富血小板纤维蛋白对牙髓细胞增殖与分化的影响

Effects of Choukroun′s Platelet-Rich Fibrin on the Osteogenic Differentiation of Dog Dental Pulp Cells in vitro

目的:评估牙髓细胞在富血小板纤维蛋白(platelet-rich fibrin,PRF)存在下的体外增殖及成骨分化的能力,为PRF作为支架材料、牙髓细胞作为种子细胞构建组织工程骨,进行前期研究。方法:3月龄比格犬拔除乳磨牙获得乳牙牙髓细胞;恒牙牙髓细胞从16月龄成年比格犬磨牙获得。静脉取血离心10 min获得PRF。实验分4组:对照组(普通培养基不加入PRF);实验组A(普通培养基加入PRF);实验组B(成骨诱导培养基不加入PRF);实验组C(成骨诱导培养基加入PRF)。分别于1、4、7和11d测定细胞数量、MTT值、半定量碱性磷酸酶值、成骨相关基因Q-PCR值,并于21d测定钙结节吸光度值。结果:PRF是一种可以促进牙髓细胞增殖的,无毒性作用的纤维网状支架结构;细胞水平上PRF促进了两种细胞的成骨分化:钙结节以及碱性磷酸酶半定量数值都明显上调(P<0.05);基因水平上,4个时间点成骨相关基因的表达量都显著增加(P<0.05),且乳牙牙髓细胞的表现均优于恒牙牙髓细胞。结论:可使用PRF和牙髓细胞复合构建组织工程骨。

Objective：To evaluate the effects of choukroun＆#39;s PRF （platelet-rich fibrin） on proliferation and differentiation of dental pulp cells in vitro and making a pre-feasibility assess hunting for the optimum scaffold. Methods：Pulp tissues of deciduous and permanent molars were harvested from 3-month and 16-month old beagle dogs respectively. Dental pulp cells were isolated and cloned. Jugular vein blood were collected from beagle dogs and centrifugation at 3 000 r/min for 10 minutes to obtain PRF. The experiments were divided into four groups：control group （ordinary medium without PRF）, test group A （ ordinary medium with PRF）, test group B （osteoinduction medium without PRF）, test group C （added PRF in osteoinduction medium ）. Cell counts and cytotoxicity tests, semi-quantitative alkaline phosphatase and real time PCR, were performed and assayed at days 1, 4, 7 and 11 of culture. The determination of calcium nodules OD value was in day 21. Results： PRF is a fibrin matrix structure and showed absence of toxic effect on two types of cells. PRF increased a significant proliferation in all cell types. Furthermore, PRF increased a strong differentiation of dental pulp cells including calcium nodules, ALP semi-quantitative（P〈0.05） and osteogenesis related genes（P〈0.05）. Dental pulp cells of exfoliated deciduous teeth showed more obvious than the permanent dental pulp cells. Conclusion： This in vitro study suggests that PRF scaffolds may be used as a specific regulator of hard-tissue engineering.