Odontoblast-like Differentiation Research Articles

Expression of matrix extracellular phosphoglycoprotein in human dental pulp cells undergoing odontoblastic differentiation

To investigate the expression of matrix extracellular phosphorylated protein (MEPE) in human dental pulp cells (hDPC) undergoing odontogenic induction and explore the role of MEPE in odontoblast-like differentiation. hDPC were isolated by enzymatic digestion and preceded to odontogenic induction for 7, 14 and 21 days respectively. hDPC before induction served as controls. The expressions of MEPE, dentin sialophosphoprotein (DSPP)/dentin sialoprotein (DSP), bone sialoprotein (BSP) and collagen type I were determined by quantitative real-time RT-PCR and Western blotting. The mRNA levels of MEPE, DSPP, BSP and type I collagen were increased in a time-dependent manner as hDPC were induced along odontoblastic lineage. Statistical differences were detected for MEPE and BSP mRNA expressions in induced hDPC compared with control group (P < 0.001). For DSPP and type I collagen, the mRNA levels in the induced groups were (12 943.33 + or - 3805.73) and (250.55 + or - 31.86) respectively, which were significantly higher than those in control group on days 21 (P < 0.05). Western blotting also revealed the increased expressions of MEPE, DSP, BSP and type I collagen in the induced DPC. hDPC showed analogously temporal expressions of MEPE, DSPP/DSP, BSP and collagen type I while differentiating along odontoblast lineage. MEPE may play an important role in the odontogenic differentiation of hDPC and may be a potential marker of odontoblast-like differentiation.

Effects of mineral trioxide aggregate on the differentiation of rat dental pulp cells

The effect of mineral trioxide aggregate (MTA) on the odontoblast-like differentiation of pulp cells was evaluated using heat-shock protein 25 (hsp25) as a marker for odontoblast differentiation. The cells were cultured with tooth-colored MTA or calcium hydroxide-containing cement (Dycal). The effects of the materials on the pulp cells were observed using a confocal laser scanning microscope. The cells were labelled immunocytochemically using polyclonal antibodies against hsp25 and actin. The mRNA expression of hsp25 and dspp in the pulp cells at 2 days were examined by quantitative reverse transcription-polymerase chain reaction (RT-PCR). Most of the cells cultured with MTA showed an intense immunolabelling for hsp25 and the mRNA expressions of hsp25 and dspp at 2 days were higher than those cultured with Dycal. These findings indicate that MTA is an effective pulp capping material and is able to induce the differentiation of odontoblast-like cells and the formation of reparative tertiary dentin with minimum apoptosis.

Differentially Expressed Protein Profile of Human Dental Pulp Cells in the Early Process of Odontoblast-like Differentiation In Vitro

Dental pulp cells (DPCs) are capable of differentiating into odontoblasts that secrete reparative dentin after pulp injury. The molecular mechanisms governing reparative dentinogenesis are yet to be fully understood. Here we investigated the differential protein profile of human DPCs undergoing odontogenic induction for 7 days. Using two-dimensional differential gel electrophoresis coupled with matrix-assisted laser adsorption ionization time of flight mass spectrometry, 23 protein spots related to the early odontogenic differentiation were identified. These proteins included cytoskeleton proteins, nuclear proteins, cell membrane-bound molecules, proteins involved in matrix synthesis, and metabolic enzymes. The expression of four identified proteins, which were heteronuclear ribonuclear proteins C, annexin VI, collagen type VI, and matrilin-2, was confirmed by Western blot and real-time real-time polymerase chain reaction analyses. This study generated a proteome reference map during odontoblast-like differentiation of human DPCs, which will be valuable to better understand the underlying molecular mechanisms in odontoblast-like differentiation.

Effects of FGF2 and TGFβ1 on the differentiation of human dental pulp stem cells in vitro

Two crucial growth factors, FGF2 and TGFbeta1, were investigated in this study to determine their inductive effects on the odontoblastic differentiation of human dental pulp stem cells (DPSCs) in vitro. DPSCs were isolated by immunomagnetic bead selection using the STRO-1 antibody, and then co-cultured respectively with FGF2, TGFbeta1 and FGF2+TGFbeta1. The results showed that FGF2 can exert a significant effect on the cell proliferation, while TGFbeta1 or FGF2+TGFbeta1 can initiate an odontoblast-like differentiation of DPSCs. Moreover, FGF2 can synergistically upregulate the effects of TGFbeta1 on the odontoblastic differentiation of DPSCs, as indicated by the increased alkaline phosphatase activity, the polarized cell appearance and secretary ultrastructural features, the formation of mineralized nodules and the gene/protein expression of dentin sialoprotein and dentin matrix protein-1. Together, FGF2 acted primarily on the cell proliferation, while TGFbeta1 and FGF2+TGFbeta1 mainly stimulated the odontoblastic differentiation of DPSCs. This study provides interesting progress in the odontoblastic differentiation of DPSCs induced by FGF2 and TGFbeta1.

Wnt/β-catenin Inhibits Dental Pulp Stem Cell Differentiation

Dental pulp stem cells (DPSCs) are a unique precursor population isolated from postnatal human dental pulp and have the ability to regenerate a reparative dentin-like complex. Canonical Wnt signaling plays a critical role in tooth development and stem cell self-renewal through beta-catenin. In this study, the regulation of odontoblast-like differentiation of DPSCs by canonical Wnt signaling was examined. DPSCs were stably transduced with canonical Wnt-1 or the active form of beta-catenin, with retrovirus-mediated infection. Northern blot analysis found that Wnt-1 strongly induced the expression of matricellular protein osteopontin, and modestly enhanced the expression of type I collagen in DPSCs. Unexpectedly, Wnt-1 inhibited alkaline phosphatase (ALP) activity and the formation of mineralized nodules in DPSCs. Moreover, over-expression of beta-catenin was also sufficient to suppress the differentiation and mineralization of DPSCs. In conclusion, our results suggest that canonical Wnt signaling negatively regulates the odontoblast-like differentiation of DPSCs.

Matrix and TGF-β-related gene expression during human dental pulp stem cell (DPSC) mineralization

We have recently reported the induction of dental pulp stem cells (DPSCs) into dentin-secreting odontoblast-like cells after stimulation by isolated dentin matrix components, thus mimicking the nature of tissue regeneration seen after tooth disease and injury. After confluency, the cells were further cultured for 21 d in the 10% fetal bovine serum (FBS) Dulbecco's modified Eagle's medium (DMEM) (control), and in this medium, with the addition of dentin extract (DE) and the mineralization supplement (MS) of ascorbic acid and beta-glycerophosphate (treatment). To identify genes associated with this process, specimens were analyzed with a HG-U133A human gene chip and Arrayassist software. A total of 425 genes, among them 21 matrix and eight TGF-beta-related genes, were either up- or downregulated in the experimental group in which the cells showed odontoblast-like differentiation and mineralization. Expression of selected genes was further confirmed by real-time polymerase chain reaction (PCR) analysis. Of the extracellular matrix (ECM)-related genes, two types of collagen genes were upregulated and seven others downregulated. Other ECM-related genes, for example fibulin-1, tenascin C, and particularly thrombospondin 1, were upregulated, and fibulin-2 was downregulated. Most noticeably, the matrix metalloproteinase 1 was induced by the treatment. In the TGF-beta superfamily, upregulation of the type II receptor, endoglin, and growth/differentiation factor 5 was coordinated with the downregulation of activin A, TGF-beta2, and TGF-beta1 itself. This study identifies the matrix and TGF-beta-related gene profiles during the DPSC cell mineralization in which several genes are reported for the first time to be associated with this process, thus greatly expanding our molecular knowledge of the induced disease repair process.

Adenovirus-mediated Recombinant Human Bone Morphogenetic Protein-7 Expression Promotes Differentiation of Human Dental Pulp Cells

Recombinant human bone morphogenetic protein-7 (BMP-7) has been shown to stimulate new reparative dentin formation in animal models. However, little is known about whether BMP-7 could promote the odontoblast-like differentiation and the formation of mineralized nodules in human dental pulp cells. Here, we reported that the infection with adenovirus-BMP-7 (Ad-BMP-7), a BMP-7-expressing adenoviral vector, induced the expression of BMP-7 in primarily cultured human dental pulp cells in the long term with little effect on their proliferation and viability. Importantly, BMP-7 expression significantly increased alkaline phosphatase activity and induced the dentin sialophosphoprotein expression in a dose- and time-dependent manner, suggesting that BMP-7 promoted the odontoblast differentiation. Furthermore, BMP-7 expression stimulated the formation of many mineralized dentin-like calcified nodules. Our data suggest that Ad-BMP-7-mediated BMP-7 expression can promote the differentiation of human pulp cells into odontoblast-like cells and mineralization in vitro, which may provide insight for the design of new gene therapy for the pulp capping in the clinic.

Culture medium modulates the behaviour of human dental pulp-derived cells: Technical Note

In vitro approaches have extensively been developed to study reparative dentinogenesis. While dental pulp is a source of unidentified progenitors able to differentiate into odontoblast-like cells, we investigated the effect of two media; MEM (1.8 mM Ca and 1 mM Pi) and RPMI 1640 (0.8 mM Ca and 5 mM Pi) on the behaviour of human dental pulp cells. Our data indicate that MEM significantly increased cell proliferation and markedly enhanced the proportion of alpha-smooth muscle actin positive cells, which represent a putative source of progenitors able to give rise to odontoblast-like cells. In addition, MEM strongly stimulated alkaline phosphatase activity and was found to induce expression of transcripts encoding dentin sialophosphoprotein, an odontoblastic marker, without affecting that of parathyroid hormone/parathyroid hormone related protein-receptor and osteonectin. In conclusion, these observations demonstrate that not only proliferation but also differentiation into odontoblast-like cells was induced by rich calcium and poor phosphate medium (MEM) as compared to RPMI 1640. This study provides important data for the determination of the optimal culture conditions allowing odontoblast-like differentiation in human pulp cell culture.

Enhancement of alkaline phosphatase synthesis in pulp cells co‐cultured with epithelial cells derived from lower rabbit incisors

Dental papilla mesenchymal cells differentiate into odontoblasts through epithelial-mesenchymal interactions. However, the mechanism by which enamel epithelial cells affect the differentiation of dental mesenchymal cells remains unknown. Alkaline phosphatase (ALPase) is a marker for odontoblast-like differentiation, because odontoblasts show much higher ALPase activity than dental undifferentiated mesenchymal cells. The continuously growing rabbit incisor is a good model for the epithelial-mesenchymal interaction during odontogenesis. In the present study, we isolated and maintained rabbit incisor-derived epithelial cells and rabbit incisor pulp-derived fibroblastic cells, and examined the effect of epithelial cells on ALPase activity in fibroblastic cells. Epithelial cells were stained with anti-cytokeratin 5 and 8 antibodies and showed the expression of tuftelin mRNA. In separate cultures of epithelial cells or fibroblastic cells, ALPase activity and mRNA levels were very low, but were upregulated in co-cultures of epithelial and fibroblastic cells. Histochemical analysis found high ALPase activity in fibroblastic cells close to epithelial cells. These findings suggest that epithelial cells play an important role in promoting ALPase expression in pulp fibroblastic cells. The co-culture system developed here will be useful for examining the role of the epithelial-mesenchymal interaction during odontoblast differentiation.