Aims. After a few passages of in vitro culture, primary human articular chondrocytes undergo senescence and loss of their phenotype. Most of the available chondrocyte cell lines have been obtained from cartilage tissues different from diarthrodial joints, and their utility for osteoarthritis (OA) research is reduced. Thus, the goal of this research was the development of immortalized chondrocyte cell lines proceeded from the articular cartilage of patients with and without OA. Methods. Using telomerase reverse transcriptase (hTERT) and SV40 large T antigen (SV40LT), we transduced primary OA articular chondrocytes. Proliferative capacity, degree of senescence, and chondrocyte surface antigen expression in transduced chondrocytes were evaluated. In addition, the capacity of transduced chondrocytes to synthesize a tissue similar to cartilage and to respond to interleukin (IL)-1β was assessed. Results. Coexpression of both transgenes (SV40 and hTERT) were observed in the nuclei of transduced chondrocytes. Generated chondrocyte cell lines showed a high proliferation capacity and less than 2% of senescent cells. These cell lines were able to form 3D aggregates analogous to those generated by primary articular chondrocytes, but were unsuccessful in synthesizing cartilage-like tissue when seeded on type I collagen sponges. However, generated chondrocyte cell lines maintained the potential to respond to IL-1β stimulation. Conclusion. Through SV40LT and hTERT transduction, we successfully immortalized chondrocytes. These immortalized chondrocytes were able to overcome senescence in vitro, but were incapable of synthesizing cartilage-like tissue under the experimental conditions. Nonetheless, these chondrocyte cell lines could be advantageous for OA investigation since, similarly to primary articular chondrocytes, they showed capacity to upregulate inflammatory mediators in response to the IL-1β cytokine. Cite this article: Bone Joint Res 2023;12(1):46–57

Aims. To explore the novel molecular mechanisms of histone deacetylase 4 (HDAC4) in chondrocytes via RNA sequencing (RNA-seq) analysis. Methods. Empty adenovirus (EP) and a HDAC4 overexpression adenovirus were transfected into cultured human chondrocytes. The cell survival rate was examined by real-time cell analysis (RTCA) and EdU and flow cytometry assays. Cell biofunction was detected by Western blotting. The expression profiles of messenger RNAs (mRNAs) in the EP and HDAC4 transfection groups were assessed using whole-transcriptome sequencing (RNA-seq). Volcano plot, Gene Ontology, and pathway analyses were performed to identify differentially expressed genes (DEGs). For verification of the results, the A289E/S246/467/632 A sites of HDAC4 were mutated to enhance the function of HDAC4 by increasing HDAC4 expression in the nucleus. RNA-seq was performed to identify the molecular mechanism of HDAC4 in chondrocytes. Finally, the top ten DEGs associated with ribosomes were verified by quantitative polymerase chain reaction (QPCR) in chondrocytes, and the top gene was verified both in vitro and in vivo. Results. HDAC4 markedly improved the survival rate and biofunction of chondrocytes. RNA-seq analysis of the EP and HDAC4 groups showed that HDAC4 induced 2,668 significant gene expression changes in chondrocytes (1,483 genes upregulated and 1,185 genes downregulated, p < 0.05), and ribosomes exhibited especially large increases. The results were confirmed by RNA-seq of the EP versus mutated HDAC4 groups and the validations in vitro and in vivo. Conclusion. The enhanced ribosome pathway plays a key role in the mechanism by which HDAC4 improves the survival rate and biofunction of chondrocytes. Cite this article: Bone Joint Res 2023;12(7):433–446

Aims. Abnormal lipid metabolism is involved in the development of osteoarthritis (OA). Growth differentiation factor 11 (GDF11) is crucial in inhibiting the differentiation of bone marrow mesenchymal stem cells into adipocytes. However, whether GDF11 participates in the abnormal adipogenesis of chondrocytes in OA cartilage is still unclear. Methods. Six-week-old female mice were subjected to unilateral anterior crossbite (UAC) to induce OA in the temporomandibular joint (TMJ). Histochemical staining, immunohistochemical staining (IHC), and quantitative real-time polymerase chain reaction (qRT-PCR) were performed. Primary condylar chondrocytes of rats were stimulated with fluid flow shear stress (FFSS) and collected for oil red staining, immunofluorescence staining, qRT-PCR, and immunoprecipitation analysis. Results. Abnormal adipogenesis, characterized by increased expression of CCAAT/enhancer-binding protein α (CEBPα), fatty acid binding protein 4 (FABP4), Perilipin1, Adiponectin (AdipoQ), and peroxisome proliferator-activated receptor γ (PPARγ), was enhanced in the degenerative cartilage of TMJ OA in UAC mice, accompanied by decreased expression of GDF11. After FFSS stimulation, there were fat droplets in the cytoplasm of cultured cells with increased expression of PPARγ, CEBPα, FABP4, Perilipin1, and AdipoQ and decreased expression of GDF11. Exogenous GDF11 inhibited increased lipid droplets and expression of AdipoQ, CEBPα, and FABP4 induced by FFSS stimulation. GDF11 did not affect the change in PPARγ expression under FFSS, but promoted its post-translational modification by small ubiquitin-related modifier (SUMOylation). Local injection of GDF11 alleviated TMJ OA-related cartilage degeneration and abnormal adipogenesis in UAC mice. Conclusion. Abnormal adipogenesis of chondrocytes and decreased GDF11 expression were observed in degenerative cartilage of TMJ OA. GDF11 supplementation effectively inhibits the adipogenesis of chondrocytes and thus alleviates TMJ condylar cartilage degeneration. GDF11 may inhibit the abnormal adipogenesis of chondrocytes by affecting the SUMOylation of PPARγ. Cite this article: Bone Joint Res 2022;11(7):453–464

Aims. Pellino1 (Peli1) has been reported to regulate various inflammatory diseases. This study aims to explore the role of Peli1 in the occurrence and development of osteoarthritis (OA), so as to find new targets for the treatment of OA. Methods. After inhibiting Peli1 expression in chondrocytes with small interfering RNA (siRNA), interleukin (IL)-1β was used to simulate inflammation, and OA-related indicators such as synthesis, decomposition, inflammation, and apoptosis were detected. Toll-like receptor (TLR) and nuclear factor-kappa B (NF-κB) signalling pathway were detected. After inhibiting the expression of Peli1 in macrophages Raw 264.7 with siRNA and intervening with lipopolysaccharide (LPS), the polarization index of macrophages was detected, and the supernatant of macrophage medium was extracted as conditioned medium to act on chondrocytes and detect the apoptosis index. The OA model of mice was established by destabilized medial meniscus (DMM) surgery, and adenovirus was injected into the knee cavity to reduce the expression of Peli1. The degree of cartilage destruction and synovitis were evaluated by haematoxylin and eosin (H&E) staining, Safranin O/Fast Green staining, and immunohistochemistry. Results. In chondrocytes, knockdown of Peli1 produced anti-inflammatory and anti-apoptotic effects by targeting the TLR and NF-κB signalling pathways. We found that in macrophages, knockdown of Peli1 can inhibit M1-type polarization of macrophages. In addition, the corresponding conditioned culture medium of macrophages applied to chondrocytes can also produce an anti-apoptotic effect. During in vivo experiments, the results have also shown that knockdown Peli1 reduces cartilage destruction and synovial inflammation. Conclusion. Knockdown of Peli1 has a therapeutic effect on OA, which therefore makes it a potential therapeutic target for OA. Cite this article: Bone Joint Res 2023;12(2):121–132

Aims. Osteoarthritis (OA) is the most common chronic pathema of human joints. The pathogenesis is complex, involving physiological and mechanical factors. In previous studies, we found that ferroptosis is intimately related to OA, while the role of Sat1 in chondrocyte ferroptosis and OA, as well as the underlying mechanism, remains unclear. Methods. In this study, interleukin-1β (IL-1β) was used to simulate inflammation and Erastin was used to simulate ferroptosis in vitro. We used small interfering RNA (siRNA) to knock down the spermidine/spermine N1-acetyltransferase 1 (Sat1) and arachidonate 15-lipoxygenase (Alox15), and examined damage-associated events including inflammation, ferroptosis, and oxidative stress of chondrocytes. In addition, a destabilization of the medial meniscus (DMM) mouse model of OA induced by surgery was established to investigate the role of Sat1 inhibition in OA progression. Results. The results showed that inhibition of Sat1 expression can reduce inflammation, ferroptosis changes, reactive oxygen species (ROS) level, and lipid-ROS accumulation induced by IL-1β and Erastin. Knockdown of Sat1 promotes nuclear factor-E2-related factor 2 (Nrf2) signalling. Additionally, knockdown Alox15 can alleviate the inflammation-related protein expression induced by IL-1β and ferroptosis-related protein expression induced by Erastin. Furthermore, knockdown Nrf2 can reverse these protein expression alterations. Finally, intra-articular injection of diminazene aceturate (DA), an inhibitor of Sat1, enhanced type II collagen (collagen II) and increased Sat1 and Alox15 expression. Conclusion. Our results demonstrate that inhibition of Sat1 could alleviate chondrocyte ferroptosis and inflammation by downregulating Alox15 activating the Nrf2 system, and delaying the progression of OA. These findings suggest that Sat1 provides a new approach for studying and treating OA. Cite this article: Bone Joint Res 2024;13(3):110–123

Aims. Exosomes (exo) are involved in the progression of osteoarthritis (OA). This study aimed to investigate the function of dysfunctional chondrocyte-derived exo (DC-exo) on OA in rats and rat macrophages. Methods. Rat-derived chondrocytes were isolated, and DCs induced with interleukin (IL)-1β were used for exo isolation. Rats with OA (n = 36) or macrophages were treated with DC-exo or phosphate-buffered saline (PBS). Macrophage polarization and autophagy, and degradation and chondrocyte activity of cartilage tissues, were examined. RNA sequencing was used to detect genes differentially expressed in DC-exo, followed by RNA pull-down and ribonucleoprotein immunoprecipitation (RIP). Long non-coding RNA osteoarthritis non-coding transcript (OANCT) and phosphoinositide-3-kinase regulatory subunit 5 (PIK3R5) were depleted in DC-exo-treated macrophages and OA rats, in order to observe macrophage polarization and cartilage degradation. The PI3K/AKT/mammalian target of rapamycin (mTOR) pathway activity in cells and tissues was measured using western blot. Results. DC-exo inhibited macrophage autophagy (p = 0.002) and promoted M1 macrophage polarization (p = 0.002). DC-exo at 20 μg/ml induced collagen degradation (p < 0.001) and inflammatory cell infiltration (p = 0.023) in rats. OANCT was elevated in DC (p < 0.001) and in cartilage tissues of OA patients (p < 0.001), and positively correlated with patients’ Kellgren-Lawrence grade (p < 0.001). PIK3R5 was increased in DC-exo-treated cartilage tissues (p < 0.001), and OANCT bound to fat mass and obesity-associated protein (FTO) (p < 0.001). FTO bound to PIK3R5 (p < 0.001) to inhibit the stability of PIK3R5 messenger RNA (mRNA) (p < 0.001) and disrupt the PI3K/AKT/mTOR pathway (p < 0.001). Conclusion. Exosomal OANCT from DC could bind to FTO protein, thereby maintaining the mRNA stability of PIK3R5, further activating the PI3K/AKT/mTOR pathway to exacerbate OA. Cite this article: Bone Joint Res 2022;11(9):652–668

Cartilage degeneration and loss are key events in the initiation and progression of osteoarthritis (OA). Changes to chondrocyte volume and morphology (in the form of cytoplasmic processes) and thus cell phenotype are implicated, as they lead to the production of a mechanically-weakened extracellular matrix. The chondrocyte cytoskeleton is intimately linked to cell volume and morphology and hence we have investigated alterations to levels and distribution of chondrocyte F-actin that occur during early OA. The femoral heads (FH) from hip joints (N=16) were obtained with ethical permission and patient consent following femoral neck fracture. Cartilage was assessed as grade 0 (non-degenerate) and grade 1 (superficial fibrillation) using OARSI criteria. In situ chondrocyte volume and F-actin distribution were assessed using the fluorescent indicators (5-chloromethyl fluorescein diacetate (CMFDA)) and phalloidin, and imaged and quantified by confocal microscopy, Imaris. TM. and ImageJ software. There were no differences between the volume or total F-actin levels of in situ chondrocytes within the superficial zone of grade 0 (n=164 cells) compared to grade 1 (n=145) cartilage (P>0.05). However, a more detailed analysis of phalloidin labelling was performed, which demonstrated significant increases in both intense punctuate (IP) or intense areas (IA) (P<0.0001; P=0.0175 respectively). A preliminary analysis of IP and IA F-actin labelling suggested that while the former did not appear to be associated with changes to chondrocyte morphology, most of the cytoplasmic processes were associated with the presence of IA at the starting point of the protrusion. These results demonstrate marked changes to F-actin distribution in chondrocytes in the very early stages of cartilage degeneration as occurs in OA. These subtle changes are probably an early indication of a change to the chondrocyte phenotype and thus worthy of further study as they may lead to deleterious alterations to matrix metabolism and ultimately cartilage weakening

Abstract. Objectives. Osteoarthritis (OA) is a painful and debilitating disorder of diarthroidal joints. Progressive degeneration of the cartilage extracellular matrix (ECM) together with abnormal chondrocyte characteristics occur leading to a switch to a fibroblast-like phenotype and production of mechanically-weak cartilage. Early changes to chondrocytes within human cartilage have been observed including chondrocyte swelling. [1]. together with the development of thin cytoplasmic processes which increase in number and length with degeneration. [2]. Changes to chondrocyte phenotype in degenerate cartilage are associated with F-actin redistribution and stress fibres (SF) formation, leading to morphologically-dedifferentiated (fibroblast-like) chondrocytes. [3,4]. It is unclear if these processes are a consequence of ‘passive’ cell swelling into a defective ECM or an ‘active’ event driven by changes in cell metabolism resulting in alterations to cell shape. To address this, we have quantified and compared the distribution and levels of F-actin, a key cytoskeletal protein involved in the formation of cytoplasmic processes, within in situ chondrocytes in non-degenerate and mildly degenerate human cartilage. Methods. Human femoral head cartilage was obtained from 21 patients [15 females, 6 males, average age 69.6yrs, (range 47–90yrs)] following femoral neck fracture, with Ethical Approval and patient's permission. Cartilage explants were removed from areas graded non-degenerate grade 0 (G0) or mildly degenerate grade 1 (G1) and cultured for up to 3wks in Dulbecco's Modified Eagle's Medium (DMEM) +/− 25% human serum (HS). In situ chondrocytes were stained with CMFDA (5-chloromethylfluoresceindiacetate, Cell-Tracker Green®) and phalloidin (F-actin labelling) and imaged by confocal microscopy and analysed quantitatively using ImageJ and Imaris® software. Results. There were significant increases in the total amount (TA) of F-actin and its distribution [intense punctuate (IP) and intense areas (IA)] between the whole chondrocyte populations of G0 and G1 cartilage (P=0.0356; 0.0112; 0.016, respectively). Where the volume of chondrocytes was divided into normal (<1000 µm³) and swollen (≥1000 µm³) cells, F-actin TA increased in swollen cells (P=0.036 within G0 and G1, and P=0.0009 between grades) compared to chondrocytes of normal volume in each grade. Moreover, IP and IA within and between G0 and G1 were higher compared to normal chondrocytes (with P<0.0001 for IP and P<0.001 for IA). In addition, tissue culture experiments demonstrated that 90% of chondrocytes with cytoplasmic processes had strong F-actin intensity (either IP or IA with P<0.0001). Furthermore, 83% of this F-actin was associated with cytoplasmic processes, with >65% situated at the base of the process (P<0.0001). Conclusions. The increases in chondrocyte F-actin levels (TA) and its localisation (IP, IA) appear to be associated with cell swelling and development of cytoplasmic processes, which are both characteristics of early OA cartilage. [1]. This suggests the formation of chondrocyte cytoplasmic processes is an ‘active’ event potentially involving changes to matrix metabolism rather than a ‘passive’ cell swelling into a defective extracellular matrix. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Introduction. Osteoarthritis (OA) often results from joint misloading, which affects chondrocyte calcium signaling through mechano-sensitive receptors such as Piezo1, -2, and TRPV4. Activation of Piezo1, especially under inflammatory conditions, can trigger premature chondrocyte apoptosis. Intra-articular glucocorticoid therapy, while beneficial against inflammation and pain in osteoarthritis, may induce oxidative stress and chondrotoxicity at higher doses. This study aims to assess the effects of glucocorticoids, particularly triamcinolone, on chondrocyte elasticity and mechanosignaling. Method. Chondrocytes isolated from articular condyles obtained from patients undergoing knee replacement surgery (n= 5) were cultured for 7 days in triamcinolone acetonide (TA) at different concentrations (0.2µM – 2mM). Cytoskeletal changes were assessed by F-actin labeling. Cell elasticity was measured using atomic force microscopy (AFM). Labeling cells (n=6 patients) with the calcium-sensitive dye (Fluo-4) enabled monitoring changes in intracellular calcium fluorescence intensity during guided single-cell mechanical indentation (500 nN) by AFM. Result. Cell exposure to 2 mM TA led to cell death and crystallization of TA in the cell culture media. However, the concentration of TA for intra-articular application is 46 times higher at 92.1 mM (40 mg/ml). The maximal pharmacological effect on viable cells was observed at 0.2 mM. AFM results showed a significant decrease of elasticity (p<0.001), alongside significantly higher calcium intensities both prior to and during mechanical stimulation in the TA-treated samples (p<0.05). Conclusion. Administration of TA significantly impacts the mechanical properties of chondrocytes, reducing cellular elasticity while simultaneously enhancing calcium-dependent mechanosensitivity. This data suggests a correlation between glucocorticoid-induced changes in cell elasticity and cell mechanosensitivity. Finding ways to minimize the effect of glucocorticoids on cell mechanosensitivity could help to make future therapies safer and reduce side effects

Aims. Autologous chondrocyte implantation (ACI) is a promising treatment for articular cartilage degeneration and injury; however, it requires a large number of human hyaline chondrocytes, which often undergo dedifferentiation during in vitro expansion. This study aimed to investigate the effect of suramin on chondrocyte differentiation and its underlying mechanism. Methods. Porcine chondrocytes were treated with vehicle or various doses of suramin. The expression of collagen, type II, alpha 1 (COL2A1), aggrecan (ACAN); COL1A1; COL10A1; SRY-box transcription factor 9 (SOX9); nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX); interleukin (IL)-1β; tumour necrosis factor alpha (TNFα); IL-8; and matrix metallopeptidase 13 (MMP-13) in chondrocytes at both messenger RNA (mRNA) and protein levels was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot. In addition, the supplementation of suramin to redifferentiation medium for the culture of expanded chondrocytes in 3D pellets was evaluated. Glycosaminoglycan (GAG) and collagen production were evaluated by biochemical analyses and immunofluorescence, as well as by immunohistochemistry. The expression of reactive oxygen species (ROS) and NOX activity were assessed by luciferase reporter gene assay, immunofluorescence analysis, and flow cytometry. Mutagenesis analysis, Alcian blue staining, reverse transcriptase polymerase chain reaction (RT-PCR), and western blot assay were used to determine whether p67. phox. was involved in suramin-enhanced chondrocyte phenotype maintenance. Results. Suramin enhanced the COL2A1 and ACAN expression and lowered COL1A1 synthesis. Also, in 3D pellet culture GAG and COL2A1 production was significantly higher in pellets consisting of chondrocytes expanded with suramin compared to controls. Surprisingly, suramin also increased ROS generation, which is largely caused by enhanced NOX (p67. phox. ) activity and membrane translocation. Overexpression of p67. phox. but not p67. phox. AD (deleting amino acid (a.a) 199 to 212) mutant, which does not support ROS production in chondrocytes, significantly enhanced chondrocyte phenotype maintenance, SOX9 expression, and AKT (S473) phosphorylation. Knockdown of p67. phox. with its specific short hairpin (sh) RNA (shRNA) abolished the suramin-induced effects. Moreover, when these cells were treated with the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) inhibitor LY294002 or shRNA of AKT1, p67. phox. -induced COL2A1 and ACAN expression was significantly inhibited. Conclusion. Suramin could redifferentiate dedifferentiated chondrocytes dependent on p67. phox. activation, which is mediated by the PI3K/AKT/SOX9 signalling pathway. Cite this article: Bone Joint Res 2022;11(10):723–738

Aims. Circular RNA (circRNA) is involved in the regulation of articular cartilage degeneration induced by inflammatory factors or oxidative stress. In a previous study, we found that the expression of circStrn3 was significantly reduced in chondrocytes of osteoarthritis (OA) patients and OA mice. Therefore, the aim of this paper was to explore the role and mechanism of circStrn3 in osteoarthritis. Methods. Minus RNA sequencing, fluorescence in situ hybridization, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of circStrn3 in human and mouse OA cartilage tissues and chondrocytes. Chondrocytes were then stimulated to secrete exosomal miR-9-5p by cyclic tensile strain. Intra-articular injection of exosomal miR-9-5p into the model induced by destabilized medial meniscus (DMM) surgery was conducted to alleviate OA progression. Results. Tensile strain could decrease the expression of circStrn3 in chondrocytes. CircStrn3 expression was significantly decreased in human and mouse OA cartilage tissues and chondrocytes. CircStrn3 could inhibit matrix metabolism of chondrocytes through competitively ‘sponging’ miRNA-9-5p targeting Kruppel-like factor 5 (KLF5), indicating that the decrease in circStrn3 might be a protective factor in mechanical instability-induced OA. The tensile strain stimulated chondrocytes to secrete exosomal miR-9-5p. Exosomes with high miR-9-5p expression from chondrocytes could inhibit osteoblast differentiation by targeting KLF5. Intra-articular injection of exosomal miR-9-5p alleviated the progression of OA induced by destabilized medial meniscus surgery in mice. Conclusion. Taken together, these results demonstrate that reduction of circStrn3 causes an increase in miR-9-5p, which acts as a protective factor in mechanical instability-induced OA, and provides a novel mechanism of communication among joint components and a potential application for the treatment of OA. Cite this article: Bone Joint Res 2023;12(1):33–45

Aims. Therapeutic agents that prevent chondrocyte loss, extracellular matrix (ECM) degradation, and osteoarthritis (OA) progression are required. The expression level of epidermal growth factor (EGF)-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) in damaged human cartilage is significantly higher than in undamaged cartilage. However, the effect of EDIL3 on cartilage is still unknown. Methods. We used human cartilage plugs (ex vivo) and mice with spontaneous OA (in vivo) to explore whether EDIL3 has a chondroprotective effect by altering OA-related indicators. Results. EDIL3 protein prevented chondrocyte clustering and maintained chondrocyte number and SOX9 expression in the human cartilage plug. Administration of EDIL3 protein prevented OA progression in STR/ort mice by maintaining the number of chondrocytes in the hyaline cartilage and the number of matrix-producing chondrocytes (MPCs). It reduced the degradation of aggrecan, the expression of matrix metalloproteinase (MMP)-13, the Osteoarthritis Research Society International (OARSI) score, and bone remodelling. It increased the porosity of the subchondral bone plate. Administration of an EDIL3 antibody increased the number of matrix-non-producing chondrocytes (MNCs) in cartilage and exacerbated the serum concentrations of OA-related pro-inflammatory cytokines, including monocyte chemotactic protein-3 (MCP-3), RANTES, interleukin (IL)-17A, IL-22, and GROα. Administration of β1 and β3 integrin agonists (CD98 protein) increased the expression of SOX9 in OA mice. Hence, EDIL3 might activate β1 and β3 integrins for chondroprotection. EDIL3 may also protect cartilage by attenuating the expression of IL-1β-enhanced phosphokinase proteins in chondrocytes, especially glycogen synthase kinase 3 alpha/beta (GSK-3α/β) and phospholipase C gamma 1 (PLC-γ1). Conclusion. EDIL3 has a role in maintaining the cartilage ECM and inhibiting the development of OA, making it a potential therapeutic drug for OA. Cite this article: Bone Joint Res 2023;12(12):734–746

Aims. Osteoarthritis (OA) is a common degenerative disease. PA28γ is a member of the 11S proteasome activator and is involved in the regulation of several important cellular processes, including cell proliferation, apoptosis, and inflammation. This study aimed to explore the role of PA28γ in the occurrence and development of OA and its potential mechanism. Methods. A total of 120 newborn male mice were employed for the isolation and culture of primary chondrocytes. OA-related indicators such as anabolism, catabolism, inflammation, and apoptosis were detected. Effects and related mechanisms of PA28γ in chondrocyte endoplasmic reticulum (ER) stress were studied using western blotting, real-time polymerase chain reaction (PCR), and immunofluorescence. The OA mouse model was established by destabilized medial meniscus (DMM) surgery, and adenovirus was injected into the knee cavity of 15 12-week-old male mice to reduce the expression of PA28γ. The degree of cartilage destruction was evaluated by haematoxylin and eosin (HE) staining, safranin O/fast green staining, toluidine blue staining, and immunohistochemistry. Results. We found that PA28γ knockdown in chondrocytes can effectively improve anabolism and catabolism and inhibit inflammation, apoptosis, and ER stress. Moreover, PA28γ knockdown affected the phosphorylation of IRE1α and the expression of TRAF2, thereby affecting the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signalling pathways, and finally affecting the inflammatory response of chondrocytes. In addition, we found that PA28γ knockdown can promote the phosphorylation of signal transducer and activator of transcription 3 (STAT3), thereby inhibiting ER stress in chondrocytes. The use of Stattic (an inhibitor of STAT3 phosphorylation) enhanced ER stress. In vivo, we found that PA28γ knockdown effectively reduced cartilage destruction in a mouse model of OA induced by the DMM surgery. Conclusion. PA28γ knockdown in chondrocytes can inhibit anabolic and catabolic dysregulation, inflammatory response, and apoptosis in OA. Moreover, PA28γ knockdown in chondrocytes can inhibit ER stress by promoting STAT3 phosphorylation. Cite this article: Bone Joint Res 2024;13(11):659–672

While cell morphology has been recognized as a fundamental regulator of cell behavior, few studies have measured the complex cell morphological changes of chondrocytes using quantitative cell morphometry descriptors in relation to inflammation and phenotypic outcome. Acute vs. persistent exposure to IL-1β and how IL-1β modulated dynamic changes in cell morphology in relation to the phenotype, donor and OA grade in healthy and osteoarthritis (OA) chondrocytes was investigated. A panel of quantitative cell morphometry descriptors was measured using an automated high-throughput method. Absolute quantification of gene expression was measured by ddPCR followed by correlation analyses. In OA chondrocytes, chronic IL-1β significantly decreased COL2A1, SOX9, and ACAN, increased IL-6 and IL-8 levels and caused chondrocytes to become less wide, smaller, longer, slimmer, less round and more circular, consistent with a de-differentiated phenotype. In healthy chondrocytes, 3 days after acute (72 h) IL-1β exposure, COL1A2 and IL-6 significantly increased but had minor effects on cell morphology. However, in healthy chondrocytes, persistent IL-1β led to more profound effects in all cell morphology descriptors and chondrocytes expressed significantly less COL2A1 and more IL-6 and IL-8 vs. controls and acutely-stimulated chondrocytes. In both OA and healthy chronically-stimulated chondrocytes, area, width and circularity were sensitive to the persistent presence of the IL-1β cytokine. Moreover, there were many significant and strong correlations among the measured parameters, with several indications of an IL-1β-mediated mechanism. Cell morphology combined with gene expression analysis could guide researchers interested in understanding inflammatory effects in the complex domain of cartilage/chondrocyte biology. Use of quantitative cell morphometry could complement classical approaches by providing numerical data on a large number of cells, thereby providing a biological fingerprint for describing chondrocyte phenotype, which could help to understand how changes in cell morphology lead to disease progression

Osteoarthritis (OA) leads to articular cartilage degradation, following complex dysregulation of chondrocyte's metabolism towards a catabolic state. Mechanical and biochemical signals are involved and need to be considered to understand the condition. Regulatory network-based models (RNM) successfully simulated the biological activity of the chondrocyte and the transduction of mechanical signals at the molecular and cell levels. However, the knowledge gap between single-cell regulation and intercellular communication in tissue volumes hinders the interpretability of such models at larger scales. Accordingly, a novel tissue-level biochemical model is proposed. We hypothesise that it is possible to simulate interacting network effects through the transport of diluted species in a finite-element model, to grasp relevant dynamics of cell and tissue regulation in OA. Chondrocyte RNM equations were translated into a reaction term of 18 multi-species diffusion model (e.g., 3 anti-inflammatory and 8 pro-inflammatory interleukins, 3 pro-anabolic and 1 pro-catabolic growth factors, 2 nociceptive factors and 2 pro-inflammatory cytokines). Elements with RNM reaction terms represented the chondrocytes and were distributed randomly through the model, according to known cellular density in the knee cartilage, and could both react to and produce diffusive entities through the pericellular matrix, associated with reduced diffusion coefficients. The model was constructed over a 2D square of 0.47 mm sides considered to be in the middle of the cartilage, so boundary conditions were settled as periodic. Different simulations were initialised with initial concentrations of either healthy or pro-OA mediators. Preliminary results showed that, independently of the initial conditions, the chondrocytes successfully evolved into anabolic states, in absence of sustained pro-catabolic external stimulations, in contrast to single-cell RNM [2]. Our intercellular model suggests that paracrine communication may increase robustness towards cartilage maintenance, and future tests shall reveal new OA dynamics. Acknowledgements: Funding was provided by the European Commission (ERC-2021-CoG-O-Health-101044828)

Aims. Staphylococcus aureus is a major cause of septic arthritis, and in vitro studies suggest α haemolysin (Hla) is responsible for chondrocyte death. We used an in vivo murine joint model to compare inoculation with wild type S. aureus 8325-4 with a Hla-deficient strain DU1090 on chondrocyte viability, tissue histology, and joint biomechanics. The aim was to compare the actions of S. aureus Hla alone with those of the animal’s immune response to infection. Methods. Adult male C57Bl/6 mice (n = 75) were randomized into three groups to receive 1.0 to 1.4 × 10. 7. colony-forming units (CFUs)/ml of 8325-4, DU1090, or saline into the right stifle joint. Chondrocyte death was assessed by confocal microscopy. Histological changes to inoculated joints were graded for inflammatory responses along with gait, weight changes, and limb swelling. Results. Chondrocyte death was greater with 8325-4 (96.2% (SD 5.5%); p < 0.001) than DU1090 (28.9% (SD 16.0%); p = 0.009) and both were higher than controls (3.8% (SD 1.2%)). Histology revealed cartilage/bone damage with 8325-4 or DU1090 compared to controls (p = 0.010). Both infected groups lost weight (p = 0.006 for both) and experienced limb swelling (p = 0.043 and p = 0.018, respectively). Joints inoculated with bacteria showed significant alterations in gait cycle with a decreased stance phase, increased swing phase, and a corresponding decrease in swing speed. Conclusion. Murine joints inoculated with Hla-producing 8325-4 experienced significantly more chondrocyte death than those with DU1090, which lack the toxin. This was despite similar immune responses, indicating that Hla was the major cause of chondrocyte death. Hla-deficient DU1090 also elevated chondrocyte death compared to controls, suggesting a smaller additional deleterious role of the immune system on cartilage. Cite this article: Bone Joint Res 2022;11(9):669–678

Cell culture on tissue culture plastic (TCP) is widely used across biomedical research to understand the in vivo environment of a targeted biological system. However, growing evidence indicates that the characteristics of cells investigated in this way differ substantially from their characteristics in the human body. The limitations of TCP monolayer cell cultures are especially relevant for chondrocytes, the cell population responsible for producing cartilage matrix, because their zonal organization in hyaline cartilage is not preserved in a flattened monolayer assay. Here, we contrast the response of primary human chondrocytes to inflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma, via transcriptional, translational, and histological profiling, when grown either on TCP or within a 3D cell pellet (scaffold-less). We focus on anti-apoptotic (Bcl2), pro-apoptotic (Bax, Mff, Fis1), and senescent (MMP13, MMP1, PCNA, p16, p21) markers. We find that the 3D environment of the chondrocyte has a profound effect on the behavior and fate of the cell; in TCP monolayer cultures, chondrocytes become anti-apoptotic and undergo senescence in response to inflammatory cytokines, whereas in 3D cell pellet cultures, they exhibit a pro-apoptotic response. Our findings demonstrate that chondrocyte culture environment plays a pivotal role in cell behavior, which has important implications for the clinical applicability of in vitro research of cartilage repair. Although there are practical advantages to 2D cell cultures, our data suggest researchers should be cautious when drawing conclusions if they intend to extrapolate findings to in vivo phenomena. Our data demonstrates opposing chondrocyte responses in relation to apoptosis and senescence, which appear to be solely reliant on the environment of the culture system. This biological observation highlights that proper experimental design is crucial to increase the clinical utility of cartilage repair experiments and streamline their translation to therapy development

Abstract. BACKGROUND. Cell culture on tissue culture plastic (TCP) is widely used across biomedical research to understand the in vivo environment of a targeted biological system. However, growing evidence indicates that the characteristics of cells investigated in this way differ substantially from their characteristics in the human body. The limitations of TCP monolayer cell cultures are especially relevant for chondrocytes, the cell population responsible for producing cartilage matrix, because their zonal organization in hyaline cartilage is not preserved in a flattened monolayer assay. OBJECTIVE. Here, we contrast the response of primary human chondrocytes to inflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma, via transcriptional, translational, and histological profiling, when grown either on TCP or within a 3D cell pellet (scaffold-less). We focus on anti-apoptotic (Bcl2), pro-apoptotic (Bax, Mff, Fis1), and senescent (MMP13, MMP1, PCNA, p16, p21) markers. RESULTS. We find that the 3D environment of the chondrocyte has a profound effect on the behavior and fate of the cell; in TCP monolayer cultures, chondrocytes become anti-apoptotic and undergo senescence in response to inflammatory cytokines, whereas in 3D cell pellet cultures, they exhibit a pro-apoptotic response. CONCLUSION. Our findings demonstrate that chondrocyte culture environment plays a pivotal role in cell behavior, which has important implications for the clinical applicability of in vitro research of cartilage repair. Although there are practical advantages to 2D cell cultures, our data suggest researchers should be cautious when drawing conclusions if they intend to extrapolate findings to in vivo phenomena. Our data demonstrates opposing chondrocyte responses in relation to apoptosis and senescence, which appear to be solely reliant on the environment of the culture system. This biological observation highlights that proper experimental design is crucial to increase the clinical utility of cartilage repair experiments and streamline their translation to therapy development. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Introduction. Piezo1 is a mechanosensitive Ca. 2+. ion channel that has been shown to transduce hyper-physiologic mechanical loads in chondrocytes. In osteoarthritic cartilage, Piezo1 expression was shown to be upregulated by interleukin-1 alpha (IL-1α) and resulted in altered calcium dynamics and actin cytoskeleton rarefication. Together these studies highlight the importance of Piezo1 channels during joint injury. However, the mechanism by which Piezo1 regulates chondrocyte physiology and mechanotransduction during homeostasis is still largely unknown. In this study, we investigate the impact of Piezo1 activation on nuclear mechanics and chromatin methylation state. Methods. Porcine chondrocytes (n=3-5 pigs) were treated with Yoda1, a Piezo1-specific agonist, for either 2, 5, 15 or 180 minutes. To characterize chromatin state, we monitored the abundance of a chromatin methylation marker (H3K9Me3) using immunofluorescence (IF). Atomic force microscopy (AFM, 25 nm cantilever) was employed to quantify the nuclear elastic modulus (NEM) of individual cell nuclei. To explore the interplay between cytoskeletal dynamics and nuclear mechanics, chondrocytes were treated with Latrunculin A (LatA), an actin polymerization inhibitor. Result. IF experiments showed chromatin methylation was the lowest 2 minutes post Yoda1 activation of Piezo1 (p=0.027). Additionally, we found that 2 or 5 minutes post-Piezo1 activation resulted in a significantly lower NEM when compared to the control (p<0.00001). The observed decrease in NEM at 2 and 5 minutes post-Piezo1 activation was not observed after knocking down Piezo1 (p>0.99). In LatA treated cells, the elevated NEM persisted even after Piezo1 activation with Yoda1 (p>0.75). Conclusion. These findings illuminate the mechanism by which Piezo1 activation and actin remodeling regulate transient mechanotransduction during homeostasis. Further research into the transient decrease in nuclear stiffness and chromatin methylation observed during the initial 5 minutes of Piezo1-induced Ca2+ signaling, may contribute to a better understanding of the role of Piezo1 channels in joint injury and development of therapeutic interventions for osteoarthritis

Aims. cAMP response element binding protein (CREB1) is involved in the progression of osteoarthritis (OA). However, available findings about the role of CREB1 in OA are inconsistent. 666-15 is a potent and selective CREB1 inhibitor, but its role in OA is unclear. This study aimed to investigate the precise role of CREB1 in OA, and whether 666-15 exerts an anti-OA effect. Methods. CREB1 activity and expression of a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) in cells and tissues were measured by immunoblotting and immunohistochemical (IHC) staining. The effect of 666-15 on chondrocyte viability and apoptosis was examined by cell counting kit-8 (CCK-8) assay, JC-10, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) staining. The effect of 666-15 on the microstructure of subchondral bone, and the synthesis and catabolism of cartilage, in anterior cruciate ligament transection mice were detected by micro-CT, safranin O and fast green (S/F), immunohistochemical staining, and enzyme-linked immunosorbent assay (ELISA). Results. CREB1 was hyperactive in osteoarthritic articular cartilage, interleukin (IL)-1β-treated cartilage explants, and IL-1β- or carbonyl cyanide 3-chlorophenylhydrazone (CCCP)-treated chondrocytes. 666-15 enhanced cell viability of OA-like chondrocytes and alleviated IL-1β- or CCCP-induced chondrocyte injury through inhibition of mitochondrial dysfunction-associated apoptosis. Moreover, inhibition of CREB1 by 666-15 suppressed expression of ADAMTS4. Additionally, 666-15 alleviated joint degeneration in an ACLT mouse model. Conclusion. Hyperactive CREB1 played a critical role in OA development, and 666-15 exerted anti-IL-1β or anti-CCCP effects in vitro as well as joint-protective effects in vivo. 666-15 may therefore be used as a promising anti-OA drug. Cite this article: Bone Joint Res 2024;13(1):4–18