牙周膜成纤维细胞-富血小板胶的合成及其成骨活性表达的研究

Synthesis of periodontal ligament fibroblasts/platelet-rich plasma gel and its osteogenic activity expression

目的:体外合成牙周膜成纤维细胞-富血小板胶(Periodontal ligament fibroblasts-Platelet-Rich Plasma gel,PDLFs/PRP-gel)并探讨其在临床应用中最佳植入时间。方法:体外合成PDLFs/PRP-gel;并测定其碱性磷酸酶(Alkaline phosphatase,ALP)含量;免疫组化法检测基质中骨涎蛋白(Bone sialoprotein,BSP)、骨桥蛋白(Osteopontin,OPN)含量;Von Kossa染色法观察沉积钙盐。结果:PDLFs/PRP-gel基质内第6d时ALP含量明显增高,第8～10d达到峰值,11d后迅速下降,与对照组比较差异有统计学意义。基质内骨桥蛋白在15～16d时表达明显,与其他时间的实验组比较有统计学差异;骨涎蛋白在4～6d和15～18d时表达明显,与其他时间的实验组比较有统计学差异。PRP-gel缓释液培养的人牙周膜成纤维细胞约在第15d时出现钙化结节。结论:临床牙周组织再生术中宜选用在体外培养8～15d的PDLFs/PRP-gel进行植入。

Objective： To synthesize the periodontal ligament fibroblasts / Platelet-Rich Plasma gel in vitro and determine the optimal cultivated time by comparing its osteogenic activity expression in different periods. Method： ALP activity in the matrix was detected： Bone sialoprotein （BSP）and osteopontin （OPN） were also tested by immunohistochemistry method in different days. Von Kossa staining was utilized to evaluate calcification capability of PDLFs in matrix. Result： （1） ALP activity in the matrix increased gradually on the 4th to 8th days and reached the maximum on the 8th day,then declined gradually. （2）The expression of bone sialoprotein （BSP）and the osteopontin （OPN） in the matrix was obvious from the 15th to 18th day （P〈0.05）. （3） Most Von Kossa positive particles were appeared under microscope from the 15th day. Conclusion： This experiment imply that the best moment to apply PDLFs / PRP-gel in periodontal tissue regeneration in vivo is from the 8th to the lgth days.