Zhao Shengli, Yin Jie, Yu Qinghe, Zhang Guowei, Min Shaoxiong
Department of Spinal Surgery, Zhujiang Hospital of Southern Medical University, Guangzhou Guangdong, 510282, P.R.China.
Department of Hand Surgery, Ningbo No.6 Hospital, Ningbo Zhejiang, 315040, P.R.China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2019 Feb 15;33(2):243-251. doi: 10.7507/1002-1892.201808099.
To observe the effect of vascular endothelial growth factor/polylactide-polyethyleneglycol-polylactic acid copolymer/basic fibroblast growth factor (VEGF/PELA/bFGF) mixed microcapsules in promoting the angiogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) .
The BMSCs were isolated by the method of whole bone marrow adherent, and sub-cultured. The passage 3 BMSCs were identified by Wright-Giemsa staining and flow cytometry, and used for subsequent experiments. VEGF/PELA/bFGF (group A), PELA/bFGF (group B), VEGF/PELA (group C), and PELA (group D) microcapsules were prepared. The biodegradable ability and cytotoxicity of PELA microcapsule were determined，and the slow-released ability of VEGF/PELA/bFGF mixed microcapsules was measured. The passage 3 BMSCs were co-cultured with the extracts of groups A, B, C, and D, separately. At 1, 3, 7, 14, and 20 days after being cultured, the morphological changes of induced BMSCs were recorded. At 21 days, the induced BMSCs were tested for DiI-labeled acetylated low density lipoprotein (Dil-ac-LDL) and FITC-labeled ulex europaeus agglutinin I (FITC-UEA-I) uptake ability. The tube-forming ability of the induced cells on Matrigel was also verified. The differences of the vascularize indexes in nodes, master junctions, master segments, and tot.master segments length in 4 groups were summarized and analyzed.
The isolated and cultured cells were identified as BMSCs. The degradation time of PELA was more than 20 days. There was no significant effect on cell viability under co-culture conditions. At 20 days, the cumulative release of VEGF in the mixed microcapsules exceeded 95%, and the quantity of bFGF exceeded 80%. The morphology of cells in groups A, B, and C were changed. The cells in groups A and B showed the typical change of cobble-stone morphology. The numbers of double fluorescent labeled cells observed by fluorescence microscope were the most in group A, and decreases from group B and group C, with the lowest in group D. The cells in groups A and B formed a grid-like structure on Matrigel. Quantitative analysis showed that the differences in the number of nodes, master junctions, master segments, and tot.master segments length between groups A, B and groups C, D were significant ( <0.05). The number of nodes and the tot.master segments length of group A were more than those of group B ( <0.05). There was no significant differences in the number of master junctions and master segments between group A and group B ( >0.05).
VEGF/PELA/bFGF mixed microcapsules have significantly ability to promote the angiogenic differentiation of rat BMSCs .
观察血管内皮生长因子/聚乳酸-聚乙二醇-聚乳酸共聚物/碱性成纤维细胞生长因子（VEGF/PELA/bFGF）混合微囊对大鼠骨髓间充质干细胞（BMSCs）血管生成分化的影响。
采用全骨髓贴壁法分离大鼠BMSCs并传代培养。对第3代BMSCs进行瑞氏-吉姆萨染色和流式细胞术鉴定，用于后续实验。制备VEGF/PELA/bFGF（A组）、PELA/bFGF（B组）、VEGF/PELA（C组）和PELA（D组）微囊。测定PELA微囊的生物降解能力和细胞毒性，检测VEGF/PELA/bFGF混合微囊的缓释能力。将第3代BMSCs分别与A、B、C、D组提取物共培养。培养1、3、7、14和20天后，记录诱导后BMSCs的形态变化。培养21天时，检测诱导后BMSCs对DiI标记的乙酰化低密度脂蛋白（Dil-ac-LDL）和FITC标记的荆豆凝集素I（FITC-UEA-I）的摄取能力。验证诱导细胞在基质胶上的成管能力。总结并分析4组在节点、主连接、主段和总主段长度方面血管化指标的差异。
分离培养的细胞被鉴定为BMSCs。PELA的降解时间超过20天。共培养条件下对细胞活力无显著影响。20天时，混合微囊中VEGF的累积释放量超过95%，bFGF的累积释放量超过80%。A、B、C组细胞形态发生改变。A、B组细胞呈现典型的鹅卵石样形态改变。荧光显微镜下观察到的双荧光标记细胞数量A组最多，B、C组依次减少，D组最少。A、B组细胞在基质胶上形成网格样结构。定量分析显示，A、B组与C、D组在节点数、主连接数、主段数和总主段长度方面的差异有统计学意义（<0.05）。A组的节点数和总主段长度多于B组（<0.05）。A组与B组在主连接数和主段数方面无显著差异（>0.05）。
VEGF/PELA/bFGF混合微囊具有显著促进大鼠BMSCs血管生成分化的能力。
