釉基质蛋白与不同化学方式联合处理根面对牙周膜细胞附着、增殖的影响

Effects of enamel matrix proteins combined with different chemical treatments on the attachment and proliferation of human periodontal ligment cells onto the root surface

目的:观察釉基质蛋白(enamel matrix proteins,EMPs)与不同的化学方式联合处理牙周炎患牙根面后对牙周膜细胞(periodontal ligament cells,PDLCs)附着和增殖的影响,为牙周炎患牙体外牙周组织构建奠定基础。方法:收集因晚期牙周炎拔除的患牙60个,经刮治和根面平整后,制备5 mm×5 mm×1 mm大小的根片并随即分成3组,分别用20 g/L盐酸四环素、240 g/L EDTA、生理盐水(对照组)处理3 min后,再使用100μg/mL EMPs处理2 h。人PDLCs体外培养后接种于各组根片并于24 h后在扫描电镜下、HE染色观察细胞的附着状况,分别在1、3、5、7 d用CCK-8试剂盒检测细胞的增殖,并对数据进行双因素方差分析,检测水准α=0.05。结果:两实验组采用化学处理与EMPs联合应用后,PDLCs在根片上的附着良好,可见细胞间大量丝状伪足相连,HE染色显示细胞沿根片呈连续排列,优于单纯使用EMPs的对照组。接种第3天开始,240 g/LEDTA+EMPs组的细胞量,比对照组有显著增多(P(0.05),接种第5天开始,20 g/L盐酸四环素+EMPs组的细胞量比对照组有显著增多(P(0.05),而两实验组之间无显著性差异。结论:EMPs与不同化学处理方式联合处理牙周炎患牙根面相比,单纯使用EMPs更有利于PDLCs在根面的附着和增殖,而240 g/L EDTA与EMPs配伍使用有助于PDLCs在根面的早期增殖。

AIM ： To study the effect of enamel matrix proteins （ EMPs ） on the attachment and proliferation of human periodontal ligment cells （PDLCs） onto the root surface of teeth with periodontitis, which were pretreated with different chemical root conditional agents. It can provide bases for the formation of cementum - periodontium in vitro. METHODS ： 60 extracted teeth because of severe periodontitis were treated by scaling and root planning （SRP）. Then the experimental surfaces of fragments （5mm× 5mm × 1 mm） were prepared. The fragments were randomly divided into three groups. The root surfaces in group 1,2 and 3 were treated by 20g/L tetracycline HCL ,240g/L EDTA and physiological saline（control） for 3 rain respectively. Then all fragments were treated with EMPs at a concentration of 100 ug/ml for 2h. Human periodontal ligament cells from healthy teeth were cultured and incubated on the surface of each fragment. The spread of PDLCs on root surfaces was studied by scaning electron microscope（SEM） and HE stain. The proliferation was quantified by the CCK - 8 assay for 1,3,5,7days respectively after incubation. The data were analysed by variance. RESULTS： PDLCs on surfaces of experimental groups under SEM were growing robust, and HE stain showed a continuous line of PDLCs along the root surface. By contrast, PDLCs on surfaces in the control group were only partly coverd with spindle - shaped cells and with filopodia appearing short and thin. CCK - 8 assay indicated that the number of adhered cells on the diseased root surfaces treated with 240g/L EDTA ＋ EMPs was significantly greater than that on the diseased root sufaces treated with saline ＋ EMPs in the third day,but the number of adhered ceils on diseased root surfaces treated with 20g/L tetracycline HCL ＋ EMPs was significantly greater than that on diseased root sufaces treated with saline ＋ EMPs in the fifth day. CONCLUSION： It was suggested that the combined utilization of chemical root conditioning agents and EMPs on diseased root surfaces after SRP could promote the attachment and proliferation of PDLCs. 240g/L EDTA combined with EMPs is benefitial to early proliferation of PDLCs on diseased root surfaces in comparison with 20g/L tetracycline HCL.