| IntroductionTendon and ligament damage are frequently encountered in sports injuries, which often result in suboptimal healing and cause significant dysfunction and disability. Presently, the main therapeutic options to treat tendon and ligament injuries include prosthetic scaffold devices and tissue grafting. Until now, no prosthetic devices have been able to adequately restore the long term function of tendons. Tissue grafting methods, including autografts, allografts, xenografts, are limited by the major disadvantages such as quality and availability of autograft tissues, compromising normal healthy tissue, and the risk of disease transmission and immune response from allografts and xenografts. Moreover, injured tendon which repaired by autograft could only reach 40% of normal mechanical strength, due to the lack of regeneration potential. It is clinically important to search for a new promising tendon regeneration methods.Recently, a novel tissue-engineering technique has emerged, which combines biodegradable biomaterials, cell, growth factors, and gene transfer methods. It has shown great potentials for tendon and ligament repairment. However, none of the key components of this technique has not been optimized. The cell source is vital for tendon and ligament tissue engineering, yet……(major problem associated with cell source) Another key issue of tendon tissue engineering is scaffold, which under optimal conditions should possesses optimal strength, a porous structure and a biocompatible microenvironment. So far, the optimal scaffold has not been developed.To provide enough seed cells, we stepwise induce hESC into tenocytes to provide seed cells. We also investigate the mechanism involve in the differentiation to provide a theoretical basis between differentiation. This study also aimed to design a new practical tendon scaffold by the synergistic incorporation of silk fibers, a knitted structure, and a collagen matrix. Silk fibers provided mechanical strength. The knitted structure provided internal connective space. Collagen matrix initially occupied the internal space of the knitted scaffold for neoligament tissue ingrowth as well as the capacity to modulate neoligament regeneration by regulating matrix gene expression and the assembly of collagen fibrils? The combination of scaffold and induced embryonic stem cells thus fomed a novel tendon tissue engineering product. We further evaluated the role of engineered tendon in promoting tendon regeneration in animal models. Our work will make tendon tissue engineering closer to the bedside and bring a new direction for treatment of tendon injuries.The current study include four stages:stage 1 to induce hESC into MSCs and investigate the potential of hESC-MSCs in the tendon tissue engineering; stage 2 to investigate the synergetic function of scleraxis and mechanical stress on the teno-lineage induction; stage 3 to fabricate scaffold that compose of the knitted silk scaffold combined with collagen matrix and evaluate the feasibility and advantages for the tendon tissue engineering; stage 4 to fabricate engineered tendon that compose of scaffold and induced embryonic stem cells and evaluate the role of engineered tendon in promoting tendon regeneration in animal modelStage 1 Stepwise Differentiation of Human Embryonic Stem Cells Promotes Tendon Regeneration by Secreting Fetal Tendon Matrix and Differentiation FactorsAim:Human embryonic stem cells (hESCs) are ideal seed cells for tissue regeneration, but no research has yet been reported concerning their potential for tendon regeneration. This study investigated the strategy and efficacy of using hESCs for tendon regeneration as well as the mechanism involved.Methods and results:hESCs were first induced to differentiate into mesenchymal stem cells (MSCs), which had the potential to differentiate into the three mesenchymal lineages and were positive for MSC surface markers. hESC-derived MSCs (hESC-MSCs) regenerated tendon tissues in both an in vitro tissue engineering model and an in vivo ectopic tendon regeneration model, as confirmed by the expression of tendon-specific genes and structure. In in-situ rat patellar tendon repair, tendon treated with hESC-MSCs had much better structural and mechanical properties than did controls. Furthermore, hESC-MSCs remained viable at the tendon wound site for at least 4 weeks and secreted human fetal tendon-specific matrix components and differentiation actors, which then activated the endogenous regeneration process in tendon.Conclusion:These findings demonstrate a safe and practical strategy of applying ESCs for tendon regeneration and may assist in future strategies to treat tendon diseases. Moreover, no teratoma was found in any samples.Stage 2 Overexpression of Scleraxis and Dynamic Mechanical Stress Regulate Tendon-lineage Differentiation of Human Embryonic Stem Cells for Tendon RepairAim:Until now no optimal induction for tenocytes differentiation and tendon regeneration has yet been achieved. In embryo development, TGF, FGF signal and ectoderm signal and mechanical stress is critical for tendon development and associate with tenocytes differentiation. Scleraxis, a bHLH transcription factor, is a highly specific marker of tendons and Scleraxis knockout cause the severe force-transmitting tendon defects suggest that the mechanical stress and scleraxis may play a synergic role in the tendon development. The signaling mechanisms that mediate force-induced tenocytes differentiation and collagen expression are not defined. In this study, we tested the hypothesis that mechanical stress interacts with the transfer growth factor-beta (TGF-beta) pathway and scleraxis transcription factor to stimulate tendon-lineage differentiation and tendon regeneration.Methods and results:Human ESCs were first induced to differentiate into mesenchymal stem cells (MSCs). The immuno-phenotype of hESC-derived MSCs (hESC-MSCs) was identified by flow cytometry. Then the hESC-MSCs were transfer with the tendon-lineage specific transcription factor scleraxis. hESC-MSCs formed cell sheet after 14 days culture and engineered tendon were formed in vitro. The engineered tendon was subjected to a dynamic mechanical stress of 1HZ for 2h/day. Then the regeneration potential of the engineered tendon tissues was evaluated in both an in vitro tissue engineering model and an in-situ rat patellar tendon window repair model.Scleraxis overexpression increased the expression of collagen I, III, XIV, reduced collagen II promoter activation and BMP induced smad activation. However, ALP activity and alizarin red staining showed scleraxis also increase the bone induction. The expression of tendon-specific genes was significantly higher in scleraxis transfected hESC-MSCs under mechanical stimulus when compare to the scleraxis transfected hESC-MSCs without mechanical stimulus or the native hESC-MSCs under mechanical stimulus. In vivo ectopic implantation also shows the synergetic function of scleraxis and mechanical stress in tendon differentiation. Tendon treated with scleraxis hESC-MSCs had much better structural and mechanical properties than did controls. Furthermore, hESC-MSCs remained viable at the tendon wound site for at least 4 week. Moreover, no teratoma was found in any samples. Conclusion:The present study demonstrates that both mechanical stress and scleraxis are not only important for the tendon differentiation but also have synergetic function on tendon regeneration. The role of scleraxis on tendon differentiation is partially by changing the activation of BMP-smad pathway. These findings may have considerable importance on understanding the roles of mechanical stress and scleraxis on tendon differentiation as well as developing therapeutics for tendon regeneration.Stage 3 Ligament Regeneration Using a Knitted Silk Scaffold Combined with Collagen MatrixAim:This study aimed to develop a new practical ligament scaffold by synergistic incorporation of silk fibers, a knitted structure, and a collagen matrix. The efficacy for ligament tissue engineering was investigated in vitro and in animal models.Methods and results:Cells cultured on a collagen substrate expressed higher levels of ligament matrix genes than those on a silk substrate. The silk scaffold elicited little inflammatory reaction and degraded slowly after subcutaneous implantation in a mouse model. In the rabbit MCL defect model, MCLs treated with a silk+collagen scaffold deposited more collagen, had better mechanical properties, and showed more native microstructure with larger diameter collagen fibrils and stronger scaffold-ligament interface healing than untreated MCLs and those treated with silk scaffolds.Conclusion:These results demonstrated that the knitted silk+collagen sponge scaffold improves structural and functional ligament repair by regulating ligament matrix gene expression and collagen fibril assembly. The findings are the first to highlight the important roles of biomaterials in ligament regeneration biology. Also, the concept of an "internal-space-preservation" scaffold is proposed for the tissue repair under physical loading.Stage 4 Experimental Study on Engineered hESC-MSC-Silk-Collagen Sponge Tissue Engineered TendonAim:This study aimed to engineer tendon by the combination of engineered hESC-MSCs with knitted silk scaffold combined with collagen matrix.Methods and results:hESC-MSCs and SCX-hESC-MSCs were induced into teno-lineage on knitted silk scaffold combined with collagen matrix under mechanical stress in vitro and in vivo. In in situ repair study, hESC-MSCs-scaffold engineered tendon were used for rat AT regeneration. The repaired tendon was used for histological examination, mechanical properties and transmission electron microscopy analysis.Tendon-like tissue was formed in vitro in the constructs with mechanical stress. And tendon-specific genes expressions were significantly higher. The results of in vivo heterotypic transplantation showed spindle-shaped and regularly aligned cells, larger collagen fibers and more deposited collagen in the mechanical stress group. These results demonstrated that engineered tendon was successfully fabricated by human ESC combine with mechanical stress and the collagen sponge-knitted silk scaffolds. Engineered tendon improveConclusion:The engineered tendon developed in this study is promising in restoring or replacing the damaged tendon in future clinical trial.
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