蚕丝-PLGA支架的生物相容性及力学性能的研究

发布时间：2018-05-16 21:49

本文选题：蚕丝-PLGA支架 + 骨髓间充质干细胞　；参考：《浙江省医学科学院》2015年硕士论文

【摘要】：前交叉韧带是膝关节内重要稳定结构,损伤后缺乏自我愈合能力,通常需要利用移植物关节镜下重建前交叉韧带。各种移植物都有其局限性,利用组织工程方法构建有望克服目前移植物的问题。理想的组织工程支架应具有良好的生物安全性、组织相容性,能为韧带/肌腱早期活动提供可靠的力学强度,为种子细胞的生长繁殖和生理功能发挥提供空间。本实验通过蚕丝-PLGA支架的细胞毒性、生物安全性,力学强度细胞相容性,支架降解液与细胞增殖的研究,得出蚕丝-PLGA支架是一种较理想的组织工程支架。第一部分：蚕丝-PLGA支架的细胞毒性及生物安全性目的：对蚕丝-PLGA混合编织支架进行体外细胞毒性实验和动物生物安全性评价,探讨蚕丝-PLGA混合编织支架材料的细胞毒性和生物安全性,为该材料临床应用提供理论依据。方法：制备蚕丝-PLGA细丝混合编织支架,并制备该支架材料的浸提液,通过MTT法检测25%、50%、100%浓度的蚕丝-PLGA混合编织支架材料浸提液对兔骨髓间充质干细胞(MSCs)的细胞毒性作用；采用蚕丝-PLGA细丝混合编织支架浸提液进行动物实验研究：急性全身毒性试验、皮内刺激试验、溶血试验、热原试验,并根据实验数据进行生物安全性的分析、评价。结果：在细胞毒性中,MSCs于不同浓度实验组及阴性对照组中生长均好,细胞呈长梭形等形态,细胞形态饱满有光泽,MTT法结果表明各时间点各浓度实验组与阴性对照组之间的A值差异不显著(P0.05),各时间点各浓度浸提液对MSCs细胞的相对增殖率均在93%以上,毒性分级为0级或1级,无细胞毒性。同时支架材料无急性全身毒性作用,无皮内刺激反应,不具溶血作用,符合生物材料溶血试验的要求。热原试验表明,3只实验兔体温升高数分别为0.4、0.2、0.3℃,均低于0.6℃,总值小于1.4℃,不具热原反应。结论：蚕丝-PLGA混合编织支架材料细胞相容性良好,无细胞毒性,符合材料毒性的安全标准,而且具有良好的生物相容性,是一种较有潜力的组织工程韧带/肌腱的支架材料。第二部分：蚕丝-PLGA支架的力学强度和生物相容性目的：通过对蚕丝-PLGA支架的力学性能及其与骨髓间充质干细胞体外共培养的细胞相容性实验,探讨使用该材料构建组织工程韧带的可行性。方法：通过捻拧编织法和纬编针织法制备蚕丝-PLGA共聚物细丝混合支架,并以纤维连接蛋白作表面修饰,检测支架材料的力学性能。将制备的兔骨髓间充质干细胞种植在蚕丝-PLGA共聚物细丝混合支架上进行体外共培养,观察细胞与支架复合生长、基质形成,以及细胞与支架结合的情况。结果：蚕丝-PLGA混合编织支架呈乳白色,质地均匀,韧性强,为螺旋上升的绳索状,直径为2.3mm。支架材料的最大负荷、拉伸强度、断点伸长率、弹性模量分别为(315.06 ±30.77)N、(75.83±7.46)MPa、(61.39±7.26)％、(213.58±23.45)MPa。扫描电镜观察显示,骨髓间充质干细胞贴附于支架表面生长,增殖良好,细胞大多呈梭形,伸出伪足匍匐于材料的表面,形态较佳,伸展良好,呈立体状生长,并分泌基质。纬编针织法制备的蚕丝-PLGA复合网状支架外观呈乳白色,质地均匀,韧性强,支架宽约为5mm。支架的最大载荷、抗拉强度、断点伸长率、弹性模量分别为(118.32± 11.21)N、(52.90±5.03)MPa、49.79%±5.16%、(177.25±19.18)MPa。支架-MSCs复合物体外培养2天的扫描电镜观察显示：MSCs细胞附于支架表面生长,黏附在支架上,呈梭形,形态较佳,呈立体状生长,增殖良好,并分泌基质。结论：表明蚕丝-PLGA共聚物绳状和网状支架都具有良好的机械性能及细胞相容性,是一种较有潜力的组织工程韧带的支架材料。第三部分：蚕丝-PLGA支架降解液与骨髓间充质干细胞的增殖目的：观察蚕丝-PLGA细丝混合编织支架体外长期降解过程中降解液对兔骨髓间充质干细胞增殖活性的影响。方法：将蚕丝-PLGA细丝混合编织支架材料置于完全培养基中体外降解14周,每周换液1次,测定各周支架降解液的pH值。将兔骨髓间充质干细胞分组培养,实验组加入各周支架降解液和新鲜完全培养基各100μL,阴性对照组加入完全培养基200μL,培养4d.MTT法检测细胞增殖、生长情况。结果：①支架降解液pH值的变化：前3周下降缓慢,从7.00降到6.89；第4周起下降较快,6-11周较低,在5.16-5.67之间；12-14周呈上升趋势,回升到6.95。②骨髓间充质干细胞形态：实验组及阴性对照组细胞增殖生长及形态状况基本相似。降解7-10周支架降解液对细胞的生长有抑制作用,细胞数量相对较少、较疏,而其余各周支架降解液对细胞生长无明显抑制作用。③骨髓间充质干细胞的增殖：1-6周及11-14周的支架降解液对细胞增殖无显著影响,细胞相对增殖率均在92.1%以上,毒性分级为0或1级；7-10周的支架降解液虽对细胞增殖有抑制作用,但细胞相对增殖率为82.5%-87.9%,毒性分级为1级,为合格。结论：表明蚕丝-PLGA共聚物混合编织支架降解液具有良好的细胞相容性。
[Abstract]:Anterior cruciate ligament (ACL) is an important stable structure in the knee joint, and it lacks self healing ability after injury. It is usually necessary to reconstruct the anterior cruciate ligament using graft arthroscopy. All kinds of grafts have its limitations. The tissue engineering scaffold should have good biological safety. Integrity and histocompatibility can provide a reliable mechanical strength for early ligament / tendon activity and provide space for the growth, reproduction and physiological function of seed cells. In this experiment, the cytotoxicity, biosafety, mechanical strength cell compatibility of the silk -PLGA scaffold, the study of the scaffold degradation liquid and cell proliferation, and the -PLGA branch of silkworm silk were obtained in this experiment. The frame is an ideal scaffold for tissue engineering. Part 1: cytotoxicity and biological safety of silk -PLGA scaffold: the cytotoxicity test and animal biological safety evaluation of silk -PLGA braided braid in vitro, and the cytotoxicity and biological safety of the silk -PLGA braid scaffold materials are discussed. The clinical application provides the theoretical basis. Methods: the silk -PLGA filaments mixed braiding stents were prepared and the extracts of the scaffold material were prepared. The cytotoxic effects of the extracts of the silk -PLGA mixed woven scaffold material on the rabbit bone marrow mesenchymal stem cells (MSCs) were detected by MTT, and the mixed braiding of the silk -PLGA filaments was used. Animal experimental study of scaffold leaching solution: acute systemic toxicity test, intradermal stimulation test, hemolysis test, pyrogen test, and biological safety analysis according to the experimental data. Results: in cytotoxicity, MSCs grew well in different concentration experimental group and negative control group, cell shape was long shuttle shape, cell shape The MTT method showed that there was no significant difference in A between the experimental group and the negative control group at each time point (P0.05). The relative proliferation rate of the extract solution to MSCs cells at each time point was above 93%, the toxicity was grade 0 or 1, and the scaffold material had no acute systemic toxicity and no intradermal spines. The irritable reaction, which was not hemolytic, accorded with the requirements of the hemolytic test of biomaterials. The thermometer test showed that the temperature rise of the 3 experimental rabbits was 0.4,0.2,0.3 C, lower than 0.6, and less than 1.4, and no thermion reaction. Conclusion: the silk -PLGA braided scaffold has good cytocompatibility and no cytotoxicity, which conforms to the toxicity of the material. Safety standards, with good biocompatibility, is a potential scaffold for tissue engineering ligaments / tendons. The second part: the mechanical strength and biocompatibility of silk -PLGA scaffold: the mechanical properties of silk -PLGA scaffold and the compatibility with bone marrow mesenchymal stem cells in vitro The feasibility of using this material to construct tissue engineering ligaments was explored. Methods: silk -PLGA copolymer filaments mixed scaffold was prepared by twisting and weft knitting method and weft knitting method, and the mechanical properties of the scaffold were detected with fibronectin as surface modification. The prepared rabbit bone marrow mesenchymal stem cells were planted in the silk -PLGA copolymer. The combined growth of cells and scaffolds, the formation of matrix, and the combination of the cells with the scaffold were observed on the mixed scaffold. Results: the silk -PLGA braided braid was milky white, with a uniform texture, strong toughness, a spiral rise rope, and the maximum load, tensile strength and breakpoint elongation of the 2.3mm. scaffold. The modulus of elasticity was (315.06 + 30.77) N, (75.83 + 7.46) MPa, (61.39 + 7.26)% and (213.58 + 23.45) MPa. scanning electron microscopy (213.58 + 23.45). The bone marrow mesenchymal stem cells were attached to the surface of the scaffold, with good proliferation, mostly spindle shaped and protruding on the surface of the material. The silkworm -PLGA composite mesh scaffold prepared by weft knitting method has a milky white appearance, uniform texture and strong toughness, and the width of the scaffold is about the maximum load of 5mm. scaffold, tensile strength, elongation at breakpoint, modulus of elasticity (118.32 + 11.21) N, (52.90 + 5.03) MPa, 49.79% + 5.16%, and (177.25 + 19.18) MPa. scaffold -MSCs compound for 2 days outside. The scanning electron microscopy (SEM) showed that MSCs cells attached to the surface of the scaffold grew on the scaffold, attached to the scaffold and were spindle shaped, with a better shape, good proliferation, and secreted matrix. Conclusion: the silk -PLGA copolymer and reticular scaffold have good mechanical properties and cell compatibility, which is a potential tissue engineering toughening. The third part: the third part: the proliferation of silkworm silk scaffold degradation solution and bone marrow mesenchymal stem cells: the effect of the degradation solution on the proliferation activity of rabbit bone marrow mesenchymal stem cells in the long term degradation process of silk -PLGA filament mixed braid. Method: the mixed silk -PLGA silk braid scaffold material is placed completely The culture medium was degraded for 14 weeks in vitro and changed 1 times a week to determine the pH value of the scaffold degradation solution in each week. The rabbit bone marrow mesenchymal stem cells were grouped into groups, the experimental group was added to the scaffold degradation solution and the fresh complete medium 100 mu L, the negative control group was added to the complete medium 200 mu L, and the cell proliferation and growth were detected by 4d.MTT method. The results were as follows: 1 The changes in the pH value of the scaffold degradation fluid: the decline in the first 3 weeks was slow, from 7 to 6.89, and the decrease was faster in the fourth week, the 6-11 week was lower, and between the 12-14 weeks. The 12-14 weeks showed an upward trend, and rose to the form of 6.95. bone marrow mesenchymal stem cells: the growth growth and morphology of the cells in the experimental group and the negative control group were basically similar. The degradation of the scaffold was reduced for 7-10 weeks. The solution has a inhibition effect on cell growth, and the number of cells is relatively small and sparse, while the rest of the scaffold degradation solution has no obvious inhibitory effect on cell growth for the rest of the week. The proliferation of bone marrow mesenchymal stem cells: 1-6 weeks and 11-14 weeks of scaffold degradation has no significant effect on cell proliferation, the relative proliferation rate of the cells is above 92.1% and the toxicity classification is 0. Or 1 grade; 7-10 weeks of scaffold degradation solution had inhibitory effect on cell proliferation, but the relative proliferation rate of cells was 82.5%-87.9% and the toxicity grade was 1, which was qualified. Conclusion: the results showed that the mixed woven scaffold degradation solution of silk -PLGA copolymer had good cytocompatibility.
【学位授予单位】：浙江省医学科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R318.08

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