BMP-7/聚乳酸-羟基乙酸共聚物缓释微球对兔BMSCs增殖和成软骨分化的影响  

Effect of bone morphogenetic protein 7/poly(lactide-co-glycolide) microspheres on the in vitro proliferation and chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells

作　　者：陈家磊[1] 刘明[1] 段鑫[1] 黄富国[1] 项舟[1] CHEN Jialei, LIU Ming, DUAN Xin, HUANG Fuguo, XIANG Zhou(Department of Orthopeadics, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P.R.Chin)

机构地区：[1]四川大学华西医院骨科,成都610041

出　　处：《中国修复重建外科杂志》2018年第4期428-433,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(31370984;81401813)~~

摘　　要：目的通过体外实验探讨载负BMP-7的聚乳酸-羟基乙酸共聚物[poly(lactide-co-glycolide),PLGA]缓释微球对兔BMSCs增殖和成软骨分化的影响。方法采用复乳化-液中干燥法制备BMP-7/PLGA缓释微球,并将BMP-7/PLGA缓释微球与软骨分化培养基混合后,在第1、3、7、14、21天收集其上清液作为体外释放液。取3～5日龄新西兰乳兔双侧股骨及胫骨分离培养BMSCs,取第3代BMSCs分为微球组和对照组,微球组在每2～3天换液过程中更换为200μL不同时间点的BMP-7/PLGA缓释微球体外释放液,对照组使用等体积软骨诱导培养液。采用MTT法测定两组培养1、3、7、14、21 d的吸光度(A)值,检测细胞增殖情况;培养21 d行番红O染色、甲苯胺蓝染色和Ⅱ型胶原免疫组织化学染色观察,并于1、3、7、14、21 d采用阿利新蓝染色法测定糖胺聚糖(glycosaminoglycan,GAG)含量,检测成软骨细胞分化能力。结果 MTT检测示,培养1、3、7 d两组细胞均迅速增殖;7 d后对照组增殖速度变缓,微球组仍呈持续快速增殖。培养1、3、7 d两组A值比较差异无统计学意义(P>0.05),14、21 d微球组A值显著高于对照组(P<0.05)。培养21 d与对照组比较,微球组番红O染色可见几乎所有细胞核被染成鲜红色,甲苯胺蓝染色可见几乎所有细胞核出现异染性紫红色,Ⅱ型胶原免疫组织化学染色可见多数细胞核呈黄色或棕黄色,Ⅱ型胶原表达为强阳性。阿利新蓝染色法测定示,培养各时间点两组细胞GAG含量呈持续增高趋势,7 d后对照组增高趋势减缓,微球组仍呈持续高分泌状态。培养1、3、7 d两组GAG含量比较差异无统计学意义(P>0.05),14、21 d微球组GAG含量显著高于对照组(P<0.05)。结论复乳化-液中干燥法制备的BMP-7/PLGA缓释微球在体外具有促进兔BMSCs增殖和向软骨细胞分化的能力。ObjectiveTo evaluate the effect of bone morphogenetic protein 7 （BMP-7）/poly （lactide-co-glycolide） （PLGA） microspheres on in vitro proliferation and chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells （BMSCs）.MethodsBMP-7/PLGA microspheres were fabricated by double emulsion-drying in liquid method. After mixing BMP-7/PLGA microspheres with the chondrogenic differentiation medium, the supernatant was collected on the 1st, 3rd, 7th, 14th, and 21st day as the releasing solution. The BMSCs were isolated from the bilateral femurs and tibias of 3-5 days old New Zealand rabbits, and the 3rd generation BMSCs were divided into 2 groups： microspheres group and control group. The BMSCs in microspheres group were cultured by 200 μL BMP-7/PLGA microspheres releasing solution in the process of changing liquid every 2-3 days, while in control group were cultured by chondrogenic medium. The cell proliferation （by MTT assay） and the glycosaminoglycan （GAG） contents （by Alician blue staining） were detected after chondrogenic cultured for 1, 3, 7, 14, and 21 days. The chondrogenic differentiation of BMSCs was observed by safranine O staining, toluidine blue staining, and collagen type Ⅱ immunohistochemistry staining at 21 days.ResultsMTT test showed that BMSCs proliferated rapidly in 2 groups at 1, 3, and 7 days; after 7 days, the proliferation of BMSCs in the control group was slow and the BMSCs in microspheres group continued to proliferate rapidly. There was no significant difference of the absorbance （A） value at 1, 3, and 7 days between 2 groups （P〉0.05）, but theA value at 14 and 21 days in microspheres group was significantly higher than that in control group （P〈0.05）. Compared with control group at 21 days, in microsphere group, almost all nuclei were dyed bright red by safranine O staining, almost all the nuclei appeared metachromatic purple red by toluidine blue staining, and the most nuclei were yellow or brown by immunohistochemical staining of colla

关 键 词：BMP-7 聚乳酸-羟基乙酸共聚物 缓释微球 BMSCS 兔 

分 类 号：R684[医药卫生—骨科学]