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Background In osteoarthritis (OA), cartilage matrix is lost despite vigorous chondrocyte anabolism. In this study, we attempted to determine whether altered matrix synthesis is involved in this paradox in disease progression through gene expression analysis and ultrastructural analysis of collagen fibrils within the cartilage matrix. Methods Cartilage tissues were obtained from 29 end-stage OA knees and 11 control knees. First, cDNA microarray analysis was performed and the expression of 9 genes involved in collagen fibrillogenesis was compared between OA and control cartilages. Then their expression was investigated in further detail by a quantitative polymerase chain reaction (qPCR) analysis combined with laser capture microdissection. Finally, collagen fibril formation was compared between OA and control cartilage by transmission electron microscopy. Results The result of the microarray analysis suggested that the expression of type IX and type XI collagens and fibrillogenesis-related small leucine-rich proteoglycans (SLRPs) may be reduced in OA cartilage relative to the type II collagen expression. The qPCR analysis confirmed these results and further indicated that the relative reduction in the minor collagen and SLRP expression may be more obvious in degenerated areas of OA cartilage. An ultrastructural analysis suggested that thicker collagen fibrils may be formed by OA chondrocytes possibly through reduction in the minor collagen and SLRP expression. Conclusions This may be the first study to report the possibility of altered collagen fibrillogenesis in OA cartilage. Disturbance in collagen fibril formation may be a previously unidentified mechanism underlying the loss of cartilage matrix in OA. Electronic supplementary material The online version of this article (10.1186/s12891-019-2596-y) contains supplementary material, which is available to authorized users.

Background: In osteoarthritis (OA), cartilage matrix is lost gradually despite enhanced matrix synthesis by chondrocytes. This paradox may be explained, at least partly, by reduced chondrocyte anabolism in degenerated area of OA cartilage. However, to date, it is not known why chondrocyte anabolism is suppressed in those areas.Methods: Cartilage was obtained from control knees and end-stage OA knees in macroscopically preserved areas and degenerated areas, and gene expression was analyzed in respective regions of cartilage using laser capture microdissection and qPCR. For the cartilage protein analysis, cartilage was obtained from preserved areas and degenerated areas of OA knees in pairs, and proteins were extracted using urea buffer. Protein concentrations were determined by Luminex and compared between the areas. Cartilage explants prepared from preserved areas and degenerated areas of OA knees were cultured in the presence or absence of an AKT inhibitor, and the gene expression was evaluated by qPCR. Finally, the expression of SP1 was evaluated in OA and control cartilage, and the significance of Sp1 on the expression of IGF1R and IRS1 was investigated in experiments using primary cultured chondrocytes.Results: Within OA cartilage, the expression of IGF-1, IGF-2, IGF1R and IRS1 was reduced in degenerated areas compared to preserved areas, while the expression of all six IGF-binding protein genes examined was enhanced in the former areas. Consistent results were obtained by a protein analysis. In explant culture, the inhibition of AKT signaling abrogated the abundant matrix gene expression in the preserved areas over the degenerated areas, indicating that suppressed matrix synthesis in degenerated areas may be ascribed, at least partly, to attenuated IGF signaling. Within OA cartilage, the expression of Sp1 was considerably reduced in severely degenerated areas compared to preserved areas, which correlated well with the expression of IGF1R and IRS1. In experiments using primary cultured chondrocytes, the expression of IGF1R and IRS1 was enhanced by the induction of Sp1 expression and reduced by the suppression of Sp1 expression. Conclusions: The results of this study suggest that attenuated IGF signaling may be responsible, at least partly, for the reduced matrix synthesis in degenerated areas of OA cartilage.

Background In osteoarthritis (OA), cartilage matrix is lost gradually despite enhanced matrix synthesis by chondrocytes. This paradox may be explained, at least partly, by reduced chondrocyte anabolism in degenerated area of OA cartilage. However, to date, it is not known why chondrocyte anabolism is suppressed in those areas. Methods Cartilage was obtained from control knees and end-stage OA knees in macroscopically preserved areas and degenerated areas, and gene expression was analyzed in respective regions of cartilage using laser capture microdissection and qPCR. For the cartilage protein analysis, cartilage was obtained from preserved areas and degenerated areas of OA knees in pairs, and proteins were extracted using urea buffer. Protein concentrations were determined by Luminex and compared between the areas. Cartilage explants prepared from preserved areas and degenerated areas of OA knees were cultured in the presence or absence of an AKT inhibitor, and the gene expression was evaluated by qPCR. Finally, the expression of SP1 was evaluated in OA and control cartilage, and the significance of Sp1 on the expression of IGF1R and IRS1 was investigated in experiments using primary cultured chondrocytes. Results Within OA cartilage, the expression of IGF-1, IGF-2, IGF1R and IRS1 was reduced in degenerated areas compared to preserved areas, while the expression of all six IGF-binding protein genes examined was enhanced in the former areas. Consistent results were obtained by a protein analysis. In explant culture, the inhibition of AKT signaling abrogated the abundant matrix gene expression in the preserved areas over the degenerated areas, indicating that suppressed matrix synthesis in degenerated areas may be ascribed, at least partly, to attenuated IGF signaling. Within OA cartilage, the expression of Sp1 was considerably reduced in severely degenerated areas compared to preserved areas, which correlated well with the expression of IGF1R and IRS1. In experiments using primary cultured chondrocytes, the expression of IGF1R and IRS1 was enhanced by the induction of Sp1 expression and reduced by the suppression of Sp1 expression. Conclusions The results of this study suggest that attenuated IGF signaling may be responsible, at least partly, for the reduced matrix synthesis in degenerated areas of OA cartilage.

Figure S1. For the cDNA microarray analysis, cartilage tissues were obtained from 10 OA knees and 9 control knees at the lateral femoral condyles in full thickness above the tidemark, in squares of approximately 10 mm per side. In OA knees, tissues were obtained from areas where no overt sings of cartilage degeneration were observed. Considering that the chondrocyte metabolism differs significantly among cartilage zones, the cartilage tissues were separated into three cartilage zones, and RNA was obtained from each of the zones as previously described (Fukui N, et al. Arthritis Rheum 2008;58:3843-53). Using extracted RNA, the gene expression profiles were determined in respective cartilage zones using the Human Gene Expression Microarray (G4112F; Agilent Technologies, Santa Clara, CA, USA), which carries probes for 19,596 human genes. RNA was used for the analysis after confirming that the A260 nm to A280 nm ration was â ¤ 1.8, and that the 28S to 18S ration was â Ľ 1.4 by an analysis using a Bioanalyzer 2100 with RNA 6000 Nano Chips (Agilent Technologies). All RNA samples were analyzed respectively, yielding 27 sets of microarray data. Using these data, the expression levels of nine cartilage procollagen genes were compared between OA (OA) and control cartilage (Control) in respective cartilage zones, and statistical significance was determined by two-tailed t-test using R (version 3.3.1 for Windows). The level of statistical significance was set at p < 0.05. In each graph, open, shaded and solid bars indicate the expression levels in the superficial, middle and deep cartilage zones, respectively. Bars indicate the mean + SD of the signal intensities of 9 (control) or 10 (OA) cartilage samples determined by the microarray analysis. **, p < 0.01 versus corresponding zone in control cartilage.. (PDF 348 kb)

Figure S2. Comparison of the expression levels of three SLRP genes in OA (OA) and control cartilage (Control) determined by the cDNA microarray analysis. The expression was compared in respective cartilage zones as described in Additional file 1: Table S1. In each graph, open, shaded and solid bars indicate the findings for the superficial, middle and deep cartilage zones, respectively. Bars indicate the mean + SD of the signal intensities of 9 (control) or 10 (OA) cartilage samples determined by the microarray analysis. *, p < 0.05 and **, p < 0.01 versus corresponding zone in control cartilage. (PDF 341 kb)

Table S1. Supplementary Table. (DOCX 22 kb)

Our aim was to explore important inflammatory mediators for synovial chondromatosis in the temporomandibular joints (TMJs) by analysing synovial fluid. Samples were collected from 10 patients with unilateral synovial chondromatosis of the TMJ. Control samples were obtained from 11 subjects with no symptoms in the TMJ. Concentrations of aggrecan, interleukin (IL)-2, IL-4, IL-5, IL-6, IL-8 (CXCL8), IL-10, interferon (IFN)-γ, tumour necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF)-A were measured in the samples of synovial fluid, and the results in the two groups compared. The tissues from the affected TMJ were examined histologically and immunohistochemically. Of the proteins evaluated, the concentrations of aggrecan, IL-6, and VEGF-A were significantly higher in the group with synovial chondromatosis. The immunohistochemical analysis showed that the synovial cells around the osteocartilaginous nodules were vigorously expressing VEGF-A. IL-6 and VEGF-A are thought to have important roles in the pathology of synovial chondromatosis of the TMJ.

Objective When cultured in monolayers, articular chondrocytes undergo an obvious phenotypic change. Although the involvement of integrins has been suggested, the exact mechanisms of the change have not been determined. This study was undertaken to clarify the mechanisms underlying the loss of chondrocyte phenotype early after plating. Methods Primary cultured human articular chondrocytes were used for the experiments. Involvement of respective integrins in the phenotypic change was investigated in RNA interference (RNAi) experiments. A signaling pathway involved in the change was identified in experiments using specific inhibitors and adenoviruses encoding mutated genes involved in the pathway. Adenoviruses carrying mutated GTPases were used to determine the involvement of small GTPases in the process. Results In monolayer-cultured chondrocytes, suppression of αv or β5 integrin expression by RNAi inhibited morphologic changes in the cells and increased (or prevented a reduction in) the expression of various cartilage matrix genes. Consistent results were obtained in experiments using a blocking antibody and a synthetic inhibitor of αvβ5 integrin. The decrease in cartilage matrix gene expression in chondrocytes after plating was mediated by ERK signaling, which was promoted primarily by αvβ5 integrin. In articular chondrocytes, the affinity of αvβ5 integrin for ligands was regulated by the small GTPase R-Ras. R-Ras was gradually activated in monolayer-cultured chondrocytes after plating, which caused a gradual decline in cartilage matrix gene expression through enhanced αvβ5 integrin activation and the subsequent increase in ERK signaling. Conclusion Our findings indicate that αvβ5 integrin may be involved in the change that occurs in monolayer-cultured chondrocytes after plating.

BackgroundOsteoarthritis (OA) is a common joint disorder with increasing impact in an aging society; however, there is no cure or effective treatments so far due to lack of sufficient understanding of its pathogenesis. While genome-wide association studies (GWAS) and DNA methylation profiling identified many non-coding loci associated to OA, the interpretation of them remains challenging.MethodsHere, we employed Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-seq) to map the accessible chromatin landscape in articular knee cartilage of OA patients and to identify the chromatin signatures relevant to OA.ResultsWe identified 109,215 accessible chromatin regions in cartilage and 71% of these regions were annotated as enhancers. We found these accessible chromatin regions are enriched for OA GWAS single nucleotide polymorphisms (SNPs) and OA differentially methylated loci, implying their relevance to OA. By linking these enhancers to their potential target genes, we have identified a list of candidate enhancers that may be relevant to OA. Through integration of ATAC-seq data with RNA-seq data, we identified genes that are altered both at epigenomic and transcriptomic levels. These genes are enriched in pathways regulating ossification and mesenchymal stem cell (MSC) differentiation. Consistently, the differentially accessible regions in OA are enriched for mesenchymal stem cell-specific enhancers and motifs of transcription factor families involved in osteoblast differentiation (e.g. bZIP and ETS).ConclusionsThis study marks the first investigation of accessible chromatin landscape on clinically relevant hard tissues and demonstrates how accessible chromatin profiling can provide comprehensive epigenetic information of a disease. Our analyses provide supportive evidence towards the model of endochondral ossification-like cartilage-to-bone conversion in OA knee cartilage, which is consistent with the OA characteristic of thicker subchondral bone. The identified OA-relevant genes and their enhancers may have a translational potential for diagnosis or drug targets.

Objective To determine the change in metabolic activity of chondrocytes in osteoarthritic (OA) cartilage, considering regional difference and degree of cartilage degeneration. Methods OA cartilage was obtained from knee joints with end-stage OA, at both macroscopically intact areas and areas with various degrees of cartilage degeneration. Control cartilage was obtained from age-matched donors. Using laser capture microdissection, cartilage samples were separated into superficial, middle, and deep zones, and gene expression was compared quantitatively in the respective zones between OA and control cartilage. Results In OA cartilage, gene expression changed markedly with the site. The expression of cartilage matrix genes was highly enhanced in macroscopically intact areas, but the enhancement was less obvious in the degenerated areas, especially in the upper regions. In contrast, in those regions, the expression of type III collagen and fibronectin was most enhanced, suggesting that chondrocytes underwent a phenotypic change there. Within OA cartilage, the expression of cartilage matrix genes was significantly correlated with SOX9 expression, but not with SOX5 or SOX6 expression. In OA cartilage, the strongest correlation was observed between the expression of type III collagen and fibronectin, suggesting the presence of a certain link(s) between their expression. Conclusion The results of this study revealed a comprehensive view of the metabolic change of the chondrocytes in OA cartilage. The change of gene expression profile was most obvious in the upper region of the degenerated cartilage. The altered gene expression at that region may be responsible for the loss of cartilage matrix associated with OA.

Ingestion of sodium bicarbonate (NaHCO3) is known to enhance athletic performance, probably via increased extracellular buffering capacity. At present, little is known about the direct effects of NaHCO3 on myogenesis, especially in vitro. Here, we examined the effects of NaHCO3 and the combined effects of NaHCO3 and continuous mild heat stress (CMHS) at 39°C on the differentiation of human skeletal muscle myoblasts (HSMMs). Levels of myosin heavy chain (MyHC) type I mRNA increased with increasing NaHCO3 concentrations; in contrast, those of MyHC IIx decreased. The NaHCO3-induced fast-to-slow shift was additively enhanced by CMHS. Likewise, intracellular calcium levels and expression of three factors, nuclear factor of activated T cells c2 (NFATc2), NFATc4, and peroxisome-proliferator-activated receptor-γ coactivator-1α, were upregulated with increasing NaHCO3 concentrations; moreover, these effects of NaHCO3 were additively enhanced by CMHS. Overexpression experiments and small interfering RNA-mediated knockdown experiments confirmed that NFATc2 and NFATc4 were involved in MyHC I regulation. The present study provided evidence that NaHCO3 and CMHS distinctly and additively induced a fast-to-slow fiber type shift through changes in intracellular calcium levels and the modulation of calcium signaling.

The integrity of articular cartilage is maintained by chondrocytes, the sole type of cell that resides within the tissue. The noncalcified region of articular cartilage can be divided into three zones based on histological features, in which the chondrocyte metabolism is known to differ obviously among the zones. In pathological cartilage, the chondrocyte metabolism may change dramatically, which could play a pivotal role in the progression of the disease. Since such change in metabolism differs obviously from site to site within cartilage, it is crucial to determine the chondrocyte metabolism in respective regions. To this end, we have employed laser-capture microdissection (LCM) to analyze chondrocyte metabolism in various regions of pathological and control cartilage. In this report, we describe our protocol for LCM on adult human cartilage tissue. With this protocol, a specific site of cartilage tissue was successfully obtained by LCM for gene expression analysis.

The integrity of articular cartilage is maintained by chondrocytes, the sole type of cell that resides within the tissue. The noncalcified region of articular cartilage can be divided into three zones based on histological features, in which the chondrocyte metabolism is known to differ obviously among the zones. In pathological cartilage, the chondrocyte metabolism may change dramatically, which could play a pivotal role in the progression of the disease. Since such change in metabolism differs obviously from site to site within cartilage, it is crucial to determine the chondrocyte metabolism in respective regions. To this end, we have employed laser-capture microdissection (LCM) to analyze chondrocyte metabolism in various regions of pathological and control cartilage. In this report, we describe our protocol for LCM on adult human cartilage tissue. With this protocol, a specific site of cartilage tissue was successfully obtained by LCM for gene expression analysis.

Objective To determine the chondrocyte metabolism in respective zones of osteoarthritic (OA) cartilage. Methods OA cartilage was obtained from macroscopically intact areas of 4 knee joints with end-stage OA. The cartilage was divided into 3 zones, and gene expression profiles were determined in the respective zones by a custom-designed microarray that focused on chondrocyte-related genes. For the genes whose expression was significantly different among the zones, the expression was compared between OA and control cartilage in the respective zones by an analysis using laser capture microdissection and real-time polymerase chain reaction (PCR). For some genes, the correlation of expression was investigated in specific cartilage zones. Results A total of 198 genes (∼40% of those investigated) were found to be expressed at significantly different levels among the zones. Expression of 26 of those genes was evaluated by laser capture microdissection and real-time PCR, which confirmed the validity of microarray analysis. The expression of cartilage matrix genes was mostly enhanced in OA cartilage, at similar levels across the zones but at different magnitudes among the genes. The expression of bone-related genes was induced either in the superficial zone or in the deep zone, and positive correlations were found among their expression in the respective zones. The expression of 5 proteinase genes was most enhanced in the superficial zone, where their expression was correlated, suggesting the presence of a common regulatory mechanism(s) for their expression. Conclusion In OA cartilage, the metabolic activity of chondrocytes differed considerably among zones. Characteristic changes were observed in the superficial and deep zones.

Introduction Articular chondrocytes undergo an obvious phenotypic change when cultured in monolayers. During this change, or dedifferentiation, the expression of type I and type III procollagen is induced where normal chondrocytes express little type I and type III procollagen. In this study, we attempted to determine the mechanism(s) for the induction of such procollagen expression in dedifferentiating chondrocytes. Methods All experiments were performed using primary-cultured human articular chondrocytes under approval of institutional review boards. Integrin(s) responsible for the induction of type I and type III procollagen expression were specified by RNAi experiments. The signal pathway(s) involved in the induction were determined by specific inhibitors and RNAi experiments. Adenovirus-mediated experiments were performed to identify a small GTPase regulating the activity of integrins in dedifferentiating chondrocytes. The effect of inhibition of integrins on dedifferentiation was investigated by experiments using echistatin, a potent disintegrin. The effect of echistatin was investigated first with monolayer-cultured chondrocytes, and then with pellet-cultured chondrocytes. Results In dedifferentiating chondrocytes, α5β1 integrin was found to be involved in the induction of type I and type III procollagen expression. The induction was known to be mediated by v-akt murine thymoma viral oncogene homolog (AKT) signaling. Among the three AKT isoforms, AKT1 seemed to be most involved in the signaling. Elated RAS viral (r-ras) oncogene homolog (RRAS) was considered to regulate the progression of dedifferentiation by modulating the affinity and avidity of α5β1 integrin to ligands. Echistatin inhibited dedifferentiation of monolayer-cultured chondrocytes. Furthermore, the matrix formed by pellet-cultured chondrocytes more closely resembled that of normal cartilage compared with the controls. Conclusions The result of this study has shown, for the first time, that α5β1 integrin may be responsible for the induction of non-cartilaginous collagen expression in chondrocytes undergoing dedifferentiation. Again, this study has shown that the inhibition of ligand ligation to integrins may be an effective strategy to inhibit phenotypic change of cultured chondrocytes, and to improve the quality of matrix synthesized by primary cultured chondrocytes.

Background Although osteoarthritis (OA) of the knee joints is the most common and debilitating joint disease in developed countries, the factors that determine the severity of symptoms are not yet understood well. Subjects with symptomatic medial knee OA were followed up prospectively to explore the relationship between radiographic changes and symptoms or physical examination findings. Methods One-hundred six OA knees in 68 subjects (mean age 71.1 years; 85% women) were followed up at 6-month intervals over 36 months. At each visit, knee radiographs were obtained, symptoms were assessed by a validated questionnaire, and the result of physical examination was recorded systematically using a specific chart. Correlations between the change of radiographs and clinical data were investigated in a longitudinal manner. Results During the study period, the narrowing of joint space width (JSW) was observed in 34 joints (32%). Although those knees were clinically or radiographically indistinguishable at baseline from those without JSW narrowing, differences became apparent at later visits during the follow-up. The subjects with knees that underwent JSW narrowing had severer symptoms, and the symptoms tended to be worse for those with higher rates of narrowing. A significant correlation was not found between the severity of symptoms and the growth of osteophytes. For the knees that did not undergo radiographic progression, the range of motion improved during the follow-up period, possibly due to the reduction of knee pain. Such improvement was not observed with the knees that underwent JSW narrowing or osteophyte growth. Conclusion The result of this study indicates that the symptoms of knee OA patients tend to be worse when JSW narrowing is underway. This finding may explain, at least partly, a known dissociation between the radiographic stage of OA and the severity of symptoms.