively similar from joint to joint across species (range of 0.5 to 5.0 MPa).¹

They also noted that evidence suggested some disorders of cartilage deterioration are associated with somewhat higher peak pressures ranging from 1-20 MPa, but overlapping the range of normal pressures. This, in part, may be explained by some evidence suggesting static contact stresses per se do not predict cartilage responses, but rather temporal aspects of the contact stress history.

Given the question of contact stress history a remarkable concept was put forth over 20 years ago. It was called the “envelope of function”. The proposal that there is a range of load that can be applied across an individual joint in a given period without supraphysiologic overload or structural failure. This is the joint “envelope of function”. On a graph with increased applied loads on the vertical axis and the frequency of loading on the horizontal axis there is a curvilinear line below which a person would be within the envelope of function. Four factors together determine the envelope of function for a given joint including anatomic, kinematic, physiologic, and treatment factors. This theory of joint function can result in a more rational clinical approach to treating patients with knee injuries and other orthopaedic conditions.²

A good clinical examples in the case of scapholunate dissociation. Using MRI contact calculation investigators found that injury to the scapholunate ligament increased joint cartilage peak force measures, suggesting a risk for onset of osteoarthritis in both the scaphocapitate and lunocapitate joints. Surgical repair appeared to restore most measures of contact mechanics to near normal values.³

This was best stated by Dr. Brand: Finally, since all articular cartilage experiences similar stresses, the concept of a “weight-bearing” versus a “non-weight-bearing” joint seems flawed, and should be abandoned.³

References

Brand RA. Joint contact stress: a reasonable surrogate for biological processes? Iowa Orthop J. 2005;25:82-94. PMID: 16089079
Dye SF. The knee as a biologic transmission with an envelope of function: a theory. Clin Orthop Relat Res. 1996 Apr;(325):10-8. PMID: 8998861
Modaresi S, Kallem MS, Lee P, McIff TE, Toby EB, Fischer KJ Evaluation of midcarpal capitate contact mechanics in normal, injured and post-operative wrists. Clin Biomech (Bristol, Avon). 2017 Aug;47:96-102. doi: 10.1016/j.clinbiomech.2017.06.008. Epub 2017 Jun 13. PMID: 28628801

Targeting Fracture Healing

The Wnt/β-catenin pathway functions by regulating the amount of the transcriptional co-activator β-catenin. This pathway controls gene expression programs for cell proliferation, cell polarity and cell fate determination during embryonic development as well as tissue homeostasis in adult life. Manipulating the pathway is a promising target to augment musculoskeletal stem cell (MSC) osteogenesis during healing. To rescue Wnt/β-catenin signaling, β-catenin agonists can be used.

Systemic drug delivery has the potential for numerous unwanted side effects since untargeted tissue can adversely respond. Physical treatments offer the advantage of focal application which avoids, in part, systemic effects. A means of circumventing unwanted side effects of systemically delivered materials is the use of nanoparticles (NPs) for drug delivery. Some NPs are more selectively taken up by cells in regions of edema and vascular extravasation, such as that seen in tumors or fracture healing. Other NPs may have proteins that have an affinity for a specific tissue. Investigators used synthesized amphiphilic diblock copolymers of PSMAb-PS were self-assembled into NPs. TRAP-binding peptide (TBP), scrambled control peptide (SCP) were attached to the NPs. β-catenin agonists were loaded into directly on NPs for in vitro MSC cell studies and linked with TBP to target fracture healing sites.

In Vitro NPs were characterized in vitro for cell uptake via fluorescence microscopy and flow cytometry and β-catenin upregulation via luciferase reporter assays. . MSCs showed ~100% internalization of Texas Red-labeled NPs and ~157-fold increase in β-catenin signaling after β-catenin agonist-loading NPs.

In vivo Saline, NP, SCP-NP and TBP-NP loaded with IR780 (a model drug and near-IR dye) were injected retroorbitally to analyze NP biodistribution 3 days after fracture. TBP-NP-β-catenin agonist treatment was analyzed via LacZ reporter mice (Tg(Fos-LacZ)34Efu/J). Furthermore, fracture healing was evaluated using X-ray. TBP-NP-IR780 (a model drug and near-IR dye) exhibited significantly higher accumulation at fractured bone compared with NP- or SCP-NP-IR780. Signal was nearly undetectable in heart, lungs and kidneys in all treatment groups, with exception to limited liver signal in mice treated with NP-IR780 and SCP-NP-IR780. Histologically there was more accumulation of TBP-NP-IR780 at fractures compared with SCP-NP-IR780. Callus and periosteum treated with TBP-NPβ-catenin agonist versus untreated controls revealed successful β-catenin activation during healing and later expedited fracture healing at 21 days.

The significance of this work is the development of targeted NPs for fracture-specific delivery of small molecule drugs. More specifically, β-catenin agonists loaded TBP-NPs and subsequent targeted delivery to fractures improve bone regeneration with reduced off-target side effects, which is not currently possible via systemic delivery of small molecule drugs.

Reference

MacDonald BT, Tamai K, He X. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell. 2009 Jul;17(1):9-26. doi: 10.1016/j.devcel.2009.06.016. Review. PMID:19619488
Wang Y, Baranello MP, Newman M, Sheu T-J, Puzas JE, Benoit DSW. Delivery of β-Catenin Agonists via Targeted Nanoparticles to Enhance Fracture Healing. ORS 2017 Paper 109

Prosthetic Coatings: Making it Stick - Depending

Staphylococcus aureus and Staphylococcus epidermidis are predominant causes of total joint infections. Investigators proposed that chemical grafting of bioactive polymers onto the surface of implants could prevent bacterial adhesion and colonization. Sulfonate groups (SO3-) can be bonded to poly(1-phenylethene) (polysterene) to from poly(sodium styrene sulfonate) (poly(NaSS)) which can be covalently linked to titanium alloy Ti6Al4V by a radical grafting process. Previous in vitro reports show poly(NaSS) resists enzymatic degradation, is very stable in physiological environments, reduces bacterial adhesion and colonization, and is capable of generating new active sites for protein binding. Grafted and ungrafted (control) Ti6Al4V surfaces were absorbed with adhesive proteins, including fibronectin (Fn), collagen type I (Col I) and fibrinogen (Fg), to study the ability of poly(NaSS) to promote bone-implant binding and prevent bacteria adhesion in the presence of these molecules at the interface, both in vitro and in vivo.

Poly(NaSS) Grafting: Successful poly(NaSS) on Ti6Al4V was confirmed with X-ray photoelectron spectroscopy (XPS), Fourier Transformed Infrared (FTIR), contact angle measurements and the toluidine blue colorimetric method.

In vitro Bone-Bonding Ability: Fn and Col I were adsorbed onto the surfaces. MC3T3-E1 osteoblastic cells were seeded and cultured up to 28 days. Fn and Col I proteins led to superior cell attachment intensified by poly(NaSS). Significant cytoplasmatic cell expansion was observed on Fn adsorbed surfaces. Col I led to increased attachment strength enhancing alkaline phosphatase production and calcium and phosphate concentrations.

In vivo Osseointegration and Implant Stability: Ti6Al4V cylinder-shaped implants were grafted with poly(NaSS) and plasma sprayed with hydroxyapatite and press-fit at both the femoral and the lateral condyles of 5 month old New Zealand white rabbits. Osseointegration was promoted on poly(NaSS) grafted implants and physiological stability was greater compared to ungrafted and HA-coated Ti6Al4V measured in periods up to 12 months.

In vitro Bacterial Adhesion: S. epidermidis (ATCC) and clinical methicillin-resistant S. aureus (MRSA, ATCC) were cultured on poly(NaSS) grafted Ti6Al4V surfaces and ungrafted Ti6Al4V surfaces. Fn, Fg, albumin (BSA), Fn/BSA, Fg/BSA, 10% plasma and serum was placed on separate surfaces. Fn and Fg increased staphylococcal adhesion on ungrafted Ti6Al4V surfaces. To the contrary, grafted surfaces reduced bacterial adhesion between 30% and 90% depending on the protein nature at the interface.

In vivo Infection: tibial Ti6Al4V prosthetic implants were used for partial knee replacement of the tibia of 12 New Zealand white rabbits and animals were infected by injection with 5×107 MRSA CFU’s. At day 14 mean log10 CFU/g of bone was significantly lower (4.21± 1.49) in the poly(NaSS) grafted versus ungrafted Ti6Al4V implant group (5.97± 0.91) (p<0.05). MRSA adhesion on grafted Ti6Al4V (4.25 ±1.55 CFU/ml) was lower than adhesion to ungrafted implants (5.32 ± 0.46 CFU/ml).

Take Home: Grafting of poly(NaSS) improves bacterial resistance and osseointegration of Ti6Al4V. This presents great potential to the biomedical field.

Reference

Felgueiras HP, Viateau V, M’Ghir AS, Crémieux A-C, Blanquaert D, Migonney V Bioactive Ti6Al4V Alloy Improves Bone Bonding and Prevents Bacterial Infection: THP Application. Trans ORS 2017. 287

Chondrocyte Transdifferentiation: Why, That’s a Chondrocyte of a Different Color

Over the years it has been accepted that hypertrophic cartilage cells in the physis die and that the residual calcified cartilage lattice is the framework upon which perivascular osteoblasts make the primary spongiosa. This decades old model acquired the additional concept of hypertrophic chondrocyte apoptosis wherein the programed death would result in the necessary matrix changes for progression of vascular invasion into the physis leaving behind expired chondrocytes. There has been a dramatic change in this concept.

Transdifferentiation is the change of a cell phenotype by the same cell. An example is a chondrocyte transdifferentiating into an osteoblasts or osteocyte¹. What cell signals could be responsible for this remarkable transition? SHP2 is a cytoplasmic protein tyrosine phosphatase required for most, if not all, receptor tyrosine kinases, cytokine receptors and extracellular matrix protein signaling. This signaling protein was knocked out in COL2a1 or COL10a1 cells with an associated reporter gene expression². This allowed evaluation of phenotypic expression as well cell source in the region of the physis. With SRT eliminated from the cell in in the COL2a1 group the mutants exhibited a cartilage pathophenotype, including dwarfism, cartilaginous exostoses and chondrodysplasia with elongated growth plate cartilage. In the COL10a1 with SRT deletion the mice appeared normal at birth but had reduced bone mineral density at adulthood. µCT analysis demonstrated that the mutants had reduced bone volume. At the cellular level, lineage tracing studies revealed a marked reduction of COL10a1+/OSTERIX+ double positive cells (yellow) in the metaphyseal trabecular bone of the Col10a1 SRT depleted cells compared to COL10a1 controls. Taken together, this suggests that SHP2 deletion in hypertrophic chondrocytes compromises their transdifferentiation into bone-forming osteoblasts.

Sox 9 is a transcription factor associated with chondrogenesis. Sox 9 expressing cells can be found in the periosteum of long bones. In a similar project to the one above it was found that Sox9+ periosteal cells have distinct characteristics of osteochondroprogenitors and can differentiate into chondrocytes, osteoblasts and osteocytes during the different stages of fracture healing³.

For decades it has been taught that endochondral ossification during fracture healing involves the formation of a cartilage template wherein vessels invade the cartilage to create the marrow space and deliver the osteoprogenitor cells that form the new bone while the chondrocytes undergo apoptosis. In a fracture healing study using similar mouse genetic technology it was found that chondrocytes are the primary, if not the sole source of osteoblasts during bone fracture healing, and that the chondrocytes provide the new osteochondral progenitor cells that populate the newly formed periosteum. The investigators also found that there was a direct contribution of these new periosteal cells to fracture callus following a second injury. More investigations will be required to determine the mechanisms that control the fates of the chondrocytes in the physis and in fracture healing.

References

Yang L, Tsang KY, Tang HC, Chan D, Cheah KS. Hypertrophic chondrocytes can become osteoblasts and osteocytes in endochondral bone formation. Proc Natl Acad Sci U S A. 2014 Aug 19;111(33):12097-102. doi: 10.1073/pnas.1302703111. PMID: 25092332
Wang L, Chunlin Zuo C, Moore DC, Wu Q, Huang J, Xie L, Warman ML, Ehrlich MG, Yang W.SHP2 Regulates Endochondral Ossification by Modulating Osteogenic Differentiation of Hypertrophic Chondrocytes. Trans ORS 2017:0075
Bougioukli S, He X, Ortega B, Arevalo E, Lieberman JR, McMahon AP. A Sox9 positive cell population in the periosteum contributes to adult long bone fracture repair. Trans ORS 2017:0188
Marcucio RS, Bahney C, Miclau T III, Yang F, Hu D. The Fate of the Chondrocyte during Bone Fracture Healing: Rebirth of the Stem Cell Compartment. Trans ORS 2017:0189

ACL Injury: It's a Slippery Slope

There are a number of risk factors for anterior cruciate injury. Variations in muscle strength and proprioception have a particular effect on female athletes. Another is increased levels of relaxin. There are conflicting reports regarding the role of osseous morphologic characteristics such as an increased tibial slope in anterior cruciate ligament (ACL) injury. Few studies have analyzed the role of a combination of osseous morphologic characteristics in a matched case control study. One study was designed to determine if there is an association of specific osseous morphologic characteristics and ACL injury in male college American-football players.

Ninety male college football players had a magnetic resonance imaging (MRI) for a knee injury between 2005 and 2014. Those with an ACL injury (ACL-injured group) were matched for age, height, weight, and body mass index to those without an ACL injury (control group). Measured osseous morphologic characteristics included medial and lateral condylar width, medial and lateral plateau width, notch width, bicondylar width, notch width index, and medial and lateral tibial slopes Conditional logistic regression was used to analyze the data. Significance was set at p < 0.05.

The investigators found several ACL rupture risks relative to controls. These were:

a narrower lateral femoral condyle (odds ratio, 0.82 [95% confidence interval (95% CI), 0.68 to 0.97])
increased medial tibial plateau slope (odds ratio, 1.42 [95% CI, 1.09 to 1.85])
increased lateral tibial plateau slope (odds ratio, 1.43 [95% CI, 1.15 to 1.78])

Multivariable analysis revealed that increased lateral tibial slope (odds ratio, 1.32 [95% CI, 1.03 to 1.70]) was the sole independent predictor of ACL injury. This information might help to improve prevention strategies to lower ACL injury.

Reference

Rahnemai-Azar AA, Yaseen Z, van Eck CF, Irrgang JJ, Fu FH, Musahl V. Increased Lateral Tibial Plateau Slope Predisposes Male College Football Players to Anterior Cruciate Ligament Injury. J Bone Joint Surg Am. 2016 Jun 15;98(12):1001-6. doi: 10.2106/JBJS.15.01163. PMID: 27307360

Interesting, No Matter How You Slice It

Digital tomosynthesis (DTS) is a radiologic imaging device that produces a multi-slice image of an object. In DTS the x-ray source and detector are moved together during exposure, rapidly producing sharply focused planes. The DTS system produces 2-dimensional slice images with a high in-plane resolution of 150-300 µm with about one-fifth of the exposure of a computed tomography examination. DTS has been used in fracture healing and the detection of occult fractures. Although approved by the FDA, DTS is not available to most centers in the US due to insurance reimbursement issues.

Investigators used a radiologic portion of the OARSI osteoarthritis scoring system to investigate the reproducibility of osteoarthritis component scores comparing tomosynthesis to plain radiographs, using CT as a standard. The OA components were:

Osteophytes: present to distinct (0 to 3)
Joint space narrowing: normal to distinct (0 to 3)
Subchondral sclerosis: none to present (0 or 1)
Lateral deformity: none to present (0 or 1)
Subchondral cysts none to present (0 or 1)
Central subchondral erosion as a sign of erosive OA: none to present (0 or 1)

6 joints were used on 12 cadaver hands: index and long finger distal and proximal interphalangeal joints (4), thumb metacarpal carpal joint, and the scaphotrapezotrapezoidal joint. There were 10 possible points for each for a minimum and maximum score of 0 to 60.

Additional wrists were evaluated for presence of calcium pyrophosphate disease (CPPD).

Comparing tomosynthesis to conventional radiographs the sensitivity for:

the presence of osteophytes was 95,7% vs 65,2%
joint space narrowing 95,8% vs 52,1%
subchondral sclerosis 61,5% vs 51,3%
lateral deformity 83.3% vs 83,3%
subchondral cysts 45,8% vs 29,2%.
Erosions were not present.

Total hand OARSI score on Tomosynthesis were similar to CT but conventional radiographs had significantly lower mean OARSI. Inter-reader agreement for OARSI scoring was excellent (ICC = 0.99). CPPD calcifications present in CT were also visible with tomosynthesis, but not with conventional radiography.

The future of tomosynthesis for musculoskeletal research remains broad.

Martini K, Becker AS, Guggenberger R, Andreisek G, Frauenfelder T. Value of tomosynthesis for lesion evaluation of small joints in osteoarthritic hands using the OARSI score. Osteoarthritis Cartilage. 2016 Jul;24(7):1167-71. doi: 10.1016/j.joca.2016.01.982. Epub 2016 Jan 30. PMID: 26828358

Cartilage Tissue Engineering: Close, But So Far

In articular cartilage tissue engineering there are five aspects: mechanical adequacy of scaffolds, cell source, cell signaling delivery (product or genes), appropriate anatomic shape, and construct homeostasis.

Most cartilage tissue engineering is aimed at focal defects since resurfacing of an entire osteoarthritic articular surfaces is difficult in the face of cartilage degeneration that occurs in osteoarthritis. The inflammatory environment of OA may inhibit chondrogenesis and induce degradation of both native and engineered cartilage. In this study adult stem cells were engineered in 22 mm hemispherical scaffolds fabricated from3D woven poly(e-caprolactone) (PCL) cartilage constructs capable of tunable and inducible expression of antiinflammatory molecules, specifically IL-1 receptor antagonist (IL-1Ra). The constructs were seeded with human adipose-derived stem cells (ASCs). Doxycycline (dox)-inducible lentiviral vectors containing a green florescent marker protein (eGFP) or IL-1Ra transgenes were immobilized to the PCL construct to transduce ASCs upon seeding. The constructs were cultured in chondrogenic conditions for 28 d.

Cartilage properties and uniform tissue growth occurred and their anatomic shape was maintained throughout culture.

IL-1Ra–expressing constructs produced nearly 1 μg/mL of IL-1Ra upon controlled induction with dox.
IL-1 treatment significantly increased matrix metalloprotease activity in the conditioned media of eGFP-expressing constructs but not in IL-1Ra–expressing constructs.

Advanced textile manufacturing combined with scaffold mediated gene delivery was used to tissue engineer large anatomically shaped cartilage constructs that possess controlled delivery of anticytokine therapy. The clinical step will be to determine the potential to provide mechanical functionality immediately upon implantation attempting to replace a majority, if not the entire joint surface to restore function.

The development this technologies and others like it is constrained by the regulatory environment that governs their deployment. Novel technologies can require a long and formidable process that may overwhelm the financial realities of health care economics. Adequate stratification and identification of patients and an altered approval process must occur before complex therapies can provide the economic benefit to make them viable.

Moutos FT, Glass KA, Compton SA, Ross AK, Gersbach CA, Guilak F, Estes BT. Anatomically shaped tissue-engineered cartilage with tunable and inducible anticytokine delivery for biological joint resurfacing. Proc Natl Acad Sci U S A. 2016 Aug 2;113(31):E4513-22. doi: 10.1073/pnas.1601639113. Epub 2016 Jul 18. PMID: 27432980

Verrier S1, Alini M, Alsberg E, Buchman SR, Kelly D, Laschke MW, Menger MD, Murphy WL, Stegemann JP, Schütz M, Miclau T, Stoddart MJ, Evans C. Tissue engineering and regenerative approaches to improving the healing of large bone defects. Eur Cell Mater. 2016 Jul 19;32:87-110. PMID: 27434267

Obesity vs. Cartilage: Fat Chance

Metabolic syndrome describes the inflammatory effect of the combination of hyperlipidemia, hypertension, diabetes, and obesity. Metabolic syndrome has been found to be important in the inflammatory process in cardiovascular disease and more recently in osteoarthritis. In the cases of osteoarthritis, some authors feel that obesity is the greatest factor, independent of any mechanical effect. Obese patients with knee osteoarthritis can benefit from diets aimed at all four aspects of metabolic syndrome. However, there are metabolic syndrome patients living into their 60’s with no evidence of disease. There is a study that may give some insight as to why this occurs.

In a study a full thickness trochlear cartilage defect was created in 12 week old mice. The filling of that defect was studied at intervals in a normal group of mice and a group on a high fat diet. The investigator’s hypothesis was that a high fat diet would negatively influence intrinsic cartilage repair. It was found that a high fat diet accelerated cartilage repair early after cartilage damage but that it did not cause major systemic or local metabolic and inflammatory changes in this DBA/1 strain of mice. Macrophage phenotype was unaffected.

In their discussion, the authors noted that prior research indicates that musculoskeletal stem cells isolated from DBA/1 mice have been reported to have a better chondrogenic potential than those isolated from C57BL/6 mice. The effect of a high fat diet on musculoskeletal stem cells chondrogenesis could be different in different strains of mice.

Obesity has been reported to negatively influence the clinical results of cartilage repair procedures and the results for this strain of mice was a surprise. This research reflects on the likelihood that not all obese patients should be excluded from cartilage regenerative treatment.

These results invite research into understanding the protective mechanisms of older grossly obese metabolic syndrome patients who have no clinical evidence of knee osteoarthritis. A more practical tool would be to define easily determined phenotypes.

Reference

Wei W, Bastiaansen-Jenniskens YM, Suijkerbuijk M, Kops N, Bos PK, Verhaar JA, Zuurmond AM, Dell’Accio F, van Osch GJ. High fat diet accelerates cartilage repair in DBA/1 mice. J Orthop Res. 2016 May 4. doi: 10.1002/jor.23280. [Epub ahead of print]

Osteoporosis Medications: The Ups and Downs of Osteoporosis Therapy

Following a major osteoporotic fracture the increased risk of a subsequent fracture is highest in the first few years after the index fracture. Bisphosphonates can be divided into non-amino bisphosphonates and amino-bisphosphonates. Both attach to bone surface, Amino-bisphosphonates result in osteoclast apoptosis as they initiate bone absorption. One alternative to bisphosphonates is Odanacatib, an inhibitor of cathepsin-K seen in osteoclast function. This way bone resorption is reduced without hindering bone formation. However there are issues with non-skeletal side effects. A fully humanized monoclonal antibody (denosumab) has been developed and shown to specifically inhibit RANK-L and, therefore, inhibit osteoclast-mediated bone destruction. Wherein bisphosphonates can be taken orally these other agents require injections. With long term use bisphosphonate may lead to bone fatigue and fracture with the recommendation for “drug holidays” for some of these agents.

Teriparatide is a recombinant form of active amino acid sequence (1-34) of parathyroid hormone. The daily subcutaneous dose increases bone formation. This is in contract to persistent high PTH levels that lead to bone absorption. Still in study Romosozumab is a monoclonal antibody that binds to sclerostin, inhibiting osteoblasts leading to increased bone formation. Both are anabolic agents, likely avoiding long term fatigue issues.

Abaloparatide is a parathyroid hormone-related protein (PTHrP) analog now in clinical trials. It appears to associate more strongly with R^G conformation of the parathyroid hormone type 1 receptor. In a recent phase II blinded study, daily subcutaneous injections of placebo, abaloparatide, or open-label teriparatide, 20 μg for 18 months. Non-vertebral fracture was lower with abaloparatide vs placebo. BMD increases were greater with abaloparatide than placebo (all P < .001). Incidence of hypercalcemia was lower with abaloparatide (3.4%) vs teriparatide (6.4%). The relative significance in vertebral fracture reduction was greater than reduction of other fractures.

In a commentary Dr. Cappola points out that for now the oral amino-bsphosphnates have a long term benefit, low risk to benefit ratio and relatively, and lower costs relative to the anabolic agents. Not mentioned is failure of physicians to identify osteoporosis and institution of vitamin D, calcium, and falls assessment in vulnerable persons.

Reference

Miller PD, Hattersley G, Riis BJ, Williams GC, Lau E, Russo LA, Alexandersen P, Zerbini CA, Hu MY, Harris AG, Fitzpatrick LA, Cosman F, Christiansen C. Effect of Abaloparatide vs Placebo on New Vertebral Fractures in Postmenopausal Women With Osteoporosis: A Randomized Clinical Trial. JAMA. 2016 Aug 16;316(7):722-33. doi: 10.1001/jama.2016.11136. PMID: 27533157

Cappola AR, Shoback DM. Osteoporosis Therapy in Postmenopausal Women With High Risk of Fracture. Comment on Effect of Abaloparatide vs Placebo on New Vertebral Fractures in Postmenopausal Women With Osteoporosis: A Randomized Clinical Trial. [JAMA. 2016] JAMA. 2016 Aug 16;316(7):715-6. doi: 10.1001/jama.2016.11032. PMID: 27533154

Aseptic Loosening: a Particle of Truth

With the birth or total joint arthroplasty came the birth of loosening. The initial assumption was that there was infection involved. However, in many cases there was no evidence of infection. Hence the term, aseptic loosening (AL) was born. Biologic response to particles of plastic, metal and “cement” consumed research efforts for decades. That included particle size, composition, and shape. Now the question arises, are these loosenings really aseptic and does it affect outcomes on revision.

In the presence of a confirmed prosthetic infection pre-operative and intraoperative confirmation of infection has a high sensitivity and specificity if the results are matched against Musculoskeletal Infection Society’s criteria. It is noteworthy that 7 to 15 percent of these cases are bacterial culture negative. Loosening that occurs in the absence of these criteria and where there is no biochemical clinical suspicion of infection are considered aseptic. The increased incidence of infection on revision joint replacement relative to primary has been ascribed to tissue environment rather than occult infection in the index procedure. That thinking is now under the proverbial microscope.

A recent review has focused on evidence of microbial involvement in some cases of “aseptic loosening”. Due to biofilm organisms can lie undeterred in the tissues and implant of a loose prosthesis. Some of the strategies to get at these organism include sonication to expose more organisms, immunofluorescent microscopy for specific bacteria, 16 S mRNA PCR, and DNA sequencing. Albeit these and other methods have detected long term infection in loosening that occurs after many years of implantation the joint surgeon is faced with an implant that does not appear to merit the staged procedures that are used in known infected implants.

There are a number of questions relative to the delivery of antibiotics at the time of surgery. Some long term registry studies reflect an apparent difference in rate of “AL” based on method of antibiotic delivery. The picture is complicated by the presence of microbial-associated molecular patterns (MAMPs) wherein some are related to biofilms but others are from sterile surface contaminants and not associated with bacteria. The role of MAMPs in prosthetic loosening results from the cascade of events following stimulation of toll like receptors or tumor necrosis factor mediated immune responses.

The path to reduction of AL will be paved in registry studies looking at occult infection prophylaxis along with improved detection of infection agents during the course of revision. It will be biofilm at 11.

Knee Osteoarthritis: A Weighty Subject

Knee osteoarthritis therapies have relied heavily on pharmacologic interventions. All have potential complications and some have controversial benefit. None extend the life of the joint. It had been recognized that that people cope differently with their knee osteoarthritis. These persons have been referred to as copers. A number of studies have shown weight loss as low as 20 pounds can have a considerable effect on pain improvement. Also, what is in a diet can have a significant effect on metabolic syndrome (obesity, diabetes, hypertension, and dyslipidemia).

In the OA LIFE study investigators studied the effects of pain coping skills training (education on the role of pain coping + training in relaxation, meditation, and activity pacing = PCST) and a lifestyle behavioral weight management program (BWM) based on LEARN (lifestyle, exercise, attitude, relationship, nutrition) on inflammatory markers and biomarker associations with pain and function. The program lasted 24 weeks. Change is biomarkers, weight, pain, and disability were measured along with BMI at the beginning and end of the program. There were 4 groups, PCST + BWM, PCST alone, BWM alone, or standard care (SC)

It took both PCST + BWM to get significant reductions in the inflammatory markers hsCRP (P = 0.0014), IL-6 (P = 0.0075), and leptin (P = 0.0001). Reductions in leptin and IL-6 were significantly correlated with reductions in weight, BMI and Western Ontario and McMaster Universities Arthritis Index (WOMAC) pain. Reductions in IL-6 were correlated with improvements in WOMAC and Arthritis Impact Measurement Scales (AIMS) physical function. By mediation analyses, weight loss was responsible for 54% of the change in IL-6 and all of the change in leptin. There was insufficient data for cytokines but pain remained improve at one year and there was some weight gained at one year.

These programs require fairly intense involvement of it attendees. It is hard to say if the improvement in pain and function will motivate the individuals to maintain their behavioral change. Should that occur this could become a disease modifying intervention that is safe and free of complications.

Reference

Huebner JL, Landerman LR, Somers TJ, Keefe FJ, Guilak F, Blumenthal JA, Caldwell DS, Kraus VB. Exploratory secondary analyses of a cognitive-behavioral intervention for knee osteoarthritis demonstrate reduction in biomarkers of adipocyte inflammation. Osteoarthritis Cartilage. 2016 Sep;24(9):1528-34. doi: 10.1016/j.joca.2016.04.002. Epub 2016 Apr 16.

Somers TJ, Blumenthal JA, Guilak F, Kraus VB, Schmitt DO, Babyak MA, Craighead LW, Caldwell DS, Rice JR, McKee DC, Shelby RA, Campbell LC, Pells JJ, Sims EL, Queen R, Carson JW, Connelly M, Dixon KE, Lacaille LJ, Huebner JL, Rejeski WJ, Keefe FJ. Pain coping skills training and lifestyle behavioral weight management in patients with knee osteoarthritis: a randomized controlled study. Pain. 2012 Jun;153(6):1199-209. doi: 10.1016/j.pain.2012.02.023. Epub 2012 Apr 12. PMID: 22503223

Nanotechnology: That’s About the Size of It

Definitions

nanoparticle: a particle that is 100 nanometers or less in its dimensions. As a point of contrast are Type I-III collagen molecules which are just over 1000 amino acids long and measure 300 X 1.5 nanometers.

endocytosis: cell wall ingestion of a protein or other small molecule, often the result of a specific receptor. This may include something as large as a nanoparticle.

Nanoparticle Imaging

In osteosarcoma a mutated p15 receptor is a marker for metastatic potential. A ligand for the mutated receptor attached to a nanoparticle loaded with on Superparamagnetic iron nanoparticles can be used to on MRI detect tumors likely to metastasize of metastases. Nanoparticles (hollow gold nanoshells, gold/gold sulfide nanoparticles, gold nanocages, carbon and titanium nanotubes, photothermal-based nanobubbles, polymeric nanoparticles, and copper-based nanocrystals) that can be detected by a surgeon using laser light to determine if tumor remains at the edges.

Nanoparticle treatment

Passive targeting relies on the increased permeability of tumor vessels to nanoparticles. Also decreased lymphatics in tumors resulting in a higher concentration of drug. However, normal tissue could be affected depending on the tag and the permeability of the tissue.

Active targeting relies on a receptor responding to a specific ligand placed on the nanoparticle thus targeting a specific cell. The drug loaded nanoparticle with its ligand triggers incorporation into the cell by endocytosis.

There are three main categories of lipid-based nanocarriers: solid lipid nanoparticles, nanostructured lipid carriers, and lipid–drug conjugate nanoparticles. Drug resistance is sometimes caused by the cell being able to reject the single molecule. By attachment on or within the lipid based nanoparticle delivery is more efficient and stable. Nanostructured lipid carriers can be loaded with silencing RNA to overcome P-glycoprotein which can be responsible for drug resistance. Lipid nanocarriers have also been used to protect exposure sensitive siRNA used for intracellular interference with fusion proteins associated with Ewings sarcoma. The use of lipid nanoparticles with methotrexate and newer antifolates allows for better absorption of lower oral doses of the agent and for decreased intracellular resistance. There is a wide range of receptors useful in targeting specific cancers in situ and in metastases.

Depending on specific types of osteosarcoma, smaller and larger hydroxyapatite nanoparticles have proved effective in making these cells apoptotic. Other nanoparticles used in tumor therapy include mesoporous silica nanoparticles and selenium-coated hydroxyapatite particles. These have also been explored in producing tumor cell apoptosis. Likewise, selinium nanoparticles have also been used on titanium and poly-L-lactic acid implants to inhibit tumor cells and to encourage normal bone formation.

Reference

Savvidou OD, Bolia IK, Chloros GD, Goumenos SD, Sakellariou VI, Galanis EC, Papagelopoulos PJ. Applied Nanotechnology and Nanoscience in Orthopedic Oncology. Orthopedics. 2016 Sep 1;39(5):280-6. doi: 10.3928/01477447-20160823-03.

A Cilia Excuse for an Osteocyte

Mechanical stimulation is known to stimulate bone formation. The mechanism of cellular response is incompletely understood. Primary cilia are single immotile organelles present on nearly every cell type. Cilia been implicated as mechonsensors in osteocytes. Disruption of a key cilia associated protein (IFT88) reduces mechanical induced bone formation due to paracrine signals that promote mesenchymal cell osteogenic differentiation. An antihypertensive, fenoldopam, increases osteocyte primary cilia length enhancing mechanosensitivity.

Osteocyte conditioned media effect on osteoblasts

MLO-Y4 osteocytes were treated with fenoldopam (lengthens cilia to enhance mechanosensing) for 16 hours, or tubastatin (stiffens cilia to impair mechanosensing) for 3 hours prior to mechanical stimulation. Osteocytes were mechanically stimulated by oscillatory fluid flow on a rocker for 12 hours (no flow for controls). The conditioned media was used to treat MC3T3 osteoblasts for 24 hours. Lysed osteoblasts had real time RT-PCR to quantify Osteopontin mRNA expression against GAPDH expression. Pro-osteogenic paracrine signaling to osteoblasts was impaired with tubastatin and augmented with fenoldopam.

In another experiment osteocytes were transfected by lipid-mediated transfection with IFT88 siRNA to disrupt ciliogenesis, (Scramble siRNA negative control). (Scramble siRNA negative control). The conditioned media was used to culture osteoblasts for 24 hours. Flow-induced paracrine signaling to osteoblasts was abrogated with IFT88 siRNA.

In vivo loading experiment

Skeletally mature, 16 week old, C57BL/6 mice were injected subcutaneously with 20 mg/kg fenoldopam, daily for 7 days. On days 5-7, mice were also subjected to compressive ulnar loading. Contralateral limbs served as non-loaded controls. Calcein and alizarin were injected and standard dynamic histomorphometric analysis was performed to assess bone adaptation. Mice injected with fenoldopam and subjected to compressive ulnar load exhibited no change in the amount of mineralizing surface but had significantly increased mineral apposition rate and bone formation rate compared to vehicle controls.

Given the effects of the manipulation of cilia this work shows that cilia have a role in mechanosensitive function independent of other mechanisms such as integrins. These studies showed that targeting primary cilia structure is a robust strategy for enhancing bone formation.

References

Milos Spasic, Michael P. Duffy, Christopher R. Jacobs. Enhanced bone formation in vivo by pharmacologically targeted primary cilia-mediated mechanotransduction ORS 2017 132

Can Biomarkers Become Phenotypes?

Whereas “genotype” is the genetic makeup of an organism, the phenotype is how genetic and environmental influences come together to create an organism’s physical appearance and behavior. Many researchers think of a biomarker as a genetic or biochemical indicator of disease or change in disease progression. Phenotypes can be used to select physical characteristics of the individual to predict more rapid disease progression or responsiveness to treatment. The use of the terms biomarkers and phenotype are noted in a recent study on osteoarthritis patients.

Using data from a nested case–control study from the Osteoarthritis Initiative (OAI) participants had at least 1 knee with a baseline Kellgren/Lawrence (K/L) grade of 1–3 on a standardized fixed-flexion radiograph. Baseline and 24-month knee radiographs were used to determine medial joint space width (JSW). These were compared to knee MRIs and clinical data. The MRI OA Knee Score (MOAKS) system focusing on changes in cartilage, osteophytes, meniscus, bone marrow lesions, Hoffa-synovitis, and effusion-synovitis. At 48 months MRI, radiographs, and pain scores were compared.

There were four knee groups:

both radiographic and pain progression

radiographic progression but not pain progression

pain progression but not radiographic progression

neither radiographic progression nor pain progression

Knees with both radiographic and pain progression were compared to all other knees (controls).

OAI pulse sequencing protocols were used for semiquantitative assessment based on MOAKS. The features included:

cartilage thickness and surface area changes to mark number of regions affected

osteophytes based on number and worsening of grade

meniscus extrusion grade and worsening in four locations

bone marrow lesions based on size and number with score based on no change, worsening in one region, or worsening in two or more regions

Hoffa fat changes were used as a surrogate for synovitis

Synovitis and effusion were based together on maximal capsular distention.

Over the four year period the number of cartilage surface regions affected or 5 6 or more correlated highly with progression. Higher grade meniscal extrusion correlated to progression but not as great as articular cartilage change. The number of areas of bone marrow lesions correlated highly to progression. More advanced Hoffa fat changes correlated to progression more so than capsular distention. Changes in the degree of changes on MRI at 24 months also had specific correlations.

Albeit the physical imaging categories are related to known biochemical changes the use of the term biomarkers to the categories is interesting in that some would consider the MR images to reflect phenotypes. One of the referenced authors (VK) notes that “I think of ‘progressor’ as the phenotype that is based on the current ‘gold’ standard biomarker of progression, i.e. the radiograph. By identifying baseline MR biomarkers that predict progression and/or change concurrently with progression based on radiograph (and in this case also pain progression) you are substituting one biomarker for another. Hopefully the subtitution is with a more sensitive biomarker (which we think is the case for many of these MR biomarkers). So I think of biomarkers as defining a phenotype and hopefully being able to distinguish and better define phenotypes with better biomarkers.”

Reference

Collins JE, Losina E, Nevitt MC, Roemer FW, Guermazi A, Lynch JA, Katz JN, Kwoh CK, Kraus VB, Hunter DJ. Semiquantitative Imaging Biomarkers of Knee Osteoarthritis Progression. Arthritis & Rheumatology Vol. 68, No. 10, October 2016, pp 2422–2431

Biofilm Formation: Adds a Whole New Meaning to Communication?

Biofilms are surface-associated bacterial communities embedded in an extracellular matrix that makes these cells more resistant to antibiotics than free-swimming planktonic cells. Biofilms are a major problem in post-operative infections, chronic infections, and in some cases of “aseptic” loosening. The cells’ elaboration of polymeric substances that form the matrix is associated with the biofilm community architecture and the spatial orientation of the cells inside the film. However, the molecular mechanism that produces the matrix, the roles of the individual matrix components, and the role the cells themselves play in biofilm architecture have not been clear until recently.

In Vibrio cholerae biofilms, the extracellular polysaccharide (Vps), matrix protein rugosity and biofilm structure modulator A (RbmA) binds mother-daughter cells together at their poles. The biofilm associated protein 1 (Bap1) adheres cells to the surface, and RbmC/Bap1proteins form an envelope around cell subclusters in conjunction with Vps. The genes for these components are controlled by the levels of the intracellular small molecule cyclic-diguanylate (c-di-GMP) and by the cell-cell communication process called quorum sensing.

Investigators studied live progression of cell orientation in V. cholerae biofilm growth using a customized spinning disk confocal microscope coupled with the photostable fluorescent protein mKO introduced into V. cholerae cells, allowing their tracking. The imaging procedure used minimum laser exposure to reduce phototoxicity to the cells and also limited photobleaching of the chromophores. Using rugose variant (denoted Rg) of V. cholerae that forms robust biofilms due to increased production of c-di-GMP, the scientists followed the development of isolated biofilm clusters from single founder cells to 10,000 cells.

Clusters initially expand radially in a branched pattern primarily in two dimensions and then transition into dense 3D domes. Cells in the central core aligned vertically side-by-side but cells at the periphery aligned radially and remained horizontal relative to the surface. This microstructure lends a mechanical advantage to early biofilm orientation. Mutants that lacked RbmA, RbmC, and/or Bap1 revealed the crucial role of cell orientation in making resilient biofilms.

Clinically, this is the first step-by-step picture of biofilm formation. The clinical relevance is a better understanding of biofilm formation mechanisms as they may impact early intervention.

Reference

Yan J, Sharo AG, Stone HA, Wingreen NS, Bassler BL. Vibrio cholerae biofilm growth program and architecture revealed by single-cell live imaging. Proc Natl Acad Sci U S A. 2016 Sep 6;113(36):E5337-43. doi: 10.1073/pnas.1611494113. Epub 2016 Aug 23. PMID: 27555592

Surface Infection Control: A Not So Evil Cover Up

The rising cost of antibiotic resistant infections has produced a plethora of non-antibiotic infection prevention strategies. Silver is known to have antimicrobial properties. Silver impregnated bandages have been used in active open wound infections. Copper has similar biocidal properties and has been found to be superior to silver on nanofilm surfaces¹. Copper oxide can be impregnated into composites that include hard surfaces and linens.

Multidrug resistant organisms (MDROs) and antibiotic resistant Clostridium difficile are major problems for inpatient acute care units. An acute care community hospital had one wing that replaced a 1970s-era clinical wing. The new wing had copper-impregnated composite hard surfaces and linens². Both the surface and linens were beige in color and distinct from the typical white hard surfaces and linens. Where clinical divisions had patients in both the new and old wings investigators assessed hospital acquired infections (HAIs) due to (MDROs) and Clostridium difficile. Intervention practices were the same in both units.

There was a baseline period of no copper surface or sheets with a total of 46,391 patient-days. That was compared to HAIs that occurred in the copper-containing new hospital wing (14,479 patient-days; 72 beds) and the unmodified hospital wing (19,177 patient-days). The new wing had 78% (P = .023) fewer HAIs due to MDROs or C difficile, 83% (P = .048) fewer cases of C difficile infection, and 68% (P = .252) fewer infections due to MDROs relative to the baseline period. No changes in rates of HAI were observed in the unmodified hospital wing.

However, there were more medical patients with comorbidities in the older unit compared to surgical patients who had fewer co-morbidities. By virtue of the design, the absence of an infection difference in the medical population between baseline and comparison to last months of the study imply a steady state of infection. Of note, for surgical patients there was a difference in the new wing with more surgical patients compared to both base line and the final comparator period.

Similar to no-touch interventions such as ultraviolet light the authors conclude that copper-impregnated composite hard surfaces and linens may be useful technologies to prevent HAIs in acute care hospital settings. Supplementary studies such as repeated surface cultures and cultures of post use copper impregnated linens compared to normal sheets may further define actual contamination. With additional studies the cost effectiveness of copper oxide impregnation can also be studied.

References

Codiţă I, Caplan DM, Drăgulescu EC, Lixandru BE, Coldea IL, Dragomirescu CC, Surdu-Bob C, Bădulescu M. Antimicrobial activity of copper and silver nanofilms on nosocomial bacterial species. Roum Arch Microbiol Immunol. 2010 Oct-Dec;69(4):204-12.
Sifri CD, Burke GH, Enfield KB. Reduced health care-associated infections in an acute care community hospital using a combination of self-disinfecting copper-impregnated composite hard surfaces and linens. Am J Infect Control. 2016 Sep 28. pii: S0196-6553(16)30696-4. doi: 10.1016/j.ajic.2016.07.007. [Epub ahead of print] PMID: 27692785

Greater Strength: Muscling in on Knee Osteoarthritis Progression

High knee extensor strength may be important to protect against the development of knee osteoarthritis (KOA) in populations at elevated risk. It is well recognized that patients with degenerative meniscal pathology are at risk for earlier development of KOA. Investigators hypothesized that individuals with asymptomatic meniscal pathology seen on MRI and who had high knee extensor muscle strength would have a decreased risk of developing radiographic or symptomatic knee compared to weaker individuals.

Two Multicenter Osteoarthritis Study populations were used to prospectively evaluation the effect of knee strength in KOA progression. 373 knees in 373 participants were found at baseline visit to have medial meniscal pathology on MRI but no radiographic knee OA. 531 knees in 531 participants had knee OA based on combined radiographic knee OA and frequent knee symptoms. Isokinetic knee extensor strength was measured at baseline for all 904 participants who were then followed for followed 84 months. Separate binomial regression analyses with robust SEs adjusted for age, history of knee surgery, physical activity level, and clinic site were conducted for men and women.

High knee extensor strength was normalized by allometric scaling. The adjusted strength was associated with a reduced risk of the development of radiographic knee OA in initially asymptomatic women (relative risk [RR] 0.52, 95% confidence interval [95% CI] 0.29-0.94) but not in men (RR 0.56, 95% CI 0.27-1.16). Initial high knee extensor strength did not protect against the KOA progression of symptomatic knee OA in either women or men.

The results only partly confirm the hypothesis that high knee extensor muscle strength protects against later development of knee OA. For existing symptomatic KOA initial muscle strength did not correlate to progression. Pain will generally reduce muscle strength by the inhibition of activities that would naturally maintain strength absent a dedicated specific exercise program. This investigation did not answer the question, “if a person maintains their increased strength, will there be less progression of KOA?” Film at 11.

Reference

Thorlund JB, Felson DT, Segal NA, Nevitt MC, Niu J, Neogi T, Lewis CE, Guermazi A, Roemer F, Englund M. Effect of Knee Extensor Strength on Incident Radiographic and Symptomatic Knee Osteoarthritis in Individuals With Meniscal Pathology: Data From the Multicenter Osteoarthritis Study. Arthritis Care Res (Hoboken). 2016 Nov;68(11):1640-1646. doi: 10.1002/acr.22889. Epub 2016 Oct 1. PMID: 26991698

The High Cost of Infection: A Silver Lining

Silver impregnated dressings have been used for wounds for six millennia. Halsted used a colloidal silver decreasing for surgical patients in the early 20^th century. Fears of argyria has set aside these dressings in large open wound care. Silver dressings were the most important antibacterial management prior to the development of antibiotics for both prevention and treatment¹.

Post-operative infection in prosthetic joint replacement surgery is disastrous. To try to prevent post-operative infections a number of non-antibiotic strategies including ultraviolet light and the use of copper surfaces and copper impregnated linins have been studied recently.

A recent study was designed to determine whether the use of a silver nylon dressings would reduce the incidence of superficial and deep PJI following primary THA and TKA². The case-control study involved 309 patients who received a silver nylon dressing immediately following surgery. These were compared to 527 preceding patients who did not have the dressing. The study took into account patient age at time of surgery, the American Society of Anesthesiologists (ASA) Physical Status classification, immunocompromised status and the use of silver dressing.

There were no deep PJIs in the silver nylon dressing group and only 12 superficial infections (3.9%). Fourteen patients (2.7%) in the control group developed deep infection and there were 35 superficial infections 35 (6.7%). The odds of any infection was significantly less in the study group compared to the control group (OR, 0.337; 95% CI, 0.224-0.506; P < 0.0001). The use of silver dressing in immunocompromised subjects was associated with lower odds of any infection (OR, 0.469; 95% CI, 0.460-0.479; P < 0.0001).

Given the financial impact of prosthetic joint infections the authors believe that a formal cost benefit analysis could be the subject of future investigation.

Reference

Alexander JW. History of the medical use of silver. Surg Infect (Larchmt). 2009 Jun;10(3):289-92. doi: 10.1089/sur.2008.9941. Review. PMID: 19566416
Tisosky AJ, Iyoha-Bello O, Quimbayo G, Demosthenes NJ, Coreanu T,Novack V, Abdeen A. Use of a Silver Nylon Dressing Following Total Hip and Knee Arthroplasty Decreases Postoperative Infection Rate. Trans Orthopaedic Research Society 2017 1064