OBJECTIVE: To detect the changes of mitochondrion DNA (mtDNA) sequence in articular chondrocytes of cartilage affected by osteoarthritis and to clarified the pathogenetic mechanism of osteoarthritis. METHODS: We analyzed the mtDNA 4,977 bp deletion mutations of articular chondrocytes in 10 patients with osteoarthritis and 3 normal cartilages using the gap-PCR amplification method. We designed a two round PCR detection method, in which total DNA was isolated from articular chondrocytes as the template of the first round PCR reaction and products from the first round were the template in the second round reaction. RESULTS: The results of the first rounds of PCR reaction showed the mtDNA 524 bp amplified products in the osteoarthritis group and in the corresponding peripheral blood samples were not detected, but the 533 bp products were detected. However,the results of the second round reaction revealed that the 524 bp zones were detected in 2 of the 10 osteoarthritis patients and the corresponding peripheral blood samples were not detected. The 533 bp products were detected in all specimens. The mtDNA 524 bp amplified products in all the normal articular chondrocytes and the corresponding peripheral white blood cells contrast were not detected in both rounds PCR. CONCLUSION This was the first study to evaluate the mtDNA 4799 bp large fragment deletion mutational accumulation between nt8,470 - nt13,447 of articular chondrocytes in osteoarthritic cartilage. Osteoarthritis may be related to mtDNA mutation of articular chondrocytes.
Adult ; Cartilage, Articular ; metabolism ; pathology ; Chondrocytes ; metabolism ; DNA, Mitochondrial ; genetics ; Female ; Gene Deletion ; Humans ; Male ; Middle Aged ; Osteoarthritis ; genetics ; Osteoarthritis, Hip ; genetics ; Osteoarthritis, Knee ; genetics ; Sequence Analysis, DNA

OBJECTIVETo investigate the gene expression and potential functions of transforming growth factor-beta1 in osteophyte development.

METHODSA total of 25 specimens were obtained from individuals undergoing total knee arthroplasty due to severe primary osteoarthritis. Tissue samples were embedded in paraffin wax and made into sections. Hematoxylin and eosin and toluidine blue stainings were performed. The expressions of collagen I, IIa, IIb, and X were detected by immunohistochemistry. Based on the histomorphology of cellularity and matrix abundance, the glycosaminoglycans content, and the differential expressions of collagen I, IIa, IIb, and X, the osteophytic tissues were classified. For each different type of osteophyte, expressions of transforming growth factor-beta1 were detected by immunohistochemistry and in situ hybridization, and results were analyzed using the image analysis system.

RESULTSFive different types of osteophytes were identified as type I, type II, type III, type IV, and type V. Transforming growth factor-beta1 mRNA was more and intensely expressed in chondrocytes of type II and III osteophytes, and was less in other types of osteophytes. The difference was significant (P<0.05, P<0.01).

CONCLUSIONTransforming growth factor-beta1 mRNA is mainly expressed in early-mid stages of osteophytes and may play an important role in promoting the proliferation and differentiation of chondrocytes in the early stages of osteophyte development.

Chondrocytes ; metabolism ; pathology ; Humans ; Osteoarthritis, Knee ; metabolism ; pathology ; Osteophyte ; metabolism ; pathology ; RNA, Messenger ; biosynthesis ; Transforming Growth Factor beta1 ; biosynthesis ; genetics

Osteoarthritis is a common cause of functional deterioration in older adults and is an immense burden on the aging population. Altered chondrogenesis is the most important pathophysiological process involved in the development of osteoarthritis. However, the molecular mechanism underlying the regulation of chondrogenesis in patients with osteoarthritis requires further elucidation, particularly with respect to the role of microRNAs. MiR-21 expression in cartilage specimens was examined in 10 patients with knee osteoarthritis and 10 traumatic amputees. The effect of miR-21 on chondrogenesis was also investigated in a chondrocyte cell line. The effect of miR-21 on the expression of growth differentiation factor 5 (GDF-5) was further assessed by luciferase reporter assay and western blot. We found that endogenous miR-21 is upregulated in osteoarthritis patients, and overexpression of miR-21 could attenuate the process of chondrogenesis. Furthermore, we identified GDF-5 as the direct target of miR-21 during the regulation of chondrogenesis. Our data suggest that miR-21 has an important role in the pathogenesis of osteoarthritis and is a potential therapeutic target.
Cartilage/metabolism/pathology ; Case-Control Studies ; Cell Line ; Chondrocytes/*metabolism/pathology ; Growth Differentiation Factor 5/genetics/*metabolism ; Humans ; MicroRNAs/genetics/*metabolism ; Osteoarthritis/*metabolism/pathology ; Up-Regulation

OBJECTIVETo study the characteristics of cellular metabolism of mandibular condylar chondrocytes in repairing state of osteoarthrosis and investigate its role in the pathogenesis of the disease.

METHODSTemporomandibular joint osteoarthrosis model of rabbits was created by the partial resection of joint disc and confirmed with histological diagnosis. The chondrocytes were harvested from osteoarthritic condylar cartilage in the repairing state and cultured in vitro under the monolayer culture condition. The cellular expression of cartilaginous matrix protein, collagenase and growth factors between the osteoarthritic chondrocytes and the normal controls were measured with RT-PCR technique to outline the basic feature of the osteoarthritic cells.

RESULTSThe cultured cells were confirmed as chondrocytes with their ability of expression of collagen type II and Aggrecan. In the reactive repairing state of osteoarthrosis, the chondrocytes showed the imbalance of expression of ECM proteins, and increased expression of collagenase and endogenous growth factors such as IGF-1 and TGF-beta1.

CONCLUSIONSThis study found the active anabolism of the chondrocytes within the osteoarthritic condylar cartilage and the imbalance synthesis of cartilage matrix. These repairing attempts by the osteoarthritic chondrocytes may be impossible to restore the primary homeostasis within the condylar cartilage.

Animals ; Cartilage, Articular ; metabolism ; pathology ; Cells, Cultured ; Chondrocytes ; metabolism ; Extracellular Matrix ; genetics ; metabolism ; Male ; Mandibular Condyle ; metabolism ; pathology ; Osteoarthritis ; metabolism ; pathology ; RNA, Messenger ; genetics ; metabolism ; Rabbits ; Temporomandibular Joint Disc ; pathology ; Temporomandibular Joint Disorders ; metabolism ; pathology

Articular cartilage is a connective tissue consisting of a specialized extracellular matrix (ECM) that dominates the bulk of its wet and dry weight. Type II collagen and aggrecan are the main ECM proteins in cartilage. However, little attention has been paid to less abundant molecular components, especially minor collagens, including type IV, VI, IX, X, XI, XII, XIII, and XIV, etc. Although accounting for only a small fraction of the mature matrix, these minor collagens not only play essential structural roles in the mechanical properties, organization, and shape of articular cartilage, but also fulfil specific biological functions. Genetic studies of these minor collagens have revealed that they are associated with multiple connective tissue diseases, especially degenerative joint disease. The progressive destruction of cartilage involves the degradation of matrix constituents including these minor collagens. The generation and release of fragmented molecules could generate novel biochemical markers with the capacity to monitor disease progression, facilitate drug development and add to the existing toolbox for in vitro studies, preclinical research and clinical trials.
Aggrecans ; chemistry ; genetics ; metabolism ; Animals ; Biomarkers ; metabolism ; Cartilage, Articular ; chemistry ; metabolism ; pathology ; Collagen ; chemistry ; classification ; genetics ; metabolism ; Extracellular Matrix Proteins ; chemistry ; genetics ; metabolism ; Gene Expression ; Humans ; Osteoarthritis ; diagnosis ; genetics ; metabolism ; pathology ; Protein Isoforms ; chemistry ; classification ; genetics ; metabolism

Animals ; Estrogens ; metabolism ; Gene Expression Regulation ; Humans ; Osteoarthritis ; complications ; genetics ; metabolism ; Pain ; etiology ; Pain Measurement ; Temporomandibular Joint ; pathology ; Temporomandibular Joint Disorders ; complications ; genetics ; metabolism ; Wnt Signaling Pathway

BACKGROUNDThere is a difficulty in evaluating the in vivo functionality of individual chondrocytes, and there is much heterogeneity among cartilage affected by osteoarthritis (OA). In this study, in vitro cultured chondrocytes harvested from varying stages of degeneration were studied as a projective model to further understand the pathogenesis of osteoarthritis.

METHODSCartilage of varying degeneration of end-stage OA was harvested, while cell yield and matrix glycosaminoglycan (GAG) content were measured. Cell morphology, proliferation, and gene expression of collagen type I, II, and X, aggrecan, matrix metalloproteinase 13 (MMP-13), and ADAMTS5 of the acquired chondrocytes were measured during subsequent in vitro culture.

RESULTSBoth the number of cells and the GAG content increased with increasing severity of OA. Cell spreading area increased and gradually showed spindle-like morphology during in vitro culture. Gene expression of collagen type II, collagen type X as well as GAG decreased with severity of cartilage degeneration, while expression of collagen type I increased. Expression of MMP-13 increased with severity of cartilage degeneration, while expression of ADAMTS-5 remained stable. Expression of collagen type II, X, GAG, and MMP-13 substantially decreased with in vitro culture. Expression of collagen type I increased with in vitro cultures, while expression of ADAMTS 5 remained stable.

CONCLUSIONSExpression of functional genes such as collagen type II and GAG decreased during severe degeneration of OA cartilage and in vitro dedifferentiation. Gene expression of collagen I and MMP-13 increased with severity of cartilage degeneration.

ADAM Proteins ; ADAMTS5 Protein ; Cartilage ; pathology ; Cell Differentiation ; genetics ; physiology ; Cells, Cultured ; Chondrocytes ; metabolism ; Collagen Type II ; genetics ; Collagen Type X ; genetics ; Glycosaminoglycans ; metabolism ; Humans ; Matrix Metalloproteinase 13 ; genetics ; Osteoarthritis ; genetics ; pathology

To investigate the mechanism by which Cangxi Tongbi Capsules promote chondrocyte autophagy to inhibit knee osteoarthritis(KOA) progression by regulating the circRNA＿0008365/miR-1271/p38 mitogen-activated protein kinase(MAPK) pathway. The cell and animal models of KOA were established and intervened with Cangxi Tongbi Capsules, si-circRNA＿0008365, si-NC, and Cangxi Tongbi Capsules combined with si-circRNA＿0008365. Flow cytometry and transmission electron microscopy were employed to determine the level of apoptosis and observe autophagosomes, respectively. Western blot was employed to reveal the changes in the protein levels of microtubule-associated protein light chain 3(LC3)Ⅱ/Ⅰ, Beclin-1, selective autophagy junction protein p62/sequestosome 1, collagen Ⅱ, a disintegrin and metalloproteinase with thrombospondin motifs 5(ADAMTS-5), and p38 MAPK. The mRNA levels of circRNA＿0008365, miR-1271, collagen Ⅱ, and ADAMTS-5 were determined by qRT-PCR. Hematoxylin-eosin staining was employed to reveal the pathological changes of the cartilage tissue of the knee, and enzyme-linked immunosorbent assay to measure the levels of interleukin-1β(IL-1β) and tumor necrosis factor-alpha(TNF-α). The chondrocytes treated with IL-1β showed down-regulated expression of circRNA＿0008365, up-regulated expression of miR-1271 and p38 MAPK, lowered autophagy level, increased apoptosis rate, and accelerated catabolism of extracellular matrix. The intervention with Cangxi Tongbi Capsules up-regulated the expression of circRNA＿0008365, down-regulated the expression of miR-1271 and p38 MAPK, increased the autophagy level, decreased the apoptosis rate, and weakened the catabolism of extracellular matrix. However, the effect of Cangxi Tongbi Capsules was suppressed after interfering with circRNA＿0008365. The in vivo experiments showed that Cangxi Tongbi Capsules dose-dependently inhibited the p38 MAPK pathway, enhanced chondrocyte autophagy, and mitigated articular cartilage damage and inflammatory response, thereby inhibiting the progression of KOA in rats. This study indicated that Cangxi Tongbi Capsules promoted chondrocyte autophagy by regulating the circRNA＿0008365/miR-1271/p38 MAPK pathway to inhibit the development of KOA.
Rats ; Animals ; Chondrocytes ; Osteoarthritis, Knee/pathology* ; RNA, Circular/pharmacology* ; p38 Mitogen-Activated Protein Kinases/metabolism* ; MicroRNAs/metabolism* ; Apoptosis ; Autophagy/genetics* ; Collagen/metabolism*

IL-6 is an inflammatory cytokine and its overexpression plays an important role in osteoarthritis (OA) pathogenesis. Expression of IL-6 is regulated post-transcriptionally by MCPIP1. The 3' untranslated region (UTR) of MCPIP1 mRNA harbors a miR-139 'seed sequence', therefore we examined the post-transcriptional regulation of MCPIP1 by miR-139 and its impact on IL-6 expression in OA chondrocytes. Expression of miR-139 was found to be high in the damaged portion of the OA cartilage compared with unaffected cartilage from the same patient and was also induced by IL-1beta in OA chondrocytes. Inhibition of miR-139 decreased the expression of IL-6 mRNA by 38% and of secreted IL-6 protein by 40%. However, overexpression of miR-139 increased the expression of IL-6 mRNA by 36% and of secreted IL-6 protein by 56%. These data correlated with altered expression profile of MCPIP1 in transfected chondrocytes. Studies with a luciferase reporter construct confirmed the interactions of miR-139 with the 'seed sequence' located in the 3' UTR of MCPIP mRNA. Furthermore, miR-139 overexpression increased the catabolic gene expression but expression of anabolic markers remained unchanged. Overexpression of miR-139 also induced apoptosis in OA chondrocytes. Importantly, we also discovered that IL-6 is a potent inducer of miR-139 expression in OA chondrocytes. These findings indicate that miR-139 functions as a post-transcriptional regulator of MCPIP1 expression and enhances IL-6 expression, which further upregulates miR-139 expression in OA chondrocytes. These results support our hypothesis that miR-139-mediated downregulation of MCPIP1 promotes IL-6 expression in OA. Therefore, targeting miR-139 could be therapeutically beneficial in the management of OA.
3' Untranslated Regions ; Aged ; *Apoptosis ; Chondrocytes/*metabolism/pathology ; Down-Regulation ; Female ; Gene Expression Regulation ; Humans ; Interleukin-6/*genetics ; Male ; MicroRNAs/*genetics ; Middle Aged ; Osteoarthritis/*genetics/pathology ; RNA, Messenger/genetics ; Ribonucleases/*genetics ; Transcription Factors/*genetics ; Up-Regulation

Rheumatoid arthritis (RA) and osteoarthritis (OA), two common types of arthritis, affect the joints mainly by targeting the synovium and cartilage. Increasing evidence indicates that a significant network connects synovitis and cartilage destruction during the progression of arthritis. We recently demonstrated that hypoxia-inducible factor (HIF)-2alpha causes RA and OA by regulating the expression of catabolic factors in fibroblast-like synoviocytes (FLS) or chondrocytes. To address the reciprocal influences of HIF-2alpha on FLS and chondrocytes, we applied an in vitro co-culture system using a transwell apparatus. When co-cultured with HIF-2alpha-overexpressing chondrocytes, FLS exhibited increased expression of matrix metalloproteinases and inflammatory mediators, similar to the effects induced by tumor-necrosis factor (TNF)-alpha treatment of FLS. Moreover, chondrocytes co-cultured with HIF-2alpha-overexpressing FLS exhibited upregulation of Mmp3 and Mmp13, which is similar to the effects induced by interleukin (IL)-6 treatment of chondrocytes. We confirmed these differential HIF-2alpha-induced effects via distinct secretory mediators using Il6-knockout cells and a TNF-alpha-blocking antibody. The FLS-co-culture-induced gene expression changes in chondrocytes were significantly abrogated by IL-6 deficiency, whereas TNF-alpha neutralization blocked the alterations in gene expression associated with co-culture of FLS with chondrocytes. Our results further suggested that the observed changes might reflect the HIF-2alpha-induced upregulation of specific receptors for TNF-alpha (in FLS) and IL-6 (in chondrocytes). This study broadens our understanding of the possible regulatory mechanisms underlying the crosstalk between the synovium and cartilage in the presence of HIF-2alpha, and may suggest potential new anti-arthritis therapies.
Animals ; Arthritis/genetics/*immunology/pathology ; Arthritis, Rheumatoid/genetics/immunology/pathology ; Basic Helix-Loop-Helix Transcription Factors/genetics/*immunology ; Cells, Cultured ; Chondrocytes/immunology/metabolism/*pathology ; Coculture Techniques ; Fibroblasts/immunology/metabolism/*pathology ; Gene Expression Regulation ; Interleukin-6/genetics/*immunology ; Male ; Mice ; Mice, Inbred C57BL ; Osteoarthritis/genetics/immunology/pathology ; Synovial Membrane/immunology/metabolism/*pathology ; Tumor Necrosis Factor-alpha/genetics/*immunology ; Up-Regulation