Objective: Tendon repair strategies are normally accompanied by pathological mineralization and/or scar tissue formation that increases the risk of re-injuries. This study aimed to establish an efficient tendon regeneration method, simultaneously with a reduced risk of ectopic bone formation. Materials and methods: Tenogenic differentiation was induced through transforming growth factor- β3 (TGFB3) treatment in combination with the inhibiting concentrations of bone morphogenetic proteins (BMP) antagonists, gremlin-2 (GREM2), and a Wnt inhibitor namely sclerostin (SOST). The efficacy of the procedure was evaluated using qPCR for expression analysis of tenogenic markers and osteo-chondrogenic marker genes. The expression level of two tenogenic markers, SCX and MKX, was also evaluated by immunocytochemistry. Sirius Red staining was performed to examine the amounts of collagen fibers. Moreover, to investigate the impact of the substrate on tenogenic differentiation, we employed the nanofibrous scaffolds highly resembling tendon extracellular matrix. Results: Aggregated features formed in spontaneous normal culture conditions, and were followed by up-regulation of tenogenic and osteogenic marker genes including SCX, MKX, COL1A1, RUNX2, and CTNNB1. TGFB3 treatment exaggerated morphological changes and markedly amplified tenogenic differentiation in a shorter period time. Along with TGFB3 treatment, inhibition of BMPs by GREM2 and SOST delayed migratory events to some extent, and dramatically reduced osteo-chondrogenic markers in a synergistic manner. Nanofibrous scaffolds increased tenogenic markers, while markedly declined the expression of osteo-chondrogenic genes. Conclusion: These findings revealed an appropriate in vitro potential of spontaneous tenogenic differentiation of eq-ASCs that can be improved by simultaneous activation of TGFB and inhibition of osteo-inductive signaling pathways.

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