ORS Basic Science Tips

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.

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

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

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

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.

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

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

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

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

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

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

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

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