中国组织工程研究

中国组织工程研究 ›› 2017, Vol. 21 ›› Issue (14): 2247-2253.doi: 10.3969/j.issn.2095-4344.2017.14.019

• 材料生物相容性 material biocompatibility • 上一篇    下一篇

丹参素涂层β-磷酸三钙支架材料的制备与细胞相容性

吴  涛1，刘英超1，南开辉2，舒  涛3，吴金辉1，卢志勤1，邢建洲1，吕  军1   

  1. 1广州医科大学附属武警广东省总队医院急诊科，广东省广州市  510507；2温州医科大学附属眼视光医院，温州市生物材料工程技术研究中心，浙江省温州市  325027； 3中山大学附属第三医院脊柱外科，广东省广州市  510630
  • 收稿日期:2016-12-23 出版日期:2017-05-18 发布日期:2017-06-10
  • 通讯作者: 吕军，副主任医师，武警广东省总队医院急诊科，广东省广州市 510507
  • 作者简介:吴涛，男，1979年生，湖南省岳阳市人，汉族，2009年南方医科大学毕业，博士，主治医师，主要从事骨组织工程研究。
  • 基金资助:

    国家自然科学基金(81303107)；广东省自然科学基金(S2012040006295)；广东省医学科研基金(A2012499，A2014499)

Preparation and cytocompatibility of beta-calcium phosphate scaffolds with tanshinol coating

Wu Tao1, Liu Ying-chao1, Nan Kai-hui2, Shu Tao3, Wu Jin-hui1, Lu Zhi-qin1, Xing Jian-zhou1, Lv Jun1   

  1. 1 Department of Emergency, Guangdong General Hospital of Chinese People’s Armed Police Force, Guangzhou Medical University, Guangzhou 510507, Guangdong Province, China; 2 Hospital of Optometry & Ophthalmology of Wenzhou Medical University, Wenzhou Engineering Research Center for Biomaterials, Wenzhou 325027, Zhejiang Province, China;3 Department of Spine Surgery, the Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China
  • Received:2016-12-23 Online:2017-05-18 Published:2017-06-10
  • Contact: Lv Jun, Associate chief physician, Department of Emergency, Guangdong General Hospital of Chinese People’s Armed Police Force, Guangzhou Medical University, Guangzhou 510507, Guangdong Province, China
  • About author:Wu Tao, M.D., Attending physician, Department of Emergency, Guangdong General Hospital of Chinese People’s Armed Police Force, Guangzhou Medical University, Guangzhou 510507, Guangdong Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81303107; the Natural Science Foundation of Guangdong Province, No. S2012040006295; the Medical Research Foundation of Guangdong Province, No. A2012499 and A2014499

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摘要：

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文题释义：
丹参素涂层β-磷酸三钙支架：实验采用负压吸附法构建了丹参素涂层的β-磷酸三钙骨支架材料，从扫描电镜结果来看，随着丹参素载药剂量的增加，β-磷酸三钙材料表面的结晶成分密度逐渐增加，当丹参素增至10-5 mol，β-磷酸三钙材料表面的结晶成分达到致密状态。红外光谱分析表明，负压吸附法构建丹参素涂层β-磷酸三钙支架材料过程中，两种材料未发生化学性质上的改变，能够较好地保留丹参素的药理活性。
载细胞组织工程骨植入体内后的“血管再生”难题：因为活体组织毛细血管供氧范围仅为100-150 μm，当生物材料较大时，内部氧供和营养的缺乏会导致种子细胞大量死亡；且当材料与血液接触时，会因补体活化和凝血级联反应形成血栓，阻碍氧和营养物质的渗透。为解决这一问题，研究者采用在预制材料内植入血管、带血管组织瓣包裹材料等方法进行了处理，但生物材料的血管再生过程仍需数周时间，依然无法解决与细胞耐受缺氧仅数小时之间的矛盾。“生长因子+生物材料”模式的组织工程骨固然规避了“血管再生”问题，但生长因子在体内半衰期短、容易失活、易于弥散。

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。

ORCID: 0000-0002-9081-6008(吕军)

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

关键词: 生物材料, 材料相容性, β-磷酸三钙, 丹参素, 血管内皮祖细胞, 细胞相容性, 支架材料, 国家自然科学基金

Abstract:

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.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

Key words: Calcium Phosphates, Endothelial Cells, Tissue Engineering

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