GTPases RhoA and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts

Morphogenesis and cytodifferentiation are distinct processes in tooth development. Cell proliferation predominates in morphogenesis; differentiation involves changes in form and gene expression. The cytoskeleton is essential for both processes, being regulated by Rho GTPases. The aim of this study was to verify the expression, distribution, and role of Rho GTPases in ameloblasts and odontoblasts during tooth development in correlation with actin and tubulin arrangements and amelogenin and dentin sialophosphoprotein (DSPP) expression. RhoA, Rac1, and Cdc42 were strongly expressed during morphogenesis; during cytodifferentiation, RhoA was present in ameloblasts and odontoblasts, Rac1 and its effector Pak3 were observed in ameloblasts; and Cdc42 was present in all cells of the tooth germ and mesenchyme. The expression of RhoA mRNA and its effectors RockI and RockII, Rac1 and Pak3, as analyzed by real-time polymerase chain reaction, increased after ameloblast and odontoblast differentiation, according to the mRNA expression of amelogenin and DSPP. The inhibition of all Rho GTPases by Clostridium difficile toxin A completely abolished amelogenin and DSPP expression in tooth germs cultured in anterior eye chamber, whereas the specific inhibition of the Rocks showed only a partial effect. Thus, both GTPases are important during tooth morphogenesis. During cytodifferentiation, Rho proteins are essential for the complete differentiation of ameloblasts and odontoblasts by regulating the expression of amelogenin and DSPP. RhoA and its effector RockI contribute to this role. A specific function for Rac1 in ameloblasts remains to be elucidated; its punctate distribution indicates its possible role in exocytosis/endocytosis.

Phototherapy up-regulates dentin matrix proteins expression and synthesis by stem cells from human-exfoliated deciduous teeth

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Survival of rat functional dental pulp cells in vascularized tissue engineering chambers

Regenerative endodontics aims to preserve, repair or regenerate the dental pulp tissue. Dental pulp stem cells, have a potential use in dental tissue generation. However, specific requirements to drive the dental tissue generation are still obscured. We established an in vivo model for studying the survival of dental pulp cells (DPC) and their potential to generate dental pulp tissue. DPC were mixed with collagen scaffold with or without slow release bone morphogenic protein 4 (BMP-4) and fibroblast growth factor 2 (FGF2). The cell suspension was transplanted into a vascularized tissue engineering chamber in the rat groin. Tissue constructs were harvested after 2, 4, 6, and 8 weeks and processed for histomorphological and immunohistochemical analysis. After 2 weeks newly formed tissue with new blood vessel formation were observed inside the chamber. DPC were found around dentin, particularly around the vascular pedicle and also close to the gelatin microspheres. Cell survival, was confirmed up to 8 weeks after transplantation. Dentin Sialophosphoprotein (DSPP) positive matrix production was detected in the chamber, indicating functionality of dental pulp progenitor cells. This study demonstrates the potential of our tissue engineering model to study rat dental pulp cells and their behavior in dental pulp regeneration, for future development of an alternative treatment using these techniques.

'Heat-sensing' TRP channel expression in human odontoblasts

Background: Thermal changes in the oral cavity are a common trigger of dental pain. Several members of the transient receptor potential (TRP) super family of ion channels are believed to play a critical role in sensory physiology, where they act as transducers for thermal, mechanical and chemical stimuli. Objectives: The present study was designed to determine the expression and functionality of the TRPV1 channel in human odontoblasts. Methods: Cultured human odontoblasts were derived from dental pulp cells induced with 2 mM beta-glycerophosphate. Molecular and protein expression of TRPV1 was confirmed by PCR, western blotting and immunohistochemistry. Functional expression of the ‘heat-sensing' TRPV1 channel was investigated using a Ca2+ microfluorimetry assay in the presence of agonists/antagonists or with appropriate adjustment of the recording chamber temperature. Results: The odontoblastic phenotype of the cells was confirmed by the expression of the odontoblast markers dentin sialophosphoprotein (DSPP) and nestin. Expression of TRPV1 in human odontoblastic cells was confirmed by PCR, western blotting and immunohistochemistry. Odontoblasts were shown to respond to pharmacological agonists and to increasing temperature by an increase in intracellular Ca2+. Both the pharmacological and temperature responses could be blocked by specific antagonists. These results indicate that odontoblasts may sense heat via TRPV1. Conclusion: This study reports that TRPV1 is expressed by human odontoblasts and is activated by specific pharmacological agonists and by heat.
This work was supported by Research Grants from the Royal College of Surgeons of Edinburgh and the British Endodontic Society

Comportamento de células pulpares humanas expostas ao TGFβ1 a ao aFGF em cultura

O propósito do presente estudo foi avaliar o comportamento de células pulpares humanas expostas ao TGFβ1 e ao aFGF, em cultura, nas seguintes concentrações: TGFβ1 a 1ng/mL, TGFβ1 a 5ng/mL, TGFβ1 a 1ng/mL + aFGF a 5ng/mL, TGFβ1 a 5ng/mL + aFGF a 5ng/mL e aFGF a 5ng/mL. Foi avaliada a morfologia celular, a atividade da fosfatase alcalina, através de ensaio com pNPP como substrato e a expressão das proteínas osteocalcina, sialoproteína óssea e sialofosfoproteína de dentina, através de RT-PCR. Após quatro dias, verificou-se que a média do número de nucléolos no grupo tratado com TGFβ1 a 1ng/mL foi significativamente maior que no grupo tratado com aFGF a 5ng/mL. A média da atividade da fosfatase alcalina no grupo tratado com TGFβ1 a 1ng/mL foi significativamente maior que no grupo tratado com TGFβ1 a 5ng/mL + aFGF a 5ng/mL. Foi observada a expressão de osteocalcina em todas as células pulpares humanas que proliferaram em cultura. Entretanto, no grupo em que foi utilizado o aFGF a 5ng/mL houve diminuição da expressão da osteocalcina. A exposição dos fatores não induziu a expressão de componentes da matriz de dentina tais como BSP e DSPP. Sugere-se que as células expostas ao TGFβ1 1ng/mL foram estimuladas, apresentando uma maior atividade celular e as células expostas ao aFGF 5ng/mL foram inibidas, apresentando uma menor atividade celular.

Estudo das células primárias dentais, viabilidade celular e bioatividade de materiais á base de silicato de cálcio

Pós-graduação em Odontologia - FOAR

Expression of phosphoproteins and amelotin in teeth

The organic material of our teeth consists of collagens and a number of calcium-binding phosphoproteins. Six of these phosphoproteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins), namely osteopontin, bone sialoprotein, dentin matrix protein (DMP1), dentin sialophosphoprotein (DSPP), matrix extracellular phosphoglycoprotein (MEPE) and enamelin. We prepared a cDNA library from rat incisors in order to identify the genes involved in tooth formation. The library was screened by subtractive hybridization with two probes; one specific for teeth, the other for bone. We found that the vast majority of the clones from our library were expressed at similar levels in bone and teeth, demonstrating the close relationship of the two tissues. Only 7% of all the clones were expressed in a tooth-specific fashion. These included clones for the enamel proteins; amelotin, amelogenin, ameloblastin and enamelin; for the dentin proteins DSPP and DMP1; and for the intermediate filament protein cytokeratin 13. Several typical bone proteins, including collagen I, osteocalcin, alkaline phosphatase and FATSO, were also expressed at significantly higher levels in teeth than in bone, probably due to the extreme growth rate of rat incisors. The amino acid sequence of rat amelotin showed 62% identity with the sequence from humans. It was expressed considerably later than the other enamel proteins, suggesting that amelotin may serve a function different from those of amelogenin, ameloblastin and enamelin.

Efeito da combinação de antibióticos e sinvastatina sobre microrganismos de interesse endodôntico e na expressão de marcadores odontoblásticos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação da produção de citocinas, expressão gênica e análise funcional de células SHED expostas ao LTA bacteriano

Pós-graduação em Odontologia Restauradora - ICT

Efeito da combinação de antibióticos e sinvastatina sobre microrganismos de interesse endodôntico e na expressão de marcadores odontoblásticos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Toxicity of dioxin to developing teeth and salivary glands : An experimental study

Dioxins are ubiquitous environmental poisons having unequivocal adverse health effects on various species. The majority of their effects are thought to be mediated by the aryl hydrocarbon receptor (AhR). Developing human teeth may be sensitive to dioxins and the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is developmentally toxic to rodent teeth. Mechanisms of TCDD toxicity can be studied only experimentally. The aim of the present thesis work was to delineate morphological end points of developmental toxicity of TCDD in rat and mouse teeth and salivary glands in vivo and in vitro and to characterize their cellular and molecular background. Mouse embryonic teeth and submandibular gland explants were grown in organ culture without/with TCDD at various concentrations, examined stereomicroscopically and processed for histological examination. The effects of TCDD on cellular mechanisms essential for organogenesis were investigated. The expression of various genes eliciting the response to TCDD exposure or involved in tooth and salivary gland development was studied at the mRNA and/or protein levels by in situ hybridization and immunohistochemistry. Association of the dental effects of TCDD with the resistance of a rat strain to TCDD acute lethality was analyzed in two lactationally exposed rat strains. The effect of TCDD on rat molar tooth mineralization was studied in tissue sections. TCDD dose- and developmental stage-dependently interfered with tooth formation. TCDD prevented early mouse molar tooth morphogenesis and altered cuspal morphology by enhancing programmend cell death, or apoptosis, in dental epithelial cells programmed to undergo apotosis. Cell proliferation was not affected. TCDD impaired mineralization of rat molar dental matrices, possibly by specifically reducing the expression of the mineralization-related dentin sialophosphoprotein gene shown in cultured mouse teeth. The impaired mineralization of rat teeth was accompanied by decreased expression of AhR and the TCDD-inducible xenobiotic-metabolozing enzyme P4501 A1 (CYP1A1), suggesting mediation of the TCDD effect by the AhR pathway. The severe interference by TCDD with rat incisor formation was independent of the genotypic variation of AhR determining the resistance of a rat strain to TCDD acute lethality. The impairment by TCDD of mouse submandibular gland branching morphogenesis was associated with CYP1A1 induction and involved blockage of EGF receptor signalling. In conclusion, TCDD exposure is likely to have activated the AhR pathway in target organs with the consequent activation of other signalling pathways involving developmentally regulated genes. The resultant phenotype is organ specific and modified by epithelial-mesenchymal interactions and dependent on dose as well as the stage of organogenesis at the time of TCDD exposure. Teeth appear to be responsive to TCDD exposure throughout their development.

TNF-alpha Promotes an Odontoblastic Phenotype in Dental Pulp Cells

Dental pulp cells can differentiate toward an odontoblastic phenotype to produce reparative dentin beneath caries lesions. However, the mechanisms involved in pulp cell differentiation under pro-inflammatory stimuli have not been well-explored. Thus, we hypothesized that the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) could be a mediator involved in dental pulp cell differentiation toward an odontoblastic phenotype. We observed that TNF-alpha-challenged pulp cells exhibited increased mineralization and early and increased expression of dentin phosphoprotein (DPP), dentin sialoprotein (DSP), dentin matrix protein-1, and osteocalcin during a phase of reduced matrix metalloproteinase (MMP) expression. We investigated whether these events were related and found that p38, a mitogen-activated protein kinase, differentially regulated MMP-1 and DSP/DPP expression and mediated mineralization upon TNF-alpha treatment. These findings indicate that TNF-alpha stimulates differentiation of dental pulp cells toward an odontoblastic phenotype via p38, while negatively regulating MMP-1 expression.

Effects of zoledronic acid on odontoblast-like cells

The aim of the study was to evaluate the effects of a highly potent bisphosphonate, zoledronic acid (ZOL), on cultured odontoblast-like cells MDPC-23. The cells (1.5 × 104 cells/cm2) were seeded for 48 h in wells of 24-well dished. Then, the plain culture medium (DMEM) was replaced by fresh medium without fetal bovine serum. After 24 h, ZOL (1 or 5 μM) was added to the medium and maintained in contact with the cells for 24 h. After this period, the succinic dehydrogenase (SDH) enzyme production (cell viability-MTT assay), total protein (TP) production, alkaline phosphatase (ALP) activity, and gene expression (qPCR) of collagen type I (Col-I) and ALP were evaluated. Cell morphology was assessed by SEM. Five μM ZOL caused a significant decrease in SDH production. Both ZOL concentrations caused a dose-dependent significant decrease in TP production and ALP activity. ZOL also produced discret morphological alterations in the MDPC-23 cells. Regarding gene expression, 1 μM ZOL caused a significant increase in Col-I expression. Although 5 μM ZOL did not affect Col-I expression, it caused a significant alteration in ALP expression (ANOVA and Tukey's test, p < 0.05). ZOL presented a dose-dependent cytotoxic effect on the odontoblast-like cells, suggesting that under clinical conditions the release of this drug from dentin could cause damage to the pulpo-dentin complex. © 2012 Elsevier Ltd.

Identification of a fibronectin interaction site in the extracellular matrix protein ameloblastin

Mammalian teeth are composed of hydroxyapatite crystals that are embedded in a rich extracellular matrix. This matrix is produced by only two cell types, the mesenchymal odontoblasts and the ectodermal ameloblasts. Ameloblasts secrete the enamel proteins amelogenin, ameloblastin, enamelin and amelotin. Odontoblasts secrete collagen type I and several calcium-binding phosphoproteins including dentin sialophosphoprotein, dentin matrix protein, bone sialoprotein and osteopontin. The latter four proteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) because they display similar gene structures and because they contain an RGD tripeptide sequence that binds to integrin receptors and thus mediates cell adhesion. We have prepared all the other tooth-specific proteins in recombinant form and examined whether they might also promote cell adhesion similar to the SIBLINGs. We found that only ameloblastin consistently mediated adhesion of osteoblastic and fibroblastic cells to plastic or titanium surfaces. The activity was dependent on the intact three-dimensional structure of ameloblastin and required de novo protein synthesis of the adhering cells. By deletion analysis and in vitro mutagenesis, the active site could be narrowed down to a sequence of 13 amino acid residues (VPIMDFADPQFPT) derived from exon 7 of the rat ameloblastin gene or exons 7-9 of the human gene. Kinetic studies and RNA interference experiments further demonstrated that this sequence does not directly bind to a cell surface receptor but that it interacts with cellular fibronectin, which in turn binds to integrin receptors. The identification of a fibronectin-binding domain in ameloblastin might permit interesting applications for dental implantology. Implants could be coated with peptides containing the active sequence, which in turn would recruit fibronectin from the patient's blood. The recruited fibronectin should then promote cell adhesion on the implant surface, thereby accelerating osseointegration of the implant.

Effects of Prolyl Hydroxylase Inhibitor L-mimosine on Dental Pulp in the Presence of Advanced Glycation End Products

INTRODUCTION Proangiogenic prolyl hydroxylase (PHD) inhibitors represent a novel approach to stimulate tissue regeneration. Diabetes mellitus involves the accumulation of advanced glycation end products (AGEs). Here we evaluated the impact of AGEs on the response of human pulp tissue to the PHD inhibitor L-mimosine (L-MIM) in monolayer cultures of dental pulp-derived cells (DPCs) and tooth slice organ cultures. METHODS In monolayer cultures, DPCs were incubated with L-MIM and AGEs. Viability was assessed based on formazan formation, live-dead staining, annexin V/propidium iodide, and trypan blue exclusion assay. Vascular endothelial growth factor (VEGF), interleukin (IL)-6, and IL-8 production was evaluated by quantitative polymerase chain reaction and immunoassays. Furthermore, expression levels of odontoblast markers were assessed, and alizarin red staining was performed. Tooth slice organ cultures were performed, and VEGF, IL-6, and IL8 levels in their supernatants were measured by immunoassays. Pulp tissue vitality and morphology were assessed by MTT assay and histology. RESULTS In monolayer cultures of DPCs, L-MIM at nontoxic concentrations increased the production of VEGF and IL-8 in the presence of AGEs. Stimulation with L-MIM decreased alkaline phosphatase levels and matrix mineralization also in the presence of AGEs, whereas no significant changes in dentin matrix protein 1 and dentin sialophosphoprotein expression were observed. In tooth slice organ cultures, L-MIM increased VEGF but not IL-6 and IL-8 production in the presence of AGEs. The pulp tissue was vital, and no signs of apoptosis or necrosis were observed. CONCLUSIONS Overall, in the presence of AGEs, L-MIM increases the proangiogenic capacity, but decreases alkaline phosphatase expression and matrix mineralization.