【作者】 金鑫；

【导师】 范志宏；

【作者基本信息】 上海交通大学 ， 外科学， 2016， 博士

【摘要】 研究背景:如何修复大面积骨缺损是临床中面临的难题,传统的方法有各自的缺点。目前利用骨组织工程技术进行修复时无论是在种子细胞还是在支架材料上都有不尽完美之处。骨髓基质细胞是骨组织工程最主要的种子细胞来源,但其有取材过程痛苦,细胞含量少等缺点。脂肪来源基质细胞易于获取、数量巨大,可以满足自体种子细胞来源的需要。但与骨髓基质细胞相比,其成骨能力相对较弱。因此,如何扬长避短,既利用脂肪来源基质细胞的优势,又进一步提高其成骨能力成为亟待研究的问题。传统的支架材料不能很好的模拟体内的三维微环境,因而天然合成物支架材料的研究方兴未艾。但如何能够大量获取并制备自体的或是同种异体的生物源支架材料仍有相当的局限。人头发来源的角蛋白支架由于来源广泛、取材方便、生物相容性好、经济环保、免疫反应小等优势为此提供了一种新的策略。本研究分别从种子细胞和三维水凝胶支架两方面分别探讨了更好地利用人体中原本被认为是废弃组织的脂肪和头发组织作为骨组织工程的原料来实现骨再生修复的可能性。实验目的:1.研究用骨髓基质细胞(BMSCs)及其分泌的细胞外基质(BM-ECM)促进脂肪来源基质细胞(ASCs)成骨能力的可行性;2.探讨人头发角蛋白(HHK)三维水凝胶支架材料在体外及动物体内作为骨组织工程支架材料的安全性和有效性;3.探讨微环境对基于细胞的骨组织工程的影响。实验方法:1.体外分离同一只大鼠的ASCs与BMSCs,培养扩增后进行鉴定并比较其增殖和成骨分化能力;2.将同一只大鼠的ASCs与BMSCs在六孔板中直接共培养和Transwell皿中间接共培养,成骨诱导14天后进行成骨相关的组化染色和相关基因的实时定量PCR检测;3.制备BM-ECM,与ASCs在六孔板中直接共培养,成骨诱导14天后进行茜素红染色;4.用来源于褐藻的藻酸盐、猪来源的胶原、人头发来源的角蛋白制备成三维水凝胶,在体外检测其相关表征;5.三种水凝胶材料包裹大鼠的BMSCs在体外进行三维共培养和诱导,比较其细胞活力(AM/PI)和成骨分化能力(ALP活性);6.三种水凝胶材料包裹大鼠的BMSCs在体外诱导14天后分别植入裸鼠皮下和大鼠颅骨极量缺损处,1个月后处死并进行MicroCT检测及相关组织学(HE、Masson、天狼星红)染色,比较其成骨能力。实验结果:1、ASCs相对于BMSCs具有更强的增殖及成集落的能力,成骨能力较差;2、ASCs及BMSCs共培养后增殖能力提高;3、ASCs及BMSCs二维共培养后,适当加入BMSCs可以显著提高ASCs的成骨能力,且在ASCs:BMSCs 比例为4:3时成骨效果最好;4、ASCs及BM-ECM二维共培养后的茜素红染色比单纯ASCs要好,但单纯BM-ECM的染色结果更好;5、包括角蛋白水凝胶在内的三种天然合成物三维水凝胶在体外作为BMSCs三维培养的载体引导其增值、分化;6、人头发角蛋白三维水凝胶在植入动物体内后,不会产生炎症及免疫反应,并能够促进骨再生修复,作为骨组织工程支架材料具有生物安全性和有效性;结论:1、可以通过将ASCs与BMSCs二维共培养的方式来提高ASCs的成骨分化能力,在ASCs:BMSCs=4:3时效能最高;2、人头发来源的角蛋白水凝胶可以单独使用与BMSCs三维共培养作为骨组织工程的支架材料,但其成骨能力相对藻酸钙水凝胶较弱;3、细胞的微环境对于基于细胞的骨组织工程有重要的影响,可以通过物理、化学、生物等因素进行模拟和调控。更多还原

【Abstract】 Background:How to repair large area bone defects is a difficult problem in clinic,and the traditional methods have their own shortcomings.At present,the use of bone tissue engineering technology for repair,whether in seed cells or in the scaffold materials are not perfect.Bone marrow stromal cells are the main source of seed cells in bone tissue engineering.However,the process of harvesting them is suffering.Adipose tissue derived stromal cells are easy to obtain and large in quantity,which can meet the need of the source of autologous seed cells.But compared with bone marrow stromal cells,their osteogenic ability is relatively weak.Therefore,how to foster strengths and circumvent weaknesses means that not only makes use of the advantages of adipose derived stromal cells but also further enhance the osteogenic ability become an urgent problem.Traditional scaffold materials can not simulate the 3D microenvironment properly in vivo.Therefore,the research on the bio-derived scaffold materials is in the ascendant.However,how to obtain and prepare a large number of autologous or allogeneic biological source scaffold material is still quite limited.The scaffold of human hair keratin is a new strategy because of its extensive sources,convenient materials,good biocompatibility,economic and environmental protection,small immune response and so on.In this study,both from seed cells and three-dimensional hydrogel scaffolds are discussed on better utilization of fat and hair tissue in the human body which had been considered as waste tissue for bone tissue engineering resources to achieve bone regeneration and repairObjects:1)To study the feasibility of bone marrow stromal cells(BMSCs)and its secretion of extracellular matrix(BM-ECM)to promote the osteogenic ability of adipose tissue derived stromal cells(ASCs);2)To investigate the safety and efficacy of human hair keratin(HHK)three-dimensional hydrogel scaffolds as scaffold material for bone tissue engineering in vitro and in vivo;3)To investigate the effect of microenvironment on cell-based bone tissue engineering;Methods:1)ASCs and BMSCs were isolated from the same rat.To compare their ability of proliferation and osteogenic differentiation after amplification2)ASCs and BMSCs from the same rat were seeded in 6-well plates for direct co-culture and Transwell dishes for indirect co-culture.Then osteogenic related histochemistry stainings were processed and related genes were detected by real-time quantitative PCR after osteogenesis induced for 14 days3)To prepare the BM-ECM and to direct co-culture with the ASCs in 6-well plates Alizarin staining were processed after osteogenesis induced for 14 days4)Brown alginate,porcine derived collagen and human hair derived keratin were prepared to form 3D hydrogel and their relevant characterization were detected in vitro;5)Rat BMSCs were encapsulated in three hydrogel materials for three-dimensional co-culture and induction.The live and dead of the cells(AM/PI)and bone differentiation ability(ALP activity)were detected6)Three kinds of hydrogel encapsulated rat BMSCs which were osteogenesis induced for 14 days in vitro were implanted subcutaneously in nude mice or at critical sized calvarial defects of SD rat respectively.They were sacrificed after 1 month and microCT detection and relevant histological(HE,Masson and Sirius Red)staining were proceeded to evaluate the ability of bone formation among them.Results:1)BMSCs relative to ASCs has better osteogenic ability,and ASCs relative to BMSCs has stronger ability of proliferation and colony formation ability;2)The proliferation ability of ASCs and BMSCs co-culture group was increased after cultured for a long period.3)ASCs and BMSCs were co-cultured with two-dimensional plates,appropriate amount addition of BMSCs can greatly improve the ability of bone fomationg for ASCs,and it will achieve the best osteogenic effect when the ratio of ASCs:BMSCs is 4:3;4)The Alizarin Red staining result of ASCs and BM-ECM co-cultured is better than simply ASCs,but simply BM-ECM is even better;5)Including keratin hydrogel,three kinds of 3D biological derived hydrogel may mimic the micro environment in vivo.All of them have the ability to carrier BMSCs in a 3D culture system to guide their proliferation and differentiation in vitro;6)The safety and eff-icacy of human hair keratin(HHK)three-dimensional hydrogel as a scaffold material for bone tissue engineering is certified since there is no inflammatory or immune responses,and they can promote the regeneration of bone tissue in vivo.Conclusion:1)It is available to improve the capability of osteogenic differentiation of ASCs by co-culture ASCs and BMSCs,and ASCs:BMSCs=4:3 in the highest effectiveness;2)Human hair derived keratin hydrogel can be used alone to encapulate BMSCs for bone tissue engineering,but its osteogenesis ability is relatively weaker than calcium alginate hydrogel;3)The microenvironment for cell-based bone tissue engineering is critical and it can be simulated and controlled by physical,chemical and biological cues.更多还原