BMP9转染的hPDLSCs与HA-TCP支架复合体促进牙槽骨再生的实验研究

BMP9-transfected human periodontal ligament stem cells and HA-TCP scaffold composite promotes alveolar bone regeneration

目的探索骨形成蛋白9(BMP9)重组腺病毒(Ad-BMP9)转染的人牙周膜干细胞(hPDLSCs)与羟基磷灰石磷酸三钙(HA-TCP)支架材料复合体在牙周组织再生中的研究。方法实验分3组,Ad-BMP9组(转染Ad-BMP9载体)、对照组(转染携带GFP荧光蛋白的空载体腺病毒Ad-GFP)和空白对照组(加入PBS),实时荧光定量PCR(RT-PCR)、Western blot检测BMP9 mRNA及蛋白水平;碱性磷酸酶(ALP)染色、ALP活性分析、茜素红染色、茜素红半定量分析检测BMP9调节hPDLSCs的早、晚期成骨分化能力。扫描电镜(SEM)观察Ad-BMP9组hPDLSCs在HA-TCP上的黏附增殖情况。Ad-BMP9/hPDLSCs复合HA-TCP支架材料后,检测ALP活性,RT-PCR检测复合体早、晚期成骨能力。将Ad-BMP9/hPDLSCs/HA-TCP复合体植入裸鼠皮下及大鼠牙周缺损区,组织学分析新骨形成水平。结果Ad-BMP9转染hPDLSCs后,BMP9表达较其他两组明显升高。ALP染色、ALP活性分析、茜素红染色、茜素红半定量分析发现Ad-BMP9组的早期和晚期成骨分化能力均明显高于其他两组(P<0.05)。SEM观察结果显示Ad-BMP9组hPDLSCs能在HA-TCP上正常黏附、生长、增殖。与Ad-GFP/hPDLSCs/HA-TCP组及hPDLSCs/HA-TCP组比较,Ad-BMP9/hPDLSCs/HA-TCP组的ALP活性明显增高(P<0.05);早期成骨标志物ALP中、中晚期成骨标志物骨钙素(OCN)、骨桥蛋白(OPN)表达均明显提高(P<0.05)。Ad-BMP9/hPDLSCs/HA-TCP复合体应用于体内实验,组织学分析证明有更多新骨形成。结论Ad-BMP9/hPDLSCs/HA-TCP复合体能促进新骨形成,修复牙槽骨缺损。

Objective To explore the role of human periodontal ligament stem cells(hPDLSCs)transfected with recombinant adenoviruses expressing BMP9(Ad-BMP9)and hydroxyapatite tricalcium phosphatel(HA-TCP)scaffold composite in periodontal tissue regeneration.Methods Three groups were set up in the experiment:the Ad-BMP9 group(transfected with Ad-BMP9 vector),the control group(transfected with empty vector adenoviruses Ad-GFP carrying GFP fluorescent protein)and the blank control group(added with PBS).Real-time PCR(RT-PCR)and Western blot were used to detect the mRNA and protein levels of BMP9.Alkaline phosphatase(ALP)staining,ALP activity test,alizarin red staining,and alizarin red semi-quantitative analysis were used to detecte the ability of early and advance osteogenic differentiation of hPDLSCs which regulated by BMP9.Scanning electron microscopy(SEM)was used to observe the adhesion and proliferation of hPDLSCs on HA-TCP.After Ad-BMP9/hPDLSCs were combined with HA-TCP scaffold material,ALP activity test and RT-PCR were used to detect the early and middle-advanced osteogenesis ability of the complex.Ad-BMP9/hPDLSCs/HA-TCP complex was implanted subcutaneously in nude mice and in the periodontal defect area of rats,and the new bone formation level was histologically analyzed.Results After Ad-BMP9 transfected with hPDLSCs,the expression level of BMP9 increased significantly when compared with the other two groups(P<0.05).The results of ALP staining,ALP activity test,alizarin red staining,and alizarin red semi-quantitative analysis showed that the early and advanced osteogenic differentiation ability of Ad-BMP9 group was significantly higher than that of the other groups(P<0.05).SEM observed that hPDLSCs in the Ad-BMP9 group could normally adhere,grow and proliferate on HA-TCP scaffold.Compared with the Ad-GFP/hPDLSCs/HA-TCP group and the hPDLSCs/HA-TCP group,the ALP activity of the Ad-BMP9/hPDLSCs/HA-TCP group was significantly increased(P<0.05),the expression levels of early osteogenic marker ALP,the advanced osteogenic marker osteocalcin(OCN),osteopontin(OPN)were significantly increased(P<0.05).Ad-BMP9/hPDLSCs/HA-TCP complex was used in vivo experiments,and histological analysis proved that there was more new bone formation.Conclusion Ad-BMP9/hPDLSCs/HA-TCP complex can promote new bone formation and repair alveolar bone defects.