Department of Prosthodontics, School of Dentistry, Lanzhou University, Lanzhou, Gansu, China.
State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China.
J Periodontal Res. 2022 Aug;57(4):869-879. doi: 10.1111/jre.13025. Epub 2022 Jun 22.
Periodontitis is a chronic progressive inflammation that invades periodontal supporting tissues, in which periodontal tissue regeneration engineering offers new hope for prevention and treatment, including seed cells, scaffolds, and growth factors. In recent years, scholars have shown that autologous teeth can be used as new bone tissue repair materials for periodontal regeneration and bone tissue repair. The aim of this study was to establish a human periodontal ligament cell line that expresses the human bone morphogenetic protein 2 gene (BMP2) in a stable manner using lentiviral mediation in order to explore the effect of BMP2 from autologous tooth on the proliferative and osteogenic capacity of human periodontal ligament cells (hPDLCs).
Human periodontal ligament cells were cultured, subcultured, and identified, and then homologous recombinant lentivirus plasmid plv-BMP2 was constructed and transfected into the third passage (P ) hPDLCs. After that, the effect of BMP2 on its proliferation was detected by CCK-8, at the same time, the osteogenic induction of hPDLCs was carried out at 7, 14, and 21 days, and then the effect of BMP2 on its osteogenic ability was detected by alizarin red staining, alkaline phosphatase activity determination, and the mRNA expression levels of osteogenic-related genes using real-time fluorescence quantitative PCR, including alkaline phosphatase, runt-related transcription factor 2, bone sialoprotein, osteocalcin, osteopontin, and collagen I. Finally, spss26.0 software was used for statistical processing.
The results showed that cells transfected with the homologous recombinant lentiviral plasmid pLV-BMP2 had a similar morphology to normal hPDLCs, showing a typical radial arrangement; the cell proliferative capacity of the pLV-BMP2 group as measured by CCK-8 was enhanced compared with the control group and the pLV-puro group (p < .05); alizarin red staining and alkaline phosphatase activity assay showed that the osteogenic ability of pLV-BMP2 was significantly enhanced compared with the control and pLV-puro groups (p < .01), and the findings of real-time fluorescence-based quantitative PCR showed high expression of osteogenic-related genes in pLV-BMP2 group (p < .01).
In conclusion, a stable periodontal ligament cell line overexpressing BMP2 was successfully established by a lentivirus-mediated method, which proved that BMP2 has a strong ability to promote the proliferation and osteogenesis of hPDLCs, thereby providing an opportunity for the study of periodontal tissue regeneration as well as providing an experimental basis for the application of autologous teeth as a new type of bone repair material for periodontal therapy and even for maxillofacial bone tissue repair.
牙周炎是一种慢性进行性炎症，侵袭牙周支持组织，牙周组织再生工程为其防治提供了新的希望，包括种子细胞、支架和生长因子。近年来，学者们发现自体牙可作为新型骨组织修复材料用于牙周再生和骨组织修复。本研究旨在通过慢病毒介导的方法建立稳定表达人骨形态发生蛋白 2 基因（BMP2）的人牙周膜细胞系，探讨自体牙来源的 BMP2 对人牙周膜细胞（hPDLCs）增殖和成骨能力的影响。
培养、传代、鉴定人牙周膜细胞，构建同源重组慢病毒质粒 plv-BMP2 并转染第 3 代（P3）hPDLCs。采用 CCK-8 法检测 BMP2 对其增殖的影响，同时对 hPDLCs 进行成骨诱导，分别于 7、14、21 d 时进行茜素红染色、碱性磷酸酶活性测定及实时荧光定量 PCR 检测成骨相关基因碱性磷酸酶、 runt 相关转录因子 2、骨涎蛋白、骨钙素、骨桥蛋白和Ⅰ型胶原的 mRNA 表达水平，采用 spss26.0 软件进行统计处理。
结果显示，转染同源重组慢病毒质粒 pLV-BMP2 的细胞形态与正常 hPDLCs 相似，呈典型的放射状排列；CCK-8 法检测 pLV-BMP2 组细胞增殖能力较对照组和 pLV-puro 组增强（p<0.05）；茜素红染色和碱性磷酸酶活性测定显示 pLV-BMP2 组成骨能力明显增强，与对照组和 pLV-puro 组相比差异有统计学意义（p<0.01），实时荧光定量 PCR 结果显示 pLV-BMP2 组成骨相关基因表达较高（p<0.01）。
综上所述，本研究通过慢病毒介导的方法成功建立了稳定过表达 BMP2 的牙周膜细胞系，证实 BMP2 具有较强的促进 hPDLCs 增殖和成骨的能力，为牙周组织再生的研究提供了机会，也为自体牙作为牙周治疗新型骨修复材料甚至颌面部骨组织修复的应用提供了实验依据。
