Cartilage Of Mice Research Articles

Bacterial nanocellulose-reinforced gelatin methacryloyl hydrogel enhances biomechanical property and glycosaminoglycan content of 3D-bioprinted cartilage.

Tissue-engineered ear cartilage scaffold based on three-dimensional (3D) bioprinting technology presents a new strategy for ear reconstruction in individuals with microtia. Natural hydrogel is a promising material due to its excellent biocompatibility and low immunogenicity. However, insufficient mechanical property required for cartilage is one of the major issues pending to be solved. In this study, the gelatin methacryloyl (GelMA) hydrogel reinforced with bacterial nanocellulose (BNC) was developed to enhance the biomechanical properties and printability of the hydrogel. The results revealed that the addition of 0.375% BNC significantly increased the mechanical properties of the hydrogel and promoted cell migration in the BNC-reinforced hydrogel. Constructs bioprinted with chondrocyte-laden BNC/GelMA hydrogel bio-ink formed mature cartilage in nude mice with higher Young's modulus and glycosaminoglycan content. Finally, an auricle equivalent with a precise shape, high mechanics, and abundant cartilage-specific matrix was developed in vivo. In this study, we developed a potentially useful hydrogel for the manufacture of auricular cartilage grafts for microtia patients.

Yap and Taz promote osteogenesis and prevent chondrogenesis in neural crest cells in vitro and in vivo.

Neural crest cells (NCCs) are multipotent stem cells that can differentiate into multiple cell types, including the osteoblasts and chondrocytes, and constitute most of the craniofacial skeleton. Here, we show through in vitro and in vivo studies that the transcriptional regulators Yap and Taz have redundant functions as key determinants of the specification and differentiation of NCCs into osteoblasts or chondrocytes. Primary and cultured NCCs deficient in Yap and Taz switched from osteogenesis to chondrogenesis, and NCC-specific deficiency for Yap and Taz resulted in bone loss and ectopic cartilage in mice. Yap bound to the regulatory elements of key genes that govern osteogenesis and chondrogenesis in NCCs and directly regulated the expression of these genes, some of which also contained binding sites for the TCF/LEF transcription factors that interact with the Wnt effector β-catenin. During differentiation of NCCs in vitro and NCC-derived osteogenesis in vivo, Yap and Taz promoted the expression of osteogenic genes such as Runx2 and Sp7 but repressed the expression of chondrogenic genes such as Sox9 and Col2a1. Furthermore, Yap and Taz interacted with β-catenin in NCCs to coordinately promote osteoblast differentiation and repress chondrogenesis. Together, our data indicate that Yap and Taz promote osteogenesis in NCCs and prevent chondrogenesis, partly through interactions with the Wnt-β-catenin pathway.

A Pilot Analysis of Genome-Wide DNA Methylation Patterns in Mouse Cartilage Reveals Overlapping Epigenetic Signatures of Aging and Osteoarthritis.

Cartilage epigenetic changes are strongly associated with human osteoarthritis (OA). However, the influence of individual environmental OA risk factors on these epigenetic patterns has not been determined; herein we characterize cartilage DNA methylation patterns associated with aging and OA in a mouse model. Murine knee cartilage DNA was extracted from healthy young (16-week, n=6), old (82-week, n=6), and young 4-week post-destabilization of the medial meniscus (DMM) OA (n=6) C57BL6/J mice. Genome-wide DNA methylation patterns were determined via Illumina BeadChip. Gene set enrichment analysis was performed by Ingenuity Pathway Analysis. The top seven most differentially methylated positions (DMPs) were confirmed by pyrosequencing in an independent animal set. Results were compared to previously published human OA methylation data. Aging was associated with 20,940 DMPs, whereas OA was associated with 761 DMPs. Merging these two conditions revealed 279 shared DMPs. All demonstrated similar directionality and magnitude of change (Δβ 1.0% ± 0.2%, mean methylation change ± SEM). Shared DMPs were enriched in OA-associated pathways, including RhoA signaling (P=1.57 × 10-4 ), protein kinase A signaling (P=3.38 × 10-4 ), and NFAT signaling (P=6.14 × 10-4 ). Upstream regulators, including TET3 (P=6.15 × 10-4 ), immunoglobulin (P=6.14 × 10-4 ), and TLR7 (P=7.53 × 10-4 ), were also enriched. Pyrosequencing confirmed six of the seven top DMPs in an independent cohort. Aging and early OA following DMM surgery induce similar DNA methylation changes within a murine OA model, suggesting that aging may induce pro-OA epigenetic "poising" within articular cartilage. Future research should focus on confirming and expanding these findings to other environmental OA risk factors, including obesity, as well as determining late OA changes in mice.

MiR-124-3p sabotages lncRNA MALAT1 stability to repress chondrocyte pyroptosis and relieve cartilage injury in osteoarthritis

BackgroundOsteoarthritis (OA) is a prevalent inflammatory joint disorder. microRNAs (miRNAs) are increasingly involved in OA.AimOur study is proposed to clarify the role of miR-124-3p in chondrocyte pyroptosis and cartilage injury in OA.MethodsOA mouse model was established via the treatment of destabilization of the medial meniscus (DMM), and the in vitro cell model was also established as mouse chondrocytes were induced by lipopolysaccharide (LPS). Mouse cartilage injury was assessed using safranin-O-fast green staining, hematoxylin–eosin staining, and OARSI grading method. Expressions of miR-124-3p, MALAT1, KLF5, and CXCL11 were determined. Cartilage injury (MMP-13, osteocalcin), inflammation (IL-6, IL-2, TNF-, IL-1β, and IL-18)- and pyroptosis-related factors (Cleaved Caspase-1 and GSDMD-N) levels were detected. Mechanically, MALAT1 subcellular localization was confirmed. The binding relationships of miR-124-3p and MALAT1 and MALAT1 and KLF5 were verified. MALAT1 half-life period was detected. Then, miR-124-3p was overexpressed using agomiR-124-3p to perform the rescue experiments with oe-MALAT1 or oe-CXCL11.ResultsmiR-124-3p was downregulated in DMM mice and LPS-induced chondrocytes where cartilage injury, and increased levels of inflammation- and pyroptosis-related factors were found. miR-124-3p overexpression relieved cartilage injury and repressed chondrocyte pyroptosis. miR-124-3p bounds to MALAT1 to downregulate its stability and expression, and MALAT1 bounds to KLF5 to enhance CXCL11 transcription. Overexpression of MALAT1 or CXCL11 annulled the repressive function of miR-124-3p in chondrocyte pyroptosis.ConclusionmiR-124-3p reduced MALAT1 stability and inhibited the binding of MALAT1 and KLF5 to downregulate CXCL11, thereby suppressing chondrocyte pyroptosis and cartilage injury in OA.

Direct Reprogramming of Mouse Subchondral Bone Osteoblasts into Chondrocyte-like Cells.

Treatment of full-thickness articular cartilage defects with exposure of subchondral bone often seen in osteoarthritic conditions has long been a great challenge, especially with a focus on the feasibility of in situ cartilage regeneration through minimally invasive procedures. Osteoblasts that situate in the subchondral bone plate may be considered a potentially vital endogenous source of cells for cartilage resurfacing through direct reprogramming into chondrocytes. Microarray-based gene expression profiles were generated to compare tissue-specific transcripts between subchondral bone and cartilage of mice and to assess age-dependent differences of chondrocytes as well. On osteoblast cell lines established from mouse proximal tibial subchondral bone, sequential screening by co-transduction of transcription factor (TF) genes that distinguish chondrocytes from osteoblasts reveals a shortlist of potential reprogramming factors exhibiting combined effects in inducing chondrogenesis of subchondral bone osteoblasts. A further combinatorial approach unexpectedly identified two 3-TF combinations containing Sox9 and Sox5 that exhibit differences in reprogramming propensity with the third TF c-Myc or Plagl1, which appeared to direct the converted chondrocytes toward either a superficial or a deeper zone phenotype. Thus, our approach demonstrates the possibility of converting osteoblasts into two major chondrocyte subpopulations with two combinations of three genes (Sox9, Sox5, and c-Myc or Plagl1). The findings may have important implications for developing novel in situ regeneration strategies for the reconstruction of full-thickness cartilage defects.

Construction and pathological characterization of 3 animal models of temporomandibular joint degenerative joint disease in mice

Objective: To explore the pathological characteristics of three mice models of temporomandibular joint degenerative joint disease (TMJDJD), including osteoarthritis and osteoarthrosis, and to provide references for animal experimental study regarding the pathological mechanism of osteoarthritis and osteoarthrosis. Methods: A total of 54 8-week-old male C57BL/6 mice were selected to construct three TMJDJD animal models, including bilateral temporomandibular joint (TMJ) Freund's complete adjuvant (FCA) injection model, bilateral TMJ monosodium iodoacetate (MIA) injection model, and right TMJ discectomy model. FCA injection model (15 mice) was divided into saline injection group, FCA injection group-1 week, FCA injection group-2 week, FCA injection group-4 week and FCA injection group-6 week, 3 mice were used at each time point, with a total of 6 TMJs on both sides. MIA injection model (15 mice) was separated into saline injection group, MIA injection group-1 week, MIA injection group-2 week, MIA injection group-4 week and MIA injection group-6 week, 3 mice were used at each time point, with a total of 6 TMJs on both sides. TMJ discectomy model (24 mice) was split into control group, discectomy group-2 week group, discectomy group-4 week and discectomy group-6 week, six mice were used at each time point, with a total of six right TMJs. General pictures of the bilateral joints area of mice were collected 1 day after drug injection, and stereoscopic images of condylar tissues were collected 4 weeks after microsurgery for discectomy. Mouse TMJ tissue sections from each time point were stained with HE and toluidine blue, respectively, synovial tissues were scored for synovial inflammation, and the percentage of proteoglycan in condylar cartilage was quantitatively analyzed. Results: One day after intra-articular FCA or MIA injection, the width of bilateral TMJ were significantly increased in FCA injection groups [(24.60±0.46) mm] compared with the saline injection group [(21.63±0.52) mm] (t=4.25, P<0.013), the width of bilateral TMJ in MIA injection groups [(24.50±0.62) mm] were also significantly higher than that in saline injection group [(21.40±0.52) mm] (t=3.82, P=0.019). The synovitis scores in FCA injection groups 1, 2, 4, 6 weeks after FCA injection were significantly higher than that of the saline injection group (F=18.09, P<0.001), with the proteoglycan of condylar cartilage increased firstly and then decreased compared with the saline injection group (F=21.59, P<0.001). Condylar cartilage proteoglycan loss in different degrees were observed 1, 2, 4 and 6 weeks after MIA injection (F=13.59, P<0.001), and synovitis scores were increased at different degrees compared with saline injection group (F=14.79, P<0.001). The morphology of condylar cartilage in discectomy groups mice were severely damaged, synovial tissues showed dense connective tissue lesions at 2, 4 and 6 weeks postoperatively, condylar cartilage tissues showed a time-dependent loss of proteoglycan compared with the control group (F=40.62, P<0.001). Conclusions: Intra-articular FCA injection establishes a mouse model of TMJ osteoarthritis with severe synovial inflammation. Intra-articular MIA injection constructs a mouse model of typical TMJ osteoarthritis. Discectomy establishes a mouse TMJ osteoarthrosis model with severe condylar cartilage destruction.

Synovial fluid mesenchymal progenitor cells from patients with juvenile idiopathic arthritis demonstrate limited self-renewal and chondrogenesis

Juvenile idiopathic arthritis (JIA) is a heterogeneous group of inflammatory diseases affecting joints with a prevalence of one in a thousand children. There is a growing body of literature examining the use of mesenchymal stem/progenitor cells (MPCs) for the treatment of adult and childhood arthritis, however, we still lack a clear understanding of how these MPC populations are impacted by arthritic disease states and how this could influence treatment efficacy. In the current study we examined the immunophenotyping, self-renewal ability and chondrogenic capacity (in vitro and in vivo) of synovial derived MPCs from normal, JIA and RA joints. Synovial MPCs from JIA patients demonstrated reduced self-renewal ability and chondrogenic differentiation capacity. Furthermore, they did not induce cartilage regeneration when xenotransplanted in a mouse cartilage injury model. Synovial MPCs from JIA patients are functionally compromised compared to MPCs from normal and/or RA joints. The molecular mechanisms behind this loss of function remain elusive. Further study is required to see if these cells can be re-functionalized and used in cell therapy strategies for these JIA patients, or if allogenic approaches should be considered.

Mesenchymal cell-derived Wnt1 signaling regulates subchondral bone remodeling but has no effects on the development of growth plate or articular cartilage in mice.

Chondrocyte differentiation is a principal progress in endochondral ossification and in the formation of secondary ossification center (SOC) during the long bone development. We have previously reported that targeted deletion of Wnt1 in mesenchymal progenitors (Wnt1Prrx-/-) leads to spontaneous fractures and severe osteopenia in mouse long bones, suggesting that Wnt1 is a key regulator of bone metabolism. However, the effect of Wnt1 on the regulation of cartilage development and chondrocyte differentiation remained unknown. In this study, WNT1 protein expression was observed in lateral superficial cartilage and growth plate pre-hypertrophic chondrocytes in mice. Wnt1 mRNA expression was detected in epiphyseal cartilage from E16.5 to 3month-old mice. Detailed histological analyses revealed that the average thickness and chondrocyte density of proximal tibial articular cartilage and growth plate were unchanged between Wnt1Prrx-/- and control mice. However, μCT analysis of tibial epiphyses showed that the subchondral bone mass was reduced in Wnt1Prrx-/- mice compared to control mice, as demonstrated by decreased bone volume, trabecular number, trabecular thickness, and increased trabecular separation in Wnt1Prrx-/- mice. Mechanistically, histomorphometric analyses showed that the reduced subchondral bone mass in Wnt1Prrx-/- mice was due to impaired bone formation and enhanced bone resorption. In vitro, exogenous Wnt1 inhibited chondrogenesis and chondrocyte hypertrophy in both cell autonomous and juxtacrine manners, while matrix mineralization and the expression of Mmp13, Mmp9 and Opn were induced in a juxtacrine manner. Taken together, mesenchymal cell-derived Wnt1 is an important regulator of subchondral bone remodeling, although it has no effect on the regulation of growth plate or articular cartilage.

Oxymatrine Protects Chondrocytes against IL-1β-triggered Apoptosis in Vitro and Inhibits Osteoarthritis in Mice Model.

Background Osteoarthritis (OA) is a multifactorial disease with various risk factors, resulting in the degeneration of articular cartilage and whole joints. However, to date, no effective disease-modifying therapy for OA has been developed. Oxymatrine (OMT) is associated with many pharmacological effects, including anti-inflammatory, antiapoptotic, and antioxidative properties. However, the role of OMT in OA remains unclear. Materials and Methods An IL-1β-induced chondrocyte model and anterior cruciate ligament transection (ACLT)-induced murine model of OA were constructed. The effect of OMT on chondrocyte viability was assessed using the CCK-8 assay. The protein level was assessed by Western blot analysis, and the apoptosis rate was assessed by flow cytometry in vitro and TUNEL staining in OA model mice. The effect of OMT on the degradation of articular cartilage in ACLT-induced OA mice was assessed by histological analysis. Results OMT at 0–2 mg/mL showed no conspicuous cytotoxicity on chondrocytes after 24 hours of incubation. OMT at 0.5, 1, and 2 mg/mL inhibited IL-1β-triggered apoptosis, upregulated MMP13, MMP9, and Col X, and upregulated Col II in chondrocytes in vitro. OMT represses the NF-κB signaling cascade in IL-1β-triggered chondrocytes in vitro. In an in vivo study, OMT decreased the apoptosis rate of chondrocytes and exerted a protective effect against the degradation of articular cartilage in ACLT-triggered OA mice. Conclusion OMT plays a protective role against chondrocyte injury induced by IL-1β in vitro or ACLT in vivo. OMT may play a role in chondrocytes during OA by inhibiting NF-κB signaling by decreasing the phosphorylation of p65 and IκB. OMT treatment may be a promising chondroprotective approach to delay OA cartilage progression.

FGF receptor inhibitor BGJ398 partially rescues osteoarthritis-like phenotype in older high molecular weight FGF2 transgenic mice via multiple mechanisms

We have used Basic Fibroblast Growth Factor (FGF2) transgenic mice as experimental models for human X-linked hypophosphatemia (XLH)-related degenerative osteoarthritis (OA) to investigate the pathogenesis of the disease and to test potential pharmacotherapies for treatment. This study tested the efficacy of BJG398, a small molecule fibroblast growth factor receptor tyrosine kinase (FGFRTK) inhibitor, to rescue the knee joint osteoarthritis phenotype in High Molecular Weight fibroblast growth factor 2 transgenic (HMWTgFGF2) mice. BJG398 was administered in vivo to 8-month-old female HMWTgFGF2 mice for six weeks. Histomorphometry, immunohistochemistry and micro-CT were used to examine the knee joints in BGJ398-treated and control mice. We assessed: Fibroblast Growth Factor 23 (FGF23) expression and FGFR1 activity; Matrix metalloproteinase 13 (MMP13) and Aggrecanase2 (ADAMTS5) expression; then signaling by SMAD1/5/8-pSMAD6, pERK1/2 and Runt-related transcription factor 2 (RUNX2). Using PrimePCR arrays, we identified a contributing role for major target genes in the TGFB/BMP2 signaling pathway that were regulated by BGJ398. BGJ398 inhibited HMWFGF2/FGF23-induced increase in bone morphogenic protein receptor-1, bone morphogenic protein-2 and 4 and Serine peptidase inhibitor, clade E, member 1. The results from Micro-CT and histology show BGJ398 treatment rescued the OA changes in subchondral bone and knee articular cartilage of HMWTgFGF2 mice. The gene expression and signal transduction results provide convincing evidence that HMWFGF2 generates OA through FGFRTK with characteristic downstream signaling that defines OA, namely: increased FGF23-FGFR1 activity with BMP-BMPR, activation of pSMAD1/5/8-RUNX2 and pERK signaling pathways, then upregulation of MMP13 and ADAMTS5 to degrade matrix. BGJ398 treatment effectively reversed these OA molecular phenotypes, providing further evidence that the OA generated by HMWFGF2 in the transgenic mice is FGFR-mediated and phenocopies the OA found in the Hyp mouse homolog of XLH with a spontaneous mutation in the Phex (phosphate regulating endopeptidase on the X chromosome) gene and human XLH-OA. Overall, the results obtained here explain how the pleotropic effects of FGF2 emanate from the different functions of HMW protein isoforms for cartilage and bone homeostasis, and the pathogenesis of XLH-degenerative osteoarthropathy. BGJ398 inhibits HMWFGF2-induced osteoarthritis via multiple mechanisms. These results provided important scientific evidence for the potential application of BGJ398 as a therapeutic agent for osteoarthritis in XLH.

Salinomycin alleviates osteoarthritis progression via inhibiting Wnt/β-catenin signaling

Osteoarthritis (OA) is the most prevalent degenerative whole-joint disease characterized by cartilage degeneration, synovial hyperplasia, osteophyte formation, and subchondral bone sclerosis. Currently there are no disease-modifying treatments available for OA because its etiology and pathogenesis are largely unknown. Here we report that a natural carboxylic polyether ionophore that is used as an anti-tumor drug, salinomycin (SAL), may be a promising therapeutic drug for OA in the future. We found that SAL showed no cytotoxicity on mouse chondrocytes and displayed a protective effect against interleukin-1β (IL-1β), in cultured mouse chondrocytes and cartilage explants. Treatment with low SAL concentrations directly upregulated the anabolism factors collagen II and aggrecan, while it inhibited the catabolic factors matrix metalloproteinase-13 (MMP13) and metalloproteinase with thrombospondin motifs-5 (ADAMTS5) to protect against extracellular matrix (ECM) degradation, and also suppressed inflammatory responses in mouse chondrocytes. Furthermore, SAL reduced the severity of OA-associated changes and delayed cartilage destruction, subchondral bone sclerosis, and osteophyte formation in a destabilized medial meniscus (DMM) surgery-induced mouse OA model. Mechanistically, a low SAL concentration induced anabolism and inhibited catabolism in chondrocytes via inhibiting Lrp6 phosphorylation and Wnt/β-catenin signaling. Our results suggested that SAL may serve as a potential disease-modifying therapeutic against OA pathogenesis.

Study on the Role of MicroRNA-214 in the Rehabilitation of Cartilage in Mice with Exercise-Induced Traumatic Osteoarthritis.

This study aimed to explore the possible relationship between the expression of Micro RNA-214 (miR-214) and the pathogenesis and recovery in mice with post-traumatic osteoarthritis (PTOA). In this study, 40 male C57BL/6 mice were randomly divided into five groups: model control (MC) group, model (M) group, rehabilitation control (RC) group, model + rehabilitation (M + R) group, and model + convalescent (M + C) group. Four weeks of high-intensity treadmill exercise (HITE) and 4 weeks of moderate-intensity treadmill exercise (MITE) were implemented for PTOA modeling and rehabilitation, respectively. In vitro, 10% elongation mechanical strain was used for IL-1β stimulated chondrocytes. We found that compared with the MC group, there was a significant increase in the aspect of inflammation and catabolism while a dramatic fall in miR-214 expression was observed in the M group. After the 4 weeks of MITE, the level of inflammation and metabolism, as well as miR-214 expression, was partially reversed in the M + R group compared with the M + C group. The expression of miR-214 decreased dramatically after chondrocyte stimulation by IL-1β and then increased significantly after 10% strain was applied to IL-1β-treated cells. These results suggest that a suitable mechanical load can increase the expression of miR-214, and that miR-214 may play a chondroprotective effect in the development of OA.

Lin28a induces SOX9 and chondrocyte reprogramming via HMGA2 and blunts cartilage loss in mice.

Articular cartilage has low regenerative capacity despite permanent stress. Irreversible cartilage lesions characterize osteoarthritis (OA); this is not followed by tissue repair. Lin28a, an RNA binding protein, is detected in damaged cartilage in humans and mice. We investigated the role of LIN28a in cartilage physiology and in osteoarthritis. Lin28a-inducible conditional cartilage deletion up-regulated Mmp13 in intact mice and exacerbated the cartilage destruction in OA mice. Lin28a-specific cartilage overexpression protected mice against cartilage breakdown, stimulated chondrocyte proliferation and the expression of Prg4 and Sox9, and down-regulated Mmp13. Lin28a overexpression inhibited Let-7b and Let-7c miRNA levels while RNA-sequencing analysis revealed five genes of transcriptional factors regulated by Let-7. Moreover, Lin28a overexpression up-regulated HMGA2 and activated SOX9 transcription, a factor required for chondrocyte reprogramming. HMGA2 siRNA knockdown inhibited the cartilage protective effect of Lin28a overexpression. This study provides insights into a new pathway including the Lin28a-Let7 axis, thus promoting chondrocyte anabolism in injured cartilage in mice.

6-Shogaol (enexasogoal) treatment improves experimental knee osteoarthritis exerting a pleiotropic effect over immune innate signalling responses in chondrocytes.

The pathogenesis of osteoarthritis implicates a low-grade inflammation associated to the innate immune system activation. Toll like receptor (TLR) stimulation triggers the release of inflammatory mediators, which aggravate osteoarthritis. We studied the preventive effect of 6-shogaol, a potential TLR4 inhibitor, on the treatment of experimental knee osteoarthritis. Osteoarthritis was induced in C57BL6 mice by surgical section of the medial meniscotibial ligament, which received 6-shogaol for eight weeks. Cartilage damage, inflammatory mediator presence and disease markers were assessed in joint tissues by immunohistochemistry. Computational modelling was used to predict binding modes of 6-shogaol into the TLR4/MD2 receptor and its permeability across cellular membranes. Employing LPS-stimulated chondrocytes and MAPK assay, we elucidated 6-shogaol action mechanisms. 6-Shogaol treatment prevented articular cartilage lesions, synovitis and the presence of pro-inflammatory mediators, and disease markers in osteoarthritis animals. Molecular modelling studies predicted 6-shogaol interaction with the TLR4/MD-2 heterodimer in an antagonist conformation through its binding into the MD-2 pocket. In cell culture, we confirmed that 6-shogaol reduced LPS-induced TLR4 inflammatory signalling pathways. Besides, MAPK assay demonstrated that 6-shogaol directly inhibits the ERK1/2 phosphorylation activity. 6-Shogaol evoked a preventive action on cartilage and synovial inflammation in osteoarthritis mice. 6-shogaol effect may take place not only by hindering the interaction between TLR4 ligands and the TLR4/MD-2 complex in chondrocytes, but also through inhibition of ERK phosphorylation, implying a pleiotropic effect on different mediators activated during osteoarthritis, which proposes it as an attractive drug for osteoarthritis treatments.

Mitochondrial double-stranded RNAs govern the stress response in chondrocytes to promote osteoarthritis development.

Protein kinase R (PKR) is an immune response protein that becomes activated by double-stranded RNAs (dsRNAs). PKR overactivation is associated with degenerative diseases with inflammation, including osteoarthritis (OA), but the dsRNA activator remains largely unknown. Here, we find that mitochondrial dsRNA (mt-dsRNA) expression and its cytosolic efflux are facilitated in chondrocytes under OA-eliciting conditions, leading to innate immune activation. Moreover, mt-dsRNAs are released to the extracellular space and activate Toll-like receptor 3 at the plasma membrane. Elevated levels of mt-dsRNAs in the synovial fluids and damaged cartilage of OA patients and in the cartilage of surgery-induced OA mice further support our data. Importantly, autophagy prevents PKR activation and protects chondrocytes from mitochondrial stress partly by removing cytosolic mtRNAs. Our study provides a comprehensive understanding of innate immune activation by mt-dsRNAs during stress responses that underlie the development of OA and suggests mt-dsRNAs as a potential target for chondroprotective intervention.

Targeted Deletion of the Claudin12 Gene in Mice Increases Articular Cartilage and Inhibits Chondrocyte Differentiation.

To study the role of Claudin (CLDN)12 in bone, we developed mice with a targeted deletion of exon2 in the Cldn12 gene for skeletal phenotype analysis. Micro-CT analysis of the secondary spongiosa of distal femurs of mice with targeted disruption of the Cldn12 gene and control littermates showed no significant genotype-specific differences in either cortical or trabecular bone parameters for either gender in 13-week-old mice. Immunohistochemistry revealed that while CLDN12 was expressed in both differentiating chondrocytes and osteoblasts of the secondary spongiosa of 3-week-old wild-type mice, its expression was restricted to differentiating chondrocytes in the articular cartilage and growth plate in adult mice. Articular cartilage area at the knee were increased by 47% in Cldn12 knockout (KO) mice compared to control littermates. Micro-CT analyses found that while the trabecular number was increased by 9% and the trabecular spacing was reduced by 9% in the femoral epiphysis of Cldn12 KO mice, neither bone volume nor bone volume adjusted for tissue volume was different between the two genotypes. The expression levels of Clusterin, Lubricin and Mmp13 were increased by 56%, 46%, and 129%, respectively, in primary articular chondrocytes derived from KO compared to control mice. Our data indicate that targeted deletion of the Cldn12 gene in mice increases articular cartilage, in part, by promoting articular chondrocyte phenotype.

Estrogen alleviates post-traumatic osteoarthritis progression and decreases p-EGFR levels in female mouse cartilage

ObjectiveTo investigate the effect of estrogen on the progression of post-traumatic osteoarthritis (PTOA) in mice and its possible mechanism.MethodsTwelve-week-old ICR mice were divided into Group A (female control group), group B (ovariectomized(OVX) group), group C (OVX group supplemented with estrogen), and group D (male group) by destabilization of the medial meniscus (DMM)or sham operation. Safranin O staining was performed at 8 weeks and 12 weeks after operation, and the degree of articular cartilage lesion was evaluated using Mankin score. Twelve weeks after the operation, tissue sections were stained to analyze the matrix metalloproteinase 13(MMP13), phosphorylated epidermal growth factor receptor (p-EGFR) expression and apoptosis of chondrocytes.ResultsDecreased estrogen can significantly increase the weight of mice in female mice. The degree of cartilage damage in the knee joint on the DMM side of female mice was significantly severer than that on the Sham side. The DMM side also showed higher MMP13 expression and increased apoptotic chondrocytes. The degree of cartilage damage in the knee joint on the DMM side of female mice was significantly reduced after estrogen supplementation, and cartilage damage in the knee joint on the DMM side of female mice was less serious than that of male mice. As estrogen levels decreased, the severity of cartilage erosion in the knee joint on the DMM side was aggravated, and p-EGFR expression in the cartilage surface was also higher in female mice contrast to that in male mice. However, minimal changes in p-EGFR expression in the cartilage surface of bilateral knee joints of male mice were observe.ConclusionEstrogen has a regulatory effect on PTOA and its inhibits the expression of p-EGFR in cartilage on the knee joint surface and has a protective effect on articular cartilage in female mice.

Increased Wnt/β-catenin signaling contributes to autophagy inhibition resulting from a dietary magnesium deficiency in injury-induced osteoarthritis

BackgroundDietary magnesium deficiency, which is common in modern diet, has been associated with osteoarthritis (OA) susceptibility. Despite this clinical association, no study has addressed if dietary magnesium deficiency accelerates OA development, especially at molecular level. This study aimed to explore aggravating effects of dietary magnesium deficiency on cartilage damage in an injury-induced murine OA model and to determine the underlying mechanism.MethodsTwelve-week-old C57BL/6J mice subject to injury-induced OA modeling were randomized into different diet groups in which the mice were fed a diet with daily recommended magnesium content (500 mg/kg) or diets with low magnesium content (100 or 300 mg/kg). Articular cartilage damage was evaluated using the OARSI score. To determine molecular mechanisms in vitro, mouse chondrocytes were treated with media of low magnesium conditions at 0.1 and 0.4 mM, compared with normal magnesium condition at 0.7 mM as control. Anabolic and catabolic factors, autophagy markers, β-catenin, Wnt ligands, and a magnesium channel transient receptor potential cation channel subfamily member 7 (TRPM7) were analyzed by quantitative real-time PCR and immunoblotting. Autolysosomes were detected by DALGreen staining via fluorescence microscopy and autophagosomes were evaluated by transmission electron microscopy. Autophagy markers, β-catenin, and TRPM7 were assessed in vivo in the mouse cartilage, comparing between dietary magnesium deficiency and normal diet, by immunohistochemistry.ResultsDietary magnesium deficiency aggravated injury-induced cartilage damage, indicated by significant higher OARSI scores. Autophagy markers LC3-II and Beclin-1 were decreased both in low magnesium diet-fed mice and low magnesium-treated chondrocytes. The number of autolysosomes and autophagosomes was also reduced under low magnesium conditions. Moreover, magnesium deficiency induced decreased anabolic and increased catabolic effect of chondrocytes which could be restored by autophagy activator rapamycin. In addition, reduced autophagy under low magnesium conditions is mediated by activated Wnt/β-catenin signaling. The expression of TRPM7 also decreased in low magnesium diet-fed mice, indicating that downstream changes could be regulated through this channel.ConclusionsDietary magnesium deficiency contributes to OA development, which is mediated by reduced autophagy through Wnt/β-catenin signaling activation. These findings indicated potential benefits of adequate dietary magnesium for OA patients or those individuals at high risk of OA.

Kindlin-2 loss in condylar chondrocytes causes spontaneous osteoarthritic lesions in the temporomandibular joint in mice

The progressive destruction of condylar cartilage is a hallmark of the temporomandibular joint (TMJ) osteoarthritis (OA); however, its mechanism is incompletely understood. Here, we show that Kindlin-2, a key focal adhesion protein, is strongly detected in cells of mandibular condylar cartilage in mice. We find that genetic ablation of Kindlin-2 in aggrecan-expressing condylar chondrocytes induces multiple spontaneous osteoarthritic lesions, including progressive cartilage loss and deformation, surface fissures, and ectopic cartilage and bone formation in TMJ. Kindlin-2 loss significantly downregulates the expression of aggrecan, Col2a1 and Proteoglycan 4 (Prg4), all anabolic extracellular matrix proteins, and promotes catabolic metabolism in TMJ cartilage by inducing expression of Runx2 and Mmp13 in condylar chondrocytes. Kindlin-2 loss decreases TMJ chondrocyte proliferation in condylar cartilages. Furthermore, Kindlin-2 loss promotes the release of cytochrome c as well as caspase 3 activation, and accelerates chondrocyte apoptosis in vitro and TMJ. Collectively, these findings reveal a crucial role of Kindlin-2 in condylar chondrocytes to maintain TMJ homeostasis.

Pentraxin 3 regulated by miR-224-5p modulates macrophage reprogramming and exacerbates osteoarthritis associated synovitis by targeting CD32

Emerging evidence has shown an imbalance in M1/M2 macrophage polarization to play an essential role in osteoarthritis (OA) progression. However, the underlying mechanistic basis for this polarization is unknown. RNA sequencing of OA M1-polarized macrophages found highly expressed levels of pentraxin 3 (PTX3), suggesting a role for PTX3 in OA occurrence and development. Herein, PTX3 was found to be increased in the synovium and articular cartilage of OA patients and OA mice. Intra-articular injection of PTX3 aggravated, while PTX3 neutralization reversed synovitis and cartilage degeneration. No metabolic disorder or proteoglycan loss were observed in cartilage explants when treated with PTX3 alone. However, cartilage explants exhibited an OA phenotype when treated with culture supernatants of macrophages stimulated with PTX3, suggesting that PTX3 did not have a direct effect on chondrocytes. Therefore, the OA anti-chondrogenic effects of PTX3 are primarily mediated through macrophages. Mechanistically, PTX3 was upregulated by miR-224-5p deficiency, which activated the p65/NF-κB pathway to promote M1 macrophage polarization by targeting CD32. CD32 was expressed by macrophages, that when stimulated with PTX3, secreted abundant pro-inflammation cytokines that induced severe articular cartilage damage. The paracrine interaction between macrophages and chondrocytes produced a feedback loop that enhanced synovitis and cartilage damage. The findings of this study identified a functional pathway important to OA development. Blockade of this pathway and PTX3 may prevent and treat OA.