中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (14): 2223-2227.doi: 10.3969/j.issn.2095-4344.1674

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

仿生大孔骨支架的制备及生物相容性评估

李 智1，谭春华1，蔡贤华1，王华松1，丁晓明2，赵艳红3   

  1. 1解放军中部战区总医院骨科/湖北省骨创伤救治临床医学研究中心，湖北省武汉市 430070；2日照市中医医院骨伤五科，山东省日照市 276800；3天津医科大学口腔医院正畸科，天津市 300070
  • 收稿日期:2018-12-26
  • 通讯作者: Zhao Yanhong, Associate chief physician, Department of Orthodontics, Stomatological Hospital of Tianjin Medical University, Tianjin 300070, China
  • 作者简介:李智，男，1975年生，新疆维吾尔自治区乌鲁木齐市人，医学博士，副主任医师，主要从事创伤修复研究。
  • 基金资助:

    湖北省自然科学基金(2017CFB569)，项目负责人：李智；国家自然科学基金(31470937)，项目参与者：丁晓明；国家自然科学基金(31300798)，项目负责人：赵艳红；天津市卫计委基金资助项目(16KG114)，项目负责人：赵艳红；天津市卫计委基金资助项目(15KG125)，项目参与者：赵艳红

Fabrication and biocompatibility assessment of the scaffold with biomimetic interconnected macropore structure

Li Zhi1, Tan Chunhua1, Cai Xianhua1, Wang Huasong1, Ding Xiaoming2, Zhao Yanhong3   

  1.  赵艳红，副主任医师，天津医科大学口腔医院正畸科，天津市 300070
  • Received:2018-12-26
  • Contact: 1Department of Orthopedics, General Hospital of Chinese PLA Central Theater Command & Hubei Provincial Center for Bone Trauma Treatment Clinical Medicine, Wuhan 430070, Hubei Province, China; 2Fifth Department of Orthopedics, Rizhao Hospital of Traditional Chinese Medicine, Rizhao 276800, Shandong Province, China; 3Department of Orthodontics, Stomatological Hospital of Tianjin Medical University, Tianjin 300070, China
  • About author:Li Zhi, MD, Associate chief physician, Department of Orthopedics, General Hospital of Chinese PLA Central Theater Command & Hubei Provincial Center for Bone Trauma Treatment Clinical Medicine, Wuhan 430070, Hubei Province, China
  • Supported by:

    the Natural Science Foundation of Hubei Province, No. 2017CFB569 (to LZ); the National Natural Science Foundation of China, No. 31470937 (to DXM) and 31300798 (to ZYH); the Foundation of Health and Family Planning Commission of Tianjin, No. 16KG114 and 15KG125 (both to ZYH)

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摘要：

文章快速阅读：

 

文题释义：
丝素蛋白/羟基磷灰石复合材料：丝素蛋白是一种天然大分子蛋白，来源于多种昆虫的茧，目前大部分研究所用的丝素蛋白由家蚕蚕茧经过脱胶、溶解、透析、浓缩而成，来源广泛。丝素蛋白含有较多的β折叠结构，该结构稳定，不溶于水，力学性能优良，可通过简单的化学修饰来改进各方面的性能；羟基磷灰石是是骨基质的主要矿物质成分广泛存在于动物的骨骼和牙齿中，本身具有生物活性和骨引导作用，将二者结合是骨组织工程支架载体的理想材料。
溶剂浇铸/粒子沥滤技术：是最简便、也是研究最广泛的一种多孔三维细胞支架制备技术，经典的制备过程是：通过筛分获得需要尺寸大小的致孔剂颗粒，将致孔剂颗粒均匀地分散在左旋聚乳酸的氯仿溶液中，然后浇铸在适当的模具中，待大量氯仿挥发后，真空干燥去除混合物中的残余溶剂，即可获得干燥的左旋聚乳酸致孔剂复合物。
 
 
背景：骨组织工程的发展，为解决骨缺损修复提供了新手段，而其中的支架载体选材和结构构建是目前的热点问题。
目的：采用石蜡微球沥滤技术制备具有仿生大孔结构的丝素蛋白/纳米羟基磷灰石支架，分析其细胞相容性。
方法：以丝素蛋白和纳米羟基磷灰石为原料，采用石蜡微球沥滤技术制成具有仿生大孔结构的丝素蛋白/纳米羟基磷灰石支架，利用体式显微镜和扫描电镜观察支架结构，测量支架的弹性模量。将第3代兔脂肪间充质干细胞接种于丝素蛋白/纳米羟基磷灰石支架上，培养48 h后，LIVE/DEAD染色观察细胞活性；培养1周，苏木精-伊红染色观察细胞黏附情况；培养3 d，扫描电镜观察支架-细胞复合物；培养1，3，5，7 d，CCK-8法检测细胞增殖，以单纯细胞培养为对照。

结果与结论：①体式显微镜下可见丝素蛋白/纳米羟基磷灰石支架呈乳白色，扫描电镜显示无论在支架横截面还是纵切面上，均可见均匀排列的大孔结构，连通性好，支架孔径为(362.23±26.52) μm，弹性模量为(54.93±5.44) kPa；②扫描电镜显示，脂肪间充质干细胞能够很好地在支架孔壁及连通孔隙内黏附和伸展，分泌大量细胞外基质，填充在丝素蛋白/纳米羟基磷灰石支架孔隙内；③苏木精-伊红染色显示，脂肪间充质干细胞均匀黏附在丝素蛋白/纳米羟基磷灰石支架孔隙内壁上，并且大量增殖；④LIVE/DEAD染色显示脂肪间充质干细胞活性良好；⑤CCK-8检测结果显示，脂肪间充质干细胞在丝素蛋白/纳米羟基磷灰石支架可良好地生长与增殖；⑥结果表明，丝素蛋白/纳米羟基磷灰石支架具有良好的孔径与细胞相容性。

ORCID: 0000-0002-9327-8110(李智)

  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: 骨缺损, 丝素蛋白/纳米羟基磷灰石支架, 生物材料, 丝素蛋白, 羟基磷灰石, 脂肪干细胞, 细胞黏附

Abstract:

BACKGROUND: Development of bone tissue engineering provides a new method to solve bone defect repair. Scaffold material and structure construction are issues of concern.

OBJECTIVE: To fabricate a silk fibroin and hydroxyapatite scaffold with biomimetic interconnected macropores structure using the paraffin-sphere leaching technology and to evaluate its possibility of cytocompatibility.
METHODS: The scaffold with biomimetic interconnected macropores structure was made by the paraffin-sphere leaching technology. The structure of scaffold was observed by the stereomicroscope and scanning electron microscope. The pore size and elasticity modulus were calculated. Passage 3 rabbit adipose-derived mesenchymal stem cells were seeded into the scaffold. The cell viability was detected by live/dead staining at 48 hours after culture. The cell adhesion was observed by hematoxylin-eosin staining at 1 week of culture. The scaffold-cell composite was observed under scanning electron microscope at 3 days of culture. The cell proliferation was detected by the cell counting-kit 8 assay at 1, 3, 5 and 7 days of culture. Those cells cultured alone were considered as control group.
RESULTS AND CONCLUSION: (1) Stereomicroscope showed the ivory silk fibroin/hydroxyapatite scaffold. Scanning electron microscope revealed pore structures in cross-section and longitudinal-section with good connectivity. The scaffold pore size was (362.23±26.52) μm and the elasticity modulus was (54.93±5.44) kPa. (2) Scanning electron microscope showed that adipose-derived mesenchymal stem cells adhered and stretched well in the pore wall and connected pore, secreted abundant extracellular matrix, and filled in the pores of silk fibroin/hydroxyapatite scaffold. (3) Hematoxylin-eosin staining results found that adipose-derived mesenchymal stem cells evenly adhered onto the inner wall of silk fibroin/hydroxyapatite scaffold, and proliferated well. (4) Live/dead staining revealed a good viability of adipose-derived mesenchymal stem cells. (5) Cell counting-kit 8 assay results showed the good proliferation of adipose-derived mesenchymal stem cells on the scaffold. (6) To conclude, the silk fibroin/hydroxyapatite scaffold possesses good pore size and cytocompatibility.

Key words: Silk, Hydroxyapatites, Cell Adhesion, Biocompatible Materials, Tissue Engineering

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