Jing Linguo, Yang Chen, Huan Zhiguang, Ke Qinfei, Chang Jiang
College of Life and Environmental Sciences, Shanghai Normal University, Shanghai, 200234, P.R.China.
Biomaterial and Tissue Engineering Research Center, State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, P.R.China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2019 Mar 15;33(3):363-369. doi: 10.7507/1002-1892.201804100.
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.
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.
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 ( <0.05). There was no significant difference in the porosity between groups ( >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 on groups 2 and 3 adhered and proliferated better and had more ALP expression than that on groups 1 and 4 ( <0.05).
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.
研究乌贼骨转化羟基磷灰石（CB-HA）多孔陶瓷纳米结构的形成以及不同纳米结构对成骨细胞黏附、增殖和碱性磷酸酶（ALP）表达的影响。
将乌贼骨加工成直径10 mm、厚度2 mm的圆板，加水后分为4组。第1-4组的CB-HA与不同磷溶液混合，然后置于120℃烘箱中24小时。此外，第4组样品进一步在1200℃烧结3小时以去除纳米结构作为对照。通过X射线衍射光谱、傅里叶变换红外光谱和电感耦合等离子体（ICP）分析CB-HA的化学成分。使用扫描电子显微镜、比表面积测试仪和孔隙率测试仪分析物理结构。将第4代MC3T3-E1细胞与4组CB-HA共培养。1天后，在扫描电子显微镜下观察细胞形态。在1、3和7天后，通过MTT法分析细胞增殖。在7和14天后，通过对硝基苯磷酸酯（pNPP）法测量ALP表达。
X射线衍射光谱显示CB-HA的四种纳米结构均由羟基磷灰石制成。红外吸收光谱显示CB-HA的红外吸收峰与羟基磷灰石一致。ICP显示所有CB-HA的钙磷比为1.68-1.76，与羟基磷灰石一致。扫描电子显微镜观察显示，第1-3组CB-HA表面的纳米结构分别为大、中、小簇状结构，第4组CB-HA无明显纳米结构。各组之间比表面积存在显著差异（<0.05）。各组之间孔隙率无显著差异（>0.05）。与第4组相比，第1-3组有更多孔径小于50 nm的孔隙。与成骨细胞共培养后，扫描电子显微镜观察和MTT法显示，第2组和第3组的细胞黏附、增殖更好，ALP表达比第1组和第4组更多（<0.05）。
通过调节磷溶液中铵离子的浓度可以控制CB-HA表面簇状纳米结构的大小，CB-HA表面引入小尺寸簇状纳米结构可显著提高材料的细胞黏附、增殖和ALP表达，这可能是由于表面积增大所致。
