NNT-AS1 enhances bladder cancer cell growth by targeting miR-1301-3p/PODXL axis and activating Wnt pathway.

As a tumor involved in the urinary system, bladder cancer (BC) seriously threatens human health. Emerging as crucial biomarkers, long noncoding RNAs (lncRNAs) play an important role in the regulation of many cancers. lncRNA NNT-AS1 has been studied in a series of cancers, whereas its role and potential molecular mechanism was poorly understood in BC. Here, we found that NNT-AS1 was upregulated in BC cells. Functionally, the silencing of NNT-AS1 inhibited cell proliferation, migration, invasion, and endothelial-mesenchymal transition. Furthermore, the apoptosis of BC cells was induced upon NNT-AS1 knockdown. Later, miR-1301-3p, the downstream gene of NNT-AS1, was found at a low level in BC cells. In addition, we found that miR-1301-3p targeted to PODXL. PODXL expression downregulated in NNT-AS1-silenced cells was restored by miR-1301-3p inhibition. Importantly, NNT-AS1 was discovered to activate Wnt pathway, and the treatment of LiCl recovered the repressive role of NNT-AS1 silencing in BC cell growth. Through restoration assays, we observed that PODXL overexpressing countervailed NNT-AS1 depletion-mediated suppression on BC cell growth and Wnt pathway. These data suggested that NNT-AS1 enhances BC cell growth and activates Wnt pathway by targeting miR-1301-3p/PODXL axis.

Effects of a triple antibiotic solution on pulpal dynamics after intentionally delayed tooth replantation in mice.

INTRODUCTION: This study analyzed the detailed biological events underlying pulpal dynamics evoked by 3Mix (the mixture of ciprofloxacin, metronidazole, and minocycline) solution after intentionally delayed tooth replantation because 3Mix improves pulpal healing after tooth injuries. METHODS: The maxillary first molars of 3-week-old mice were extracted and immersed in 3Mix solution for 30 minutes in comparison with phosphate buffered saline (PBS) alone. Cell proliferation, apoptosis, and differentiation were assessed in extracted/replanted teeth during days 0-14 using immunohistochemistry, apoptosis assay, and reverse-transcriptase polymerase chain reaction. RESULTS: 3Mix solution accelerated odontoblast differentiation in the coronal pulp on day 7 and tertiary dentin formation on day 14, whereas the regenerative process was delayed in the PBS group. Cell proliferation and apoptosis occurred in the pulp of the 3Mix group during days 5-7 and subsequently decreased from days 7-14. On day 5, dentin sialophosphoprotein and nestin were first recovered in the 3Mix group, whereas expression levels for alkaline phosphatase, osteopontin, and osteocalcin increased in the PBS group. The expression levels for octamer-binding factor 3/4A and 3/4B reached the maximum level on day 1 and were sharply decreased on day 3 in both groups. High expression levels of Cd11c were first observed in the 3Mix group on day 1 and later at days 5 and 7. CONCLUSIONS: The results suggest that the application of 3Mix may suppress osteoblast differentiation by the migration of dendritic cells to the injury site and via the activation of stem/progenitor cells, resulting in the acceleration of odontoblastlike cell differentiation.

The role of integrin αv in proliferation and differentiation of human dental pulp cell response to calcium silicate cement.

INTRODUCTION: It has been proved that integrin αv activity is related to cell proliferation, differentiation, migration, and organ development. However, the biological functions of integrin αv in human dental pulp cells (hDPCs) cultured on silicate-based materials have not been explored. The aim of this study was to investigate the role of integrin αv in the proliferation and odontogenic differentiation of hDPCs cultured with the effect of calcium silicate (CS) cement and ß-tricalcium phosphate (TCP) cement. METHODS: In this study, hDPCs were cultured on CS and TCP materials, and we evaluated fibronectin (FN) secretion and integrin αv expression during the cell attachment stage. After small interfering RNA transfection targeting integrin αv, the proliferation and odontogenesis differentiation behavior of hDPCs were analyzed. RESULTS: The results indicate that CS releases Si ion-increased FN secretion and adsorption, which promote cell attachment more effectively than TCP. The CS cement facilitates FN and αv subintegrin expression. However, the FN adsorption and integrin expression of TCP are similar to that observed in the control dish. Integrin αv small interfering RNA inhibited odontogenic differentiation of hDPCs with the decreased formation of mineralized nodules on CS. It also down-regulated the protein expression of multiple markers of odontogenesis and the expression of dentin sialophosphoprotein protein. CONCLUSIONS: These results establish composition-dependent differences in integrin binding and its effectiveness as a mechanism regulating cellular responses to biomaterial surface.

Mineral trioxide aggregate promotes the odonto/osteogenic differentiation and dentinogenesis of stem cells from apical papilla via nuclear factor kappa B signaling pathway.

INTRODUCTION: Mineral trioxide aggregate (MTA) has been widely used in clinical apexification and apexogenesis. However, the effects of MTA on the stem cells from apical papilla (SCAPs) and the precise mechanism of apexogenesis have not been elucidated in detail. METHODS: Multiple colony-derived stem cells were isolated from the apical papillae, and the effects of MTA on the proliferation and differentiation of SCAPs were investigated both in vitro and in vivo. Activation of nuclear factor kappa B (NFκB) pathway in MTA-treated SCAPs was analyzed by immunofluorescence assay and Western blot. RESULTS: MTA at the concentration of 2 mg/mL did not affect the proliferation activity of SCAPs. However, 2 mg/mL MTA-treated SCAPs presented the ultrastructural changes, up-regulated alkaline phosphatase, increased calcium deposition, up-regulated expression of odontoblast markers (dentin sialoprotein and dentin sialophosphoprotein) and odonto/osteoblast markers (runt-related transcription factor 2 and osteocalcin), suggesting that MTA enhanced the odonto/osteoblastic differentiation of SCAPs in vitro. In vivo results confirmed that MTA can promote the regular dentinogenesis of SCAPs. Moreover, MTA-treated SCAPs exhibited the up-regulated cytoplasmic phos-IκBα and phos-P65, enhanced nuclear P65, and increased nuclear translocation of P65. When co-treated with BMS345541 (the specific NFκB inhibitor), MTA-mediated odonto/osteoblastic differentiation was significantly attenuated. CONCLUSIONS: MTA at the concentration of 2 mg/mL can improve the odonto/osteogenic capacity of SCAPs via the activation of NFκB pathway.

Gene expression and cytokine release during odontogenic differentiation of human dental pulp stem cells induced by 2 endodontic biomaterials.

INTRODUCTION: Mineral trioxide aggregate (MTA) and calcium-enriched mixture (CEM) have shown osteogenic/cementogenic/dentinogenic activities; however, their mechanism of action is not fully understood. We aimed to evaluate the effect of these biomaterials on odontogenic differentiation of human dental pulp stem cells (DPSCs). METHODS: Flow cytometry with stem cell markers for the confirmation of stemness and homogeneity was first performed. Then isolated DPSCs were seeded on prepared discs of MTA, CEM, differentiation medium (DM), and growth medium (GM) and incubated up to 14 days. Concentrations of transforming growth factor-ß1, bone morphogenetic protein (BMP)2, BMP4, and fibroblast growth factor 4 were measured at each interval using an enzyme-linked immunosorbent assay reader. Gene expression of dentin sialophosphoprotein, dentin matrix protein 1, and the cytokines were evaluated by reverse-transcription polymerase chain reaction. To evaluate the cell morphology, scanning electron micrographs were taken; mineralization potential was evaluated using alizarin red S staining. RESULTS: Scanning electron micrographs showed that DPSCs spread/adhered/proliferated similarly on MTA and CEM. On day 14, alizarin red S staining confirmed that mineralization occurred in all groups except GM. Expressions of dentin matrix protein 1 and dentin sialophosphoprotein genes were similar in the CEM, MTA, and DM groups; they were significantly higher compared with the GM group (P < .05). A greater amount of transforming growth factor-ß1 gene was expressed in MTA compared with the other groups (P < .05). However, the expression of fibroblast growth factor 4 and BMP2 genes was significantly greater in the CEM group (P < .05). In all the tested groups, the expression of BMP4 was less than GM (P < .01); however, CEM and DM were similar but more than MTA (P < .05). Concentrations of protein product detected using an enzyme-linked immunosorbent assay reader confirmed these gene expressions. CONCLUSIONS: MTA and CEM can induce osteo-/odontogenic-like phenotype differentiation of human DPSCs; however, they stimulate different gene expressions and growth factor release.

Concentration-dependent effect of sodium hypochlorite on stem cells of apical papilla survival and differentiation.

INTRODUCTION: Intracanal disinfection is a crucial step in regenerative endodontic procedures. Most published cases suggest the use of sodium hypochlorite (NaOCl) as the primary irrigant. However, the effect of clinically used concentrations of NaOCl on the survival and differentiation of stem cells is largely unknown. In this study, we tested the effect of various concentrations of NaOCl on the stem cells of the apical papilla (SCAPs) survival and dentin sialophosphoprotein (DSPP) expression. METHODS: Standardized root canals were created in extracted human teeth and irrigated with NaOCl (0.5%, 1.5%, 3%, or 6%) followed by 17% EDTA or sterile saline. SCAPs in a hyaluronic acid-based scaffold were seeded into the canals and cultured for 7 days. Next, viable cells were quantified using a luminescence assay, and DSPP expression was evaluated using quantitative real-time polymerase chain reaction. RESULTS: There was a significant reduction in survival and DSPP expression in the group treated with 6% NaOCl compared with the untreated control group. Comparable survival was observed in the groups treated with the lower concentrations of NaOCl, but greater DSPP expression was observed in the 1.5% NaOCl group. In addition, 17% EDTA resulted in increased survival and DSPP expression partially reversing the deleterious effects of NaOCl. CONCLUSIONS: Collectively, the results suggest that dentin conditioning with high concentrations of NaOCl has a profound negative effect on the survival and differentiation of SCAPs. However, this effect can be prevented with the use of 1.5% NaOCl followed by 17% EDTA. The inclusion of this irrigation regimen might be beneficial in regenerative endodontic procedures.

Effects of ProRoot MTA, Bioaggregate, and Micromega MTA on odontoblastic differentiation in human dental pulp cells.

INTRODUCTION: The aim of this study was to compare the biocompatibility and odontogenic potential of newly developed Bioaggregate (BA) and Micromega MTA (MMTA) with ProRoot MTA (PMTA) and intermediate restorative material (IRM) by using human dental pulp cells. METHODS: Biocompatibility was assessed by an 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide assay and scanning electron microscopy. Differentiation was evaluated by alkaline phosphatase (ALP) activity, alizarin red staining, and reverse transcriptase-polymerase chain reaction for the maker genes. The levels of inflammatory mediators and cytokines were measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: PMTA, BA, and MMTA exhibited equally good biocompatibility, whereas IRM showed cytotoxicity compared with these materials. PMTA, BA, and MMTA increased the ALP activity, promoted mineralization nodule formation, and enhanced the mRNA expression level of the osteogenic/odontogenic markers (ALP, osteopontin, osteocalcin, dentin sialophosphoprotein, and dentin matrix protein-1) compared with IRM. The levels of proinflammatory mediators and proinflammatory cytokines were lower in PMTA, BA, and MMTA compared with the IRM group. CONCLUSIONS: Collectively, the biocompatibility, odontogenic potentials, and inflammatory response of BA and MMTA are equal to those of PMTA and superior to those of IRM.

EphB-EphrinB interaction controls odontogenic/osteogenic differentiation with calcium hydroxide.

INTRODUCTION: Calcium hydroxide is used in direct pulp capping of uncontaminated exposed vital pulps caused by mechanical or traumatic injury. Calcium hydroxide creates a high alkaline pH environment and initiates a mineralized tissue formation in the pulp. The exact mechanism by which calcium hydroxide induces the reparative dentin formation is unknown. Because Eph receptors and ephrin ligands play a role in pulp stem cell migration and proliferation, our hypothesis is that calcium hydroxide-related odontogenic/osteogenic differentiation may be associated with Eph-ephrin interaction. The aim of this study was to investigate whether Eph-ephrin interaction regulates odontogenic/osteogenic differentiation with calcium hydroxide. METHODS: Primary pulp cells were harvested from the molars of C57BL/6 mice. The cells were treated with calcium hydroxide. Immunofluorescence was used to detect protein expression. A knockout of the ephrinB1 or EphB2 gene was performed with short hairpin RNAs. Cell migration, proliferation, and gene expression were then analyzed. RESULTS: Calcium hydroxide stimulated EphB2 gene expression but suppressed ephrinB1 gene expression at the proliferation stage. However, calcium hydroxide stimulated both ephrinB1 and EphB2 gene expression at the differentiation stage. In addition, EphB2 localized at ephrinB1-positive cells at the area of Dentin sialoprotein (DSP) staining, which increased with calcium hydroxide treatment. Knockdown of ephrinB1-EphB2 significantly suppressed cell proliferation. Additionally, knockdown of the ephrinB1 gene caused cell migration, whereas a lack of the EphB2 gene suppressed calcium hydroxide-induced mineralization from primary pulp cells. CONCLUSIONS: EphrinB1-EphB2 interaction contributes to calcium hydroxide-induced odontogenic/osteogenic differentiation. This observation is the first finding of the mechanism of calcium hydroxide-induced odontogenic/osteogenic differentiation.

Effect of F68, F127, and P85 pluronic block copolymers on odontogenic differentiation of human tooth germ stem cells.

INTRODUCTION: The major challenge in dental pulp engineering is to make a successful combination of stem cells and biomaterials with the aim of providing the differentiation of stem cells into odontogenic cell types. Among biomaterials, some types of pluronics have been reported to increase bone formation of stem cells. The effect of these pluronics on odontogenic differentiation has not been addressed yet. This study aimed to examine the effect of pluronics F68, F127, and P85 on odontogenic differentiation of stem cells derived from third molar tooth germs of young adults. METHODS: Human tooth germ stem cells (hTGSCs) were induced to differentiate into odontogenic cells in the presence of different concentrations of pluronics. Differentiation efficiency was assessed by quantitative real-time polymerase chain reaction for determining expression messenger RNA levels and by immunocytostaining for determining the protein expression of odontogenic markers (ie, dentin sialoprotein, dentin matrix protein 1, bone morphogenic protein 2, bone morphogenic protein 7) by measuring alkaline phosphatase enzyme activity and lastly by von Kossa staining for determining mineralization. RESULTS: The results revealed for the first time that F68 has a great potential to boost odontogenic differentiation of hTGSCs. P85 was found to reduce cell viability during differentiation. F127 was nontoxic to hTGSCs but did not have any effect on differentiation. CONCLUSIONS: The positive effect of F68 on odontogenic differentiation might enable more efficient pulp regeneration. Yet, the exact mechanism of how F68 alters the differentiation pattern of hTGSCs remains to be investigated in the future studies.

In vitro osteogenic/dentinogenic potential of an experimental calcium aluminosilicate cement.

INTRODUCTION: Calcium aluminosilicate cements are fast-setting, acid-resistant, bioactive cements that may be used as root-repair materials. This study examined the osteogenic/dentinogenic potential of an experimental calcium aluminosilicate cement (Quick-Set) by using a murine odontoblast-like cell model. METHODS: Quick-Set and white ProRoot MTA (WMTA) were mixed with the proprietary gel or deionized water, allowed to set completely in 100% relative humidity, and aged in complete growth medium for 2 weeks until rendered non-cytotoxic. Similarly aged Teflon disks were used as negative control. The MDPC-23 cell line was used for evaluating changes in mRNA expressions of genes associated with osteogenic/dentinogenic differentiation and mineralization (quantitative reverse transcription polymerase chain reaction), alkaline phosphatase enzyme production, and extracellular matrix mineralization (alizarin red S staining). RESULTS: After MDPC-23 cells were incubated with the materials in osteogenic differentiation medium for 1 week, both cements showed up-regulation in ALP and DSPP expression. Fold increases in these 2 genes were not significantly different between Quick-Set and WMTA. Both cements showed no statistically significant up-regulation/down-regulation in RUNX2, OCN, BSP, and DMP1 gene expression compared with Teflon. Alkaline phosphatase activity of cells cultured on Quick-Set and WMTA were not significantly different at 1 week or 2 weeks but were significantly higher (P < .05) than Teflon in both weeks. Both cements showed significantly higher calcium deposition compared with Teflon after 3 weeks of incubation in mineralizing medium (P < .001). Differences between Quick-Set and WMTA were not statistically significant. CONCLUSIONS: The experimental calcium aluminosilicate cement exhibits similar osteogenic/dentinogenic properties to WMTA and may be a potential substitute for commercially available tricalcium silicate cements.

Role of the extracellular signal-regulated kinase 1/2 pathway in driving tricalcium silicate-induced proliferation and biomineralization of human dental pulp cells in vitro.

INTRODUCTION: The aim of this study was to investigate the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in regulating tricalcium silicate (C3S)-driven proliferation and biomineralization of human dental pulp cells (hDPCs) in vitro. METHODS: Human DPCs were cultured in C3S-containing medium and compared with untreated controls. Cell viability was measured by the methyl-thiazol-tetrazolium assay. Biomineralization was assessed by staining calcium deposits on the extracellular matrix with von Kossa and alizarin red S stains. Phosphorylated ERK1/2 was evaluated by immunoblotting. The ERK1/2 inhibitor U0126 was used to assess the role of this pathway on stage of the cell cycle and mineralization-dependent gene expressions of hDPCs by using flow cytometry and real-time polymerase chain reaction, respectively. Data were analyzed by analysis of variance followed by the Student-Newman-Keuls post hoc test, with significance set at P < .05. RESULTS: The viability and biomineralization of hDPCs were promoted by C3S extracts (P < .05). Phosphorylated ERK1/2 strongly appeared after hDPCs were cultured in the C3S extracts for 30 minutes. Moreover, inhibition of the ERK1/2 pathway in C3S-treated hDPCs decreased proliferation and the expression of mineralization-dependent genes, including collagen type I, dentin sialophosphoprotein, osteopontin, and osteocalcin (P < .05). CONCLUSIONS: C3S stimulated the proliferation and biomineralization of hDPCs in vitro, with the ERK1/2 pathway playing a key role in the regulation of these effects.

In Vitro biocompatibility evaluation of a root canal filling material that expands on water sorption.

INTRODUCTION: CPoint is a polymeric endodontic point that takes advantage of water-induced, non-isotropic radial expansion to adapt to canal irregularities. This study evaluated the effects of CPoint on the viability and mineralization potential of odontoblast-like cells. METHODS: The biocompatibility of CPoint and commercially available gutta-percha points was evaluated by using a rat odontoblast-like cell line (MDPC-23). Cell viability was evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, and confocal laser scanning microscopy. The mineralization potential of MDPC-23 cells, in the presence of the root-filling materials, was evaluated by examining the changes in osteogenic gene marker expression (quantitative real-time polymerase chain reaction), alkaline phosphatase activity, alizarin red S assay, and transmission electron microscopy. RESULTS: CPoint showed higher initial cytotoxicity compared with gutta-percha and Teflon (P < .05), which became nonsignificant after 4 immersion cycles. Significant differences were also found between eluents from CPoint and gutta-percha at 1:1 concentration (P < .05) but not at 1:10 or 1:100 concentration. Both materials induced minimal apoptosis-induced alteration in plasma membrane permeability, as evidenced by flow cytometry and confocal laser scanning microscopy. Compared with the Teflon negative control, CPoint and gutta-percha groups showed up-regulation of most osteogenic gene markers except for dentin sialophosphoprotein, which was down-regulated. Alkaline phosphatase activity and alizarin red assay for CPoint and gutta-percha were both significantly higher than for Teflon but not significantly different from each other (P > .05). Transmission electron microscopy showed discrete nodular electron-dense mineralization foci in all 3 groups. CONCLUSIONS: The in vitro biocompatibility of CPoint is comparable to gutta-percha with minimal adverse effects on osteogenesis after elution of potentially toxic components.

Hypotonic-induced stretching of plasma membrane activates transient receptor potential vanilloid channels and sodium-calcium exchangers in mouse odontoblasts.

INTRODUCTION: A number of transient receptor potential (TRP) channels have been identified as membrane-bound sensory proteins in odontoblasts. However, the activation properties of these channels remain to be clarified. The purpose of this study was to investigate hypotonic stimulation-induced Ca(2+) entry via TRP vanilloid subfamily member (TRPV) 1, TRPV2, and TRPV4 channels, which are sensitive to osmotic and mechanical stimuli, and their functional coupling with Na(+)-Ca(2+) exchangers (NCXs) in mouse odontoblast lineage cells. METHODS: We examined TRP channel activity by measuring intracellular-free Ca(2+) concentration by using fura-2 fluorescence and ionic current recordings with whole-cell patch-clamp methods. Protein localization and messenger RNA expression were characterized using immunofluorescence and reverse-transcription polymerase chain reaction analyses. RESULTS: Extracellular hypotonic solution-induced stretching of plasma membrane resulted in the activation of Ca(2+) influx and inward currents. TRPV1, TRPV2, and TRPV4 channel antagonists inhibited the hypotonic stimulation-induced Ca(2+) entry and currents. Their respective agonists activated Ca(2+) entry. Although the increase in the intracellular free Ca(2+) concentration decayed rapidly after the applications of these TRPV channel agonists, NCX inhibitors significantly prolonged the decay time constant. The messenger RNA expression of TRPV1, TRPV2, and TRPV4 channels; NCX isoforms 2 and 3; and dentin sialophosphoprotein were up-regulated after 24 hours of exposure to the hypotonic culture medium. CONCLUSIONS: These results indicate that stretching of the odontoblast membrane activates TRPV1-, TRPV2-, and TRPV4-mediated Ca(2+) entry, and increased intracellular-free Ca(2+) concentration is extruded via NCXs. These results suggest that odontoblasts can act as sensors that detect stimuli applied to exposed dentin and drive a number of cellular functions including dentinogenesis and/or sensory transduction.

Effect of statins with α-tricalcium phosphate on proliferation, differentiation, and mineralization of human dental pulp cells.

INTRODUCTION: The aim of this study was to investigate the combined effect of statin and α-tricalcium phosphate (α-TCP) on odontoblastic differentiation of human dental pulp cells and to compare them with mineral trioxide aggregate (MTA). METHODS: Experimental cements were prepared with TCP containing simvastatin and atorvastatin. Cell proliferation, cell adherence on a dentin disc, alkaline phosphatase (ALP) activity, expression of osteogenic/odontoblastic markers, and mineralization of the human dental pulp cells on experimental cement and MTA were assessed. RESULTS: The cell growth and ALP activity of TCP containing simvastatin-treated cells was greater than MTA-treated cells. The mineralization and messenger RNA expression of markers (ie, dentin sialophosphoprotein, dentin matrix protein 1, bone morphogenetic protein 2, ALP, and osteonectin) of TCP containing simvastatin- and TCP containing atorvastatin-treated cells were comparable with MTA-treated cells. The enhanced cell proliferation and similar level of ALP of TCP-treated cells compared with the control indicate that α-TCP is an effective osteoconductive material. The differentiation effect observed in TCP containing simvastatin- and TCP containing atorvastatin-treated cells is attributed to the effect of statin. CONCLUSIONS: The results suggest that α-TCP can be used for local delivery of statin as a pulp capping material to accelerate reparative dentin formation.

Fluocinolone acetonide promotes the proliferation and mineralization of dental pulp cells.

INTRODUCTION: The aim of this study was to investigate the role of the steroid fluocinolone acetonide on the proliferation and mineralization of human dental pulp cells (DPCs). The potential effect of fluocinolone acetonide on reparative dentin formation and the recovery of injured dental pulp were evaluated. METHODS: The proliferative effect of fluocinolone acetonide on DPCs was analyzed by cholecystokinin octapeptide assay and flow cytometry. The mineralized effect of fluocinolone acetonide was investigated by the detection of mineralization-related biomarkers including alkaline phosphatase (ALP), bone sialoprotein, and osteocalcin by using ALP histochemical staining, ALP activity, immunostaining, alizarin red staining, and reverse-transcriptase polymerase chain reaction. The molecules, including dentin sialophosphoprotein and Wnt4, involved in the process of mineralization were detected by real-time polymerase chain reaction and Western blot analysis. RESULTS: Low concentrations of fluocinolone acetonide (0.1-40 µmol/L) promoted the proliferation of DPCs. The flow cytometry results showed that the CD146-positive subpopulation of DPCs was significantly increased after treatment with fluocinolone acetonide at 1 and 10 µmol/L for 48 hours, respectively. The messenger RNA expression and activity of the early-stage mineralization marker ALP were evidently increased in fluocinolone acetonide-treated DPCs compared with the untreated control group, so did the middle-stage mineralization marker bone sialoprotein and the late-stage mineralization marker osteocalcin. Meanwhile, Wnt4 and the dentin-specific marker dentin sialophosphoprotein were obviously up-regulated by fluocinolone acetonide compared with the untreated controls. CONCLUSIONS: Fluocinolone acetonide can promote the proliferation of DPCs, especially for the CD146+ subpopulation. Fluocinolone acetonide can initiate the mineralization of DPCs and has the potential role in repairing injured pulp tissues.

The effect of mineral trioxide aggregate on odontogenic differentiation in dental pulp stem cells.

INTRODUCTION: This study aimed to identify the early genetic changes related to odontogenic differentiation when mineral trioxide aggregate (MTA) is applied to dental pulp stem cells (DPSCs). METHODS: Odontogenic-differentiated cells (induced DPSCs) were obtained by culturing DPSCs in odontoinduction medium for 14 days. Thereafter, MTA in Teflon tubes was applied to the induced DPSCs and uninduced cells. Cells exposed to empty tubes were used as negative controls. The total RNA was extracted from the MTA treated and MTA untreated cells 1 and 3 days after tube application and assessed by microarray analysis. The key results were confirmed selectively by reverse-transcription polymerase chain reaction. We also performed a gene set enrichment analysis. RESULTS: In microarray analysis, although the expression levels of 460 genes were changed more than 2-fold in MTA-treated, uninduced DPSCs after 1 day, only 39 genes were altered in MTA-treated, induced DPSCs. In the odontoinduction medium-induced, MTA-treated DPCs, the value of correlation was 0.993 on 1 day and 0.986 on 3 day compared with 0.970 on 1 day and 0.975 on 3 day in the uninduced, MTA-treated DPSCs. Gene set enrichment analysis revealed that MTA significantly up-regulated gene sets involved in cell migration, the response to transforming growth factor ß1, and the inflammation pathway in the uninduced DPSCs, whereas in the induced DPSCs it only up-regulated genes involved in cell migration after 1 day. CONCLUSIONS: This result shows that MTA stimulates the odontogenic differentiation of DPSCs, and the effects of MTA are drastically increased in uninduced pulp cells compared with odontogenic-differentiated cells.

Evaluation of the effects of transient or continuous PTH administration to odontoblast-like cells.

Parathyroid hormone participates in the metabolism of mineralized tissue. Its role in the formation of dentine is, as yet, incompletely understood. In the present study we analyzed the effect of transient (1 and 24-h/cycle) or continuous hPTH (1-34) treatment in odontoblast-like cells (MDPC-23) to the following parameters: mineral deposition detected by alizarin red, mRNA expression of the type I collagen (COL1), alkaline phosphatase (ALP), biglycan (BGN), matrix metalloproteinase 2 (MMP-2) and dentine sialophosphoprotein (DSPP) quantified by qRT-PCR. MMP-2 and ALP activities were quantified by zymography and colorimetric assay, respectively. The results showed that compared to Control group: intermittent PTH administration (1 and 24-h/cycle) decreased the mineral deposition and ALP activity. DSPP gene expression was not detected in both control and PTH treated cells. The PTH administration for 24-h/cycle increased the ALP, BGN and COL1 mRNA expression and continuous PTH treatment increased BGN and COL1 mRNA expression. Zymography assays showed that compared to Control group: PTH treatment for 1-h/cycle increased the total MMP-2 secretion and the continuous treatment decreased the secreted levels of MMP-2 active-form. Taken together, the results shown that PTH may regulate the odontoblast-like cells-induced secretion, and potentially this hormone can affect in vivo odontoblasts functions.

Direct contact with mineral trioxide aggregate activates and differentiates human dental pulp cells.

INTRODUCTION: Mineral trioxide aggregate (MTA) is routinely used for pulp-capping procedures. However, little is known about its direct interaction with the cells or whether MTA is capable of releasing soluble factors that could help in differentiating cells. There have been no previous studies showing this aspect of MTA. Hence, the aim of this study was to determine whether direct contact of the cells with MTA was necessary to help differentiate the pulp cells into odontoblast-like cells. METHODS: Human dental pulp cells were cultured on gray MTA, either in direct contact or away from the cells on a cell culture insert, and the levels of gene expression, secretion of vascular endothelial growth factor (VEGF), and the rates of cell proliferation were analyzed. RESULTS: MTA when placed in direct contact with the cells promoted up-regulated the expression of important odontoblastic genes like osteocalcin and dentin sialoprotein, thereby showing that direct contact of the cells with the MTA is necessary to promote differentiation of the pulp cells into odontoblast-like cells, which in turn are responsible for dentin bridge formation. MTA also induced an increase in the secretion of VEGF when placed in direct contact with the cells. CONCLUSIONS: Overall, our study supports the fact that direct contact of the cells with the MTA is necessary to help differentiate them into odontoblast-like cells, which in turn will lead to a successful treatment outcome.

Blockade of TLR2 inhibits Porphyromonas gingivalis suppression of mineralized matrix formation by human dental pulp stem cells.

INTRODUCTION: Human dental pulp stem/progenitor cells (hDPSC) can differentiate into odontoblast-like cells and express dentin sialophosphoprotein (DSPP) and osteocalcin (OCN); thus, they may be used to regenerate dentin. However, residual bacterial components in the root canal may suppress this activity. PURPOSE: This study investigated the effect of a Porphyromonas gingivalis component on the expression of DSPP and OCN by stimulated hDPSCs and the influence of blockade of TLR2-mediated P. gingivalis host recognition. METHODS: Stimulated hDPSCs were exposed to varying concentrations of P. gingivalis lipopolysaccharide (LPS), and the expression of DSPP and OCN was measured. Similar groups of stimulated hDPSCs were exposed to TLR2 blocking agents before exposure to LPS. RESULTS: hDPSCs exposed to 5, 10, and 20 µg/mL LPS exhibited a dose-dependent reduction in the expression of DSPP (3.19 ± 0.18, 2.60 ± 0.49, and 1.15 ± 0.29, respectively) and OCN (3.51 ± 1.18, 2.60 ± 0.67 and 1.66 ± 0.89, respectively). The expression of DSPP and OCN after exposure to 20 µg/mL of LPS was significantly lower than measured for unexposed stimulated cells (analysis of variance and post hoc Tukey test, P < .05). The blockade of TLR2 using an extra- and intracellular agent affected DSPP (4.67 ± 0.97 and 5.29 ± 1.66, respectively) and OCN (5.25 ± 1.69 and 5.82 ± 2.38, respectively) expression at levels comparable to stimulated cells unexposed to 20 µg/mL LPS (6.32 ± 2.47 and 4.70 ± 1.60 for DSPP and OCN, respectively). CONCLUSIONS: The suppressing effect of P. gingivalis on mineralized matrix formation by hDPSCs is confirmed, and this suppression can be moderated by TLR2 blockade.

Dentine sialoprotein and collagen I expression after experimental pulp capping in humans using emdogain gel.

AIM: To characterize the hard tissue formed in human teeth experimentally pulp capped either with calcium hydroxide or with Emdogain Gel (Biora AB, Malmö, Sweden) - a derivative of enamel matrix (EMD), using two markers for dentine; dentine sialoprotein (DSP) and type 1 collagen (Col I). METHODOLOGY: Affinity-purified rabbit anti-Col I and anti-DSP polyclonal antibodies were used to stain histological sections from nine pairs of contra-lateral premolars that had been experimentally pulp amputated and randomly capped with EMDgel or calcium hydroxide. Twelve weeks after the teeth had been pulp capped, they were extracted, fixed, demineralized and serially sectioned prior to immunohistochemical staining. RESULTS: In the calcium hydroxide treated teeth DSP was seen in the new hard tissue which formed a bridge. DSP was also seen in the newly formed hard tissue in the EMDgel-treated teeth. Proliferated pulp tissue partly filled the space initially occupied by EMDgel and DSP-stained hard tissue was observed alongside exposed dentine surfaces as well as in isolated masses within the proliferated pulp tissue, although the new hard tissue did not cover the pulp exposure. DSP staining was also seen in the cells lining the hard tissue in both groups. Col I staining was seen in the newly formed hard tissue in both groups. CONCLUSIONS: The new hard tissue formed after pulp capping with EMDgel or calcium hydroxide contained DSP and Col I, considered to be markers for dentine. Thus, the newly formed hard tissue can be characterized as dentine rather than unspecific hard tissue.