Rho/ROCK Inhibition Promotes TGF-β3-Induced Tenogenic Differentiation in Mesenchymal Stromal Cells

Abstract

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 <i>in vivo</i>. The transforming growth factor (TGF)-<i>β</i>, 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-<i>β</i>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-<i>β</i>3-induced tenogenic differentiation. The MSC were incubated with and without TGF-<i>β</i>3 (10 ng/ml), Y-27632 (10 <i>μ</i>M), or both. On day 1 and day 3, the signalling pathway of TGF-<i>β</i> 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-<i>β</i>3 stimulation. Interestingly, this effect was most pronounced with additional ROCK inhibition in both species (<i>p</i> < 0.05 in equine MSC). In line with that, the tendon marker scleraxis showed the strongest upregulation when TGF-<i>β</i>3 and ROCK inhibition were combined (<i>p</i> < 0.05 in human MSC). The regulation pattern of tendon extracellular matrix components and the signalling molecules TGF-<i>β</i>3 and Smad 8 showed differences between human and equine MSC. The obtained results showed that ROCK inhibition promotes the TGF-<i>β</i>3/Smad 2/3 axis, with possible implications for future MSC priming regimes in tendon therapy.