模拟[牙合]力对不同根吸收时期乳牙牙周膜干细胞的影响

Effect of simulated occlusal force on periodontal ligament stem cells of the deciduous teeth at different stages of root absorption

目的研究[牙合]力对不同根吸收时期乳牙和恒牙牙周膜干细胞(periodontal ligament stem cell,PDLSC)生物学特性的影响,探索乳牙生理性根吸收的相关机制。方法收集2019年4月至2020年4月第四军医大学口腔医学院儿童口腔科因乳牙滞留拔除的健康乳切牙12颗和口腔颌面外科因正畸需要拔除的健康第一前磨牙6颗,根据牙齿类型及牙根吸收程度分为:乳牙牙根未吸收组(the unresorbed group,UN组)、乳牙牙根吸收组(the resorbed group,R组)和恒牙组(the permanent teeth group,P组),每组各6颗。从各组牙周膜分离原代PDLSC并进行传代培养,对各组PDLSC分别加载0~45、0~90、0~135、0~180、0~225、0~270 kPa的流体动态压力,细胞计数试剂盒(cell counting kit-8,CCK-8)法分别检测加载压力后1~7 d的细胞增殖能力;对各组PDLSC分别加载0~45、0~90、0~135、0~180、0~225 kPa的流体动态压力,流式细胞技术检测细胞凋亡水平,各时间各组均以未加载压力作为对照。加载0~90 kPa的流体动态压力作用于细胞骨架,纤维型肌动蛋白(fibrous actin,F-actin)染色方法观察细胞微丝的改变。结果不同流体动态压力下,各组PDLSC均具有一定自我增殖能力。UN组和R组在0~180 kPa及以下动态压力作用3~7 d的A值均显著高于P组(P<0.05),但在0~270 kPa压力作用下1~7 d,UN组、R组和P组A值差异均无统计学意义(P>0.05)。UN组和R组PDLSC在0~180 kPa及以下动态压力作用下的A值与各自对照组相比差异均无统计学意义(P>0.05),但在0~225及0~270 kPa压力作用下,3~7 d的A值与各自对照组相比差异均有统计学意义(P<0.05);P组PDLSC在0~225 kPa及以下压力作用下1~7 d的A值与其对照组相比差异均无统计学意义(P>0.05),在0~270 kPa压力作用下,仅3 d(1.386±0.131)和5~7 d的A值(分别为:1.728±0.226、2.029±0.168、2.263±0.210)分别与各自对照组相比差异有统计学意义(P<0.05)。细胞凋亡结果显示,相比各组对照组,UN组、R组和P组PDLSC分别在0~90 kPa及以上、0~135 kPa及以上和0~180 kPa及以上流体动态压力作用下A值差异有统计学意义,细胞凋亡显著升高(P<0.05)。F-actin染色结果显示,在0~90 kPa压力下,各组细胞微丝均呈绿色丝状,R组和P组细胞微丝沿细胞长轴排列,UN组细胞微丝部分纤维结构改变,排列更紧密。结论不同根吸收时期PDLSC受到力学刺激后的生物学特性均发生改变,牙根未吸收期乳牙PDLSC对力学刺激更敏感。

Objective To explore the related mechanisms of biological root resorption in decidual teeth by studying the biological effect of simulated occlusal force on the periodontal ligament stem cells(PDLSC)at different stages of root absorption.Methods According to the tooth type and root absorption degree,healthy retained deciduous incisors and healthy first premolars that needed to be removed for orthodontic treatment were collected and divided into three groups with six teeth in each group:the deciduous unabsorbed group(UN group),the absorbed group(R group)and the permanent teeth group(P group).PDLSC was isolated from periodontal ligament and cultured.PDLSC of three groups were loaded with dynamic pressure of 0-45,0-90,0-135,0-180,0-225 and 0-270 kPa,respectively.The proliferation ability was detected by cell counting kit-8(CCK-8)technique on day 1 to day 7,respectively.The apoptosis levels of PDLSC after loading with dynamic pressure of 0-45,0-90,0-135,0-180 and 0-225 kPa were observed by the flow cytometry.The changes of microfilaments were observed by fibrous actin(F-actin)staining after the cytokeleton was subjected to dynamic pressure of 0-90 kPa.Results PDLSC of three groups exhibited various proliferation abilities to dynamic pressure.The A values in the UN group and R group were significantly higher than those in the P group and the difference was statistically significant(P<0.05).However,there was no significant difference between the UN group and the R group(P>0.05).The A values of PDLSC in UN group and R group under dynamic pressures of 0-45,0-90,0-135 and 0-180 kPa had no statistical significance compared with the control group unloading dynamic pressure(P>0.05).However,under 0-225 and 0-270 kPa dynamic pressures,the A values at the day 3 to day 7 were statistically significant(P<0.05).The A values of PDLSC in P group under 0-45,0-90,0-135,0-180 and 0-225 kPa dynamic pressures for 1 to 7 days were no statistically significant difference compared with the control group(P>0.05).The A value in P group under the 0-270 kPa was statistically significant only on day 3(1.386±0.131)and day 5 to day 7(1.728±0.226,2.029±0.168 and 2.263±0.210,respectively)(P<0.05).The result of apoptosis showed that the A values of PDLSC in UN group,R group and P group were significantly increased under 0-90,0-135,0-180 kPa and above dynamic pressures,respectively(P<0.05)compared with the control group unloading dynamic pressure.Under 0-90 kPa dynamic pressure,F-actin fluorescence staining samples in three groups all showed green filaments which were arranged along the long axis of the cells in the R group and the P group,while some fibers in the UN group were closely arranged and promoted stress fiber assembly.Conclusions The biological characteristics of PDLSC at different root absorption stages were changed when they were stimulated by mechanical stress,and PDLSC of the deciduous teeth at the root unabsorption stage were more sensitive to mechanical stress stimulation.