四种仿生骨组织工程支架材料的细胞相容性

Compatibility of osteoblasts on four kinds of biomimetic composite scaffolds of bone tissue engineering

背景:模拟生理条件体外细胞培养可排除生物材料受体内多种因素的影响,控制实验条件,方法简便,重复性好。目的:以改性生物活性玻璃粉体和胶原为基础,配以透明质酸钠、磷酸丝氨酸制备4种仿生复合支架材料,观察其与体外成骨细胞的细胞相容性。设计、时间及地点:对比观察实验,于2006-03/08在南方医科大学珠江医院临床医学基础研究所完成。材料和方法:小鼠胚胎成骨细胞株MC3T3-E1由浙江大学医学院病原生物学教研室提供。采用冷冻干燥和仿生矿化技术自制4种仿生复合骨组织工程三维支架材料。将小鼠胚胎成骨细胞种植于自制4种支架及58S生物玻璃支架上。主要观察指标:培养1,3,5d后,MTT法测定各组材料上成骨细胞活性,铁氰化钾法检测各组材料上细胞的碱性磷酸酶活性。倒置相差显微镜观察细胞动态变化,扫描电镜观察细胞形貌特征及其在材料上的生长情形。结果:成骨细胞活性和碱性磷酸酶活性测定结果表明,生物活性玻璃/胶原蛋白/透明质酸/磷酸丝氨酸组>生物活性玻璃/胶原蛋白/磷酸丝氨酸组>生物活性玻璃/胶原蛋白/透明质酸组>生物活性玻璃/胶原蛋白组>58S生物玻璃组,各组间差异均有显著性意义(P均<0.05),生物活性玻璃/胶原蛋白/透明质酸/磷酸丝氨酸组和生物活性玻璃/胶原蛋白/磷酸丝氨酸组的碱性磷酸酶活性比较差异无显著性意义(P>0.05);成骨细胞活性和碱性磷酸酶活性随着培养时间增长而增加,各时间点间比较差异均有显著性意义(P均<0.05)。成骨细胞在4种仿生复合材料上能良好黏附、增殖,尤其在生物活性玻璃/胶原蛋白/透明质酸/磷酸丝氨酸复合支架上生长活力最旺盛,在58S生物玻璃上黏附差、细胞逐渐死亡。结论:仿生复合支架生物活性玻璃/胶原蛋白/透明质酸/磷酸丝氨酸具有促进成骨细胞黏附、生长、分泌与迁移等作用,细胞相容性好,与天然骨基质的生物效应相类似,优于其他3种复合支架材料。

BACKGROUND： In vitro cell culture in the biomimetic physiological condition is a convenient method to prevent the biomaterials fi-om multiple factors in vivo, and to control the experimental conditions with potent reproducibility. OBJECTIVE： To explore the compatibility of osteoblasts culturing on four kinds of biomimetic composite scaffolds, which are prepared modified bioacfive glass （BG）, collagen （COL）, hyaluronic acid （HYA） and phosphoserine （PS）. DESIGN, TIME AND SETTING： The controlled experiment was conducted in the Clinical Medicine Research Institute of Zhujiang Hospital, Southern Medical University （Guangzhou, Guangdong, China） from March to August in 2006. MATERIALS AND METHODS： Monse embryo osteoblasts were offered by the Department of Pathogenic Biology in Zhejiang University Medical College. Four kinds of biomimetic three-dimensional composite scaffolds of bone tissue engineering were prepared by means of freeze drying and biomimetic mineralization. Mouse embryo osteoblasts were seeded on the four kinds of composite scaffolds and 58S BG scaffold, Tespecfively. MAIN OUTCOME MEASURES： At 1, 3 and 5 days of the culture, MTT assay was employed to detect the activity of osteoblasts, alkaline phosphatase activity was assayed with potassium ferricyanide method. Inverted phase contrast microscopy was performed to analyze the dynamic change of osteoblasts. Effects of biomimetic composite scaffolds on cell morphology, growth and proliferation were observed under scanning electron microscope. RESUI,TS： The osteoblasts activity and alkaline phosphatase activity showed the best outcomes in the BG-COL-HYA-PS, and then BG-COL-PS, BG-COL-HYA, BG-COL, but poor in 58S BG group. There were significant differences among the groups （P 〈 0.05）. The alkaline phosphatase activity was of insignificant differences between BG-COL-FIYA-PS scaffold and BG-COL-PS scaffold （P 〉 0.05）. The activities of osteoblasts and alkaline phosphatase were increased as time going on, with the significant differences at each time point （P 〈 0.05）. Osteoblasts adhered well on the BG-COL-HYA-PS scaffolds, but poorly on 58S BG scaffold and even gradually died. CONCI.USION： The biomimetic BG-COL-HYA-PS composite scaffold has a remarkable promotion on the osteoblasts adhesion, growth, secretion and migration, indicating well comp-atibility. This composite scaffold has similar biological effects to natural bones and is superior to other three materials.