Histological and immunohistochemical analyses of articular cartilage during onset and progression of pre- and early-stage osteoarthritis in a rodent model.

Early diagnosis and treatment of pre- and early-stage osteoarthritis (OA) is important. However, the cellular and cartilaginous changes occurring during these stages remain unclear. We investigated the histological and immunohistochemical changes over time between pre- and early-stage OA in a rat model of traumatic injury. Thirty-six male rats were divided into two groups, control and OA groups, based on destabilization of the medial meniscus. Histological and immunohistochemical analyses of articular cartilage were performed on days 1, 3, 7, 10, and 14 postoperatively. Cell density of proteins associated with cartilage degradation increased from postoperative day one. On postoperative day three, histological changes, including chondrocyte death, reduced matrix staining, and superficial fibrillation, were observed. Simultaneously, a compensatory increase in matrix staining was observed. The Osteoarthritis Research Society International score increased from postoperative day seven, indicating thinner cartilage. On postoperative day 10, the positive cell density decreased, whereas histological changes progressed with fissuring and matrix loss. The proteoglycan 4-positive cell density increased on postoperative day seven. These findings will help establish an experimental model and clarify the mechanism of the onset and progression of pre- and early-stage traumatic OA.

SKP2-mediated ubiquitination and degradation of KLF11 promotes osteoarthritis via modulation of JMJD3/NOTCH1 pathway.

Osteoarthritis (OA) is the main cause of cartilage damage and disability. This study explored the biological function of S-phase kinase-associated protein 2 (SKP2) and Kruppel-like factor 11 (KLF11) in OA progression and its underlying mechanisms. C28/I2 chondrocytes were stimulated with IL-1ß to mimic OA in vitro. We found that SKP2, Jumonji domain-containing protein D3 (JMJD3), and Notch receptor 1 (NOTCH1) were upregulated, while KLF11 was downregulated in IL-1ß-stimulated chondrocytes. SKP2/JMJD3 silencing or KLF11 overexpression repressed apoptosis and extracellular matrix (ECM) degradation in chondrocytes. Mechanistically, SKP2 triggered the ubiquitination and degradation of KLF11 to transcriptionally activate JMJD3, which resulted in activation of NOTCH1 through inhibiting H3K27me3. What's more, the in vivo study found that KLF11 overexpression delayed OA development in rats via restraining apoptosis and maintaining the balance of ECM metabolism. Taken together, ubiquitination and degradation of KLF11 regulated by SKP2 contributed to OA progression by activation of JMJD3/NOTCH1 pathway. Our findings provide promising therapeutic targets for OA.

CHMP5 attenuates osteoarthritis via inhibiting chondrocyte apoptosis and extracellular matrix degradation: involvement of NF-κB pathway.

BACKGROUND: Osteoarthritis (OA), the most common joint disease, is linked with chondrocyte apoptosis and extracellular matrix (ECM) degradation. Charged multivesicular body protein 5 (CHMP5), a member of the multivesicular body, has been reported to serve as an anti-apoptotic protein to participate in leukemia development. However, the effects of CHMP5 on apoptosis and ECM degradation in OA remain unclear. METHODS: In this study, quantitative proteomics was performed to analyze differential proteins between normal and OA patient articular cartilages. The OA mouse model was constructed by the destabilization of the medial meniscus (DMM). In vitro, interleukin-1 beta (IL-1ß) was used to induce OA in human chondrocytes. CHMP5 overexpression and silencing vectors were created using an adenovirus system. The effects of CHMP5 on IL-1ß-induced chondrocyte apoptosis were investigated by CCK-8, flow cytometry, and western blot. The effects on ECM degradation were examined by western blot and immunofluorescence. The potential mechanism was explored by western blot and Co-IP assays. RESULTS: Downregulated CHMP5 was identified by proteomics in OA patient cartilages, which was verified in human and mouse articular cartilages. CHMP5 overexpression repressed cell apoptosis and ECM degradation in OA chondrocytes. However, silencing CHMP5 exacerbated OA chondrocyte apoptosis and ECM degradation. Furthermore, we found that the protective effect of CHMP5 against OA was involved in nuclear factor kappa B (NF-κB) signaling pathway. CONCLUSIONS: This study demonstrated that CHMP5 repressed IL-1ß-induced chondrocyte apoptosis and ECM degradation and blocked NF-κB activation. It was shown that CHMP5 might be a novel potential therapeutic target for OA in the future.

Esculin suppresses the PERK-eIF2α-CHOP pathway by enhancing SIRT1 expression in oxidative stress-induced rat chondrocytes, mitigating osteoarthritis progression in a rat model.

OBJECTIVE: Osteoarthritis (OA) is a degenerative disease characterized by the gradual degeneration of chondrocytes, involving endoplasmic reticulum (ER) stress. Esculin is a natural compound with antioxidant, anti-inflammatory and anti-tumor properties. However, its impact on ER stress in OA therapy has not been thoroughly investigated. We aim to determine the efficiency of Esculin in OA treatment and its underlying mechanism. METHODS: We utilized the tert-butyl hydroperoxide (TBHP) to establish OA model in chondrocytes. The expression of SIRT1, PERK/eIF2α pathway-related proteins, apoptosis-associated proteins and ER stress-related proteins were detected by Western blot and Real-time PCR. The apoptosis was evaluated by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. X-ray imaging, Hematoxylin & Eosin staining, Safranin O staining and immunohistochemistry were used to assess the pharmacological effects of Esculin in the anterior cruciate ligament transection (ACLT) rat OA model. RESULTS: Esculin downregulated the expression of PERK/eIF2α pathway-related proteins, apoptosis-associated proteins and ER stress-related proteins, while upregulated the expression of SIRT1 and Bcl2 in the TBHP-induced OA model in vitro. It was coincident with the results of TUNEL staining and flow cytometry. We further confirmed the protective effect of Esculin in the rat ACLT-related model. CONCLUSION: Our results suggest the potential therapeutic value of Esculin on osteoarthritis. It probably inhibits the PERK-eIF2α-ATF4-CHOP pathway by upregulating SIRT1, thereby mitigating endoplasmic reticulum stress and protecting chondrocytes from apoptosis.

Thyroid hormone induces ossification and terminal maturation in a preserved OA cartilage biomimetic model.

OBJECTIVE: To characterize aspects of triiodothyronine (T3) induced chondrocyte terminal maturation within the molecular osteoarthritis pathophysiology using the previously established T3 human ex vivo osteochondral explant model. DESIGNS: RNA-sequencing was performed on explant cartilage obtained from OA patients (n = 8), that was cultured ex vivo with or without T3 (10 ng/ml), and main findings were validated using RT-qPCR in an independent sample set (n = 22). Enrichment analysis was used for functional clustering and comparisons with available OA patient RNA-sequencing and GWAS datasets were used to establish relevance for OA pathophysiology by linking to OA patient genomic profiles. RESULTS: Besides the upregulation of known hypertrophic genes EPAS1 and ANKH, T3 treatment resulted in differential expression of 247 genes with main pathways linked to extracellular matrix and ossification. CCDC80, CDON, ANKH and ATOH8 were among the genes found to consistently mark early, ongoing and terminal maturational OA processes in patients. Furthermore, among the 37 OA risk genes that were significantly affected in cartilage by T3 were COL12A1, TNC, SPARC and PAPPA. CONCLUSIONS: RNA-sequencing results show that metabolic activation and recuperation of growth plate morphology are induced by T3 in OA chondrocytes, indicating terminal maturation is accelerated. The molecular mechanisms involved in hypertrophy were linked to all stages of OA pathophysiology and will be used to validate disease models for drug testing.

Molecular mechanism of thiram-induced abnormal chondrocyte proliferation via lncRNA MSTRG.74.1-BNIP3 axis.

Thiram, a widely used organic pesticide in agriculture, exhibits both bactericidal and insecticidal effects. However, prolonged exposure to thiram has been linked to bone deformities and cartilage damage, contributing to the development of tibial dyschondroplasia (TD) in broilers and posing a significant threat to global agricultural production. TD, a prevalent nutritional metabolic disease, manifests as clinical symptoms like unstable standing, claudication, and sluggish movement in affected broilers. In recent years, there has been growing recognition of the regulatory role of long non-coding RNA (lncRNA) in tibial cartilage formation among broilers through diverse signaling pathways. This study employs in vitro experimental models, growth performance analysis, and clinical observation to assess broilers' susceptibility to thiram pollution. Transcriptome sequencing analysis revealed a significant elevation in the expression of lncRNA MSTRG.74.1 in both the con group and the thiram-induced in vitro group. The results showed that lncRNA MSTRG.74.1 plays a pivotal role in influencing the proliferation and abnormal differentiation of chondrocytes. This regulation occurs through the negative modulation of apoptotic genes, including Bax, Cytc, Bcl2, Apaf1, and Caspase3, along with genes Atg5, Beclin1, LC3b, and protein p62. Moreover, the overexpression of lncRNA MSTRG.74.1 was found to regulate broiler chondrocyte development by upregulating BNIP3. In summary, this research sheds light on thiram-induced abnormal chondrocyte proliferation in TD broilers, emphasizing the significant regulatory role of the lncRNA MSTRG.74.1-BNIP3 axis, which will contribute to our understanding of the molecular mechanisms underlying TD development in broilers exposed to thiram.

Single-cell RNA-seq analysis reveals the Wnt/Ca2+ signaling pathway with inflammation, apoptosis in nucleus pulposus degeneration.

BACKGROUND: Increasing studies have shown degeneration of nucleus pulposus cells (NPCs) as an critical part of the progression of intervertebral disc degeneration (IVDD). However, there are relatively few studies on single-cell transcriptome contrasts in human degenerated NPCs. Moreover, differences in Wnt/Ca2+ signaling in human degenerated nucleus pulposus cells have not been elucidated. The aim of this study is to investigate the differential expression of Wnt/Ca2+ signaling pathway between normal and degenerated nucleus pulposus cells in humans and try to investigate its mechanism. METHODS: We performed bioinformatics analysis using our previously published findings to construct single cell expression profiles of normal and degenerated nucleus pulposus. Then, in-depth differential analysis was used to characterize the expression of Wnt/Ca2+ signaling pathway between normal and degenerated nucleus pulposus cells in humans. RESULTS: The obtained cell data were clustered into five different chondrocytes clusters, which chondrocyte 4 and chondrocyte 5 mainly accounted for a high proportion in degenerated nucleus pulposus tissues, but rarely in normal nucleus pulposus tissues. Genes associated within the Wnt/Ca2+ signaling pathway, such as Wnt5B, FZD1, PLC (PLCB1), CaN (PPP3CA) and NAFATC1 are mainly present in chondrocyte 3, chondrocyte 4 and chondrocyte 5 from degenerated nucleus pulposus tissues. In addition, as a receptor that activates Wnt signaling pathway, LRP5 is mainly highly expressed in chondrocyte 5 of degenerated nucleus pulposus cells. Six genes, ANGPTL4, PTGES, IGFBP3, GDF15, TRIB3 and TNFRSF10B, which are associated with apoptosis and inflammatory responses, and are widespread in chondrocyte 4 and chondrocyte 5, may be closely related to degenerative of nucleus pulposus cells. CONCLUSIONS: Single-cell RNA sequencing revealed differential expression of Wnt/Ca2+ signaling in human normal and degenerated nucleus pulposus cells, and this differential expression may be closely related to the abundance of chondrocyte 4 and chondrocyte 5 in degenerated nucleus pulposus cells. In degenerated nucleus pulposus cells, LRP5 activate Wnt5B, which promotes nucleus pulposus cell apoptosis and inflammatory response by regulating the Wnt/Ca2+ signaling pathway, thereby promoting disc degeneration. ANGPTL4, IGFBP3, PTGES in chondrocyte 4 and TRIB3, GDF15, TNFRSF10B in chondrocyte 5 may play an important role in this process.

The Role of Copper-Induced M2 Macrophage Polarization in Protecting Cartilage Matrix in Osteoarthritis.

BACKGROUND The pathological mechanism of osteoarthritis is still unclear. The regulation of the immune microenvironment has been of growing interest in the progression and treatment of osteoarthritis. Macrophages with different phenotypes, producing different cytokines, have been linked to the mechanism of cartilage injury in osteoarthritis. Copper ions play a role in the immune response and are involved in the pathological mechanisms of osteoarthritis by affecting the metabolism of the cartilage matrix. Bioactive glass (BG) is an osteogenic material with superior biocompatibility. Here, we report on the regulatory behavior of macrophages using a copper-based composite BG material. MATERIAL AND METHODS Cu-BGC powder was prepared by sol-gel method, and scaffolds were fabricated and characterized using 3D printing. Macrophage cultures grown with Cu-BGC were examined for cell culture and proliferation. The effect of Cu-BGC on the degradation metabolism of chondrocytes, cultured in the environment of inflammatory cytokine IL-1ß, was determined. In addition, the morphology of macrophages, secretion of inflammatory cytokines, and expression of surface markers were examined. RESULTS The results show that Cu-BGC promotes macrophage proliferation at a range of concentrations and increases the secretion of anti-inflammatory cytokines while inhibiting proinflammatory cytokines. At the same time, M2-type cell surface markers are definitely expressed and the morphology of macrophages is altered. In addition, Cu-BGC inhibited the degradation metabolism of chondrocytes in the inflammatory environment induced by IL-1ß. CONCLUSIONS These results suggest that Cu-BGC induced macrophage polarization into an M2 type anti-inflammatory phenotype, and inhibition of immune injury response may play a role in delaying cartilage matrix damage in osteoarthritis.

HIF-1α in cartilage homeostasis, apoptosis, and glycolysis in mice with steroid-induced osteonecrosis of the femoral head.

With the prevalence of coronavirus disease 2019, the administration of glucocorticoids (GCs) has become more widespread. Treatment with high-dose GCs leads to a variety of problems, of which steroid-induced osteonecrosis of the femoral head (SONFH) is the most concerning. Since hypoxia-inducible factor 1α (HIF-1α) is a key factor in cartilage development and homeostasis, it may play an important role in the development of SONFH. In this study, SONFH models were established using methylprednisolone (MPS) in mouse and its proliferating chondrocytes to investigate the role of HIF-1α in cartilage differentiation, extracellular matrix (ECM) homeostasis, apoptosis and glycolysis in SONFH mice. The results showed that MPS successfully induced SONFH in vivo and vitro, and MPS-treated cartilage and chondrocytes demonstrated disturbed ECM homeostasis, significantly increased chondrocyte apoptosis rate and glycolysis level. However, compared with normal mice, not only the expression of genes related to collagens and glycolysis, but also chondrocyte apoptosis did not demonstrate significant differences in mice co-treated with MPS and HIF-1α inhibitor. And the effects observed in HIF-1α activator-treated chondrocytes were similar to those induced by MPS. And HIF-1α degraded collagens in cartilage by upregulating its downstream target genes matrix metalloproteinases. The results of activator/inhibitor of endoplasmic reticulum stress (ERS) pathway revealed that the high apoptosis rate induced by MPS was related to the ERS pathway, which was also affected by HIF-1α. Furthermore, HIF-1α affected glucose metabolism in cartilage by increasing the expression of glycolysis-related genes. In conclusion, HIF-1α plays a vital role in the pathogenesis of SONFH by regulating ECM homeostasis, chondrocyte apoptosis, and glycolysis.

Targeted inhibition of STAT3 (Tyr705) by xanthatin alleviates osteoarthritis progression through the NF-κB signaling pathway.

The transcription factor, signal transducer, and stimulator of transcription 3 (STAT3) is a potential target in osteoarthritis (OA) treatment. Although xanthatin (XA), a biologically active substance derived from Xanthium strumarium L, specifically inhibits STAT3 phosphorylation at Tyr705, the mechanism underlying its inhibitory effect on OA progression remains unclear. In this study, our objective was to explore the therapeutic effects exerted by XA on OA and the underlying molecular mechanisms. The effects of XA treatment on mouse OA models subjected to destabilization of the medial meniscus using medial collateral ligament transection, as well as on interleukin-1ß (IL-1ß)-induced mouse chondrocytes, were examined. Histological changes in cartilage and subchondral bone (SCB), as well as changes in the expression levels of osteophytes, cartilage degeneration- and osteoclast differentiation-related factors, and the role of XA-related signaling pathways in human cartilage tissue, were studied using different techniques. XA inhibited STAT3 phosphorylation at Tyr705 and further attenuated the activity of nuclear factor-κB (NF-κB) in chondrocytes and osteoclasts. In vitro, XA administration alleviated pro-inflammatory cytokine release, extracellular matrix catabolism, and RANKL-mediated osteoclast differentiation. In vivo, intraperitoneal injection of XA exerted a protective effect on cartilage degeneration and SCB loss. Similarly, XA exerted a protective effect on human cartilage tissue by inhibiting the STAT3/NF-κB signaling pathway. Overall, our study elucidated the therapeutic potential of XA as a small-molecule inhibitor of STAT3-driven OA progression. This discovery may help enhance innovative clinical interventions against OA.

An injectable cartilage-coating composite with long-term protection, effective lubrication and chondrocyte nourishment for osteoarthritis treatment.

The osteoarthritic (OA) environment within articular cartilage poses significant challenges, resulting in chondrocyte dysfunction and cartilage matrix degradation. While intra-articular injections of anti-inflammatory drugs, biomaterials, or bioactive agents have demonstrated some effectiveness, they primarily provide temporary relief from OA pain without arresting OA progression. This study presents an injectable cartilage-coating composite, comprising hyaluronic acid and decellularized cartilage matrix integrated with specific linker polymers. It enhances the material retention, protection, and lubrication on the cartilage surface, thereby providing an effective physical barrier against inflammatory factors and reducing the friction and shear force associated with OA joint movement. Moreover, the composite gradually releases nutrients, nourishing OA chondrocytes, aiding in the recovery of cellular function, promoting cartilage-specific matrix production, and mitigating OA progression in a rat model. Overall, this injectable cartilage-coating composite offers promising potential as an effective cell-free treatment for OA. STATEMENT OF SIGNIFICANCE: Osteoarthritis (OA) in the articular cartilage leads to chondrocyte dysfunction and cartilage matrix degradation. This study introduces an intra-articular injectable composite material (HDC), composed of decellularized cartilage matrix (dECMs), hyaluronan (HA), and specially designed linker polymers to provide an effective cell-free OA treatment. The linker polymers bind HA and dECMs to form an integrated HDC structure with an enhanced degradation rate, potentially reducing the need for frequent injections and associated trauma. They also enable HDC to specifically coat the cartilage surface, forming a protective and lubricating layer that enhances long-term retention, acts as a barrier against inflammatory factors, and reduces joint movement friction. Furthermore, HDC nourishes OA chondrocytes through gradual nutrient release, aiding cellular function recovery, promoting cartilage-specific matrix production, and mitigating OA progression.

LIPUS regulates the progression of knee osteoarthritis in mice through primary cilia-mediated TRPV4 channels.

Osteoarthritis (OA) is a common disease in middle-aged and elderly people. An imbalance in calcium ion homeostasis will contribute to chondrocyte apoptosis and ultimately lead to the progression of OA. Transient receptor potential channel 4 (TRPV4) is involved in the regulation of intracellular calcium homeostasis. TRPV4 is expressed in primary cilia, which can sense mechanical stimuli from outside the cell, and its abnormal expression is closely related to the development of OA. Low-intensity pulsed ultrasound (LIPUS) can alleviate chondrocyte apoptosis while the exact mechanism is unclear. In this project, with the aim of revealing the mechanism of action of LIPUS, we proposed to use OA chondrocytes and animal models, LIPUS intervention, inhibition of primary cilia, use TRPV4 inhibitors or TRPV4 agonist, and use Immunofluorescence (IF), Immunohistochemistry (IHC), Western Blot (WB), Quantitative Real-time PCR (QP) to detect the expression of cartilage synthetic matrix and endoplasmic reticulum stress markers. The results revealed that LIPUS altered primary cilia expression, promoted synthetic matrix metabolism in articular chondrocytes and was associated with primary cilia. In addition, LIPUS exerted a active effect on OA by activating TRPV4, inducing calcium inward flow, and facilitating the entry of NF-κB into the nucleus to regulate synthetic matrix gene transcription. Inhibition of TRPV4 altered primary cilia expression in response to LIPUS stimulation, and knockdown of primary cilia similarly inhibited TRPV4 function. These results suggest that LIPUS mediates TRPV4 channels through primary cilia to regulate the process of knee osteoarthritis in mice.

TMF inhibits extracellular matrix degradation by regulating the C/EBPß/ADAMTS5 signaling pathway in osteoarthritis.

Osteoarthritis (OA) is a chronic joint disease, characterized by degenerative destruction of articular cartilage. Chondrocytes, the unique cell type in cartilage, mediate the metabolism of extracellular matrix (ECM), which is mainly constituted by aggrecan and type II collagen. A disintegrin and metalloproteinase with thrombospondin 5 (ADAMTS5) is an aggrecanase responsible for the degradation of aggrecan in OA cartilage. CCAAT/enhancer binding protein ß (C/EBPß), a transcription factor in the C/EBP family, has been reported to mediate the expression of ADAMTS5. Our previous study showed that 5,7,3',4'-tetramethoxyflavone (TMF) could activate the Sirt1/FOXO3a signaling in OA chondrocytes. However, whether TMF protected against ECM degradation by down-regulating C/EBPß expression was unknown. In this study, we found that aggrecan expression was down-regulated, and ADAMTS5 expression was up-regulated. Knockdown of C/EBPß could up-regulate aggrecan expression and down-regulate ADAMTS5 expression in IL-1ß-treated C28/I2 cells. TMF could compromise the effects of C/EBPß on OA chondrocytes by activating the Sirt1/FOXO3a signaling. Conclusively, TMF exhibited protective activity against ECM degradation by mediating the Sirt1/FOXO3a/C/EBPß pathway in OA chondrocytes.

Engineered human osteoarthritic cartilage organoids.

The availability of human cell-based models capturing molecular processes of cartilage degeneration can facilitate development of disease-modifying therapies for osteoarthritis [1], a currently unmet clinical need. Here, by imposing specific inflammatory challenges upon mesenchymal stromal cells at a defined stage of chondrogenesis, we engineered a human organotypic model which recapitulates main OA pathological traits such as chondrocyte hypertrophy, cartilage matrix mineralization, enhanced catabolism and mechanical stiffening. To exemplify the utility of the model, we exposed the engineered OA cartilage organoids to factors known to attenuate pathological features, including IL-1Ra, and carried out mass spectrometry-based proteomics. We identified that IL-1Ra strongly reduced production of the transcription factor CCAAT/enhancer-binding protein beta [2] and demonstrated that inhibition of the C/EBPß-activating kinases could revert the degradative processes. Human OA cartilage organoids thus represent a relevant tool towards the discovery of new molecular drivers of cartilage degeneration and the assessment of therapeutics targeting associated pathways.

Genetically inspired organoids prevent joint degeneration and alleviate chondrocyte senescence via Col11a1-HIF1α-mediated glycolysis-OXPHOS metabolism shift.

INTRODUCTION: Developmental dysplasia of hip (DDH) is a hip joint disorder leading to subsequent osteoarthritis. Previous studies suggested collagen XI alpha 1 (COL11A1) as a potential gene in hip dysplasia and chondrocyte degeneration. However, no genetic association has reported COL11A1-related cellular therapy as treatment of DDH and joint degeneration. METHODS AND RESULTS: We report identified genetic association between COL11A1 locus and DDH with genome-wide association study (GWAS). Further exome sequencing for familial DDH patients was conducted in different populations to identify potential pathogenic Col11A1 variants for familiar DDH. Further studies demonstrated involvement of COL11A1 expression was down-regulated in femoral head cartilage of DDH patients and Col11a1-KO mice with induced DDH. Col11a1-KO mice demonstrated aggravated joint degeneration and severe OA phenotype. To explore the underlying mechanism of Col11a1 in cartilage and DDH development, we generated scRNA-seq profiles for DDH and Col11a1-KO cartilage, demonstrating disrupted chondrocyte homeostasis and cellular senescence caused by Col11a1-HIF1α-mediated glycolysis-OXPHOS shift in chondrocytes. Genetically and biologically inspired, we further fabricated an intra-articular injection therapy to preventing cartilage degeneration by generating a Col11a1-over-expressed (OE) SMSC mini-organoids. Col11a1-OE organoids demonstrated superior chondrogenesis and ameliorated cartilage degeneration in DDH mice via regulating cellular senescence by up-regulated Col11a1/HIF1α-mediated glycolysis in chondrocytes. CONCLUSION: We reported association between COL11A1 loci and DDH with GWAS and exome sequencing. Further studies demonstrated involvement of COL11A1 in DDH patients and Col11a1-KO mice. ScRNA-seq for DDH and Col11a1-KO cartilage demonstrated disrupted chondrocyte homeostasis and cellular senescence caused by Col11a1-HIF1α-mediated glycolysis-OXPHOS shift in chondrocytes. Genetically and biologically inspired, an intra-articular injection therapy was fabricated to prevent cartilage degeneration with Col11a1-OE SMSC organoids. Col11a1-OE organoids ameliorated cartilage degeneration in DDH mice via regulating cellular senescence by up-regulated Col11a1/HIF1α-mediated glycolysis in chondrocytes.

Collagen 1 gel may improve the regenerative capacity of minced adult and preosteoarthritic cartilage.

PURPOSE: Minced cartilage implantation (MCI) is an evolving technique for the treatment of osteochondral lesions. It was hypothesised that mincing of cartilage may affect chondrocyte viability and phenotype and that embedding in collagen 1 gel results in an improved outcome. The objective of this study was to evaluate the impact of cartilage mincing and whether collagen 1 gel mediates beneficial effects on the chondrocyte phenotype and viability. METHODS: Human cartilage samples from 11 patients undergoing total knee arthroplasty were collected and minced according to the MCI protocol. Minced cartilage was cultured for 1 week with and without embedding in collagen 1 gel and was compared with unminced cartilage flakes as control. Quantitative reverse transcription-PCR and immunohistochemical staining for the chondrocyte marker genes SOX9, COL2, ACAN, COL10 and MMP13 were used to examine the chondrocyte phenotype. Cell death was assessed by the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. RESULTS: Increased chondrocyte cell death of cultured cartilage after mincing was observed. Chondrocytes from minced cartilage exhibited significantly decreased expression and protein levels of homeostatic and hypertrophic chondrocyte markers. Embedding in collagen 1 gel showed no positive effect on viability. However, remarkable is the increased expression of ACAN and the preserved protein level of SOX9 in the collagen 1-embedded minced cartilage. CONCLUSIONS: This study shows that the mincing of cartilage leads to increased chondrocyte death and decreased expression of chondrocyte phenotypic marker genes after 7 days. The use of collagen 1 gel may improve the stability of the phenotype, which needs to be further elucidated. LEVEL OF EVIDENCE: Level III (therapeutic).

Sirt6 promotes DNA damage repair in osteoarthritis chondrocytes by activating the Keap1/Nrf2/HO-1 signaling pathway.

The aim of this study was to explore the effect and mechanism of Sirt6 on DNA damage repair in OA chondrocytes. Cartilage tissues were collected from OA patients with knee arthroplasty and traumatic amputation patients without OA. Besides, 7-week-old male C57BL/6 mice were randomly divided into Control and OA groups; CHON-001 cells of corresponding groups were treated with 10 ng/ml interleukin (IL)-1ß, respectively. Subsequently, Sirt6 or siNrf2 was over-expressed in CHON-001 cells to observe the effect of Sirt6 on DNA damage and senescence of chondrocytes by IL-1ß through the nuclear factor E2-related factor 2 (Nrf2) signaling pathway. The expression level of Sirt6 in human and mouse OA cartilage tissues was significantly decreased. However, 24 h of treatment with IL-1ß significantly decreased the expression of Sirt6 in chondrocytes, induced DNA damage, and promoted cellular senescence. In addition, over-expression of Sirt6 promoted DNA damage repair and inhibited cellular senescence in IL-1ß-induced chondrocytes. Moreover, the overexpression of Sirt6 activated the Keap1/Nrf2/HO-1 signaling pathway in chondrocytes, while knockdown of Nrf2 expression inhibited the DNA damage repair and anti-senescence effects of Sirt6 on IL-1ß-treated chondrocytes. Sirt6 may reduce DNA damage and cellular senescence in OA chondrocytes induced by IL-1ß through activating the Keap1/Nrf2/HO-1 signaling pathway.

Osmolarity-Induced Altered Intracellular Molecular Crowding Drives Osteoarthritis Pathology.

Osteoarthritis (OA) is a multifactorial degenerative joint disease of which the underlying mechanisms are yet to be fully understood. At the molecular level, multiple factors including altered signaling pathways, epigenetics, metabolic imbalance, extracellular matrix degradation, production of matrix metalloproteinases, and inflammatory cytokines, are known to play a detrimental role in OA. However, these factors do not initiate OA, but are mediators or consequences of the disease, while many other factors causing the etiology of OA are still unknown. Here, it is revealed that microenvironmental osmolarity can induce and reverse osteoarthritis-related behavior of chondrocytes via altered intracellular molecular crowding, which represents a previously unknown mechanism underlying OA pathophysiology. Decreased intracellular crowding is associated with increased sensitivity to proinflammatory triggers and decreased responsiveness to anabolic stimuli. OA-induced lowered intracellular molecular crowding could be renormalized via exposure to higher extracellular osmolarity such as those found in healthy joints, which reverse OA chondrocyte's sensitivity to catabolic stimuli as well as its glycolytic metabolism.

Role of Apoptosis in the Pathogenesis of Osteoarthritis: An Explicative Review.

Apoptosis is a complex regulatory, active cell death process that plays a role in cell development, homeostasis, and ageing. Cancer, developmental defects, and degenerative diseases are all pathogenic disorders caused by apoptosis dysregulation. Osteoarthritis (OA) is by far the most frequently diagnosed joint disease in the aged, and it is characterized by the ongoing breakdown of articular cartilage, which causes severe disability. Multiple variables regulate the anabolic and catabolic pathways of the cartilage matrix, which either directly or indirectly contribute to cartilage degeneration in osteoarthritis. Articular cartilage is a highly specialized tissue made up of an extracellular matrix of cells that are tightly packed together. As a result, chondrocyte survival is crucial for the preservation of an optimal cartilage matrix, and chondrocyte characteristics and survival compromise may result in articular cartilage failure. Inflammatory cytokines can either promote or inhibit apoptosis, the process of programmed cell death. Pro-apoptotic cytokines like TNF-α can induce cell death, while anti-apoptotic cytokines like IL-4 and IL-10 protect against apoptosis. The balance between these cytokines plays a critical role in determining cell fate and has implications for tissue damage and disease progression. Similarly, they contribute to the progression of OA by disrupting the metabolic balance in joint tissues by promoting catabolic and anabolic pathways. Their impact on cell joints, as well as the impacts of cell signalling pathways on cytokines and inflammatory substances, determines their function in osteoarthritis development. Apoptosis is evident in osteoarthritic cartilage; however, determining the relative role of chondrocyte apoptosis in the aetiology of OA is difficult, and the rate of apoptotic chondrocytes in osteoarthritic cartilage is inconsistent. The current study summarises the role of apoptosis in the development of osteoarthritis, the mediators, and signalling pathways that trigger the cascade of events, and the other inflammatory features involved.

Liproxstatin-1 alleviates cartilage degradation by inhibiting chondrocyte ferroptosis in the temporomandibular joint.

BGROUND INFORMATION: Ferroptosis contributes to temporomandibular joint osteoarthritis (TMJOA) lesion development and is still poorly understood. RESULTS: In this study, we used different TMJOA animal models to examine whether ferroptosis was related to disease onset in TMJOA induced by monosodium iodoacetate (MIA), IL-1ß, occlusion disorder (OD), and unilateral anterior crossbite (UAC). Immunohistochemical staining and Western blot analysis were used to detect ferroptosis- and cartilage degradation-related protein expression. Our results revealed reduced levels of the ferroptosis-related protein GPX4 in the cartilage layer, but the levels of ACSL4 and P53 were increased in the condyle. Injection of the ferroptosis inhibitor liproxstatin-1 (Lip-1) effectively decreased ACSL4, P53 and TRF expression. In vitro, IL-1ß reduced cartilage extracellular matrix expression in mandibular condylar chondrocytes (MCCs). Lip-1 maintained the morphology and function of mitochondria and ameliorated the exacerbation of lipid peroxidation and reactive oxygen species (ROS) production induced by IL-1ß. CONCLUSION: These results suggest that chondrocyte ferroptosis plays an important role in the development and progression of TMJOA. SIGNIFICANCE: Inhibiting condylar chondrocyte ferroptosis could be a promising therapeutic strategy for TMJOA.