丹参素涂层β-磷酸三钙支架材料的制备与细胞相容性

摘要

BACKGROUND: Revascularization is a challenge for the tissue-engineered bone carrying cells after implanted into human body. Previous studies have found that tanshinol can improve the functions of endothelial progenitor cells and exert vascular protective effects.OBJECTIVE: To prepare the β-calcium phosphate (β-TCP) scaffold with tanshinol coating, and to observe its cytocompatibility.METHODS: The β-TCP scaffolds coated with 10-7, 10-6 and 10-5 mol of tanshinol were constructed by negative pressure absorption method. The distribution of tanshinol coating on the scaffold was observed using scanning electron microscopy,and the inner ingredients were analyzed by infrared spectrum. Human endothelial progenitor cells (hEPCs) were cultured in the extracts of β-TCP and β-TCP scaffolds with 10-7, 10-6, 10-5 mol of tanshinol coatings, respectively. The cell proliferation was detected at 2, 4, 6, 8 and 10 days of culture; the levels of nitric oxide and vascular endothelial growth factor in the supernatants were detected at 1, 7 and 14 days of culture; the lumen formation on the matrigel was observed after 14-day culture. hEPCs were respectively seeded onto the β-TCP and β-TCP scaffolds with different dosages of tanshinol coating,and then the cell growth was observed under scanning electron microscope at 7 days.RESULTS AND CONCLUSION: The tanshinol coating evenly distributed on the inner surface of the pores, and its crystalline structure became dense with dosage increasing. Infrared spectrum analysis revealed no changes in the characteristic absorption peak of tanshinol and TCP in the scaffold. The β-TCP scaffolds with tanshinol coating could promote the proliferation of hEPCs, especially the scaffolds with 10-6 and 10-5 mol tanshinol coating. Compared with the β-TCP scaffold, the scaffolds with 10-6 and 10-5 mol tanshinol coating significantly upregulated the nitric oxide level at 14 days of culture, and significantly increased the level of vascular endothelial growth factor at 7 and 14 days of culture (P <0.05). Although it could be found in all β-TCP scaffolds with tanshinol coating, the lumen formation was the maturest in the scaffold with 10-5 mol tanshinol coating. These results suggest the β-TCP scaffolds with tanshinol coating can promote the proliferation and endothelial differentiation of hEPCs, and hold a good cytocompatibility.%背景:研究发现载细胞的组织工程骨植入体内后面临严峻的"血管再生"难题;前期研究发现丹参素可改善血管内皮祖细胞功能,发挥血管保护作用.目的:制备丹参素涂层β-磷酸三钙支架材料,观察其细胞相容性.方法:采用负压吸附法构建载药剂量分别为10-7,10-6,10-5 mol的丹参素涂层β-磷酸三钙支架材料,采用扫描电镜观察材料表面丹参素涂层分布,采用红外光谱分析材料内部成分.分别以β-磷酸三钙支架材料浸提液与不同载药量丹参素涂层β-磷酸三钙支架材料浸提液培养人血管内皮祖细胞,培养2,4,6,8,10 d,检测细胞增殖;培养1,7,14 d,检测细胞上清液中NO和血管内皮生长因子水平;培养14 d,观察细胞在血管基质胶上的管腔形成.将人血管内皮祖细胞接种于β-磷酸三钙支架与不同载药量丹参素涂层β-磷酸三钙支架上,培养7 d,扫描电镜观察细胞生长.结果与结论:①丹参素涂层均匀一致地平铺于材料孔隙内表面,随载药剂量增加,丹参素涂层的结晶结构呈现出更加致密状态;②丹参素涂层β-磷酸三钙支架中丹参素和磷酸三钙的红外光谱特征吸收峰位置均未发生变化;③丹参素涂层β-磷酸三钙支架对人血管内皮祖细胞具有促增殖作用,以10-6,10-5 mol丹参素涂层β-磷酸三钙支架为有效;④与β-磷酸三钙比较,10-6,10-5 mol丹参素涂层β-磷酸三钙支架可在第14天显著提高细胞上清液中NO水平(P<0.05),在第7,14 d显著提高细胞上清液中血管内皮生长因子水平(P<0.05);⑤不同载药量丹参素涂层β-磷酸三钙支架组浸提液培养细胞均有形成管腔结构趋势,以10-5 mol丹参素涂层β-磷酸三钙支架组形成的管腔结构最为成熟;⑥结果表明,丹参素涂层β-磷酸三钙支架可促进人血管内皮祖细胞增殖及向成熟内皮细胞分化,具有良好的细胞相容性.