生物玻璃/P-15复合骨替代材料的实验研究

The study of a bioglass and peptide /P-15 combined bone substitude material

目的:将P-15多肽粘附肽与生物玻璃复合,联合二者各自的优点,得到一种成骨活性更高的人工骨材料,并评价其在体外体内实验中的成骨活性。方法:Wistar大鼠体内分离并培养骨髓基质细胞,接种在已经铺好生物玻璃/P-15复合材料的24孔板内。并以未复合多肽的生物玻璃做为对照,分别在第1d、4d、7d、10d测定培养液中的细胞的碱性磷酸酶(ALP)活性和蛋白质含量,观察共培养后BMSCs的成骨活性变化。筛选出理想的多肽浓度,将相应浓度复合材料植入兔颅骨缺损模型内,在4w、6W、8w通过软X线和组织学观察新骨形成的情况。结果:体外实验表明,细胞在材料周围生长、铺展良好,可复层排列。当多肽液浓度为200μg/ml时,与复合材料共培养的BMSCs的ALP活性及总蛋白含量明显高于未复合多肽组和其他浓度组(P<0.01)。各组之间总蛋白含量差异无统计学意义。软X线和组织切片显示,在4w、6W、8w植入的颅骨模型标本中,实验组材料降解速度和新骨形成面积高于同期对照组标本。结论:复合多肽后的生物玻璃对大鼠BMSCs的细胞相容性良好,材料周围细胞生长、铺展正常。复合材料能增强BMSCs的ALP表达,增强其成骨活性。在修复兔颅骨缺损中,能促进更多的新骨形成。因而将P-15多肽与其它材料复合不失为提高骨替代材料活性的一个有效途径。

Purpouse:Combine P-15 peptide with bioglass,union their advantages,get a new bone substitude material with better bone formation performance.Test its bone formation ability in vitro and vivo experiments.Method:We use the bone marrow stromal cells which are separated from rat and then the third passage cells are plated at a density of 2×104/well in 24-well plates that have been filled with Bioglass/P-15 material.Bioglass without P-15 serves as a control group.We detect the total protein and alkaline phosphatase(ALP)activity on 1d,4d,7d,10d.Then choose the optimum concentration of P-15,which will be uesed in vivo experiment at the skull of rabbit.After the 4w,6w and 8w,rabbit were executed,we used the soft X ray and specimen slices to analyze the quantity and quality of new bone formation.Result:in vitro study shows,cells' shaps are favourable near the glass particles and perform a multilayer structures.When the concentrations of solution of the P-15 in phosphate buffered saline is 200μg/ml,the BMSCs which are cultured with the Bioglass/P-15 show the better alkaline phosphatase(ALP)activity than other groups,and the difference had statistical significance(P<0.01).The total protein level of each groups shows differences that are lack of statistical significance.Soft X ray and specimen slices suggest in the experiment group,newly formed bone and residual grafted materials are favorable than other groups and the difference had statistical significance.Conclusion:after the combination with P-15,Bioglass/P-15 maintains a good biocompatibility and enhances bone marrow stromal cells' alkaline phosphatase(ALP)activity.Cells' shaps are favourable near the glass particles and perform a multilayer structures.In vivo experiment showes that Bioglass/P-15 has a better performance in new bone formation.So,combing P-15 peptide with other scaffold could be a effective way of bone substitude material enhancement