Primary Articular Chondrocytes Research Articles

The Effects of Seleno-Methionine in Cadmium-Challenged Human Primary Chondrocytes.

Cadmium (Cd) is a potentially toxic element able to interfere with cellular functions and lead to disease or even death. Cd accumulation has been demonstrated in cartilage, where it can induce damage in joints. The aim of this study was to evaluate the effect of CdCl2 on primary cultures of human chondrocytes and the possible protective effect of seleno-methionine (Se-Met). Human primary articular chondrocytes were cultured and treated as follows: control groups, cells challenged with 7.5 μM and 10 μM CdCl2 alone, and cells pretreated with 10 and 20 μM Se-Met and then challenged with 7.5 μM and 10 μM CdCl2. Twenty-four hours after incubation, cell viability, histological evaluation with hematoxylin-eosin stain, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were performed. Furthermore, reverse transcription-PCR was carried out to evaluate mRNA levels of BAX, BAK1, CASP-3, and CASP-9. After CdCl2 challenge at both doses, a reduced cell viability and an overexpression of BAX, BAK1, CASP-3, and CASP-9 genes, as well as a high number of TUNEL-positive cells, were demonstrated, all parameters becoming higher as the dose of CdCl2 was increased. The pretreatment with Se-Met lowered the expression of all considered genes, improved cell viability and morphological changes, and reduced the number of TUNEL-positive cells. It was concluded that Se-Met plays a protective role against CdCl2-induced structural and functional changes in chondrocytes in vitro, as it improved cell viability and showed a positive role in the context of the apoptotic pathways. It is therefore suggested that a translational, multifaceted approach, with plant-based diets, bioactive functional foods, nutraceuticals, micronutrients, and drugs, is possibly advisable in situations of environmental pollution caused by potentially toxic elements.

Reproducible Synthesis of Biocompatible Albumin Nanoparticles Designed for Intra-articular Administration of Celecoxib to Treat Osteoarthritis.

Osteoarthritis (OA) is the most common form of arthritis, with intra-articular (IA) delivery of therapeutics being the current best option to treat pain and inflammation. However, IA delivery is challenging due to the rapid clearance of therapeutics from the joint and the need for repeated injections. Thus, there is a need for long-acting delivery systems that increase the drug retention time in joints with the capacity to penetrate OA cartilage. As pharmaceutical utility also demands that this is achieved using biocompatible materials that provide colloidal stability, our aim was to develop a nanoparticle (NP) delivery system loaded with the COX-2 inhibitor celecoxib that can meet these criteria. We devised a reproducible and economical method to synthesize the colloidally stable albumin NPs loaded with celecoxib without the use of any of the following conditions: high temperatures at which albumin denaturation occurs, polymer coatings, oils, Class 1/2 solvents, and chemical protein cross-linkers. The spherical NP suspensions were biocompatible, monodisperse with average diameters of 72 nm (ideal for OA cartilage penetration), and they were stable over 6 months at 4 °C. Moreover, the NPs loaded celecoxib at higher levels than those required for the therapeutic response in arthritic joints. For these reasons, they are the first of their kind. Labeled NPs were internalized by primary human articular chondrocytes cultured from the knee joints of OA patients. The NPs reduced the concentration of inflammatory mediator prostaglandin E2 released by the primaries, an indication of retained bioactivity following NP synthesis. Similar results were observed in lipopolysaccharide-stimulated human THP-1 monocytes. The IA administration of these NPs is expected to avoid side-effects associated with oral administration of celecoxib and to maintain a high local concentration in the knee joint over a sustained period. They are now ready for evaluation by IA administration in animal models of OA.

Single-stage autologous chondrocyte coimplantation on a hyaluronan scaffold for the treatment of knee cartilage lesions: a case series of 16 patients with clinical outcomes up to 5 years

IntroductionThe direct reimplantation of autologous primary articular chondrocytes in a single-stage procedure for knee cartilage lesions is a novel approach, yet to be extensively reported. This study aimed to evaluate the clinical outcomes and reinterventions over a 5-year period post-surgery. MethodsA prospective case series involving 16 patients (4 female, 12 male) with single or multiple focal knee cartilage lesions was conducted. The mean age at baseline was 36.8 (±11.5) years. The mean total lesion size was 4.5 (±2.3) cm2 Patients underwent surgery where articular chondrocytes and mononuclear bone marrow cells were isolated, mixed, and seeded onto a hyaluronan-based scaffold within the lesion. A structured physical therapy regimen was followed, and patients were assessed using Knee injury and Osteoarthritis Outcome Score (KOOS) and International Knee Documentation Committee (IKDC) Subjective questionnaires at various intervals. Overall, patient-reported outcomes improved over the first 3 years post-surgery, with slight declines thereafter. ResultsSignificant improvements over baseline were noted for various KOOS parameters and IKDC Subjective scores at different follow-up points. Reinterventions were required for two patients, one receiving intra-articular injections with mesenchymal stromal cells and another undergoing knee washout for septic arthritis while a meniscus implant was removed. Patient satisfaction at final follow-up was generally favorable. Despite a significant intraoperative cell isolation time of approximately 1.5 hours, the procedure demonstrated safety and efficacy, with an average of ±0.9 million articular chondrocytes obtained per case. ConclusionsThis study underscores the potential of coimplantation of intraoperatively isolated articular chondrocytes and bone marrow cells on a hyaluronan scaffold as a promising strategy for treating symptomatic knee cartilage lesions.

A COMPARATIVE STUDY OF THREE DIFFERENT BIOACTIVE GLASS SCAFFOLDS TAILORED FOR CARTILAGE TISSUE ENGINEERING

The regenerative capacity of hyaline cartilage is greatly limited. To prevent the onset of osteoarthritis, cartilage defects have to be properly treated. Cartilage, tissue engineered by mean of bioactive glass (BG) scaffolds presents a promising approach. Until now, conventional BGs have been used mostly for bone regeneration, as they are able to form a hydroxyapatite (HA) layer and are therefore, less suited for cartilage reconstruction. The aim of this study is to compare two BGs based on a novel BG composition tailored specifically for cartilage (CAR12N) and patented by us with conventional BG (BG1393) with a similar topology. The highly porous scaffolds consisting of 100% BG (CAR12N, CAR12N with low Ca2+/Mg2+ and BG1393) were characterized and dynamically seeded with primary porcine articular chondrocytes (pACs) or primary human mesenchymal stem cells (hMSCs) for up to 21 days. Subsequently, cell viability, DNA and glycosaminoglycan contents, cartilage-specific gene and protein expression were evaluated. The manufacturing process led to a comparable high (over 80%) porosity in all scaffold variants. Ion release and pH profiles confirmed bioactivity for them. After both, 7 and 21 days, more than 60% of the total surfaces of all three glass scaffold variants was densely colonized by cells with a vitality rate of more than 80%. The GAG content was significantly higher in BG1393 colonized with pACs. In general, the GAG content was higher in pAC colonized scaffolds in comparison to those seeded with hMSCs. The gene expression of cartilage-specific collagen type II, aggrecan, SOX9 and FOXO1 could be detected in all scaffold variants, irrespectively whether seeded with pACs or hMSCs. Cartilage-specific ECM components could also be detected at the protein level. In conclusion, all three BGs allow the maintenance of the chondrogenic phenotype or chondrogenic differentiation of hMSCs and thus, they present a high potential for cartilage regeneration.

Exploring the translational potential of PLGA nanoparticles for intra-articular rapamycin delivery in osteoarthritis therapy

Osteoarthritis (OA) is a prevalent joint disease that affects all the tissues within the joint and currently lacks disease-modifying treatments in clinical practice. Despite the potential of rapamycin for OA disease alleviation, its clinical application is hindered by the challenge of achieving therapeutic concentrations, which necessitates multiple injections per week. To address this issue, rapamycin was loaded into poly(lactic-co-glycolic acid) nanoparticles (RNPs), which are nontoxic, have a high encapsulation efficiency and exhibit sustained release properties for OA treatment. The RNPs were found to promote chondrogenic differentiation of ATDC5 cells and prevent senescence caused by oxidative stress in primary mouse articular chondrocytes. Moreover, RNPs were capable to alleviate metabolism homeostatic imbalance of primary mouse articular chondrocytes in both monolayer and 3D cultures under inflammatory or oxidative stress. In the mouse destabilization of the medial meniscus (DMM) model, intra-articular injection of RNPs effectively mitigated joint cartilage destruction, osteophyte formation, chondrocytes hypertrophy, synovial inflammation, and pain. Our study demonstrates the feasibility of using RNPs as a potential clinically translational therapy to prevent the progression of post-traumatic OA.Graphical abstract

Augmenting mesenchymal stem cell therapy for osteoarthritis via inflammatory priming: a comparative study on mesenchymal stem cells derived from various perinatal tissue sources.

Background: Osteoarthritis (OA), a degenerative disease prevalent among the elderly, poses significant challenges due to its high incidence and disability rates. Regrettably, there exists a lack of effective regenerative therapies for the irreversible degradation of cartilage in OA. Mesenchymal stem cells (MSCs), known for their robust differentiation and immune regulatory capabilities, have emerged as promising candidates for OA treatment. MSCs sourced from perinatal tissues offer the dual advantage of convenience in extraction and ethical non-controversy. However, the heterogeneous nature of MSCs derived from different perinatal tissue sources gives rise to varying therapeutic indications. Moreover, the immune response of MSCs may be modulated under the influence of inflammatory factors. Methods: In this study, we isolated mesenchymal stem cells from distinct parts of human perinatal tissue: umbilical cord-derived MSCs (UC-MSCs), fetal placenta-derived MSCs (FP-MSCs), and umbilical cord placental junction-derived MSCs (CPJ-MSCs). These cells were cultured in vitro and subjected to a 24-hour treatment with the inflammatory mediator Interleukin-1β (IL-1β). Subsequently, the MSCs were evaluated for changes in proliferation, migration, and regulatory capabilities. To assess the comparative anti-injury potential of MSCs from different sources, primary articular chondrocytes (ACs) were exposed to H2O2-induced injury and co-cultured with IL-1β-primed MSCs. Changes in the proliferation, migration, and regulatory abilities of ACs resembling those observed in OA were examined. Results: Following IL-1β treatment, all three types of MSCs displayed decreased rates of proliferation and migration. Notably, their chondrogenic differentiation capacities exhibited an enhancement. Additionally, diverse MSCs exhibited a degree of efficacy in restoring damaged ACs in vitro. Among these, CPJ-MSCs demonstrated superior potential in promoting cartilage cell proliferation, while FP-MSCs displayed notable anti-inflammatory effects. Conclusion: Our findings underscore the substantial capacity of primed FP-MSCs and CPJ-MSCs to alleviate the injury in OA-like ACs. Consequently, this study advocates for the prospective use of preconditioning strategies involving FP-MSCs and CPJ-MSCs in forthcoming OA therapies.

Piezo1 activation accelerates osteoarthritis progression and the targeted therapy effect of artemisinin

IntroductionOsteoarthritis (OA) is a devastating whole-joint disease affecting a large population worldwide with no cure; its mechanism remains poorly defined. Abnormal mechanical stress is the main pathological factor of OA. ObjectivesTo investigate the effects of Piezo1 activation on OA development and progression and to explore Piezo1-targeting OA treatment. MethodsThe expression levels of Piezo1 were determined in human OA cartilage and experimental OA mice. Mice with genetic Piezo1 deletion in chondrocytes or intra-articular injection of the Piezo1 activator Yoda1 were utilized to determine the effects on DMM-induced OA progression. Effects of artemisinin (ART), a potent antimalarial drug, on Piezo1 activation, chondrocyte metabolism and OA lesions were determined. ResultsPiezo1 expression was elevated in articular chondrocytes in human OA and DMM-induced mouse OA cartilage. Piezo1 deletion in chondrocytes largely attenuates DMM-induced OA-like phenotypes. In contrast, intra-articular injection of Yoda1 aggravates the knee joint OA lesions in mice. PIEZO1 activation increases, while PIEZO1 siRNA knockdown decreases, expression of RUNX2 and catabolic enzymes MMP13 and ADAMTS5 in primary human articular chondrocytes in a PI3K-AKT dependent manner. We have provided strong evidence supporting that ART is a novel and potent inhibitor of Piezo1 activation in primary OA-HACs and all cell lines examined, including human endothelial HUVEC cells, ATDC5 chondrocyte-like cells and MLO-Y4 osteocytes-like cells. Results from in vitro experiments confirmed that ART decreases the Yoda1-induced increases in the levels of OA-related genes and p-PI3K and p-AKT proteins in OA-HACs and alleviates DMM-induced OA lesions in mice. ConclusionsWe establish a critical role of Piezo1 in promoting OA development and progression and define ART as a potential OA treatment.

SNORC knockdown alleviates inflammation, autophagy defect and matrix degradation of chondrocytes in osteoarthritis development.

Excessive inflammation and autophagy defect of chondrocytes play important roles in the pathological process of osteoarthritis (OA). The present study aimed to clarify the roles of small novel rich in cartilage (SNORC) in these pathological changes of chondrocytes in OA. Bioinformatics analysis of GEO dataset GSE207881 displayed that SNORC was a potential biomarker for OA. As confirmed by quantitative real-time PCR, immunohistochemical staining and western blotting, SNORC was significantly up-regulated in cartilage of OA rat model and interleukin (IL)-1β-stimulated primary rat articular chondrocytes in contrast to their corresponding normal control. Knocking down SNORC in IL-1β-induced chondrocytes obviously suppressed the production of nitric oxide (NO), IL-6, tumor necrosis factor (TNF)-α and prostaglandin E2 (PGE2) to alleviate inflammation, and reduced the protein levels of a disintegrin and metalloproteinase with thrombospondin 5 (ADAMTS5) and matrix metallopeptidase (MMP)13 and elevated collagen type 2 alpha 1 (COL2A1) level to improve matrix degradation. Down-regulation of SNORC increased Beclin1 expression and LC3II/LC3I ratio, but suppressed p62 expression to restore impaired autophagy in IL-1β-induced chondrocytes. Moreover, down-regulating SNORC mitigated mitochondrial dysfunction and apoptosis in IL-1β-stimulated chondrocytes. Mechanically, SNORC simultaneously activated the phosphatidylinositol-3-kinase/serine threonine kinase (PI3K/AKT) and c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway in the IL-1β-induced chondrocyte, while re-activating the PI3K and JNK signals abolished the suppressive effect of down-regulating SNORC on IL-1β-induced chondrocyte damage. In a word, SNORC knockdown alleviates inflammation, matrix degradation, autophagy defect and excessive apoptosis of chondrocytes during OA development via suppressing the PI3K and JNK signaling pathway.

Depletion of SNORA33 Abolishes ψ of 28S-U4966 and Affects the Ribosome Translational Apparatus.

Eukaryotic ribosomes are complex molecular nanomachines translating genetic information from mRNAs into proteins. There is natural heterogeneity in ribosome composition. The pseudouridylation (ψ) of ribosomal RNAs (rRNAs) is one of the key sources of ribosome heterogeneity. Nevertheless, the functional consequences of ψ-based ribosome heterogeneity and its relevance for human disease are yet to be understood. Using HydraPsiSeq and a chronic disease model of non-osteoarthritic primary human articular chondrocytes exposed to osteoarthritic synovial fluid, we demonstrated that the disease microenvironment is capable of instigating site-specific changes in rRNA ψ profiles. To investigate one of the identified differential rRNA ψ sites (28S-ψ4966), we generated SNORA22 and SNORA33 KO SW1353 cell pools using LentiCRISPRv2/Cas9 and evaluated the ribosome translational capacity by 35S-Met/Cys incorporation, assessed the mode of translation initiation and ribosomal fidelity using dual luciferase reporters, and assessed cellular and ribosomal proteomes by LC-MS/MS. We uncovered that the depletion of SNORA33, but not SNORA22, reduced 28S-ψ4966 levels. The resulting loss of 28S-ψ4966 affected ribosomal protein composition and function and led to specific changes in the cellular proteome. Overall, our pioneering findings demonstrate that cells dynamically respond to disease-relevant changes in their environment by altering their rRNA pseudouridylation profiles, with consequences for ribosome function and the cellular proteome relevant to human disease.

ADRM1/RPN13 attenuates cartilage extracellular matrix degradation via enhancing UCH37-mediated ALK5 deubiquitination

Osteoarthritis (OA) is the most common age-related joint disorder with no effective therapy, and its specific pathological mechanism remains to be fully clarified. Adhesion-regulating molecule 1 (ADRM1) has been proven to be involved in OA progression as a favorable gene. However, the exact mechanism of ADRM1 involved in OA were unknown. Here, we showed that the ADRM1 expression decreased in human OA cartilage, destabilization of the medial meniscus (DMM)-induced mouse OA cartilage, and interleukin (IL)-1β-induced primary mouse articular chondrocytes. Global knockout (KO) ADRM1 in cartilage or ADRM1 inhibitor (RA190) could accelerate the disorders of extracellular matrix (ECM) homeostasis, thereby accelerated DMM-induced cartilage degeneration, whereas overexpression of ADRM1 protected mice from DMM-induced OA development by maintaining the homeostasis of articular cartilage. The molecular mechanism study revealed that ADRM1 could upregulate ubiquitin carboxy-terminal hydrolase 37 (UCH37) expression and bind to UCH37 to activate its deubiquitination activity. Subsequently, increased and activated UCH37 enhanced activin receptor-like kinase 5 (ALK5) deubiquitination to stabilize ALK5 expression, thereby maintaining ECM homeostasis and attenuating cartilage degeneration. These findings indicated that ADRM1 could attenuate cartilage degeneration via enhancing UCH37-mediated ALK5 deubiquitination. Overexpression of ADRM1 in OA cartilage may provide a promising OA therapeutic strategy.

Cytotoxic effects of different mouthwash solutions on primary human articular chondrocytes and normal human articular cartilage – an in vitro study

ObjectivesTo compare the cytotoxicity of octenidine dihydrochloride and chlorhexidine gluconate at different concentrations on primary human articular chondrocytes and cartilage.Materials and methodsPrimary cultures of human normal adult articular chondrocytes were exposed to octenidine dihydrochloride (0.001562%, 0.003125%, 0.00625%, 0.0125%, 0.025%, 0.05%, and 0.1%), chlorhexidine gluconate (0.003125%, 0.00625%, 0.0125%, 0.025%, 0.05%, 0.1%, and 0.2%), and control (Dulbecco’s modified Eagle medium or phosphate-buffered saline) for 30 s. Normal human articular cartilage explants were exposed to octenidine dihydrochloride (0.1% versus control) and chlorhexidine gluconate (0.1% versus control) for 30 s. The viability of human articular chondrocytes was measured by Trypan blue staining, Cell Proliferation Reagent WST-1, and Live/Dead staining. The proliferation of human chondrocytes was measured using the Cell Proliferation Reagent WST-1. The viability of human articular cartilage explants was measured by using Live/Dead staining.ResultsOctenidine dihydrochloride and chlorhexidine gluconate exposure decreased cell viability and proliferation in a dose-dependent manner in primary human articular chondrocytes. Octenidine dihydrochloride and chlorhexidine gluconate exposure decreased cell viability in human articular cartilage explant cultures.ConclusionThe degree of toxicity varied between octenidine dihydrochloride and chlorhexidine gluconate, with chlorhexidine gluconate being less toxic than octenidine dihydrochloride at the same concentration. Additionally, both octenidine dihydrochloride and chlorhexidine gluconate evaluation had cytotoxic effects on human articular cartilage. Therefore, dosing for the antimicrobial mouthwash ingredients administration would ideally be determined to remain below IC50.Clinical relevanceThese data support the in vitro safety of antimicrobial mouthwashes on primary adult human articular chondrocytes.

Stevioside protects primary articular chondrocytes against IL-1β-induced inflammation and catabolism by targeting integrin.

Osteoarthritis (OA) is a common, progressive, and chronic disorder of the joints that is characterized by the inflammation and degradation of articular cartilage and is known to significantly impair quality of daily life. Stevioside (SVS) is a natural diterpenoid glycoside that has anti-inflammatory benefits. Hence, in the current research, it was hypothesized that SVS might exert anti-inflammatory effects on articular chondrocytes and alleviate cartilage degradation in mice with OA. The expression of inflammatory cytokines, like inducible nitric oxide synthase (iNOS), NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), and cyclooxygenase-2 (COX-2) in chondrocytes after interleukin-1β (IL-1β) exposure, was inhibited by the pretreatment of SVS. As well, SVS inhibited the reduction of collagen II and sry-box transcription factor 9 (SOX9) in chondrocytes stimulated by IL-1β and suppressed the expression of MMP3 and MMP13. Further, after treatment with SVS, cell cytometry, autophagy flux, and related protein expression showed diminished cell apoptosis and reduced autophagy impairment. Moreover, SVS blocked the activation of phosphoinositide-3-kinase/Akt/nuclear factor-kappa beta (PI3K/Akt/NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways stimulated by IL-1β. This resulted in decreased cellular inflammation. In vivo experiments with intra-articular injections of SVS in mice with the DMM mouse model demonstrated a decrease in cartilage degradation and an improvement in subchondral bone remodeling. After the integrin αVβ3-related knockdown using siRNA, a reversed effect was observed on the anti-inflammatory, anabolic promoting, catabolic blocking, and NF-κB and MAPK signaling pathway inhibition of SVS on chondrocytes treated with IL-1β. The above findings highlighted that SVS blocked IL-1β, triggered an inflammatory response in mice chondrocytes, and prevented cartilage degradation in vivo through integrin αVβ3. This suggested that SVS might serve as a novel therapeutic option for OA.

Long-Term Culture Performance of a Polyelectrolyte Complex Microcapsule Platform for Hyaline Cartilage Repair.

Articular cartilage (AC) tissue repair and regeneration remains an ongoing challenge. One component of the challenge is the limited ability to scale an engineered cartilage graft to clinically relevant sizes while maintaining uniform properties. In this paper, we report on the evaluation of our polyelectrolyte complex microcapsule (PECM) platform technology as a technique for generating cartilage-like spherical modules. Bone marrow-derived mesenchymal stem cells (bMSCs) or primary articular chondrocytes were encapsulated within PECMs composed of methacrylated hyaluronan, collagen I, and chitosan. The formation of cartilage-like tissue in the PECMs over a 90-day culture was characterized. The results showed that chondrocytes exhibited superior growth and matrix deposition compared to either chondrogenically-induced bMSCs or a mixed PECM culture containing both chondrocytes and bMSCs. The chondrocyte-generated matrix filled the PECM and produced substantial increases in capsule compressive strength. The PECM system thus appears to support intracapsular cartilage tissue formation and the capsule approach promotes efficient culture and handling of these micro tissues. Since previous studies have proven the feasibility of fusing such capsules into large tissue constructs, the results suggest that encapsulating primary chondrocytes in PECM modules may be a viable route toward achieving a functional articular cartilage graft.

E3 Ubiquitin Ligase Constitutive Photomorphogenic 1 Regulates Differentiation and Inflammation via MAPK Signaling Pathway in Rabbit Articular Chondrocytes

Constitutive photomorphogenic 1 (COP1), is an E3 ubiquitin ligase that plays a role in the regulation of various cellular processes including cell growth, differentiation, and survival in mammals. In certain conditions such as overexpression or loss of function, COP1 acts either as an oncogenic protein or as a tumor suppressor by targeting specific proteins for ubiquitination-mediated degradation. However, the precise role of COP1 has not been well studied in primary articular chondrocytes. In this study, we investigated the role of COP1 in chondrocyte differentiation. Western blotting and reverse transcription-polymerase chain reaction analysis demonstrated that COP1 overexpression reduced type II collagen expression, promoted cyclooxygenase 2 (COX-2) expression, and reduced sulfated proteoglycan synthesis, as detected by Alcian blue staining. Upon siRNA treatment, revived type II collagen, sulfated proteoglycan production, and decreased COX-2 expression. Phosphorylation of p38 kinase and ERK-1/-2 signaling pathways was regulated by COP1 upon cDNA and siRNA transfection in chondrocytes. The inhibition of the p38 kinase and ERK-1/-2 signaling pathways with SB203580 and PD98059 ameliorated the expression of type II collagen and COX-2 in transfected chondrocytes, thus suggesting that COP1 regulates differentiation and inflammation in rabbit articular chondrocytes via the p38 kinase and ERK-1/-2 signaling pathway.

Zinc finger E-box binding homebox 2 alleviated experimental osteoarthritis in rats

ABSTRACT Aims Osteoarthritis (OA) is a chronic inflammatory disease, characterized by degradation of extracellular matrix, apoptosis of chondrocytes and inflammation in articular cartilage. Zinc finger E-box binding homebox 2 (ZEB2), a transcription repressor, has been demonstrated with anti-inflammatory role in some cells. The analysis from GEO data reveals that ZEB2 expression is upregulated in articular cartilage of OA patients and experimental OA rodents. This study aims to confirm the function of ZEB2 in OA process. Material and methods The experimental OA was induced by anterior cruciate ligament transaction (ACLT) in rats, and the adenovirus loaded with ZEB2 coding sequence was intra-articularly injected into rats (1 × 10 PFU). The primary articular chondrocytes were stimulated by interleukin-1β (IL-1β) (10 ng/ml) to mimic the osteoarthritic injury, and transfected with the adenovirus carrying ZEB2 coding or silencing sequence. The apoptosis, content of extracellular matrix, inflammation, and the activity of NFκB signaling in chondrocytes and cartilage were determined. Results ZEB2 was highly expressed in osteoarthritic cartilage tissues and IL-1β-treated chondrocytes. The overexpression of ZEB2 restrained ACLT- or IL-1β administration-induced apoptosis, matrix degradation and inflammation in vivo or in vitro, evidenced by changed levels of cleaved caspase-3/PARP, collagen-II, aggrecan, matrix metalloproteinase 3/13, tumor necrosis factor-α and interleukin-6. Additionally, the phosphorylation of NFκB p65, IκBα and IKKα/β, and the nuclear translocation of p65 was blocked by ZEB2, implying inactivation of this signaling. Conclusions ZEB2 mitigated osteoarthritic symptoms in rats and chondrocytes, and NFκB signaling might be involved. These findings may provide novel insights for clinical treatment of OA.

Hydroquinone, an Environmental Pollutant, Affects Cartilage Homeostasis through the Activation of the Aryl Hydrocarbon Receptor Pathway.

Exposure to environmental pollutants has a proven detrimental impact on different aspects of human health. Increasing evidence has linked pollution to the degeneration of tissues in the joints, although through vastly uncharacterised mechanisms. We have previously shown that exposure to hydroquinone (HQ), a benzene metabolite that can be found in motor fuels and cigarette smoke, exacerbates synovial hypertrophy and oxidative stress in the synovium. To further understand the impact of the pollutant on joint health, here we investigated the effect of HQ on the articular cartilage. HQ exposure aggravated cartilage damage in rats in which inflammatory arthritis was induced by injection of Collagen type II. Cell viability, cell phenotypic changes and oxidative stress were quantified in primary bovine articular chondrocytes exposed to HQ in the presence or absence of IL-1β. HQ stimulation downregulated phenotypic markers genes SOX-9 and Col2a1, whereas it upregulated the expression of the catabolic enzymes MMP-3 and ADAMTS5 at the mRNA level. HQ also reduced proteoglycan content and promoted oxidative stress alone and in synergy with IL-1β. Finally, we showed that HQ-degenerative effects were mediated by the activation of the Aryl Hydrocarbon Receptor. Together, our findings describe the harmful effects of HQ on articular cartilage health, providing novel evidence surrounding the toxic mechanisms of environmental pollutants underlying the onset of articular diseases.

Silencing miR-146a-5p Protects against Injury-Induced Osteoarthritis in Mice.

Osteoarthritis (OA), the most prevalent joint disease and the leading cause of disability, remains an incurable disease largely because the etiology and pathogenesis underlying this degenerative process are poorly understood. Low-grade inflammation within joints is a well-established factor that disturbs joint homeostasis and leads to an imbalance between anabolic and catabolic processes in articular cartilage; however, the complexity of the network between inflammatory factors that often involves positive and negative feedback loops makes current anti-cytokine therapy ineffective. MicroRNAs (miRNAs) have emerged as key regulators to control inflammation, and aberrant miRNAs expression has recently been linked to OA pathophysiology. In the present study, we characterized transcriptomic profiles of miRNAs in primary murine articular chondrocytes in response to a proinflammatory cytokine, IL-1β, and identified miR-146a-5p as the most responsive miRNA to IL-1β. miR-146a-5p was also found to be upregulated in human OA cartilage. We further demonstrated that knockdown of miR-146a-5p antagonized IL-1β-mediated inflammatory responses and IL-1β-induced catabolism in vitro, and silencing of miR-146a in chondrocytes ameliorated articular cartilage destruction and reduced OA-evoked pain in an injury-induced murine OA model. Moreover, parallel RNA sequencing revealed that differentially expressed genes in response to IL-1β were enriched in pathways related to inflammatory processes, cartilage matrix homeostasis, and cell metabolism. Bioinformatic analyses of putative miR-146a-5p gene targets and following prediction of protein-protein interactions suggest a functional role of miR-146a-5p in mediating inflammatory processes and regulation of cartilage homeostasis. Our genetic and transcriptomic data define a crucial role of miR-146a-5p in OA pathogenesis and implicate modulation of miR-146a-5p in articular chondrocytes as a potential therapeutic strategy to alleviate OA.

Ribosomal RNA-based epitranscriptomic regulation of chondrocyte translation and proteome in osteoarthritis

Osteoarthritis-related cartilage extracellular matrix remodelling is dependent on changes in chondrocyte protein expression. Yet, the role of ribosomes in chondrocyte translation regulation is unknown. In this exploratory study, we investigated ribosomal RNA (rRNA) epitranscriptomic-based ribosome heterogeneity in human articular chondrocytes and its relevance for osteoarthritis. Sequencing-based rRNA 2'-O-methylation profiling analysis (RiboMethSeq) was performed on non-OA primary human articular chondrocytes (n=5) exposed for 14 days to osteoarthritic synovial fluid (14 donors, pooled, 20% v/v). The SW1353 SNORD71 KO cell pool was generated using LentiCRISPRv2/Cas9. The mode of translation initiation and fidelity were determined by dual-luciferase reporters. The cellular proteome was analysed by LC-MS/MS and collagen type I protein expression was evaluated by immunoblotting. Loading of COL1A1 mRNA into polysomes was determined by sucrose gradient ultracentrifugation and fractionation. We discovered that osteoarthritic synovial fluid instigates site-specific changes in the rRNA 2'-O-me profile of primary human articular chondrocytes. We identified five sites with differential 2'-O-me levels. The 2'-O-me status of 5.8S-U14 (one of identified differential 2'-O-me sites; decreased by 7.7%, 95% CI [0.9% to 14.5%]) was targeted by depleting the level of its guide snoRNA SNORD71 (50% decrease, 95% CI [33% to 64%]). This resulted in an altered ribosome translation modus (e.g CrPV IRES, FC 3, 95% CI [2.2 to 4.1]) and promoted translation of COL1A1 mRNA which led to increased levels of COL1A1 protein (FC 1.7, 95% CI [1.3 to 2.0]). Our data identify a novel concept suggesting that articular chondrocytes employ rRNA epitranscriptomic mechanisms in osteoarthritis development.

Generation of human immortalized chondrocytes from osteoarthritic and healthy cartilage

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. 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. 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. 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 Res2023;12(1):46-57.

RUNX2 stabilization by long non-coding RNAs contributes to hypertrophic changes in human chondrocytes.

Background: Chondrocyte hypertrophy has been implicated in endochondral ossification and osteoarthritis (OA). In OA, hypertrophic chondrocytes contribute to the destruction and focal calcification of the joint cartilage. Although studies in this field have remarkably developed the modulation of joint inflammation using gene therapy and regeneration of damaged articular cartilage using cell therapy, studies that can modulate or prevent hypertrophic changes in articular chondrocytes are still lacking. Methods: In vitro hypertrophic differentiation and inflammation assays were conducted using human normal chondrocyte cell lines, TC28a2 cells. Human cartilage tissues and primary articular chondrocytes were obtained from OA patients undergoing total knee arthroplasty. Long non-coding RNAs (lncRNAs), LINC02035 and LOC100130207, were selected through RNA-sequencing analysis using RNAs extracted from TC28a2 cells cultured in hypertrophic medium. The regulatory mechanism was evaluated using western blotting, real-time quantitative polymerase chain reaction, osteocalcin reporter assay, RNA-immunoprecipitation (RNA-IP), RNA-in situ hybridization, and IP. Results: LncRNAs are crucial regulators of various biological processes. In this study, we identified two important lncRNAs, LINC02035 and LOC100130207, which play important roles in hypertrophic changes in normal chondrocytes, through RNA sequencing. Interestingly, the expression level of RUNX2, a master regulator of chondrocyte hypertrophy, was regulated at the post-translational level during hypertrophic differentiation of the normal human chondrocyte cell line, TC28a2. RNA-immunoprecipitation proved the potential interaction between RUNX2 protein and both lncRNAs. Knockdown (KD) of LINC02035 or LOC100130207 promoted ubiquitin-mediated proteasomal degradation of RUNX2 and prevented hypertrophic differentiation of normal chondrocyte cell lines, whereas overexpression of both lncRNAs stabilized RUNX2 protein and generated hypertrophic changes. Furthermore, the KD of the two lncRNAs mitigated the destruction of important cartilage matrix proteins, COL2A1 and ACAN, by hypertrophic differentiation or inflammatory conditions. We also confirmed that the phenotypic changes raised by the two lncRNAs could be rescued by modulating RUNX2 expression. In addition, the KD of these two lncRNAs suppressed hypertrophic changes during chondrogenic differentiation of mesenchymal stem cells. Conclusion: Therefore, this study suggests that LINC02035 and LOC100130207 contribute to hypertrophic changes in normal chondrocytes by regulating RUNX2, suggesting that these two novel lncRNAs could be potential therapeutic targets for delaying or preventing OA development, especially for preventing chondrocyte hypertrophy.