Downregulation of MicroRNA-495 Alleviates IL-1β Responses among Chondrocytes by Preventing SOX9 Reduction

Purpose@#Our previous work demonstrated that miRNA-495 targets SOX9 to inhibit chondrogenesis of mesenchymal stem cells.In this study, we aimed to investigate whether miRNA-495-mediated SOX9 regulation could be a novel therapeutic target for osteoarthritis (OA) using an in vitro cell culture model. @*Materials and Methods@#An in vitro model mimicking the OA environment was established using TC28a2 normal human chondrocyte cells. Interleukin-1β (IL-1β, 10 ng/mL) was utilized to induce inflammation-related changes in TC28a2 cells. Safranin O staining and glycosaminoglycan assay were used to detect changes in proteoglycans among TC28a2 cells. Expression levels of COX-2, ADAMTS5, MMP13, SOX9, CCL4, and COL2A1 were examined by qRT-PCR and/or Western blotting. Immunohistochemistry was performed to detect SOX9 and CCL4 proteins in human cartilage tissues obtained from patients with OA. @*Results@#miRNA-495 was upregulated in IL-1β-treated TC28a2 cells and chondrocytes from damaged cartilage tissues of patients with OA. Anti-miR-495 abolished the effect of IL-1β in TC28a2 cells and rescued the protein levels of SOX9 and COL2A1, which were reduced by IL-1β. SOX9 was downregulated in the damaged cartilage tissues of patients with OA, and knockdown of SOX9 abolished the effect of anti-miR-495 on IL-1β-treated TC28a2 cells. @*Conclusion@#We demonstrated that inhibition of miRNA-495 alleviates IL-1β-induced inflammatory responses in chondrocytes by rescuing SOX9 expression. Accordingly, miRNA-495 could be a potential novel target for OA therapy, and the application of anti-miR-495 to chondrocytes could be a therapeutic strategy for treating OA.

Downregulation of MicroRNA-495 Alleviates IL-1β Responses among Chondrocytes by Preventing SOX9 Reduction

Purpose@#Our previous work demonstrated that miRNA-495 targets SOX9 to inhibit chondrogenesis of mesenchymal stem cells.In this study, we aimed to investigate whether miRNA-495-mediated SOX9 regulation could be a novel therapeutic target for osteoarthritis (OA) using an in vitro cell culture model. @*Materials and Methods@#An in vitro model mimicking the OA environment was established using TC28a2 normal human chondrocyte cells. Interleukin-1β (IL-1β, 10 ng/mL) was utilized to induce inflammation-related changes in TC28a2 cells. Safranin O staining and glycosaminoglycan assay were used to detect changes in proteoglycans among TC28a2 cells. Expression levels of COX-2, ADAMTS5, MMP13, SOX9, CCL4, and COL2A1 were examined by qRT-PCR and/or Western blotting. Immunohistochemistry was performed to detect SOX9 and CCL4 proteins in human cartilage tissues obtained from patients with OA. @*Results@#miRNA-495 was upregulated in IL-1β-treated TC28a2 cells and chondrocytes from damaged cartilage tissues of patients with OA. Anti-miR-495 abolished the effect of IL-1β in TC28a2 cells and rescued the protein levels of SOX9 and COL2A1, which were reduced by IL-1β. SOX9 was downregulated in the damaged cartilage tissues of patients with OA, and knockdown of SOX9 abolished the effect of anti-miR-495 on IL-1β-treated TC28a2 cells. @*Conclusion@#We demonstrated that inhibition of miRNA-495 alleviates IL-1β-induced inflammatory responses in chondrocytes by rescuing SOX9 expression. Accordingly, miRNA-495 could be a potential novel target for OA therapy, and the application of anti-miR-495 to chondrocytes could be a therapeutic strategy for treating OA.

In Vivo Cartilage Formation Using Human Bone Marrow-Derived Mesenchymal Stem Cells Mixed with Fibrin Glue / 대한정형외과연구학회지

PURPOSE: To investigate the feasibility of in vivo cartilage formation by direct injection the chondrogenic undifferentiated human bone marrow-derived mesenchymal stem cells (hBMSCs) mixed with fibrin glue including TGF-beta3. MATERIALS AND METHODS: Chondrogenic differentiation induced hBMSCs for 14 days (control group-2) and undifferentiated hBMSCs combined with TGF-beta3 mixed (experimental group-3) with fibrin glue and fibrin glue only (control group-1) were injected subcutanteously into the back of nude mouse. For evaluation of the cartilage-like tissue formed after 8 weeks after injection, real time PCR, histological analysis and immunohistochemical analysis were used. RESULTS: Control group-1 did not form any visible mass. Control group-2 as well as experimental group-3 could form new cartilage-like tissue which were demonstrated expression of type II collagen by real-time PCR, histology analysis such as H&E staining, MT staining and type II collagen specific immunohistologic analysis. As results, expression of type II collagen was shown in the both groups. CONCLUSION: Our study confirmed that cartilage-like tissues could be formed in subcutaneous layer of nude mouse by direct injection mixed with fibrin glue including TGF-beta3 without chondrogenic-induction of hBMSCs, suggesting that these model could be suitable for preliminary studies or optimizing experiments to evaluate reconstruction of cartilage.

Comparison of Efficiency of Self-renewal and Differentiation Potential in Tendon-derived Mesenchymal Stem Cells Isolated by Magnetic-activated Cell Sorting Method or Colony Picking Method / 대한족부족관절학회지

PURPOSE: The purpose of this study is to evaluate the efficacy of mesenchymal stem cell (MSC) isolation by the magnetic-activated cell sorting (MACS) method in tendon tissue-derived cells compared to the colony picking method for isolation of MSCs by picking colonyforming cells. MATERIALS AND METHODS: Human tendon-derived cells were isolated by enzyme digestion using normal tendon tissues from three donors. We used the magnetic kit and well-known MSC markers (CD90 or CD105) to isolate MSCs in tendon-derived cells using MACS. Cloning cylinders were used to isolate colony-forming cells having MSC characteristics in tendon-derived cells. Colony-forming unitfibroblast (CFU-F) assay was used to evaluate the self-renewal capacity of cells isolated using the colony picking method or MACS. For comparison of differentiation potentials into osteogenic or adipogenic lineage between two groups, alizarin red S and oil red O staining were performed at 14 days after induction of differentiation in vitro. RESULTS: Flow cytometry results showed that early passage tendon-derived cells expressed CD44 in 99.13%, CD90 in 56.51%, and CD105 in 86.19%. In the CFU-F assay, CD90+ or CD105+ cells isolated with MACS showed larger colony formation in size than cells isolated using the colony picking method. We also observed that CD90+ or CD105+ cells were constantly differentiated into both osteogenic and adipogenic lineages in cells from all donors, whereas cells isolated using the colony picking method were heterogeneous in differentiation potentials to the osteogenic and adipogenic lineages. CONCLUSION: CD90+ or CD105+ cells isolated using MACS showed superior MSC characteristics in the self-renewal and multi-differentiation capacities compared with cells isolated using the colony picking method.