Normal Chondrocytes Research Articles

Transcriptomic Changes During the Replicative Senescence of Human Articular Chondrocytes

Aging is a major risk factor for osteoarthritis (OA), but the specific mechanisms connecting aging and OA remain unclear. Although chondrocytes rarely divide in adult articular cartilage, they undergo replicative senescence in vitro, offering a model to study aging-related changes under controlled conditions. OA cartilage was obtained from an 80-year-old male and a 72-year-old female, while normal cartilage was sourced from a 26-year-old male. Chondrocyte cultures were established and sub-cultured to their Hayflick limit. Bulk RNA sequencing on early- and late-passage human articular chondrocytes identified transcriptomic changes associated with cellular aging. Early-passage OA chondrocytes already showed senescent phenotypes, unlike normal chondrocytes. All three cultures underwent 30 population doublings before replicative exhaustion, at which point all cells displayed senescence. During this process, cells lost their ability to form cartilaginous pellets. Differential gene expression analysis revealed distinct transcriptomic profiles between early- and late-passage chondrocytes and between normal and OA-derived cells. Genes related to matrix synthesis, degradation, inflammation, and the senescence-associated secretory phenotype (SASP) showed significant expression changes. Despite being a small pilot study, these findings suggest that further research into the molecular and metabolic changes during chondrocyte senescence could provide valuable insights into OA pathobiology.

In-situ hydrogen-generating injectable short fibers for osteoarthritis treatment by alleviating oxidative stress

Hydrogen (H₂) has great potential in the treatment of osteoarthritis, but its rapid diffusion and short retention time make it difficult to exert stable therapeutic effects. This study developed a short-fiber injectable material that can continuously generate hydrogen in situ to eliminate reactive oxygen species (ROS), alleviate oxidative stress and inflammation, and promote tissue repair. We prepared H–Si nanosheets with high hydrogen generation efficiency using a wet chemical exfoliation method and combined them with GelMA short fibers via electrospinning technology, achieving the in situ delivery of H–Si nanosheets and regulated hydrogen generation rate through the encapsulation and degradation of GelMA, ultimately achieving continuous and controlled hydrogen supply and stable therapeutic effects for osteoarthritis. In vitro and in vivo experiments confirmed the safety and efficacy of this material. The results showed that the material could continuously and efficiently generate hydrogen in simulated physiological environments (100 mg of material could generate 8.6 % hydrogen), effectively eliminate cellular reactive oxygen species (ROS positive rate reduced by 85.89 %), reduce cellular senescence and apoptosis (cell death rate decreased by 52 %, SA-βgal expression decreased by 78.3 %), promote normal chondrocyte function (Col II expression increased by 67.4 %, Ki67 expression increased by 87.5 %), and improve osteoarthritis in rats (OARSI score increased by 216 %). The in situ hydrogen generation and control system designed in this study provides a new method for the hydrogen's local and stable treatment of osteoarthritis. Statement of significanceHydrogen (H₂) has great potential in the treatment of osteoarthritis by alleviating oxidative stress, but its rapid diffusion and short retention time make it difficult to exert stable therapeutic effects. This study introduces an innovative injectable material combining H–Si nanosheets and GelMA short fibers to address this issue. By enabling continuous in situ hydrogen generation, this material effectively eliminates reactive oxygen species, reduces oxidative stress and inflammation, and promotes tissue repair. In vitro and in vivo experiments demonstrate its high hydrogen generation efficiency, safety, and therapeutic efficacy, offering a promising new approach for osteoarthritis management.

Investigating cryopreservation techniques for maintaining morphology and in vitro viability of cartilage and skin from Spix's yellow-toothed cavies (Galea spixii Wagler, 1831) for conservation through biobanks.

Conservation of the genetic diversity through skin and cartilage biobanks represents an essential strategy for maintaining biodiversity. Biobanks for the wild species of the order Rodentia have been little studied. Considering that the cryopreservation technique has specific relationships with the tissue and species of interest, we propose investigating different techniques for preserving tissue integrity and cell viability after cartilage and skin culture from Spix's yellow-toothed cavies. Subsequently, two techniques [solid-surface vitrification (SSV) vs. slow freezing (SF)] were used for cartilage and skin cryopreservation. Tissues not subjected to cryopreservation were used as controls. All tissues were evaluated for morphology and proliferation by histological techniques. Moreover, fragments were cultured, and cells were evaluated for viability, proliferation, metabolism, and apoptosis. Regardless of the cryopreservation technique, no differences were observed for the thickness of the epidermis, dermis, skin, spinous and basal layers, fibroblasts, and proliferative activity regarding the number of nucleolar organizer regions (NOR). SSV ensured better maintenance of epidermal cells, normal chondrocytes, filled gaps, collagen fibers, proliferative activity by NOR area/cell, and reduced perinuclear halos and empty gaps compared to SF. SF ensured the conservation of corneum thickness compared to the control. Although both techniques promoted cell recovery after culture, cells from SF resulted in better subconfluence time and day with cell growth around fragments compared to SSV. In conclusion, both cryopreservation techniques resulted in viable cells after culture. However, SSV promoted better maintenance of tissue morphological integrity, and SF ensured the preservation of all cell quality parameters in Spix's yellow-toothed cavies.

Memantine attenuates the development of osteoarthritis by blocking NMDA receptor mediated calcium overload and chondrocyte senescence

BackgroundMemantine, which is an FDA-proven drug for the treatment of dementia, exerts its function by blocking the function of NMDA (N-methyl-D-aspartate) receptor, a calcium-permeable ion channel that reduces cytotoxic calcium overload. Chondrocyte senescence is a crucial cellular event that contributes to articular cartilage degeneration during osteoarthritis (OA) development. To date, the effects of memantine and its downstream NMDA receptor on chondrocyte senescence and OA have been rarely reported. MethodsThe protein levels of NMDA receptor and its agonistic ligand, glutamate, were compared between normal and OA chondrocytes. The quantity of intracellular calcium ions and the level of mitochondrial damage were evaluated using specific fluorescent probes and transmission electron microscopy (TEM), respectively. Chondrocyte senescence was evaluated by senescence-associated β-galactosidase (SA-β-Gal) staining and p16INK4a analysis. The function of NMDA receptor in chondrocyte senescence and OA was tested via agonists activation and gene knockdown experiments. The therapeutic effects of memantine on OA were examined both in vitro and in vivo. Additionally, to verify the findings from animal samples, a propensity score-matched cohort study was conducted using data from a United Kingdom primary care database (i.e., IQVIA Medical Research Database [IMRD]) to compare the risk of OA-related joint replacement involved in memantine initiators versus active comparators (i.e., acetylcholinesterase [AchE] initiators) in patients with dementia. ResultsThe protein expression of NMDA receptor and the secretion of glutamate were both significantly increased in OA chondrocytes. NMDA receptor activation was found to stimulate chondrocyte calcium overload, which further led to mitochondrial fragmentation and chondrocyte senescence. Blocking the NMDA receptor with memantine and N-methyl-D-aspartate receptor subunit 1(NR1, the gene encoding NMDA receptor) knockdown resulted in reduced calcium influx, mitochondrial fragmentation, as well as cellular senescence in OA chondrocytes. Intra-articular injection of memantine in OA mice also exhibited protective effects against cartilage degeneration. Moreover, in the 1:5 propensity score-matched cohort study consisting of 6218 patients (n = 1435 in the memantine cohort; n = 4783 in the AchE cohort), the memantine initiator was associated with a lower risk of OA-related joint replacement than AchE initiators (Hazard ratio = 0.56, 95 % confidence interval: 0.34 to 0.99). ConclusionNMDA receptor plays an important role in inflammatory-induced cytotoxic calcium overload in chondrocytes, while memantine can effectively block the NMDA receptor to reduce chondrocyte senescence and retard the development of OA. The translational potential of this articleAs a clinically licensed drug used for the treatment of dementia, memantine has shown promising therapeutic effects on OA. Mechanistically, it functions by blocking NMDA receptor from mediating chondrocyte senescence. The protective effects of memantine against OA were verified not only by in vivo and in vitro experiments but also via a propensity score-matched human cohort study. These findings presented robust evidence for repurposing memantine for the treatment of OA.

ELIXCYTE®, an Allogenic Adipose-Derived Stem Cell Product, Mitigates Osteoarthritis by Reducing Inflammation and Preventing Cartilage Degradation In Vitro.

Adipose-derived stem cells (ADSCs) comprise a promising therapy for osteoarthritis (OA). The therapeutic potential of ELIXCYTE®, an allogeneic human ADSC (hADSC) product, was demonstrated in a phase I/II OA clinical trial. However, the exact mechanism underlying such effects is not clear. Moreover, studies suggest that interleukin-11 (IL-11) has anti-inflammatory, tissue-regenerative, and immune-regulatory functions. Our aim was to unravel the mechanism associated with the therapeutic effects of ELIXCYTE® on OA and its relationship with IL-11. We cocultured ELIXCYTE® with normal human articular chondrocytes (NHACs) in synovial fluid obtained from individuals with OA (OA-SF) to investigate its effect on chondrocyte matrix synthesis and degradation and inflammation by assessing gene expression and cytokine levels. NHACs exposed to OA-SF exhibited increased MMP13 expression. However, coculturing ELIXCYTE® with chondrocytes in OA-SF reduced MMP13 expression in chondrocytes and downregulated PTGS2 and FGF2 expression in ELIXCYTE®. ELIXCYTE® treatment elevated anti-inflammatory cytokine (IL-1RA, IL-10, and IL-13) levels, and the reduction in MMP13 was positively correlated with IL-11 concentrations in OA-SF. These findings indicate that IL-11 in OA-SF might serve as a predictive biomarker for the ELIXCYTE® treatment response in OA, emphasizing the therapeutic potential of ELIXCYTE® to mitigate OA progression and provide insights into its immunomodulatory effects.

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.

A novel mechanism behind irreversible development of cartilage degradation driven articular cartilage defects revealed by rat model: The chain reaction initiated by extracellular vesicles delivered LOC102546541

BackgroundArticular cartilage defects (ACD) are injuries with a diameter greater than 3 mm, resulting from wear and tear on joints. When the diameter of the defect exceeds 6 mm, it can further damage the surrounding joint cartilage, causing osteoarthritis (OA). Try to explain why OA is an irreversible disease, we hypothesize that damaged articular chondrocytes (DAC) may have reduced capacities to repair cartilage because its extracellular vesicle (EVs) that might directly contribute to OA formation. MethodsIn this study, DAC-EVs and AC-EVs were isolated using ultracentrifugation. Next-generation sequencing was employed to screen for a pathogenic long non-coding RNA (lncRNA). After verifying its function in vitro, the corresponding small interfering RNA (siRNA) was constructed and loaded into extracellular vesicles, which were then injected into the knee joint cavities of rats. ResultsThe results revealed that DAC-EVs packaged lncRNA LOC102546541 acts as a competitive endogenous RNA (ceRNA) of MMP13, down-regulating miR-632. Consequently, the function of MMP13 in degrading the extracellular matrix is enhanced, promoting the development of osteoarthritis. ConclusionsThis study uncovered a novel mode of OA pathogenesis using rat models, which DAC deliver pathogenic LOC102546541 packaged EVs to normal articular chondrocytes, amplifying the degradation of the extracellular matrix. Nonetheless, the functions of highly homologous human gene of LOC102546541 need to be verified in the future.

Multi-omics analysis of a case of congenital microtia reveals aldob and oxidative stress associated with microtia etiology.

Microtia is reported to be one of the most common congenital craniofacial malformations. Due to the complex etiology and the ethical barrier of embryonic study, the precise mechanisms of microtia remain unclear. Here we report a rare case of microtia with costal chondrodysplasia based on bioinformatics analysis and further verifications on other sporadic microtia patients. One hundred fourteen deleterious insert and deletion (InDel) and 646 deleterious SNPs were screened out by WES, candidate genes were ranked in descending order according to their relative impact with microtia. Label-free proteomic analysis showed that proteins significantly different between the groups were related with oxidative stress and energy metabolism. By real-time PCR and immunohistochemistry, we further verified the candidate genes between other sporadic microtia and normal ear chondrocytes, which showed threonine aspartase, cadherin-13, aldolase B and adiponectin were significantly upregulated in mRNA levels but were significantly lower in protein levels. ROS detection and mitochondrial membrane potential (∆ Ψ m) detection proved that oxidative stress exists in microtia chondrocytes. Our results not only spot new candidate genes by WES and label-free proteomics, but also speculate for the first time that metabolism and oxidative stress may disturb cartilage development and this might become therapeutic targets and potential biomarkers with clinical usefulness in the future.

Autophagy in Osteoarthritis: A Double-Edged Sword in Cartilage Aging and Mechanical Stress Response: A Systematic Review.

Background: Although osteoarthritis (OA) development is epidemiologically multifactorial, a primary underlying mechanism is still under debate. Understanding the pathophysiology of OA remains challenging. Recently, experts have focused on autophagy as a contributor to OA development. Method: To better understand the pathogenesis of OA, we survey the literature on the role of autophagy and the molecular mechanisms of OA development. To identify relevant studies, we used controlled vocabulary and free text keywords to search the MEDLINE, EMBASE, the Cochrane Central Register of Controlled Trials, Web of Science, and SCOPUS database. Thirty-one studies were included for data extraction and systematic review. Among these studies, twenty-five studies investigated the effects of autophagy in aging and OA chondrocytes, six studies examined the effects of autophagy in normal human chondrocytes, and only one study investigated the effects of mechanical stress-induced autophagy on the development of OA in normal chondrocytes. Results: The studies suggest that autophagy activation prevents OA by exerting cell-protective effects in normal human chondrocytes. However, in aging and osteoarthritis (OA) chondrocytes, the role of autophagy is intricate, as certain studies indicate that stimulating autophagy in these cells can have a cytotoxic effect, while others propose that it may have a protective (cytoprotective) effect against damage or degeneration. Conclusions: Mechanical stress-induced autophagy is also thought to be involved in the development of OA, but further research is required to identify the precise mechanism. Thus, autophagy contributions should be interpreted with caution in aging and the types of OA cartilage.

Targeted treatment of chondrosarcoma with a bacteriophage-based particle delivering a secreted tumor necrosis factor-related apoptosis-inducing ligand

Chondrosarcoma is a malignant cartilage-forming bone tumor which is inherently resistant to chemotherapy and radiotherapy leaving surgery as the only treatment option. We have designed a tumor-targeted bacteriophage (phage)-derived particle (PDP), for targeted systemic delivery of cytokine-encoding transgenes to solid tumors. Phage has no intrinsic tropism for mammalian cells; therefore, it was engineered to display a double cyclic RGD4C ligand on the capsid to target tumors. To induce cancer cell death, we constructed a transgene cassette expressing a secreted form of the cytokine tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). We detected high expression of αvβ3 and αvβ5 integrin receptors of the RGD4C ligand, and of the TRAIL receptor-2 in human chondrosarcoma cells (SW1353) but not in primary normal chondrocytes. The RGD4C.PDP-Luc particle carrying a luciferase reporter gene, Luc, effectively and selectively mediated gene delivery to SW1353 cells but not primary chondrocytes. Transduction of SW1353 cells with RGD4C.PDP-sTRAIL encoding a human sTRAIL, resulted in expression of TRAIL and subsequent cell dead without harming the normal chondrocytes. Intravenous administration of RGD4C.PDP-sTRAIL to mice with established human chondrosarcoma resulted in reduction of tumor size and tumor viability. Altogether, RGD4C.PDP-sTRAIL can be used to target systemic treatment of chondrosarcoma with the sTRAIL.

Response of Articular Cartilage to Hyperosmolar Stress: Report of an Ex Vivo Injury Model

Background: Physiological 0.9% saline is commonly used as an irrigation fluid in modern arthroscopy. There is a growing body of evidence that a hyperosmolar saline solution has chondroprotective effects, especially if iatrogenic injury occurs. Purpose: To (1) corroborate the superiority of a hyperosmolar saline solution regarding chondrocyte survival after mechanical injury and (2) observe the modulatory response of articular cartilage to osmotic stress and injury. Study Design: Controlled laboratory study. Methods: Osteochondral explants were isolated from bovine stifle joints and exposed to either 0.9% saline (308 mOsm) or hyperosmolar saline (600 mOsm) and then damaged with a sharp dermatome blade to attain a confined full-thickness cartilage injury site, incubated in the same fluids for another 3 hours, and transferred to chondropermissive medium for further culture for 1 week. Chondrocyte survival was assessed by confocal imaging, while the cellular response was evaluated over 1 week by relative gene expression for apoptotic and inflammatory markers and mediator release into the medium. Results: The full-thickness cartilage cut resulted in a confined zone of cell death that mainly affected superficial zone chondrocytes. Injured samples that were exposed to hyperosmolar saline showed less expansion of cell death in both the axial (P < .007) and the coronal (P < .004) plane. There was no progression of cell death during the following week of culture. Histological assessment revealed an intact cartilage matrix and normal chondrocyte morphology. Inflammatory and proapoptotic genes were upregulated on the first days postexposure with a notable downregulation toward day 7. Mediator release into the medium was concentrated on day 3. Conclusion: This in vitro cartilage injury model provides further evidence for the chondroprotective effect of a hyperosmolar saline irrigation fluid, as well as novel data on the capability of articular cartilage to quickly regain joint homeostasis after osmotic stress and injury. Clinical Relevance: Raising the osmolarity of an irrigating solution may be a simple and safe strategy to protect articular cartilage during arthroscopic surgery.

In Situ Remodeling of Efferocytosis via Lesion-Localized Microspheres to Reverse Cartilage Senescence.

Efferocytosis, an intrinsic regulatory mechanism to eliminate apoptotic cells, will be suppressed due to the delayed apoptosis process in aging-related diseases, such as osteoarthritis (OA). In this study, cartilage lesion-localized hydrogel microspheres are developed to remodel the in situ efferocytosis to reverse cartilage senescence and recruit endogenous stem cells to accelerate cartilage repair. Specifically, aldehyde- and methacrylic anhydride (MA)-modified hyaluronic acid hydrogel microspheres (AHM), loaded with pro-apoptotic liposomes (liposomes encapsulating ABT263, A-Lipo) and PDGF-BB, namely A-Lipo/PAHM, are prepared by microfluidic and photo-cross-linking techniques. By a degraded porcine cartilage explant OA model, the in situ cartilage lesion location experiment illustrated that aldehyde-functionalized microspheres promote affinity for degraded cartilage. In vitro data showed that A-Lipo induced apoptosis of senescent chondrocytes (Sn-chondrocytes), which can then be phagocytosed by the efferocytosis of macrophages, and remodeling efferocytosis facilitated the protection of normal chondrocytes and maintained the chondrogenic differentiation capacity of MSCs. In vivo experiments confirmed that hydrogel microspheres localized to cartilage lesion reversed cartilage senescence and promoted cartilage repair in OA. It is believed this in situ efferocytosis remodeling strategy can be of great significance for tissue regeneration in aging-related diseases.

Saikosaponin D alleviates inflammatory response of osteoarthritis and mediates autophagy via elevating microRNA-199-3p to target transcription Factor-4

ObjectiveThis study was to investigate the underlying mechanism by which Saikosaponin D (SSD) mitigates the inflammatory response associated with osteoarthritis (OA) and regulates autophagy through upregulation of microRNA (miR)-199-3p and downregulation of transcription Factor-4 (TCF4).MethodsA mouse OA model was established. Mice were intragastrically administered with SSD (0, 5, 10 μmol/L) or injected with miR-199-3p antagomir into the knee. Then, pathological changes in cartilage tissues were observed. Normal chondrocytes and OA chondrocytes were isolated and identified. Chondrocytes were treated with SSD and/or transfected with oligonucleotides or plasmid vectors targeting miR-199-3p and TCF4. Cell viability, apoptosis, inflammation, and autophagy were assessed. miR-199-3p and TCF4 expressions were measured, and their targeting relationship was analyzed.ResultsIn in vivo experiments, SSD ameliorated cartilage histopathological damage, decreased inflammatory factor content and promoted autophagy in OA mice. miR-199-3p expression was downregulated and TCF4 expression was upregulated in cartilage tissues of OA mice. miR-199-3p expression was upregulated and TCF4 expression was downregulated after SSD treatment. Downregulation of miR-199-3p attenuated the effect of SSD on OA mice. In in vitro experiments, SSD inhibited the inflammatory response and promoted autophagy in OA chondrocytes. Downregulation of miR-199-3p attenuated the effect of SSD on OA chondrocytes. In addition, upregulation of miR-199-3p alone inhibited inflammatory responses and promoted autophagy in OA chondrocytes. miR-199-3p targeted TCF4. Upregulation of TCF4 attenuated the effects of miR-199-3p upregulation on OA chondrocytes.ConclusionsSSD alleviates inflammatory response and mediates autophagy in OA via elevating miR-199-3p to target TCF4.

Protective Effect of Knee Postoperative Fluid on Oxidative-Induced Damage in Human Knee Articular Chondrocytes.

The oxidative-stress-elicited deterioration of chondrocyte function is the initial stage of changes leading to the disruption of cartilage homeostasis. These changes entail a series of catabolic damages mediated by proinflammatory cytokines, MMPs, and aggrecanases, which increase ROS generation. Such uncontrolled ROS production, inadequately balanced by the cellular antioxidant capacity, eventually contributes to the development and progression of chondropathies. Several pieces of evidence show that different growth factors, single or combined, as well as anti-inflammatory cytokines and chemokines, can stimulate chondrogenesis and improve cartilage repair and regeneration. In this view, hypothesizing a potential growth-factor-associated action, we investigate the possible protective effect of post-operation knee fluid from patients undergoing prosthesis replacement surgery against ROS-induced damage on normal human knee articular chondrocytes (HKACs). To this end, HKACs were pre-treated with post-operation knee fluid and then exposed to H2O2 to mimic oxidative stress. Intracellular ROS levels were measured by using the molecular probe H2DCFDA; cytosolic and mitochondrial oxidative status were assessed by using HKACs infected with lentiviral particles harboring the redox-sensing green fluorescent protein (roGFP); and cell proliferation was determined by measuring the rate of DNA synthesis with BrdU incorporation. Moreover, superoxide dismutase (SOD), catalase, and glutathione levels from the cell lysates of treated cells were also measured. Postoperative peripheral blood sera from the same patients were used as controls. Our study shows that post-operation knee fluid can counteract H2O2-elicited oxidative stress by decreasing the intracellular ROS levels, preserving the cytosolic and mitochondrial redox status, maintaining the proliferation of oxidatively stressed HKACs, and upregulating chondrocyte antioxidant defense. Overall, our results support and propose an important effect of post-operation knee fluid substances in maintaining HKAC function by mediating cell antioxidative system upregulation and protecting cells from oxidative stress.

Exploring m6A-linked aging genes in osteoarthritis and broad cancer spectrum: Prospects for diagnostic and therapeutic advancements.

Osteoarthritis (OA) is a prevalent degenerative joint disease that significantly impacts individuals and healthcare systems worldwide. However, the exploration of N6-methyladenosine (m6A)-related aging genes in OA pathogenesis remains largely underexplored. This study aimed to elucidate the role of m6A-related aging genes in OA and to develop a robust diagnostic model based on their expression profiles. Leveraging publicly available gene expression datasets, we conducted consensus clustering to categorize OA into distinct subtypes, guided by the expression patterns of m6A-related aging genes. Utilizing XGBoost, a cutting-edge machine learning approach, we identified key diagnostic genes and constructed a predictive model. Our investigation extended to the immune functions of these genes, shedding light on potential therapeutic targets and underlying regulatory mechanisms. Our analysis unveiled specific OA subtypes, each marked by unique expression profiles of m6A-related aging genes. We pinpointed a set of pivotal diagnostic genes, offering potential therapeutic avenues. The developed diagnostic model exhibited exceptional capability in distinguishing OA patients from healthy controls. To corroborate our computational findings, we performed quantitative real-time polymerase chain reaction analyses on two cell lines: HC-OA (representing adult osteoarthritis cells) and C-28/I2 (representative of normal human chondrocytes). The gene expression patterns observed were consistent with our bioinformatics predictions, further validating our initial results. In conclusion, this study underscores the significance of m6A-related aging genes as promising biomarkers for diagnosis and prognosis, as well as potential therapeutic targets in OA. Although these findings are encouraging, further validation and functional analyses are crucial for their clinical application.

Identification and Verification of Hub Mitochondrial Dysfunction Genes in Osteoarthritis Based on Bioinformatics Analysis.

Age-related mitochondrial dysfunction and associated oxidative stress may contribute to the development of osteoarthritis. The aim of this study was to identify hub genes associated with mitochondrial dysfunction in osteoarthritis (OA) patients, helping predict the risk of OA, and revealing the mechanism of OA progression. OA expression data and mitochondrial dysfunction genes were downloaded from GEO (GSE55235, GSE82107, and GSE114007) and GeneCard databases. The differentially expressed mitochondrial dysfunction genes (DEMDFGs) between OA and control samples were screened. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes pathways were analyzed for DEMDFGs. The hub genes were determined by WGCNA and LASSO regression analysis. ROC curves manifested the diagnostic efficacy of each hub gene. A nomogram model was constructed and validated to predict OA risk. The expression of hub genes in OA and normal chondrocytes was verified by external datasets, qRT-PCR and western blotting. A total of 31 DEMDFGs were identified, with 15 genes upregulated and 16 genes downregulated. GO functional enrichment analysis revealed that DEMDFGs were enriched in biological processes related to energy metabolism and cellular respiration. By employing weighted gene coexpression network analysis, we identified four distinct coexpression modules, among which the blue module exhibited the strongest correlation with OA. The intersection between DEMDFGs and this module yielded eight candidate genes. After LASSO analysis of the data, four hub genes (ACADL, CYBA, SLC19A2, and UCP2) were identified as potential biomarkers for OA. The expression levels of these four genes were externally validated in the GSE114007 dataset. And the biologically differential expression of these four genes has been verified in OA and normal chondrocytes. Moreover, the four hub genes had good sensitivity and specificity by ROC curve analysis, and the risk model constructed with these four genes showed promising performance. In conclusion, our study may provide novel mitochondrial dysfunction hub genes with potential clinical applications for understanding the pathology, diagnosis, and treatment of OA.

DOES INFLAMMATORY AND METABOLOMIC ACTIVITY ON NORMAL AND OSTEOARTHRITIC CHONDROCYTES DIFFER BETWEEN MESENCHYMAL STEM CELLS (MSCS) DERIVED FROM THE INFRAPATELLAR FAT, SYNOVIUM, AND SUBCUTANEOUS TISSUES?

Osteoarthritis (OA) is a disabling disease depriving the quality of life of patients. Mesenchymal stem cells (MSCs) are recently used to modify the inflammatory and degenerative cascade of the disease. Source of MSCs could change the progression and symptoms of OA due to their different metabolomic activities. We asked whether MSCs derived from the infrapatellar fat (IPF), synovium (Sy) and subcutaneous (SC) tissues will decrease inflammatory and degenerative markers of normal and OA chondrocytes and improve regeneration in culture. Tissues were obtained from three male patients undergoing arthroscopic knee surgery due to sports injuries after ethical board approval. TNFa concentration decreased in all MSC groups (Sy=156,6±79, SC=42,1±6 and IPF=35,5±3 pg/ml; p=0,036) on day 14 in culture. On day seven (Sy=87,4±43,7, SC=23±8,9 and IPF=14,7±3,3 pg/ml, p=0,043) and 14 (Sy=29,1±11,2, SC=28,3±18,5 and IPF=20,3±16,2 pg/ml, p=0,043), MMP3 concentration decreased in all groups. COMP concentration changes however were not significant. Plot scores of tissues for PC2-13,4% were significantly different. Based on the results of liquid chromatography-mass spectrometry (LC-MS) metabolomics coupled with recent data processing strategies, clinically relevant seven metabolites (L-fructose, a-tocotrienol, coproporphyrin, nicotinamide, bilirubin, tauro-deoxycholic acid and galactose-sphingosine) were found statistically different (p&lt;0.05 and fold change&gt;1.5) ratios in tissue samples. Focusing on these metabolites as potential therapeutics could enhance MSC therapies.Acknowledgment: Hacettepe University, Scientific Research Projects Coordination Unit (#THD-2020-18692) and Turkish Society of Orthopedics and Traumatology (#TOTBID-89) funded this project. Feza Korkusuz MD is a member of the Turkish Academy of Sciences (TÜBA).

Blocking TXNIP reduced IL-1β Induced chondrocyte cell inflammation.

Osteoarthritis (OA) is the most common joint disease in the elderly and is characterized by progressive and irreversible degeneration of articular cartilage, particularly cartilage loss and callus formation. This study would like to investigate the important role and the molecular mechanism of OA progression following interleukin 1β (IL-1β)-induced chondrocyte injury regulated by TXNIP. In this study, high-purity mouse chondrocytes were obtained by enzymatic two-step digestion for primary culture. Toluidine blue staining and type II collagen immunofluorescence were used to identify cells through histochemical staining after slide mounting. The relative expression of TXNIP was detected by immunohistochemical staining and qRT-PCR.Aiming at the shRNA sequence of the TXNIP gene, the shRNA expression vector was constructed and packaged with lentivirus to form the lentiviral vector shTXNIP. After inhibiting the expression of TXNIP by transfecting shTXNIP into normal mouse chondrocytes, the CCK-8 kit was used for detecting its effect on cell proliferation after transfection, and the effect on chondrocyte apoptosis was detected by flow cytometry. The staining kit was used to detect the effect of TXNIP knockout on chondrocyte aging, and the differential expression of TNF, IL-6, MMP3, MMP13, ADAMTS-5 and type II collagen genes in chondrocytes was detected by RT-PCR and Western-bolt. Western blot was used to detect the expression of upstream-related protein P-ERK, downstream-related protein NLRP3 and Caspase1 after inflammatory injury of mouse articular chondrocytes. Results showed that the expression level of TXNIP in chondrocytes induced by different concentrations of il-1β was proportional to the concentration. After silencing TXNIP by shRNA, cell proliferation increased, chondrocyte apoptosis was weakened, and chondrocyte aging was weakened. The differential expression of genes such as TNF, IL-6, MMP3, MMP13, ADAMTS-5 and type II collagen and the differential expression of protein levels were relatively decreased. In addition, the expression of the upstream-related protein P-ERK did not change much when TXNIP was silenced, and the expression levels of the downstream-related proteins NLRP3 and Caspase1 were slightly reduced. In conclusion, silencing TXNIP can inhibit il-1β-induced chondrocyte apoptosis and aging, and has a positive effect on cell proliferation. However, this study has not clarified the molecular mechanism involved in TXNIP and the process of its signaling expression pathway.

The silencing of NREP aggravates OA cartilage damage through the TGF-β1/Smad2/3 pathway in chondrocytes

BackgroundOsteoarthritis (OA) is a common chronic degenerative joint disease. Due to the limited understanding of its complex pathological mechanism, there is currently no effective treatment that can alleviate or even reverse cartilage damage associated with OA. With improvement in public databases, researchers have successfully identified the key factors involved in the occurrence and development of OA through bioinformatics analysis. The aim of this study was to screen for the differentially expressed genes (DEGs) between the normal and OA cartilage through bioinformatics, and validate the function of the TGF-β1/Smad2/3 pathway-related neuron regeneration related protein (NREP) in the articular cartilage. MethodsThe DEGs between the cartilage tissues of OA patients and healthy controls were screened by bioinformatics, and functionally annotated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The expression levels of the DEG in human and murine OA cartilage was verified by reverse transcription-quantitative PCR (RT-qPCR), Western blotting and immunohistochemistry (IHC). RT-qPCR, Western-blotting, Cell Counting Kit-8(CCK8) and EdU assays were used to evaluate the effects of knocking down NREP in normal chondrocytes, and the molecular mechanisms were investigated by RT-qPCR, Western blotting and IHC. ResultsIn this study, we identified NREP as a DEG in OA through bioinformatics analysis, and found that NREP was downregulated in the damaged articular cartilage of OA patients and mouse model with surgically-induced OA. In addition, knockdown of NREP in normal chondrocytes reduced their proliferative capacity, which is the pathological basis of OA. At the molecular level, knock-down of NREP inactivated the TGF-β1/Smad2/3 pathway, resulting in the downregulation of the anabolic markers Col2a1 and Sox9, and an increase in the expression of the catabolic markers MMP3 and MMP13. ConclusionNREP plays a key role in the progression of OA by regulating the TGF-β1/Smad2/3 pathway in chondrocytes, and warrants further study as a potential therapeutic target.

MiR-217 Regulates SIRT1 Expression and Promotes Inflammatory and Apoptotic Responses in Osteoarthritis.

Previous studies have reported miR-217 uregulation in age-related pathologies. We investigated the impact of miR-217-5p on sirtuin 1 (SIRT1) regulation in human osteoarthritic (OA) chondrocytes. MiR-217 target enrichment analyses were performed using three public databases, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. MiR-217-5p expression levels were quantified in normal and OA chondrocytes. SIRT1 expression levels, nuclear factor kappa-B p65 subunit (NF-κBp65) and p53 acetylation levels, and expression levels of OA-related pro-inflammatory markers [tumor necrosis factor α (TNFα), interleukin 1β (IL-1β), IL-6], pro-apoptotic markers [Bax, pro-caspase 3, cleaved caspase 3] and matrix regulators [matrix metalloproteinase (MMP)-1, MMP-13, MMP-9, Collagen 2 (COL2A1), Aggrecan (ACAN)] were evaluated in miR-217 mimic-treated and/or miR-217 inhibitor-treated OA chondrocytes, with/without subsequent treatment with siRNA against SIRT1 (siSIRT1). MiR-217-5p was upregulated in OA chondrocytes, while target prediction/enrichment analyses revealed SIRT1 as miR-217 target-gene. Deacetylation of NF-κBp65 and p53 in miR-217 inhibitor-treated OA chondrocytes was reversed by siSIRT1 treatment. MiR-217 inhibitor-treated OA chondrocytes showed increased COL2A1, ACAN and decreased IL-1β, IL-6, TNFα, Bax, cleaved caspase 3 and MMPs expression levels, which were reversed following miR-217 inhibitor/siSIRT1 treatment. Our findings highlight the impact of miR-217-5p on SIRT1 downregulation contributing to OA pathogenesis.