Degradation of Proteoglycans and Collagen in Equine Meniscal Tissues.

Investigate meniscal extracellular matrix degradation. Equine menisci (n = 34 from 17 horses) were studied. Site-matched sections were cut and scored from three regions (ROIs; n = 102) and stained for histology, proteoglycan (safranin O and fast green), aggrecan, and collagen cleavage (NITEGE, DIPEN, and C1,2C antibodies, respectively). Picrosirius red and second harmonic generation microscopy were performed to investigate collagen ultrastructure. A total of 42 ROIs met the inclusion criteria and were included in the final analysis. The median (range) ROI histological score was 3 (0-9), providing a large spectrum of pathology. The median (range) proteoglycan score was 1 (0-3), representing superficial and central meniscal loss. The median (range) of DIPEN, NITEGE, and C1,2C scores was 1 (0-3), revealing immunostaining of the femoral and tibial surfaces. The proteoglycan scores exhibited significant positive associations with both histologic evaluation (p = 0.03) and DIPEN scores (p = 0.02). Additionally, a robust positive association (p = 0.007) was observed between the two aggrecanolysis indicators, NITEGE and DIPEN scores. A negative association (p = 0.008) was identified between NITEGE and histological scores. The C1,2C scores were not associated with any other scores. Picrosirius red and second harmonic generation microscopy (SHGM) illustrated the loss of the collagen matrix and structure centrally. Proteoglycan and collagen degradation commonly occur superficially in menisci and less frequently centrally. The identification of central meniscal proteoglycan and collagen degradation provides novel insight into central meniscal degeneration. However, further research is needed to elucidate the etiology and sequence of degradative events.

Correlation of meniscus tear type with synovial inflammation and the therapeutic potential of docosapentaenoic acid.

BACKGROUND: Synovitis, characterized by inflammation of the synovial membrane, is commonly induced by meniscus tears. However, significant differences in inflammatory responses and the key inflammatory mediators of synovium induced by different types of meniscal tears remain unclear. METHODS: Magnetic resonance imaging (MRI) was employed to identify the type of meniscus tear, and the quantification of synovial inflammation was assessed through H&E staining assay. Transcription and expression levels of IL-1ß and IL-6 were evaluated using bioinformatics, ELISA, RT-qPCR, and IHC of CD68 staining assays. The therapeutic potential of Docosapentaenoic Acid (DPA) was determined through network pharmacology, ELISA, and RT-qPCR assays. The safety of DPA was assessed using colony formation and EdU staining assays. RESULTS: The results indicate that both IL-1ß and IL-6 play pivotal roles in synovitis pathogenesis, with distinct expression levels across various subtypes. Among tested meniscus tears, oblique tear and bucket handle tear induced the most severe inflammation, followed by radial tear and longitudinal tear, while horizontal tear resulted in the least inflammation. Furthermore, in synovial inflammation induced by specific meniscus tears, the anterior medial tissues exhibited significantly higher local inflammation than the anterior lateral and suprapatellar regions, highlighting the clinical relevance and practical guidance of anterior medial tissues' inflammatory levels. Additionally, we identified the essential omega-3 fatty acid DPA as a potential therapeutic agent for synovitis, demonstrating efficacy in blocking the transcription and expression of IL-1ß and IL-6 with minimal side effects. CONCLUSION: These findings provide valuable insights into the nuanced nature of synovial inflammation induced by various meniscal tear classifications and contribute to the development of new adjunctive therapeutic agents in the management of synovitis.

Shedding light on the effects of blood on meniscus tissue: the role of mononuclear leukocytes in mediating meniscus catabolism.

OBJECTIVE: Traumatic meniscal injuries can cause acute pain, hemarthrosis (bleeding into the joint), joint immobility, and post-traumatic osteoarthritis (PTOA). However, the exact mechanism(s) by which PTOA develops following meniscal injuries is unknown. Since meniscus tears commonly coincide with hemarthrosis, investigating the direct effects of blood and its constituents on meniscus tissue is warranted. The goal of this study was to determine the direct effects of blood and blood components on meniscus tissue catabolism. METHODS: Porcine meniscus explants or primary meniscus cells were exposed to whole blood or various fractions of blood for 3 days to simulate blood exposure following injury. Explants were then washed and cultured for an additional 3 days prior to collection for biochemical analyses. RESULTS: Whole blood increased matrix metalloproteinase (MMP) activity. Fractionation experiments revealed blood-derived red blood cells did not affect meniscus catabolism. Conversely, viable mononuclear leukocytes induced MMP activity, nitric oxide (NO) production, and loss of tissue sulfated glycosaminoglycan (sGAG) content, suggesting that these cells are mediating meniscus catabolism. CONCLUSIONS: These findings highlight the potential challenges of meniscus healing in the presence of hemarthrosis and the need for further research to elucidate the in vivo effects of blood and blood-derived mononuclear leukocytes due to both hemarthrosis and blood-derived therapeutics.

Increased Wnt5a/ROR2 signaling is associated with chondrogenesis in meniscal degeneration.

The aim of the present study was to investigate the association between chondrogenic differentiation and Wnt signal expression in the degenerative process of the human meniscus. Menisci were obtained from patients with and without knee osteoarthritis (OA), and degeneration was histologically assessed using a grading system. Immunohistochemistry, real-time polymerase chain reaction (PCR), and Western blot analysis were performed to examine the expressions of chondrogenic markers and of the components of Wnt signaling. Histological analyses showed that meniscal degeneration involved a transition from a fibroblastic to a chondrogenic phenotype with the upregulation of SOX9, collagen type II, collagen type XI, and aggrecan, which were associated with increased Wnt5a and ROR2 and decreased TCF7 expressions. OA menisci showed significantly higher expressions of Wnt5a and ROR2 and significantly lower expressions of AXIN2 and TCF7 than non-OA menisci on real-time PCR and Western blot analysis. These results potentially demonstrated that increased expression of Wnt5a/ROR2 signaling promoted chondrogenesis with decreased expression in downstream Wnt/ß-catenin signaling. This study provides insights into the role of Wnt signaling in the process of meniscal degeneration, shifting to a chondrogenic phenotype. The findings suggested that the increased expression of Wnt5a/ROR2 and decreased expression of the downstream target of Wnt/ß-catenin signaling are associated with chondrogenesis in meniscal degeneration.

Age-dependent changes in collagen crosslinks reduce the mechanical toughness of human meniscus.

The mechanical resilience of the knee meniscus is provided by a group of structural proteins in the extracellular matrix. Aging can alter the quantity and molecular structure of these proteins making the meniscus more susceptible to debilitating tears. In this study, we determined the effect of aging on the quantity of structural proteins and collagen crosslinks in human lateral meniscus, and examined whether the quantity of these molecules was predictive of tensile toughness (area under the stress-strain curve). Two age groups were tested: a young group under 40 and an older group over 65 years old. Using mass spectrometry, we quantified the abundance of proteins and collagen crosslinks in meniscal tissue that was adjacent to the dumbbell-shaped specimens used to measure uniaxial tensile toughness parallel or perpendicular to the circumferential fiber orientation. We found that the enzymatic collagen crosslink deoxypyridinoline had a significant positive correlation with toughness, and reductions in the quantity of this crosslink with aging were associated with a loss of toughness in the ground substance and fibers. The non-enzymatic collagen crosslink carboxymethyl-lysine increased in quantity with aging, and these increases corresponded to reductions in ground substance toughness. For the collagenous (Types I, II, IV, VI, VIII) and non-collagenous structural proteins (elastin, decorin, biglycan, prolargin) analyzed in this study, only the quantity of collagen VIII was predictive of toughness. This study provides valuable insights on the structure-function relationships of the human meniscus, and how aging causes structural adaptations that weaken the tissue's mechanical integrity.

Comparative Effects of Intra-Articular versus Intravenous Mesenchymal Stromal Cells Therapy in a Rat Model of Osteoarthritis by Destabilization of Medial Meniscus.

Transplanted mesenchymal stromal cells (MSCs) exhibit a robust anti-inflammatory and homing capacity in response to high inflammatory signals, as observed in studies focused on rheumatic diseases that target articular cartilage (AC) health. However, AC degradation in osteoarthritis (OA) does not necessarily coincide with a highly inflammatory joint profile. Often, by the time patients seek medical attention, they already have damaged AC. In this study, we examined the therapeutic potential of a single bone marrow MSC transplant (2 × 106 cells/kgbw) through two different routes: intra-articular (MSCs-IAt) and intravenous (MSCs-IVt) in a preclinical model of low-grade inflammatory OA with an established AC degeneration. OA was induced through the destabilization of the medial meniscus (DMM) in female Wistar Kyoto rats. The animals received MSCs 9 weeks after surgery and were euthanized 4 and 12 weeks post-transplant. In vivo and ex vivo tracking of MSCs were analyzed via bioluminescence and imaging flow cytometry, respectively. Cytokine/chemokine modulation in serum and synovial fluid was measured using a multiplex panel. AC degeneration was quantified through histology, and hindlimb muscle balance was assessed with precision weighing. To our knowledge, we are the first group to show the in vivo (8 h) and ex vivo (12 h) homing of cells to the DMM-OA joint following MSCs-IVt. In the case of MSCs-IAt, the detection of cellular bioluminescence at the knee joint persisted for up to 1 week. Intriguingly, intra-articular saline injection (placebo-IAt) resulted in a worse prognosis of OA when compared to a non-invasive control (placebo-IVt) without joint injection. The systemic cytokines/chemokines profile exhibited a time-dependent variation between transplant routes, displaying a transient anti-inflammatory systemic response for both MSCs-IVt and MSCs-IAt. A single injection of MSCs, whether administered via the intra-articular or intravenous route, performed 9 weeks after DMM surgery, did not effectively inhibit AC degeneration when compared to a non-invasive control.

Deletion of the chondrocyte glucocorticoid receptor attenuates cartilage degradation through suppression of early synovial activation in murine posttraumatic osteoarthritis.

OBJECTIVE: Disruption of endogenous glucocorticoid signalling in bone cells attenuates osteoarthritis (OA) in aged mice, however, the role of endogenous glucocorticoids in chondrocytes is unknown. Here, we investigated whether deletion of the glucocorticoid receptor, specifically in chondrocytes, also alters OA progression. DESIGN: Knee OA was induced by surgical destabilisation of the medial meniscus (DMM) in male 22-week-old tamoxifen-inducible glucocorticoid receptor knockout (chGRKO) mice and their wild-type (WT) littermates (n = 7-9/group). Mice were harvested 2, 4, 8 and 16 weeks after surgery to examine the spatiotemporal changes in molecular, cellular, and histological characteristics. RESULTS: At all time points following DMM, cartilage damage was significantly attenuated in chGRKO compared to WT mice. Two weeks after DMM, WT mice exhibited increased chondrocyte and synoviocyte hypoxia inducible factor (HIF)-2α expression resulting in extensive synovial activation characterised by synovial thickening and increased interleukin-1 beta expression. At 2 and 4 weeks after DMM, WT mice displayed pronounced chondrocyte senescence and elevated catabolic signalling (reduced Yes-associated protein 1 (YAP1) and increased matrix metalloprotease [MMP]-13 expression). Contrastingly, at 2 weeks after DMM, HIF-2α expression and synovial activation were much less pronounced in chGRKO than in WT mice. Furthermore, chondrocyte YAP1 and MMP-13 expression, as well as chondrocyte senescence were similar in chGRKO-DMM mice and sham-operated controls. CONCLUSION: Endogenous glucocorticoid signalling in chondrocytes promotes synovial activation, chondrocyte senescence and cartilage degradation by upregulation of catabolic signalling through HIF-2α in murine posttraumatic OA. These findings indicate that inhibition of glucocorticoid signalling early after injury may present a promising way to slow osteoarthritic cartilage degeneration.

Oral Administration of Bovine Milk-Derived Extracellular Vesicles Attenuates Cartilage Degeneration via Modulating Gut Microbiota in DMM-Induced Mice.

Osteoarthritis (OA) is the most common joint disease primarily characterized by cartilage degeneration. Milk-derived extracellular vesicles (mEVs) were reported to inhibit catabolic and inflammatory processes in the cartilage of OA patients. However, the current therapies target the advanced symptoms of OA, and it is significant to develop a novel strategy to inhibit the processes driving OA pathology. In this study, we investigated the therapeutic potential of mEVs in alleviating OA in vivo. The results revealed that mEVs ameliorated cartilage degeneration by increasing hyaline cartilage thickness, decreasing histological Osteoarthritis Research Society International (OARSI) scores, enhancing matrix synthesis, and reducing the expression of cartilage destructive enzymes in the destabilization of medial meniscus (DMM) mice. In addition, the disturbed gut microbiota in DMM mice was partially improved upon treatment with mEVs. It was observed that the pro-inflammatory bacteria (Proteobacteria) were reduced and the potential beneficial bacteria (Firmicutes, Ruminococcaceae, Akkermansiaceae) were increased. mEVs could alleviate the progression of OA by restoring matrix homeostasis and reshaping the gut microbiota. These findings suggested that mEVs might be a potential therapeutic dietary supplement for the treatment of OA.

Advanced glycation end-product accumulation differs by location and sex in aged osteoarthritic human menisci.

OBJECTIVE: There is a clear link between increasing age and meniscus degeneration, leading to increased injury, osteoarthritis (OA) progression, and often total knee replacement. Advanced glycation end-products (AGEs) are non-enzymatic crosslinks and adducts that accumulate in collagen with age, altering tissue mechanics and cell function, ultimately leading to increased injury and inflammation. AGEs, both fluorescent and non-fluorescent, play a central role in age-related degradation of tissues throughout the body; however, little is known about their role in meniscus degeneration. The objective of this study was to characterize changes in aged OA menisci, specifically evaluating zonal AGE accumulation, to gain a better understanding of changes that may lead to age-related meniscal degeneration. METHOD: Deidentified human menisci (N = 48, 52-84 years old) were obtained from subjects undergoing total knee replacement. Changes in extracellular matrix (ECM) were assessed by gross morphology, confocal analysis, and biochemical assays. Deoxyribonucleic acid (DNA), glycosaminoglycan (GAG), collagen, and AGE accumulation were compared with patient age, zonal region, and patient sex. RESULTS: There were minimal changes in DNA, GAG, and collagen concentration with age or zone. However, collagen fraying and AGEs increased with age, with more AGEs accumulating in the meniscal horns compared to the central body and in male menisci compared to females. CONCLUSIONS: Overall, this work provides greater insights into regional changes that occur in human menisci with age and OA. These results suggest AGEs may play a role in the degeneration of the meniscus, with AGEs being a possible target to reduce age-related tears, degeneration, and OA progression.

Induced inactivation of Wnt16 in young adult mice has no impact on osteoarthritis development.

Osteoarthritis (OA) is a common disorder and a major cause of disability in the elderly population. WNT16 has been suggested to play important roles in joint formation, bone homeostasis and OA development, but the mechanism of action is not clear. Transgenic mice lacking Wnt16 expression (Wnt16-/-) have a more severe experimental OA than control mice. In addition, Wnt16-/- mice have a reduced cortical thickness and develop spontaneous fractures. Herein, we have used Cre-Wnt16flox/flox mice in which Wnt16 can be conditionally ablated at any age through tamoxifen-inducible Cre-mediated recombination. Wnt16 deletion was induced in 7-week-old mice to study if the Cre-Wnt16flox/flox mice have a more severe OA phenotype after destabilizing the medial meniscus (DMM surgery) than littermate controls with normal Wnt16 expression (Wnt16flox/flox). WNT16 deletion was confirmed in articular cartilage and cortical bone in Cre-Wnt16flox/flox mice, shown by immunohistochemistry and reduced cortical bone area compared to Wnt16flox/flox mice. After DMM surgery, there was no difference in OA severity in the articular cartilage in the knee joint between the Cre-Wnt16flox/flox and Wnt16flox/flox mice in neither female nor male mice. In addition, there was no difference in osteophyte size in the DMM-operated tibia between the genotypes. In conclusion, inactivation of Wnt16 in adult mice do not result in a more severe OA phenotype after DMM surgery. Thus, presence of WNT16 in adult mice does not have an impact on experimental OA development. Taken together, our results from Cre-Wnt16flox/flox mice and previous results from Wnt16-/- mice suggest that WNT16 is crucial during synovial joint establishment leading to limited joint degradation also later in life, after onset of OA. This may be important when developing new therapeutics for OA treatment.

Evaluating the Efficacy of Combined P188 Treatment and Surgical Intervention in Preventing Post-Traumatic Osteoarthritis Following a Traumatic Knee Injury.

Previous studies have shown that reconstructive surgery alone following injury to the anterior cruciate ligament (ACL) does not prevent the development of post-traumatic osteoarthritis (PTOA). Poloxamer 188 (P188) has been shown to prevent cell death following trauma in both articular cartilage and meniscal tissue. This study aims to test the efficacy of single or multiple administrations of P188 in conjunction with reconstructive surgery to help prevent or delay the onset of the disease. Thirty skeletally mature rabbits underwent closed-joint trauma that resulted in ACL rupture and meniscal damage and were randomly assigned to one of four treatment groups with varying doses of P188. ACL reconstruction was then performed using an autograft from the semitendinosus tendon. Animals were euthanized 1-month following trauma, meniscal tissue was assessed for changes in morphology, mechanical properties, and proteoglycan content. Femurs and tibias were scanned using microcomputed tomography to determine changes in bone quality, architecture, and osteophyte formation. The medial meniscus experienced more damage and a decrease in the instantaneous modulus regardless of treatment group, while P188 treatment tended to limit degenerative changes in the lateral meniscus. Both lateral and medial menisci had documented decreases in the equilibrium modulus and inconsistent changes in proteoglycan content. Minimal changes were documented in the tibias and femurs, with the only significant change being the formation of osteophytes in both bones regardless of treatment group. The data suggest that P188 was able to limit some degenerative changes in the meniscus associated with PTOA and may warrant future studies.

Testing Hypoxia in Pig Meniscal Culture: Biological Role of the Vascular-Related Factors in the Differentiation and Viability of Neonatal Meniscus.

Menisci play an essential role in shock absorption, joint stability, load resistance and its transmission thanks to their conformation. Adult menisci can be divided in three zones based on the vascularization: an avascular inner zone with no blood supply, a fully vascularized outer zone, and an intermediate zone. This organization, in addition to the incomplete knowledge about meniscal biology, composition, and gene expression, makes meniscal regeneration still one of the major challenges both in orthopedics and in tissue engineering. To overcome this issue, we aimed to investigate the role of hypoxia in the differentiation of the three anatomical areas of newborn piglet menisci (anterior horn (A), central body (C), and posterior horn (P)) and its effects on vascular factors. After sample collection, menisci were divided in A, C, P, and they were cultured in vitro under hypoxic (1% O2) and normoxic (21% O2) conditions at four different experimental time points (T0 = day of explant; T7 = day 7; T10 = day 10; T14 = day 14); samples were then evaluated through immune, histological, and molecular analyses, cell morpho-functional characteristics; with particular focus on matrix composition and expression of vascular factors. It was observed that hypoxia retained the initial phenotype of cells and induced extracellular matrix production resembling a mature tissue. Hypoxia also modulated the expression of angiogenic factors, especially in the early phase of the study. Thus, we observed that hypoxia contributes to the fibro-chondrogenic differentiation with the involvement of angiogenic factors, especially in the posterior horn, which corresponds to the predominant weight-bearing portion.

Knee Osteoarthritis Progression Is Delayed in Silent Information Regulator 2 Ortholog 1 Knock-in Mice.

Overexpression of silent information regulator 2 ortholog 1 (SIRT1) is associated with beneficial roles in aging-related diseases; however, the effects of SIRT1 overexpression on osteoarthritis (OA) progression have not yet been studied. The aim of this study was to investigate OA progression in SIRT1-KI mice using a mouse OA model. OA was induced via destabilization of the medial meniscus using 12-week-old SIRT1-KI and wild type (control) mice. OA progression was evaluated histologically based on the Osteoarthritis Research Society International (OARSI) score at 4, 8, 12, and 16 weeks after surgery. The production of SIRT1, type II collagen, MMP-13, ADAMTS-5, cleaved caspase 3, Poly (ADP-ribose) polymerase (PARP) p85, acetylated NF-κB p65, interleukin 1 beta (IL-1ß), and IL-6 was examined via immunostaining. The OARSI scores were significantly lower in SIRT1-KI mice than those in control mice at 8, 12, and 16 weeks after surgery. The proportion of SIRT1 and type II collagen-positive-chondrocytes was significantly higher in SIRT1-KI mice than that in control mice. Moreover, the proportion of MMP-13-, ADAMTS-5-, cleaved caspase 3-, PARP p85-, acetylated NF-κB p65-, IL-1ß-, and IL-6-positive chondrocytes was significantly lower in SIRT1-KI mice than that in control mice. The mechanically induced OA progression was delayed in SIRT1-KI mice compared to that in control mice. Therefore, overexpression of SIRT1 may represent a mechanism for delaying OA progression.

BNIP3-Dependent Mitophagy via PGC1α Promotes Cartilage Degradation.

Since mitochondria are suggested to be important regulators in maintaining cartilage homeostasis, turnover of mitochondria through mitochondrial biogenesis and mitochondrial degradation may play an important role in the pathogenesis of osteoarthritis (OA). Here, we found that mitochondrial dysfunction is closely associated with OA pathogenesis and identified the peroxisome proliferator-activated receptor-gamma co-activator 1-alpha (PGC1α) as a potent regulator. The expression level of PGC1α was significantly decreased under OA conditions, and knockdown of PGC1α dramatically elevated the cartilage degradation by upregulating cartilage degrading enzymes and apoptotic cell death. Interestingly, the knockdown of PGC1α activated the parkin RBR E3 ubiquitin protein ligase (PRKN)-independent selective mitochondria autophagy (mitophagy) pathway through the upregulation of BCL2 and adenovirus E1B 19-kDa-interacting protein 3 (BNIP3). The overexpression of BNIP3 stimulated mitophagy and cartilage degradation by upregulating cartilage-degrading enzymes and chondrocyte death. We identified microRNA (miR)-126-5p as an upstream regulator for PGC1α and confirmed the direct binding between miR-126-5p and 3' untranslated region (UTR) of PGC1α. An in vivo OA mouse model induced by the destabilization of medial meniscus (DMM) surgery, and the delivery of antago-miR-126 via intra-articular injection significantly decreased cartilage degradation. In sum, the loss of PGC1α in chondrocytes due to upregulation of miR-126-5p during OA pathogenesis resulted in the activation of PRKN-independent mitophagy through the upregulation of BNIP3 and stimulated cartilage degradation and apoptotic death of chondrocytes. Therefore, the regulation of PGC1α:BNIP3 mitophagy axis could be of therapeutic benefit to cartilage-degrading diseases.

Curcumenol mitigates chondrocyte inflammation by inhibiting the NF­κB and MAPK pathways, and ameliorates DMM­induced OA in mice.

At present, an increasing number of individuals are affected by osteoarthritis (OA), resulting in a heavy socioeconomic burden. OA in knee joints is caused by the release of inflammatory cytokines and subsequent biomechanical and structural deterioration. To determine its anti­inflammatory function, the current study investigated the use of the plant­derived medicine, curcumenol, in OA treatment. Curcumenol was not cytotoxic to ATDC5 chondrocytes and primary chondrocytes, as determined using a cell viability test. When these cells were treated with TNF­α and IL­1ß to induce inflammation, curcumenol treatment inhibited the progression of inflammation by inactivating the NF­κB and MAPK signaling pathways, as well as decreasing the expression levels of MMP3 (as indicated by reverse transcription­quantitative PCR and western blotting). Moreover, to analyze metabolic and catabolic status in high­density and pellet culture, catalytic changes and the degradation of the extracellular matrix induced by TNF­α and IL­1ß, were evaluated by alcian blue staining. These catalytic deteriorations were ameliorated by curcumenol. Using curcumenol in disease management, the mechanical and metabolic disruption of cartilage caused in the destabilization of medial meniscus (DMM) model was prevented in vivo. Thus, curcumenol mitigated inflammation in ATDC5 chondrocytes and primary mice chondrocytes, and also ameliorated OA in a DMM­induced mouse model.

The critical role of Hedgehog-responsive mesenchymal progenitors in meniscus development and injury repair.

Meniscal tears are associated with a high risk of osteoarthritis but currently have no disease-modifying therapies. Using a Gli1 reporter line, we found that Gli1+ cells contribute to the development of meniscus horns from 2 weeks of age. In adult mice, Gli1+ cells resided at the superficial layer of meniscus and expressed known mesenchymal progenitor markers. In culture, meniscal Gli1+ cells possessed high progenitor activities under the control of Hh signal. Meniscus injury at the anterior horn induced a quick expansion of Gli1-lineage cells. Normally, meniscal tissue healed slowly, leading to cartilage degeneration. Ablation of Gli1+ cells further hindered this repair process. Strikingly, intra-articular injection of Gli1+ meniscal cells or an Hh agonist right after injury accelerated the bridging of the interrupted ends and attenuated signs of osteoarthritis. Taken together, our work identified a novel progenitor population in meniscus and proposes a new treatment for repairing injured meniscus and preventing osteoarthritis.

Involvement of Transient Receptor Potential Vanilloid Channel 2 in the Induction of Lubricin and Suppression of Ectopic Endochondral Ossification in Mouse Articular Cartilage.

OBJECTIVE: Transient receptor potential vanilloid channel 2 (TRPV2) is a Ca2+ -permeable channel and plays a role in mediating intracellular Ca2+ current via mechanical stimuli. This study was undertaken to examine the expression and role of TRPV2 in adult articular cartilage and the development of osteoarthritis (OA). METHODS: We examined TRPV2 expression in mouse and human articular cartilage. We analyzed the development of OA in Col2a1-CreERt2 ;Trpv2fl/fl mice and Trpv2fl/fl littermates in the resection of the medial meniscus and medial collateral ligament model (n = 5 each), the destabilization of the medial meniscus model (n = 5 each), and the aging mouse model (n = 8-9 each). We examined marker protein expression in these joints, Ca2+ influx by mechanical stimuli, and downstream pathways in vitro. RESULTS: TRPV2 was expressed in mouse and human articular cartilage and ectopic ossification lesions. In all mouse models of OA examined, Col2a1-CreERt2 ;Trpv2fl/fl mice were observed to have enhanced degradation of articular cartilage accompanied by decreased expression of lubricin/Prg4, and marked formation of periarticular ectopic ossification. Mechanical stress-induced Ca2+ influx was decreased by Trpv2 knockout (KO). Prg4 induction by fluid-flow shear stress was diminished in Trpv2-KO mouse chondrocytes, and this was mediated by the Ca2+ /calmodulin-dependent protein kinase kinase-cyclic AMP response element binding protein axis. Hypertrophic differentiation was enhanced in Trpv2-KO mouse chondrocytes. Increased activity of calcineurin and nuclear translocation of nuclear factor in activated T cells 1 induced by fluid-flow shear stress or TRP agonist treatment was reversed by Trpv2 knockout. CONCLUSION: Our findings demonstrate regulation of articular cartilage by TRPV2 through Prg4 induction and suppression of ectopic ossification.

Lipopolysaccharide Binding Protein and CD14, Cofactors of Toll-like Receptors, Are Essential for Low-Grade Inflammation-Induced Exacerbation of Cartilage Damage in Mouse Models of Posttraumatic Osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is initiated by pathogenic factors produced by multiple stimuli, including mechanical stress, metabolic stress, and/or inflammaging. This study was undertaken to identify novel low-grade inflammation-associated pathogenic mediators of OA. METHODS: Candidate pathogenic molecules were screened using microarray data obtained from chondrocytes exposed to OA-associated catabolic factors. In mice with OA generated by destabilization of the medial meniscus (DMM), low-grade inflammation was induced by a high-fat diet or endotoxemia. Functions of candidate molecules in OA pathogenesis were examined using primary-culture chondrocytes from mice with DMM-induced OA, following intraarticular injection of adenovirus expressing the candidate gene. Specific functions of candidate genes were evaluated using whole-body gene-knockout mice. RESULTS: Bioinformatics analysis identified multiple candidate pathogenic factors that were associated with low-grade inflammation, including components of the Toll-like receptor (TLR) signaling pathways (e.g., TLR-2, TLR-4, lipopolysaccharide binding protein [LBP], and CD14). Overexpression of the individual TLR signaling components in mouse joint tissue did not alter cartilage homeostasis. However, the low-grade inflammation induced by a high-fat diet or endotoxemia markedly enhanced posttraumatic OA cartilage destruction in mice, and this exacerbation of cartilage destruction was significantly abrogated in LBP-/- and CD14-/- mice. Additionally, LBP and CD14 were found to be necessary for the expression of matrix-degrading enzymes in mouse chondrocytes treated with proinflammatory cytokines. CONCLUSION: LBP and CD14, which are accessory molecules of TLRs, are necessary for the exacerbation of posttraumatic OA cartilage destruction resulting from low-grade inflammation, such as that triggered by a high-fat diet or endotoxemia.

Identification of tissue-dependent proteins in knee OA synovial fluid.

OBJECTIVE: For many proteins from osteoarthritic synovial fluid, their intra-articular tissue of origin remains unknown. In this study we performed comparative proteomics to identify osteoarthritis-specific and joint tissue-dependent secreted proteins that may serve as candidates for osteoarthritis biomarker development on a tissue-specific basis. DESIGN: Protein secretomes of cartilage, synovium, Hoffa's fat pad and meniscus from knee osteoarthritis patients were determined using liquid chromatography tandem mass spectrometry, followed by label-free quantification. Validation of tissue-dependent protein species was conducted by ELISA on independent samples. Differential proteomes of osteoarthritic and non-osteoarthritic knee synovial fluids were obtained via similar proteomics approach, followed by ELISA validation. RESULTS: Proteomics revealed 64 proteins highly secreted from cartilage, 94 from synovium, 37 from Hoffa's fat pad and 21 from meniscus. Proteomic analyses of osteoarthritic vs non-osteoarthritic knee synovial fluid revealed 70 proteins with a relatively higher abundance and 264 proteins with a relatively lower abundance in osteoarthritic synovial fluid. Of the 70 higher abundance proteins, 23 were amongst the most highly expressed in the secretomes of a specific intra-articular tissue measured. Tissue-dependent release was validated for SLPI, C8, CLU, FN1, RARRES2, MATN3, MMP3 and TNC. Abundance in synovial fluid of tissue-dependent proteins was validated for IGF2, AHSG, FN1, CFB, KNG and C8. CONCLUSIONS: We identified proteins with a tissue-dependent release from intra-articular human knee OA tissues. A number of these proteins also had an osteoarthritis-specific abundance in knee synovial fluid. These proteins may serve as novel candidates for osteoarthritis biomarker development on a tissue-specific basis.

Degeneration alters the biomechanical properties and structural composition of lateral human menisci.

OBJECTIVE: Because the literature relating to the influence of degeneration on the viscoelasticity and tissue composition of human lateral menisci remains contradictory or completely lacking, the aim of this study was to fill these gaps by comprehensively characterising the biomechanical properties of menisci with regard to the degree of degeneration. DESIGN: Meniscal tissue from 24 patients undergoing a total knee replacement was collected and the degeneration of each region classified according to Pauli et al. For biomechanical characterisation, compression and tensile tests were performed. Additionally, the water content was determined and infrared (IR) spectroscopy was applied to detect changes in the structural composition, particularly of the proteoglycan and collagen content. RESULTS: With an increasing degree of degeneration, a significant decrease of the equilibrium modulus was detected, while simultaneously the water content and the hydraulic permeability significantly increased. However, the tensile modulus displayed a tendency to decrease with increasing degeneration, which might be due to the significantly decreasing amount of collagen content identified by the IR measurements. CONCLUSION: The findings of the current study may contribute to the understanding of meniscus degeneration, showing that degenerative processes appear to mainly worsen viscoelastic properties of the inner circumference by disrupting the collagen integrity.