Abstract Objective: This study evaluated the angiogenesis-enhancing potential of a tricalcium silicate-based mineral trioxide aggregate (ProRoot MTA), Biodentine, and a novel bioceramic root canal sealer (Well-Root ST) in human dental pulp stem cells (hDPSCs), human periodontal ligament stem cells (hPLSCs), and human tooth germ stem cells (hTGSCs). Methodology: Dulbecco's modified Eagle's medium was conditioned for 24 h by exposure to ProRoot MTA, Biodentine, or Well-Root ST specimens (prepared according to the manufacturers' instructions). The cells were cultured in these conditioned media and their viability was assessed with 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H tetrazolium (MTS) on days 1, 3, 7, 10, and 14. Angiogenic growth factors [platelet-derived growth factor (PDGF), basic ﬁbroblast growth factor (FGF-2), and vascular endothelial growth factor (VEGF)] were assayed by sandwich enzyme-linked immunosorbent assay (ELISA) on days 1, 7, and 14. Human umbilical vein endothelial cell (HUVEC) migration assays were used to evaluate the vascular effects of the tested materials at 6-8 h. Statistical analyses included Kruskal-Wallis, Mann-Whitney U, and Friedman and Wilcoxon signed rank tests. Results: None of tricalcium silicate-based materials were cytotoxic and all induced a similar release of angiogenic growth factors (PDGF, FGF-2, and VEGF) (p>0.05). The best cell viability was observed for hDPSCs (p<0.05) with all tricalcium silicate-based materials at day 14. Tube formation by HUVECs showed a significant increase with all tested materials (p<0.05). Conclusion: The tricalcium silicate-based materials showed potential for angiogenic stimulation of all stem cell types and significantly enhanced tube formation by HUVECs.
Subject(s)Humans , Root Canal Filling Materials/pharmacology , Stem Cells/drug effects , Ceramics/pharmacology , Silicates/pharmacology , Calcium Compounds/pharmacology , Angiogenesis Inducing Agents/pharmacology , Periodontal Ligament/cytology , Periodontal Ligament/drug effects , Tooth Germ/cytology , Tooth Germ/drug effects , Biocompatible Materials/pharmacology , Materials Testing , Platelet-Derived Growth Factor/analysis , Platelet-Derived Growth Factor/drug effects , Enzyme-Linked Immunosorbent Assay , Cell Survival/drug effects , Reproducibility of Results , Fibroblast Growth Factor 2/analysis , Fibroblast Growth Factor 2/drug effects , Statistics, Nonparametric , Neovascularization, Physiologic/drug effects , Dental Pulp/cytology , Dental Pulp/drug effects , Vascular Endothelial Growth Factor A/analysis , Vascular Endothelial Growth Factor A/drug effects , Human Umbilical Vein Endothelial Cells/drug effects , Flow Cytometry
OBJECTIVE: The aim of this study was to compare the cytotoxic effects of endodontic cements on human tooth germ stem cells (hTGSCs). MTA Fillapex, a mineral trioxide aggregate (MTA)-based, salicylate resin containing root canal sealer, was compared with iRoot SP, a bioceramic sealer, and AH Plus Jet, an epoxy resin-based root canal sealer. MATERIAL AND METHODS: To evaluate cytotoxicity, all materials were packed into Teflon rings (4 mmµ3 mm) and co-cultured with hTGSCs with the aid of 24-well Transwell permeable supports, which had a pore size of 0.4 µm. Coverslips were coated with MTA Fillapex, iRoot SP and AH Plus Jet and each coverslip was placed onto the bottom of one well of a six-well plate for scanning electron microscopy (SEM) analysis. Before the cytotoxicity and SEM analysis, all samples were stored at 37ºC and at 95% humidity and 5% CO2 for 24 hours to set. The cellular viability was analyzed using MTS test (3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium). The cytotoxic effects and SEM visualization of the tested materials were analyzed at 24-hour, 72-hour, one-week and two-week periods. RESULTS: On the 1st day, only MTA Fillapex caused cytotoxicity compared to negative control (NC) group (p<0.008). No significant difference was observed between the other tested materials at this period (p>0.05). After 14 days of incubation with the test materials, MTA Fillapex exhibited significantly higher cytotoxicity compared with iRoot SP, AH Plus Jet and the NC group (P<0.008). In the SEM analysis, the highest levels of cell attachment were observed for iRoot SP and the control group. After 24 hours, MTA Fillapex reduced the number of cells attached to the surface. CONCLUSIONS: Within the limitations of this study, sealers exerted different cytotoxic effects on hTGSCs. Although all materials ...
Subject(s)Humans , Calcium Compounds/toxicity , Dental Cements/toxicity , Silicates/toxicity , Stem Cells/drug effects , Tooth Germ/cytology , Aluminum Compounds/toxicity , Biocompatible Materials/toxicity , Cells, Cultured , Cell Survival/drug effects , Drug Combinations , Epoxy Resins/toxicity , Materials Testing , Microscopy, Electron, Scanning , Oxides/toxicity , Root Canal Filling Materials/toxicity , Statistics, Nonparametric , Surface Properties/drug effects , Time Factors
The Notch signaling pathway is an evolutionary conserved mechanism that plays an important role in cell-cell communication and cell fate in a wide range of tissues. The mammalian family of Notch receptors consists of 4 members: Notch1/2/3/4. The Notch ligand family consists of 5 members: Delta1/3/4 and Jagged1/2. Math1 encodes a murine Basic helix-loop-helix (bHLH) transcription factor that acts as positive regulator of cell differentiation. Recently, links between Notch and Math1 pathways were demonstrated in various tissues. Expression of Notch1, Jagged2 and Math1 were analyzed in the mouse molar tooth germ during embryonic stage (E) 13 and E15 and during postnatal stage (PN) 1, PN3, PN5, PN10 and PN14 by using in situ hybridization. Positive Notch1 expression was found at the tooth bud during embryonic stages, but its expression was absent from the basal cells in contact with the dental mesenchyme. Jagged2 and Math1 were strongly expressed in differentiated ameloblasts and odontoblasts and Math1 strong expression was even maintained until PN14 stage. Math1 showed the strongest expression. Our results suggest that the Notch1 signaling pathway through Jagged2 could be importantly related to Math1, directing the process of odontogenesis toward cell differentiation.
Subject(s)Animals , Rats , Basic Helix-Loop-Helix Transcription Factors , Gene Expression Regulation, Developmental , Tooth Germ/cytology , Tooth Germ/physiology , Mice, Inbred BALB C , Membrane Proteins/genetics , Membrane Proteins/metabolism , Signal Transduction/physiology , Molar/anatomy & histology , Molar/physiology , Odontogenesis/physiology , Receptor, Notch1/genetics , Receptor, Notch1/metabolism
A comparative nonisotopic in situ hybridization (ISH) analysis was carried out for the detection of Bmp-4, Shh and Wnt-5a transcripts during mice odontogenesis from initiation to cap stage. Bmp-4 was expressed early in the epithelium and then in the underlying mesenchyme. Shh expression was seen in the odontogenic epithelial lining thickening, being stronger in the enamel knot area, during the cap stage. Wnt-5a transcripts were expressed only in the mesenchyme during the initiation, bud and cap stages, with strong expression in the dental mesenchyme during the bud stage. The present results showed that Bmp-4, Shh and Wnt-5a are expressed since the very early stages of tooth development, and they suggest that the Wnt-5a gene is expressed in different cell populations than Bmp-4 and Shh.
No presente trabalho, realizou-se uma análise comparativa não isotópica por hibridização in situ a fim de se detectar a presença de transcritos de Bmp-4, Shh e Wnt-5a durante as fases iniciais da odontogênese em camundongos, desde a iniciação até o estágio de capuz. No estágio de iniciação, observou-se expressão precoce de Bmp-4 no epitélio e no mesênquima subjacente, enquanto que a expressão de Shh ocorreu durante o estágio de capuz, na região de espessamento do revestimento epitelial odontogênico, tornando-se mais intensa na área de nó do esmalte. Os transcritos de Wnt-5a foram expressos somente no mesênquima durante os estágios de iniciação, botão e capuz, com intenso sinal na região no mesênquima na fase de botão. Estes resultados mostraram que Bmp-4, Shh e Wnt-5a são expressos desde os estágios mais precoces do desenvolvimento dentário, sugerindo que o gene Wnt-5a seja expresso em populações celulares distintas daquelas que expressam Bmp-4 e Shh.