化学修饰三维多孔支架及其应用于间充质干细胞和软骨细胞培养的研究

发布时间：2018-03-04 03:17

本文选题：组织工程　切入点：多孔支架　出处：《华东理工大学》2015年硕士论文　论文类型：学位论文

【摘要】：组织工程方法的基本要素包括种子细胞、支架材料和细胞因子。间充质干细胞(Mesenchymal stem cells, MSCs)和成体细胞如软骨细胞等都是组织工程研究中重要的种子细胞。三维多孔支架的孔径大小以及不同化学基团的表面修饰都可能对细胞行为有重要影响,但文献报道主要集中在对材料单一性质的研究,而将材料理化性质相结合系统考察生物材料与细胞之间相互作用的研究相对匮乏。为此,本论文首先选用聚己内酯(P olycaprolactone, PCL)材料,制备得到表面形貌光滑的PCL膜和不同孔径(100-200μ、200-300 μm和300-450μm)的PCL三维多孔支架；然后考察不同表面化学修饰的PCL薄膜对人MSCs (human MSCs, hMSCs)贴附和生长的影响；进一步重点考察了不同孔径和表面化学修饰的PCL多孔支架材料对hMSCs的生长、迁移、增殖和分化的影响；最后,将不同密度的兔关节软骨细胞(1abbit articular chondrocytes, rACs)接种在不同孔径的PCL支架材料上,考察接种密度和孔径对软骨细胞表型维持的影响。结果发现：(1)通过选择合适的化学修饰条件能够维持PCL薄膜表面形貌,而接触角均有降低；(2)水解处理的PCL薄膜促进了hMSCs的贴附、铺展和增殖,而过于剧烈的氨解处理则使PCL表面不利于hMSC s的生长；(3)化学修饰的PCL多孔支架接触角降低,连通性良好,hMSCs可以贴着孔壁生长,也能跨孔生长,修饰后的支架更有利于细胞的贴附和增殖,而且200-300μm孔径的支架较其它孔径更有利于细胞贴附和增殖；(4)整体上,修饰后的支架有利于hMSCs成骨分化,PCL-H支架更能促进hMSCs成软骨分化,而且孔径为200-450μm的支架更能促进hMSCs成软骨分化；(5)软骨细胞在支架内都出现了不同程度的去分化现象,接种密度为5×104 cells/scaffold比30×104 cells/scaffold的增殖速度更快,接种密度为30×104 cells/scaffold比5×104cells/scaffod和15×104 cells/scaffod对P1rACs表型的维持更有利；(6)300-450μm孔径支架上的P1rACs的生长和糖胺聚糖(Glycosaminoglycans, GAG)的分泌较其它孔径略大,而且细胞向支架内部迁移较其它孔径更深。综上所述,多孔支架材料的表面化学性质和孔径尺寸都会对hMSCs的粘附、增殖和分化以及软骨细胞表型的维持都有显著影响,而且存在显著的协同作用。该研究结果将可能为今后的组织构建过程和软骨组织工程提供科学的指导。
[Abstract]:The basic elements of tissue engineering methods including seed cells, scaffolds and cytokine. Mesenchymal stem cells (Mesenchymal stem cells, MSCs) and adult cells such as cartilage cells are important seed cells in the tissue engineering. Three dimensional porous scaffolds with different pore size and surface modification of chemical groups may have an important influence on the behavior of cells, but the literature reports mainly focus on research on single material properties, and the material physical and chemical properties of the system combining investigation study of the interaction between biomaterials and cells is relatively scarce. Therefore, this paper chooses polycaprolactone (P olycaprolactone, PCL) materials, preparation of PCL film surface the morphology of smooth and different aperture (100-200, 200-300 m and 300-450 m) PCL three-dimensional porous scaffold; and the effects of different surface chemical modification of PCL films on MSCs (human MSCs, HMSCs) the adhesion and growth effect; the further study on the growth of PCL porous scaffolds with different pore sizes and surface chemical modification on hMSCs migration, proliferation and differentiation; finally, the different density of rabbit articular chondrocytes (1abbit articular, chondrocytes, rACs) were seeded on PCL scaffolds with different pore sizes, influence effects of inoculum density and pore size on the phenotype of chondrocytes maintained. Results showed that: (1) by choosing appropriate chemical modification conditions to maintain the PCL film surface morphology, and the contact angle decreased; (2) PCL films attached to promote the hydrolysis of hMSCs, spreading and proliferation, and severe ammonia solution the PCL surface is not conducive to the growth of s hMSC; (3) the chemical modification of porous PCL contact angle decreased, good connectivity, hMSCs may be close to the hole wall growth, can also cross hole growth, support the modified more For cell adhesion and proliferation, but also support 200-300 m pore diameter is more conducive to cell adhesion and proliferation of other aperture; (4) on the whole, the modified scaffold for hMSCs osteogenic differentiation, PCL-H scaffolds can promote chondrogenic differentiation of hMSCs, and the aperture of 200-450 m stents can promote hMSCs chondrogenic differentiation of cartilage cells; (5) there are different degrees of dedifferentiation in stent, inoculation density of 5 * 104 cells/scaffold 30 * 104 faster than the cells/scaffold growth rate and inoculation density of 30 * 104 cells/scaffold 5 * 104cells/scaffod and 15 * 104 cells/scaffod to maintain the phenotype of P1rACs (more favorable; 6) 300-450 m aperture bracket on the growth of P1rACs and glycosaminoglycans (Glycosaminoglycans, GAG) secretion than the other slightly larger aperture, and the cell to support internal migration aperture deeper than the other. In summary, many The surface chemical properties and pore size of scaffold materials will adhere to hMSCs, have a significant impact on the proliferation and differentiation and maintain the phenotype of chondrocytes, and there is a significant synergistic effect. The results of this study may provide scientific guidance and cartilage tissue engineering for the future of the organization.

【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R318.08 ;TQ317

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