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” is now shared in ORS Connect, the society’s e-newsletter. Did you miss one? Read past tips here:
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.
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.
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?
- 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 (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.
- 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
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.
- 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
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.
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.
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.
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.
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
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.
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.
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
- 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
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.
- 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
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.
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
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.
- 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
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.
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
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
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
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.
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]
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 RG 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.
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
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 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.
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
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.
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.
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.
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.
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.
Milos Spasic, Michael P. Duffy, Christopher R. Jacobs. Enhanced bone formation in vivo by pharmacologically targeted primary cilia-mediated mechanotransduction ORS 2017 132
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.”
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
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.
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
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.
- 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
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.
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
Silver impregnated dressings have been used for wounds for six millennia. Halsted used a colloidal silver decreasing for surgical patients in the early 20th 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.
- 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