兼具抗炎及血管化微球型可塑性骨支架材料的制备及其对HPMSCs生物学活性的影响

发布时间：2018-04-16 21:25

本文选题：血管内皮生长因子 + 万古霉素　；参考：《吉林大学》2017年硕士论文

【摘要】：研究目的:1.以海藻酸盐及壳聚糖为载体,研制同时装载VEGF及万古霉素的微球型、可塑性骨支架材料,并对其性能进行分析。2.用无菌环境中制备的双重载药多层海藻酸盐-壳聚糖微球型支架材料完全细胞培养液浸出液培养第3代HPMSCs,并检测用支架材料浸出液培养后,HPMSCs增殖活性及成骨活性的变化。研究方法:1.采用组织块培养法从人足月胎盘组织中分离培养HPMSCs,采用MTT法检测并绘制细胞生长曲线,对第3代HPMSCs进行成骨诱导以及成脂诱导,验证其多向分化潜能;2.按照前期实验在无菌环境中制备多层载药缓释微球的方法,灵活运用海藻酸盐和壳聚糖交联原理,利用海藻酸盐和壳聚糖交联产物的可塑性,将缓释与支架支撑功能相结合,选择微球与支架最优配比,探索在无菌环境中制备双重载药多层海藻酸盐-壳聚糖微球型支架材料;3.将制备好的支架材料与第3代HPMSCs共培养,采用CCK-8试剂盒检测细胞增殖能力,采用茜素红染色及ALP试剂盒检测细胞成骨能力的变化。研究结果:1.倒置显微镜下观察到从人足月胎盘组织中分离培养出的细胞呈成纤维细胞样贴壁生长,细胞生长曲线呈“S”形,并且细胞经过成骨诱导后可以见到胞外有钙盐沉积、茜素红染色阳性,经成脂诱导后油红O染色阳性;2.无菌环境下制得的双重载药多层海藻酸盐-壳聚糖微球型支架材料形态可塑,并且兼具缓释与支架功能;3.与支架材料浸出液共培养后,HPMSCs的增殖能力无显著变化(P0.05);成骨诱导后,茜素红染色显示载药微球支架组钙盐沉积多于空载微球支架组、无支架组和空白对照组,ALP试剂盒测试结果证实加入载药微球支架后细胞内碱性磷酸酶含量明显增高(P0.05),空载微球支架组与无支架组相比也有显著性差异(P0.05)。研究结论:1.成功从人足月胎盘组织中分离培养出HPMSCs,对其生物学特征鉴定后证明其符合干细胞特性;2.成功制备出形态可塑的双重载药多层海藻酸盐-壳聚糖微球型支架材料;3.通过双重载药多层海藻酸盐-壳聚糖微球型支架浸出液与HPMSCs共培养,证明其对HPMSCs增殖活性无明显影响,但能显著改善HPMSCs的成骨活性。
[Abstract]:Objective: 1.Using alginate and chitosan as carriers, microspheres and plastic bone scaffolds loaded with VEGF and vancomycin were prepared and their properties were analyzed.The third generation of HPMSCs was cultured in the third generation of HPMSCs from the multi-layer alginate-chitosan microsphere scaffolds prepared in aseptic environment. The proliferative and osteogenic activities of HPMSCs were detected after the culture of the scaffold extracts.Research method: 1.HPMSCs were isolated and cultured from human full-term placenta by tissue mass culture method. The cell growth curves were measured and plotted by MTT method. The third generation HPMSCs was induced by osteogenesis and adipogenesis to verify its multidirectional differentiation potential.According to the method of preparing multilayer drug loaded sustained-release microspheres in aseptic environment in previous experiments, the principle of alginate and chitosan crosslinking was used flexibly, and the plasticity of alginate and chitosan crosslinking products was used to combine the sustained release with the support function of the scaffold.The optimum ratio of microspheres and scaffolds was chosen to prepare multilayer alginate chitosan microsphere scaffold material in aseptic environment.The prepared scaffolds were co-cultured with the third generation of HPMSCs. The proliferation of the cells was detected by CCK-8 kit, and the osteogenic ability of the cells was detected by alizarin red staining and ALP kit.The result of the study was: 1.The cells isolated from human placenta were observed to be fibroblast-like growth under inverted microscope. The growth curve of the cells was "S", and the extracellular calcium deposits could be seen after osteogenesis induction.Alizarin red staining was positive, and oil red O staining was positive after lipogenesis.The multilayer alginate chitosan microsphere scaffold material prepared under sterile environment has the function of both sustained release and scaffold.The proliferative ability of HPMSCs was not significantly changed after co-culture with scaffold material leaching solution (P 0.05). After osteogenesis induction, alizarin red staining showed that calcium salt deposition in drug-loaded microsphere scaffold group was more than that in no-loaded microsphere scaffold group, while that in drug-loaded microsphere scaffold group was higher than that in non-loaded microsphere scaffold group.The results of ALP kit test in the no stent group and the blank control group confirmed that the content of alkaline phosphatase in the cells increased significantly with the addition of the drug loaded microsphere stent, and there was also a significant difference between the no loaded microsphere stent group and the no stent group (P 0. 05).Conclusion: 1.HPMSCs were isolated and cultured successfully from human full-term placenta. The biological characteristics of HPMSCs were confirmed to be in accordance with stem cell characteristics.The multilayer alginate-chitosan microsphere scaffold material with plastic form was successfully prepared.By co-culture of multi-layer alginate-chitosan microsphere scaffolds with double drug loading with HPMSCs, it was proved that it had no obvious effect on the proliferation of HPMSCs, but could significantly improve the osteogenic activity of HPMSCs.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08

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