【作者】 陈晓；

【导师】 欧阳宏伟；

【作者基本信息】 浙江大学 ， 临床医学， 2010， 博士

【摘要】 中文摘要背景：随着国民素质的提高以及人们对健康的追求,体育锻炼和竞赛活动明显增加,以及由于意外事故不断发生和社会老龄化趋势日益明显,运动损伤也越来越多,其中韧带肌腱损伤占50%以上。统计表明,每年至少有3000万的肌腱损伤病例。目前,临床上对肌腱损伤的治疗主要停留在理疗、手术缝合以及自体或异体移植阶段,虽有一定效果,但是疗效有限,即使是自体肌腱移植修复也只能达到正常肌腱力学性能的40%左右,且伴随有大量疤痕组织增生。这主要是因为成体肌腱不具备完全再生能力,所以修复后肌腱的质量远不如正常肌腱,易出现肌腱粘连,肌腱结构和力学性能低下,常重复断裂。因此,寻找新型的、可促进肌腱生理性再生修复的方法具有极其重要的临床意义。随着最近10年组织工程和干细胞研究的兴起,使临床医学步入了“再生医学”的新阶段。通过利用干细胞复合支架材料的组织工程手段,促使身体自主再生已损伤组织,为提高肌腱等软组织的修复质量带来了全新的机会。肌腱组织工程技术中包含三个重要的迫切需要解决的关键问题：1)获得足够分化成熟的肌腱种子细胞；2)能够提供足够生长空间的生物支架材料；3)种子细胞分化的力学刺激以及其他转录因子的调控。然而,目前这些问题尚未解决,影响了肌腱组织工程的发展,从而制约了组织工程肌腱在临床用于肌腱缺损再生修复治疗。本研究针对目前这些现状,旨在系统的研究组织工程肌腱所面临的三个迫切问题,最终目的为复合诱导后的胚胎干细胞与蚕丝-胶原海绵支架构建组织工程肌腱并促进肌腱损伤的再生。研究内容包含：在种子细胞选择、分化和调控方面,拟采用分子生物学手段调控诱导人胚胎干细胞阶段性向肌腱细胞分化以提供足够的种子细胞,并阐明力学和转录因子因素调控干细胞肌腱分化的机理和协同效果,获得有效的肌腱分化条件；本方向的进一步研究将为肌腱分化及再生提供全新的思路和知识,为认知肌腱再生的生物机理提供实验基础和理论数据,为运用特定分化后肌腱干细胞用于肌腱再生打下基础。在组织工程支架方面,拟研发一个既符合组织工程支架需要,又具有生理承载功能的可抗拉力的功能性肌腱支架--蚕丝-胶原海绵复合支架,使之更符合肌腱生物学特性和力学特性。最终将利用研发的支架复合诱导的人胚胎干细胞,构建组织工程肌腱,通过动物模型评估组织工程肌腱对肌腱损伤修复的再生作用。本研究课题,将最终为组织工程肌腱应用于临床打下基础,为肌腱损伤治疗带来新的方向。由于肌腱与韧带在结构与功能上类似,因此,在本课题中肌腱组织工程泛指肌腱与韧带组织工程。本研究分为四个部分：(1)人胚胎干细胞(hESCs)诱导成为间质干细胞(MSCs),并应用于肌腱组织工程研究,探索hESCs促进肌腱再生的可行性和功效性；(2)研究肌腱发育特征转录因子SCX对hESCs来源MSCs的肌腱分化影响。阐明SCX对hESC-MSCs的肌腱分化调控,并获得SCX+的肌腱祖细胞。在获得SCX+肌腱祖细胞的基础上研究外加力学刺激与转录因子对肌腱分化的协同效应以及对相关信号通路的协同调控机理,明确肌腱祖细胞向肌腱分化调控机制；(3)构建网状蚕丝-胶原海绵复合支架,评估该支架用于肌腱／韧带组织工程的可行性与优势。(4)支架复合诱导后的胚胎干细胞构建组织工程肌腱,在动物模型中评估工程化的细胞与支架构成的组织工程肌腱对肌腱损伤修复的促进作用。第一章人胚胎干细胞的阶段性分化通过分泌胚胎肌腱基质和分化因子促进肌腱再生目的：人胚胎干细胞(hESCs)是组织再生的理想种子细胞,但是至今尚未有研究报道人胚胎干细胞用于肌腱再生的可能性。本课题主要研究人胚胎干细胞用于肌腱再生的策略和功效,以及相关的机理。方法与结果：首先,将人胚胎干细胞诱导分化成间充质干细胞(MSC),这种MSC具有三系分化潜能并且表达MSC的表面标记。通过肌腱特异性基因的表达和肌腱结构鉴定证明人胚胎干细胞来源的间充质干细胞(hESC-MSCs)不仅在体外组织工程模型还是在体内异位肌腱再生模型中都可以再生肌腱组织。在大鼠原位髌腱修复实验中,用hESC-MSCs处理组与对照组相比具有较好的组织结构和力学性能。另外,hESC-MSCs可以在肌腱损伤区域存活至少4周,并且分泌人胚胎肌腱特异的胞外基质成分和分化因子,这些因子进一步激活肌腱内源的再生过程。并且,在所有样本中均未发现畸胎瘤形成。结论：本研究开创了一种安全有效的将胚胎干细胞用于肌腱再生的方法,并有助于发展未来应对肌腱疾病的手段。第二章转录因子scleraxis与力学刺激协同调控干细胞腱系分化机理和效应研究目的：缺乏肌腱分化调控知识是实现肌腱再生的根本性难题。发育生物学是分化研究的线索。已有肌腱发育分化研究发现：1)SCX是肌腱祖细胞的标志,TGF, FGF是肌腱发育的关键通路,而目前从SCX+祖细胞到肌腱的分化调控知识缺乏；2)SCX是肌腱发育的必要但不充分条件,敲除SCX出现严重的肌腱缺失；3)肌腱发育过程提示存在力学刺激促肌腱成熟现象。而力学与SCX在肌腱成熟和分化中的协同作用及相应机制仍未清楚。本章主要研究SCX+祖细胞向成熟肌腱细胞分化调控机制,即以SCX+干细胞为起点复合力学刺激诱导肌腱分化,明确SCX与力学刺激对肌腱分化是否有协同作用并探索其机制。方法与结果：第一阶段将肌腱发育特征转录因子SCX转入hESC来源MSCs,获得SCX+的肌腱祖细胞。第二阶段将在SCX+肌腱祖细胞构建的细胞片的基础上研究外加周期性力学刺激(1Hz),并揭示力学与转录因子对肌腱分化的协同效应以及对相关信号通路的协同调控机理,明确肌腱祖细胞向肌腱分化调控机制。第三阶段,评估SCX复合力学分化的肌腱细胞修复再生修复肌腱的效率。实验表明：1)SCX可增加肌腱细胞外基质(胶原Ⅰ,ⅩⅣ)表达,并降低胶原Ⅱ表达及BMP活性。然而,SCX亦能增加runx2表达及骨诱导效果。2)在力学协同刺激下,SCX可增加肌腱特异性基因表达而诱导肌腱分化。3)体内异位种植及原位修复结果显示,SCX与力学有协同诱导分化及修复作用,可诱导胶原纤维成熟,并促进肌腱修复。结论：该研究表明SCX与力学不仅在肌腱分化中有重要作用,并且能够协同作用诱导肌腱分化与修复。SCX与力学刺激的协同作用部分是通过调控BMP-Smad通路及runx2的功能。该研究发现将为肌腱分化调控提控新知识,并将可能通过更准确调控干细胞腱系分化为肌腱再生的新的种子细胞和手段。第三章网状蚕丝-胶原海绵复合支架促肌腱再生研究目的：本课题致力于发明一种力学性能和生物学性能良好的生物材料,为肌腱韧带组织工程提供一种同时具备有良好力学性能和足够相通的细胞组织容纳空间的支架,即网状蚕丝复合胶原海绵支架。方法与结果：该支架用于韧带肌腱组织工程的功效性通过体外和动物体内进行评估。在胶原基质上培养的细胞与生长在蚕丝上的细胞高表达肌腱韧带的胞外基质基因。小鼠皮下移植实验结果显示蚕丝支架有很好的生物相容性,并会缓慢的降解。兔内侧副韧带损伤模型显示,将网状蚕丝复合胶原海绵支架用于内侧副韧带的修复得到更多的胶原沉积和更好的力学性能,与未修复和单纯蚕丝支架修复效果相比,实验组超微结构的胶原直径更大且支架和肌腱的交界点修复更强。结论：本研究结果证明了网状蚕丝复合胶原海绵支架通过调控肌腱韧带胞外基质基因的表达和胶原纤维的聚合促进了肌腱结构和功能的修复。这些发现第一次强调了生物材料在肌腱韧带再生生物学中的重要角色。另外,“内部空间预留”的支架概念的提出有利于处于力学张力下组织的修复。第四章基因工程化hESC-MSC与蚕丝-胶原海绵复合支架构建组织工程肌腱研究目的：我们之前的研究中已证明复合了胶原的蚕丝支架在肌腱再生中拥有良好的潜能,并证实hESC-MSCs及SCX+hESC-MSCs在肌腱分化及促进修复肌腱再生中的潜能。本研究旨在应用支架复合诱导后的胚胎干细胞构建组织工程肌腱,在动物模型中评估工程化的细胞与支架构成的组织工程肌腱对肌腱损伤修复的促进作用。方法与结果：体外实验将hESC-MSC及工程化SCX+hESC-MSC种在复合支架上在体外施以动态力学刺激(DM)或无力学刺激(NM)达14天。体内异位种植则埋入裸鼠皮下4周,部分通过将构建物缝合在裸鼠背部脊上韧带施以天然的力学刺激(DM),其他的则不受力(NM)。原位跟腱修复分别于固定后2周,4周,进行大体、组织学、超微结构、生化组成、生物力学检测。力学刺激诱导了复合胶原蚕丝支架上的hESC-MSC及SCX+hESC-MSC向肌腱方向分化,体内异位移植肌腱的组织学、生化组成和胶原表达、胶原纤维大小均大于无力学组,两种细胞间差异并不明显。肌腱修复结果显示细胞促进形成更成熟的胶原纤维,并促进损伤肌腱的修复。另外SCX+hESC-MSC可促进修复肌腱的力学性能,并增加胶原纤维成熟。结论：hESC-MSC及工程化SCX+hESC-MSC复合网状蚕丝-胶原海绵支架可构建组织工程肌腱,并促进损伤肌腱修复。SCX+hESC-MSC可进一步促进肌腱修复及胶原纤维成熟。更多还原

【Abstract】 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.更多还原