Optimizing Tenogenic Differentiation of Equine Adipose-Derived Mesenchymal Stem Cells (eq-ASC) Using TGFB3 Along with BMP Antagonists

Document Type : Original Article


1 Division of Physiology, Department of Basic Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran

2 Department of Animal Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran

3 Stem Cell Biology and Regenerative Medicine Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran


Objective: Tendon repair strategies usually are accompanied by pathological mineralization and 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: In this experimental study, 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 procedure’s efficacy was evaluated using real-time polymerase chain reaction (qPCR) for expression analysis of tenogenic markers and osteochondrogenic 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, the nanofibrous scaffolds that highly resemble tendon extracellular matrix was employed.
Results: Aggregated features formed in spontaneous normal culture conditions 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 of time. Along with TGFB3 treatment, inhibition of BMPs by GREM2 and SOST delayed migratory events to some extent and dramatically reduced osteo-chondrogenic markers synergistically. Nanofibrous scaffolds increased tenogenic markers while declining 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 osteoinductive signaling pathways.