富血小板纤维蛋白提取液对钛表面成骨细胞黏附、增殖及分化的影响

The effects of platelet-rich fibrin extract on MC3T3-E1 cells cultured on the titanium discs

目的：探讨富血小板纤维蛋白提取液（platelet-rich fibrin extract，PRFe）对钛表面成骨细胞黏附、增殖、分化的影响，探讨PRFe作为生长因子来源对种植体-骨结合的促进作用。方法实验组和对照组分别用含0.5%PRFe和不含PRFe的α-最低必须培养基，在钛片表面接种成骨细胞（每组每时间点设3个重复样本）。扫描电镜及激光扫描共聚焦显微镜分别观察接种24、72 h后细胞黏附及12、24 h后细胞骨架形态。甲基噻唑基四唑法测定培养1、3、5、7 d的细胞增殖情况；碱性磷酸酶活性检测诱导1、3、5、7 d的细胞分化情况；荧光实时定量PCR（quantitative real-time PCR，qRT-PCR）测定诱导3和7 d成骨细胞核心结合因子α1（core-binding factorα1，Cbfα1）和骨钙蛋白的基因表达。结果扫描电镜及激光扫描共聚焦显微镜显示，随时间延长，实验组细胞形态较对照组更伸展，肌动蛋白排列更致密。培养1、3、5、7 d实验组细胞增殖（A值分别为0.299±0.002、0.517±0.004、0.810±0.002和1.203±0.011）均显著大于对照组（分别为0.198±0.003、0.399±0.002、0.588±0.002和0.897±0.005）（P〈0.05）。培养1、3、5、7 d实验组碱性磷酸酶A值（分别为0.162±0.004、0.289±0.001、0.491±0.006和0.647±0.005）均显著大于对照组（分别为0.121±0.003、0.191±0.006、0.252±0.004和0.365±0.012）（P〈0.05）。qRT-PCR显示，实验组Cbfα1和骨钙蛋白基因表达量（3 d：分别为1.50±0.04和3.37±0.17；7 d：分别为1.94±0.06和3.92±0.04）均显著高于对照组（3 d：均为1；7 d：分别为1.18±0.13和2.34±0.09）（P〈0.05）。结论 PRFe能有效促进成骨细胞在钛表面的黏附、增殖和分化。

Objective To evaluate the effect of platelet-rich fi brin extract （PRFe） on the adhesion, proliferation and differentiation of MC3T3-E1 cells cultured on the titanium discs. Methods Samples were divided into experimental group （P） and control group （D）. Group P used the α-minimal essential medium （α-MEM） containing PRFe （0.5%）, while group D used only the α-MEM. Cell adhesion and cytoskeleton were observed using scanning electron microscopy （SEM） and laser scanning confocal microscopy （LSCM）. Methyl thiazolyl tetrazolium （MTT） assay to detect the number of the osteoblasts at 1, 3, 5, 7 d;the activity of alkaline phosphatase （ALP） to detect the differentiation of osteoblast at 1, 3, 5, 7 d;the level of osteogenetic biomarkers core-binding factorα1 （cbfα1） and osteocalcin （OCN） were quantified by quantitative real-time PCR （qRT-PCR） at 3 and 7 d. Results SEM and LSCM showed that the adhesion and filaments of group P were higher than those of group D at each time point. MTT assay showed that the absorbance were significantly increased in group P （1 d：0.299±0.002, 3 d：0.517±0.004, 5 d：0.810±0.002, 7 d：1.203±0.011） compared with group D （1 d：0.198±0.003, 3 d：0.399±0.002, 5 d：0.588±0.002, 7 d：0.897±0.005） at each time points （P〈0.05）. Furthermore, the ALP activity of group P （1 d：0.162 ± 0.004, 3 d：0.289 ± 0.001, 5 d： 0.491±0.006, 7 d：0.647±0.005） was significantly higher than that of group D （1 d：0.121±0.003, 3 d：0.191± 0.006, 5 d：0.252 ± 0.004, 7 d：0.365 ± 0.012）, （P〈0.05）. Moreover, the qRT-PCR showed that the Cbfα1 and OCN gene expression in group P （Cfbα1, 3 d：1.50±0.04, 7 d：1.94±0.06;OCN, 3 d：3.37±0.17, 7 d：3.92± 0.04） were significantly higher than that in group D（Cfbα1, 3 d：1, 7 d：1.18 ± 0.13;OCN, 3 d：1, 7 d：2.34 ± 0.09） （P〈0.05）. Conclusions PRFe promoted the adhension, proliferation and differentiation of MC3T3-E1 cells on the titanium discs.