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Effects of core binding factor alpha1 or bone morphogenic protein-2 overexpression on osteoblast/cementoblast-related gene expressions in NIH3T3 mouse cells and dental follicle cells.

ABSTRACT: OBJECTIVES:Bone morphogenic protein-2 (BMP-2) has long been used to promote bone and periodontal regeneration, while core binding factor alpha1 (CBFA1) plays important roles in both osteogenic differentiation and tooth morphogenesis. The aim of this study was to evaluate the effects of CBFA1 or BMP-2 overexpression on osteoblast/cementoblast-related gene expressions in NIH3T3 cells and dental follicle cells (DFCs). MATERIALS AND METHODS:CBFA1 or BMP-2 overexpression in NIH3T3 and DFCs was achieved by infection with retroviral vectors containing CBFA1 or BMP-2 cDNA. Cells stably integrated with CBFA1 or BMP-2 cDNA were selected with G418 for 14 days. Western blotting, real-time reverse transcriptase-polymerase chain reaction, and in vitro mineralization assay were performed to evaluate effects of CBFA1 or BMP-2 overexpression in cells undergoing osteoblast/cementoblast differentiation. RESULTS:Our results demonstrated that osteoblast/cementoblast-related gene expression levels in CBFA1-overexpressing NIH3T3 cells were higher than those in BMP-2-overexpressing cells. More mineral nodules were observed in CBFA1-overexpressing NIH3T3 cells than in BMP-2-overexpressing cells. CBFA1 overexpression in DFCs also increased osteoblast/cementoblast-related gene expression and promoted mineral nodule formation. However, no significant changes in gene expression levels nor mineral nodule formation were found in BMP-2-overexpressing DFCs when compared with empty vector transduced DFCs. CONCLUSIONS:CBFA1 overexpression up-regulated expression levels of osteoblast/cementoblast-related genes and enhanced in vitro osteogenic differentiation more efficiently than BMP-2 in both NIH3T3 cells and DFCs.

SUBMITTER: Pan K

PROVIDER: S-EPMC6496175 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Effects of core binding factor alpha1 or bone morphogenic protein-2 overexpression on osteoblast/cementoblast-related gene expressions in NIH3T3 mouse cells and dental follicle cells.

Pan K K Yan S S Ge S S Li S S Zhao Y Y Yang P P

Cell proliferation 20090424 3

<h4>Objectives</h4>Bone morphogenic protein-2 (BMP-2) has long been used to promote bone and periodontal regeneration, while core binding factor alpha1 (CBFA1) plays important roles in both osteogenic differentiation and tooth morphogenesis. The aim of this study was to evaluate the effects of CBFA1 or BMP-2 overexpression on osteoblast/cementoblast-related gene expressions in NIH3T3 cells and dental follicle cells (DFCs).<h4>Materials and methods</h4>CBFA1 or BMP-2 overexpression in NIH3T3 and ...[more]

PMID: 19397592

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Project description:Dental follicle cells (DFCs) are a heterogeneous population that exhibit a variety of phenotypes. However, it remains unclear whether DFCs can maintain stem cell characteristics, or mediate tissue-regeneration to form single or complex tissues in the periodontium, after long-term culturing. Therefore, DFCs were isolated from human impacted molars (HIM-DFCs), passaged >30 times, and then evaluated for their heterogeneity and multipotential differentiation. Morphology, proliferation, epitope profile, and mineralization characteristics of clones derived from single HIM-DFCs in vitro were also assayed. HIM-DFCs (passage #30) were found to be positive for the heterogeneous markers, Notch-1, stro-1, alkaline phosphomonoesterase (ALP), type I collagen (COL-I), type III collagen (COL-III), and osteocalcine. Moreover, passage #30 of the HDF1, 2, and 3 subclone classes identified in this study were found to express high levels of the mesenchymal stem cells markers, CD146 and Stro1. HDF3 subclones were also associated with the strongest ALP staining detected, and strongly expressed osteoblast and cementoblast markers, including COL-I, COL-III, bone sialoprotein (BSP), and Runx2. In contrast, HDF1 subclone analyzed strongly expressed COL-I and COL-III, yet weakly expressed BSP and Runx2. The HDF2 subclone was associated with the strongest proliferative capacity. To evaluate differentiation characteristics in vivo, these various cell populations were combined with ceramic bovine bone and implanted into subcutaneous pockets of nude mice. The 30th passage of subclone HDF1 and 3 were observed to contribute to fiber collagens and the mineralized matrix present, respectively, whereas HDF2 subclones were found to have a minimal role in these formations. The formation of a cementum-periodontal ligament (PDL) complex was observed 6 weeks after HIM-DFCs (passage #30) were implanted in vivo, thus suggesting that these cells maintain stem cell characteristics. Therefore, subclone HDF1-3 may be related to the differentiation of fibroblasts in the PDL, undifferentiated cells, and osteoblasts and cementoblasts, respectively. Overall, this study is the first to amplify HIM-DFCs and associated subclones with the goal of reconstructing complex or single periodontium. Moreover, our results demonstrate the potential for this treatment approach to address periodontal defects that result from periodontitis, or for the regeneration of teeth.

Project description:Bone formation is important for the reconstruction of bone-related structures in areas that have been damaged by inflammation. Inflammatory conditions such as those that occur in patients with rheumatoid arthritis, cystic fibrosis, and periodontitis have been shown to inhibit osteoblastic differentiation. This study focussed on dental follicle stem cells (DFSCs), which are found in developing tooth germ and participate in the reconstruction of alveolar bone and periodontal tissue in periodontal disease. After bacterial infection of inflamed dental tissue, the destruction of bone was observed. Currently, little is known about the relationship between the inflammatory environment and bone formation. Osteogenic differentiation of inflamed DFSCs resulted in decreased alkaline phosphatase (ALP) activity and alizarin red S staining compared to normal DFSCs. Additionally, in vivo transplantation of inflamed and normal DFSCs demonstrated severe impairment of osteogenesis by inflamed DFSCs. Protein profile analysis via liquid chromatography coupled with tandem mass spectrometry was performed to analyse the differences in protein expression in inflamed and normal tissue. Comparison of inflamed and normal DFSCs showed significant changes in the level of expression of transforming growth factor (TGF)-β2. Porphyromonas gingivalis (P.g.)-derived lipopolysaccharide (LPS) was used to create in vitro inflammatory conditions similar to periodontitis. The osteogenic differentiation of LPS-treated DFSCs was suppressed, and the cells displayed low levels of TGF-β1 and high levels of TGF-β2. DFSCs treated with TGF-β2 inhibitors showed significant increases in alizarin red S staining and ALP activity. TGF-β1 expression was also increased after inhibition of TGF-β2. By examining inflamed DFSCs and LPS-triggered DFSCs, these studies showed both clinically and experimentally that the increase in TGF-β2 levels that occurs under inflammatory conditions inhibits bone formation.

Dataset Information

Effects of core binding factor alpha1 or bone morphogenic protein-2 overexpression on osteoblast/cementoblast-related gene expressions in NIH3T3 mouse cells and dental follicle cells.

Publications

Effects of core binding factor alpha1 or bone morphogenic protein-2 overexpression on osteoblast/cementoblast-related gene expressions in NIH3T3 mouse cells and dental follicle cells.

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