Experimental findings have suggested that the metabolic activities of articular cartilage can be influenced by mechanical stimuli. Our recent mathematical analysis predicted that cyclic compressive loading may create periods of intermittent negative hydrostatic pressure within the cartilage extracellular matrix. Therefore, we hypothesize that intermittent negative hydrostatic pressure, created in the cartilage extracellular matrix during dynamic compression, has a stimulative effect on the biosynthesis of chondrocytes. In order to test this hypothesis, the present study developed a custom designed negative pressure generator to subject a monolayer culture of chondrocytes to an intermittent negative pressure. It was found that the intermittent negative pressure produced a 40% increase in proteoglycan and a l7% increase in non-collagenous protein synthesis during the pressurization period(p (0.05). The collagenous protein synthesis was not affected by the intermittent negative pressure regimen used in this study. After the intermittent negative pressurization, the metabolic activities of the chondrocytes returned to normal(control level). The intermittent negative pressure also produced an increase in the mRNA signals for aggrecan. Therefore, we conclude that intermittent negative pressure may be one of the major mechanical stimulators of chondrocytes in articular cartilage during dynamic compression.
Aggrecans ; Cartilage ; Cartilage, Articular ; Chondrocytes* ; Collagen ; Extracellular Matrix ; Hydrostatic Pressure* ; Metabolism* ; Proteoglycans ; RNA, Messenger

The progressive destruction of condylar cartilage is a hallmark of the temporomandibular joint (TMJ) osteoarthritis (OA); however, its mechanism is incompletely understood. Here, we show that Kindlin-2, a key focal adhesion protein, is strongly detected in cells of mandibular condylar cartilage in mice. We find that genetic ablation of Kindlin-2 in aggrecan-expressing condylar chondrocytes induces multiple spontaneous osteoarthritic lesions, including progressive cartilage loss and deformation, surface fissures, and ectopic cartilage and bone formation in TMJ. Kindlin-2 loss significantly downregulates the expression of aggrecan, Col2a1 and Proteoglycan 4 (Prg4), all anabolic extracellular matrix proteins, and promotes catabolic metabolism in TMJ cartilage by inducing expression of Runx2 and Mmp13 in condylar chondrocytes. Kindlin-2 loss decreases TMJ chondrocyte proliferation in condylar cartilages. Furthermore, Kindlin-2 loss promotes the release of cytochrome c as well as caspase 3 activation, and accelerates chondrocyte apoptosis in vitro and TMJ. Collectively, these findings reveal a crucial role of Kindlin-2 in condylar chondrocytes to maintain TMJ homeostasis.
Aggrecans/metabolism* ; Animals ; Cartilage, Articular/metabolism* ; Chondrocytes/pathology* ; Cytoskeletal Proteins/metabolism* ; Mice ; Muscle Proteins/metabolism* ; Osteoarthritis/pathology* ; Temporomandibular Joint/pathology*

This paper aims to investigate the effects and mechanisms of adipose-derived stem cells-exosomes(ADSCs-exos) toge-ther with aucubin in protecting human-derived nucleus pulposus cells(NPCs) from inflammatory injury, senescence, and apoptosis. The tert-butyl hydroperoxide(TBHP)-induced NPCs were assigned into normal, model, aucubin, ADSCs-exos, and aucubin+ADSCs-exos groups. The cell viability was examined by cell counting kit-8(CCK-8), cell proliferation by EdU staining, cell senescence by senescence-associated-β-galactosidase(SA-β-Gal), and cell cycle and apoptosis by flow cytometry. Enzyme-linked immunosorbent assay was employed to examine the expression of interleukin-1β(IL-1β), IL-10, and tumor necrosis factor-α(TNF-α). Real-time fluorescence quantitative PCR and Western blot were employed to determine the mRNA and protein levels of aggregated proteoglycan(aggrecan), type Ⅱ collagen alpha 1(COL2A1), Toll-like receptor 4(TLR4), and nuclear factor-kappa B(NF-κB). The results showed that compared with the model group, the aucubin or ADSCs-exos group showed enhanced viability and proliferation of NPCs, decreased proportion of G_0/G_1 phase cells, increased proportion of S phase cells, reduced apoptosis and proportion of cells in senescence, lowered IL-1β and TNF-α levels, elevated IL-10 level, down-regulated mRNA and protein levels of TLR4 and NF-κB, and up-regulated mRNA and protein levels of aggrecan and COL2A1. Compared with the aucubin or ADSCs-exos group, the aucubin+ADSCs-exos combination further increased the viability and proliferation of NPCs, decreased the proportion of G_0/G_1 phase cells, increased the proportion of S phase cells, reduced the apoptosis and proportion of cells in senescence, lowered the IL-1β and TNF-α levels, elevated the IL-10 level, down-regulated the mRNA and protein levels of TLR4 and NF-κB, and up-regulated the mRNA and protein levels of aggrecan and COL2A1. In summary, both aucubin and ADSCs-exos could exert protective effects by inhibiting inflammatory responses, reducing apoptosis and senescence of NPCs, improving cell viability and proliferation as well as extracellular matrix synthesis, which may be associated with the inhibition of TLR4/NF-κB signaling pathway activation. The combination of both plays a synergistic role in the protective effects.
Humans ; NF-kappa B/metabolism* ; Interleukin-10 ; Nucleus Pulposus/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Aggrecans/metabolism* ; Toll-Like Receptor 4/metabolism* ; RNA, Messenger/metabolism*

OBJECTIVE: To explore and verify that transient receptor potential vanilloid 4(TRPV4) affects chondrocyte degeneration. METHODS: Neonatal SD rats were selected, primary chondrocytes were extracted, and identified by toluidine blue staining and alcian blue staining;an in vitro chondrocyte inflammation model was constructed by IL-1β, and TRPV4 inhibitor was used to treat chondrocytes under inflammatory conditions, and the chondrocytes were treated by RT-PCR method was used to detect matrix metallopeptidase 13(MMP-13), a disintegrin and metalloproteinase with thrombospondin 5, (ADAMTS-5)、nitric oxide synthase 2(NOS2)、Collagen, type II alpha 1(Col2α1)and aggrecan (Acan) mRNA in chondrocytes; primary chondrocytes were treated with different concentrations of TRPV4 overexpression plasmid, and the optimal overexpression dose was screened. The mRNA expressions of TRPV4, MMP-13, ADAMTS-5, NOS2, Col2α1 and Acan in chondrocytes under the optimal TRPV4 overexpression dose were detected. RESULTS: Toluidine blue staining and Alcian blue staining identified the extracted cells as primary chondrocytes;RT-PCR showed that TRPV4, MMP-13, ADAMTS-5, NOS2 mRNA in chondrocytes treated with TRPV4 inhibitor under inflammatory conditions. The expression of Col2α1 mRNA was significantly decreased (P<0.05), and the expression of Col2α1 mRNA was increased (P<0.05). Although there was no significant difference in the expression of Acan mRNA, the overall trend was also increasing. The expression of Col2α1 and Acan mRNA in chondrocytes was significantly decreased (P<0.05), and the expression of NOS2 mRNA was increased(P<0.05), but there was no significant difference in MMP-13 and ADAMTS-5 (P>0.05). CONCLUSION Inhibiting the expression of TRPV4 can down-regulate the expression of genes related to chondrocyte degeneration.
Animals ; Rats ; Aggrecans/metabolism* ; Cartilage, Articular ; Cells, Cultured ; Chondrocytes ; Interleukin-1beta/metabolism* ; Matrix Metalloproteinase 13/metabolism* ; Rats, Sprague-Dawley ; RNA, Messenger/metabolism* ; TRPV Cation Channels/metabolism*

OBJECTIVETo investigate the feasibility of chondrogenesis in vitro with bone marrow stromal cells (BMSCs) induced by the co-cultured chondrocytes.

METHODSThe BMSCs and chondrocytes were separated from pig and cultured. The supernatant of chondrocytes was used as the inducing solution for BMSCs from the 2nd generation. 7 days later, samples were taken and underwent immunohistochemistry and RT-PCR for detection of the expression of specific type II cartilage collagen, type II collagen and aggrecan mRNA. The cultured BMSCs and chondrocytes were mixed at a ratio of 8:2 (BMSC: cartilage cell) and were inoculated into a polyglycolic acid/polylactic acid (PGA/PLA) scaffold at the final concentration of 5.0 x 10(7)/ml. The cartilage cells and BMSCs were also inoculated separately at the same concentration as the positive and negative control. Pure cartilage cells at 20% of the above mentioned concentration (1.0 x 10(7)/ml) were used as the low concentration cartilage cell control group. Samples were collected 8 weeks later. General observations, wet weight, glycosaminoglycans (GAGs) determination and histological and immunohistochemistry examinations were performed.

RESULTSThe expression of type II collagen, type II collagen and aggrecan mRNA were positive in induced BMSCs. In the co-cultured group and the positive control group, pure mature cartilage was formed after 8 weeks of culture in vitro, and the size and shape of the scaffold were maintained. The newly formed cartilage in the two groups were almost the same in appearance and histological properties. The immunohistochemistry results indicated that the cartilage cells of the two groups all expressed ample cartilage-specific type II collagen. The average wet weight and GAG content in the co-cultured group reached more than 70% of those in positive control group. Only an extremely small amount of immature cartilage tissues was formed in local regions in pure BMSC group, and the scaffold was obviously shrunk and deformed. Although the wet weight of newly generated cartilage tissue in the low concentration cartilage cell group reached 30% of that in positive control group, the scaffold was obviously shrunken and deformed. Only regional and discontinuous cartilage tissues were formed, and the amount of newly formed cartilage was obviously less than that in the co-culture group and the positive control group.

CONCLUSIONSChondrocytes can provide a micro-environment for the formation of cartilage, and also effectively induce BMSC to differentiate into chondrocytes and form tissue-engineered cartilage in vitro.

Aggrecans ; metabolism ; Animals ; Cell Differentiation ; Cells, Cultured ; Chondrocytes ; cytology ; Coculture Techniques ; Collagen Type II ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Swine ; Tissue Scaffolds

BACKGROUND: The mechanism by which mutant cartilage oligomeric matrix protein (COMP) induces a pseudoachondroplasia phenotype remains unknown, and the reason why a mutation of a minor protein of the growth plate cartilage causes total disruption of endochondral bone formation has not yet been determined. The current study was performed to investigate the effects of mutated COMP on the synthesis of the cartilage-specific major matrix proteins of Swarm rat chondrosarcoma chondrocytes. METHODS: The Swarm rat chondrosarcoma chondrocytes transfected with a chimeric construct, which consisted of a mutant gene of human COMP and an amino acid FLAG tag sequence, were cultured in agarose gel. Formation of extracellular proteoglycan and type-II collagen by the cells was evaluated by immunohistochemical staining and measuring the (35)S-sulfate incorporation. RESULTS: No difference was observed for the detection of type-II collagen among the cell lines expressing mutant COMP and the control cell lines. Histochemical staining of sulfated proteoglycans with safranin-O showed that lesser amounts of proteoglycans were incorporated into the extracellular matrix of the chondrocytes transfected with the mutant gene. (35)S-sulfate incorporation into the cell/matrix fractions demonstrated markedly lower radiolabel incorporation, as compared to that of the control cells. CONCLUSIONS: Mutation of COMP has an important impact on the processing of proteoglycans, rather than type-II collagen, in the three-dimensional culture of Swarm rat chondrosarcoma chondrocytes.
Aggrecans/analysis/*biosynthesis ; Animals ; Cells, Cultured ; Chondrocytes/*metabolism ; Chondrosarcoma/metabolism ; Collagen Type II/*biosynthesis ; Extracellular Matrix/*metabolism ; Extracellular Matrix Proteins/*genetics ; Glycoproteins/*genetics ; Humans ; Mutation ; Rats ; Transfection

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

The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family consists of 19 proteases. These enzymes are known to play important roles in development, angiogenesis and coagulation; dysregulation and mutation of these enzymes have been implicated in many disease processes, such as inflammation, cancer, arthritis and atherosclerosis. This review briefly summarizes the structural organization and functional roles of ADAMTSs in normal and pathological conditions, focusing on members that are known to be involved in the degradation of extracellular matrix and loss of cartilage in arthritis, including the aggrecanases (ADAMTS-4 and ADAMTS-5), ADAMTS-7 and ADAMTS-12, the latter two are associated with cartilage oligomeric matrix protein (COMP), a component of the cartilage extracellular matrix (ECM). We will discuss the expression pattern and the regulation of these metalloproteinases at multiple levels, including their interaction with substrates, induction by pro-inflammatory cytokines, protein processing, inhibition (e.g., TIMP-3, alpha-2-macroglobulin, GEP), and activation (e.g., syndecan-4, PACE-4).
ADAM Proteins ; antagonists & inhibitors ; chemistry ; genetics ; physiology ; Aggrecans ; metabolism ; Alternative Splicing ; Arthritis ; enzymology ; genetics ; Cartilage ; enzymology ; Endopeptidases ; genetics ; physiology ; Extracellular Matrix ; enzymology ; Humans ; Protein Structure, Tertiary

OBJECTIVETo study the effect of RNA interference (RNAi)-mediated aggrecanase-1 gene silencing on extracellular matrix metabolism of cultured rat costochondral chondrocytes.

METHODSRat costochondral chondrocyte monolayers were obtained by microdissection and digestion. The growth and morphological changes of the chondrocytes were observed after RNAi of aggrecanase-1 gene. The mRNA expression of aggrecanase-1 was detected by RT-PCR method, and aggrecan content was determined by Western blotting.

RESULTSThe specific inhibition of aggrecanase-1 by RNAi produced no adverse effect on the morphology and growth of the chondrocytes. The mRNA of aggrecanase-1 decreased and aggrecan content increased significantly after transfection of the chondrocytes.

CONCLUSIONInhibition of aggrecanase-1 decreases aggrecan degradation in cultured rat chondrocytes. RNAi technique can be a useful means for studying extracellular matrix metabolism in the cartilage.

ADAM Proteins ; genetics ; metabolism ; ADAMTS4 Protein ; Aggrecans ; metabolism ; Animals ; Cells, Cultured ; Chondrocytes ; cytology ; metabolism ; Extracellular Matrix ; metabolism ; Female ; Procollagen N-Endopeptidase ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Transfection

OBJECTIVETo investigate the role of GATA4 gene in the endocardial cushions development.

METHODSTarget gene eukaryote expression vectors were constructed by pcDNA3.1(-) vector plasmid, and were identified by DNA sequence analysis. Recombinant plasmids were transfected into Hela cells with lipofectamine 2000, meanwhile Hela cells transfected with empty vector or those without transfection served as transfection control group and blank control group, respectively. Real-time PCR and Western blot were performed to detect the relative expression of mRNA and protein of transcription factors GATA4, Sox9, Scleraxis and ECM proteins Aggrecan, Tenascin in each group.

RESULTSThe relative mRNA expression of GATA4 in experimental group was significantly higher than in transfection control group and blank control group. GATA4 mRNA expression in Hela(GATA4), Hela(H436Y), Hela(Null) and Hela group was 310.83 ± 2.39, 146.35 ± 1.74, 0.94 ± 0.32, 1.00 ± 0.28, respectively (F = 72.508, P < 0.05). Western blot results were consistent with the results obtained by qRT-PCR. The relative mRNA and protein expressions of Sox9, Scleraxis, Aggrecan and Tenascin in both experimental groups were significantly higher than that in transfection control group and blank control group (P < 0.05), and above gene expressions were significantly downregulated in GATA4(H436Y) group, while they were similar between transfection control group and blank control group (all P > 0.05).

CONCLUSIONSGATA4 H436Y mutation reduces it's transcriptional activation, which might serve as a theoretical framework to demonstrate the roles of GATA4 gene in endocardial cushion development.

Aggrecans ; metabolism ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Down-Regulation ; Endocardial Cushions ; embryology ; GATA4 Transcription Factor ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; HeLa Cells ; Humans ; RNA, Messenger ; SOX9 Transcription Factor ; metabolism ; Tenascin ; metabolism ; Transfection