皮肤组织工程种子细胞与支架材料的生物学研究
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摘要
组织工程是利用生命科学与工程科学的原理和方法,研究和开发具有修复或改善人体组织或器官功能的新一代临床应用替代物,以用于替代组织或器官的一部分或全部功能。组织工程的典型方法是从组织中分离出种子细胞并在体外进行培养扩增,然后,将扩增的细胞种植于生物支架材料中,通过在体外培养扩增形成新组织后植入患者的组织或器官病损部位,从而达到修复创伤和重建功能的目的。其中种子细胞和支架材料是组织工程最基本的要素,细胞种植于支架材料上构建组织是组织工程技术的核心,也是形成组织工程化生物活性组织的关键所在。
本研究应用组织工程学的原理和方法,从大鼠皮肤中分离出具有稳定生物学性状的真皮成纤维细胞(Fibroblasts,Fbs);用本实验室提取的Ⅰ型胶原及壳聚糖制备出适于组织工程皮肤构建的胶原.壳聚糖海绵支架,并将Fbs种植在该支架中进行联合培养来研究组织构建过程中种子细胞与支架材料的相互作用,为组织工程皮肤制备技术的改进和完善提供理论依据。本文主要研究结果如下:
1.用DispaseⅡ-胶原酶联合消化法进行SD大鼠皮肤Fbs的分离和培养。该方法获得的Fbs生长迅速、增殖能力强、生物学性状稳定,适于构建组织工程皮肤。
2.以胶原和壳聚糖为原料,戊二醛为交联剂,采用冷冻干燥法制备出的胶原-壳聚糖组织工程支架,孔隙率大于90%、平均孔径大小为116.5μm,具备一定的柔韧性和力学强度。细胞相容性实验表明,该支架无细胞毒性,具有良好的细胞粘附性能。
3.将Fbs种植于胶原-壳聚糖组织工程支架中进行联合培养,并对支架中细胞的生长特性和生物学活性进行检测。用MTT实验和流式细胞术检测细胞周期和凋亡情况结果表明,Fbs在支架中能够缓慢而稳定地增殖。用EILISA法检测IL-6的分泌情况发现,该支架能促进Fbs细胞因子的分泌。
实验结果表明,组织工程皮肤构建过程中种子细胞与三维多孔支架间的相互作用类似于体内正常组织中细胞-细胞外基质间的动态相互作用。以上各实验结果数据对于人工细胞外基质-支架材料的改进和体外人工组织器官构建的改进和完善具有重要的参考意义。
Tissue engineering utilizes the principles and methods of life science and engineering science, researching and developing new clinic substitute for human tissue and apparatus.Its typical method is seeding cells in the three-dimensional biomaterial scaffolds with excellent biocompatibility to form artificial tissue constructs after isolating and proliferating seed-cells in vitro. Thereinto, seed-cells and scaffold materials are two basal elements in tissue engineering. Planting the seed-cells into scaffolds to construct artificial tissue is tissue engineering technology's core and even more the key to successful construction.
According to tissue engineering's principles and methods, we isolated dermal fibroblasts(Fbs) with stable biological character from SD rat skin and fabricated collagen-chitosan sponge scaffolds which were suitable for tissue engineering skin construction, then transplanted the Fbs on the sponge scaffolds. We studied the two elements' biological characters'changes in the process of tissue construction in order to provide theory data for the tissue engineering technology's improvement and perfection. The main results of the research are as follows:
1. We successfully isolated SD rat's skin Fbs by using Dispase II- type I collagenase digestion. The Fbs are suitable for tissue engineering skin construction research for their strong proliferation ability and stable biological characteristic.
2. The collagen-chitosan sponge scaffolds were fabricated with type I collagen and chitosan by freeze-drying and then cross-linked with glutaraldehyde(GA). The scaffolds have interconnected pore structure, suitable pore sizes and good mechanical property. The cell compatibility experiments indicated that the scaffolds had no cytotoxicity and showed good cell adhesion property.
3. We transplanted the Fbs on the collagen-chitosan scaffolds and studied the seed-cells' proliferation and biological activity. MTT test and flow cytometry indicated that cells on the scaffolds proliferated slowly and steadily. IL-6 concentration measurement by ELISA test indicated that the scaffolds could promote Fbs' cytokines secretion.
In summary, Fbs and the 3D-scaffolds interacted with each other in the process of tissue construction. This process was similar with dynamic effection of cells and extracellularmatrix (ECM) in vivo. These experiment data are important for scaffold materials' improvement and artificial tissues' construction in vitro.
本研究应用组织工程学的原理和方法,从大鼠皮肤中分离出具有稳定生物学性状的真皮成纤维细胞(Fibroblasts,Fbs);用本实验室提取的Ⅰ型胶原及壳聚糖制备出适于组织工程皮肤构建的胶原.壳聚糖海绵支架,并将Fbs种植在该支架中进行联合培养来研究组织构建过程中种子细胞与支架材料的相互作用,为组织工程皮肤制备技术的改进和完善提供理论依据。本文主要研究结果如下:
1.用DispaseⅡ-胶原酶联合消化法进行SD大鼠皮肤Fbs的分离和培养。该方法获得的Fbs生长迅速、增殖能力强、生物学性状稳定,适于构建组织工程皮肤。
2.以胶原和壳聚糖为原料,戊二醛为交联剂,采用冷冻干燥法制备出的胶原-壳聚糖组织工程支架,孔隙率大于90%、平均孔径大小为116.5μm,具备一定的柔韧性和力学强度。细胞相容性实验表明,该支架无细胞毒性,具有良好的细胞粘附性能。
3.将Fbs种植于胶原-壳聚糖组织工程支架中进行联合培养,并对支架中细胞的生长特性和生物学活性进行检测。用MTT实验和流式细胞术检测细胞周期和凋亡情况结果表明,Fbs在支架中能够缓慢而稳定地增殖。用EILISA法检测IL-6的分泌情况发现,该支架能促进Fbs细胞因子的分泌。
实验结果表明,组织工程皮肤构建过程中种子细胞与三维多孔支架间的相互作用类似于体内正常组织中细胞-细胞外基质间的动态相互作用。以上各实验结果数据对于人工细胞外基质-支架材料的改进和体外人工组织器官构建的改进和完善具有重要的参考意义。
Tissue engineering utilizes the principles and methods of life science and engineering science, researching and developing new clinic substitute for human tissue and apparatus.Its typical method is seeding cells in the three-dimensional biomaterial scaffolds with excellent biocompatibility to form artificial tissue constructs after isolating and proliferating seed-cells in vitro. Thereinto, seed-cells and scaffold materials are two basal elements in tissue engineering. Planting the seed-cells into scaffolds to construct artificial tissue is tissue engineering technology's core and even more the key to successful construction.
According to tissue engineering's principles and methods, we isolated dermal fibroblasts(Fbs) with stable biological character from SD rat skin and fabricated collagen-chitosan sponge scaffolds which were suitable for tissue engineering skin construction, then transplanted the Fbs on the sponge scaffolds. We studied the two elements' biological characters'changes in the process of tissue construction in order to provide theory data for the tissue engineering technology's improvement and perfection. The main results of the research are as follows:
1. We successfully isolated SD rat's skin Fbs by using Dispase II- type I collagenase digestion. The Fbs are suitable for tissue engineering skin construction research for their strong proliferation ability and stable biological characteristic.
2. The collagen-chitosan sponge scaffolds were fabricated with type I collagen and chitosan by freeze-drying and then cross-linked with glutaraldehyde(GA). The scaffolds have interconnected pore structure, suitable pore sizes and good mechanical property. The cell compatibility experiments indicated that the scaffolds had no cytotoxicity and showed good cell adhesion property.
3. We transplanted the Fbs on the collagen-chitosan scaffolds and studied the seed-cells' proliferation and biological activity. MTT test and flow cytometry indicated that cells on the scaffolds proliferated slowly and steadily. IL-6 concentration measurement by ELISA test indicated that the scaffolds could promote Fbs' cytokines secretion.
In summary, Fbs and the 3D-scaffolds interacted with each other in the process of tissue construction. This process was similar with dynamic effection of cells and extracellularmatrix (ECM) in vivo. These experiment data are important for scaffold materials' improvement and artificial tissues' construction in vitro.
引文
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