Expression Of Dentin Sialophosphoprotein mRNA Research Articles

Translated Mutant DSPP mRNA Expression Level Impacts the Severity of Dentin Defects.

Hereditary dentin defects are conventionally classified into three types of dentinogenesis imperfecta (DGI) and two types of dentin dysplasia (DD). Mutations in the dentin sialophosphoprotein (DSPP) gene have been identified to cause DGI type II and III and DD type II; therefore, these are not three different conditions, but rather allelic disorders. In this study, we recruited three families with varying clinical phenotypes from DGI-III to DD-II and performed mutational analysis by candidate gene analysis or whole-exome sequencing. Three novel mutations including a silent mutation (NM_014208.3: c.52-2del, c.135+1G>C, and c.135G>A; p.(Gln45=)) were identified, all of which affected pre-mRNA splicing. Comparison of the splicing assay results revealed that the expression level of the DSPP exon 3 deletion transcript correlated with the severity of the dentin defects. This study did not only expand the mutational spectrum of DSPP gene, but also advanced our understanding of the molecular pathogenesis impacting the severity of hereditary dentin defects.

DSPP dosage affects tooth development and dentin mineralization.

Dentin Sialoprotein (DSP) and phosphophoryn (PP) are two most dominant non-collagenous proteins in dentin, which are the cleavage products of the DSPP (dentin sialophosphoprotein) precursor protein. The absence of the DSPP gene in DSPP knock-out (KO) mice results in characteristics that are consistent with dentinogenesis imperfecta type III in humans. Symptoms include thin dentin, bigger pulp chamber with frequent pulp exposure as well as abnormal epithelial-mesenchymal interactions, and the appearance of chondrocyte-like cells in dental pulp. To better understand how DSPP influences tooth development and dentin formation, we used a bacterial artificial chromosome transgene construct (BAC-DSPP) that contained the complete DSPP gene and promoter to generate BAC-DSPP transgenic mice directly in a mouse DSPP KO background. Two BAC-DSPP transgenic mouse strains were generated and characterized. DSPP mRNA expression in BAC-DSPP Strain A incisors was similar to that from wild-type (wt) mice. DSPP mRNA expression in BAC-DSPP Strain B animals was only 10% that of wt mice. PP protein content in Strain A incisors was 25% of that found in wt mice, which was sufficient to completely rescue the DSPP KO defect in mineral density, since microCT dentin mineral density analysis in 21-day postnatal animal molars showed essentially identical mineral density in both strain A and wt mice. Strain B mouse incisors, with 5% PP expression, only partially rescued the DSPP KO defect in mineral density, as microCT scans of 21-day postnatal animal molars indicated a reduced dentin mineral density compared to wt mice, though the mineral density was still increased over that of DSPP KO. Furthermore, our findings showed that DSPP dosage in Strain A was sufficient to rescue the DSPP KO defect in terms of epithelial-mesenchymal interactions, odontoblast lineage maintenance, along with normal dentin thickness and normal mineral density while DSPP gene dosage in Strain B only partially rescued the aforementioned DSPP KO defect.

Inhibition of odontogenic differentiation of human dental pulp cells by dental resin monomers.

BackgroundDental resin monomers that are leached from the resin matrix due to incomplete polymerization can affect the viability and various functions of oral tissues and cells. In this study, the effects of triethylene glycol dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) on odontogenic differentiation of human dental pulp cells (HDPCs) were examined. To mimic clinical situations, dental pulp cells were treated with resin monomers for 24 h prior to the analysis of alkaline phosphatase (ALP) activity and mRNA expression of genes related to pulp cell differentiation. To elucidate the underlying signaling pathways, regulation of mitogen-activated protein (MAP) kinases by resin monomers was also investigated.ResultsThe ALP activity of HDPCs was reduced by TEGDMA and HEMA at noncytotoxic concentrations. The mRNA expression of dentin sialophosphoprotein (DSPP), osteocalcin (OCN), and osteopontin (OPN) was also downregulated by resin monomers. However, DSPP expression was not affected by hydrogen peroxide (H2O2). Among the MAP kinases examined, ERK activation (ERK phosphorylation) was not affected by either resin monomers or H2O2, whereas JNK was phosphorylated by TEGDMA and HEMA. Phospho-p38 was upregulated by HEMA, while TEGDMA and H2O2 suppressed p38 phosphorylation.ConclusionsExposure to TEGDMA and HEMA for a limited period suppresses differentiation of HDPCs via different signaling pathways.

The Effects of Tumor Necrosis Factor-α on Mineralization of Human Dental Apical Papilla Cells

IntroductionHuman dental apical papilla cells (APCs) have mineralization potential, which plays a key role in the root development of young permanent teeth. Limited literature is available about APC mineralization in the presence of inflammatory cytokines. The purpose of this study was to investigate the effects of tumor necrosis factor-α (TNF-α) on APC mineralization. MethodsAPC cultures were established with the enzymatic dissociation method in vitro. The viability of APCs treated with TNF-α was investigated using methyl-thiazol-tetrazolium assays. Cells were then cultured in osteo-/dentinogenic medium with TNF-α, and mineralization was assessed by alizarin red S staining. Bone sialoprotein (BSP) and dentin sialoprotein (DSP) were analyzed using immunocytochemistry. Mineralization genes such as BSP, dentin sialophosphoprotein (DSPP), osteocalcin (OCN), and dentin matrix acidicphosphoprotein-1 (DMP1) were determined with real-time polymerase chain reaction analyses. ResultsThe viability of cultured cells was higher with TNF-α concentrations of 10 ng/mL and 50 ng/mL than with 5 ng/mL or in the control group. Alizarin red S staining showed that APCs had a higher mineralization activity when the osteo-/dentinogenic culture medium contained 10 ng/mL TNF-α. Immunocytochemical detection showed that the expression of BSP and DSP was positive in APCs after they were induced in osteo-/dentinogenic medium. The expression of mineralization genes differed when treated with 10 ng/mL TNF-α (ie, the expression of DSPP mRNA increased on days 7 and 14, whereas the expression of DSPP mRNA decreased on day 21). ConclusionsTNF-α may promote APC mineralization in short-term cultures and inhibit the mineralization in long-term cultures.

Natural Mineralized Scaffolds Promote the Dentinogenic Potential of Dental Pulp Stem Cells Via the Mitogen-Activated Protein Kinase Signaling Pathway

The selection of a suitable scaffold material is important for dentin tissue regeneration, as the characteristics of biomaterials can potentially influence cell proliferation and differentiation. We compared the effects of different scaffolds on dentin regeneration based on dental pulp stem cells (DPSCs) and investigated the regulatory mechanisms of odontogenic differentiation of DPSCs by these scaffolds. Five different scaffolds were tested: demineralized dentin matrix (DDM), ceramic bovine bone (CBB), small intestinal submucosa (SIS), poly-L-lactate-co-glycolate, and collagen-chondroitin sulfate-hyaluronic acid. DPSCs cultured on DDM and CBB exhibited higher levels of alkaline phosphatase (ALP) activity and mRNA expression of bone sialoprotein, osteocalcin, dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) than those cultured on the other three scaffolds. Further, the phosphorylation levels of mitogen-activated protein kinase (MAPK) ERK1/2 and p38 in DPSCs cultured on DDM and CBB were also significantly enhanced compared with the other three scaffolds, and their inhibitors significantly inhibited odontogenic differentiation as assessed by ALP activity and mRNA expression of DSPP and DMP-1. The implantation experiment confirmed these results and showed a large amount of regular-shaped dentin-pulp complex tissues, including dentin, predentin, and odontoblasts only in the DDM and CBB groups. The results indicated that natural mineralized scaffolds (DDM and CBB) have potential as attractive scaffolds for dentin tissue-engineering-promoted odontogenic differentiation of DPSCs through the MAPK signaling pathway.

Effect of Dental Materials Calcium Hydroxide–containing Cement, Mineral Trioxide Aggregate, and Enamel Matrix Derivative on Proliferation and Differentiation of Human Tooth Germ Stem Cells

Introduction Biocompatibility of pulp capping materials is important for successful use in dentistry. These materials should be nontoxic and permissive for proliferation and induction of odontogenic differentiation of pulp cells. The aim of our study was to evaluate the effects of enamel matrix derivative (EMD), mineral trioxide aggregate (MTA), and calcium hydroxide–containing cement (DYCAL) on proliferation and odontogenic differentiation of human tooth germ stem cells (hTGSCs) in which cells belonging to both pulp tissue and dental follicle exist. Methods The 96-well plates, 24-well plates, and special chamber slides were coated with biomaterials for cell proliferation, differentiation, and scanning electron microscopy analysis. Odontogenic differentiation of hTGSCs was evaluated by analyzing mRNA expression of dentin sialophosphoprotein (DSPP) by real-time polymerase chain reaction expression analysis, measurement of alkaline phosphatase activity, and visualization of calcium depositions by von Kossa staining. Results Our results demonstrate that EMD is the best material in terms of inducing differentiation and proliferation of hTGSCs. DYCAL was found to be toxic to hTGSCs; however, EMD-coated DYCAL showed less toxicity. EMD-coated MTA was not efficient at inducing proliferation and differentiation. Conclusions Pulp capping materials come in direct contact with dental pulp cells; thus, they require comprehensive evaluation of interactions between cells and biomaterials. Therefore, we cultured hTGSCs, capable of odontogenic differentiation, on pulp capping materials directly. Our results suggest that combination of capping materials with EMD would increase the quality of capping by increasing biocompatibility of capping materials.

Effect of radiopaque Portland cement on mineralization in human dental pulp cells

The aim of this study was to investigate whether radiopaque Portland cement (RPC) facilitates the mineralization process in human dental pulp cells (HDPCs) compared with pure Portland cement (PC). Under a scanning electron microscope (SEM), cellular morphology was evaluated. Alkaline phosphatase (ALP) activity was analyzed, and nodule formation was assessed by performing Alizarin Red S staining. In addition, the mRNA expressions of mineralization-related proteins were evaluated by performing a real-time polymerase chain reaction. On SEM evaluation, healthy HDPCs were found adhering to the surfaces of PC and RPC. The ALP activity increased in the PC and RPC groups compared with the control group at 1 day. Alizarin Red stain increased in the PC and RPC groups compared with the control group at 2 and 3 weeks. The mRNA expression of dentin sialophosphoprotein increased at 14 days in the PC and RPC groups. These results show that PC and RPC have similar effects in terms of mineralization and suggest that RPC also has the potential to be used as a clinically suitable pulp-capping material.

Effects of mineral trioxide aggregate and calcium hydroxide on the proliferation and differentiation capacity of pulp cells of primary teeth

To investigate the effects of mineral trioxide aggregate (MTA) and calcium hydroxide on the proliferation and differentiation capacity of pulp cells of primary teeth. Pulp cells were isolated from the retained primary teeth without apparent root resorption and cultured. The cells of 4 - 8 passages were used in the study. Cell proliferation was detected by MTT array, von Kossa staining employed to observe the formation of mineralized nodules and mRNA expression of alkaline phosphatase (ALP) and dentin sialophosphoprotein (DSPP) determined by real time PCR. MTA-treated cells proliferated significantly faster than the other two groups (F = 1835.065, P < 0.01), while calcium hydroxide-treated cells grew slower than the control significantly (F = 1792.301, P < 0.01). The formation of mineralized nodules was found in both MTA-treated and calcium hydroxide-treated pulp cells. The number of mineralized nodules showed no significant difference between the two groups (P > 0.05). Either ALP or DSPP mRNA expression showed significant difference among the three groups (F = 349.651, P < 0.01; F = 1653.001, P < 0.01). MTA increased mRNA expression of ALP and DSPP in pulp cells (P < 0.01), whereas calcium hydroxide down-regulated them (P < 0.01). MTA is more suitable than calcium hydroxide as pulp-capping agent in primary teeth.

Cell pellets from dental papillae can reexhibit dental morphogenesis and dentinogenesis

We isolated dental papilla mesenchymal cells (DPMCs) from different rat incisor germs at the late bell stage and incubated them as cell pellets in polypropylene tubes. In vitro pellet culture of DPMCs presented several crucial characteristics of odontoblasts, as indicated by accelerated mineralization, positive immunostaining for dentin sialophosphoprotein and dentin matrix protein 1, and expression of dentin sialophosphoprotein mRNA. The allotransplantation of these pellets into renal capsules was also performed. Despite the absence of dental epithelial components, dissociated DPMCs with a complete loss of positional information rapidly underwent dentinogenesis and morphogenesis, and formed a cusp-like dentin-pulp complex containing distinctive odontoblasts, predentin, dentin, and dentinal tubules. These results imply that DPMCs at the late bell stage can reexhibit the dental morphogenesis and dentinogenesis by themselves, and epithelial–mesenchymal interactions at this stage may not be indispensable. Furthermore, different DPMC populations from the similar stage may keep the same developmental pattern.

Acceleration Effect of Human Recombinant Bone Morphogenetic Protein-2 on Differentiation of Human Pulp Cells Into Odontoblasts

Predictable pulp capping procedures remain problematic, possibly because of the lack of appropriate stimulating factors for dentin formation. The present study examines the ability of one such stimulating factor, bone morphogenetic protein-2, to accelerate the differentiation of human dental pulp cells into odontoblasts. The number and morphology of cells between groups treated with 0 and 100 ng/ml of human recombinant bone morphogenetic protein-2 (rhBMP-2) did not significantly differ. However, ALPase activity (a marker for biomineralization) in the group stimulated with rhBMP-2 was more than double that of the control group. We then measured the expression of mRNA encoding dentin sialophosphoprotein (DSPP) as a marker of odontoblasts in rhBMP-2-stimulated human pulp cells using a quantitative polymerase chain reaction. The expression of DSPP mRNA in cells stimulated for 24 h by 1000 ng/ml of rhBMP-2 was approximately 20-fold and 5-fold higher than that by stimulated by 10 and 100 ng/ml, respectively. These findings show that rhBMP-2 promoted the differentiation of human dental pulp cells into odontoblasts but did not affect cell proliferation, suggesting that rhBMP-2 may have therapeutic utility in vital pulp therapy.