MTA和三种改良盖髓剂对人牙髓干细胞增殖及分化为成牙本质细胞的促进作用观察  

Promoting effects of MTA and three modified pulp-capping agents on proliferation and differentiation of human dental pulp stem cells into odontoblasts

作　　者：黄子璇 王小聪 乐曼妮 张慧琳 张晓月 龚伶玲 朱毅男 李明 HUANG Zixuan;WANG Xiaocong;LE Manni;ZHANG Huilin;ZHANG Xiaoyue;GONG Lingling;ZHU Yinan;LI Ming(VIP Dental Center,Changsha Stomatological Hospital,Changsha 410004,China;不详)

机构地区：[1]长沙市口腔医院特诊中心,长沙410004 [2]湖南中医药大学口腔医(学)院 [3]长沙市口腔医院口腔修复科

出　　处：《山东医药》2024年第30期44-49,共6页Shandong Medical Journal

基　　金：湖南省科技厅创新引导课题(2021SK53301);湖南省卫生健康委重点指导课题(202108051626);长沙市自然科学基金(kq2208458);湖南省教育厅重点项目(22A0249);湖南省自然科学基金面上基金项目(2022JJ30630);湖南省自然科学基金(2023JJ60401)。

摘　　要：目的观察矿物三氧化物凝聚体(Mineral Trioxide Aggregate,MTA)和三种改良盖髓剂(nRoot、Vitapex、iRoot BP Plus)对人牙髓干细胞(hDPSCs)增殖、分化为成牙本质细胞的促进作用。方法①不同浓度四种盖髓剂对hDPSCs增殖的促进作用观察。取第5代hDPSCs细胞分为MTA组、iRoot BP Plus组、nRoot组、Vitapex组及空白组,MTA组、iRoot BP Plus组、nRoot组、Vitapex组分别加入不同浓度(0.02、0.2、1、2 mg/mL)的MTA、iRoot BP Plus、nRoot和Vitapex培养液,空白组加入含10%FBS的DMEM/F12培养液。分别于培养24、48 h时,采用CCK-8法测算各组细胞增殖活性。②四种盖髓剂对hDPSCs分化为成牙本质细胞的促进作用观察。通过ALP活性检测筛选出四种材料对hDPSCs最适诱导浓度。取第4代hDPSCs分为五组:空白组、MTA组、iRoot BP Plus组、nRoot组和Vitapex组换为0.2 mg/mL的MTA、iRoot BP Plus、nRoot和Vitapex成骨培养基,空白组为正常成骨培养基。培养第21天时采用茜素红染色和半定量分析法观察各组细胞矿化结节形成情况,培养第7天时采用Western Blotting法检测细胞相关蛋白类核转录因子(Runt-related transcription factor 2,RUNX-2)、骨钙素(Osteocalcin,OCN)、牙本质涎磷蛋白(Dentin sialophosphoprotein,DSPP)以及牙本质基质蛋白1(Dentin matrix protein 1,DMP-1)。结果与空白组相比,培养第2天时0.02、0.2、1 mg/mL的MTA组、iRoot BP Plus在和nRoot组细胞增殖活性高(P均<0.05);与培养第1天时相比,培养第2天时2 mg/mL的MTA组和Vitapex组细胞增殖活性低(P均<0.05)。筛选0.2 mg/mL为后续受试浓度。与nRoot组和Vitapex组相比,iRoot BP Plus组和MTA组细胞钙沉积量高(P均<0.05)。与空白组相比,MTA组、iRoot BP Plus组、nRoot组和Vitapex组细胞OCN、RUNX-2、DSPP、DMP-1相对表达量均升高(P均<0.05);与nRoot组、Vitapex组相比,iRoot BP Plus组细胞OCN、RUNX-2、DSPP、DMP-1相对表达量均高(P均<0.05)。结论相比于MTA、Vitapex,nRoot和iRoot BP Plus更能Objective To observe the promoting effects of mineral trioxide aggregate(MTA)and three modified pulp-capping agents(namely nRoot,Vitapex,and iRoot BP Plus)on the proliferation and differentiation of human dental pulp stem cells(hDPSCs)into odontoblasts.Methods①Observation on the promoting effects of four different concentrations of pulp-capping agents on proliferation of hDPSCs:In this study,the fifth-passage hDPSCs were divided into the MTA,iRoot BP Plus,nRoot,Vitapex,and control groups.The MTA,iRoot BP Plus,nRoot,and Vitapex groups were treated with various concentrations(0.02,0.2,1,and 2 mg/mL)of MTA,iRoot BP Plus,nRoot,and Vitapex culture media,respectively;the control group was treated with DMEM/F12 culture medium containing 10%fetal bovine serum(FBS).Cell proliferation activity was assessed using the CCK-8 method at 24 and 48 h after incubation.②Observation on the promoting effects of four pulp-capping agents on differentiation of hDPSCs into odontoblasts:The optimal inducing concentrations of the four materials for hDPSCs were screened through alkaline phosphatase(ALP)activity detection.The fourth-passage hDPSCs were divided into five groups:control,MTA,iRoot BP Plus,nRoot,and Vitapex groups.The cells in the MTA,iRoot BP Plus,nRoot,and Vitapex groups were treated with osteogenic media containing 0.2 mg/mL of MTA,iRoot BP Plus,nRoot,and Vitapex,respectively,while the control group was treated with normal osteogenic media.On day 21 of culture,alizarin red staining and semi-quantitative analysis were used to observe the formation of mineralized nodules in each group.On day 7 of culture,Western blotting was employed to detect the expression levels of runt-related transcription factor 2(RUNX-2),osteocalcin(OCN),dentin sialophosphoprotein(DSPP),and dentin matrix protein 1(DMP-1).Results On the second day of culture,compared with the control group,the cell proliferation activity was significantly higher in the MTA group,iRoot BP Plus group and nRoot group at concentrations of 0.02,0.2,and 1 mg/mL(all P<0.05).

关 键 词：盖髓剂 nRoot盖髓剂 Vitapex盖髓剂 iRoot BP Plus盖髓剂 矿物三氧化物凝聚体 人牙髓干细胞 细胞增殖 成牙分化 

分 类 号：R788.2[医药卫生—口腔医学]