Rho/ROCK Inhibition Promotes TGF-3-Induced Tenogenic Differentiation in Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSC) represent a promising therapeutic tool for tendon regeneration. Their tenogenic differentiation is crucial for tissue engineering approaches and may support their beneficial effects after cell transplantation . The transforming growth factor (TGF)-, signalling via intracellular Smad molecules, is a potent paracrine mediator of tenogenic induction. Moreover, scaffold topography or tendon matrix components induced tenogenesis via activation of the Rho/ROCK cascade, which, however, is also involved in pathological adaptations in extracellular matrix pathologies. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-3/Smad signalling in tenogenic differentiation in both human and equine MSC. Primary equine and human MSC isolated from adipose tissue were cultured as monolayers or on tendon-derived decellularized scaffolds to evaluate the influence of the ROCK inhibitor Y-27632 on TGF-3-induced tenogenic differentiation. The MSC were incubated with and without TGF-3 (10 ng/ml), Y-27632 (10 M), or both. On day 1 and day 3, the signalling pathway of TGF- and the actin cytoskeleton were visualized by Smad 2/3 and phalloidin staining, and gene expression of signalling molecules and tendon markers was assessed. ROCK inhibition was confirmed by disruption of the actin cytoskeleton. Activation of Smad 2/3 with nuclear translocation was evident upon TGF-3 stimulation. Interestingly, this effect was most pronounced with additional ROCK inhibition in both species ( < 0.05 in equine MSC). In line with that, the tendon marker scleraxis showed the strongest upregulation when TGF-3 and ROCK inhibition were combined ( < 0.05 in human MSC). The regulation pattern of tendon extracellular matrix components and the signalling molecules TGF-3 and Smad 8 showed differences between human and equine MSC. The obtained results showed that ROCK inhibition promotes the TGF-3/Smad 2/3 axis, with possible implications for future MSC priming regimes in tendon therapy.