摘要
背景:富血小板纤维蛋白在临床上已被用于骨及软组织修复,但是由于其具有生物活性,仅限于短期临床应用。如何稳定保存富血小板纤维蛋白及延长临床应用时间,显得尤为重要。目的:探讨低温冷冻干燥技术对富血小板纤维蛋白形态学以及对富血小板纤维蛋白中血小板释放生长因子活力及释放规律的影响。方法:制备新鲜及冻干富血小板纤维蛋白,将其浸泡于无血清的DMEM基础培养基,收集1,7,14,21 d各个时间点冻干及新鲜富血小板纤维蛋白析出液,进行成纤维细胞培养及血小板衍生生长因子BB含量测定,比较冻干前后富血小板纤维蛋白形态结构、生长因子活力及释放规律;通过苏木精-伊红染色进行形态学观察;通过CCK8法测定细胞增殖;通过细胞划痕实验评价对细胞迁移能力的影响;通过ELISA试剂盒检测不同时点两组析出液中血小板衍生生长因子BB含量。结果与结论:①苏木精-伊红染色显示,冻干富血小板纤维蛋白结构呈疏松呈海绵状,孔隙直径较大,蓝染有核细胞数目明显减少;②与无血清基础培养基相比,第1天和第7天收集的新鲜及冻干富血小板纤维蛋白析出液明显促进成纤维细胞增殖(P<0.05),其中冻干富血小板纤维蛋白析出液对成纤维细胞增殖能力效果更显著(P<0.05);③与无血清培养基相比,新鲜、冻干富血小板纤维蛋白析出液及含10%胎牛血清的完全培养基,在12 h及24 h两个时间点均能有效促进成纤维细胞迁移,差异有显著性意义(P<0.05);④与新鲜富血小板纤维蛋白析出液相比,冻干富血小板纤维蛋白析出液在12 h及24 h两个时间点促进成纤维细胞的迁移作用更明显(P<0.05),差异有显著性意义(P<0.05);⑤新鲜富血小板纤维蛋白组血小板衍生生长因子BB在第7天释放量最大,随着时间推移,生长因子释放量逐渐减少;冻干富血小板纤维蛋白组血小板衍生生长因子BB在第1天释放量最大,随着时间推移生长因子释放量逐渐减少,差异有显著性意义(P<0.05);两组生长因子释放总量进行比较,差异无显著性意义(P>0.05);⑥结果表明,冻干技术保存富血小板纤维蛋白具有可行性,可以为组织修复重建提供理论依据。
BACKGROUND:Platelet-rich fibrin has been used in bone and soft tissue repair in clinic.However,due to its biological activity,it is limited to short-term clinical application.Therefore,how to keep platelet-rich fibrin stably and prolong the time of clinical application is very important.OBJECTIVE:To explore the effect of freeze-drying technology on the morphology of platelet-rich fibrin and the activity and release regularity of growth factor releasing from platelets in platelet-rich fibrin.METHODS:Fresh and freeze-dried platelet-rich fibrin were prepared and then soaked in DMEM basic medium without fetal bovine serum.The supernatant of freeze-dried or fresh platelet-rich fibrin was collected at 1,7,14 and 21 days and used for fibroblast culture and platelet-derived growth factor-BB content determination.The morphological structure,activity and release regularity of growth factor releasing from platelets in platelet-rich fibrin were compared before and after freeze-drying.Morphological observation was detected by hematoxylin-eosin staining.Cell proliferation was measured by CCK8 assay.The cell migration ability was evaluated by cell scratch test.Platelet-derived growth factor-BB content of collected supernatant at different time points in the two groups was detected by using ELISA kit.RESULTS AND CONCLUSION:(1)Hematoxylin-eosin staining showed that the structure of freeze-dried platelet-rich fibrin was loose and spongy,and the pore diameter was large.The number of nuclear cells in blue staining was significantly reduced.(2)Compared with the serum-free medium,the fresh and freezedried platelet-rich fibrin extracts collected on the 1 and 7 days significantly promoted the proliferation of fibroblasts(P<0.05),and freeze-dried platelet-rich fibrin extracts strongly promoted the proliferation capacity of fibroblasts(P<0.05).(3)Compared with the serum-free medium,the fresh and freeze-dried platelet-rich fibrin extract,and the complete medium containing 10%fetal bovine serum could effectively promote the migration of fibroblasts at 12 and 24 hours,and the difference was statistically significant(P<0.05).(4)Compared with the fresh platelet-rich fibrin,the freeze-dried platelet-rich fibrin showed higher migration ability of fibroblasts at 12 and 24 hours(P<0.05),and the difference was statistically significant(P<0.05).(5)The content of platelet-derived growth factor-BB in fresh platelet-rich fibrin group was the largest at 7 days,and the release of growth factor gradually decreased.The content of plateletderived growth factor-BB in freeze-dried platelet-rich fibrin group was the largest at 1 day,and the release of growth factor gradually decreased with time,and the difference was statistically significant(P<0.05).The total content of platelet-derived growth factor-BB showed no significant difference between the two groups(P>0.05).(6)The results show that freeze-drying technology is feasible for platelet-rich fibrin preservation,which can provide theoretical basis for tissue repair and reconstruction.
作者
刘磊
狄海萍
郭海娜
曹大勇
牛希华
夏成德
Liu Lei;Di Haiping;Guo Haina;Cao Dayong;Niu Xihua;Xia Chengde(Department of Burn,First People’s Hospital of Zhengzhou City,Zhengzhou 450000,Henan Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2021年第31期4995-4999,共5页
Chinese Journal of Tissue Engineering Research