Aims. Circular RNA (circRNA) S-phase cyclin A-associated protein in the endoplasmic reticulum (ER) (circSCAPER, ID: hsa_circ_0104595) has been found to be highly expressed in osteoarthritis (OA) patients and has been associated with the severity of OA. Hence, the role and mechanisms underlying circSCAPER in OA were investigated in this study. Methods. In vitro cultured human normal chondrocyte C28/I2 was exposed to interleukin (IL)-1β to mimic the microenvironment of OA. The expression of circSCAPER, microRNA (miR)-140-3p, and enhancer of zeste homolog 2 (EZH2) was detected using quantitative real-time polymerase chain reaction and Western blot assays. The extracellular matrix (ECM) degradation, proliferation, and apoptosis of chondrocytes were determined using Western blot, cell counting kit-8, and flow cytometry assays. Targeted relationships were predicted by bioinformatic analysis and verified using dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. The levels of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway-related protein were detected using Western blot assays. Results. CircSCAPER was highly expressed in OA cartilage tissues and IL-1β-induced chondrocytes. Knockdown of circSCAPER reduced IL-1β-evoked ECM degradation, proliferation arrest, and apoptosis enhancement in chondrocytes. Mechanistically, circSCAPER directly bound to miR-140-3p, and miR-140-3p inhibition reversed the effects of circSCAPER knockdown on IL-1β-induced chondrocytes. miR-140-3p was verified to target EZH2, and overexpression of miR-140-3p protected chondrocytes against IL-1β-induced dysfunction via targeting EZH2. Additionally, we confirmed that circSCAPER could regulate EZH2 through sponging miR-140-3p, and the circSCAPER/miR-140-3p/EZH2 axis could activate the PI3K/AKT pathway. Conclusion. CircSCAPER promoted IL-1β-evoked ECM degradation, proliferation arrest, and apoptosis enhancement in chondrocytes via regulating miR-140-3p/EZH2 axis, which gained a new insight into the pathogenesis of OA. Cite this article: Bone Joint Res 2022;11(2):61–72

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. Excessive chondrocyte hypertrophy is a common feature in cartilage degeneration which is susceptible to joint overloading, but the relationship between mechanical overloading and chondrocyte hypertrophy still remains elusive. The aim of our study was to explore the mechanism of mechanical compression-induced chondrocyte hypertrophy. Methods. In this study, the temporomandibular joint (TMJ) degeneration model was built through forced mandibular retrusion (FMR)-induced compression in TMJ. Chondrocytes were also mechanically compressed in vitro. The role of O-GlcNAcylation in mechanical compression-induced chondrocyte hypertrophy manifested through specific activator Thiamet G and inhibitor OSMI-1. Results. Both in vivo and in vitro data revealed that chondrocyte hypertrophic differentiation is promoted by compression. Immunofluorescent and immunoblotting results showed that protein pan-O-GlcNAcylation levels were elevated in these hypertrophic chondrocytes. Pharmacologically inhibiting protein pan-O-GlcNAcylation by OSMI-1 partially mitigated the compression-induced hypertrophic differentiation of chondrocytes. Specifically, runt-related transcription factor 2 (Runx2) and SRY-Box 9 transcription factor (Sox9) were subjected to modification of O-GlcNAcylation under mechanical compression, and pharmacological activation or inhibition of O-GlcNAcylation affected the transcriptional activity of Runx2 but not Sox9. Furthermore, compression-induced protein pan-O-GlcNAcylation in chondrocytes was induced by enhanced expression of glucose transporter 1 (GLUT1), and depletion of GLUT1 by WZB117 dampened the effect of compression on chondrocyte hypertrophy. Conclusion. Our study proposes a novel function of GLUT1-mediated protein O-GlcNAcylation in driving compression-induced hypertrophic differentiation of chondrocytes by O-GlcNAc modification of Runx2, which promoted its transcriptional activity and strengthened the expressions of downstream hypertrophic marker. Cite this article: Bone Joint Res 2025;14(3):209–222

Aims. Osteoarthritis (OA) is a common degenerative disease that leads to pain, disability, and reduced quality of life. Orientin exhibits considerable anti-inflammatory and antioxidative properties, but its role in chondrocyte senescence and OA progress has not yet been fully characterized. The aim of this study was to evaluate the protective effects of orientin on OA. Methods. The role of orientin in extracellular matrix (ECM) degradation, mitochondrial homeostasis, and chondrocyte senescence was investigated in vitro. Meanwhile, we used molecular docking, small molecular inhibitors, and RNA interference to screen and validate candidate proteins regulated by orientin. In an anterior cruciate ligament transection (ACLT) rat model, radiograph, micro-CT, and various histological examinations were applied to evaluate the therapeutic effects of orientin on OA. Results. We found that orientin inhibited ECM degradation and senescence-associated secretory phenotype (SASP) factor expression in interleukin (IL)-1β-treated chondrocytes. Additionally, orientin reduced the level of reactive oxygen species (ROS) and improved mitochondrial homeostasis. Furthermore, orientin suppressed IL-1β-induced activation of the nuclear factor kappa B (NF-κB) signalling pathway. We also found that orientin bound to phosphoinositide 3-kinase (PI3K) and inhibited NF-κB cascades via the PI3K/AKT pathway. In vivo, we demonstrated that orientin improved cartilage wear and reduced synovial inflammation and osteophyte in an ACLT rat model. Conclusion. Orientin improves mitochondrial homeostasis, inhibits chondrocyte senescence, and alleviates OA progress via the PI3K/AKT/NF-κB axis, which suggests that orientin is a potential effective therapeutic agent for OA. Cite this article: Bone Joint Res 2025;14(3):245–258

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

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

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. 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

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

Aims. Osteoporosis (OP) is a metabolic bone disease, characterized by a decrease in bone mineral density (BMD). However, the research of regulatory variants has been limited for BMD. In this study, we aimed to explore novel regulatory genetic variants associated with BMD. Methods. We conducted an integrative analysis of BMD genome-wide association study (GWAS) and regulatory single nucleotide polymorphism (rSNP) annotation information. Firstly, the discovery GWAS dataset and replication GWAS dataset were integrated with rSNP annotation database to obtain BMD associated SNP regulatory elements and SNP regulatory element-target gene (E-G) pairs, respectively. Then, the common genes were further subjected to HumanNet v2 to explore the biological effects. Results. Through discovery and replication integrative analysis for BMD GWAS and rSNP annotation database, we identified 36 common BMD-associated genes for BMD irrespective of regulatory elements, such as FAM3C (p. discovery GWAS. = 1.21 × 10. -25. , p. replication GWAS. = 1.80 × 10. -12. ), CCDC170 (p. discovery GWAS. = 1.23 × 10. -11. , p. replication GWAS. = 3.22 × 10. -9. ), and SOX6 (p. discovery GWAS. = 4.41 × 10. -15. , p. replication GWAS. = 6.57 × 10. -14. ). Then, for the 36 common target genes, multiple gene ontology (GO) terms were detected for BMD such as positive regulation of cartilage development (p = 9.27 × 10. -3. ) and positive regulation of chondrocyte differentiation (p = 9.27 × 10. -3. ). Conclusion. We explored the potential roles of rSNP in the genetic mechanisms of BMD and identified multiple candidate genes. Our study results support the implication of regulatory genetic variants in the development of OP. Cite this article: Bone Joint Res 2023;12(2):147–154

Osteoarthritis (OA) is a highly prevalent and disabling disease with an unmet therapeutic need. The characteristic cartilage loss and alteration of other joint structures result from a complex interaction of multiple risk factors, with mechanical overload consistently playing a central role. This overload generates an inflammatory response in the cartilage due to the activation of the innate immune response in chondrocytes, which occurs through various cellular mechanisms. Moreover, risk factors associated with obesity, being overweight, and metabolic syndrome enhance the inflammatory response both locally and systemically. OA chondrocytes, the only cells present in articular cartilage, are therefore inflamed and initiate an anabolic process in an attempt to repair the damaged tissue, which ultimately results in an aberrant and dysfunctional process. Under these circumstances, where the cartilage continues to be subjected to chronic mechanical stress, proposing a treatment that stimulates the chondrocytes’ anabolic response to restore tissue structure does not appear to be a therapeutic target with a high likelihood of success. In fact, anabolic drugs proposed for the treatment of OA have yet to demonstrate efficacy. By contrast, multiple therapeutic strategies focused on pharmacologically managing the inflammatory component, both at the joint and systemic levels, have shown promise. Therefore, prioritizing the control of chronic innate pro-inflammatory pathways presents the most viable and promising therapeutic strategy for the effective management of OA. As research continues, this approach may offer the best opportunity to alleviate the burden of this incapacitating disease. Cite this article: Bone Joint Res 2025;14(3):199–207

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. To examine how eukaryotic translation initiation factor 5A (eIF5A) regulates osteoarthritis (OA) during mechanical overload and the specific mechanism. Methods. Histological experiments used human bone samples and C57BL/6J mice knee samples. All cell experiments were performed using mice primary chondrocytes. Messenger RNA (mRNA) sequencing was performed on chondrocytes treated with 20% cyclic tensile strain for 24 hours. Western blot (WB) and quantitative polymerase chain reaction were employed to detect relevant indicators of cartilage function in chondrocytes. We created the destabilization of the medial meniscus (DMM) model and the mechanical overload-induced OA model and injected with overexpressing eIF5A adenovirus (eIF5A-ADV). Cartilage degeneration was evaluated using Safranin O/Fast Green staining. Relative protein levels were ascertained by immunohistochemistry (IHC) and immunofluorescence (IF) staining. Results. After OA initiation, eIF5A caused an upregulation of type II collagen (COL2) and a downregulation of matrix metalloproteinase 13 (MMP13), P16, and P21, which postponed the aggravation of OA. Further sequencing and experimental findings revealed that eIF5A knockdown accelerated the progression of OA by boosting the expression of histone acetyltransferase cyclic-adenosine monophosphate response element binding protein (CREB)-binding protein (CREBBP) to mediate activation of the Notch pathway. Conclusion. Our findings identified a crucial functional mechanism for the onset of OA, and suggest that intra-articular eIF5A injections might be a useful therapeutic strategy for OA treatment. Cite this article: Bone Joint Res 2025;14(2):124–135

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

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)

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

Abstract. Objectives. Osteoarthritis (OA) is a complex joint disorder characterised by the loss of extracellular matrix (ECM) leading to cartilage degeneration. Changes to cartilage cell (chondrocyte) behaviour occur including cell swelling, the development of fine cytoplasmic processes and cell clustering leading to changes in cell phenotype and development of focal areas of mechanically-weak fibrocartilaginous matrix. [1]. To study the sequence of events in more detail, we have investigated the changes to in situ chondrocytes within human cartilage which has been lightly scraped and then cultured with serum. Methods. Human femoral heads were obtained with Ethical permission and consent from four female patients (mean age 74 yrs) undergoing hip arthroplasty following femoral neck fracture. Osteochondral explants of macroscopically-normal cartilage were cultured as a non-scraped control, or scraped gently six times with a scalpel blade and both maintained in culture for up to 2wks in Dulbecco's Modified Eagle's Medium (DMEM) with 25% human serum (HS). Explants were then labelled with CMFDA (5-chloromethylfluorescein-diacetate) and PI (propidium iodide) (10μM each) to identify the morphology of living or dead chondrocytes respectively. Explants were imaged using confocal microscopy and in situ chondrocyte morphology, volume and clustering assessed quantitatively within standardised regions of interest (ROI) using Imaris. ®. imaging software. Results. Within 2wks of culture with HS, chondrocyte volume increased significantly from 412±9.3µm. 3. (unscraped) at day 0 to 724±16.6 µm. 3. (scraped) [N(n) = 4(380)] (P=0.0002). Chondrocyte clustering was a prominent feature of HS culture as the percentage of clusters in the cell population increased with scraping from 4.8±1.4% to 14.9±3.9% [N(n) = 4(999)] at week 2 (P=0.0116). In addition, the % of the chondrocyte population within clusters increased from approximately 38% to 60%, and the number of cells per cluster increased significantly from 3.2±0.08 to 4±0.22 (P=0.031). The development of abnormal ‘fibroblastic-like’ chondrocyte morphology demonstrating long (>5µm) cytoplasmic processes also occurred, however the time course of this was more variable. For some samples, clustering occurred before abnormal morphology, but for others the opposite occurred. Typically, by the second week, 17±2.64% of the cell population had processes and this increased to 22±4.02% [N(n) = 4(759)] with scraping. Conclusions. Scraping the cartilage will remove surface constituents including lubricants (e.g. lubricin, hyaluronic acid, phospholipids), extracellular matrix constituents (collagen, proteoglycans – potentially the ‘lamina splendens’) and cells (chondrocytes and mesenchymal stromal cells (MSCs)). Although we do not know which of these component(s) is important, the effect is to dramatically increase the permeation of serum factors into the cartilage matrix and signal the development of cytoplasmic processes, cell clustering and swelling. It is notable that these cellular changes are similar to those occurring in early OA. [1]. This raises the interesting possibility that scraped cartilage cultured with human serum recapitulates some of the changes to in situ chondrocytes during early stages of cartilage degeneration and as such, could be a useful model for following the deleterious changes to matrix metabolism. 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