牙髓干细胞对牙周炎中破骨细胞的作用

The effect of dental pulp stem cells on osteoclasts in periodontitis

目的研究牙髓干细胞(DPSC)对牙周炎中破骨细胞形成及牙槽骨再生的影响,并初步探索DPSC对小鼠破骨细胞的作用机制。方法体外诱导小鼠骨髓单核细胞破骨分化,观察破骨细胞组(OC组)及其与DPSC共培养组(OC+DPSC组)的抗酒石酸酸性磷酸酶(TRAP)染色情况,实时荧光定量聚合酶链反应(PCR)检测破骨分化相关基因包括活化T细胞核因子(NFATc1)、基质金属蛋白酶9(MMP-9)及TRAP的表达差异。体内构建小鼠慢性牙周炎模型,通过微计算机体层摄影(microCT)扫描后三维重建,比较慢性牙周炎+0.9%氯化钠溶液注射组(NS组)和慢性牙周炎+DPSC注射组(DPSC组)釉牙骨质界至牙槽嵴顶(CEJ-ABC)距离,并对标本进行苏木精-伊红和TRAP染色,观察DPSC对小鼠破骨细胞及牙槽骨再生的影响。采用SPSS 20.0软件进行数据统计分析,采用独立样本t检验及校正t检验分析组间差异。结果体外TRAP染色发现,与DPSC共培养明显抑制成熟破骨细胞形成,OC+DPSC组成熟破骨细胞均数(4.2±0.2)少于OC组均数(6.8±0.2),差异有统计学意义(t=15.922,P<0.001);破骨细胞表面积均数(0.046±0.007)mm2也明显小于OC组(0.763±0.015)mm2,差异有统计学意义(t=83.174,P<0.001)。相对OC组,OC+DPSC共培养组的MMP-9、NFATc1及TRAP的m RNA相对表达量明显降低,均值分别为0.38±0.17(t=6.217,P=0.003)、0.24±0.12(t=10.569,P=0.003)和0.55±0.13(t=6.077,P=0.026)。micro-CT扫描结果显示,DPSC注射组CEJ-ABC的平均距离为(0.215±0.017)mm,明显小于0.9%氯化钠溶液组(0.311±0.022)mm,差异有统计学意义(t=10.921,P<0.001),组织学观察下DPSC组炎症反应较0.9%氯化钠溶液组轻,且破骨细胞更少。结论 DPSC可通过抑制牙周炎破骨细胞的形成从而促进牙槽骨再生,有望作为一种可局部注射的骨代谢双向调节生物制剂,治疗临床上包括牙周炎等因骨代谢失衡引起的炎症性骨吸收疾病。

Objective To explore the effect of dental pulp stem cells(DPSC)on osteoclasts and the regeneration of the alveolar bone in periodontitis,and to preliminarily investigate the underlying machanism.Methods Monocytes were co-cultured with DPSC on osteoclastogenesis.TRAP staining were performed to observe the osteoclastogenesis of osteoclast group and DPSC co-culture group,and RT-PCR was applied to analyze the osteoclast-related genes,including NFATc1,MMP-9 and TRAP.Periodontitis models were established.Micro-CT scanning was performed to evaluate the distance from cementum-enamel junction to alveolar bone crest(CEI-ABC)of DPSC injection group and 0.9%sodium chloride solution injection group.HE and TRAP staining were also performed to compare the inflammatory response and osteoclast formation.The data were processed by the SPSS 20.0 software.T test and t′test were used to analyze the difference between the groups.Results It′s found in TRAP staining that co-culture with DPSC successfully inhibited osteoclast formation.The mean osteoclast number in DPSC co-culture group(4.2±0.2)was significantly less than osteoclast group(6.8±0.2)(t=15.922,P<0.001),and the mean cell size(0.046±0.007)mm2 was also less than osteoclast group(0.763±0.015)mm2(t=83.174,P<0.001).Compared with those of osteoclast group,the expression of osteoclastogenesis-related genes including NFATc1,MMP-9 and TRAP of DPSC co-culture group were statistically lower,the value of which were 0.38±0.17(t=6.217,P=0.003),0.24±0.12(t=10.569,P=0.003),0.55±0.13(t=6.077,P=0.026)respectively.Micro-CT scanning showed that the CEI-ABC distance of DPSC group(0.215±0.017)mm was lower than that of NS group(0.311±0.022)mm(t=10.921,P<0.001).Histologic examination showed a less sever inflammatory response and a less osteoclast formation in DPSC group than those in NS group.Conclusions DPSC may promote the alveolar bone regeneration via inhibiting the osteoclast formation in chronic periodontitis.As a promising bi-directional biological agent for bone metabolism,DPSC may show good probability for the treatment of inflammatory bone resorption diseases including periodontitis in the future.