富血小板纤维蛋白对犬牙髓细胞的体外作用

Effects of platelet-rich fibrin on canine dental pulp cells in vitro

目的:检测比格犬静脉血制取的富血小板纤维蛋白(platelet-rich fibrin,PRF)对自体牙髓细胞(canine dental pulp cells,cDPCs)增殖和趋化的作用,探讨PRF作为自体来源生物材料在临床活髓治疗中应用的可行性。方法:用酶消化法分离培养cDPCs;用Choukroun一步离心法制取PRF,将其浸泡于纯净的最小必需培养基α(minimum essential medium alpha medium,α-MEM)中,于第7天取浸出液,即为PRF浸出液。细胞增殖作用采用细胞计数试剂盒-8(cell counting kit-8,CCK-8)检测,对照组为含2%(体积分数)胎牛血清(fetal bovine serum,FBS)的α-MEM培养基,实验组为含2%FBS的PRF浸出液,并按PRF浸出液浓度(体积分数)分为20%、40%、60%、80%、100%共5组,分别记为PRF1、PRF2、PRF3、PRF4、PRF5。趋化实验采用Transwell模型,实验组PRF浸出液浓度选择对增殖促进作用最显著的浓度,阴性对照组为不含FBS的α-MEM培养基,阳性对照组为含30%(体积分数)FBS的α-MEM培养基,各组上室均接种1×105个细胞。结果:PRF2组的光密度值(1.45±0.06)显著高于对照组(1.21±0.11),差异具有统计学意义(P<0.001),PRF1组、PRF3组、PRF4组、PRF5组光密度值分别为1.20±0.02、1.28±0.04、1.19±0.02、1.22±0.02,与对照组差异无统计学意义(P值分别为0.902、0.084、0.726、0.779),即40%浓度的PRF浸出液对自体cDPCs的增殖具有显著的促进作用,在该浓度下,PRF组细胞迁移数目为55.89±18.42,与阴性对照组(6.52±1.97)比较差异具有统计学意义(P<0.001),而与阳性对照组(59.25±29.17)差异无统计学意义(P=0.970)。结论:PRF与cDPCs有良好的生物相容性,40%浓度的PRF浸出液可促进cDPCs的增殖、趋化作用,提示PRF可作为活髓治疗中牙髓修复的盖髓材料使用。

Objective：To investigate the effect of platelet-rich fibrin （PRF） on the proliferation and cbemotaxis capacity of autogenous canine dental pulp cells （cDPCs） , and to evaluate the use of PRF as a pulp capping material in vital pulp therapy. Methods： cDPCs were isolated and cultured from perma- nent anterior teeth of Beagle dogs by enzymatic methods. PRF was attained by Choukroun＇ s protocols and the exudates of PRF were collected at the time point of the 7th day. Cell counting kit-8 （ CCK-8 ） was ap- plied to analyze cell proliferation. The medium of the control group was minimum essential medium alpha medium （a-MEM） containing 2% （volume fraction） fetal bovine serum （FBS）. The experiment group was the exudates of PRF containing 2% FBS, and was divided into 5 groups （20% , 40% , 60% , 80% , 100% ） by the volume fraction of the exudates of PRF. The 5 groups were named as PRF1, PRF2, PRF3, PRF4 and PRF5 respectively. Transwell model was used to evaluate cell chemotaxis capacity. The exudates of PRF which was most effectively in promoting cDPCs proliferation was added in the lower chamber of the experimental group; The positive control group was ct-MEM containing 30% FBS and the negative control group was fresh a-MEM ; The upper chamber of each group was added with 1 × 105 cells. Results ： The optical density of group PRF2 （ 1.45 ± 0.06） was significantly higher than that of the con- trol group（l. 21±0.11, P 〈0. 001 ）. The optical density of groups PRF1, PRF3, PRF4, and PRF5 were 1.20 ±0.02, 1.28 ±0.04, 1.19 ±0.02, 1.22 ±0.02, respectively, and there was no significant difference between these groups and the control group （ PRF1 ： P = 0. 902 ; PRF3 ： P = 0. 084 ; PRF4 ： P = O. 726 ; PRF5 ： P = 0.779）. Therefore, the volume fraction which was most effective in promoting cell proliferation was 40%. At these concentrations the number of migrated cells was higher in PRF group