具有不同孔径的三维多孔支架材料的制备以及孔径对细胞体外生长和分化调节作用的研究
发布时间：2019-01-23 15:51
【摘要】：组织工程概念的提出为修复重建病损组织或器官带来了希望,其基本原理是基于细胞、材料以及生物因子的有机结合,构建出具有与天然组织结构和功能类似的组织器官替代物。细胞在体内处于特定的微环境之中,受到严格的调控；而生物材料则需要通过模拟细胞的体内微环境,来精确调控细胞行为,才可能最终促进组织结构的形成。近年来的研究发现,生物材料的物理结构及力学特性都是决定体外细胞行为的关键因素。孔径作为多孔生物支架材料的关键参数,对于组织结构的形成有着决定性的影响。但是,目前的认识还不具系统性,依然有待深入研究。 为此,本论文基于生物相容性好且可降解的聚己内酯(Polycaprolactone, PCL)材料,制备了具有不同孔径(100-200μm,200-300μm,300-450μm和450-600μm)的三维多孔支架；考察了支架孔径的变化对一系列组织工程种子细胞生物学行为的影响,包括对人羊膜来源的间充质干细胞(human aminotic membrane-derived mesenchymal stem cells, hAMSCs)的粘附、增殖及分化的调节作用,对兔关节软骨细胞(rabbit articular chondrocytes, rACs)表型维持和分化的影响,以及对人脐静脉内皮细胞(human umbilical endothelial cells, HUVEC)与hAMSCs共培养条件下]hAMSCs成骨分化能力的影响作用。 通过以上实验研究,我们发现：①结合溶剂浇注/粒子浸出和热致相分离技术能成功制备四种不同孔径的三维多孔支架,且支架的连通性良好,孔隙率均在95%以上；②hAMSCs和rACs在三维多孔支架上均能很好的粘附和增殖,但是hAMSCs增殖会随着孔径的增加而不断降低,同时hAMSCs往支架中迁移的深度随着孔径的增加而增加；③在成骨诱导培养条件下,孔径较小(100-300μm)的支架更有利于hAMSCs的成骨分化,而过大的孔径不利于hAMSCs的成骨分化；④在成软骨诱导培养条件下,孔径为200-450μm的支架更有利于hAMSCs和P3rACs的成软骨分化,而孔径过大或过小都不利于细胞的成软骨分化,相反,稍大的孔径对P1rACs软骨表型的维持比较有利；⑤hAMSCs和HUVEC以hAMSCs:HUVEC=3:1的数量比在混合培养基中直接混合共培养更利于hAMSCs的成骨分化和血管化。 因此,基于本研究工作,三维多孔支架的孔径对1hAMSCs的粘附、增殖和分化以及rACs的表型都有显著影响,这揭示了支架微结构对细胞生物学行为的调控规律,对指导组织构建具有重要的意义。但是,对于孔径调节细胞行为的机制还有待深入研究,同时HUVEC是如何促进hAMSCs成骨诱导分化的,也需要进一步的考察。
[Abstract]:The concept of tissue engineering offers hope for the repair and reconstruction of diseased tissues or organs, and its basic principle is based on the organic combination of cells, materials and biological factors. A tissue-organ substitute with a similar structure and function to the natural tissue was constructed. Cells in a specific microenvironment are strictly regulated, while biomaterials need to accurately regulate cell behavior by mimicking the microenvironment of cells in order to ultimately promote the formation of tissue structures. In recent years, it has been found that the physical structure and mechanical properties of biomaterials are the key factors to determine cell behavior in vitro. Pore size, as a key parameter of porous biological scaffolds, has a decisive effect on the formation of tissue structure. However, the current understanding is not systematic, still need to be further studied. Therefore, based on the biocompatible and degradable poly (caprolactone) (Polycaprolactone, PCL) materials, three dimensional porous scaffolds with different pore sizes (100-200 渭 m, 200-300 渭 m, 300-450 渭 m and 450-600 渭 m) were prepared. The effects of scaffold pore size on the biological behavior of a series of tissue engineering seed cells were investigated, including the regulation of (human aminotic membrane-derived mesenchymal stem cells, hAMSCs) adhesion, proliferation and differentiation of human amniotic membrane derived mesenchymal stem cells. The effects on the maintenance and differentiation of (rabbit articular chondrocytes, rACs) phenotype in rabbit articular chondrocytes and on the osteogenic differentiation of hAMSCs in human umbilical vein endothelial cells co-cultured with (human umbilical endothelial cells, HUVEC) and hAMSCs. Through the above experiments, we found that: 1 combined with solvent-pouring / particle leaching and thermo-induced phase separation technology, four kinds of three-dimensional porous scaffolds with different pore sizes can be successfully prepared, and the scaffolds have good connectivity with porosity above 95%; Both 2hAMSCs and rACs could adhere and proliferate well on the three-dimensional porous scaffold, but the hAMSCs proliferation decreased with the increase of pore size, and the depth of hAMSCs migration into the scaffold increased with the increase of pore size. 3 under the condition of osteogenic culture, the scaffold with small pore diameter (100-300 渭 m) was more favorable to the osteogenic differentiation of hAMSCs, but too large pore diameter was not conducive to the osteogenic differentiation of hAMSCs. (4) under the condition of chondrogenic culture, the scaffolds with a diameter of 200-450 渭 m were more favorable for the chondrogenic differentiation of hAMSCs and P3rACs, while too large or too small aperture was not conducive to the differentiation of chondrogenic cells. Larger aperture is beneficial to the maintenance of P1rACs cartilage phenotype. 5hAMSCs and HUVEC were more conducive to osteogenic differentiation and vascularization of hAMSCs than direct co-culture in mixed medium with the quantity of hAMSCs:HUVEC=3:1. Therefore, based on this work, the pore size of three-dimensional porous scaffolds has a significant effect on the adhesion, proliferation and differentiation of 1hAMSCs, as well as the phenotype of rACs, which reveals the regulation of scaffold microstructure on the biological behavior of cells. It is of great significance to guide the construction of organization. However, the mechanism of pore size regulation of cell behavior remains to be further studied, and how HUVEC promotes the differentiation of hAMSCs osteogenesis needs further investigation.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R318.08
本文编号：2413962
[Abstract]:The concept of tissue engineering offers hope for the repair and reconstruction of diseased tissues or organs, and its basic principle is based on the organic combination of cells, materials and biological factors. A tissue-organ substitute with a similar structure and function to the natural tissue was constructed. Cells in a specific microenvironment are strictly regulated, while biomaterials need to accurately regulate cell behavior by mimicking the microenvironment of cells in order to ultimately promote the formation of tissue structures. In recent years, it has been found that the physical structure and mechanical properties of biomaterials are the key factors to determine cell behavior in vitro. Pore size, as a key parameter of porous biological scaffolds, has a decisive effect on the formation of tissue structure. However, the current understanding is not systematic, still need to be further studied. Therefore, based on the biocompatible and degradable poly (caprolactone) (Polycaprolactone, PCL) materials, three dimensional porous scaffolds with different pore sizes (100-200 渭 m, 200-300 渭 m, 300-450 渭 m and 450-600 渭 m) were prepared. The effects of scaffold pore size on the biological behavior of a series of tissue engineering seed cells were investigated, including the regulation of (human aminotic membrane-derived mesenchymal stem cells, hAMSCs) adhesion, proliferation and differentiation of human amniotic membrane derived mesenchymal stem cells. The effects on the maintenance and differentiation of (rabbit articular chondrocytes, rACs) phenotype in rabbit articular chondrocytes and on the osteogenic differentiation of hAMSCs in human umbilical vein endothelial cells co-cultured with (human umbilical endothelial cells, HUVEC) and hAMSCs. Through the above experiments, we found that: 1 combined with solvent-pouring / particle leaching and thermo-induced phase separation technology, four kinds of three-dimensional porous scaffolds with different pore sizes can be successfully prepared, and the scaffolds have good connectivity with porosity above 95%; Both 2hAMSCs and rACs could adhere and proliferate well on the three-dimensional porous scaffold, but the hAMSCs proliferation decreased with the increase of pore size, and the depth of hAMSCs migration into the scaffold increased with the increase of pore size. 3 under the condition of osteogenic culture, the scaffold with small pore diameter (100-300 渭 m) was more favorable to the osteogenic differentiation of hAMSCs, but too large pore diameter was not conducive to the osteogenic differentiation of hAMSCs. (4) under the condition of chondrogenic culture, the scaffolds with a diameter of 200-450 渭 m were more favorable for the chondrogenic differentiation of hAMSCs and P3rACs, while too large or too small aperture was not conducive to the differentiation of chondrogenic cells. Larger aperture is beneficial to the maintenance of P1rACs cartilage phenotype. 5hAMSCs and HUVEC were more conducive to osteogenic differentiation and vascularization of hAMSCs than direct co-culture in mixed medium with the quantity of hAMSCs:HUVEC=3:1. Therefore, based on this work, the pore size of three-dimensional porous scaffolds has a significant effect on the adhesion, proliferation and differentiation of 1hAMSCs, as well as the phenotype of rACs, which reveals the regulation of scaffold microstructure on the biological behavior of cells. It is of great significance to guide the construction of organization. However, the mechanism of pore size regulation of cell behavior remains to be further studied, and how HUVEC promotes the differentiation of hAMSCs osteogenesis needs further investigation.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R318.08
【参考文献】
相关期刊论文 前2条
1 孙安科,裴国献;组织工程研究面临的问题和发展方向[J];国外医学.生物医学工程分册;2003年05期
2 张睿婷;韩之波;王涛;孟磊;刘志鹏;李奎;及月茹;杨洲鑫;李扬秋;韩忠朝;;人胎盘绒毛膜来源间充质干细胞的生物学特性[J];中国组织工程研究与临床康复;2011年10期
，本文编号：2413962
本文链接：https://www.wllwen.com/yixuelunwen/swyx/2413962.html
