Induction Of Tenogenesis Osteogenesis Of Mesenchymal Stem Cells By Defined Factors

Part Ⅰ: The Role and mechanism of Mohawk on the tenogenesis of mesenchymal stem cellsThe repair of injured tendons remains a great challenge, largely owing to a lack of understanding of the molecular interactions in tendon development. Here, we investigated the role of the transcription factor Mohawk (Mkx) in the tenogenesis of mesenchymal stem cells (MSCs) and evaluated the application of Mkx to tendon regeneration.We showed that Mkx expression was dramatically decreased in human tendinopathy than normal tendon tissue, indicating that Mkx is required for maintenance of tendon physiologic function. We also established a rat model of Achilles tendon injury and found that Mkx was highly expressed in the injured tendons2weeks after injury and was reduced at4weeks, indicating that Mkx is involved in the early stage of tendon healing. Then we analyzed tenogenic genes expression at embryogenesis and after birth and found that Mkx expression was increased from E10.5to E13.5in forelimb and hindlimb.We showed that Mkx was highly expressed in tendon stem/progenitor cells (TSPCs) and silencing Mkx levels leaded to marked decreases in the expression of critical tenogenic genes, suggesting that Mkx is necessary for the maintenance of TSPCs characteristics.Furthermore, we analyzed the data from GEO datasets and found Mkx expression was increased from E11.5to E14.5in forelimb tendon cells, however, Sex expression was decreased from E11.5, indicating their different roles in tendon development. So we next investigated the specific role of Mkx and Sex in tenogenesis. We found that forced Mkx expression remarkably induced the tenogenesis of MSCs even more effectively than Scleraxis (Sex). Several tenogenic genes were significantly up-regulated especially in Mkx-MSC cell-sheets, such as Sex, Bgn (biglycan), Collal, Col3al, Col5al, Coll4, Den (decorin), Fmod, Tnc (tenascin-C) and Tnmd (tenomodulin). In addition, the collagen fibril diameter in Mkx-MSC cell-sheets was increased by1.8-fold over the control group (p<0.01) and1.35-fold over the Sex group (p<0.01).What’s more, it was revealed by ChIP-squencing and ChIP-PCR that MKX bind to Tgfb2promoter. Then we further demonstrated that Mkx regulates Sex though TGF-β signaling pathway.Finally, the application of Mkx-MSC cell-sheets promoted the formation of tendon-like tissues in a mouse model of Achilles tendon defect; the Mkx-expressing group had better histological features, larger collagen fibrils, and improved biomechanical properties.Therefore, to our knowledge, this study is the first to show a particular role of Mkx in promoting the tenogenesis of MSCs via activating Sex and tendon extracellular matrix molecules both in vitro and in vivo, and Mkx had more profound effects than Sex in promoting tenogenic gene expression and collagen fibril growth, which offering new insights into tendon development and providing an alternative way of obtaining cells suitable for tendon regeneration. Part II: Efficacy and mechanism of nanofibrous hydroxyapatite/chitosan scaffold on the osteogenesis of mesenchymal stem cellsIn bone tissue engineering, a combination of biomimetic nanofibrous scaffolds with renewable stem cells has recently emerged as a new strategy for promoting bone regeneration. We have previously developed a biomimetic nanocomposite nanofibrous scaffold of hydroxyapatite/chitosan (nHAp/CTS)[1]. However, the mechanism behind the supportive function of the scaffolds has not yet been adequately explored. Here, we evaluated the effect of nHAp/CTS seeded with bone marrow mesenchymal stem cells (BMSCs) on bone regeneration and examined the underlying mechanism in vitro and in vivo.The scaffolds of nHAp/CTS induced higher proliferation of BMSCs than membranous hydroxyapatite/chitosan (mHAp/CTS) and electrospun nanofibrous chitosan (nCTS) did. Interestingly, regardless the nanfibrous effect, nHAp/CTS and mHAp/CTS supported the spindle-shaped morphology, in contrast to the spherical shape of BMSCs on nCTS, indicating that HAp supports cell adhesion. Furthermore, the levels of the mRNA for Smadl, BMP2/4, Runx2, ALP, collagen I, integrin subunits together with myosins were significantly upregulated on nHAp/CTS whereas these genes were expressed at markedly low levels on mHAp/CTS and nCTS even in osteogenic medium. In addition, the critical proteins pSmad1/5/8in BMP pathway showed clear nuclear localization and osteocalcin were significantly elevated on nHAp/CTS than mHAp/CTS (P<O.01) and nCTS (P<O.01). Similarly, the cells exhibited higher ALP activity on nHAp/CTS than mHAp/CTS (P<O.01) and nCTS (P<O.05). Therefore, the findings indicated the activating of intergrin-BMP/Smad signaling pathway of BMSCs on nHAp/CTS. Finally, in vivo, nHAp/CTS/BMSCs had a superior ability of bone reconstruction than other groups for cranial bone defects.In conclusion, our results demonstrated that nHAp/CTS scaffold promotes bone regeneration by supporting the adhesion, proliferation and activating integrin-BMP/Smad signaling pathway of BMSCs both in vitro and in vivo.