富血小板血浆对人牙周组织细胞矿化作用的影响

Mineralization effects of human platelet-rich plasma on human periodontium cells

目的观察富血小板血浆(PRP)对人牙龈成纤维细胞(GF)、牙周膜细胞(PDLC)和牙槽骨成骨细胞(AOB)的矿化诱导作用。方法抽取健康成人的新鲜全血,经过两次离心得到富血小板血浆。培养GF、PDLC和AOB,取第4代细胞用于实验,3种细胞均以4×107个/L接种于6孔培养板内,标准条件下培养96h后换不同的培养液,实验组换用含有矿化液(10nmol/L地塞米松、10mmol/Lβ-甘油磷酸钠和新鲜制备的0·2mmol/L抗坏血酸)和50ml/LPRP的DMEM(100ml/LFBS)培养液,对照组换用含有矿化液的DMEM培养液,空白组换用普通DMEM培养液。隔天换液,培养至第30天进行vonKossa染色,显微镜下观察并记录结果,用图像分析软件半定量分析染色阳性面积比及细胞衰减的面积比。结果对于PDLC和AOB,实验组及对照组的矿化量多于空白组(P<0·05),同时实验组的矿化量高于对照组(P<0·05);对于GF,3个组之间的矿化量差异无统计学意义(P>0·05)。AOB实验组在形成基质矿化时的衰减比对照组和空白组少(P<0·05)。结论PRP可促进PDLC和AOB在体外形成矿化小结,并能减少HAOB在矿化形成时的衰减,提示PRP可提高牙周骨组织的矿化和再生。

Objective To observe the effects of platelet-rich plasma （PRP） on inducing mineralization of human gingival fibroblasts （ GF ） , periodontal ligament cells （ PDLC ） and alveolar bone osteoblasts （AOB）. Methods Human whole blood from healthy subjects was collected and PRP was obtained after twice centrifuged. GF, PDLC and AOB established from tissue explants were used at 4th passage in culture. All cells were seeded at density of 4×10^7/L at 6 well tissue culture plate and cultured at standard condition for 96 hours, and then the different media was used. In the experimental gToup, the cells continued to be cultured in 50 ml/L PRP in DMEM media with mineral solution （10 mmol/L β- glycerophosphate, 0. 2 mmol/L freshly prepared ascorbic acid, 10 nmol/L dexamethasone）, in the control group, the cells to be cultured in DMEM media with mineral solution, and in the blank group DMEM media was used. The media was changed every other day until day 30, the cells were dyed with von Kossa. Pictures were taken and analyzed by the image analysis software to semiquantitate the proportion of the positive area of von Kossa dye and attenuation aera of AOB. Results In PDLC and AOB, the quantity of mineralization nodule in the experimental group and the control group was more than that in the blank group （P 〈0.05 ）, and there were more mineralization nodules in the experimental group than in the control group （ P 〈 0. 05）, while the amount of mineralization nodules in GF was low in the three groups （ P 〉 0. 05 ）. Attenuation of AOB in the experimental group was less than the control group （ P 〈 0. 05 ）. Conclusions PRP can enhance the quantity of mineralization in PDLC and AOB in vitro and reduce attenuation in AOB, which suggests that PRP may be helpful to periodontal alveolar bone mineralization and regeneration.