3D打印技术构建不同比例聚己内酯磷酸三钙支架及其体外诱导成骨性能

《中国组织工程研究》 Chinese Journal of Tissue Engineering Research文章编号:2095-4344(2019)06-00821-06 821www.CRTER .org·研究原著·苑博，男，1991年生，山东省淄博市人，汉族，解放军第二军医大学在读博士，主要从事骨组织工程方面的研究。

通讯作者：陈雄生，博士，博士生导师，主任医师，教授，解放军第二军医大学上海长征医院骨科医院脊柱外科，上海市 200003文献标识码:A稿件接受：2018-10-19Yuan Bo, Doctorate candidate, Spine Center, Department of Orthopedics, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai 200003, ChinaCorresponding author: Chen Xiongsheng, MD, PhD, Chief physician, Professor, Doctoral supervisor, Spine Center, Department of Orthopedics, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai 200003, China3D 打印技术构建不同比例聚己内酯-磷酸三钙支架及其体外 诱导成骨性能苑 博，王智巍，唐一钒，周盛源，陈雄生，贾连顺(解放军第二军医大学上海长征医院骨科医院脊柱外科，上海市 200003)DOI:10.3969/j.issn.2095-4344.1578 ORCID: 0000-0001-9880-4864(苑博)文章快速阅读：文题释义：3D 打印技术：是一种材料加工方法，首先数码方法获取或者创建一个物理实体的CAD 模型；然后将CAD 模型转换为可以进行虚拟切割的电脑模型；再将其切割为一层一层的横断面，也成为“预加工”；随后RP 机器按照预先切割的横断面层层打印，每完成一层，工作台就会向上或者向下移动一层的厚度，进行下一层打印，直到整个打印过程结束；最后，根据制造工艺和制造目的的不同，进行辅助硬化、表面处理等“后加工”。

聚己内酯：是一种人工合成高分子有机化合物，作为美国FDA 批准的用于组织工程的生物材料，其具有良好的生物相容性、生物可降解性和可塑性，但其本身不具有生物活性，机械强度差，降解速度过慢，表面光滑，疏水性强，不适宜成骨细胞黏附和骨组织再生，常与其他一种或多种生物材料复合使用，以增强诱导成骨特性或生物力学强度。

摘要背景：骨组织工程支架的复合材料选择及混合比例对于支架成骨诱导作用的影响巨大。

目的：借助3D 打印技术构建不同混合比例的聚己内酯-磷酸三钙复合支架，探究其物理特征和成骨诱导能力。

方法：将聚己内酯(polycaprolactone ，PCL)和磷酸三钙(tricalcium phosphate ，TCP)分别按照10∶0、9∶1、8∶2、7∶3的质量比混合，借助3D 打印技术构建单纯聚己内酯支架和聚己内酯-磷酸三钙复合支架，分别命名为单纯PCL 支架、PCL-10%TCP 支架、PCL-20%TCP 支架、PCL-30%TCP 支架，利用扫描电子显微镜和疲劳试验机表征支架的微观结构和力学性能。

将兔骨髓间充质干细胞分别接种于单纯PCL 支架、PCL-10%TCP 支架、PCL-20%TCP 支架上，接种后第1，4，7天检测细胞增殖，第1，7，14天检测细胞内碱性磷酸酶活性，第7，14，21天检测支架上清液内的钙含量。

结果与结论：①PCL-30%TCP 支架无法通过该方式构建支架；②与单纯PCL 支架相比，PCL-10%TCP 支架、PCL-20%TCP 支架的抗压强度和弹性模量更强(P < 0.05)，孔隙率无明显改变；③单纯PCL 支架、PCL-10%TCP 支架、PCL-20%TCP 支架均可观察到预先设计的孔隙结构，支架内部孔隙相互连通；④单纯PCL 支架、PCL-10%TCP 支架、PCL-20%TCP 支架均可促进骨髓间充质干细胞增殖，PCL-20%TCP 支架组接种第4，7天的细胞增殖效果优于其余两组支架(P < 0.05)；⑤PCL-20%TCP 支架组接种第7，14天的细胞内碱性磷酸酶活性高于其余两组支架(P < 0.05)；⑥接种第14天，PCL-20%TCP 支架组钙含量高于其余两组支架(P < 0.05)；⑦结果表明，相比于其他支架，PCL-20%TCP 支架具有更好的力学性能，更能促进骨髓间充质干细胞的增殖和成骨分化。

关键词：3D 打印；聚己内酯；磷酸三钙；复合支架；力学性能；成骨分化；细胞内碱性磷酸酶活性 主题词：生物相容性材料；间质干细胞；细胞增殖；碱性磷酸酶；组织工程 中图分类号：R459.9；R318.08 基金资助：上海市科委基础研究重点项目(15JC1491003)，项目负责人：陈雄生Construction of polycaprolactone-tricalcium phosphate with different mixture ratios using three-dimensional printing technology and its osteoinductivity in vitroYuan Bo, Wang Zhiwei, Tang Yifan, Zhou Shengyuan, Chen Xiongsheng, Jia Lianshun (Spine Center, Department of Orthopedics, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai 200003, China)苑博，王智巍，唐一钒，周盛源，陈雄生，贾连顺. 3D 打印技术构建不同比例聚己内酯-磷酸三钙支架及其体外诱导成骨性能[J]. 中国组织工程研究，2019，23(6):821-826. DOI:10.3969/j.issn.2095-4344.1578ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH822AbstractBACKGROUND: The choice and mixture ratio of composite materials for bone tissue engineering scaffolds have a great influence on the osteoinductivity of scaffolds.OBJECTIVE: To construct the polycaprolactone-tricalcium phosphate composite scaffolds with different mixture ratios using 3D printing technology, and to test their physiochemical characteristics and osteoinductivity.METHODS: Polycaprolactone and tricalcium phosphate were mixed in different ratios of 10:0, 9:1, 8:2, and 7:3, and the polycaprolactone and polycaprolactone-tricalcium phosphate scaffolds were constructed by the 3D printing technology. The scaffolds were named as PCL scaffold, PCL-10% TCP scaffold, PCL-20% TCP scaffold, PCL-30% TCP scaffold. The microstructure and mechanical properties of the scaffolds were characterized by scanning electron microscopy and fatigue testing machine. The proliferation and alkaline phosphatase activity of rabbit bone marrow mesenchymal stem cells seeded on different scaffolds were detected at 1, 4, 7 days of culture and at 1, 7 and 14 days of culture, respectively, by in vitro experiments. The content of calcium in the supernatant of the scaffolds was detected at 7, 14, and 21 days of culture RESULTS AND CONCLUSION: The PCL-30%TCP scaffold could not be constructed by this method. Compared with the PCL scaffold, the compressive strength and elastic modulus of the PCL-10% TCP and PCL-20% TCP composite scaffolds were stronger (P < 0.05), and the porosity did not change significantly. Pre-designed pore structure could be observed in the PCL, PCL-10%TCP and PCL-20%TCP scaffolds, and the internal pores of the scaffolds were interconnected. Bone marrow mesenchymal stem cells could proliferate on all the three scaffolds. The proliferation rate of the cells on the PCL-20% TCP scaffold was better than that of the other two scaffolds at 4 and 7 days of culture (P < 0.05). The alkaline phosphatase activity (7 and 14 days of culture) and calcium content (14 days of culture) of bone marrow mesenchymal stem cells on the PCL-20% TCP scaffold were significantly higher than those of the PCL and PCL-10%TCP scaffolds (P < 0.05). These results suggest that the PCL-20% TCP scaffold has better mechanical properties, and can promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.Subject headings: Biocompatible Materials; Mesenchymal Stem Cells; Cell Proliferation; Alkaline Phosphatase; Tissue Engineering Funding: the Major Project of the Science and Technology Commission of Shanghai Municipality, No. 15JC1491003 (to CXS)0 引言 Introduction创伤、肿瘤、畸形、退变等原因引起的需要骨组织重建治疗疾患正逐年增加。