Melzer Michaela, Schubert Susanna, Müller Simon Franz, Geyer Joachim, Hagen Alina, Niebert Sabine, Burk Janina
Equine Clinic (Surgery, Orthopedics), Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, 35392 Giessen, Germany.
Saxon Incubator for Clinical Translation, University of Leipzig, 04103 Leipzig, Germany.
Stem Cells Int. 2021 Oct 8;2021:8284690. doi: 10.1155/2021/8284690. eCollection 2021.
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.
间充质基质细胞（MSC）是肌腱再生的一种有前景的治疗工具。它们向肌腱细胞的分化对于组织工程方法至关重要，并且可能支持细胞移植后的有益效果。转化生长因子（TGF）-β通过细胞内Smad分子发出信号，是肌腱诱导的一种有效的旁分泌介质。此外，支架拓扑结构或肌腱基质成分通过Rho/ROCK级联的激活诱导肌腱形成，然而，其也参与细胞外基质病理中的病理适应过程。本研究的目的是研究Rho/ROCK和TGF-β/Smad信号在人和马MSC的肌腱形成分化中的相互作用。从脂肪组织分离的原代马和人MSC以单层形式或在肌腱来源的脱细胞支架上培养，以评估ROCK抑制剂Y-27632对TGF-β诱导的肌腱形成分化的影响。将MSC与有或无TGF-β（10 ng/ml）、Y-27632（10 μM）或两者一起孵育。在第1天和第3天，通过Smad 2/3和鬼笔环肽染色观察TGF-β的信号通路和肌动蛋白细胞骨架，并评估信号分子和肌腱标志物的基因表达。通过肌动蛋白细胞骨架的破坏证实了ROCK抑制。TGF-β刺激后，Smad 2/3激活并伴有核转位明显可见。有趣的是，在两个物种中，这种效应在额外的ROCK抑制时最为明显（马MSC中P<0.05）。与此一致，当TGF-β和ROCK抑制联合时，肌腱标志物硬骨素显示出最强的上调（人MSC中P<0.05）。肌腱细胞外基质成分以及信号分子TGF-β和Smad 8的调节模式在人和马MSC之间存在差异。获得的结果表明，ROCK抑制促进TGF-β/Smad 2/3轴，这可能对肌腱治疗中未来的MSC预处理方案有影响。
