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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 pre-clinical 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.

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.

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.

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.

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.

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., Summary: Ablation of S1P in the osterix-lineage downregulates bone development due to hematopoietic progenitor alterations that increase bone marrow neutrophils with accompanying increases in stefin proteins with osteogenic inhibitory activity.

ObjectiveTo query the transcript-level changes in the medial and lateral tibial plateau cartilage in tandem with obesity in patients with end-stage osteoarthritis (OA).DesignCartilage was obtained from 23 patients (20 obese [body mass index > 30 kg/m2], 3 overweight [body mass index < 30 kg/m2]) 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.ResultsScoring 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.ConclusionsOur 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.

Intervertebral disc degeneration (IDD) causes back pain and sciatica, affecting quality of life and resulting in high economic/social burden. The etiology of IDD is not well understood. Along with aging and environmental factors, genetic factors also influence the onset, progression and severity of IDD. Genetic studies of risk factors for IDD using human cohorts are limited by small sample size and low statistical power. Animal models amenable to genetic and functional studies of IDD provide desirable alternatives. Despite differences in size and cellular content as compared to human intervertebral discs (IVDs), the mouse is a powerful model for genetics and assessment of cellular changes relevant to human biology. Here, we provide evidence for early onset disc degeneration in SM/J relative to LG/J mice with poor and good tissue healing capacity respectively. In the first few months of life, LG/J mice maintain a relatively constant pool of notochordal-like cells in the nucleus pulposus (NP) of the IVD. In contrast, chondrogenic events are observed in SM/J mice beginning as early as one-week-old, with progressive fibrotic changes. Further, the extracellular matrix changes in the NP are consistent with IVD degeneration. Leveraging on the genomic data of two parental and two recombinant inbred lines, we assessed the genetic contribution to the NP changes and identified processes linked to the regulation of ion transport systems. Significantly, "transport" system is also in the top three gene ontology (GO) terms from a comparative proteomic analysis of the mouse NP. These findings support the potential of the SM/J, LG/J and their recombinant inbred lines for future genetic and biological analysis in mice and validation of candidate genes and biological relevance in human cohort studies. The proteomic data has been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD008784.

The mission of the Orthopaedic Research Society is to promote and advance musculoskeletal research worldwide. With this in mind, the Annual Meeting Program Committee sought to establish a debate as a key component of the meeting. Our purpose was to provoke discussion on topics that are core to our mission and to engage all constituencies within the society by examining questions of broad relevance. To this end, the topic "Regenerative medicine will make orthopaedic implants obsolete in our time" was selected as the title of the inaugural debate. The arguments for and against the motion are presented in this perspectives article. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2579-2585, 2018.

Summary 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.

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.

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.

Objectives: While meniscus tears are associated with an increased risk of developing knee osteoarthritis (OA), it is not possible to predict which patients are at the highest risk for developing OA. Gene signature in meniscus resected at the time of arthroscopic partial meniscectomy may provide insight into an individual’s risk of OA progression. The purpose of this study is to test the hypothesis that gene signatures at the time of arthroscopic partial meniscectomy predict radiographic progression of OA. Methods: Meniscus fragments were collected from 40 patients at the time of clinically indicated isolated arthroscopic partial medial meniscectomy. Gene expression was assessed by quantitative real-time PCR using a panel of 28 candidate genes with known roles in cartilage homeostasis, OA, extracellular-matrix degradation and obesity. Radiographs obtained prior to surgery and at final follow-up were graded by a musculoskeletal radiologist. The association of meniscus gene expression at the time of surgery with progression of radiographic OA was determined. Results: Gene expression in meniscus resected at arthroscopic partial medial meniscectomy and radiographic data at baseline and mean follow-up of 6.2 ± 1.3 years were available for 31 patients (77.5%). The cohort consisted of 45.2% females with a mean age of 48.5 ± 9.2 years and mean body mass index of 28.1 ± 5.1 kg/m2. Gene expression was associated with radiographic progression of OA (Table 1). Patients without OA progression had significantly higher expression of seven genes, namely MMP9 (5.1-fold, P=0.002), IL8 (2.9-fold, P=0.016), CCL3 (3.7-fold, P=0.032), CCL3L1 (4.5-fold, P=0.008), CXCL6 (6.2-fold, P=0.010), LEP (5.2-fold, P=0.004) and RETN (46-fold, P=0.008). Patients with progression showed very low expression of these genes (Figure 1). When segregating patients based on severity of OA progression, there was significantly increased expression of three genes (MMP9, CXCL6, LEP) among patients without any radiographic OA progression. There was an increased expression of CXCL6 (9.5-fold, P=0.042), LEP (4.3-fold, P=0.047) and MMP9 (5.0-fold, P=0.023) among those without any radiographic OA progression compared to those with rapid progression in the medial compartment. There was also an increased expression of MMP9 among patients without progression compared to those with progression in the lateral and/or patellofemoral compartments (7.2-fold, P=0.038). Conclusions: Our findings suggest that gene expression in the meniscus at the time of arthroscopic partial meniscectomy is associated with the risk for OA progression. Elevated expression of the aforementioned genes in non-progressors may reflect a potentially chondroprotective response. Further studies including additional genes, larger cohorts and extended follow-up are warranted to better characterize this association. Ultimately, this line of investigation could facilitate the development of more sensitive methods to determine the risk for OA progression among patients with meniscal injury and approaches to delay or prevent OA in this population. [Table: see text]

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.

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.

Summary 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.

We report the results of a symposium aimed at identifying validated biomarkers that can be used to complement clinical observations for diagnosis and prognosis of joint injury leading to equine osteoarthritis (OA). Biomarkers might also predict pre-fracture change that could lead to catastrophic bone failure in equine athletes. The workshop was attended by leading scientists in the fields of equine and human musculoskeletal biomarkers to enable cross-disciplinary exchange and improve knowledge in both. Detailed proceedings with strategic planning was written, added to, edited and referenced to develop this manuscript. The most recent information from work in equine and human osteoarthritic biomarkers was accumulated, including the use of personalized healthcare to stratify OA phenotypes, transcriptome analysis of anterior cruciate ligament (ACL) and meniscal injuries in the human knee. The spectrum of "wet" biomarker assays that are antibody based that have achieved usefulness in both humans and horses, imaging biomarkers and the role they can play in equine and human OA was discussed. Prediction of musculoskeletal injury in the horse remains a challenge, and the potential usefulness of spectroscopy, metabolomics, proteomics, and development of biobanks to classify biomarkers in different stages of equine and human OA were reviewed. The participants concluded that new information and studies in equine musculoskeletal biomarkers have potential translational value for humans and vice versa. OA is equally important in humans and horses, and the welfare issues associated with catastrophic musculoskeletal injury in horses add further emphasis to the need for good validated biomarkers in the horse. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:823-831, 2018.

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., Summary: S1P governs a fundamental aspect of skeletal development and homeostasis, mainly the maintenance and osteogenic differentiation of skeletogenic stem cells that are a source of osteoblast and chondrocyte lineages.

This chapter will summarize the field as seen from the viewpoint of a biologist and cite some examples of challenges in the field and a perspective on where the field is moving. Secondly, it will provide some examples of how developmental principles have been used to understand and potentially treat orthopedic diseases in a broader view. An excellent review from both the tissue engineering and biology sides was published in 2017 (Marcucio et al., 2017). Classically, the goal of tissue engineering was to treat orthopedic disease by making a tissue replacement as good as or better than the biologic one, often using principles derived from developmental biology. The tissue could be manufactured from components such as plastics or ceramics or made biologically by cells such as stem cells differentiated into tissues or mature cells 3D-printed into the shape of an organ. Interestingly, few of these tissue-engineered products are available for clinical use. There is certainly no lack of ingenuity and creativity in the tissue engineering field; however, making tissues as good as the original, keeping them in place and functioning over time, and overcoming regulatory hurdles has proven a higher bar than expected. However, there are some successes to point to. In 2011, scientists at the Karolinska Institute were able to implant an artificial trachea made from a synthetic scaffold seeded with patient's stem cells (Jungebluth and Macchiarini, 2011). This was based on an established proof of concept by Langer where they grew an outer ear from bovine chondrocytes and an ear-shaped scaffold on the back of a mouse (Cao et al., 1997). Cancedda and colleagues (2001, 2007) used bone marrow–derived stem cells in an osteoconductive scaffold to repair a large segmental bone defect. The reconstruction of a human mandible was reported using a bone-muscle flap in vivo where the patient served as his/her own bioreactor (Warnke et al., 2006). These early trials relied on the differentiated cells (i.e., chondrocytes making more chondrocytes), porous scaffolds that provided a substrate for cell differentiation from adult stem cells (osteoconductive or osteoinductive scaffolds), or induction of unknown tissue differentiation events. Most tissue engineering was impeded by a lack of knowledge of the native developmental processes. Now much more attention is paid to understanding the developmental processes involved in differentiation of cells and tissues, renewal or repair of mature tissues, stem cell generation, stem cell reservoirs, and stem cell differentiation to specific tissues. The focus on developmental processes has also had an impact on potential treatment of disease. For example, one theory of the pathogenesis of osteoarthritis is that the cells in articular cartilage continue to differentiate into hypertrophic cells, a characteristic of growth plate hypertrophic chondrocytes. This theory has directed studies geared toward inhibition of this growth plate hypertrophic phenotype, maintaining the cells in a mature chondrocyte phenotype (Nishimura et al., 2017).

Summary 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.

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.

Summary Objective C–C chemokine receptor type 5 (CCR5) has been implicated in rheumatoid arthritis and several inflammatory diseases, where its blockade resulted in reduced joint destruction. However, its role in modulating cartilage and bone changes in post-traumatic osteoarthritis (OA) has not yet been investigated. In this study, we investigated changes in articular cartilage, synovium and bone in a post-traumatic OA model using CCR5-deficient (CCR5 −/− ) mice. Method Destabilization of the medial meniscus (DMM) was performed on the right knee of 10-week old CCR5 −/− and C57BL/6J wild-type (WT) mice to induce post-traumatic OA. The contralateral left knee served as sham-operated control. Knee joints were analyzed at 4-, 8- and 12-weeks after surgery to evaluate cartilage degeneration and synovitis by histology, and bone changes via micro-CT. Results Our findings showed that CCR5 −/− mice exhibited significantly less cartilage degeneration than WT mice at 8- and 12-weeks post-surgery. CCR5 −/− mice showed some altered bone parameters 18- and 22-weeks of age, but body size and weight were not affected. The effect of CCR5-ablation was insignificant at all time points post-surgery for synovitis and for bone parameters such as bone volume/total volume, connectivity density index (CDI), structure model index (SMI), subchondral bone plate thickness, and trabecular bone number, thickness and spacing. Conclusion These findings suggest that CCR5 −/− mice developed less cartilage degeneration, which may indicate a potential protective role of CCR5-ablation in cartilage homeostasis. There were no differences in bone or synovial response to surgery suggesting that CCR5 functions primarily in cartilage during the development of post-traumatic OA.

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. There was a higher correlation between the PCR data and the RNA-seq as well as the microarrays data. These findings demonstrate that RNA-seq has advantages over microarrays for transcriptome profiling of ligament tissues when available and affordable. This article is protected by copyright. All rights reserved

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.

Mammals rarely regenerate their lost or injured tissues into adulthood. MRL/MpJ mouse strain initially identified to heal full-thickness ear wounds now represents a classical example of mammalian wound regeneration since it can heal a spectrum of injuries such as skin and cardiac wounds, nerve injuries and knee articular cartilage lesions. In addition to MRL/MpJ, a few other mouse strains such as LG/J (a parent of MRL/MpJ) and LGXSM-6 (arising from an intercross between LG/J and SM/J mouse strains) have now been recognized to possess regenerative/healing abilities for articular cartilage and ear wound injuries that are similar, if not superior, to MRL/MpJ mice. While some mechanisms underlying regenerative potential have been begun to emerge, a complete set of biological processes and pathways still needs to be elucidated. Using a panel of healer and non-healer mouse strains, our recent work has provided some insights into the genes that could potentially be associated with healing potential. Future mechanistic studies can help seek the Holy Grail of regenerative medicine. This review highlights the regenerative capacity of selected mouse strains for articular cartilage, in particular, and lessons from other body tissues, in general.

Objective Higher body mass index (BMI) increases the risk of meniscus injury and knee osteoarthritis (OA). However, it is unknown if and how obesity affects meniscus biology. We analyzed transcriptome-wide gene expression profiles of injured human menisci to test the hypothesis that meniscal gene expression signatures relate to patient BMI. Methods Meniscus samples were obtained from patients undergoing arthroscopic partial meniscectomy. Transcriptome-wide analysis of gene expression followed by validation of selected transcripts by QuantiGene Plex assay was performed. Correlations of gene expression with BMI and relative fold changes (≥1.5-fold) in 3 BMI categories (lean [BMI 18.5–24.9 kg/m2], overweight [BMI 25.0–29.9 kg/m2], and obese [BMI >30.0 kg/m2]) were analyzed, and integrated functional classifications were probed computationally. Results The obese versus overweight comparison resulted in the largest set of differences (565 transcripts) followed by obese versus lean (280 transcripts) and overweight versus lean (125 transcripts). Biologic reproducibility was confirmed by cluster analysis of expressed transcripts. Differentially regulated transcripts represented important functional classifications. Transcripts associated with oxygen transport, calcium ion binding, and cell homeostasis were elevated with BMI, while those related to extracellular matrix deposition, cell migration, and glucosamine metabolic processes were repressed. While these functional classifications may play key roles in cartilage/meniscus homeostasis, failure of extracellular matrix deposition and increase in calcium ion binding likely contribute to OA development following meniscal injury. Conclusion Our results indicate greater differences in gene expression between obese and overweight groups than between overweight and lean groups. This may indicate that there is a weight threshold at which injured meniscus responds severely to increased BMI. BMI-related changes in gene expression present a plausible explanation for the role of meniscal injury in OA development among obese patients.

Objective Joint trauma can lead to a spectrum of acute lesions, including cartilage degradation, ligament or meniscus tears, and synovitis, all potentially associated with osteoarthritis (OA). This study was undertaken to generate and validate a murine model of knee joint trauma following noninvasive controlled injurious compression in vivo. Methods The right knees of 8-week-old mice were placed in a hyperflexed position and subjected to compressive joint loading at 1 of 3 peak forces (3N, 6N, or 9N) for 60 cycles in a single loading period and harvested on days 5, 9, and 14 after loading (n = 3–5 for each time point and for each loading). The left knees were not loaded and were used as the contralateral control. Histologic, immunohistochemical, and enzyme-linked immunosorbent assay analyses were performed to evaluate acute pathologic features in chondrocyte viability, cartilage matrix metabolism, synovial reaction, and serum cartilage oligomeric matrix protein (COMP) levels. Results Acute joint pathology was associated with increased injurious loads. All loading regimens induced chondrocyte apoptosis, cartilage matrix degradation, disruption of cartilage collagen fibril arrangement, and increased levels of serum COMP. We also observed that 6N loading induced mild synovitis by day 5, whereas at 9N, with tearing of the anterior cruciate ligament, severe posttraumatic synovitis and ectopic cartilage formation were observed. Conclusion We have established a murine model of knee joint trauma with different degrees of overloading in vivo. Our results suggest that immediate therapies particularly targeted to apoptosis and synovial cell proliferation could affect the acute posttraumatic reaction to potentially limit chronic consequences and OA.

Type II collagen is the major collagenous component of the cartilage extracellular matrix; formation of a covalently cross-linked type II collagen network provides cartilage with important tensile properties. The Col2a1 gene is encoded by 54 exons, of which exon 2 is subject to alternative splicing, resulting in different isoforms named IIA, IIB, IIC and IID. The two major procollagen protein isoforms are type IIA and type IIB procollagen. Type IIA procollagen mRNA contains exon 2 and is generated predominantly by chondroprogenitor cells and other non-cartilaginous tissues. Differentiated chondrocytes generate type IIB procollagen, devoid of exon 2. Although type IIA procollagen is produced in certain non-collagenous tissues during development, this developmentally-regulated alternative splicing switch to type IIB procollagen is restricted to cartilage cells. Though a much studied and characterized molecule, the importance of the various type II collagen protein isoforms in cartilage development and homeostasis is still not completely understood. Effective antibodies against specific epitopes of these isoforms can be useful tools to decipher function. However, most type II collagen antibodies to date recognize either all isoforms or the IIA procollagen isoform. To specifically identify the murine type IIB procollagen, we have generated a rabbit antibody (termed IIBN) directed to a peptide sequence that spans the murine exon 1–3 peptide junction. Characterization of the affinity-purified antibody by western blotting of collagens extracted from wild type murine cartilage or cartilage from Col2a1 + ex2 knock-in mice (which generates predominantly the type IIA procollagen isoform) demonstrated that the IIBN antibody is specific to the type IIB procollagen isoform. IIBN antibody was also able to detect the native type IIB procollagen in the hypertrophic chondrocytes of the wild type growth plate, but not in those of the Col2a1 + ex2 homozygous knock-in mice, by both immunofluorescence and immunohistochemical studies. Thus the IIBN antibody will permit an in-depth characterization of the distribution of IIB procollagen isoform in mouse skeletal tissues. In addition, this antibody will be an important reagent for characterizing mutant type II collagen phenotypes and for monitoring type II procollagen processing and trafficking.

Osteoarthritis affects the whole joint structure with progressive changes in cartilage, menisci, ligaments and subchondral bone, and synovial inflammation. Biomarkers are being developed to quantify joint remodelling and disease progression. This article was prepared following a working meeting of the European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis convened to discuss the value of biochemical markers of matrix metabolism in drug development in osteoarthritis. The best candidates are generally molecules or molecular fragments present in cartilage, bone or synovium and may be specific to one type of joint tissue or common to them all. Many currently investigated biomarkers are associated with collagen metabolism in cartilage or bone, or aggrecan metabolism in cartilage. Other biomarkers are related to non-collagenous proteins, inflammation and/or fibrosis. Biomarkers in osteoarthritis can be categorised using the burden of disease, investigative, prognostic, efficacy of intervention, diagnostic and safety classification. There are a number of promising candidates, notably urinary C-terminal telopeptide of collagen type II and serum cartilage oligomeric protein, although none is sufficiently discriminating to differentiate between individual patients and controls (diagnostic) or between patients with different disease severities (burden of disease), predict prognosis in individuals with or without osteoarthritis (prognostic) or perform so consistently that it could function as a surrogate outcome in clinical trials (efficacy of intervention). Future avenues for research include exploration of underlying mechanisms of disease and development of new biomarkers; technological development; the ‘omics’ (genomics, metabolomics, proteomics and lipidomics); design of aggregate scores combining a panel of biomarkers and/or imaging markers into single diagnostic algorithms; and investigation into the relationship between biomarkers and prognosis.

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.

The meniscus is an essential component of the tibiofemoral articulation that contributes to the complex biomechanics of the knee joint, protecting the underlying articular cartilage. The role of the meniscus in the initiation and progression of osteoarthritis (OA) is thought to be minimal unless it is injured (1, 2). The location of the meniscus and its role in shock absorption and load transmission predispose the meniscus to traumatic and degenerative injuries, thus, meniscus tears are one of the most common intra-articular injuries of the knee (3, 4). There is an age bias in terms of mechanisms of meniscal injury (5): younger people are more likely to have acute tears due to trauma whereas older people are more likely to have tears due to degeneration (6-9). Since the majority of meniscus tears are unsuitable for repair (3, 10, 11), partial meniscectomy is used to redress most meniscus tears (7, 12). A large body of information suggests that damage to or loss of meniscus is associated with changes in cartilage (13-16). It has also been reported that following partial meniscectomy, there is an elevated risk for functional deterioration of cartilage (11, 17, 18), often resulting in the development of OA and irreparable joint damage (2, 10, 14, 17, 19-21). Despite the fact that meniscus and knee articular cartilage have several characteristics in common and the mechanisms of meniscus and knee cartilage degeneration have been demonstrated (22, 23), the molecular pathogenesis of traumatic and degenerative meniscus tears and their association with degenerative changes in the articular cartilage are not known. We have previously shown, through a candidate gene approach, that the expression of various OA- and obesity-related genes diverged with age (24, 25) and to some extent with body mass index (BMI) (25) in the injured meniscus. As these studies provided information on only a handful of genes (known for their specific role in OA and obesity), we sought to determine the transcriptome-wide gene expression profile in meniscus tears in patients undergoing arthroscopic partial meniscectomy to identify a comprehensive set of targets for future studies. The goal is to identify changes in the gene expression profile of the torn meniscus that may be relevant to age-associated meniscal and cartilage degeneration. We believe that this information will allow for a better understanding of why and how degenerative changes occur in the meniscus and how injury to the meniscus, whether traumatic or degenerative, contributes to the initiation and progression of OA in the knee.

Summary The sixth Osteoarthritis Research Society International (OARSI) Workshop on Imaging in Osteoarthritis combined with the third osteoarthritis (OA) Biomarkers Workshop is the first to bring together the imaging and molecular biomarker communities to focus on clinical validation and qualification of OA biomarkers. The workshop was held in Hilton Head, SC, USA, from June 12–14, 2012; 138 attendees participated, including representatives from academia, pharmaceutical and magnetic resonance imaging (MRI) industries, Food and Drug Administration (FDA), and National Institutes of Health (NIH). Presentations and discussions raised awareness, consolidated knowledge, and identified strategies to overcome challenges for the development and application of imaging and biochemical biomarkers in OA research studies and clinical trials. Conclusion The OA research communities need to work alongside regulatory agencies across the world, to qualify and validate new chemical and imaging biomarkers for future research and clinical trials.

Future clinical trials investigating the natural history and treatment of femoroacetabular impingement (FAI) will require multimodal staging systems for hip osteoarthritis because the optimal system will differ based on the size of the study population, the specific objective in question, and the time frame in which the investigator expects to see the specified end point. Plain radiographs are readily available, low in cost, and of unquestioned validity, but they are relatively insensitive to early joint damage. MRI allows assessment of both bony and soft-tissue pathology within the joint, and it is much more sensitive for early joint damage because cartilage is visualized directly. Biochemical imaging techniques such as delayed gadolinium-enhanced MRI of cartilage, T2 mapping, and T1rho offer the potential to identify biochemical damage to cartilage before the onset of irreversible tissue loss. In the future, biomarkers may allow earlier detection of osteoarthritis before the development of radiographic evidence of disease.