新型多孔β磷酸三钙作为骨组织工程支架材料的实验研究

目的探讨新型多孔β磷酸三钙（β tricalcium phosphate,β-TCP）作为骨组织工程支架材料的应用效果。方法将兔骨髓间充质干细胞（marrow mesenchymal stem cells,MSCs）与β-TCP复合培养,实验组于24孔培养板中每孔加入3mm×3mm×3mm的β-TCP小块与细胞混合培养,对照组单纯接种细胞。培养后至10d内行倒置相差显微镜观察,第6天行扫描电镜观察细胞生长情况并与对照组比较;复合培养3、6、9、12d MTT法测定细胞增殖情况并与对照组比较以判断其细胞相容性。通过不同浓度（100%、50%、10%、1%、0）的β-TCP浸提液对细胞增殖的影响检验其细胞毒性。将MSCs诱导为成骨细胞,并与β-TCP复合修复兔桡骨1.5cm大段骨缺损,术后2、6、12周分别取材通过组织学、X线片和放射性核素骨扫描（emission computed tomograph,ECT）检验其成骨效果。结果MSCs细胞接种后4h可见部分细胞贴壁,12h后完全贴壁,细胞呈多角形、梭形;8～10d后汇成单层,实验组细胞生长与对照组相似。第6天倒置相差显微镜和扫描电镜观察可见细胞黏附性良好。MTT法测定示实验组与对照组各时间点吸光度（A）值比较差异无统计学意义（P〉0.05）。各时间点不同浓度β-TCP的细胞毒性均为0级。组织学、X线片和ECT均显示复合材料能够修复兔桡骨的大段骨缺损,且体内降解速率与骨的形成速率一致。结论新型多孔β-TCP具有独特的三维立体结构,优良的理化性质,是一种良好的骨组织工程支架材料。

NEW POROUS β-TRICALCIUM PHOSPHATE AS SCAFFOLD FOR BONE TISSUE ENGINEERING

OBJECTIVE: To investigate the feasibility of a new kind of porous beta tricalcium phosphate (P-TCP) as a scaffold for the bone tissue engineering. METHODS: The inverted phase contrast microscope was used to observe the growth of the marrow mesenchymal stem cells (MSCs) in the experimental group and the control group at 10 days. In the experimental group, the MSCs were cultured with beta-TCP(3 mm x 3 mm x 3 mm) in the 24-hole cultivation board, and in the control to control group, only MSCs were cultivated. The scanning electron microscope was used to observe growth of MSCs at 6 days. Cultivated with beta-TCP at 3, 6, 9, 12 days, the MTT assay was used to judge the biocompatibility. The cytotoxicity was analyzed with the method that used the different density (100%, 50%, 10%, 1%, 0%) leaching liquor gained from beta-TCP to raise MSCs. MSCs were induced into the osteoblasts and were mixed with beta-TCP, and the composite was used to repair a large radius bone defect in the rabbit. The specimens were made at 2, 6, 12 weeks. The histology imageology, and the radionuclide bone scan were used to analyze the bone formation. RESULTS: Some MSCs had a good adherence 4 hours after MSCs were inoculated and had a complete adherence at 12 hours. The cells were shaped like poly-angle, spindle or converge monolayer after 8-10 days. The cells in the two groups had no difference. The cell adhesion was good, when observed by the inverted phase contrast microscope and the scanning electron microscope at 6 days. MTT showed that the absorbance (A) was not statistically different between the experimental group and the control group (P > 0.05); the different density leaching liquor had no cytotoxicity at the different time points. Histology, X-ray, and CT tomograph showed that it could repair the large radius bone defect in the rabbit and its in vivo degradation rate was the same as the bone formation rate. CONCLUSION: The new porous beta-TCP has a unique three dimensional (3D) stereochemical structure and superordinary physico-chemical property, and so it is a good scaffold for the bone tissue engineering.