纳米羟基磷灰石/明胶仿生复合材料的制备及其细胞相容性

目的：评价纳米羟基磷灰石/明胶仿生复合材料的一般理化性质及细胞相容性。方法：采用冷冻干燥技术 将纳米羟基磷灰石与明胶复合物制成3D多孔支架材料，并用扫描电镜、傅里叶红外光谱、万能试验机等对复合材料 进行表征；原代培养BALB/c小鼠成骨细胞，取第3代与复合材料共培养，采用扫描电镜、细胞毒性实验、细胞增殖实 验和碱性磷酸酶活性测定法观察支架材料对细胞分化、增殖的影响。结果：扫描电镜显示复合材料为三维多孔状， 孔径大小约150~400 μm；傅里叶变换红外光谱显示复合材料无机相和有机相之间有较强的化学键合；复合材料抗压 强度为(3.28±0.51)MPa，孔隙率为(80.6±4.1)%，接近人松质骨。扫描电镜观察和MTT实验结果显示复合材料具有良好 的细胞相容性，小鼠成骨细胞在材料上黏附和伸展情况良好；细胞毒性I~II级；与材料共培养第5，7天的细胞增殖 率与对照组差异有统计学意义(P<0.05)；第1，3天的碱性磷酸酶活性与对照组之间的差异有统计学意义(P<0.05)。结 论：明胶与纳米羟基磷灰石在一定比例和条件下，采用冷冻干燥法可制备出具有较高孔隙率、适宜孔径大小的三维 连通多孔结构的仿生支架材料，具有良好的细胞相容性，有望成为一种新型骨组织工程支架材料。

Biomimetic nanohydroxyapatite/gelatin composite material preparation and in vitro study

Objective: To prepare nHA/gelatin porous scaffold and to evaluate its physical and chemical properties and biocompatibility. Methods: We used nanopowders of HA and gelatin to prepare 3D porous composite scaffold by freeze-drying technique, and used scanning electron microscope, fourier transform infrared spectroscopy and universal testing machine to characterize the composite material. Osteoblasts were primarily cultured, and the third-passage osteoblasts were co-cultured with the composite material. The cell adhesion and morphology were examined under scanning electron microscope. The cell viability analysis was performed by MTT assay, and the alkaline phosphatase activity was measured with alkaline phosphatase kit. Results: Scanning electron microscope showed that the scaffold possessed a 3-dimensional interconnected homogenous porous structure with pore sizes ranging from 150 to 400 μm. Fourier transform infrared spectroscopy showed that the composite material had a strong chemical bond between the inorganic phase and organic phase. The scaffold presented the compressive strength of (3.28±0.51) MPa and porosities of (80.6±4.1)%. Composite materials showed features of had good biocompatibility. Mouse osteoblasts were well adhered and spread on the materials. The grade of the cell toxicity ranged from I to II. On the 5th and 7th day the proliferative rate of osteoblasts on scaffolds in the composite materials was significantly higher than that in the control group. The activity of alkaline phosphatase was obviously higher than that in the control group on Day 1 and 3. Conclusion: Nano-hydroxyapatite and gelatin in certain proportions and under certain conditions can be prepared into a composite biomimetic porous scaffolds with high porosity and threedimensional structure using freeze-drying method. The scaffold shows good biocompatibility with mouse osteoblasts and may be a novel scaffolds for bone tissue engineering.