中国修复重建外科杂志

中国修复重建外科杂志

墨鱼骨转化羟基磷灰石多孔陶瓷表面纳米结构调控及其对成骨细胞作用的研究

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目的 探索墨鱼骨转化羟基磷灰石多孔陶瓷(cuttlefish bone transformed hydroxyapatite,CB-HA)表面纳米结构的制备与调控,以及不同尺寸纳米结构对成骨细胞黏附、增殖以及 ALP 表达的影响。 方法 取墨鱼骨制备成直径 10 mm、厚 2 mm 的注水骨片后分为 4 组,分别与不同浓度磷源溶液混合,置于水热釜,放入烘箱于 120℃ 反应 24 h 后,组 1~3 样品不作处理,组 4 进一步于 1 200℃ 下烧结 3 h,去除表面结构作为对照。采用 X 射线衍射仪、傅里叶红外光谱仪测试、电感耦合等离子体原子发射光谱分析各组 CB-HA 化学成分,扫描电镜、比表面测试仪、孔隙率测试仪分析其物理结构。取第 4 代 MC3T3-E1 细胞分别与 4 组 CB-HA 共培养,培养 1 d 后扫描电镜观察细胞形态,1、3、7 d 后用 MTT 法分析细胞增殖情况,7、14 d 后用 pNPP 法测试细胞 ALP 表达情况。 结果 4 组 CB-HA X 射线衍射光谱均见羟基磷灰石的峰形,红外线吸收光谱示红外吸收峰与羟基磷灰石一致,电感耦合等离子体原子发射光谱分析显示钙磷比为 1.68~1.76。扫描电镜观察见组 1~3 CB-HA 表面分别为大、中、小尺寸团簇结构,组 4 表面没有明显纳米结构。4 组 CB-HA 比表面积比较差异有统计学意义(P<0.05),孔隙率比较差异无统计学意义(P>0.05)。与组 4 相比,组 1~3 孔径<50 nm 的微孔更多,并且随着纳米团簇结构尺寸的减小,微孔数量逐渐增多。与细胞共培养后,扫描电镜及 MTT 检测示,与组 1、4 相比,组 2、3 CB-HA 表面细胞黏附及增殖均更好,并且细胞有更多的 ALP 表达(P<0.05)。 结论 通过控制磷源溶液中铵根离子浓度能够调控 CB-HA 表面纳米团簇结构的尺寸,并且在 CB-HA 表面引入小尺寸团簇纳米结构能够改善材料的细胞黏附、增殖以及 ALP 表达,这种作用可能与纳米结构引起比表面积增加有关。

Objective To investigate the formation of nanostructure on cuttlefish bone transformed hydroxyapatite (CB-HA) porous ceramics and the effects of different nanostructures on the osteoblasts adhesion, proliferation, and alkaline phosphatase (ALP) expression. Methods The cuttlefish bone was shaped as plate with diameter of 10 mm and thickness of 2 mm, filled with water, and divided into 4 groups. The CB-HA in groups 1-4 were mixed with different phosphorous solutions and then placed in an oven at 120℃ for 24 hours. In addition, the samples in group 4 were further sintered at 1 200℃ for 3 hours to remove nanostructure as controls. The chemical composition of CB-HA were analyzed by X-ray diffraction spectroscopy, Fourier transform infrared spectrum, and inductively coupled plasma (ICP). The physical structure was analyzed using scanning electron microscopy, specific surface tester, and porosity tester. The MC3T3-E1 cells of 4th generation were co-cultured with 4 groups of CB-HA. After 1 day, the morphology of the cells was observed under scanning electron microscopy. After 1, 3, and 7 days, the cell proliferation was analyzed by MTT assay. After 7 and 14 days, the ALP expression was measured by pNPP method. Results X-ray diffraction spectrum showed that the four nanostructures of CB-HA were made of hydroxyapatite. The infrared absorption spectrum showed that the infrared absorption peak of CB-HA was consistent with hydroxyapatite. ICP showed that the ratio of calcium to phosphorus of all CB-HA was 1.68-1.76, which was consistent with hydroxyapatite. Scanning electron microscopy observation showed that the nanostructure on the surface of CB-HA in groups 1-3 were large, medium, and small cluster-like structures, respectively, and CB-HA in group 4 had no obvious nanostructure. There were significant differences in the specific surface areas between groups (P<0.05). There was no significant difference in the porosity between groups (P>0.05). Compared with group 4, groups 1-3 have more pores with pore size less than 50 nm. After co-cultured with osteoblasts, scanning electron microscopy observation and MTT assay showed that the cells in groups 2 and 3 adhered and proliferated better and had more ALP expression than that in groups 1 and 4 (P<0.05). Conclusion The size of cluster-like nanostructure on the surface of CB-HA can be controlled by adjusting the concentration of ammonium ions in the phosphorous solution, and the introduction of small-sized cluster-like nanostructure on the surface of CB-HA can significantly improve the cell adhesion, proliferation, and ALP expression of the material which might be resulted from the enlarged surface area.

关键词: 骨组织工程; 墨鱼骨; 羟基磷灰石; 多孔陶瓷; 纳米结构

Key words: Bone tissue engineering; cuttlefish bone; hydroxyapatite; porous ceramics; nanostructure

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