Shojaee Asiyeh, Ejeian Fatemeh, Parham Abbas, Nasr Esfahani Mohammad Hossein
Division of Physiology, Department of Basic Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.
Department of Animal Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
Cell J. 2022 Jul 27;24(7):370-379. doi: 10.22074/cellj.2022.7892.
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.
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.
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.
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.
肌腱修复策略通常伴随着病理性矿化和瘢痕组织形成，这增加了再次受伤的风险。本研究旨在建立一种有效的肌腱再生方法，同时降低异位骨形成的风险。
在本实验研究中，通过转化生长因子-β3（TGFB3）处理并结合抑制浓度的骨形态发生蛋白（BMP）拮抗剂gremlin-2（GREM2）和一种Wnt抑制剂即硬化蛋白（SOST）来诱导成腱分化。使用实时聚合酶链反应（qPCR）对成腱标志物和骨软骨生成标志物基因进行表达分析，以评估该方法的有效性。还通过免疫细胞化学评估了两种成腱标志物SCX和MKX的表达水平。进行天狼星红染色以检查胶原纤维的数量。此外，为了研究底物对成腱分化的影响，采用了高度类似于肌腱细胞外基质的纳米纤维支架。
在自发正常培养条件下形成聚集特征，随后成腱和成骨标志物基因上调，包括SCX、MKX.COL1A1、RUNX2和CTNNB1。TGFB3处理加剧了形态学变化，并在更短的时间内显著增强了成腱分化。与TGFB3处理一起，GREM2和SOST对BMP的抑制在一定程度上延迟了迁移事件，并协同显著降低了骨软骨生成标志物。纳米纤维支架增加了成腱标志物，同时降低了骨软骨生成基因的表达。
这些发现揭示了等轴羊膜干细胞自发成腱分化在体外具有适当的潜力，通过同时激活TGFB和抑制骨诱导信号通路可以使其得到改善。
