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Proliferation and odontogenic differentiation of BMP2 gene?transfected stem cells from human tooth apical papilla: an in vitro study.

ABSTRACT: Stem cells from the apical papilla (SCAP) have odontogenic potential, which plays a pivotal role in the root dentin development of permanent teeth. Human bone morphogenetic protein 2 (BMP2) is a well-known gene that participates in regulating the odontogenic differentiation of dental tissue?derived stem cells. However, little is known regarding the effects of the BMP2 gene on the proliferation and odontogenic differentiation of SCAP. This study aimed to evaluate the odontogenic differentiation potential of lentiviral?mediated BMP2 gene?transfected human SCAP (SCAP/BMP2) in vitro. SCAP were isolated by enzymatic dissociation of human teeth apical papillae. The multipotential of SCAP was verified by their osteogenic and adipogenic differentiation characteristics. The phenotype of SCAP was evaluated by flow cytometry (FCM). The proliferation status of the blank vector?transfected SCAP (SCAP/Vector) and SCAP/BMP2 was analyzed by a cell counting kit-8 (CCK?8). Odontogenic genes, including alkaline phosphatase (ALP), osteocalcin (OCN), dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) of the two groups of cells were evaluated by quantitative polymerase chain reaction (qPCR). ALP staining and alizarin red (AR) staining of the cells was performed on the 16th day after transfection. In vitro results of CCK-8, qPCR, ALP and AR staining demonstrated that: ?) SCAP/BMP2 had a comparable proliferation rate to SCAP/Vector; ?) SCAP/BMP2 presented significantly better potential to differentiate into odontoblasts compared to SCAP/Vector by upregulating ALP, OCN, DSPP and DMP1 genes; ?) more ALP granules and mineralized deposits were formed by SCAP/BMP2 as compared to SCAP/Vector. The results suggested that lentiviral-mediated BMP2 gene transfection enhances the odontogenic differentiation capacity of human SCAP in vitro.

SUBMITTER: Zhang W

PROVIDER: S-EPMC4152145 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Proliferation and odontogenic differentiation of BMP2 gene‑transfected stem cells from human tooth apical papilla: an in vitro study.

Zhang Wen W Zhang Xiaolei X Ling Junqi J Liu Wei W Zhang Xinchun X Ma Jinglei J Zheng Jianmao J

International journal of molecular medicine 20140724 4

Stem cells from the apical papilla (SCAP) have odontogenic potential, which plays a pivotal role in the root dentin development of permanent teeth. Human bone morphogenetic protein 2 (BMP2) is a well-known gene that participates in regulating the odontogenic differentiation of dental tissue‑derived stem cells. However, little is known regarding the effects of the BMP2 gene on the proliferation and odontogenic differentiation of SCAP. This study aimed to evaluate the odontogenic differentiation p ...[more]

PMID: 25070743

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Project description:Stem cells from apical papilla (SCAP) possess clear osteo?/odontogenic differentiation capabilities, and are regarded as the major cellular source for root dentin development. Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) serve pivotal roles in the modulation of tooth development and dentin formation. However, the synergistic effects of BMP2 and VEGF on osteo?/odontogenic differentiation of SCAP remain unclear. The current study aimed to investigate the proliferative and osteo?/odontogenic differentiating capabilities of BMP2 and VEGF gene-co-transfected SCAP (SCAP-BMP2-VEGF) in vitro. The basic characteristics of the isolated SCAP were identified by the induction of multipotent differentiation and by flow cytometry. Lentiviral vector?mediated gene transfection was conducted with SCAP in order to construct blank vector?transfected SCAP (SCAP-green fluorescent protein), BMP2 gene-transfected SCAP (SCAP-BMP2), VEGF gene?transfected SCAP (SCAP?VEGF) and SCAP-BMP2-VEGF. The Cell Counting Kit 8 assay was used to analyze the proliferative capacities of the four groups of cells. The expression of osteo-/odontogenic genes and proteins in the cells were evaluated by reverse transcription-quantitative polymerase chain reaction and western blotting. The mineralized nodules formed by the four group cells were visualized by alkaline phosphatase (ALP) staining. Among the four groups of cells, SCAP?VEGF was demonstrated to exhibit increased proliferation, and SCAP?BMP2?VEGF exhibited reduced proliferation during eight days observation. SCAP?BMP2?VEGF exhibited significantly increased expression levels of ALP, osteocalcin, dentin sialophosphoprotein, dentin matrix acidic phosphoprotein gene 1 and dentin sialoprotein than the other three groups at the majority of the time points. Furthermore, the SCAP?BMP2?VEGF group exhibited a significantly greater number of ALP?positive mineralized nodules than the other groups following 16 days culture in vitro. In conclusion, lentiviral vector-mediated BMP2 and VEGF gene co-transfection significantly activated the osteo?/odontogenic differentiation of human SCAP.

Project description:IntroductionPeriapical alveolar bone loss is the common consequence of apical periodontitis (AP) caused by persistent local inflammation around the apical area. Human stem cells from apical papilla (hSCAPs) play a crucial role in the restoration of bone lesions during AP. Studies have recently identified the critical role of microRNAs (miRNAs) involved in AP pathogenesis, but little is known about their function and potential molecular mechanism, especially in the osteogenesis of hSCAPs during AP. Here, we investigated the role of clinical sample-based specific miRNAs in the osteogenesis of hSCAPs.MethodsDifferential expression of miRNAs were detected in the periapical tissues of normal and patients with AP via transcriptomic analysis, and the expression of miR-199a-5p was confirmed by qRT-PCR. Treatment of hSCAPs with miR-199a-5p mimics while loaded onto beta-tricalcium phosphate (β-TCP) ceramic particle scaffold to explore its effect on osteogenesis in vivo. RNA binding protein immunoprecipitation (RIP) and Luciferase reporter assay were conducted to identify the target gene of miR-199a-5p.ResultsThe expression of miR-199a-5p was decreased in the periapical tissues of AP patients, and miR-199a-5p mimics markedly enhanced cell proliferation and osteogenic differentiation of hSCAPs, while miR-199a-5p antagomir dramatically attenuated hSCAPs osteogenesis. Moreover, we identified and confirmed Interferon Induced Protein with Tetratricopeptide Repeats 2 (IFIT2) as a specific target of miR-199a-5p, and silencing endogenous IFIT2 expression alleviated the inhibitory effect of miR-199a-5p antagomir on the osteogenic differentiation of hSCAPs. Furthermore, miR-199a-5p mimics transfected hSCAPs loaded onto beta-tricalcium phosphate (β-TCP) scaffolds induced robust subcutaneous ectopic bone formation in vivo.DiscussionThese results strengthen our understanding of predictors and facilitators of the key AP miRNAs (miR-199a-5p) in bone lesion repair under periapical inflammatory conditions. And the regulatory networks will be instrumental in exploring the underlying mechanisms of AP and lay the foundation for future regenerative medicine based on dental mesenchymal stem cells.

Dataset Information

Proliferation and odontogenic differentiation of BMP2 gene?transfected stem cells from human tooth apical papilla: an in vitro study.

Publications

Proliferation and odontogenic differentiation of BMP2 gene‑transfected stem cells from human tooth apical papilla: an in vitro study.

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