中国修复重建外科杂志

中国修复重建外科杂志

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

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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 200 μL 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 collagen type Ⅱ. Alcian blue staining showed that the content of GAG in 2 groups increased continuously at different time points; after 7 days, the increasing trend of the control group was slow and the microspheres group continued hypersecretion. There was no significant difference of the GAG content at 1, 3, and 7 days between 2 groups (P>0.05), but the GAG content at 14 and 21 days in microspheres group was significantly higher than that in control group (P<0.05).ConclusionBMP-7/PLGA microspheres prepared by double emulsion-drying in liquid method in vitro can promote proliferation and chondrogenic differentiation of rabbit BMSCs.

关键词: BMP-7; 聚乳酸-羟基乙酸共聚物; 缓释微球; BMSCs;

Key words: Bone morphogenetic protein 7; poly (lactide-co-glycolide); microspheres; bone marrow mesenchymal stem cells; rabbit

引用本文: 陈家磊, 刘明, 段鑫, 黄富国, 项舟. 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. 中国修复重建外科杂志, 2018, 32(4): 428-433. doi: 10.7507/1002-1892.201711093 复制

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