Background: In healthy dentin conditions, odontoblasts have an important role such as protection from invasion of pathogens. In mammalian teeth, progenitors such as mesenchymal stem cells (MSCs) can migrate and differentiate into odontoblast-like cells, leading to the formation of reparative dentin. For differentiation using stem cells, it is crucial to provide conditions similar to the complex and intricate in vivo environment. The purpose of this study was to evaluate the potential of differentiation into odonto/ osteoblasts, and compare co-culture with/without epithelial cells. Methods: MSCs and epithelial cells were successfully isolated from dental tissues. We investigated the influences of epithelial cells on the differentiation process of dental pulp stem cells into odonto/osteoblasts using co-culture systems. The differentiation potential with/without epithelial cells was analyzed for the expression of specific markers and calcium contents. Results: Differentiated odonto/osteoblast derived from dental pulp tissue-derived mesenchymal stem cells with/without epithelial cells were evaluated by qRT-PCR, immunostaining, calcium content, and ALP staining. The expression of odonto/ osteoblast-specific markers, calcium content, and ALP staining intensity were significantly increased in differentiated cells. Moreover, the odonto/osteogenic differentiation capacity with epithelial cells co-culture was significantly higher than without epithelial cells co-culture. Conclusions: These results suggest that odonto/osteogenic differentiation co-cultured with epithelial cells has a more efficient application.

Bioactive flavonoids have been shown to improve the biological activity of stem cells derived from different sources in tissue regeneration. The goal of this study was to see how naringin, a natural flavonoid discovered in citrus fruits, affected the biological properties of human dental pulp stem cells (HDPSCs). In this study, we found that naringin increases the migratory ability of HDPSCs. Naringin increased matrix metalloproteinase-2 (MMP-2) and C-X-C chemokine receptor type 4 (CXCR4) mRNA and protein expression in HDPSCs. ARP100, a selective MMP-2 inhibitor, and AMD3100, a CXCR4 antagonist, both inhibited the naringin-induced migration of HDPSCs. Furthermore, naringin increased osteogenic differentiation of HDPSCs and the expression of the osteogenic-related marker, alkaline phosphatase in HDPSCs. Taken together, our findings suggest that naringin may be beneficial on dental tissue or bone regeneration by increasing the biological activities of HDPSCs.

Cyclooxygenase-2 (COX-2)-mediated prostaglandin E2 (PGE2) plays a key role in development and progression of inflammatory responses and Porphyromonas gingivalis is a common endodontic pathogen. In this study, we investigated induction of COX-2 and PGE2 by P. gingivalis in human dental pulp cells (HDPCs). P. gingivalis increased expression of COX-2, but not that of COX-1. Increased levels of PGE2 were released from P. gingivalis-infected HDPCs and this PGE2 increase was blocked by celecoxib, a selective COX-2 inhibitor. P. gingivalis activated all three types of mitogen-activated protein kinases (MAPKs). P. gingivalis-induced activation of nuclear factor-κB (NF-κB) was demonstrated by the results of phosphorylation of NF-κ B p65 and degradation of inhibitor of κB-α (IκB-α). Pharmacological inhibition of each of the three types of MAPKs and NF-κB substantially attenuated P. gingivalisinduced PGE2 production. These results suggest that P. gingivalis should promote endodontic inflammation by stimulating dental pulp cells to produce PGE2.

Although anti-aging activities of melatonin, a hormone secreted by the pineal gland, have been reported in senescence-accelerated mouse models and several types of cells, its impact and mechanism on the senescence of human dental pulp cells (HDPCs) remains unknown. In this study, we examined the impact of melatonin on cellular premature senescence of HDPCs. Here, we found that melatonin markedly inhibited senescent characteristics of HDPCs after exposure to hydrogen peroxide (H2O2), including the increase in senescence-associated β-galactosidase (SA-β-gal)-positive HDPCs and the upregulation of p21 protein, an indicator for senescence. In addition, as melatonin attenuated H2O2-stimulated phosphorylation of c-Jun N-terminal kinase (JNK), while selective inhibition of JNK activity with SP600125 significantly attenuated H2O2-induced increase in SA-beta-gal activity. Results reveal that melatonin antagonizes premature senescence of HDPCs via JNK pathway. Thus, melatonin may have therapeutic potential to prevent stress-induced premature senescence, possibly correlated with development of dental pulp diseases, and to maintain oral health across the life span.

To gain insights into the role of purinergic receptors in human dental pulp cells (hDPCs) differentiation, we characterized the expression and functional activity of P2Y1 receptors and investigated the effects of ADP on the proliferation and differentiation of this pulp stem-like cell population. Our data showed that ADP did not induce cell proliferation to expose the various ADP concentrations for 72 hours, but the proliferative capacity of hDPCs was inhibited at higher ATP concentrations (100 μM). Using RT-PCR analysis, we found that ADP induced several P2Y receptors including P2Y1 as well as odontoblastic differentiation genes, dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) in a dose-dependent manner. The effects of ADP on the expression of DMP-1 and DSPP mRNA were prevented by the P2Y1 antagonist MRS2179. The extracellular matrix calcium deposits were clearly observed in ADP-treated hDPCs by alizarin red S staining. Quantitative measurement of mineralization induced by ADP was significantly inhibited in MRS2179-treated hDPCs. These results may provide new insights into the molecular regulation of the differentiation of hDPCs.

Dental pulp is a highly vascularized tissue with high regenerative potential. Revascularization of severed vasculature in the tooth is required for pulp healing during avulsed tooth treatment. In this study, the relative expression of angiogenesis-related proteins was determined in human dental pulp cells using a human angiogenesis proteome profiler array. The proteome profiler array detected differentially expressed angiogenesis-related factors under conditions of hypoxia, which enhances the angiogenic potential of dental pulp cells. We confirmed that hypoxia regulates the mRNA expression of angiogenesis-related factors, including CXCL16 in dental pulp cells. Furthermore, conditioned media of hypoxic pulp cells induced tube-like structures of vascular endothelial cells, which were reduced by the neutralization of CXCL16 function. In conclusion, our data show that angiogenesisrelated factors are differentially expressed by hypoxia in dental pulp cells and suggest that CXCL16 may involve in the revascularization of hypoxic dental pulp.

This study investigated the genes involved in the dif- ferentiation of odontoblasts derived from human dental pulp stem cells (hDPSCs). hDPSCs isolated from human tooth pulp were validated by fluorescence activated cell sor- ting (FACS). After odontogenic induction, hDPSCs were analyzed investigated by Alizaline red-S staining, ALP assay, ALP staining and RT-PCR. Differential display-poly- me rase chain reac tion (DD-PCR) was pe rformed to s c re en differentially expressed genes involved in the differentia- tion of hDPSCs. By FACS analysis, the stem cell markers CD24 and CD44 were found to be highly expressed in hDPSCs. When hDPSCs were treated with agents such as β- glycerophosphate (β-GP) and ascorbic acid (AA), nodule formation was exhibited within six weeks. The ALP activity of hDPSCs was found to elevate over time, with a detectable up-regulation at 14 days after odontogenic induc- tion. RT-PCR analysis revealed that dentin sialophospho- protein (DSPP) and osteocalcin (OC) expression had inc- reased in a time-dependent manner in the induction culture. Through the use of DD-PCR, several genes were diffe- rentially detected following the odontogenic induction. These results suggest that these genes may possibly be linked to a variety of cellular process during odontogenesis. Further-more, the characterization of these regulated genes during odontogenic induction will likely provide valuable new in- sights into the functions of odontoblasts.

Dlx3 and Dlx5 are homeobox domain proteins and are well-known regulators of osteoblastic differentiation. Since possible reciprocal relationships between osteogenic and adipogenic differentiation in mesenchymal stem cells exist, we examined the regulatory role of Dlx3 and Dlx5 on adipogenic differentiation using human dental pulp stem cells. Over-expression of Dlx3 and Dlx5 stimulated osteogenic differentiation but inhibited adipogenic differentiation of human dental pulp stem cells. Dlx3 and Dlx5 suppressed the expression of adipogenic marker genes such as C/EBPα, PPARγ, aP2 and lipoprotein lipase. Adipogenic stimuli suppressed the mRNA levels of Dlx3 and Dlx5, whereas osteogenic stimuli enhanced the expression of Dlx3 and Dlx5 in 3T3-L1 preadipocytes. These results suggest that Dlx3 and Dlx5 exert a stimulatory effect on osteogenic differentiation of stem cells through the inhibition of adipogenic differ¬entiation as well as direct stimulation.

The aim of t his study was to investigate the cytotoxic and ni t ric oxide (NO)-inducing effects of bismuth oxide (Bi203)-containi ng Portland cement (BPC) on human dental pulp cells. We also assessed whether heme oxygenase-l (HO-l) is involved in BPC-induced cytotox.icity in dental pulp cells Cytotoxicity and NO production induced by BPC were higher than those induced by Portland cement (PC) at 12 and 24 hours, and the former grad ua lly decreased to the level observed for PC. HO- l and inducible nitric oxide synthase (iNOS) mRNA expressions in the BPC group showed maximal increase at 24 hours. and it gradually decreased with increasing cultivation tlme Hemin treatment reversed the BPC-induced cytotoxicity ‘ whereas zinc protoporphyrin IX treatment increased the cytotoxicity. These results suggested that NO production by BPC correlates with HO-l exp1'ession in dental pulp cells Moreover ‘ BPC- induced HO-l expression in dental pulp cells plays a protective 1'ole against the cytotox.ic effects of BPC.

Tissue stem cells are used for the regenerative medicine. In previous study we observed hard tissue formation of human dental pulp-derived cells using alginate scaffold. In this study, we explore the ability to differentiate of the 13th passage cells with glycerol 2-phosphate disodium salt hydrate (β-GP) which accelerate calcification. Reverse transcriptase Polymerase Chain Reaction (RT-PCR), transplants using alginate scaffold and histological examination were performed. We observed the expression of DSPP mRNA on day 10 cultured cells with β-GP. In conclusion, the 13th passage cells still have an ability to differentiate into odontoblast-like cells and alginate supports the differentiation of cultured cells in the transplants.

Background : Tooth vitality is reflected by the health of dental pulp. Schisandrin C is a natural compound extracted from the fruit of Schisandra chinensis which has anti-inflammatory and anti-oxidant properties. The role of Schisandrin C on human dental pulp cells (HDPCs) has not been studied yet. This study examined the properties of Schisandrin C as an anti-inflammatory and anti-oxidant compound, and whether its characteristics promote mitochondrial biogenesis in HDPCs. Methods and Results : HDPCs were extracted from fresh third molars and cultured. Reactive oxidative stress (ROS) and nitric oxide (NO) formation were analyzed by a Muse cell analyzer. Western blotting and gelatin zymography were used to identify the presence of anti-oxidants, as well as inflammatory and mitochondrial biogenesis. Confocal microscopy was used for the detection of mitochondrial activity. Schisandrin C inhibited lipopolysaccharide (LPS)-stimulated inflammatory molecules; intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), matrix metalloproteinase-2 and -9 (MMP-2/9), NO production, ROS formation and the mitogen-activated protein (MAPK) pathway through minimizing the nuclear factor kappa B (NF-kB) translocation. Schisandrin C increased the expression of superoxide dismutase (SOD) enzymes as well as heme oxygenase-1 (HO-1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a) through the phosphorylated-protein kinase B (p-AKT) and nuclear factor erythroid 2-related factor-2 (Nrf-2) pathways. The anti-inflammatory and anti-oxidant properties of Schisandrin C promoted mitochondrial biogenesis. Conclusions : These results suggest that Schisandrin C may be used as an anti-inflammatory compound to reduce oral inflammation such as pulpitis.