山羊半月板脱细胞基质支架的制备及扩大孔径方法的研究

发布时间：2018-02-10 20:39

本文关键词： 半月板脱细胞基质支架山羊　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：半月板对于维持膝关节的生理功能是很重要的,半月板损伤与膝关节骨关节炎(OA)的发生密切相关。由于半月板的自我修复能力有限,半月板部分或全部切除是治疗半月板损伤的常用手段。随着医学的发展,组织工程对损伤半月板缺损的再生修复带来了希望,而找寻合适的半月板支架在半月板组织工程中起着至关重要的作用。脱细胞半月板支架不仅具有良好的三维结构、生物相容性和生物力学性能。本文旨在研究山羊半月板脱细胞基质支架的制备方法、观察它们的形态结构、测定其生物力学性能,并且进一步研究扩大支架孔径地方法,为构建组织工程半月板研究打下基础。方法:取淘汰山羊半月板,采用不同脱细胞方法进行山羊半月板脱细胞基质支架的制备。方法一:采用高渗-低渗反复冻融、Tris-HCl溶液联合胰蛋白酶溶液进行半月板基质的脱细胞处理;方法二:用高渗-低渗反复冻融、双氧水和胰蛋白酶溶液轮流浸泡后,再用化学去垢剂TrionX-100溶液和脱氧胆酸钠溶液交替使用进行半月板基质的脱细胞处理。组织学观察:用HE染色、天狼猩红染色、甲苯胺蓝染色、番红O染色和番红-固绿染色方法对两种不同方法制备的脱细胞基质进行组织学检测,并将无细胞残留组与天然半月板进行扫描电镜观察和生物力学测定。最后分别用甲酸、I型胶原酶、胰蛋白酶+I型胶原酶对制备好的山羊半月板脱细胞基质支架用进行处理,在扫描电镜下观察并进行生物力学测定。结果:方法一制备的脱细胞支架组织学观察有细胞残留,方法二制备的脱细胞支架组织学检测无任何细胞残留,基质中保存了大量的胶原纤维。扫描电镜结果显示脱细胞支架组的孔径和孔隙率比天然半月板略大,生物力学性能上二者无显著性差异。扩大孔径研究结果显示:甲酸组、I型胶原酶组和胰蛋白酶+I型胶原酶组的孔径和孔隙率较脱细胞基质支架的大,生物力学测定显示甲酸组、I型胶原酶组和胰蛋白酶+I型胶原酶组与脱细胞基质支架组均存在显著性差异。结论:1.用蒸馏水-10%NaCl溶液反复冻融、0.1%双氧水和0.5%的胰蛋白酶轮流浸泡后,再用化学去垢剂TrionX-100溶液和脱氧胆酸钠溶液交替使用进行半月板基质脱细胞方法可以将羊半月板中的细胞成分除去,并保留了半月板的三维立体结构,生物力学特性与正常半月板接近,可以作为半月板支架材料;2.甲酸、I型胶原酶、胰蛋白酶+I型胶原酶对山羊脱细胞基质支架处理结果显示,支架的孔径和孔隙率均有所提升,但会造成生物力学性能的下降。
[Abstract]:Meniscus is very important to maintain the physiological function of knee joint. Meniscus injury is closely related to the occurrence of osteoarthritis of knee joint (OAA). Partial or total meniscal excision is a common method in the treatment of meniscus injury. With the development of medicine, tissue engineering brings hope to the regeneration and repair of injured meniscus defect. Finding suitable meniscus scaffolds plays an important role in meniscus tissue engineering. Acellular meniscus scaffolds not only have a good three-dimensional structure. The purpose of this paper is to study the preparation methods of goat meniscus acellular matrix scaffolds, observe their morphological structure, determine their biomechanical properties, and further study the method of expanding the pore size of the scaffolds. Methods: goat meniscus was eliminated. Preparation of goat meniscus acellular matrix scaffolds with different acellular methods. Methods: 1. Hyperosmotic and hypotonic thawing Tris-HCl solution combined with trypsin solution were used to acellular the meniscus matrix. Method two: after repeated freezing and thawing with hyperosmotic and hypotonic solution, the solution of hydrogen peroxide and trypsin alternately soaked, Then acellular treatment of meniscus matrix was carried out with chemical defouling agent TrionX-100 solution and sodium deoxycholate solution alternately. Histological observation: stained with HE, Sirius red, toluidine blue. The acellular substrates prepared by two different methods were detected by the methods of phannin O staining and fast green staining. Scanning electron microscopy and biomechanical analysis were carried out between the cells free residual group and the natural meniscus. Finally, the prepared scaffolds of goat meniscus acellular matrix were treated with formic acid type I collagenase and trypsin type I collagenase, respectively. Results: methods: cell residues were observed in the acellular scaffolds, and no cell residues were observed in the acellular stents prepared in the second method. A large number of collagen fibers were preserved in the matrix. SEM results showed that the pore size and porosity of the acellular scaffolds were slightly larger than those of the natural meniscus. There was no significant difference in biomechanical properties between the two groups. The results of expanded pore size study showed that the pore size and porosity of formic acid group and trypsin type I collagenase group were larger than those of acellular matrix scaffold. Biomechanical measurements showed that there were significant differences between formic acid group and trypsin type I collagenase group and acellular matrix scaffold group. Conclusion: 1. Frozen and thawed with distilled water -10 NaCl solution, 0.1% hydrogen peroxide and 0.5% pancreatic eggs were repeatedly frozen and thawed with distilled water. After the white enzyme was soaked in turns, Using the chemical descaling agent TrionX-100 solution and sodium deoxycholate solution alternately to carry on the meniscus matrix acellular method can remove the cell composition from the sheep meniscus and retain the three-dimensional structure of the meniscus. The biomechanical properties were close to the normal meniscus and could be used as meniscus scaffolds. The results of treatment of goat acellular matrix scaffolds with formate type I collagenase and trypsin type I collagenase showed that the pore size and porosity of the scaffolds were increased. But the biomechanical properties will decrease.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.0

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