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Animal models of osteoarthritis (OA) are essential tools for investigating the development of the disease on a more rapid timeline than human OA. Mice are particularly useful due to the plethora of genetically modified or inbred mouse strains available. The majority of available mouse models of OA use a joint injury or other acute insult to initiate joint degeneration, representing post-traumatic osteoarthritis (PTOA). However, no consensus exists on which injury methods are most translatable to human OA. Currently, surgical injury methods are most commonly used for studies of OA in mice; however, these methods may have confounding effects due to the surgical/invasive injury procedure itself, rather than the targeted joint injury. Non-invasive injury methods avoid this complication by mechanically inducing a joint injury externally, without breaking the skin or disrupting the joint. In this regard, non-invasive injury models may be crucial for investigating early adaptive processes initiated at the time of injury, and may be more representative of human OA in which injury is induced mechanically. A small number of non-invasive mouse models of PTOA have been described within the last few years, including intra-articular fracture of tibial subchondral bone, cyclic tibial compression loading of articular cartilage, and anterior cruciate ligament (ACL) rupture via tibial compression overload. This review describes the methods used to induce joint injury in each of these non-invasive models, and presents the findings of studies utilizing these models. Altogether, these non-invasive mouse models represent a unique and important spectrum of animal models for studying different aspects of PTOA.

In our recent studies, we have demonstrated that monolayer cultures of bovine aortic endothelial (BAE) cells that do not express type I collagen also fail to express and synthesize decorin, a small chondroitin/dermatan sulfate proteoglycan that interacts with type I collagen and regulates collagen fibrillogenesis in vitro. However, BAE cells exhibiting a spontaneous sprouting phenotype and a predisposition toward the formation of cords and tube-like structures (an in vitro model for angiogenesis) initiate the synthesis of type I collagen during their morphological transition from a polygonal monolayer to an angiogenic phenotype. In the present study, we examined whether BAE cells also initiate the synthesis of the proteoglycan decorin during this morphological transition. We show by Northern blot analysis and by immunochemical methods that BAE cell cultures containing sprouting cells and cords, but not monolayer cultures of these cells, express and synthesize decorin (M(r) approximately 100,000). We also show that type I collagen expression by BAE cell cultures is initiated concomitantly. However, the localization of decorin and type I collagen in cord and tube-forming BAE cell cultures is not completely identical. Type I collagen is detected only in sprouting BAE cells and in endothelial cords, whereas decorin is also apparent in BAE cells surrounding the cords and tubes. Our results indicate that the synthesis of decorin as well as type I collagen is associated with endothelial cord and tube formation in vitro.

Osteoarthritis (OA) has a considerable hereditary component and is considered to be a polygenic disease. Data derived from genetic analyses and genome-wide screening of individuals with this disease have revealed a surprising trend: genes associated with OA tend to be related to the process of synovial joint development. Mutations in these genes might directly cause OA. In addition, they could also determine the age at which OA becomes apparent, the joint sites involved, the severity of the disease and how rapidly it progresses. In this Review, I propose that genetic mutations associated with OA can be placed on a continuum. Early-onset OA is caused by mutations in matrix molecules often associated with chondrodysplasias, whereas less destructive structural abnormalities or mutations confer increased susceptibility to injury or malalignment that can result in middle-age onset. Finally, mutations in molecules that regulate subtle aspects of joint development and structure lead to late-onset OA. In this Review, I discuss the genetics of OA in general, but focus on the potential effect of genetic mutations associated with OA on joint structure, the role of joint structure in the development of OA--using hip abnormalities as a model--and how understanding the etiology of the disease could influence treatment. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) biomarkers that can measure and predict the full spectrum of disease progression and outcomes are needed, but few, if any, such biomarkers have been validated for this purpose. The Osteoarthritis Research Society International (OARSI) has organized an OA Biomarkers Global Initiative. As a part of this Initiative, three workshops have been planned to occur over the next 4 years to focus on identifying and removing obstacles to progress in the field and planning the way forward. In addition to OARSI, the National Institute of Arthritis, Musculoskeletal, and Skin Disease, the Arthritis Foundation, the Orthopaedic Research Society, and the American Orthopaedic Sports Medicine Society cosponsored the first meeting April 23-24, 2009. Organizers brought together thought and research leaders in the field, young investigators, biomarkers researchers with insights from other fields, clinical investigators with a responsibility for OA sample and resource management, funding agencies, and commercial entities with an interest in the commercial propagation as well as the application of markers in OA. [ABSTRACT FROM AUTHOR]

Objective: To provide a more complete picture of the effect of interleukin-1 beta (IL-1beta) on adult human articular chondrocyte gene expression, in contrast to the candidate gene approach.Design: Chondrocytes from human knee cartilage were cultured in medium containing IL-1beta. Changes in gene expression were analyzed by microarray and reverse transcriptase-polymerase chain reaction analysis. The ability of transforming growth factor beta-1 (TGF-beta1), fibroblast growth factor (FGF)-18, and bone morphogenetic protein 2 (BMP-2) to alter the effects of IL-1beta was analyzed. Computational analysis of the promoter regions of differentially expressed genes for transcription factor binding motifs was performed.Results: IL-1beta-treated human chondrocytes showed significant increases in the expression of granulocyte colony stimulating factor-3, endothelial leukocyte adhesion molecule 1 and leukemia inhibitory factor as well as for a large group of chemokines that include CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, CCL2, CCL3, CCL4, CCL5, CCL8, CCL20, CCL3L1, CX3CL1 and the cytokine IL-6. As expected, the mRNA for matrix metalloproteinase (MMP)-13 and BMP-2 also increased while mRNA for the matrix genes COL2A1 and aggrecan was down-regulated. A subset of chemokines increased rapidly at very low levels of IL-1beta. The phenotype induced by IL-1beta was partially reversed by TGF-beta1, but not by BMP-2. In the presence of IL-1beta, FGF-18 increased expression of ADAMTS-4, aggrecan, BMP-2, COL2A1, CCL3, CCL4, CCL20, CXCL1, CXCL3, CXCL6, IL-1beta, IL-6, and IL-8 and decreased ADAMTS-5, MMP-13, CCL2, and CCL8. Computational analysis revealed a high likelihood that the most up-regulated chemokines are regulated by the transcription factors myocyte enhancer binding factor-3 (MEF-3), CCAAT/enhancer binding protein (C/EBP) and nuclear factor-kappa B (NF-kappaB).Conclusion: IL-1beta has a diverse effect on gene expression profile in human chondrocytes affecting matrix genes as well as chemokines and cytokines. TGF-beta1 has the ability to antagonize some of the phenotype induced by IL-1beta. [ABSTRACT FROM AUTHOR]

Objective: Oxidative stress occurs when the metabolic balance of a cell is disrupted through exposure to excess pro-oxidant. Whilst it is known that unregulated production or exposure to exogenous sources of pro-oxidants induces chondrocyte cell death and degrades matrix components in vitro, relatively little is known of the effects of pro-oxidants on articular cartilage in situ. The objective of this study was to determine if a single exposure to the pro-oxidant hydrogen peroxide (H(2)O(2)) induces a degenerative phenotype.Methods: Articular cartilage explants were obtained from skeletally mature bovine steers and exposed to a single dose of hydrogen peroxide (0.1-1.0 mM) and cultured for up to 21 days. Cell death, and sulfated glycosaminoglycan loss into the medium and gene expression were quantitatively determined. Adoption of an abnormal chondrocyte phenotype was analyzed through the expression of 3B3(-), nitrotyrosine and procollagen type IIA epitopes in cartilage explants.Results: Cell death occurred primarily at the surface zone of cartilage in a dose-dependent manner in H(2)O(2) treated explants, and supplementation of standard serum-free medium with insulin-selenium-transferrin significantly reduced cell death (>fourfold). Nitric oxide synthase-2 gene expression and proteoglycan loss increased in oxidant treated explants in a concentration-dependent manner. Antibody labeling to 3B3(-), procollagen type IIA and nitrotyrosine was present in all treated explants but absent in untreated explants.Conclusions: This study demonstrates that a single exposure to high levels of pro-oxidant causes the expression of genes and antibody epitopes that are associated with early degenerative changes observed in experimental osteoarthritis. [ABSTRACT FROM AUTHOR]

Background: Chemotherapy is associated with decreased quality of life (QOL), fatigue, depression, and weight gain in patients with breast cancer. Weight gain is associated with poorer prognosis. Yoga improves QOL, fatigue, and mood in women with breast cancer but its effect on treatment-related weight gain has not been studied. The aim of this trial was to determine the feasibility of personalized yoga therapy in women receiving treatment for early-stage or locally advanced breast cancer and assess its impact on weight gain., Methods: Thirty women were randomized 1:1 to receive yoga therapy by a certified yoga therapist during treatment or a control group. Participants in the yoga arm were asked to complete three 30 minute yoga sessions weekly (which included movement, breath work, mindfulness, and relaxation) throughout adjuvant or neoadjuvant chemotherapy (N = 29) or endocrine (N = 1); the control arm received breast cancer treatment without yoga. For comparability between participants randomized to yoga therapy, the single patient treated with endocrine therapy was excluded from the analysis. Primary outcomes were feasibility and weight change. Additional outcomes were mood, fatigue, QOL, serum tumor necrosis factor-alpha (TNF-alpha), and C-reactive protein (CRP) as immune mediator biomarkers., Results: Mean age was 51.6 years, 75.9% were white and 24.1% were people of color, reflecting the cancer center population. 80% had stage II-III disease. Enrollment was completed in 9 months. Compliance was lower than predicted; however, participants participated in on average 1.7 yoga sessions/week for a mean 15.6 weeks duration. There were no adverse events. Control arm participants gained on average 2.63% body weight during treatment while yoga participants lost 0.14% body weight (weight change = -0.36 in yoga arm vs. 2.89 in standard of care arm, Wilcoxon rank sum test P  = .024). Control participants reported increased fatigue and decreased QOL, while yoga participants reported no change in QOL. No significant change in TNF-alpha or CRP was noted in either arm., Conclusion: This feasibility study suggests that personalized yoga therapy is beneficial for QOL and weight maintenance among women undergoing chemotherapy for early-stage or locally advanced breast cancer. Weight maintenance associated with yoga therapy may be of clinical significance in this population given the poorer prognosis associated with weight gain in breast cancer survivors., Trial Registration: NIH Clinicaltrials.gov #NCT03262831; August 25, 2017. https://clinicaltrials.gov/ct2/show/NCT03262831.

We provide a detailed account of the origin and establishment of the Osteoarthritis Research Society International (OARSI) and celebrate its history from inception to the current day. We discuss the mission, vision and strategic objectives of OARSI and how these have developed and evolved over the last 3 decades. We celebrate the achievements of the society as we approach its 30th birthday, honor the entire presidential line and respectfully pay tribute to the past presidents who are no longer with us. We reflect on the strong foundations of our society, OARSI's efforts to disseminate understanding of the health, disability and economic burdens of osteoarthritis (OA) to policymakers, and the exciting initiatives to make the society inclusive and international. We thank our corporate and industrial sponsors, who have supported us over many years, without whom our annual congresses would not have been possible. We celebrate our longstanding strategic partnership with our publisher, Elsevier, and the successful launch of our new journal Osteoarthritis and Cartilage Open, the most significant new development in our dissemination toolbox. For the first time in the history of the organization, our annual congress was cancelled in April 2020 and the 2021 meeting will be virtual. Despite the numerous challenges posed by the ongoing COVID-19 pandemic and the need to adapt quickly to a rapidly changing landscape, we must remain optimistic about the future. We will take advantage of new exciting opportunities to advance our mission and vision to enhance the quality of life of persons with OA., Competing Interests: The authors have no conflicts to declare in connection with this article., (© 2021 The Author(s).)

Objective: To query the transcript-level changes in the medial and lateral tibial plateau cartilage in tandem with obesity in patients with end-stage osteoarthritis (OA)., Design: Cartilage was obtained from 23 patients (20 obese [body mass index > 30 kg/m 2 ], 3 overweight [body mass index < 30 kg/m 2 ]) at the time of total knee replacement. Cartilage integrity was assessed using Outerbridge scale, while radiographic changes were scored on preoperative X-rays using Kellgren-Lawrence (K-L) classification. RNA was probed for differentially expressed transcripts between medial and lateral compartments using Affymetrix Gene 2.0 ST Array and validated via real-time polymerase chain reaction. Gene ontology and pathway analyses were also queried., Results: Scoring of cartilage integrity by the Outerbridge scale indicated that the medial and lateral compartments were similar, while scoring by the K-L classification indicated that the medial compartment was more severely damaged than the lateral compartment. We observed a distinct transcript profile with >50% of transcripts unique between medial and lateral compartments. MMP13 and COL2A1 were more highly expressed in medial versus lateral compartment. Polymerase chain reaction confirmed expression of 4 differentially expressed transcripts. Numerous transcripts, biological processes, and pathways were significantly different between overweight and obese patients with a differential response of obesity on medial and lateral compartments., Conclusions: Our findings support molecular differences between medial and lateral compartments reflective of the greater severity of OA in the medial compartment. The K-L system better reflected the molecular results than did the Outerbridge. Moreover, the molecular effect of obesity was different between the medial and lateral compartments of the same knee plausibly reflecting the molecular effects of differential biomechanical loading.

Background: While knees with meniscal tears are associated with a heightened risk of developing osteoarthritis (OA), it is difficult to predict which patients are at the greatest risk for OA. Gene signatures in menisci that are resected during arthroscopic partial meniscectomy (APM) may provide insight into the risk of OA progression., Hypothesis: Meniscal gene signatures at the time of APM will predict radiographic OA progression., Study Design: Case series; Level of evidence, 4., Methods: Meniscal fragments were collected from 38 patients without OA during clinically indicated APM of the medial meniscus. The expression of 28 candidate genes with known roles in cartilage homeostasis, OA, extracellular matrix degradation, and obesity was assessed by quantitative real-time polymerase chain reaction. Weightbearing radiographs obtained before surgery and at final follow-up were graded by a musculoskeletal radiologist using the Kellgren-Lawrence classification of OA. The association of meniscal gene expression at baseline with the progression of radiographic OA was determined., Results: Gene expression and baseline and follow-up radiographic data were available from 31 patients (81.6%) at a mean follow-up of 6.2 ± 1.3 years. Patients without OA progression had significantly higher expression of 7 genes: MMP9 (5.1-fold; P = .002), IL8 (2.9-fold; P = .016), CCL3 (3.7-fold; P = .032), CCL3L1 (4.5-fold; P = .008), CXCL6 (6.2-fold; P = .010), LEP (5.2-fold; P = .004), and RETN (46-fold; P = .008)., Conclusion: Gene expression in the meniscus at the time of APM may be associated with the risk for progression of OA after surgery. Elevated expression of the aforementioned genes may reflect a chondroprotective response. Stratifying the risk for OA progression after APM could facilitate targeted interventions to delay or prevent the development of OA. Further studies in a larger cohort with an extended follow-up, and inclusion of additional genes, are warranted to better characterize this association., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: This study received support from an OREF Resident Clinician Scientist Training Grant (award 18-014 to J.D.L.). J.D.L. has received educational support from Elite Orthopaedics. R.H.B. has received educational support from Arthrex and Elite Orthopaedics, speaking fees from Smith & Nephew, and consulting fees from ISTO Technologies and Sanofi. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto., (© The Author(s) 2020.)

Objective: The etiology of osteoarthritis (OA) is unknown, however, there appears to be a significant contribution from genetics. We have identified recombinant inbred strains of mice derived from LG/J (large) and SM/J (small) strains that vary significantly in their ability to repair articular cartilage and susceptibility to post-traumatic OA due to their genetic composition. Here, we report cartilage repair phenotypes in the same strains of mice in which OA susceptibility was analyzed previously, and determine the genetic correlations between phenotypes., Design: We used 12 recombinant inbred strains, including the parental strains, to test three phenotypes: ear-wound healing (n = 263), knee articular cartilage repair (n = 131), and post-traumatic OA (n = 53) induced by the surgical destabilization of the medial meniscus (DMM). Genetic correlations between various traits were calculated as Pearson's correlation coefficients of strain means., Results: We found a significant positive correlation between ear-wound healing and articular cartilage regeneration (r = 0.71; P = 0.005). We observed a strong inverse correlation between articular cartilage regeneration and susceptibility to OA based on maximum (r = -0.54; P = 0.036) and summed Osteoarthritis Research Society International (OARSI) scores (r = -0.56; P = 0.028). Synovitis was not significantly correlated with articular cartilage regeneration but was significantly positively correlated with maximum (r = 0.63; P = 0.014) and summed (r = 0.70; P = 0.005) OARSI scores. Ectopic calcification was significantly positively correlated with articular cartilage regeneration (r = 0.59; P = 0.021)., Conclusions: Using recombinant inbred strains, our study allows, for the first time, the measurement of genetic correlations of regeneration phenotypes with degeneration phenotypes, characteristic of OA (cartilage degeneration, synovitis). We demonstrate that OA is positively correlated with synovitis and inversely correlated with the ability to repair cartilage. These results suggest an addition to the risk paradigm for OA from a focus on degeneration to regeneration., (Copyright © 2020 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Site-1 protease (S1P) ablation in the osterix-lineage in mice drastically reduces bone development and downregulates bone marrow-derived skeletal stem cells. Here we show that these mice also suffer from spina bifida occulta with a characteristic lack of bone fusion in the posterior neural arches. Molecular analysis of bone marrow-derived non-red blood cell cells, via single-cell RNA-Seq and protein mass spectrometry, demonstrate that these mice have a much-altered bone marrow with a significant increase in neutrophils and Ly6C -expressing leukocytes. The molecular composition of bone marrow neutrophils is also different as they express more and additional members of the stefin A (Stfa) family of proteins. In vitro , recombinant Stfa1 and Stfa2 proteins have the ability to drastically inhibit osteogenic differentiation of bone marrow stromal cells, with no effect on adipogenic differentiation. FACS analysis of hematopoietic stem cells show that despite a decrease in hematopoietic stem cells, S1P ablation results in an increased production of granulocyte-macrophage progenitors, the precursors to neutrophils. These observations indicate that S1P has a role in the lineage specification of hematopoietic stem cells and/or their progenitors for development of a normal hematopoietic niche. Our study designates a fundamental requirement of S1P for maintaining a balanced regenerative capacity of the bone marrow niche., Competing Interests: Competing interestsDr Linda Sandell serves as the editor of the Journal of Orthopaedic Research. All other authors declare no competing interests., (© 2020. Published by The Company of Biologists Ltd.)

RNA interference (RNAi) is a cellular mechanism for post-transcriptional gene regulation mediated by small interfering RNA (siRNA) and microRNA. siRNA-based therapy holds significant promise for the treatment of a wide-range of arthritic diseases. siRNA selectively suppresses the expression of a gene product and can thus achieve the specificity that is lacking in small molecule inhibitors. The potential use of siRNA-based therapy in arthritis, however, has not progressed to clinical trials despite ample evidence for efficacy in preclinical studies. One of the main challenges to clinical translation is the lack of a suitable delivery vehicle to efficiently and safely access diverse pathologies. Moreover, the ideal targets in treatment of arthritides remain elusive given the complexity and heterogeneity of these disease pathogeneses. Herein, we review recent preclinical studies that use RNAi-based drug delivery systems to mitigate inflammation in models of rheumatoid arthritis and osteoarthritis. We discuss a self-assembling peptide-based nanostructure that demonstrates the potential of overcoming many of the critical barriers preventing the translation of this technology to the clinic., (Published by Elsevier Inc.)

Osteoarthritis (OA) is a debilitating joint disease resulting from chronic joint inflammation and erosion of articular cartilage. A promising biological treatment for OA is intra-articular administration of platelet-rich plasma (PRP). However, immediate bolus release of growth factors limits beneficial therapeutic effects of PRP, thus necessitating the demand for sustained release platforms. In this study, we evaluated the therapeutic value of PRP released from a polyethylene glycol (PEG) hydrogel on articular chondrocytes/cartilage explants derived from OA patients. Lyophilized PRP (PRGF) was encapsulated in PEG hydrogels at 10% w/v and hydrogel swelling, storage modulus and degradation and PRGF release kinetics were determined. PRGF releasate from the hydrogels was collected on day 1, 4, and 11. Encapsulation of PRGF at 10% w/v in PEG hydrogels had minimal effect on hydrogel properties. PRGF was released with an initial burst followed by sustained release until complete hydrogel degradation. Effect of PRGF releasates and bolus PRGF (1% w/v PRGF) on patient-derived cartilage explants or chondrocytes was assessed by chondrocyte proliferation (pico-green assay), gene expression for COL1A1, COL2A1, MMP13, COX2, and NFKB1 (real-time polymerase chain reaction), and measurement of nitric oxide concentration (Griess' assay). Compared to bolus PRGF, PRGF releasates enhanced chondrocyte proliferation, suppressed the expression of genes like MMP13, NFKB1, COL1A1, and COL2A1 and reduced levels of nitric oxide. Taken together, these results indicate that release of PRGF from PEG hydrogels may improve the therapeutic efficacy of PRP and merits further investigation in an animal model of OA. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2401-2410, 2019., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Orthopaedic surgery has a rich history of publication of the science that supports the practice of our specialty, which dates from 1887. Orthopaedic publishing has evolved since that time, expanding from print to online access, with increasing variation in publication models, including open-access journals and article repositories, and methods of information delivery that include video, data archives, and commentary. This symposium provides an overview of the changes and challenges in the publication of orthopaedic science.

Objective: Recombinant inbred mouse strains generated from an LG/J and SM/J intercross offer a unique resource to study complex genetic traits such as osteoarthritis (OA). We undertook this study to determine the susceptibility of 14 strains to various phenotypes characteristic of posttraumatic OA. We hypothesized that phenotypic variability is associated with genetic variability., Methods: Ten-week-old male mice underwent surgical destabilization of the medial meniscus (DMM) to induce posttraumatic OA. Mice were killed 8 weeks after surgery, and knee joints were processed for histology to score cartilage degeneration and synovitis. Micro-computed tomography was used to analyze trabecular bone parameters including subchondral bone plate thickness and synovial ectopic calcifications. Gene expression in the knees was assessed using a QuantiGene Plex assay., Results: Broad-sense heritability ranged from 0.18 to 0.58, which suggested that the responses to surgery were moderately heritable. The LGXSM-33, LGXSM-5, LGXSM-46, and SM/J strains were highly susceptible to OA, while the LGXSM-131b, LGXSM-163, LGXSM-35, LGXSM-128a, LGXSM-6, and LG/J strains were relatively OA resistant. This study was the first to accomplish measurement of genetic correlations of phenotypes that are characteristic of posttraumatic OA. Cartilage degeneration was significantly positively associated with synovitis (r = 0.83-0.92), and subchondral bone plate thickness was negatively correlated with ectopic calcifications (r = -0.59). Moreover, we showed that 40 of the 78 genes tested were significantly correlated with various OA phenotypes. However, unlike the OA phenotypes, there was no evidence for genetic variation in differences in gene expression levels between DMM-operated and sham-operated knees., Conclusion: For these mouse strains, various characteristics of posttraumatic OA varied with genetic composition, which demonstrated a genetic basis for susceptibility to posttraumatic OA. The heritability of posttraumatic OA was established. Phenotypes exhibited various degrees of correlations; cartilage degeneration was positively correlated with synovitis, but not with the formation of ectopic calcifications. Further investigation of the genome regions that contain genes implicated in OA, as well as further investigation of gene expression data, will be useful for studying mechanisms of OA and identifying therapeutic targets., (© 2018, American College of Rheumatology.)

Delivery of therapeutic small interfering RNAs (siRNAs) in an effective dose to articular cartilage is very challenging as the cartilage dense extracellular matrix renders the chondrocytes inaccessible, even to intra-articular injections. Herein, we used a self-assembling peptidic nanoparticle (NP) platform featuring a cell penetrating peptide complexed to NF-κB p65 siRNA. We show that it efficiently and deeply penetrated human cartilage to deliver its siRNA cargo up to a depth of at least 700 μm. To simulate osteoarthritis in vitro, human articular cartilage explants were placed in culture and treated with IL-1β, a cytokine with known cartilage catabolic and pro-inflammatory effects. Exposure of peptide-siRNA NP to cartilage explants markedly suppressed p65 activation, an effect that persisted up to 3 weeks after an initial 48 h exposure to NP and in the presence of continuous IL-1β stimulation. Suppression of IL-1β-induced p65 activity attenuated chondrocyte apoptosis and maintained cartilage homeostasis. These findings confirm our previous in vivo studies in a murine model of post-traumatic osteoarthritis and suggest that the ability of peptide-siRNA NP to specifically modulate NF-κB pathway, a central regulator of the inflammatory responses in chondrocytes, may potentially mitigate the progression of cartilage degeneration.

Purpose of Review: The interaction between joint injuries and posttraumatic osteoarthritis (PTOA) is generally thought to be mechanical in nature, however, surgical intervention has little effect on the development of PTOA. Little is known about the biological underpinning of how meniscus and anterior cruciate ligament (ACL) tears lead to cartilage degeneration. This review summarizes the latest findings regarding biological factors that influence how the knee responds to meniscus and ligament injuries, how meniscus and/or ACL tears turn the joint in the direction of PTOA and whether patient risk for PTOA after meniscus/ACL injury can be predicted., Recent Findings: Literature indicates that numerous intrinsic and extrinsic factors are associated with the biological response of the knee to injuries associated with PTOA. Gene/protein biomarkers provide insight into the biologic response of the knee to meniscus/ACL tears and the relationship to osteoarthritis in at-risk patients. Animal studies detail the time-course of disease pathogenesis and inform about the molecules that potentially alter the course of disease., Summary: The molecular metabolic state of the meniscus/ACL after injury is associated with several biological factors. The limited studies to date provide initial evidence on the early molecular manifestations of injury, suggesting possible mechanisms for further study.

Objective: Anterior cruciate ligament (ACL) injury initiates a cascade of events often leading to osteoarthritis (OA). ACL reconstruction does not alter the course of OA, suggesting that heightened OA risk is likely due to factors in addition to the joint instability. We showed that torn ACL remnants express periostin (POSTN) in the acute phase of injury. Considering that ACL injury predisposes to OA and that POSTN is associated with cartilage metabolism, we hypothesize that ACL injury affects chondrocytes via POSTN., Design: Cartilage was obtained from osteoarthritic patients and ACL remnants were collected from patients undergoing ACL reconstruction. Crosstalk between ACL remnants and chondrocytes was studied in a transwell co-culture system. Expression of POSTN and other anabolic and catabolic genes was assessed via real-time polymerase chain reaction (PCR). Immunostaining for periostin was performed in human and mouse cartilage. The impact of exogenous periostin and siRNA-mediated ablation of periostin on matrix metabolism and cell migration was examined. Furthermore, the effect of anabolic (transforming growth factor beta 1 [TGF-β1]) and catabolic (interleukin 1 beta [IL-1β]) factors on POSTN expression was investigated., Results: ACL remnants induced expression of POSTN, MMP13 and ADAMTS4. Periostin levels were significantly higher in osteoarthritic compared to normal cartilage. Exogenous periostin induced MMP13 expression and cell migration, and repressed COL1A1 expression while POSTN knockdown inhibited expression of both anabolic and catabolic genes and impeded cell migration. TGF-β1 and IL-1β treatment did not alter POSTN expression but influenced chondrocyte metabolism as determined by quantification of anabolic and catabolic genes via real-time PCR., Conclusions: ACL remnants can exert paracrine effects on cartilage, altering cellular homeostasis. Over time, this metabolic imbalance could contribute to OA development., (Copyright © 2018 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: To assess the impact of osteoarthritis (OA) on the meniscus by comparing transcripts and biological processes in the meniscus between patients with and without OA., Design: RNA microarrays were used to identify transcripts differentially expressed (DE) in meniscus obtained from 12 OA and 12 non-OA patients. The non-OA specimens were obtained at the time of arthroscopic partial meniscectomy. Real-time PCR was performed on selected transcripts. Biological processes and gene-networking was examined computationally. Transcriptome signatures were mapped with 37 OA-related transcripts to evaluate how meniscus gene expression relates to that of OA cartilage., Results: We identified 168 transcripts significantly DE between OA (75 elevated, 93 repressed) and non-OA samples (≥1.5-fold). Among these, CSN1S1, COL10A1, WIF1, and SPARCL1 were the most prominent transcripts elevated in OA meniscus, POSTN and VEGFA were most highly repressed in OA meniscus. Transcripts elevated in OA meniscus represented response to external stimuli, cell migration and cell localization while those repressed in OA meniscus represented histone deacetylase activity (related to epigenetics) and skeletal development. Numerous long non-coding RNAs (lncRNAs) were DE between the two groups. When segregated by OA-related transcripts, two distinct clustering patterns appeared: OA meniscus appeared to be more inflammatory while non-OA meniscus exhibited a "repair" phenotype., Conclusions: Numerous transcripts with potential relevance to the pathogenesis of OA are DE in OA and non-OA meniscus. These data suggest an involvement of epigenetically regulated histone deacetylation in meniscus tears as well as expression of lncRNAs. Patient clustering based on transcripts related to OA in articular cartilage confirmed distinct phenotypes between injured (non-OA) and OA meniscus., (Copyright © 2017. Published by Elsevier Ltd.)

Site-1 protease (S1P) is a proprotein convertase with essential functions in the conversion of precursor proteins to their active form. In earlier studies, we demonstrated that S1P ablation in the chondrocyte lineage results in a drastic reduction in endochondral bone formation. To investigate the mechanistic contribution of S1P to bone development we ablated S1P in the osterix lineage in mice. S1P ablation in this lineage results in osteochondrodysplasia and variable degrees of early postnatal scoliosis. Embryonically, even though Runx2 and osterix expression are normal, S1P ablation results in a delay in vascular invasion and endochondral bone development. Mice appear normal when born, but by day 7 display pronounced dwarfism with fragile bones that exhibit significantly reduced mineral density, mineral apposition rate, bone formation rate and reduced osteoblasts indicating severe osteopenia. Mice suffer from a drastic reduction in bone marrow mesenchymal progenitors as analyzed by colony-forming unit-fibroblast assay. Fluorescence-activated cell sorting analysis of the skeletal mesenchyme harvested from bone marrow and collagenase-digested bone show a drastic reduction in hematopoietic lineage-negative, endothelial-negative, CD105 + skeletal stem cells. Bone marrow mesenchymal progenitors are unable to differentiate into osteoblasts in vitro , with no effect on adipogenic differentiation. Postnatal mice have smaller growth plates with reduced hypertrophic zone. Thus, S1P controls bone development directly by regulating the skeletal progenitor population and their differentiation into osteoblasts.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Microarrays and RNA-seq are at the forefront of high throughput transcriptome analyses. Since these methodologies are based on different principles, there are concerns about the concordance of data between the two techniques. The concordance of RNA-seq and microarrays for genome-wide analysis of differential gene expression has not been rigorously assessed in clinically derived ligament tissues. To demonstrate the concordance between RNA-seq and microarrays and to assess potential benefits of RNA-seq over microarrays, we assessed differences in transcript expression in anterior cruciate ligament (ACL) tissues based on time-from-injury. ACL remnants were collected from patients with an ACL tear at the time of ACL reconstruction. RNA prepared from torn ACL remnants was subjected to Agilent microarrays (N = 24) and RNA-seq (N = 8). The correlation of biological replicates in RNA-seq and microarrays data was similar (0.98 vs. 0.97), demonstrating that each platform has high internal reproducibility. Correlations between the RNA-seq data and the individual microarrays were low, but correlations between the RNA-seq values and the geometric mean of the microarrays values were moderate. The cross-platform concordance for differentially expressed transcripts or enriched pathways was linearly correlated (r = 0.64). RNA-Seq was superior in detecting low abundance transcripts and differentiating biologically critical isoforms. Additional independent validation of transcript expression was undertaken using microfluidic PCR for selected genes. PCR data showed 100% concordance (in expression pattern) with RNA-seq and microarrays data. These findings demonstrate that RNA-seq has advantages over microarrays for transcriptome profiling of ligament tissues when available and affordable. Furthermore, these findings are likely transferable to other musculoskeletal tissues where tissue collection is challenging and cells are in low abundance. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:484-497, 2018., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Purpose/Aim: Meniscus tears are a common injury to the knee associated with the development of osteoarthritis. Gene expression in the injured meniscus may be associated with early degeneration in the articular cartilage. The purpose of this study was to test the hypothesis that gene expression in meniscus tears is associated with early degenerative changes in the articular cartilage at the time of partial meniscectomy., Materials and Methods: Torn meniscus was removed at the time of partial meniscectomy in 68 patients without radiographic osteoarthritis. Meniscal mRNA expression was measured by quantitative PCR for multiple molecular markers of osteoarthritis and cartilage homeostasis. The presence of early degenerative changes in the knee was recorded by X-ray (N = 63), magnetic resonance imaging (MRI, N = 48), and arthroscopy (N = 63). Gene expression was tested for correlation with the presence/absence of degenerative changes after adjusting for age, sex, and body mass index., Results: Overall gene expression varied significantly with degenerative changes based on X-ray (P = 0.047) and MRI (P = 0.018). The linear combination of gene variation was also significant. However, only adiponectin (ADIPOQ) (P = 0.015) was expressed at a significantly lower level in patients with chondrosis on MRI, while the expression of ADIPOQ (P = 0.035) and resistin (RETN) (P = 0.017) was higher in patients with early degenerative changes on X-ray. None of the genes varied significantly with presence/absence of chondrosis as measured by arthroscopy., Conclusions: There is an overall association of gene expression in meniscal tears to early degenerative changes in the knee, but only a limited number of specific genes demonstrate this relationship. The roles of adiponectin and resistin in knee injury and osteoarthritis deserve further study.

Objective: To investigate the therapeutic potential of intra-articular hyaluronan-derivative HYADD® 4-G and/or platelet-rich plasma (PRP) in a mouse model of non-invasive joint injury., Methods: Non-invasive axial tibial loading was used to induce joint injury in 10-week-old C57BL/6J mice (n = 86). Mice underwent a single loading of either 6 Newton (N) or 9N axial tibial compression. HYADD® 4-G was injected intra-articularly at 8 mg/mL or 15 mg/mL either before or after loading with or without PRP. Phosphate-buffered-saline was injected as control. Knee joints were harvested at 5 or 56 days post-loading and prepared for micro-computed tomography scanning and subsequently processed for histology. Immunostaining was performed for aggrecan to monitor its distribution, for CD44 to monitor chondrocyte reactive changes and for COMP (cartilage oligomeric matrix protein) as an index for cartilage matrix changes related to loading and cartilage injury. TUNEL assay was performed to identify chondrocyte apoptosis., Results: Loading initiated cartilage proteoglycan loss and chondrocyte apoptosis within 5 days with slowly progressive post-traumatic osteoarthritis (no cartilage degeneration, but increased synovitis and ectopic calcification after 9N loading) at 56 days. Mice treated with repeated HYADD® 4-G (15 mg/mL) or HYADD® 4-G (8 mg/mL) ± PRP or PRP alone exhibited no significant improvement in the short-term (5 days) and long-term (56 days) consequences of joint loading except for a trend for improved bone changes compared to non-loaded joints., Conclusion: While we failed to show an overall effect of intra-articular delivery of hyaluronan-derivative and/or PRP in reversing/protecting the pathological events in cartilage and synovium following joint injury, some bone alterations were relatively less severe with hyaluronan-derivative at higher concentration or in association with PRP.

We investigated the spectrum of lesions characteristic of post-traumatic osteoarthritis (PTOA) across the knee joint in response to mechanical injury. We hypothesized that alteration in knee joint stability in mice reproduces molecular and structural features of PTOA that would suggest potential therapeutic targets in humans. The right knees of eight-week old male mice from two recombinant inbred lines (LGXSM-6 and LGXSM-33) were subjected to axial tibial compression. Three separate loading magnitudes were applied: 6N, 9N, and 12N. Left knees served as non-loaded controls. Mice were sacrificed at 5, 9, 14, 28, and 56 days post-loading and whole knee joint changes were assessed by histology, immunostaining, micro-CT, and magnetic resonance imaging. We observed that tibial compression disrupted joint stability by rupturing the anterior cruciate ligament (except for 6N) and instigated a cascade of temporal and topographical features of PTOA. These features included cartilage extracellular matrix loss without proteoglycan replacement, chondrocyte apoptosis at day 5, synovitis present at day 14, osteophytes, ectopic calcification, and meniscus pathology. These findings provide a plausible model and a whole-joint approach for how joint injury in humans leads to PTOA. Chondrocyte apoptosis, synovitis, and ectopic calcification appear to be targets for potential therapeutic intervention.

In this study, we examined early time-dependent changes in articular cartilage and synovium in response to tibial compression and sought the plausible origin of cells that respond to compression in the healer (LGXSM-6) and non-healer (LGXSM-33) recombinant inbred mouse strains. The right knee of 13-week old male mice was subjected to tibial compression using 9N axial loading. The contralateral left knee served as a control. Knees were harvested at 5, 9, and 14 days post-injury. Histological changes in cartilage and synovium, immunofluorescence pattern of CD44, aggrecan, type-II collagen, cartilage oligomeric matrix protein and the aggrecan neo-epitope NITEGE, and cell apoptosis (by TUNEL) were examined. We used a double nucleoside analog cell-labeling strategy to trace cells responsive to injury. We showed that tibial compression resulted in rupture of anterior cruciate ligament, cartilage matrix loss and chondrocyte apoptosis at the injury site. LGXSM-33 showed higher synovitis and ectopic synovial chondrogenesis than LGXSM-6 with no differences for articular cartilage lesions. With loading, an altered pattern of CD44 and NITEGE was observed: cells in the impacted area underwent apoptosis, cells closely surrounding the injured area expressed CD44, and cells in the intact area expressed NITEGE. Cells responding to injury were found in the synovium, subchondral bone marrow and the Groove of Ranvier. Taken together, we found no strain differences in chondrocytes in the early response to injury. However, the synovial response was greater in LGXSM-33 indicating that, at early time points, there is a genetic difference in synovial cell reaction to injury. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:524-536, 2017., (© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Objective: To investigate a targeted set of biochemical biomarkers as predictors of clinically relevant osteoarthritis (OA) progression., Methods: Eighteen biomarkers were measured at baseline, 12 months (M) and 24 M in serum (s) and/or urine (u) of cases (n=194) from the OA initiative cohort with knee OA and radiographic and persistent pain worsening from 24 to 48 M and controls (n=406) not meeting both end point criteria. Primary analyses used multivariable regression models to evaluate the association between biomarkers (baseline and time-integrated concentrations (TICs) over 12 and 24 M, transposed to z values) and case status, adjusted for age, sex, body mass index, race, baseline radiographic joint space width, Kellgren-Lawrence grade, pain and pain medication use. For biomarkers with adjusted p<0.1, the c-statistic (area under the curve (AUC)), net reclassification index and the integrated discrimination improvement index were used to further select for hierarchical multivariable discriminative analysis and to determine the most predictive and parsimonious model., Results: The 24 M TIC of eight biomarkers significantly predicted case status (ORs per 1 SD change in biomarker): sCTXI 1.28, sHA 1.22, sNTXI 1.25, uC2C-HUSA 1.27, uCTXII, 1.37, uNTXI 1.29, uCTXIα 1.32, uCTXIβ 1.27. 24 M TIC of uCTXII (1.47-1.72) and uC2C-Human Urine Sandwich Assay (HUSA) (1.36-1.50) both predicted individual group status (pain worsening, joint space loss and their combination). The most predictive and parsimonious combinatorial model for case status consisted of 24 M TIC uCTXII, sHA and sNTXI (AUC 0.667 adjusted). Baseline uCTXII and uCTXIα both significantly predicted case status (OR 1.29 and 1.20, respectively)., Conclusions: Several systemic candidate biomarkers hold promise as predictors of pain and structural worsening of OA., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Background: Meniscus tears are classified as traumatic or degenerative based on the tear pattern. There is little evidence demonstrating biological differences between the 2 tear types., Hypothesis: Gene expression signatures in the injured meniscus are different between traumatic (vertical) and degenerative (complex, horizontal, or flap) tears., Study Design: Controlled laboratory study., Methods: Samples of the torn meniscus from the white-white zone were removed at the time of clinically indicated partial meniscectomy from 48 patients (37 with degenerative tears and 11 with traumatic tears). mRNA expression in the injured menisci was measured by quantitative real-time polymerase chain reaction for selected molecular markers of osteoarthritis, inflammation, and cartilage homeostasis (eg, cytokines/chemokines, aggrecanases/metalloproteinases, transcription factors, cartilage matrix genes, and adipokines). The tear pattern (traumatic or degenerative) and location (medial or lateral) were recorded for each patient. Gene expression differences between degenerative and traumatic tears were computed after adjusting for patients' age, sex, and body mass index and for location of the resected meniscus (medial/lateral)., Results: Gene expression in meniscus tears varied by pattern. Chemokines ( IL8 [ P < .001] and CXCL6 [ P < .001]) and matrix metalloproteinases ( MMP1 [ P = .011] and MMP3 [ P = .016]) were expressed at a significantly higher level in traumatic tears compared with degenerative tears. In contrast, COL1A1 was expressed at a lower level in traumatic tears compared with degenerative tears ( P = .058). None of the genes tested demonstrated significant differences between medial and lateral meniscus tears., Conclusion: Traumatic meniscus tears overall exhibited a higher inflammatory/catabolic response as evidenced by higher levels of chemokine and matrix metalloproteinase expression than degenerative tears. These findings suggest that there is a (molecular) biological distinction between traumatic and degenerative tears., Clinical Relevance: The catabolic/inflammatory differences between traumatic and degenerative tears may be relevant to treatment decisions regarding the meniscus as well as advance our understanding of how meniscus tears relate to the development of knee osteoarthritis.

Osteoarthritis (OA) is a major cause of disability and morbidity in the aging population. Joint injury leads to cartilage damage, a known determinant for subsequent development of posttraumatic OA, which accounts for 12% of all OA. Understanding the early molecular and cellular responses postinjury may provide targets for therapeutic interventions that limit articular degeneration. Using a murine model of controlled knee joint impact injury that allows the examination of cartilage responses to injury at specific time points, we show that intraarticular delivery of a peptidic nanoparticle complexed to NF-κB siRNA significantly reduces early chondrocyte apoptosis and reactive synovitis. Our data suggest that NF-κB siRNA nanotherapy maintains cartilage homeostasis by enhancing AMPK signaling while suppressing mTORC1 and Wnt/β-catenin activity. These findings delineate an extensive crosstalk between NF-κB and signaling pathways that govern cartilage responses postinjury and suggest that delivery of NF-κB siRNA nanotherapy to attenuate early inflammation may limit the chronic consequences of joint injury. Therapeutic benefits of siRNA nanotherapy may also apply to primary OA in which NF-κB activation mediates chondrocyte catabolic responses. Additionally, a critical barrier to the successful development of OA treatment includes ineffective delivery of therapeutic agents to the resident chondrocytes in the avascular cartilage. Here, we show that the peptide-siRNA nanocomplexes are nonimmunogenic, are freely and deeply penetrant to human OA cartilage, and persist in chondrocyte lacunae for at least 2 wk. The peptide-siRNA platform thus provides a clinically relevant and promising approach to overcoming the obstacles of drug delivery to the highly inaccessible chondrocytes., Competing Interests: Samuel Wickline has equity in Trasir Therapeutics, Inc.

Background: Anterior cruciate ligament (ACL) tears are a common injury. The healing potential of the injured ACL is poorly understood and is considered limited. Therefore, most ACL tears that are treated surgically undergo reconstruction rather than repair. However, there has been renewed interest recently in repairing ACL tears despite unanswered questions regarding the healing capacity of the ACL., Hypothesis: Gene expression in the injured ACL varies with time from injury., Study Design: Descriptive laboratory study., Methods: Transcriptome-wide expression profiles of 24 human ACL remnants recovered at the time of surgical reconstruction were analyzed using the Agilent human 8x60K microarray platform. Gene ontology was performed on differentially expressed transcripts based on time from injury (acute, <3 months; intermediate, 3-12 months; chronic, >12 months). A subset of transcripts with large fold changes in expression between any 2 categories was validated via microfluidic digital polymerase chain reaction., Results: Numerous transcripts representing important biological processes were differentially expressed by time from injury. The most significant changes were noted between the acute and chronic groups. Expression of several extracellular matrix genes- namely, POSTN, COL5A1, COL1A1, and COL12A1-was lower in the chronic tears compared with acute and intermediate tears. In acute tears, processes representing angiogenesis and stem cell differentiation were affected. In intermediate tears, processes representing stem cell proliferation concomitant with cellular component organization/cellular localization were altered. In ACL tears more than 12 months out from injury, processes denoting myosin filament organization, cellular component organization/cell localization, and extracellular matrix organization were affected., Conclusion: These findings are consistent with initial repair activity in the injured ACL, which declines with time from injury. Individual genes identified in this study, such as periostin, deserve further investigation into their role in tissue repair., Clinical Relevance: The decreased healing capacity of ACL tears over time is relevant to the development of effective techniques for repairing ACL tears and may have some significance for ACL reconstruction techniques as well. The potential for healing appears to be greatest in acute ACL tears, suggesting this window should be the focus of research for ACL repair., (© 2016 The Author(s).)

Objectives: (i) To provide baseline knowledge of gene expression in macroscopically normal articular cartilage, (ii) to test the hypothesis that age, body-mass-index (BMI), and sex are associated with cartilage RNA transcriptome, and (iii) to predict individuals at potential risk for developing "pre-osteoarthritis" (OA) based on screening of genetic risk-alleles associated with OA and gene transcripts differentially expressed between normal and OA cartilage., Design: Healthy-appearing cartilage was obtained from the medial femoral notch of 12 knees with a meniscus tear undergoing arthroscopic partial meniscectomy. Cartilage had no radiographic, magnetic-resonance-imaging or arthroscopic evidence for degeneration. RNA was subjected to Affymetrix microarrays followed by validation of selected transcripts by microfluidic digital polymerase-chain-reaction. The underlying biological processes were explored computationally. Transcriptome-wide gene expression was probed for association with known OA genetic risk-alleles assembled from published literature and for comparison with gene transcripts differentially expressed between healthy and OA cartilage from other studies., Results: We generated a list of 27,641 gene transcripts in healthy cartilage. Several gene transcripts representing numerous biological processes were correlated with age and BMI and differentially expressed by sex. Based on disease-specific Ingenuity Pathways Analysis, gene transcripts associated with aging were enriched for bone/cartilage disease while the gene expression profile associated with BMI was enriched for growth-plate calcification and OA. When segregated by genetic risk-alleles, two clusters of study patients emerged, one cluster containing transcripts predicted by risk studies. When segregated by OA-associated gene transcripts, three clusters of study patients emerged, one of which is remarkably similar to gene expression pattern in OA., Conclusions: Our study provides a list of gene transcripts in healthy-appearing cartilage. Preliminary analysis into groupings based on OA risk-alleles and OA-associated gene transcripts reveals a subset of patients expressing OA transcripts. Prospective studies in larger cohorts are needed to assess whether these patterns are predictive for OA.

This study reports on genetic susceptibility to ectopic calcification in the LG/J and SM/J advanced intercross mice. Using 347 mice in 98 full-sibships, destabilization of medial meniscus (DMM) was performed to induce joint injury. We found that joint destabilization instigated ectopic calcifications as detected and quantified by micro-CT. We performed quantitative trait locus (QTL) analysis to map ectopic calcification phenotypes to discrete genomic locations. To validate the functional significance of the selected QTL candidate genes, we compared mRNA expression between parental LG/J and SM/J inbred strains. Overall, we detected 20 QTLs affecting synovial and meniscus calcification phenotypes with 11 QTLs linked to synovial calcification. Functional and bioinformatic analyses of single nucleotide polymorphism (SNP) identified functional classifications relevant to angiogenesis (Myo1e, Kif26b, Nprl3, Stab2, Fam105b), bone metabolism/calcification (Tle3, Tgfb2, Lipc, Nfe2l1, Ank, Fam105b), arthritis (Stab2, Tbx21, Map4k4, Hoxb9, Larp6, Col1a2, Adam10, Timp3, Nfe2l1, Trpm3), and ankylosing-spondylitis (Ank, Pon1, Il1r2, Tbkbp1) indicating that ectopic calcification involves multiple mechanisms. Furthermore, the expression of 11 out of 78 candidate genes was significantly different between LG/J and SM/J. Correlation analysis showed that Aff3, Fam81a, Syn3, and Ank were correlated with synovial calcification. Taken together, our findings of multiple genetic loci suggest the involvement of multiple genes contributing to ectopic calcification., Competing Interests: Dr. Sandell is Editor in Chief of Journal of Orthopaedic Research. Dr. Sandell owns stock or stock options in ISTO Technologies and receives royalties from Merck/Millipore for a type IIA collagen N-propeptide enzyme-linked immunosorbent assay. All other authors have nothing to disclose. None of the authors has any competing interests with regard to this manuscript., (© 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)