Human Pulp Cells Research Articles

Cytotoxicity and genotoxicity of pulp capping materials in two cell lines

The aim of this study was to evaluate the cytotoxicity and genotoxicity of the new castor oil bean cement (COB) material in comparison to commonly used pulp capping materials. Specimens of COB, calcium hydroxide (Hydro C), and mineral trioxide aggregate (white and gray MTA) were extracted in culture medium (91.6 mm(2) sample surface mL(-1)). Transfected human pulp cells (tHPCs) were exposed to dilutions of the extracts for 1 h, and the generation of reactive oxygen species (ROS) was determined by flow cytometry (FACS) using H(2)DCF-DA as a dye. Survival of tHPCs was measured photometrically using a crystal violet assay after a 24-h exposure period. Genotoxicity as indicated by the formation of micronuclei in V79 cells, and the modification of the normal cell cycle by extracts of the materials was analysed by FACS. Clear cytotoxic effects were detected only with extracts of Hydro C under the current experimental conditions. The two MTA preparations induced an insignificant reduction in the number of cells. In contrast, the extracts of COB slightly induced cell proliferation. Extracts of Hydro C caused a twofold increase in ROS production, whilst the other tested materials were ineffective. An increase in the number of micronuclei was not detected with any material tested; Hydro C slightly increased the number of cells in G1 and G2. The COB and the two MTA preparations did not negatively influence cell survival or ROS production and may thus be further considered for pulp capping studies.

A proteomic analysis of human dental pulp cells undergoing odontoblast differentiation

To analyze the difference in protein profiles between human dental pulp cells (DPC) and odontogenic differentiated DPC by using proteomic approach. Human DPC were induced to odontoblast differentiation and total proteins in the cell lysates before and after induction were prepared. Proteins spots were isolated by two-dimensional gel electrophoresis. DeCyder V6.0 software was applied to gel image analysis. Differential protein spots were identified by peptide mass fingerprinting technique. Forty-six protein spots were determined to be differentially expressed with twenty identified protein spots. Expression changes of the identified proteins revealed the involvement of various regulation mechanisms in odontoblast differentiation, such as cell cycle, cellular energy regulation and signal transduction. The proteomic approach is a high throughput method to screen the candidate proteins involved in odontoblast differentiation of DPC.

A bioactivity study of Portland cement mixed with β-glycerophosphosphate on human pulp cell

The purpose of this study is to investigate the response of human pulp cell on Portland cement mixed with -glycerophosphate. To investigate the effect of -glycerophosphate and/or dexamethasone on human pulp cell, ALP activity on various concentration of -glycerophosphate and dexamethasone was measured and mineral nodule of human pulp cell was stained with Alizarin red S. MTS assay and ALP activity of human pulp cell on Portland cement mixed with various concentration of -glycerophosphate (10 mM, 100mM, 1M) was measured and the specimens were examined under SEM. Addition of -glycerophosphate or dexamethasone alone had no effect however, the addition of 5 mM -glycerophosphate and 100 nM dexamethasone had the largest increasement in ALP activity. There was no toxicity in all samples and the data showed that Portland cement mixed with 10 mM -glycerophosphate had more increase in ALP activity compared with control. In conclusion, Portland cement mixed with -glycerophosphate has no toxicity and promotes differentiation and mineralization of pulp cell compared with additive-free Portland cement. This implicated that application of Portland cement mixed with -glycerophosphate might form more reparative dentin and in turn it would bring direct pulp capping to success.

Substance P influenced gelatinolytic activity via reactive oxygen species in human pulp cells

To investigate the effects of substance P (SP) on gelatinolytic activity of matrix metalloproteinases (MMPs) in human pulp cells. Human dental pulp cells were isolated and cultured. Subconfluent cells, between the third and sixth passages, were maintained under serum deprivation for 18 h followed by the treatment of varying doses of SP (1 pmol L(-1), 100 pmol L(-1), 10 nmol L(-1), 1 micromol L(-1) and 100 micromol L(-1)). Conditioned media were then subjected to gelatin zymography using 8% sodium dodecyl sulphate polyacrylamide gel electrophoresis minigels containing 1.5 g L(-1) gelatin. The effect of SP on intracellular reactive oxygen species (ROS) was also examined by confocal microscopy. ROS scavenger N-Acetyl-L-cysteine (NAC, 5 mmol L(-1)) was utilized to evaluate the roles of ROS pathway in mediating the impact of SP on cellular gelatinolytic activity. Data were analysed using analysis of variance with Bonferroni correction for multiple comparisons or an unpaired Student's t-test. Substance P, at levels above 1 micromol L(-1), remarkably enhanced MMP-2 activity reflected by the band migrating at 66 kDa (P < 0.05). A gelatinolytic band at approximately 44 kDa appeared to be intensified in a SP dose-dependent manner. In addition, it was demonstrated that SP could induce ROS production in pulp cells and ROS scavenger NAC was further found to significantly reduce MMP-2 activity (P < 0.05), as well as other bands of gelatinolytic proteinases. Substance P can influence gelatinolytic activity in human pulp cells via ROS pathway.

Hydrogen Peroxide Induces Heme Oxygenase–1 and Dentin Sialophosphoprotein mRNA in Human Pulp Cells

Although the induction of heme oxygenase–1 (HO-1) by hydrogen peroxide (H 2O 2) has been reported, the HO-1 and odontoblastic differentiation-inducing effects against H 2O 2have not been clarified in human pulp cells. In this study, we investigated whether HO-1 is involved in the protective mechanisms against the cytotoxic effects of H 2O 2 by using a cell viability assay, and we examined the production of dentin sialophosphoprotein (DSPP) and other mineralization markers by using reverse transcriptase–polymerase chain reaction in human pulp cells. H 2O 2decreased cell viability but increased HO-1 and DSPP expression in a concentration- and time-dependent manner. Antioxidants and inhibitors of HO-1, phosphatidylinositol-3′-kinase, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase blocked H 2O 2-induced cytotoxicity and the expression of HO-1 and DSPP mRNA in pulp cells. These data suggest that the induction of HO-1 by H 2O 2 in pulp cells plays a protective role against the cytotoxic effects of H 2O 2 and stimulates DSPP expression, resulting in premature odontoblast differentiation through pathways that involve phosphatidylinositol-3′-kinase, p38, and extracellular signal-regulated kinase.

Involvement of mitogen-activated protein kinases and nuclear factor-kappa B activation in nitric oxide–induced interleukin-8 expression in human pulp cells

This study examined the effect of nitric oxide (NO) on interleukin-8 (IL-8) production and the involvement of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-kappaB) signaling pathways in primary cultured human pulp cells. IL-8 production was measured using enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. MAPK activation and IkappaB degradation and phosphorylation were determined by western blotting. Sodium nitroprusside (SNP), an NO donor, has increased IL-8 secretion and mRNA expression in a dose- and time-dependent manner. SNP induced the phosphorylation of p38 MAPK and extracellular-regulated kinase (ERK), degradation and phosphorylation of IkappaB, and activation of NF-kappaB. Furthermore, inhibition of the ERK, p38, and NF-kappaB pathways blocked SNP-induced IL-8 secretion. Human pulp cells showed NO-induced IL-8 expression via the MAPK and NF-kappaB pathways, which may play an important role in the inflammatory responses of pulp and periapical lesions.

Effect of 2-Hydroxyethyl Methacrylate on Human Pulp Cell Survival Pathways ERK and AKT

Previous investigations have revealed that dental monomers could affect intracellular pathways leading to cell survival or cell death. Mitogen-activated protein kinase (MAPK) and protein kinase B (AKT) might mediate cell responses as well as cell survival and apoptosis. The purpose of this study was to evaluate the effects of 2-hydroxyethyl methacrylate (HEMA) on the ERK1/2 and AKT pathways in human primary pulp fibroblasts (HPCs). HPCs were treated with various concentrations of HEMA, after which viability and reactive oxygen species levels were determined by flow cytometry with Annexin V-PI staining and 2,7-dichlorofluorescine diacetate, respectively. Whole-cell extracts were immunoblotted with anti-P-Akt or anti–P-ERK1/2. Cell viability decreased in a dose-dependent manner after HEMA exposure, showing a significant decrease with 10 mmol/L HEMA (p < .05). HEMA treatment resulted in a 4-fold increase in reactive oxygen species formation (p < .05). A short HEMA exposure (30–90 minutes) increased ERK1/2 phosphorylation, whereas a decrease in the AKT phosphorylation was observed. Selective inhibitors of the ERK (PD98059) and AKT ( LY294002) pathways amplified HPC cell damage after HEMA exposure. Our findings demonstrated that HEMA exposure modulates the ERK and AKT pathways in different manners, and that in turn, they function in parallel to mediate pro-survival signaling in pulp cells subjected to HEMA cytotoxicity.

Effects of LL37 on expression of inflammatory cytokines in human pulp cells

Effects of LL37 on expression of inflammatory cytokines in human pulp cells

Effect of neurotrophins on differentiation, calcification and proliferation in cultures of human pulp cells

Neurotrophins (NTs) are expressed during tooth development. However, little is known about a role of NTs in differentiation of pulp cells into mineralizing cells. In this study, mRNA expressions of hard tissue-related proteins, calcification and proliferation are examined in cultures of human pulp (HP) cells. Nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin (NT)-3 and NT-4/5 increased the mRNA levels of dentin sialophsphoprotein, alkaline phosphatase, osteopontin, type I collagen and bone morphogenetic protein-2 and mineral deposition in cultures of HP cells. The increased levels and manners varied, depending on the concentrations of NTs and hard-tissue related protein tested. On the other hand, only NGF significantly stimulated DNA synthesis in cultures of HP cells. These findings suggest that NTs characteristically regulate hard-tissue related protein expression, calcification and proliferation in pulp cells. NTs may accelerate pulp cell differentiation.

Influence of TGF-β1 on the expression of BSP, DSP, TGF-β1 receptor I and Smad proteins during reparative dentinogenesis

Reparative dentin has a wide variety of manifestations ranging from a regular, tubular form to an irregular, atubular form. However, the characteristics of reparative dentin have not been clarified. This study hypothesized that the level of bone sialoprotein (BSP) expression will increase if the newly formed reparative dentin is bone-like but the dentin sialophosphoprotein (DSPP) level will decrease. In order to test this hypothesis, the expression of BSP and DSP was examined by immunohistochemistry and the expression of BSP was measured by in situ hybridization in an animal model. The pulps of 12 maxillary right first molars from twelve male rats were exposed and capped with MTA. In addition, in order to understand the role of transforming growth factor-beta 1 (TGF-beta1) during reparative dentinogenesis, the expression of BSP and DSPP mRNA was analyzed by RT-PCR in a human dental pulp cell culture, and the transforming growth factor-beta 1 receptors (TbetaRI) and Smad 2/3 were examined by immunofluorescence in an animal model. DSP was expressed in the normal odontoblasts and odontoblast-like cells of the reparative dentin. Interestingly, BSP was strongly expressed in the odontoblast-like cells of reparative dentin. The level of the TbetaRI and Smad 2/3 proteins was higher in the reparative dentin than in the normal dentin. TGF-beta1 up-regulated BSP in the human pulp cell cultures. This suggests that reparative dentin has both dentinogenic and osteogenic characteristics that are mediated by TGF-beta1.

Different Roles of Dexamethasone on Transforming Growth Factor-β1–induced Fibronectin and Nerve Growth Factor Expression in Dental Pulp Cells

During pulp injury, it has been hypothesized that transforming growth factor-beta1 (TGF-beta1) is released from dentin into pulp tissue and promotes pulp tissue healing. Dexamethasone is a glucocorticoid that has been used to treat pulp injury and shown to induce differentiation of hard tissue forming cells. However, the interaction between dexamethasone and TGF-beta1 is still unknown. This study aimed to examine the effects of dexamethasone on human pulp cells in the presence of TGF-beta1. TGF-beta1 increased expression and synthesis of both fibronectin and nerve growth factor (NGF), whereas dexamethasone stimulated fibronectin synthesis but inhibited NGF expression. The application of both TGF-beta1 and dexamethasone resulted in an additional effect on fibronectin; however, dexamethasone inhibited the TGF-beta1-induced NGF expression. Dexamethasone promotes fibronectin synthesis and suppresses NGF secretion, suggesting that this reagent could be used clinically to reduce pain and promote dental pulp tissue healing.

Behavior of Human Dental Pulp Cells Exposed to Transforming Growth Factor-Beta1 and Acidic Fibroblast Growth Factor in Culture

The development of methods for regenerative endodontic procedures requires an understanding of the factors regulating the development of odontoblasts from adult cell populations such as pulpal cell lines. In this study, we exposed cultures of human pulp cells (7th passage) to growth factors including transforming growth factor-beta1 (TGF-beta1, at 1 or 5 ng/mL), acidic fibroblast growth factor (aFGF, 5 ng/mL), or a combination of the 2 growth factors and evaluated cellular morphology and markers of cell phenotype including alkaline phosphatase activity, osteocalcin, bone sialoprotein (BSP), and dentin sialophosprotein (DSPP). The mean number of nucleoli in the 1 ng/mL TGF-beta1 group was significantly higher than with 5 ng/mL aFGF. Alkaline phosphatase activity was significantly greater with 1 ng/mL TGF-beta1 versus 5 ng/mL TGF-beta1 + 5 ng/mL aFGF (P < .05). Osteocalcin mRNA was expressed in all samples. The cells exposed to 1 ng/mL TGF-beta1 were stimulated; however, exposure to growth factors for 8 days was not sufficient for expression of BSP and DSPP mRNA. Cells treated with 1 ng/mL TGF-beta1 exhibited higher activity, whereas 5 ng/mL aFGF-treated cells were inhibited. Although osteocalcin was observed in all cultures, suggestive of the potential for odontoblast formation, under the present conditions, the exposure to TGF-beta1 and aFGF was not sufficient to induce expression of the dentin matrix components BSP and DSPP.

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.

Human Pulp Cells Response to Portland Cement In Vitro

The aim of this study was to investigate the cellular effects of Portland cement on cultured human pulp cells. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, no cytotoxicity was observed in the Portland cement group in comparison with the negative control group, whereas the glass ionomer cement, intermediate restorative material, and Dycal groups showed a survival rate of less than 40% at 12 hours. Scanning electron microscopy revealed that human pulp cells attached to the Portland cement were flat and had numerous cytoplasmic extensions. In the groups in which other materials were used, a few rounded cells were observed on the material but no living cells were observed. The expression of both osteonectin and dentin sialophosphoprotein mRNAs was induced in the Portland cement–treated group. These results suggest that Portland cement is biocompatible, allows the expression of mineralization-related genes on cultured human pulp cells, and has the potential to be used as a proper pulp-capping material.

Effects of adenovirus-mediated human bone morphogenetic protein-7 on proliferation and differentiation of human dental pulp cells

To investigate the effect of adenovirus expressing human bone morphogenetic protein-7 (hBMP-7) on proliferation and differentiation of human dental pulp cells. The replication-deficient adenoviral vector encoding hBMP-7 gene was constructed by using homologous recombinant modality. The efficiency of transfection was evaluated by fluorescent microscopy and flow cytometry. The expression of hBMP-7 protein in adenovirus-infected dental pulp cells was determined by Western blot. The proliferation of cells was tested by MTT method, the activity of alkaline phosphatase was assayed, von Kossa staining was used to detect mineralized nodule formation, and the expression of DSPPmRNA in cells was detected using semi-quantitative RT-PCR. Green fluorescent protein was visible under fluorescent microscopy. Higher transfection efficiency (91.1 +/- 1.0)% could be obtained at MOI of 75. Western blot from dental pulp cells infected with Ad-hBMP-7 for 48h detected protein expression of a hBMP-7 gene. The activity of alkaline phosphatase in cells was significantly higher than those of the control groups (P < 0.05). The cells infected with Ad-hBMP-7 had the ability of mineralization. DSPP mRNA expression of cells was in a time- and dose- dependent manner. Ad-hBMP-7 can induce human pulp cells into odontoblasts, but has no obvious effect on their proliferation.

Genetic and Cellular Toxicology of Dental Resin Monomers

Monomers are released from dental resin materials, and thus cause adverse biological effects in mammalian cells. Cytotoxicity and genotoxicity of some of these methacrylates have been identified in a vast number of investigations during the last decade. It has been well-established that the co-monomer triethylene glycol dimethacrylate (TEGDMA) causes gene mutations in vitro. The formation of micronuclei is indicative of chromosomal damage and the induction of DNA strand breaks detected with monomers like TEGDMA and 2-hydroxyethyl methacrylate (HEMA). As a consequence of DNA damage, the mammalian cell cycle was delayed in both G1 and G2/M phases, depending on the concentrations of the monomers. Yet, the mechanisms underlying the genetic and cellular toxicology of resin monomers have remained obscure until recently. New findings indicate that increased oxidative stress results in an impairment of the cellular pro- and anti-oxidant redox balance caused by monomers. It has been demonstrated that monomers reduced the levels of the natural radical scavenger glutathione (GSH), which protects cell structures from damage caused by reactive oxygen species (ROS). Depletion of the intracellular GSH pool may then significantly contribute to cytotoxicity, because a related increase in ROS levels can activate pathways leading to apoptosis. Complementary, cytotoxic, and genotoxic effects of TEGDMA and HEMA are inhibited in the presence of ROS scavengers like N-acetylcysteine (NAC), ascorbate, and Trolox (vitamin E). Elevated intracellular levels of ROS can also activate a complex network of redox-responsive macromolecules, including redox-sensitive transcription factors like nuclear factor kappaB (NF-kappaB). It has been shown that NF-kappaB is activated probably to counteract HEMA-induced apoptosis. The induction of apoptosis by TEGDMA in human pulp cells has been associated with an inhibition of the phosphatidylinositol 3-kinase (PI3-K) cell-survival signaling pathway. Although the details of the mechanisms leading to cell death, genotoxicity, and cell-cycle delay are not completely understood, resin monomers may be able to alter the functions of the cells of the oral cavity. Pathways regulating cellular homeostasis, dentinogenesis, or tissue repair may be modified by monomers at concentrations well below those which cause acute cytotoxicity.

New Oleanane Glycosides from the Roots of Gomphrena macrocephala

Six new (1–6) and one known (7) oleanane glycosides have been isolated from the roots of Gomphrena macrocephala. On the basis of 1D- and 2D-NMR spectroscopic data and the results of hydrolysis, the structures of 1–6 were determined to be 3β-[(O-β-D-glucuronopyranosyl)oxy]oleana-11,13-dien-28-oic acid (1), 3β-[(O-β-D-galactopyranosyl-(1→3)-O-[β-D-glucopyranosyl-(1→2)]-β-D-glucuronopyranosyl)oxy]oleana-11,13-dien-28-oic acid (2), 3β-[(O-β-D-glucuronopyranosyl)oxy]oleana-9,12-dien-28-oic acid β-D-glucopyranosyl ester (3), 3β-[(O-β-D-galactopyranosyl-(1→3)-O-[β-D-glucopyranosyl-(1→2)]-β-D-glucuronopyranosyl)oxy]oleana-9,12-dien-28-oic acid β-D-glucopyranosyl ester (4), 3β-[(O-β-D-galactopyranosyl-(1→3)-O-[β-D-glucopyranosyl-(1→2)]-β-D-glucuronopyranosyl)oxy]-11-oxo-olean-12-en-28-oic acid β-D-glucopyranosyl ester (5), and 3β-[(O-β-D-galactopyranosyl-(1→3)-O-[β-D-glucopyranosyl-(1→2)]-β-D-glucuronopyranosyl)oxy]-11α-hydroxyolean-12-en-28-oic acid β-D-glucopyranosyl ester (6), respectively. Although 1–7 did not show apparent cytotoxicity against human oral squamous cell carcinoma (HSC-2) cells and normal human pulp cells (HPC), the aglycones 1a and 3a of 1 and 3 exhibited tumor-specific cytotoxicity against HSC-2 cells, and the IC50 value of 3a against HSC-2 cells was almost equal to that of etoposide, used as a positive control.

Heme oxygenase-1 mediates cytoprotection against nitric oxide-induced cytotoxicity via the cGMP pathway in human pulp cells

This study examined the effects of exogenous nitric oxide (NO) on human pulp cells and the involvement of cyclic 3',5'-monophosphate (cGMP) in pulpal protection induced by heme oxygenase-1 (HO-1) against NO-induced cytotoxicity. This study investigated cytotoxicity and HO-1 induction in pulp cells induced by the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP), by using Western blotting and a cell viability assay. It also investigated whether HO-1 contributes to the cytoprotective effect against the cytotoxicity caused by NO and the relationship between HO-1 and cGMP in the signaling pathway. S-nitroso-N-acetyl-D,L-penicillamine decreased cell viability, but increased HO-1 expression in a concentration- and time-dependent manner in human pulp cells. NO-induced cytotoxicity was inhibited in the presence of hemin (inducer of HO-1), whereas it was enhanced in the presence of zinc protoporphyrin IX (ZnPP IX, HO-1 inhibitor); therefore, the NO-induced cytotoxicity was correlated with HO-1 expression. Pretreatment with a membrane-permeable cGMP analog, 8-bromo-cGMP, restored cell death and enhanced the HO-1 protein expression induced by SNAP. By contrast, 1 mM SNAP inhibited guanylate cyclase in pulp cells pretreated with 1H-[1,2,4]oxadiazole[4,3-alpha]quinoxalin-1-one (ODQ), resulting in marked cytotoxicity. These findings of a link between HO-1, regulated via the cGMP system and NO-induced cytotoxicity in human pulp cells, suggest a protective role for HO-1 in pulpal inflammation.

Fluoride promotes human pulp cell proliferation through p38 kinase pathway

Fluoride promotes human pulp cell proliferation through p38 kinase pathway

Human pulp‐derived cells immortalized with Simian Virus 40 T‐antigen

Primary cells in culture have a limited capacity to divide and soon reach a non-proliferative state. This cellular senescence limits the investigation of cells derived from human pulp concerning cellular pathways, gene regulation, mechanisms of dentin formation, or responses to material exposure. To overcome this problem, primary human pulp-derived cells were established and transfected with a plasmid containing coding sequences of Simian Virus 40 (SV40) large T-antigen. This resulted in the establishment of several cell clones showing an extension of life span. Expression of T-antigen transcripts and protein was verified by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Primary human pulp cells were cultured until senescence (i.e. up to passage 7) and transfected cells could be cultured to passage 18 after transfection, when a cellular crisis with massive cell death occurred. One clone escaped from crisis and has been maintained in culture for 55 wk. Experiments were performed to characterize transfected cells in comparison to primary cells. Cell morphology and proliferation were analyzed, and expression of cell-specific gene transcripts and proteins (including collagen types I and III, alkaline phosphatase, bone sialoprotein, osteocalcin, and dentin sialophosphoprotein and dentin matrix protein I) was detected by RT-PCR and immunohistochemistry. Transfection of human pulp-derived cells resulted in an immortalized cell line retaining many of the phenotypic characteristics observed in primary cells.