Selective STAT3 inhibitor STX-0119 alleviates osteoarthritis progression by modulating the STAT3/PPARγ signaling pathway.

Osteoarthritis (OA), characterized by chronic pain, significantly affects the quality of life of affected individuals. Key factors in OA pathogenesis include cartilage degradation and inflammation. Signal transducer and activator of transcription 3 (STAT3), a member of the STAT protein family, plays a pivotal role in mediating inflammation. STX-0119 has been verified as a small molecular compound that can specifically inhibit STAT3. However, the efficacy of STX-0119 in the treatment of OA remains to be evaluated. Therefore, the aim of this study was to explore the therapeutic effects and molecular mechanisms of STX-0119 in the treatment of OA. We found that the expression of phosphorylated STAT3 is upregulated in human OA cartilage as well as in the cartilage of a mouse model of OA. In vivo, joint injection of STX-0119 into OA mice alleviated cartilage degeneration without affecting the subchondral bone. Additionally, STX-0119 could inhibit the phosphorylation of STAT3 in the cartilage. In vitro, STX-0119 suppressed inflammatory responses in chondrocytes and promoted anabolic metabolism in an interleukin-1ß-induced chondrocyte inflammation model. Additionally, the results of transcriptome sequencing and lentiviral infection assays demonstrated that in chondrocytes, STX-0119 induces the upregulation of peroxisome proliferators-activated receptor gamma (PPARγ) expression by inhibiting STAT3 phosphorylation. Finally, in ex vivo cultures of human cartilage samples, STX-0119 was reaffirmed to inhibit cartilage degeneration via the STAT3/PPARγ signaling pathway. Together, our findings support the potential of STX-0119 for development as a therapeutic agent targeting STAT3 for the treatment of OA.

Bioceramic-mediated chondrocyte hypertrophy promotes calcified cartilage formation for rabbit osteochondral defect repair.

Osteochondral tissue is a highly specialized and complex tissue composed of articular cartilage and subchondral bone that are separated by a calcified cartilage interface. Multilayered or gradient scaffolds, often in conjunction with stem cells and growth factors, have been developed to mimic the respective layers for osteochondral defect repair. In this study, we designed a hyaline cartilage-hypertrophic cartilage bilayer graft (RGD/RGDW) with chondrocytes. Previously, we demonstrated that RGD peptide-modified chondroitin sulfate cryogel (RGD group) is chondro-conductive and capable of hyaline cartilage formation. Here, we incorporated whitlockite (WH), a Mg2+-containing calcium phosphate, into RGD cryogel (RGDW group) to induce chondrocyte hypertrophy and form collagen X-rich hypertrophic cartilage. This is the first study to use WH to produce hypertrophic cartilage. Chondrocytes-laden RGDW cryogel exhibited significantly upregulated expression of hypertrophy markers in vitro and formed ectopic hypertrophic cartilage in vivo, which mineralized into calcified cartilage in bone microenvironment. Subsequently, RGD cryogel and RGDW cryogel were combined into bilayer (RGD/RGDW group) and implanted into rabbit osteochondral defect, where RGD layer supports hyaline cartilage regeneration and bioceramic-containing RGDW layer promotes calcified cartilage formation. While the RGD group (monolayer) formed hyaline-like neotissue that extends into the subchondral bone, the RGD/RGDW group (bilayer) regenerated hyaline cartilage tissue confined to its respective layer and promoted osseointegration for integrative defect repair.

Spatiotemporal analysis of multi-scale cell structure in spheroid culture reveals hypertrophic chondrocyte differentiation.

3D cell culture has emerged as a promising approach to replicate the complex behaviors of cells within living organisms. This study aims to analyze spatiotemporal behavior of the morphological characteristics of cell structure at multiscale in 3D scaffold-free spheroids using chondrogenic progenitor ATDC5 cells. Over a 14-day culture period, it exhibited cell hypertrophy in the spheroids regarding cellular and nuclear size as well as changes in morphology. Moreover, biological analysis indicated a signification up-regulation of normal chondrocyte as well as hypertrophic chondrocyte markers, suggesting early hypertrophic chondrocyte differentiation. Cell nuclei underwent changes in volume, sphericity, and distribution in spheroid over time, indicating alterations in chromatin organization. The ratio of chromatin condensation volume to cell nuclear volume decreased as the cell nuclei enlarged, potentially signifying changes in chromatin state during hypertrophic chondrocyte differentiation. Our image analysis techniques in this present study enabled detailed morphological measurement of cell structure at multi-scale, which can be applied to various 3D culture models for in-depth investigation.

TBK1 pharmacological inhibition mitigates osteoarthritis through attenuating inflammation and cellular senescence in chondrocytes.

Objectives: TANK-binding kinase 1 (TBK1) is pivotal in autoimmune and inflammatory diseases, yet its role in osteoarthritis (OA) remains elusive. This study sought to elucidate the effect of the TBK1 inhibitor BX795 on OA and to delineate the underlying mechanism by which it mitigates OA. Methods: Interleukin-1 Beta (IL-1ß) was utilized to simulate inflammatory responses and extracellular matrix degradation in vitro. In vivo, OA was induced in 8-week-old mice through destabilization of the medial meniscus surgery. The impact of BX795 on OA was evaluated using histological analysis, X-ray, micro-CT, and the von Frey test. Additionally, Western blot, RT-qPCR, and immunofluorescence assays were conducted to investigate the underlying mechanisms of BX795. Results: Phosphorylated TBK1 (P-TBK1) levels were found to be elevated in OA knee cartilage of both human and mice. Furthermore, intra-articular injection of BX795 ameliorated cartilage degeneration and alleviated OA-associated pain. BX795 also counteracted the suppression of anabolic processes and the augmentation of catabolic activity, inflammation, and senescence observed in the OA mice. In vitro studies revealed that BX795 reduced P-TBK1 levels and reversed the effects of anabolism inhibition, catabolism promotion, and senescence induction triggered by IL-1ß. Mechanistically, BX795 inhibited the IL-1ß-induced activation of the cGAS-STING and TLR3-TRIF signaling pathways in chondrocytes. Conclusions: Pharmacological inhibition of TBK1 with BX795 protects articular cartilage by inhibiting the activation of the cGAS-STING and TLR3-TRIF signaling pathways. This action attenuates inflammatory responses and cellular senescence, positioning BX795 as a promising therapeutic candidate for OA treatment. The translational potential of this article: This study furnishes experimental evidence and offers a potential mechanistic explanation supporting the efficacy of BX795 as a promising candidate for OA treatment.

Effect of biological sex and short-term high-fat diet on cellular proliferation, ribosomal biogenesis, and targeted protein abundance in murine articular cartilage.

Objective: To identify factors contributing to sex-differences in OA risk by evaluating the short-term effect of high-fat (HF) diet on sex-specific changes in cartilage cell proliferation, ribosomal biogenesis, and targeted extra-cellular and cellular protein abundance. Materials and methods: Knee cartilage was harvested to the subchondral bone from 20-week-old female and male C57BL/6J mice fed a low-fat or HF diet for 4 weeks and labeled with deuterium oxide for 1, 3, 5, 7, 15, or 21 days. Deuterium enrichment was quantified in isolated DNA and RNA to measure cell proliferation and ribosomal biogenesis, respectively. Protein concentration was measured using targeted high resolution accurate mass spectrometry. Results: HF diet increased the maximal deuterium incorporation into DNA from approximately 40 to 50%, albeit at a slower rate. These findings, which were magnified in female versus male mice, indicate a greater number of proliferating cells with longer half-lives under HF diet conditions. HF diet caused distinct sex-dependent effects on deuterium incorporation into RNA, increasing the fraction of ribosomes undergoing biogenesis in male mice and doubling the rate of ribosome biogenesis in female mice. HF diet altered cartilage protein abundance similarly in both sexes, except for matrilin-3, which was more abundant in HF versus LF conditions in female mice only. Overall, HF diet treatment had a stronger effect than sex on cartilage protein abundance, with most changes involving extracellular matrix and matrix-associated proteins. Conclusions: Short-term HF diet broadly altered cartilage matrix protein abundance, while sex-dependent effects primarily involved differences in cell proliferation and ribosomal biogenesis.

Reduced Cell Migration in Human Chondrocyte Sheets Increases Tissue Stiffness and Cartilage Protein Production.

BACKGROUND: Chondrogenic differentiation medium (CDM) is usually used to maintain chondrogenic activity during chondrocyte sheet production. However, tissue qualities remain to be determined as to what factors improve cell functions. Moreover, the relationship between CDM and cell migration proteins has not been reported. METHOD: In this study, the effect of CDM on the behavior of chondrocyte sheets was investigated. Structural analysis, mechanical testing and proteomics were performed to observe tissue qualities. The relationship between CDM and cell migration proteins were investigated using time-lapse observations and bioinformatic analysis. RESULTS: During 48 h, CDM affected the chondrocyte behaviors by reducing cell migration. Compared to the basal medium, CDM impacted the contraction of monolayered chondrocyte sheets. At day 7, the contracted sheets increased tissue thickness and improved tissue stiffness. Cartilage specific proteins were also upregulated. Remarkedly, the chondrocyte sheets in CDM displayed downregulated proteins related to cell migration. Bioinformatic analysis revealed that TGFß1 was shown to be associated with cartilage functions and cell migration. Pathway analysis of chondrocyte sheets in CDM also revealed the presence of a TGFß pathway without activating actin production, which might be involved in synthesizing cartilage-specific proteins. Cell migration pathway showed MAPK signaling in both cultures of the chondrocyte sheets. CONCLUSION: Reduced cell migration in the chondrocyte sheet affected the tissue quality. Using CDM, TGFß1 might trigger cartilage protein production through the TGFß pathway and be involved in cell migration via the MAPK signaling pathway. Understanding cell behaviors and their protein expression would be beneficial for developing high-quality tissue-engineered cartilage.

Metabolic Profiles of Encapsulated Chondrocytes Exposed to Short-Term Simulated Microgravity.

The mechanism by which chondrocytes respond to reduced mechanical loading environments and the subsequent risk of developing osteoarthritis remains unclear. This is of particular concern for astronauts. In space the reduced joint loading forces during prolonged microgravity (10-6 g) exposure could lead to osteoarthritis (OA), compromising quality of life post-spaceflight. In this study, we encapsulated human chondrocytes in an agarose gel of similar stiffness to the pericellular matrix to mimic the cartilage microenvironment. We then exposed agarose-chondrocyte constructs to simulated microgravity (SM) using a rotating wall vessel (RWV) bioreactor to better assess the cartilage health risks associated with spaceflight. Global metabolomic profiling detected a total of 1205 metabolite features across all samples, with 497 significant metabolite features identified by ANOVA (FDR-corrected p-value < 0.05). Specific metabolic shifts detected in response to SM exposure resulted in clusters of co-regulated metabolites, as well as key metabolites identified by variable importance in projection scores. Microgravity-induced metabolic shifts in gel constructs and media were indicative of protein synthesis, energy metabolism, nucleotide metabolism, and oxidative catabolism. The microgravity associated-metabolic shifts were consistent with early osteoarthritic metabolomic profiles in human synovial fluid, which suggests that even short-term exposure to microgravity (or other reduced mechanical loading environments) may lead to the development of OA.

Synoviocytes assist in modulating the effect of Ross River virus infection in micromass-cultured primary human chondrocytes.

Introduction. Ross River virus (RRV) is a mosquito-borne virus prevalent in Australia and the islands of the South Pacific, where it causes an arthritogenic illness with a hallmark feature of severe joint pain. The joint space is a unique microenvironment that contains cartilage and synovial fluid. Chondrocytes and synoviocytes are crucial components of the joint space and are known targets of RRV infection.Hypothesis/Gap statement. Understanding the relationship between synoviocytes and chondrocytes during RRV infection will provide further insights into RRV-induced joint pathology.Methodology. To better understand the unique dynamics of these cells during RRV infection, we used primary chondrocytes cultured in physiologically relevant micromasses. We then directly infected micromass chondrocytes or infected primary fibroblast-like synoviocytes (FLS), co-cultured with micromass chondrocytes. Micromass cultures and supernatants were collected and analysed for viral load with a PCR array of target genes known to play a role in arthritis.Results. We show that RRV through direct or secondary infection in micromass chondrocytes modulates the expression of cellular factors that likely contribute to joint inflammation and disease pathology, as well as symptoms such as pain. More importantly, while we show that RRV can infect micromass-cultured chondrocytes via FLS infection, FLS themselves affect the regulation of cellular genes known to contribute to arthritis.Conclusion. Single-cell culture systems lack the complexity of in vivo systems, and understanding the interaction between cell populations is crucial for deciphering disease pathology, including for the development of effective therapeutic strategies.

A proteomic study of the downregulation of TRIM37 on chondrocytes: Implications for the MULIBREY syndrome.

MULIBREY nanism which results from autosomal recessive mutations in TRIM37 impacts skeletal development, leading to growth delay with complications in multiple organs. In this study, we employed a combined proteomics and qPCR screening approach to investigate the molecular alterations in the CHON-002 cell line by comparing CHON-002 wild-type (WT) cells to CHON-002 TRIM37 knockdown (KD) cells. Our proteomic analysis demonstrated that TRIM37 depletion predominantly affects the expression of extracellular matrix proteins (ECM). Specifically, nanoLC-MS/MS experiments revealed an upregulation of SPARC, and collagen products (COL1A1, COL3A1, COL5A1) in response to TRIM37 KD. Concurrently, large-scale qPCR assays targeting osteogenesis-related genes corroborated these dysregulations of SPARC at the mRNA level. Gene ontology enrichment analysis highlighted the involvement of dysregulated proteins in ECM organization and TGF-ß signaling pathways, indicating a role for TRIM37 in maintaining ECM integrity and regulating chondrocyte proliferation. These findings suggest that TRIM37 deficiency in chondrocytes change ECM protein composition and could impairs long bone growth, contributing to the pathophysiology of MULIBREY nanism.

IRF1 regulation of ZBP1 links mitochondrial DNA and chondrocyte damage in osteoarthritis.

BACKGROUND: Z-DNA binding protein 1 (ZBP1) is a nucleic acid sensor that is involved in multiple inflammatory diseases, but whether and how it contributes to osteoarthritis (OA) are unclear. METHODS: Cartilage tissues were harvested from patients with OA and a murine model of OA to evaluate ZBP1 expression. Subsequently, the functional role and mechanism of ZBP1 were examined in primary chondrocytes, and the role of ZBP1 in OA was explored in mouse models. RESULTS: We showed the upregulation of ZBP1 in articular cartilage originating from OA patients and mice with OA after destabilization of the medial meniscus (DMM) surgery. Specifically, knockdown of ZBP1 alleviated chondrocyte damage and protected mice from DMM-induced OA. Mechanistically, tumor necrosis factor alpha induced ZBP1 overexpression in an interferon regulatory factor 1 (IRF1)-dependent manner and elicited the activation of ZBP1 via mitochondrial DNA (mtDNA) release and ZBP1 binding. The upregulated and activated ZBP1 could interact with receptor-interacting protein kinase 1 and activate the transforming growth factor-beta-activated kinase 1-NF-κB signaling pathway, which led to chondrocyte inflammation and extracellular matrix degradation. Moreover, inhibition of the mtDNA-IRF1-ZBP1 axis with Cyclosporine A, a blocker of mtDNA release, could delay the progression of DMM-induced OA. CONCLUSIONS: Our data revealed the pathological role of the mtDNA-IRF1-ZBP1 axis in OA chondrocytes, suggesting that inhibition of this axis could be a viable therapeutic approach for OA.

Foxc1 and Foxc2 regulate growth plate chondrocyte maturation towards hypertrophy in the embryonic mouse limb skeleton.

The Forkhead box transcription factors Foxc1 and Foxc2 are expressed in condensing mesenchyme cells at the onset of endochondral ossification. We used the Prx1-cre mouse to ablate Foxc1 and Foxc2 in limb skeletal progenitor cells. Prx1-cre;Foxc1Δ/ Δ;Foxc2Δ/Δ limbs were shorter than controls, with worsening phenotypes in distal structures. Cartilage formation and mineralization was severely disrupted in the paws. The radius and tibia were malformed, while the fibula and ulna remained unmineralized. Chondrocyte maturation was delayed with fewer Indian Hedgehog-expressing, prehypertrophic chondrocytes forming and a smaller hypertrophic chondrocyte zone. Later, progression out of chondrocyte hypertrophy was slowed, leading to an accumulation of COLX-expressing hypertrophic chondrocyte zone and formation of a smaller primary ossification center with fewer osteoblast progenitor cells populating this region. Targeting Foxc1 and Foxc2 in hypertrophic chondrocytes with Col10a1-cre also resulted in an expanded hypertrophic chondrocyte zone and smaller primary ossification center. Our findings suggest that Foxc1 and Foxc2 direct chondrocyte maturation towards hypertrophic chondrocyte formation. At later stages, Foxc1 and Foxc2 regulate function in hypertrophic chondrocyte remodelling to allow primary ossification center formation and osteoblast recruitment.

Deferoxamine alleviates chondrocyte senescence and osteoarthritis progression by maintaining iron homeostasis.

BACKGROUND: Osteoarthritis (OA) is a prevalent age-related disease characterized by the gradual deterioration of cartilage. The involvement of chondrocyte senescence is crucial in the pathogenesis of OA. Desferoxamine (DFO) is an iron chelator with therapeutic potential in various diseases. However, the relationship of chondrocyte senescence and iron homeostasis is largely unknown. METHODS: Chondrocyte senescence was induced using tert-butyl hydroperoxide (TBHP), and the impact of DFO on chondrocyte senescence and iron metabolism was assessed through techniques such as western blotting, qRT-PCR, and ß-Galactosidase staining. To assess the impact of DFO on chondrocyte senescence and the progression of osteoarthritis (OA), the surgical destabilization of the medial meniscus model was established. RESULTS: In chondrocytes, TBHP administration resulted in elevated expression of P16, P21, and P53, as well as alterations in SA-ß-gal staining. Nevertheless, DFO effectively mitigated chondrocyte senescence induced by TBHP, and reversed the decrease in collagen II expression and increase in MMP13 expression caused by TBHP. Mechanismly, TBHP induced NCOA4 expression and iron release in chondrocytes. Excessive iron could induce chondrocyte senescence, whereas, DFO could inhibit NCOA4 expression and restore ferritin level, and chelate excessive iron. Importantly, intra-articular injection of DFO enhanced collagen II expression and reduced expression of P16, P21, and MMP13 of cartilage in OA mice, and delayed cartilage degeneration. CONCLUSIONS: Overall, this study provides evidence that DFO has the potential to alleviate chondrocyte senescence induced by TBHP and slow down the progression of osteoarthritis (OA) by effectively chelating excessive iron. These findings suggest that iron chelation could be a promising therapeutic strategy for treating OA.

Latest Advances in Chondrocyte-Based Cartilage Repair.

Chondrocyte-based cell therapy has been used for more than 30 years and is still considered to be a promising method of cartilage repair despite some limitations. This review introduces the latest developments of four generations of autologous chondrocyte implantation and current autologous chondrocyte products. The regeneration of cartilage from adult chondrocytes is limited by culture-induced dedifferentiation and patient age. Cartibeads is an innovative three-step method to produce high-quality hyaline cartilage microtissues, and it is developed from adult dedifferentiated chondrocytes with a high number of cell passages. In addition, allogeneic chondrocyte therapies using the Quantum hollow-fiber bioreactor and several signaling pathways involved in chondrocyte-based cartilage repair are mentioned, such as WNT signaling, the BMP-2/WISP1 pathway, and the FGF19 pathway.

Melatonin Delays Arthritis Inflammation and Reduces Cartilage Matrix Degradation through the SIRT1-Mediated NF-κB/Nrf2/TGF-ß/BMPs Pathway.

Cartilage, a flexible and smooth connective tissue that envelops the surfaces of synovial joints, relies on chondrocytes for extracellular matrix (ECM) production and the maintenance of its structural and functional integrity. Melatonin (MT), renowned for its anti-inflammatory and antioxidant properties, holds the potential to modulate cartilage regeneration and degradation. Therefore, the present study was devoted to elucidating the mechanism of MT on chondrocytes. The in vivo experiment consisted of three groups: Sham (only the skin tissue was incised), Model (using the anterior cruciate ligament transection (ACLT) method), and MT (30 mg/kg), with sample extraction following 12 weeks of administration. Pathological alterations in articular cartilage, synovium, and subchondral bone were evaluated using Safranin O-fast green staining. Immunohistochemistry (ICH) analysis was employed to assess the expression of matrix degradation-related markers. The levels of serum cytokines were quantified via Enzyme-linked immunosorbent assay (ELISA) assays. In in vitro experiments, primary chondrocytes were divided into Control, Model, MT, negative control, and inhibitor groups. Western blotting (WB) and Quantitative RT-PCR (q-PCR) were used to detect Silent information regulator transcript-1 (SIRT1)/Nuclear factor kappa-B (NF-κB)/Nuclear factor erythroid-2-related factor 2 (Nrf2)/Transforming growth factor-beta (TGF-ß)/Bone morphogenetic proteins (BMPs)-related indicators. Immunofluorescence (IF) analysis was employed to examine the status of type II collagen (COL2A1), SIRT1, phosphorylated NF-κB p65 (p-p65), and phosphorylated mothers against decapentaplegic homolog 2 (p-Smad2). In vivo results revealed that the MT group exhibited a relatively smooth cartilage surface, modest chondrocyte loss, mild synovial hyperplasia, and increased subchondral bone thickness. ICH results showed that MT downregulated the expression of components related to matrix degradation. ELISA results showed that MT reduced serum inflammatory cytokine levels. In vitro experiments confirmed that MT upregulated the expression of SIRT1/Nrf2/TGF-ß/BMPs while inhibiting the NF-κB pathway and matrix degradation-related components. The introduction of the SIRT1 inhibitor Selisistat (EX527) reversed the effects of MT. Together, these findings suggest that MT has the potential to ameliorate inflammation, inhibit the release of matrix-degrading enzymes, and improve the cartilage condition. This study provides a new theoretical basis for understanding the role of MT in decelerating cartilage degradation and promoting chondrocyte repair in in vivo and in vitro cultured chondrocytes.

A Reactive Oxygen Species "Sweeper" Based on Hollow Mesopore Cerium Oxide Nanospheres for Targeted and Anti-Inflammatory Management of Osteoarthritis.

Osteoarthritis (OA) is a progressive joint disorder characterized by sustained oxidative stress, chronic inflammation, and the degradation of cartilage. Despite extensive research on nanocarrier treatment strategies, the therapeutic efficacy remains limited due to the lack of satisfactory vehicles that can simultaneously exhibit excellent ROS scavenging capabilities and high drug loading capacity for effective nonsurgical management of OA. In this work, we propose an innovative strategy utilizing hollow mesoporous cerium oxide nanospheres coated with membranes derived from apoptotic chondrocytes as a reactive oxygen species "sweeper" for targeted and anti-inflammatory therapy of OA. The developed DEX@HMCeNs@M demonstrates superior drug loading capacity, notable antioxidant properties, favorable biocompatibility, and controlled drug release. By leveraging the camouflage provided by apoptotic chondrocyte membranes, the engineered DEX@HMCeNs@M, which bear natural "eat me" signals, can effectively mimic chondrocyte apoptotic bodies within the joints, thereby enabling targeted delivery of the anti-inflammatory drug DEX and subsequent controlled release triggered by the acidic environment of OA. Both in vitro and in vivo experiments validate the enhanced therapeutic efficacy of our DEX@HMCeNs@M sweeper, which operates through a synergistic mechanism involving scavenging of ROS overproduction, inhibition of inflammation, restoration of mitochondrial damage, and reduction of chondrocyte apoptosis. These findings underscore the potential and efficiency of our developed DEX@HMCeNs@M strategy as an encouraging interventional approach for the progressive treatment of OA.

Impact of Tissue Handling and Size Modification on Septal Chondrocyte Viability.

INTRODUCTION: The physical modification of cartilage grafts during rhinoplasty risks chondrocyte death at the margins where the tissue is cut. This study compares chondrocyte viability between diced, scaled, and pate samples in human models, and further computes percent chondrocyte viability as a function of sequential dicing size in a computational model. METHODS: Septal cartilage from 11 individuals was prepared as follows: diced (1 mm cubic), scaled (shaved to <1 mm thickness ~ translucent), pate (0.02 g of scraped cartilage surface), positive control (2 × 2 mm diced), and negative control (2 × 2 mm diced soaked in 70% EtOH). Viability analysis was performed using Live/Dead assay™ and confocal microscopy. Numerical simulation of cartilage dicing in 0.05 mm increments was performed using MATLAB assuming 250 chondrocytes/mm3 with each average chondrocyte size of 65 µm2. RESULTS: Chondrocyte viability was similar between 1 mm diced cartilage, scaled cartilage, and positive control samples (p > 0.05). Conversely, pate samples had significantly less viability compared to positive controls, diced samples, and scaled samples (all p < 0.01 after Bonferroni correction). Pate samples had similar chondrocyte viability compared to negative controls (p = 0.36). On computational modeling, cartilage viability decreased to 50% as the diced sample was cut from 1 mm edge length to 0.7-0.8 mm. Similarly, cartilage viability decreased to 26% at 0.55-0.65 mm, 11% at 0.4-0.5 mm, and <5% at <0.4 mm edge length. CONCLUSION: Modifying septal cartilage grafts into 1 mm diced or scaled samples maintains ideal chondrocyte viability whereas pate preparations result in significant chondrocyte death. According to computational analysis, chondrocyte viability sharply decreases as the cartilage is diced below 0.7-0.8 mm. LEVEL OF EVIDENCE: N/A Laryngoscope, 2024.

Single-cell RNA sequencing reveals different chondrocyte states in femoral cartilage between osteoarthritis and healthy individuals.

Background: Cartilage injury is the main pathological manifestation of osteoarthritis (OA). Healthy chondrocyte is a prerequisite for cartilage regeneration and repair. Differences between healthy and OA chondrocyte types and the role these types play in cartilage regeneration and OA progression are unclear. Method: This study conducted single-cell RNA sequencing (scRNA-seq) on the cartilage from normal distal femur of the knee (NC group) and OA femur (OA group) cartilage, the chondrocyte atlas was constructed, and the differences of cell subtypes between the two groups were compared. Pseudo-time and RNA velocity analysis were both performed to verify the possible differentiation sequence of cell subtypes. GO and KEGG pathway enrichment analysis were used to explore the potential functional characteristics of each cell subtype, and to predict the functional changes during cell differentiation. Differences in transcriptional regulation in subtypes were explored by single-cell regulatory network inference and clustering (SCENIC). The distribution of each cell subtype in cartilage tissue was identified by immunohistochemical staining (IHC). Result: A total of 75,104 cells were included, they were divided into 19 clusters and annotated as 11 chondrocyte subtypes, including two new chondrocyte subtypes: METRNL+ and PRG4+ subtype. METRNL+ is in an early stage during chondrocyte differentiation, and RegC-B is in an intermediate state before chondrocyte dedifferentiation. With cell differentiation, cell subtypes shift from genetic expression to extracellular matrix adhesion and collagen remodeling, and signal pathways shift from HIF-1 to Hippo. The 11 subtypes were finally classified as intrinsic chondrocytes, effector chondrocytes, abnormally differentiated chondrocytes and dedifferentiated chondrocytes. IHC was used to verify the presence and distribution of each chondrocyte subtype. Conclusion: This study screened two new chondrocyte subtypes, and a novel classification of each subtype was proposed. METRNL+ subtype is in an early stage during chondrocyte differentiation, and its transcriptomic characteristics and specific pathways provide a foundation for cartilage regeneration. EC-B, PRG4+ RegC-B, and FC are typical subtypes in the OA group, and the HippO-Taz pathway enriched by these cell subtypes may play a role in cartilage repair and OA progression. RegC-B is in the intermediate state before chondrocyte dedifferentiation, and its transcriptomic characteristics may provide a theoretical basis for intervening chondrocyte dedifferentiation.

Outcomes following gel-based autologous chondrocyte implantation for articular cartilage defects of the knee.

BACKGROUND: Gel-based autologous chondrocyte implantation (GACI) enables a simpler and more effective delivery of chondrocytes with reproducible three-dimensional structural restoration of the articular cartilage surface. There is limited documentation of medium-term outcomes. This study assessed safety and effectiveness of GACI for treatment of cartilage defects of the knee. METHODS: This multicentric retrospective study was conducted across eight hospitals in India. Patients who had undergone GACI (CARTIGROW®) between 2008 and 2014 for the treatment of focal articular cartilage defects of the knee (mean defect size 4.5 ± 5.8 cm2) in limbs with normal alignment were analyzed. Primary outcomes were changes in Lysholm Knee Scoring Scale score, and Knee Outcome Sports Activity Scale (SAS). RESULTS: A total of 107 patients (110 knee joints) with mean age 31.0 ± 10.5 years were included. The mean follow-up was 9.8 ± 1.5 years (range 7.85-13.43). Majority had osteochondritis dissecans (n = 51; 46.4%). The mean Lysholm Knee Scoring Scale score (81.23 ± 13.21 vs. 51.32 ± 17.89; p < 0.0001) and SAS score (80.93 ± 8.26 vs. 28.11 ± 12.28; p < 0.0001) improved significantly at follow-up as compared to pre-operative. Magnetic Resonance Observation of Cartilage Repair Tissue score in 39 patients at minimum 2 years follow-up was 84.5 ± 4.3. Among 30 patients who were playing sports before treatment, 17 patients (56.7%) could return to the same or higher level of sports post-transplantation. No major intra-operative or post-operative complications were noted. Four patients warranted revision surgery. CONCLUSION: GACI is an effective treatment option for large focal articular cartilage defects of the knee with a low complication rate and revision rate and significant improvement in functional scores.

Biomechanism of abnormal stress on promoting osteoarthritis of temporomandibular joint through Piezo1 ion channel.

OBJECTIVE: This study aimed to investigate whether flow fluid shear stress (FFSS)-mediated signal transduction affects the function of Piezo1 ion channel in chondrocyte and to further explore the role of mechanical overloading in development of temporomandibular joint osteoarthritis (TMJ OA). METHODS: Immunohistochemical staining was used to determine the expression of Piezo1 in TMJ OA tissue collected from rat unilateral anterior crossbite (UAC) models. Chondrocytes harvested from normal adult SD rats were treated with FFSS (0, 4, 8, 12 dyn/cm2) in vitro. Immunofluorescent staining, real-time polymerase chain reaction, western blotting, flow cytometry and phalloidin assay were performed to detect the changes of cellular morphology as well as the expression of Piezo1 and certain pro-inflammatory and degradative factors in chondrocyte. RESULTS: Immunohistochemical analysis revealed that significantly increased Piezo1 expression was associated with UAC stimulation (p < .05). As applied FFSS escalated (4, 8 and 12 dyn/cm2), the expression levels of Piezo1, ADAMTS-5, MMP-13 and Col-X gradually increased, compared with the non-FFSS group (p < .05). Administering Piezo1 ion channel inhibitor to chondrocytes beforehand, it was observed that expression of ADAMTS-5, MMP-13 and Col-X was substantially decreased following FFSS treatment (p < .05) and the effect of cytoskeletal thinning was counteracted. The activated Piezo1 ion channel enhanced intracellular Ca2+ excess in chondrocytes during abnormal mechanical stimulation and the increased intracellular Ca2+ thinned the cytoskeleton of F-actin. CONCLUSIONS: Mechanical overloading activates Piezo1 ion channel to promote pro-inflammation and degradation and to increase Ca2+ concentration in chondrocyte, which may eventually result in TMJ OA.

Denosumab ameliorates osteoarthritis by protecting cartilage against degradation and modulating subchondral bone remodeling.

Osteoarthritis (OA) is the most prevalent degenerative joint disease worldwide. Effective management for early-stage OA is crucial. Denosumab (DS) has been widely used to treat osteoporosis (OP) and rheumatoid arthritis, but its potential for managing OA remains clear. We assessed the effects of DS on osteoclast activity and chondrocyte apoptosis using tartrate-resistant acid phosphatase (TRAP) assay, quantitative real-time polymerase chain reaction (qRT-PCR), flow cytometry, and TUNEL staining. To assess the impact of DS on the NF-κB pathway, we performed Western blot and immunofluorescence staining. Additionally, we used an OA model to explore the influence of DS on subchondral bone remodeling and cartilage degeneration in vivo. We found that DS hindered receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis by inhibiting the activity of the NF-κB pathway. Besides, DS alleviated reactive oxygen species (ROS)-induced apoptosis in chondrocytes by regulating the expression of genes related to apoptosis. Moreover, we observed an attenuation of OA-related subchondral bone remodeling and cartilage degeneration in vivo. Our findings indicate that DS could effectively suppress osteoclast activity and chondrocyte apoptosis, thereby mitigating OA-related subchondral bone remodeling and cartilage degeneration. These results provide a mechanistic basis for using DS to treat OA.