【作者】 张颖；

【导师】 刘杰；

【作者基本信息】 青岛大学 ， 口腔修复学， 2021， 硕士

【摘要】 目的:分别以氯化钙、氯化锶、氯化镁和氯化锌为交联剂,制备含有不同种类离子为交联剂的复合水凝胶,检测水凝胶的理化性质,检测小鼠成骨前体细胞在水凝胶上黏附、增殖的情况,为藻酸盐凝胶作为组织工程支架材料的应用提供理论依据。方法:在交联剂中根据不同的离子种类,制备5组钙、锶、镁、锌离子质量比分别为A(2:0:0:0)、B(2:2:0:0)、C(2:2:2:0)、D(2:2:0:0.5)、E(2:2:2:0.5)5组的电解液(A组为对照组),分别与终浓度为2%wt的藻酸钠溶液进行等比例混合交联,制备出5组含有不同离子的藻酸盐凝胶支架材料。使用MTT细胞毒性检测在第1、2、3、4、5天藻酸盐水凝胶浸出液对成骨前体细胞的毒性;检测各组水凝胶在双蒸水和磷酸盐缓冲溶液(PBS)中的溶胀率及各组水凝胶降解速率;用扫描电镜(SEM)观察各组水凝胶的微观结构;通过电感耦合等离子体光谱仪(ICP)检测第1、3、5天水凝胶浸出液中锶、镁、锌离子的析出浓度;在各组水凝胶表面接种小鼠成骨前体MC3T3-E1细胞的1天后,通过扫描电镜观察细胞黏附形态;用CCK-8法检测在第1、3、5天时各组凝胶表面细胞增殖情况;各组水凝胶接种MC3T3-E1细胞在第12 h、24 h、48 h时,分别采用钙黄绿素-AM(Calcein-AM)和碘化丙啶(PI)染色,观察细胞在凝胶表面的黏附、增殖情况;采用实时荧光光定量PCR技术(RT-q PCR)检测各组凝胶对细胞黏附、增殖相关基因表达的影响。结果:各组藻酸钠水凝胶浸出液对成骨前体细胞未发现明显毒性;多离子交联剂的引入可以改变海藻酸盐水凝胶在双蒸水以及PBS中的溶胀率,其差异均有统计学意义(P<0.05);随着锶、锌、镁离子的引入,水凝胶降解率逐渐下降,其差异均有统计学意义(P<0.05);随着多离子的引入,凝胶的微观形貌发生明显变化,各组凝胶孔隙逐渐增大,而E组的微观结构相对较均一;凝胶中的锶、锌、镁三种离子在第1天均快速大量释放,随着时间的延长锶离子和锌离子释放浓度逐渐稳定,而镁离子释放迅速,到第5天时镁离子基本无析出;体外实验结果表明,多离子交联剂的引入能够有效改善成骨前体细胞在凝胶表面的黏附和增殖,而E组表现出更为显著的促进细胞黏附和增殖的作用。同时,E组的黏附、增殖相关基因与其他组相比呈现出高表达,差异均有统计学意义(P<0.05)。结论:多离子交联的方式可以有效改善藻酸盐凝胶的理化性能,进而改善成骨前体细胞在凝胶表面的黏附和增殖。结果表明,当钙、锶、镁、锌质量比为2:2:2:0.5时形成的复合水凝胶促进细胞黏附和增殖的作用最为显著。这一结果为藻酸盐凝胶为新型可注射骨组织工程支架的应用提供了一定的理论依据。更多还原

【Abstract】 OBJECTIVE: Strontium chloride,magnesium chloride and zinc chloride were used as cross-linking agents to synthesize the composite alginate brine gel containing strontium,magnesium and zinc.The physical and chemical properties of the alginate brine gel containing strontium,magnesium and zinc were detected,and the adhesion and proliferation of mouse MC3T3-E1 cells inoculated on the cross-linked alginate brine gel containing strontium,magnesium and zinc were detected.METHODS: According to different ion types in the crosslinking agent,5 groups of calcium,strontium,magnesium and zinc were prepared with the mass ratio of respectively A(2:0:0:0),B(2:2:0:0),C(2:2:2:0),D(2:2:0:0.5),E(2:2:2:0.5)electrolyte(group A for reference group),and were mixed and cross-linked with sodium alginate solution with the final concentration of 2%wt in equal proportion,respectively,to prepare5 groups with a diameter of 1.5 cm round hydrogel.The toxicity of alginate saline gel extract to osteogenic precursor cells was detected using MTT cytotoxicity assay kit at day1,2,3,4,and 5.The swelling rate of hydrogels in each group in double steam water and phosphate buffer solution(PBS)was detected.Degradation rate of hydrogels soaked in lysozyme containing PBS was detected.The microstructures of hydrogels were observed by scanning electron microscopy(SEM).Strontium,zinc and magnesium ions in the gel were all released rapidly and in large quantities on the first day.With the extension of time,the release concentrations of strontium and zinc ions gradually stabilized,while magnesium ions were released rapidly,and there was almost no precipitation of magnesium ions on the fifth day.One day after inoculating mouse pre-osteogenesis precursor MC3T3-E1 cells on the hydrogel surface of each group,the cell adhesion morphology was observed by scanning electron microscopy.MC3T3-E1 cells were inoculated with hydrogel in each group,and cell proliferation was detected by CCK8 kit at day 1,3 and 5.MC3T3-E1 cells in each group were inoculated with hydrogel.At the12 h,24h and 48 h,the cells were stained with calcein-AM and propidium iodide(PI),respectively,to observe the adhesion and proliferation of the cells on the surface of the gel.MC3T3-E1 cells in each group were inoculated with hydrogel,and the expressions of adhesion and proliferation specific genes in each group were detected by real-time fluorescent quantitative PCR(RT-q PCR)RESULTS: The results of cytotoxicity test showed that the sodium alginate hydrogel extracts of each group had no cytotoxicity to osteogenic precursor cells.The introduction of multi-ion crosslinking agent could change the swelling rate of alginate brine gel in double steam water and PBS,and the differences were statistically significant(P<0.05).The results of hydrogel degradation showed that with the introduction of strontium,zinc and magnesium ions,the hydrogel degradation rate decreased gradually.With the introduction of multi-ions,the microstructure of the gel changed significantly,and the pore size of each group increased gradually,while the microstructure of group E was relatively uniform.ICP results show that each hydrogel leaching liquid of strontium ions in 1,3,5 days to release concentration decreases gradually,and released into the leach liquor of strontium ion content and the original is proportional to the introduction of the strontium ions in water gel content,C and E group of hydrogel leaching liquid of magnesium ions quick release in 1 day,3 days to release concentration fell sharply,to 5days,there was no precipitation of magnesium ions.The release of zinc ions in the hydrogel leaching solution of group D and E was faster on the first day,and the release concentration gradually decreased on the third and fifth day.The results of in vitro experiments showed that the introduction of multi-ion crosslinking agent could effectively improve the adhesion and proliferation of osteogenic precursor cells on the gel surface,and group E showed a more significant effect on promoting the adhesion and proliferation of cells.At the same time,compared with the other groups,adhesion and proliferation-related genes in group E were highly expressed with statistical significance(P<0.05).Conclusion: Multi-ion crosslinking can effectively improve the physical and chemical properties of alginate gel,and then improve the adhesion and proliferation of osteogenic precursor cells on the gel surface.The results showed that when the mass ratio of Ca,Sr,Mg and Zn was 2:2:2:0.5,the composite hydrogel had the most significant effect on promoting cell adhesion and proliferation.The results provide a theoretical basis for the application of alginate gel as a new injectable scaffold for bone tissue engineering.更多还原