【摘要】：組織工程概念的提出為修復(fù)重建病損組織或器官帶來了希望,其基本原理是基于細(xì)胞、材料以及生物因子的有機結(jié)合,構(gòu)建出具有與天然組織結(jié)構(gòu)和功能類似的組織器官替代物。細(xì)胞在體內(nèi)處于特定的微環(huán)境之中,受到嚴(yán)格的調(diào)控；而生物材料則需要通過模擬細(xì)胞的體內(nèi)微環(huán)境,來精確調(diào)控細(xì)胞行為,才可能最終促進(jìn)組織結(jié)構(gòu)的形成。近年來的研究發(fā)現(xiàn),生物材料的物理結(jié)構(gòu)及力學(xué)特性都是決定體外細(xì)胞行為的關(guān)鍵因素。孔徑作為多孔生物支架材料的關(guān)鍵參數(shù),對于組織結(jié)構(gòu)的形成有著決定性的影響。但是,目前的認(rèn)識還不具系統(tǒng)性,依然有待深入研究。 為此,本論文基于生物相容性好且可降解的聚己內(nèi)酯(Polycaprolactone, PCL)材料,制備了具有不同孔徑(100-200μm,200-300μm,300-450μm和450-600μm)的三維多孔支架；考察了支架孔徑的變化對一系列組織工程種子細(xì)胞生物學(xué)行為的影響,包括對人羊膜來源的間充質(zhì)干細(xì)胞(human aminotic membrane-derived mesenchymal stem cells, hAMSCs)的粘附、增殖及分化的調(diào)節(jié)作用,對兔關(guān)節(jié)軟骨細(xì)胞(rabbit articular chondrocytes, rACs)表型維持和分化的影響,以及對人臍靜脈內(nèi)皮細(xì)胞(human umbilical endothelial cells, HUVEC)與hAMSCs共培養(yǎng)條件下]hAMSCs成骨分化能力的影響作用。 通過以上實驗研究,我們發(fā)現(xiàn)：①結(jié)合溶劑澆注/粒子浸出和熱致相分離技術(shù)能成功制備四種不同孔徑的三維多孔支架,且支架的連通性良好,孔隙率均在95%以上；②hAMSCs和rACs在三維多孔支架上均能很好的粘附和增殖,但是hAMSCs增殖會隨著孔徑的增加而不斷降低,同時hAMSCs往支架中遷移的深度隨著孔徑的增加而增加；③在成骨誘導(dǎo)培養(yǎng)條件下,孔徑較小(100-300μm)的支架更有利于hAMSCs的成骨分化,而過大的孔徑不利于hAMSCs的成骨分化；④在成軟骨誘導(dǎo)培養(yǎng)條件下,孔徑為200-450μm的支架更有利于hAMSCs和P3rACs的成軟骨分化,而孔徑過大或過小都不利于細(xì)胞的成軟骨分化,相反,稍大的孔徑對P1rACs軟骨表型的維持比較有利；⑤hAMSCs和HUVEC以hAMSCs:HUVEC=3:1的數(shù)量比在混合培養(yǎng)基中直接混合共培養(yǎng)更利于hAMSCs的成骨分化和血管化。 因此,基于本研究工作,三維多孔支架的孔徑對1hAMSCs的粘附、增殖和分化以及rACs的表型都有顯著影響,這揭示了支架微結(jié)構(gòu)對細(xì)胞生物學(xué)行為的調(diào)控規(guī)律,對指導(dǎo)組織構(gòu)建具有重要的意義。但是,對于孔徑調(diào)節(jié)細(xì)胞行為的機制還有待深入研究,同時HUVEC是如何促進(jìn)hAMSCs成骨誘導(dǎo)分化的,也需要進(jìn)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.
【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R318.08

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