碱性成纤维细胞生长因子/壳聚糖/聚乳酸支架用于牙周组织再生工程的体外实验  

In vitro experimental research of basic fibroblast growth factor/chitosan/polylactic acid scaffolds in periodontal tissue regeneration

作　　者：陈立红 雷志敏[1] 王莉莉 

机构地区：[1]武汉大学人民医院口腔科,湖北省武汉市430060 [2]天津市口腔医院修复科,天津市300000 [3]锦州医科大学附属口腔医院修复科,辽宁省锦州市121000

出　　处：《中国组织工程研究》2017年第26期4106-4112,共7页Chinese Journal of Tissue Engineering Research

基　　金：辽宁省大学生创新训练项目(201510160000045);辽宁省科学计划项目(2014022003)~~

摘　　要：背景:碱性成纤维细胞生长因子(basic fibroblast growth factor,b FGF)可促进牙周膜成纤维细胞的有丝分裂和趋化,有利于细胞外基质及牙周膜新生血管网的生成,但其易降解、半衰期短,单独局部应用代谢迅速。目的:探讨b FGF/壳聚糖/聚乳酸复合支架对牙周膜细胞生长和增殖的影响。方法:采用冷冻干燥法制备不同比例(聚乳酸与壳聚糖的质量比分别为4∶1、3∶2、1∶1、2∶3、1∶4)的壳聚糖/聚乳酸复合支架,以孔隙率、力学强度筛选最优配比的复合支架,用于制备含0,0.1,1,10,100μg/L bF GF的壳聚糖/聚乳酸支架。(1)细胞毒性实验:分别以含0,0.1,1,10,100μg/L bF GF的壳聚糖/聚乳酸支架浸提液(实验组)及DMEM培养液(空白对照组)培养大鼠牙周膜细胞,24,48,72 h后,MTT法检测细胞增殖;培养5 d后,流式细胞仪检测细胞周期;(2)细胞相容性实验:将含0.1,1,10,100μg/L bF GF的壳聚糖/聚乳酸支架分别与大鼠牙周膜细胞共培养,1,3,5,7 d后进行细胞计数;培养3 d后,扫描电镜观察细胞生长情况。结果与结论:(1)从孔隙率、力学强度指标综合考虑,聚乳酸与壳聚糖的最佳质量比为2∶3;(2)随着培养时间的延长,各组细胞增殖A值逐渐增加;培养48,72 h时,1,10,100μg/L实验组细胞增殖A值高于空白对照组(P<0.05),且10μg/L实验组细胞增殖A值最高(P<0.05);(3)0.1,1,10,100μg/L实验组G0/G1期细胞百分比低于空白对照组(P<0.05),以10μg/L实验组最低;0.1,1,10,100μg/L实验组S期、G2/M+S期细胞比例均高于空白对照组(P<0.05),以10μg/L实验组最高(P<0.05);(4)随着培养时间的延长,各组细胞计数逐渐增多;培养3,5 d时,10μg/L实验组细胞计数高于其余各组(P<0.05)。培养3 d后,大鼠牙周膜细胞在10μg/L实验组支架上黏附生长,状态良好;(5)结果表明,bF GF/壳聚糖/聚乳酸复合支架可促进牙周膜细胞的增殖。BACKGROUND： Basic fibroblast growth factor （bFGF） can promote mitosis and chemotaxis of periodontal ligament fibroblasts （PDLCs）, and help the generation of extracellular matrix and new blood capillaries. But it is easy to degrade,has short half-life, and metabolizes rapidly.OBJECTIVE： To evaluate the influence of bFGF/chitosan/polylactic acid scaffolds on PDLCs growth and proliferation.METHODS： Polylactic acid/chitosan composite scaffolds in different proportions （4：1, 3：2, 1：1, 2：3, 1：4） were prepared through freeze-drying method, to study their microstructure, porosity and mechanical strength and then choose the optimal ratio of chitosan/polylactic acid scaffold that was used to prepare the composite scaffolds which contained different concentrations （0, 0.1, 1, 10, 100 μg/L） of bFGF. （1） Cytotoxicity test： PDLCs were cultured with leaching liquid of polylactic acid/chitosan scaffolds which contained different concentrations （0, 0.1, 1, 10, 100 μg/L） of bFGF and DMEM culture medium respectively. The effects on cell proliferation were tested by MTT after 24, 48, 72 hours. Cell cycles were tested using flow cytometry at 5 days of culture. （2） Cytocompatibility test： PDLCs were co-cultured with the polylactic acid/chitosan scaffolds which contained different concentrations （0, 0.1, 1, 10, 100 μg/L） of bFGF. The number of PDLCs was counted at 1, 3, 5 and 7 days. The growing status of PDLCs on the scaffolds was observed by scanning electron microscope at 3 days of culture.RESULTS AND CONCLUSION： （1） The best mass ratio of polylactic acid and chitosan was 2：3 by test of porosity and mechanical strength. （2） The absorbance value of each group was increased over time. The absorbance values of 0.1, 1, 10, 100 μg/L bFGF groups were higher than that of the control group （0 μg/L bGFG）, and the A value of 10 μg/L group was highest in all groups at 48 and 72 hours after co-culture （P 〈 0.05）. （3） Cell percentages at G0/G1 phase of 0.1, 1, 10

关 键 词：生物材料 材料相容性 牙周组织工程 牙周膜细胞 壳聚糖 聚乳酸 碱性成纤维细胞生长因子 

分 类 号：R318[医药卫生—生物医学工程]