Osteoarthritis Cartilage Samples Research Articles

Epigenomic differences between osteoarthritis grades in primary cartilage

ObjectiveOsteoarthritis is a common and complex joint disorder that shows higher prevalence and greater disease severity in women. Here, we investigate genome-wide methylation profiles of primary chondrocytes from osteoarthritis patients. DesignWe compare genome-wide methylation profiles of macroscopically intact (low-grade) and degraded (high-grade) osteoarthritis cartilage samples matched from osteoarthritis patients undergoing knee replacement surgery. We perform an epigenome-wide association study (EWAS) for cartilage degeneration across 170 patients and separately in 96 women and 74 men. ResultsWe reveal widespread epigenetic differences with enrichments of nervous system and apoptosis-related processes. We further identify substantial similarities between sexes, but also sex-specific markers and pathways. ConclusionsTogether, we provide the largest genome-wide methylation profiles of primary cartilage to date with enhanced and sex-specific insights into epigenetic processes underlying osteoarthritis progression.

Comparative assessment of chondral defect repair using migratory chondroprogenitors suspended in either gelled or freeze-dried platelet-rich plasma: An in vitro and ex vivo human osteochondral unit model study

BackgroundChondroprogenitors, with enhanced chondrogenic potential, have emerged to be a promising alternative for cell-based therapy in cartilage repair. Platelet-rich plasma (PRP), widely used for intra-articular treatment, has a short half-life. Freeze-dried PRP (FD-PRP), with an extended half-life and retained growth factors, is gaining attention. This study compares the efficacy of Migratory Chondroprogenitors (MCPs) in gelled PRP and FD-PRP using in-vitro and ex-vivo models, assessing FD-PRP as a potential off-the-shelf option for effective cartilage repair. MethodologyMCPs were isolated from osteoarthritic cartilage samples (n = 3), characterized through FACS and RT-PCR. For in-vitro analysis, cells were loaded into gelled PRP and FD-PRP scaffolds at a density of 1x106 cells per scaffold. Trilineage differentiation studies and live-dead assays were conducted on MCPs using Calcein AM/Propidium Homodimer-1. In ex-vivo analysis, MCPs of the same density were added to Osteochondral Units (OCU) with chondral defects containing PRP gel and FD-PRP scaffolds, harvested on the 15th and 35th days for histological examination. Controls included cell-free scaffolds. ResultsOur in-vitro analysis demonstrates the robust viability of MCPs in both scaffolds, with no discernible impact on their differentiation capacity. Ex-vivo analysis of the OCU for cartilage repair showed that the chondrogenic potential characterized by the accumulation of extracellular matrix containing glycosaminoglycans and collagen type II production (with no alteration in collagen type X), was observed to be better with the gel PRP and the gel PRP containing MCP groups. ConclusionsThese findings support the preference for gel PRP as a superior synergistic scaffold for chondroprogenitor delivery.

Sortilin Is Upregulated in Osteoarthritis-Dependent Cartilage Calcification and Associated with Cellular Senescence.

Osteoarthritis (OA) is a chronic joint disease characterized by articular cartilage calcification, loss of articular cartilage, bone changes, pain, and disability. Cartilage calcification is one hallmark of OA and is predominantly caused by basic calcium crystals formed due to an imbalance of the pyrophosphate pathway. Sortilin is a transmembrane protein that contributes to vascular calcification in atherosclerosis by externalizing alkaline phosphatase (ALP)-containing vesicles. Calcification in atherosclerosis and osteoarthritis has been associated with cellular senescence. The aim of this study was to investigate the potential role of sortilin and senescence in osteoarthritis-dependent cartilage calcification. Osteoarthritic cartilage from human knee joints was collected after joint replacement, and samples were analyzed by immunohistochemistry and quantitative RT-PCR analysis. Human chondrocytes were treated with osteogenic medium for up to 21 days to induce calcification. Western blots for sortilin and ALP, as well as an ALP activity assay, were performed. Human chondrocytes were treated with mitomycin C to induce senescence, and sortilin expression was quantified at the protein and gene levels. Sections of knee joints from a murine model of osteoarthritis were stained for sortilin and p16 and analyzed by immunohistochemistry. Treatment of wild-type chondrocytes using an osteogenic medium similar to human chondrocytes was performed. Osteoarthritic cartilage from mouse and human knee joints showed an increased number of sortilin and p16-positive chondrocytes compared to healthy cartilage. This observation was corroborated by increased gene expression of sortilin and p16 in mild and moderate osteoarthritic cartilage samples. To investigate the mechanism of sortilin regulation, human chondrocytes were treated with osteogenic medium to induce calcification. Sortilin protein levels and expression were increased after 7 days of stimulation, whereas ALP levels and activity were upregulated after 21 days of stimulation. Similar observations were made in a murine osteoarthritis model. Mechanistically, senescent chondrocytes induced by mitomycin C showed an upregulation of sortilin and ALP gene expression compared to non-senescent chondrocytes. Our data indicate that sortilin and ALP are upregulated during cartilage calcification, which is associated with chondrocyte senescence and thus might contribute to the pathogenesis of osteoarthritis. Cellular senescence seems to induce sortilin expression.

Circular RNA CircDHRS3 Aggravates IL-1β-induced ECM Degradation, Apoptosis, and Inflammatory Response via Mediating MECP2 Expression.

Previous studies have reported that circular RNA hsa_circ_0010024 (circDHRS3), microRNA (miR)-193a-3p, and Methyl CpG binding protein 2 (MECP2) are unconventionally expressed in osteoarthritis (OA) cartilage samples. However, the regulatory mechanisms among circDHRS3, miR-193a-3p, and MECP2 in OA pathogenesis are unclear. Changes of circDHRS3, miR-193a-3p, and MECP2 mRNA were detected by qRT-PCR. Several protein levels were evaluated using western blotting. Cell proliferation was analyzed by 5-Ethynyl-2'-deoxyuridine (EdU) and cell counting assays. Cell apoptosis was determined by flow cytometry assay. Detection of pro-inflammatory cytokines was conducted using ELISA. The relationship between circDHRS3 or MECP2 and miR-193a-3p was validated by dual-luciferase reporter assay. We verified that circDHRS3 and MECP2 were overexpressed in OA cartilage samples, whereas miR-193a-3p was downregulated. CircDHRS3 silencing weakened IL-1β-induced chondrocyte cartilage extracellular matrix (ECM) degradation, apoptosis, and inflammatory response. CircDHRS3 adsorbed miR-193a-3p to modulate MECP2 expression. Also, silencing of miR-193a-3p impaired circDHRS3 silencing-mediated suppression on IL-1β-induced chondrocyte injury. Also, MECP2 overexpression alleviated miR-193a-3p mimic-mediated inhibition on IL-1β-prompted chondrocyte injury. CircDHRS3 silencing reduced MECP2 expression via sponging miR-193a-3p, thereby weakening IL-1β-induced chondrocyte ECM degradation, apoptosis, and inflammatory response.

Knockdown of circSOD2 ameliorates osteoarthritis progression via the miR-224-5p/PRDX3 axis

BackgroundAlthough the implications of circular RNAs (circRNAs) with the progression of diverse pathological conditions have been reported, the circRNA players in osteoarthritis (OA) are barely studied.MethodsIn this study, twenty-five OA patients who received arthroplasty were recruited for cartilage tissue collection. Public circRNA microarray data from Gene Expression Omnibus was retrieved for circRNA identification. An in vitro cell model of OA-related damages was constructed by treating human chondrocytes (CHON-001 cell line) with IL-1β, and circSOD2 siRNA was used to silence circSOD2 expression to study its functional role in apoptosis, inflammatory responses, and extracellular matrix (ECM) degradation. Besides, we investigated the functional interactions among circSOD2, miR-224-5p, and peroxiredoxin 3 (PRDX3) by luciferase reporter assay, RNA-immunoprecipitation assay, and quantitative reverse transcription polymerase chain reaction.ResultsOur findings revealed the overexpression of circSOD2 in the OA cartilage and cell samples, and circSOD2 knockdown alleviated ECM degradation, inflammation, and apoptosis in CHON-001 cell model. In addition, our findings suggested the regulatory function of circSOD2 knockdown on miR-224-5p expression, while miR-224-5p was capable of downregulating PRDX3 expression. The co-transfection of miR-224-5p inhibitor or pcDNA-PRDX3 could prevent the effect of circSOD2 knockdown.ConclusionHence, our results demonstrated that knockdown of circSOD2 may serve as an intervention strategy to alleviate OA progression through modulating miR-224-5p/PRDX3 signaling axis.

Mediation of the Same Epigenetic and Transcriptional Effect by Independent Osteoarthritis Risk-Conferring Alleles on a Shared Target Gene, COLGALT2.

Over 100 DNA variants have been associated with osteoarthritis (OA), including rs1046934, located within a linkage disequilibrium block encompassing part of COLGALT2 and TSEN15. The present study was undertaken to determine the target gene(s) and the mechanism of action of the OA locus using human fetal cartilage, cartilage from OA and femoral neck fracture arthroplasty patients, and a chondrocyte cell model. Genotyping and methylation array data of DNA from human OA cartilage samples (n=87) were used to determine whether the rs1046934 genotype is associated with differential DNA methylation at proximal CpGs. Results were replicated in DNA from human arthroplasty (n=132) and fetal (n=77) cartilage samples using pyrosequencing. Allelic expression imbalance (AEI) measured the effects of genotype on COLGALT2 and TSEN15 expression. Reporter gene assays and epigenetic editing determined the functional role of regions harboring differentially methylated CpGs. In silico analyses complemented these experiments. Three differentially methylated CpGs residing within regulatory regions were detected in the human OA cartilage array data, and 2 of these were replicated in human arthroplasty and fetal cartilage. AEI was detected for COLGALT2 and TSEN15, with associations between expression and methylation for COLGALT2. Reporter gene assays confirmed that the CpGs are in chondrocyte enhancers, with epigenetic editing results directly linking methylation with COLGALT2 expression. COLGALT2 is a target of this OA locus. We previously characterized another OA locus, marked by rs11583641, that independently targets COLGALT2. The genotype of rs1046934, like rs11583641, mediates its effect by modulating expression of COLGALT2 via methylation changes to CpGs located in enhancers. Although the single-nucleotide polymorphisms, CpGs, and enhancers are distinct between the 2 independent OA risk loci, their effect on COLGALT2 is the same. COLGALT2 is the target of independent OA risk loci sharing a common mechanism of action.

IRF1 promotes the chondrogenesis of human adipose-derived stem cells through regulating HILPDA

BackgroundOsteoarthritis is a main cause of deformity in aging people. The chondrogenesis of human adipose-derived stem cells (hADSCs) has a positive effect on the cure of osteoarthritis. However, the regulatory mechanism of hADSC chondrogenesis still needs further exploration. This research investigates the role of interferon regulatory factor 1 (IRF1) in the chondrogenesis of hADSCs. MethodshADSCs were purchased and cultured. The interaction between IRF1 and hypoxia inducible lipid droplet associated (HILPDA) was predicted by bioinformatics analysis, and verified through dual-luciferase reporter and chromatin immunoprecipitation assays. The expressions of IRF1 and HILPDA in osteoarthritis cartilage samples were measured through qRT-PCR. After hADSCs were transfected or further induced for chondrogenesis, the chondrogenesis was visualized by Alcian blue staining, and the expressions of IRF1, HILPDA and chondrogenesis-related factors (SOX9, Aggrecan, COL2A1, MMP13, MMP3) were determined through qRT-PCR or Western blot. ResultsHILPDA bound to IRF1 in hADSCs. IRF1 and HILPDA levels were up-regulated during the chondrogenesis of hADSCs. Overexpressions of IRF1 and HILPDA promoted the chondrogenesis of hADSCs with the up-regulation of SOX9, Aggrecan and COL2A1 and the down-regulation of MMP13 and MMP3; however, IRF1 silencing generated the opposite effects. Besides, HILPDA overexpression reversed the effects of IRF1 silencing on inhibiting chondrogenesis of hADSCs and regulating the expressions of chondrogenesis-related factors. ConclusionIRF1 promotes the chondrogenesis of hADSCs through up-regulating HILPDA level, providing novel biomarkers for treating osteoarthritis.

Knockdown of Circ_0037658 Alleviates IL-1β-Induced Osteoarthritis Progression by Serving as a Sponge of miR-665 to Regulate ADAMTS5.

Background: Osteoarthritis (OA) is a chronic musculoskeletal degeneration disease which brings great pain to patients and a tremendous burden on the world’s medical resources. Previous reports have indicated that circular RNAs (circRNAs) are involved in the pathogenesis of OA. The purpose of this study was to explore the role and mechanism of circ_0037658 in the OA cell model. Methods: The content of interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) was measured using enzyme-linked immunosorbent assay (ELISA). Cell proliferation ability and apoptosis were detected using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EDU), and flow cytometry assays. Western blot assay was used to measure the protein levels of Bcl-2-related X protein (Bax), cleaved-caspase-3, MMP13, Aggrecan, and ADAMTS5. The expression of circ_0037658, microRNA-665 (miR-665), and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 5 was detected using real-time quantitative polymerase chain reaction (RT-qPCR). Dual-luciferase reporter assay and RNA Immunoprecipitation (RIP) assay were manipulated to analyze the relationships of circ_0037658, miR-665, and ADAMTS5. Results: Human chondrocytes (CHON-001 cells) were treated with interleukin-1β (IL-1β) to establish an OA cell model. Circ_0037658 and ADAMTS5 levels were increased, and miR-665 was decreased in OA cartilage samples and IL-1β-treated chondrocyte cells. Moreover, circ_0037658 silencing promoted proliferation and impaired inflammation, apoptosis, and ECM degradation in IL-1β-treated CHON-001 cells. Mechanically, circ_0037658 acted as a sponge for miR-665 to regulate ADAMTS5 expression. Conclusion: Circ_0037658 knockdown relieved IL-1β-triggered chondrocyte injury via regulating the miR-665/ADAMTS5 axis, promising an underlying therapeutic strategy for OA.

Blocking chondrocyte hypertrophy in conditional Evc knockout mice does not modify cartilage damage in osteoarthritis.

Chondrocytes in osteoarthritic (OA) cartilage acquire a hypertrophic-like phenotype, where Hedgehog (Hh) signaling is pivotal. Hh overexpression causes OA-like cartilage lesions, whereas its downregulation prevents articular destruction in mouse models. Mutations in EVC and EVC2genes disrupt Hh signaling, and are responsible for the Ellis-van Creveld syndrome skeletal dysplasia. Since Ellis-van Creveld syndrome protein (Evc) deletion is expected to hamper Hh target gene expression we hypothesized that it would also prevent OA progression avoiding chondrocyte hypertrophy. Our aim was to study Evc as a new therapeutic target in OA, and whether Evc deletion restrains chondrocyte hypertrophy and prevents joint damage in an Evc tamoxifen induced knockout (EvccKO ) model of OA. For this purpose, OA was induced by surgical knee destabilization in wild-type (WT) and EvccKO adult mice, and healthy WT mice were used as controls (n=10knees/group). Hypertrophic markers and Hh genes were measured by qRT-PCR, and metalloproteinases (MMP) levels assessed by western blot. Human OA chondrocytes and cartilage samples were obtained from patients undergoing knee joint replacement surgery. Cyclopamine (CPA) was used for Hh pharmacological inhibition and IL-1 beta as an inflammatory insult. Our results showed that tamoxifen induced inactivation of Evc inhibited Hh overexpression and partially prevented chondrocyte hypertrophy during OA, although it did not ameliorate cartilage damage in DMM-EvccKO mice. Hh pathway inhibition did not modify the expression of proinflammatory mediators induced by IL-1 beta in human OA chondrocytes in culture. We found that hypertrophic-IHH-and inflammatory-COX-2-markers co-localized in OA cartilage samples. We concluded that tamoxifen induced inactivation of Evc partially prevented chondrocyte hypertrophy in DMM-EvccKO mice, but it did not ameliorate cartilage damage. Overall, our results suggest that chondrocyte hypertrophy per se is not a pathogenic event in the progression of OA.

Effect of micro-strain stress on in vitro proliferation and functional expression of human osteoarthritic chondrocytes

BackgroundThis study aimed to analyze the in vitro effect of micro-strain stress on the proliferation and functional marker expression in chondrocytes isolated from human osteoarthritis cartilage samples.MethodsChondrocytes isolated from human osteoarthritis cartilage samples were subjected to loading with different types of micro-strain stress. The proliferation activity was assessed by flow cytometry, and the functional expression of chondrocyte markers was detected by qRT-PCR and western blot.ResultsFlow cytometry results showed stimulation of proliferation of human osteoarthritic chondrocytes when an adequate micro-strain stress was applied. qRT-PCR and western blot results showed that micro-strain stress promotes human osteoarthritic chondrocyte functional marker expression. These features coincide with the upregulation of multiple proteins and genes affecting cell proliferation and functional chondrocyte marker expression, including cyclin D1, collagen II, and Rock.ConclusionAdequate micro-strain stress could activate the Rho/Rock signaling pathway in osteoarthritic chondrocytes, thus transmitting mechanical signals to the cytoskeleton. This process leads to cytoskeleton reorganization, and transmission of the mechanical signals to the downstream effectors to promote proliferation and functional marker expression of osteoarthritic chondrocytes.

CircPhc3 sponging microRNA‑93‑3p is involved in the regulation of chondrocyte function by mechanical instability in osteoarthritis.

Cartilage extracellular matrix (ECM) metabolism disorder caused by mechanical instability is a leading cause of osteoarthritis (OA), but the exact mechanisms have not been fully elucidated. Recent studies have suggested an important role of circular RNAs (circRNAs/circs) in OA. The present study aimed to investigate whether circRNAs might have a role in mechanical instability-regulated chondrocyte matrix metabolism in OA. The expression levels of circPhc3 in human and mouse OA cartilage samples were measured using reverse transcription-quantitative PCR and fluorescence in situ hybridization. The effects of circPhc3 on chondrocyte ECM metabolism were further investigated by overexpressing and knocking down circPhc3 in OA chondrocytes. The downstream target of circPhc3 was examined by performing a luciferase reporter assay. The results showed that the expression of circPhc3 was reduced in human and mouse OA cartilage. Moreover, circPhc3 was involved in mechanical loading-regulated production of ECM and cartilage-degrading enzymes. Further studies showed that circPhc3 regulated chondrocyte matrix metabolism primarily by binding to microRNA (miR)-93-3p, and mechanistic studies found that miR-93-3p targeting of FoxO1 was involved in chondrocyte matrix metabolism. Taken together, these results indicated that circPhc3 may serve an important role in the progression of OA and may be a good target for the treatment of OA.

Zinc finger protein A20 regulates the development and progression of osteoarthritis by affecting the activity of NF-κB p65

Objective To investigate the role of Zinc finger protein A20 in osteoarthritis (OA) by regulating NF-κB p65. Methods A20, MMP1, MMP13 and IL-1β expressions in human OA cartilage samples were detected by qRT-PCR. IL-1β-induced chondrocyte was treated with A20 lentivirus activation particle, pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor) with/without A20 siRNA. IL-6, TNF-α, and PGE2 levels were measured by ELISA, and NO production by Greiss reaction. Destabilization of the medial meniscus (DMM) surgery was used to construct the OA models, followed by injection of A20 adenovirus. MMP1 and MMP13 expression was measured by immunohistochemistry. The mRNA and protein expression were performed by qRT-PCR and western blotting, respectively. Results A20 was down-regulated in human OA cartilage samples, and negatively correlated with the expressions of MMP1, MMP13 and IL-1β. The IL-1β-induced chondrocyte manifested decreased A20 with increased NF-κB p65 activity. A20 overexpression suppressed the NF-κB p65 activity in IL-1β-induced chondrocyte. Furthermore, PDTC decreased IL-1β-induced chondrocyte apoptosis with the upregulated COL1A1, COL2A1, COL10A1 and ACAN, as well as the down-regulated MMP1, MMP13, COX2, iNOS, IL-6, TNF-α, NO and PGE2, which was reversed by A20 siRNA. In vivo, OA mice gained higher OARSI score and Mankin’s score, exhibited up-regulations of MMP1 and MMP13, and decreased NF-κB p65 activity, which was improved after injection of A20 adenovirus. Conclusion A20 was reduced in OA cartilage samples, and its overexpression, by suppressing the activity of NF-κB p65, could improve IL-1β-induced chondrocyte degradation and apoptosis in vitro, as well as mitigate the inflammation in OA mice.

Evaluation of Three Growth Factors with the Most Controversial Expression Pattern in Osteoarthritic and Non-osteoarthritic Cartilage

There is no clear consensus regarding the expression pattern of many growth factors in healthy and osteoarthritic cartilage. In this study, the expression pattern of three controversial cartilage growth factors was compared between osteoarthritic (n=27) ‎and non-osteoarthritic cartilage samples (n=14). The candidate growth factor included Insulin-like ‎growth factor 1 (IGF-1), Fibroblast Growth Factor 2 (FGF-2), and Bone Morphogenetic Protein 2 (BMP-2). The candidate markers' gene and protein expression were analyzed using relative quantification relative to real-time PCR and ELISA, respectively. The mean IGF-I ‎mRNA and protein expression were 11.1, and 2 folds greater in osteoarthritic cartilage, respectively (p<0.001 for both). The mean FGF-2 ‎mRNA and protein expression were 6.7 and 1.3 folds greater in osteoarthritic cartilage, respectively (p<0.001 and p=0.003). The mean BMP-2 ‎mRNA and protein expression were 3.48 and 1.51 folds greater in osteoarthritic cartilage, respectively (p=0.001 and p<0.001). A significant positive correlation was found between the mRNA and protein expression of all candidate growth factors. In conclusion, the gene and protein expression of IGF-I, FGF-2, and BMP-2 were significantly higher in the ‎osteoarthritic cartilages when compared with non-osteoarthritic, suggesting them as‎‏ a therapeutic ‏target in OA treatment.

RIP1 Perturbation Induces Chondrocyte Necroptosis and Promotes Osteoarthritis Pathogenesis via Targeting BMP7.

Osteoarthritis (OA) is a highly prevalent and debilitating joint disorder that characterized by progressive destruction of articular cartilage. There is no effective disease-modifying therapy for the condition due to limited understanding of the molecular mechanisms on cartilage maintenance and destruction. Receptor-interacting protein kinase 1 (RIP1)-mediated necroptosis plays a vital role in various diseases, but the involvement of RIP1 in OA pathogenesis remains largely unknown. Here we show that typical necrotic cell morphology is observed within human OA cartilage samples in situ, and that RIP1 is significantly upregulated in cartilage from both OA patients and experimental OA rat models. Intra-articular RIP1 overexpression is sufficient to induce structural and functional defects of cartilage in rats, highlighting the crucial role of RIP1 during OA onset and progression by mediating chondrocyte necroptosis and disrupting extracellular matrix (ECM) metabolism homeostasis. Inhibition of RIP1 activity by its inhibitor necrostatin-1 protects the rats from trauma-induced cartilage degradation as well as limb pain. More importantly, we identify bone morphogenetic protein 7 (BMP7) as a novel downstream target that mediates RIP1-induced chondrocyte necroptosis and OA manifestations, thereby representing a non-canonical regulation mode of necroptosis. Our study supports a model whereby the activation of RIP1-BMP7 functional axis promotes chondrocyte necroptosis and subsequent OA pathogenesis, thus providing a new therapeutic target for OA.

MicroRNA-324 regulates bone remodelling and cartilage maintenance

Purpose: Osteoarthritis is the most common musculoskeletal condition in the elderly population of the UK and is characterised primarily by the degradation of articular cartilage and remodelling of subchondral bone. microRNAs, short noncoding RNA molecules that act to downregulate a specific set of messenger RNA targets, are an important component of molecular networks involved in the musculoskeletal system and therefore also in the onset of osteoarthritis. miR-324 is a microRNA that is significantly upregulated in human osteoarthritic cartilage samples relative to neck of femur fracture controls and as such we hypothesised that it would play a role in disease.

Protease activated receptor 2 and matriptase expression in the joints of cats with and without osteoarthritis.

The aim of this study was to determine the presence of protease-activated receptor 2 (PAR2) and matriptase proteins and quantify PAR2 and matriptase mRNA expression in the articular cartilage and synovial membrane of cats with and without osteoarthritis (OA). A total of 28 articular cartilage samples from adult cats (14 OA and 14 normal), 10 synovial membranes from adult cats (five OA and five normal) and three cartilage samples from 9-week-old fetal cats were used. The presence of PAR2 and matriptase in the cartilage and synovial membrane of the adult samples was detected by immunohistochemical (IHC) staining, while real-time PCR was used for mRNA expression analyses in all samples. PAR2 was detected in all OA and normal articular cartilage and synovial membrane samples but confined to only a few superficial chondrocytes in the normal samples. Matriptase was only detected in OA articular cartilage and synovial membrane samples. PAR2 and matriptase mRNA expression were, however, detected in all cartilage and synovial membrane samples. PAR2 and matriptase mRNA expression levels in OA articular cartilage were five (P <0.001) and 3.3 (P <0.001) times higher than that of the healthy group, respectively. There was no significant difference (P = 0.05) in the OA synovial membrane PAR2 and matriptase mRNA expression compared with the normal samples. Detection of PAR2 and matriptase proteins and gene expression in feline articular tissues is a novel and important finding, and supports the hypothesis that serine proteases are involved in the pathogenesis of feline OA. The consistent presence of PAR2 and matriptase protein in the cytoplasm of OA chondrocytes suggests a possible involvement of proteases in cartilage degradation. Further investigations into the PAR2 and matriptase pathobiology could enhance our understanding of the proteolytic cascades in feline OA, which might lead to the development of novel therapeutic strategies.

Rheometer enabled study of cartilage frequency-dependent properties

Despite the well-established dependence of cartilage mechanical properties on the frequency of the applied load, most research in the field is carried out in either load-free or constant load conditions because of the complexity of the equipment required for the determination of time-dependent properties. These simpler analyses provide a limited representation of cartilage properties thus greatly reducing the impact of the information gathered hindering the understanding of the mechanisms involved in this tissue replacement, development and pathology. More complex techniques could represent better investigative methods, but their uptake in cartilage research is limited by the highly specialised training required and cost of the equipment. There is, therefore, a clear need for alternative experimental approaches to cartilage testing to be deployed in research and clinical settings using more user-friendly and financial accessible devices. Frequency dependent material properties can be determined through rheometry that is an easy to use requiring a relatively inexpensive device; we present how a commercial rheometer can be adapted to determine the viscoelastic properties of articular cartilage. Frequency-sweep tests were run at various applied normal loads on immature, mature and trypsinased (as model of osteoarthritis) cartilage samples to determine the dynamic shear moduli (G*, G′ G″) of the tissues. Moduli increased with increasing frequency and applied load; mature cartilage had generally the highest moduli and GAG depleted samples the lowest. Hydraulic permeability (KH) was estimated from the rheological data and decreased with applied load; GAG depleted cartilage exhibited higher hydraulic permeability than either immature or mature tissues. The rheometer-based methodology developed was validated by the close comparison of the rheometer-obtained cartilage characteristics (G*, G′, G″, KH) with results obtained with more complex testing techniques available in literature. Rheometry is relatively simpler and does not require highly capital intensive machinery and staff training is more accessible; thus the use of a rheometer would represent a cost-effective approach for the determination of frequency-dependent properties of cartilage for more comprehensive and impactful results for both healthcare professional and R&D.

Global knockout of MIR-324 affects bone structure and cartilage maintenance in mice

Purpose: Osteoarthritis is the most common musculoskeletal condition in the elderly population of the UK and is characterised primarily by the degradation of articular cartilage and remodelling of subchondral bone. Genetics is thought to play a key role in osteoarthritis onset and microRNAs, molecules that act to regulate gene expression by repressing translation of messenger RNA, make up a significant part of this. miR-324 is a microRNA that has previously been demonstrated to be significantly upregulated in human osteoarthritic cartilage samples relative to neck of femur fracture controls.

Single-cell mass cytometry reveals cross-talk between inflammation-dampening and inflammation-amplifying cells in osteoarthritic cartilage.

Aging or injury leads to degradation of the cartilage matrix and the development of osteoarthritis (OA). Because of a paucity of single-cell studies of OA cartilage, little is known about the interpatient variability in its cellular composition and, more importantly, about the cell subpopulations that drive the disease. Here, we profiled healthy and OA cartilage samples using mass cytometry to establish a single-cell atlas, revealing distinct chondrocyte progenitor and inflammation-modulating subpopulations. These rare populations include an inflammation-amplifying (Inf-A) population, marked by interleukin-1 receptor 1 and tumor necrosis factor receptor II, whose inhibition decreased inflammation, and an inflammation-dampening (Inf-D) population, marked by CD24, which is resistant to inflammation. We devised a pharmacological strategy targeting Inf-A and Inf-D cells that significantly decreased inflammation in OA chondrocytes. Using our atlas, we stratified patients with OA in three groups that are distinguished by the relative proportions of inflammatory to regenerative cells, making it possible to devise precision therapeutic approaches.

SIRT2 inhibits oxidative stress and inflammatory response in diabetic osteoarthritis.

Diabetes mellitus is involved in inflammation, immunity, and metabolism during osteoarthritis (OA). It destroys the normal synthesis and degradation balance of chondrocytes (CHs) and extracellular matrix (ECM). The purpose of this study was to explore the possible way of SIRT2 influencing the progress of diabetic OA. Proteins of diabetic OA and normal OA cartilage samples were extracted from patients undergoing knee joint operation. CHs were also isolated from the cartilage exempted from diabetes for cell culture. Glucose was used to treat CHs for imitating the microenvironment of diabetes. The expressions of SIRT2, acetylated H3K9, H3K14, and H3K56 protein were determined by Western blotting. SIRT2, 8-hydroxy-2' deoxyguanosine (8-OH), and MMP-13 expressions were analyzed using immunofluorescence. RT-PCR was performed to measure the mRNA levels of SOD1, SOD2, CAT, MMP-13, ADAMTS-4, and ADAMTS-5. Total ROS level was performed by flow cytometry assay. SIRT2 expression was reduced, whereas acetylated H3K9, H3K14, and H3K56 were upregulated in diabetic cartilage compared to normal. High glucose suppressed the expression of SIRT2 but accelerated the acetylation of H3K9, H3K14, and H3K56. Besides, high glucose promoted the expression of 8-OH, and inhibited SOD1, SOD2, and CAT mRNA expressions, resulting in the up-regulated ROS level of CHs. In addition, high glucose activated the inflammatory response by upregulation of MMP-13, ADAMTS-4, and ADAMTS-5 expressions. SirReal2 suppressed SIRT2 and resulted in several acetylations of H3, more ROS, less antioxidant enzymes, and stronger inflammatory response caused by high glucose. However, supplied rh-SIRT2 reversed these negative effects of high glucose in CHs. SIRT2 expression is reduced along with the diabetic OA process with increased acetylation of H3, oxidative stress, and inflammatory response. Suppression of SIRT2 accelerates the progress of diabetic OA and upregulation of SIRT2 alleviates diabetic OA development by suppressing oxidative stress and inflammatory response that are likely to be related to the deacetylation of H3.