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Article in Chinese | WPRIM | ID: wpr-879535


OBJECTIVE@#To compare the mRNA level of cell proliferation-related genes Twist1, SIRT1, FGF2 and TGF-β3 in placenta mesenchymal stem cells (PA-MSCs), umbilical cord mensenchymals (UC-MSCs) and dental pulp mesenchymal stem cells (DP-MSCs).@*METHODS@#The morphology of various passages of PA-MSCs, UC-MSCs and DP-MSCs were observed by microscopy. Proliferation and promoting ability of the three cell lines were detected with the MTT method. Real-time PCR (RT-PCR) was used to determine the mRNA levels of Twist1, SIRT1, FGF2, TGF-β3.@*RESULTS@#The morphology of UC-MSCs and DP-MSCs was different from that of PA-MSCs. Proliferation ability and promoting ability of the PA-MSCs was superior to that of UC-MSCs and DP-MSCs. In PA-MSCs, expression level of Twist1 and TGF-β3 was the highest and FGF2 was the lowest. SIRT1 was highly expressed in UC-MSCs. With the cell subcultured, different expression levels of Twist1, SIRT1, FGF2, TGF-β3 was observed in PA-MSCs, UC-MSCs and DP-MSCs.@*CONCLUSION@#Up-regulated expression of the Twist1, SIRT1 and TGF-β3 genes can promote proliferation of PA-MSCs, UC-MSCs and DP-MSCs, whilst TGF-β3 may inhibit these. The regulatory effect of Twist1, SIRT1, FGF2 and TGF-β3 genes on PA-MSCs, UC-MSCs and DP-MSCs are different.

Cell Differentiation , Cell Proliferation/genetics , Cells, Cultured , Dental Pulp/cytology , Female , Fibroblast Growth Factor 2/genetics , Humans , Mesenchymal Stem Cells/cytology , Nuclear Proteins/genetics , Placenta/cytology , Pregnancy , Sirtuin 1/genetics , Transforming Growth Factor beta3/genetics , Twist-Related Protein 1/genetics , Umbilical Cord/cytology
J. appl. oral sci ; 28: e20190105, 2020. tab, graf
Article in English | LILACS | ID: biblio-1056578


Abstract Calcium aluminate cement (CAC) has been highlighted as a promising alternative for endodontic use aiming at periapical tissue repair. However, its effects on dental pulp cells have been poorly explored. Objective: This study assessed the impact of calcium chloride (CaCl2) and bismuth oxide (Bi2O3) or zinc oxide (ZnO) additives on odontoblast cell response to CAC. Methodology: MDPC-23 cells were exposed for up to 14 d: 1) CAC with 2.8% CaCl2 and 25% ZnO (CACz); 2) CAC with 2.8% CaCl2 and 25% Bi2O3 (CACb); 3) CAC with 10% CaCl2 and 25% Bi2O3 (CACb+); or 4) mineral trioxide aggregate (MTA), placed on inserts. Non-exposed cultures served as control. Cell morphology, cell viability, gene expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), and dentin matrix protein 1 (DMP-1), ALP activity, and extracellular matrix mineralization were evaluated. Data were compared using ANOVA (α=5%). Results: Lower cell density was detected only for MTA and CACb+ compared with Control, with areas showing reduced cell spreading. Cell viability was similar among groups at days one and three (p>0.05). CACb+ and MTA showed the lowest cell viability values at day seven (p>0.05). CACb and CACb+ promoted higher ALP and BSP expression compared with CACz (p<0.05); despite that, all cements supported ALP activity. Matrix mineralization were enhanced in CACb+ and MTA. Conclusion: In conclusion, CAC with Bi2O3, but not with ZnO, supported the expression of odontoblastic phenotype, but only the composition with 10% CaCl2 promoted mineralized matrix formation, rendering it suitable for dentin-pulp complex repair.

Humans , Mice , Calcium Compounds/pharmacology , Calcium Compounds/chemistry , Aluminum Compounds/pharmacology , Aluminum Compounds/chemistry , Dental Cements/pharmacology , Dental Cements/chemistry , Dental Pulp/cytology , Dental Pulp/drug effects , Oxides/pharmacology , Oxides/chemistry , Time Factors , Zinc Oxide/pharmacology , Zinc Oxide/chemistry , Bismuth/pharmacology , Bismuth/chemistry , Materials Testing , Calcium Chloride/pharmacology , Calcium Chloride/chemistry , Gene Expression/drug effects , Cell Survival/drug effects , Cells, Cultured , Reproducibility of Results , Silicates/pharmacology , Silicates/chemistry , Drug Combinations , Alkaline Phosphatase/analysis , Alkaline Phosphatase/drug effects , Odontoblasts/drug effects
J. appl. oral sci ; 28: e20190215, 2020. graf
Article in English | LILACS, BBO | ID: biblio-1056582


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.

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
Arq. bras. oftalmol ; 82(1): 32-37, Jan.-Feb. 2019. graf
Article in English | LILACS | ID: biblio-973879


ABSTRACT Purpose: To evaluate the ability of human immature dental pulp stem cells, which are mesenchymal stem cells of neural crest origin, to differentiate into the corneal epithelium for purposes of corneal transplantation and tissue engineering when cultured on de-epithelized amniotic membranes. Methods: We compared the immunophenotypes (ABCG2, K3/12, and vimentin) of cells grown on amniotic membranes or plastic surfaces under serum-free conditions or in culture media containing serum or serum replacement components. Results: Immature dental pulp stem cells grown on amniotic membranes under basal conditions are able to maintain their undifferentiated state. Our data also suggest that the culture medium used in the present work can modulate the expression of immature dental pulp stem cell markers, thus inducing epithelial differentiation of these cells in vitro. Conclusions: Our results suggest that the amniotic membrane is a good choice for the growth and transplantation of mesenchymal stem cells, particularly immature dental pulp stem cells, in clinical ocular surface reconstruction.

RESUMO Objetivos: Avaliar a capacidade das células-tronco imaturas da polpa do dente de leite que são células-tronco mesenquimais de origem da crista neural, de se diferenciarem no epitélio corneano para fins de transplante de córnea e engenharia de tecidos quando cultivadas em membrana amnióticas desepitelizadas. Métodos: Foram comparamos so imunofenótipo (ABCG2, CK3/12 e vimentina) de células cultivadas em membranas amnióticas ou em superfícies plásticas sob condições livres de soro ou em meios de cultura contendo soro ou componentes de substituição de soro. Resultados: Células-tronco imaturas da polpa do dente de leite cultivadas sobre membrana amniótica em condições basais são capazes de manter seu estado indiferenciado. Nossos dados também sugerem que o meio de cultura utilizado no presente trabalho pode modular a expressão de marcadores de células-tronco imaturas da polpa do dente de leite, induzindo a diferenciação epitelial destas células in vitro. Conclusão: Nossos resultados sugerem que a membrana amniótica é uma boa escolha para o crescimento e transplante de células-tronco mesenquimais, particularmente as células-tronco imaturas da polpa do dente de leite, na reconstrução da superfície ocular.

Humans , Epithelium, Corneal/transplantation , Dental Pulp/cytology , Mesenchymal Stem Cell Transplantation/methods , Tissue Scaffolds , Mesenchymal Stem Cells/cytology , Amnion , Time Factors , Cells, Cultured , Reproducibility of Results , Fluorescent Antibody Technique , Cell Culture Techniques/methods , Corneal Diseases/surgery , Cell Proliferation
Braz. oral res. (Online) ; 33: e059, 2019. graf
Article in English | LILACS | ID: biblio-1039303


Abstract We recently demonstrated that a co-culture system of human umbilical vein endothelial cells (HUVECs) and human dental pulp stem cells (hDPSCs) could enhance angiogenesis ability in vitro. However, whether tumor necrosis factor α (TNF-α) could promote blood vessel formation during pulp regeneration remained unknown. The aim of this study was to investigate the effects of TNF-α on the formation of endothelial tubules and vascular networks in a co-culture system of hDPSCs and HUVECs. hDPSCs were co-cultured with HUVECs at a ratio of 1:5. The Matrigel assay was performed to detect the total tubule branching lengths and numbers of branches, and the Cell-Counting Kit 8 assay was performed to examine the effect of TNF-α on cell proliferation. Real-time polymerase chain reactions and western blot were used to detect vascular endothelial growth factor (VEGF) mRNA and protein expression. The Matrigel assay showed significantly greater total branching lengths and numbers of branches formed in the experimental groups treated with different concentrations of TNF-α compared with the control group. The decomposition times of the tubule structures were also significantly prolonged (P < 0.05). Treatment with 50 ng/ml TNF-α did not significantly change the proliferation of co-cultured cells, but it significantly increased the VEGF mRNA and protein expression levels (p < 0.05). In addition, the migration abilities of HUVECs and hDPSCs increased after co-culture with TNF-α (p < 0.05). TNF-α enhanced angiogenic ability in vitro in the co-culture system of hDPSCs and HUVECs.

Humans , Adolescent , Adult , Young Adult , Tumor Necrosis Factor-alpha/pharmacology , Neovascularization, Physiologic/drug effects , Dental Pulp/cytology , Dental Pulp/drug effects , Angiogenesis Inducing Agents/pharmacology , Human Umbilical Vein Endothelial Cells/drug effects , Proteoglycans , Reference Values , Time Factors , Cell Count , Cells, Cultured , Blotting, Western , Reproducibility of Results , Collagen , Laminin , Neovascularization, Physiologic/physiology , Dental Pulp/physiology , Vascular Endothelial Growth Factor A/analysis , Vascular Endothelial Growth Factor A/drug effects , Cell Proliferation/drug effects , Cell Proliferation/physiology , Drug Combinations , Cell Migration Assays , Human Umbilical Vein Endothelial Cells/physiology , Real-Time Polymerase Chain Reaction
Braz. oral res. (Online) ; 33: e013, 2019. graf
Article in English | LILACS | ID: biblio-989479


Abstract Recent studies on functional tissue regeneration have focused on substances that favor cell proliferation and differentiation, including the bioactive phenolic compounds present in grape seed extract (GSE). The aim of this investigation was to evaluate the stimulatory potential of GSE in the functional activity of undifferentiated pulp cells and odontoblast-like cells. OD-21 and MDPC-23 cell lines were cultivated in odontogenic medium until subconfluence, seeded in 24-well culture plates in a concentration of 2x104/well and divided into: 1) OD-21 without GSE; 2) OD-21+10 µg/mL of GSE; 3) MDPC-23 without GSE; 4) MDPC-23+10 µg/mL of GSE. Cell proliferation, in situ detection of alkaline phosphatase (ALP) and total protein content were assessed after 3, 7 and 10 days, and mineralization was evaluated after 14 days. The data were analyzed by ANOVA statistical tests set at a 5% level of significance. Results revealed that cell proliferation increased after 10 days, and protein content, after 7 days of culture in MDPC-23 cells. In situ ALP staining intensity was higher in undifferentiated pulp cells and odontoblast-like cells after 7 and 10 days, respectively. A discrete increase in MDPC-23 mineralization after GSE treatment was observed despite OD-21 cells presenting a decrease in mineralized nodule deposits. Data suggest that GSE favors functional activity of differentiated cells more broadly than undifferentiated cells (OD-21). More studies with different concentrations of GSE must be conducted to confirm its benefits to cells regarding dentin regeneration.

Animals , Mice , Dental Pulp/cytology , Dental Pulp/drug effects , Cell Proliferation/drug effects , Grape Seed Extract/pharmacology , Odontoblasts/drug effects , Reference Values , Time Factors , Cell Differentiation/drug effects , Cell Line , Cells, Cultured , Reproducibility of Results , Dentin/cytology , Dentin/drug effects , Odontogenesis/drug effects
Braz. oral res. (Online) ; 33: e084, 2019. graf
Article in English | LILACS | ID: biblio-1019612


Abstract This study aimed to evaluate the role of photobiomodulation (PBM) in apexification and apexogenesis of necrotic rat molars with an open apex. Rat molars were exposed to the oral environment for 3 weeks. Canals were rinsed with 2.5% NaOCl and 17% EDTA, filled with antibiotic paste and sealed. After 7 days, canals were rinsed and divided into six groups (n=6): mineral trioxide aggregate (MTA); blood clot (BC); human dental pulp stem cells (hDPSC); MTA+PBM; BC+PBM; and hDPSC+PBM. In hDPSC groups, a 1% agarose gel scaffold was used. Two groups were not exposed: healthy tooth+PBM (n = 6), healthy tooth (n = 3); and one was exposed throughout the experiment: necrotic tooth (n = 3). In PBM groups, irradiation was performed with aluminum gallium indium phosphide (InGaAlP) diode laser for 30 days within 24-h intervals. After that, the specimens were processed for histological and immunohistochemical analyses. Necrotic tooth showed greater neutrophil infiltrate (p < 0.05). Necrotic tooth, healthy tooth, and healthy tooth+PBM groups showed absence of a thin layer of fibrous condensation in the periapical area. All the other groups stimulated the formation of a thicker layer of fibers (p < 0.05). All groups formed more mineralized tissue than necrotic tooth (p < 0.05). PBM associated with MTA, BC, or hDPSC formed more mineralized tissue (p < 0.05). MTA+PBM induced apexification (p < 0.05). Rabbit polyclonal anti-bone sialoprotein (BSP) antibody confirmed the histological findings of mineralized tissue formation, and hDPSC groups exhibited higher percentage of BSP-positive cells. It can be concluded that PBM improved apexification and favored apexogenesis in necrotic rat molars with an open apex.

Animals , Tooth Diseases/radiotherapy , Dental Pulp Necrosis/radiotherapy , Tooth Apex/radiation effects , Low-Level Light Therapy/methods , Dental Pulp Cavity/radiation effects , Lasers, Semiconductor/therapeutic use , Apexification/methods , Oxides/therapeutic use , Stem Cells , Tooth Diseases/pathology , Immunohistochemistry , Random Allocation , Reproducibility of Results , Treatment Outcome , Rats, Wistar , Silicates/therapeutic use , Calcium Compounds/therapeutic use , Aluminum Compounds/therapeutic use , Dental Pulp Necrosis/pathology , Tooth Apex/pathology , Dental Pulp/cytology , Dental Pulp Cavity/pathology , Drug Combinations , Integrin-Binding Sialoprotein/analysis
Braz. oral res. (Online) ; 33: e117, 2019. tab, graf
Article in English | LILACS, BBO | ID: biblio-1132651


Abstract: The aim of this study was to evaluate the effect of mineral trioxide aggregate (MTA) and Brazilian propolis on the cell viability, mineralization, anti-inflammatory ability, and migration of human dental pulp cells (hDPCs). The cell viability was evaluated with CCK-8 kit after 1, 5, 7, and 9 days. The deposition of calcified matrix and the expression of osteogenesis-related genes were evaluated by Alizarin Red staining and real-time PCR after incubation in osteogenic medium for 21 days. The expression of inflammation-related genes in cells was determined after exposure to 1 μg/mL LPS for 3 h. Finally, the numbers of cells that migrated through the permeable membranes were compared during 15 h. Propolis and MTA significantly increased the viability of hDPCscompared to the control group on days 7 and 9. In the propolis group, significant enhancement of osteogenic potential and suppressed expression of IL-1β and IL-6 was observed after LPS exposure compared to the MTA and control groups. The number of migration cells in the propolis group was similar to that of the control group, while MTA significantly promoted cell migration. Propolis showed comparable cell viability to that of MTA and exhibited significantly higher anti-inflammatory and mineralization promotion effects on hDPCs.

Humans , Oxides/pharmacology , Propolis/pharmacology , Silicates/pharmacology , Calcium Compounds/pharmacology , Aluminum Compounds/pharmacology , Dental Pulp/cytology , Dental Pulp/drug effects , Anti-Inflammatory Agents/pharmacology , Brazil , Cell Movement/drug effects , Cell Survival/drug effects , Cells, Cultured , Reproducibility of Results , Anthraquinones , Interleukin-6/analysis , Tumor Necrosis Factor-alpha , Statistics, Nonparametric , Drug Combinations , Interleukin-1beta/analysis , Real-Time Polymerase Chain Reaction , Odontoblasts/drug effects
J. appl. oral sci ; 27: e20180103, 2019. graf
Article in English | LILACS, BBO | ID: biblio-1002400


Abstract Objective This study aimed to evaluate the inflammatory effect and bone formation in sterile surgical failures after implantation of a collagen sponge with mesenchymal stem cells from human dental pulp (hDPSCs) and Aloe vera. Material and Methods Rattus norvegicus (n=75) were divided into five experimental groups according to treatment: G1) control (blood clot); G2) Hemospon®; G3) Hemospon® in a culture medium enriched with 8% Aloe vera; G4) Hemospon® in a culture medium containing hDPSCs and G5) Hemospon® in a culture medium enriched with 8% Aloe vera and hDPSCs. On days 7, 15 and 30, the animals were euthanized, and the tibia was dissected for histological, immunohistochemistry and immunofluorescence analyses. The results were analyzed using nonparametric Kruskal-Wallis test and Dunn's post-test. Results On days 7 and 15, the groups with Aloe vera had less average acute inflammatory infiltrate compared to the control group and the group with Hemospon® (p<0.05). No statistically significant difference was found between the groups regarding bone formation at the three experimental points in time. Osteopontin expression corroborated the intensity of bone formation. Fluorescence microscopy revealed positive labeling with Q-Tracker® in hDPSCs before transplantation and tissue repair. Conclusion The results suggest that the combination of Hemospon®, Aloe vera and hDPSCs is a form of clinical treatment for the repair of non-critical bone defects that reduces the inflammatory cascade's effects.

Humans , Animals , Male , Rats , Bone Regeneration/drug effects , Bone Regeneration/physiology , Plant Extracts/pharmacology , Dental Pulp/cytology , Mesenchymal Stem Cell Transplantation/methods , Aloe/chemistry , Osteogenesis/drug effects , Osteogenesis/physiology , Tibia/drug effects , Tibia/physiology , Tibia/pathology , Time Factors , Immunohistochemistry , Hemostatics/pharmacology , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Reproducibility of Results , Collagen/pharmacology , Treatment Outcome , Osteopontin/analysis , Flow Cytometry , Microscopy, Fluorescence
Braz. dent. j ; 29(5): 419-426, Sept.-Oct. 2018. graf
Article in English | LILACS | ID: biblio-974185


Abstract This study evaluated in vitro cell viability and metabolism, nitric oxide release and production of chemokines by cultured human dental pulp fibroblasts (DPF) under contact with HEMA and Single Bond. Cultures of DPF were established by means of an explant technique. Once plated, cells were kept under contact with increasing concentrations of HEMA (10, 100 and 1000 nM) or Single Bond (SB) [10-fold serially diluted in culture medium (10-4, 10-3 and 10-2 v/v)] and also with polymerized SB components. Cytotoxicity was assessed by Trypan Blue exclusion method and MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Nitric oxide release on cell supernatant was detected by Griess Method whereas chemokines (CXCL12 and CXCL8) were detected by ELISA. RT-qPCR was employed for chemokines gene expression analysis. Cytotoxic tests showed significant differences for SB 10-2. None of the tested materials significantly altered NO levels. Protein levels of CXCL12 were significantly decreased only by HEMA. On the other hand, while CXCL12 mRNA remained unaltered, gene expression of CXCL8 had significant decrease with all materials, except for polymerized SB. In conclusion, Single Bond and HEMA at various concentrations, decreased expression and production of molecules involved in inflammatory processes and, therefore, the use of adhesive systems such as pulp capping materials must be viewed with caution due to its large cytotoxic effect when in close contact with the pulp.

Resumo Este estudo avaliou in vitro a viabilidade e metabolismo celular, liberação de óxido nítrico e produção de quimiocinas em cultura de fibroblastos de polpa dental humana (DPF) em contato com HEMA e Single Bond. Culturas de DPF foram estabelecidas por meio de uma técnica de explante. Uma vez plaqueadas, as células foram mantidas em contato com concentrações crescentes de HEMA (10, 100 e 1000 nM) ou Single Bond (SB) [10 vezes diluídas em série em meio de cultura (10-4, 10-3 e 10-2 v/v)] e também com SB polimerizado. A citotoxicidade foi avaliada pelo método de exclusão de Trypan Blue e pelo ensaio de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio brometo (MTT). A liberação de óxido nítrico no sobrenadante celular foi detectada pelo método de Griess, enquanto as quimiocinas (CXCL12 e CXCL8) foram detectadas por ELISA. RT-qPCR foi empregada para análise de expressão gênica de quimiocinas. Testes citotóxicos mostraram diferenças significativas para SB 10-2. Nenhum dos materiais testados alterou significativamente os níveis de NO. Os níveis de proteína de CXCL12 foram significativamente diminuídos apenas pelo HEMA. Por outro lado, enquanto o RNAm de CXCL12 permaneceu inalterado, a expressão gênica de CXCL8 teve redução significativa com todos os materiais, com exceção do SB polimerizado. Em conclusão, Single Bond e HEMA, em várias concentrações, diminuíram a expressão e produção de moléculas envolvidas em processos inflamatórios e, portanto, o uso de sistemas adesivos, como o material protetor da polpa, deve ser visto com cautela devido ao seu grande efeito citotóxico quando em contato com a polpa.

Humans , Bisphenol A-Glycidyl Methacrylate/pharmacology , Dental Pulp/cytology , Fibroblasts/drug effects , Methacrylates/pharmacology , In Vitro Techniques , Materials Testing , Cell Survival , Cells, Cultured , Polymerase Chain Reaction , Chemokines/metabolism , Nitric Oxide/metabolism
Braz. dent. j ; 29(5): 427-434, Sept.-Oct. 2018. graf
Article in English | LILACS | ID: biblio-974184


Abstract Recently, human natal dental pulp stem cells (hNDP-SCs) have been characterized in vitro and it has been shown that they satisfy criteria defining human mesenchymal stromal cells (MSCs), as proposed by the International Society for Cellular Therapy. However, these results were reached in the presence of xenogeneic expansion medium, which has the potential to alter the cells' functional capacity. To determine the validity of the previously reported hNDP-SCs characteristics for human cell therapy, we have cultured hNDP-SCs in allogeneic expansion medium. Two hNDP-SC lineages were isolated from vital natal teeth, donated by a healthy newborn female and cultured in 2% platelet rich plasma (PRP). Analysis of the phenotypic expressions, proliferation rates, viability, telomerase length and in vitro adipogenic, osteogenic and chondrogenic differentiation potentials of two hNDP-SCs lineages (Zn001 and Zn002) were performed. Both lineages displayed similar morphology, proliferation rates, adipogenic, chondrogenic and osteogenic differentiation potential. Telomere shortening by 41.0% and 13.49% occurred from 3rd till 14th passage for lineages Zn001 and Zn002 respectively. Viability of both lineages was higher than 90%. Flow cytometry demonstrated that both lineages were positive to the majority of tested markers, including markers, which were negatively, expressed when hNDP-SCs were cultured previously in xenogeneic medium. Using immune-cytochemistry the cells were shown to express beta III-tubulin, nestin, neurofilaments and Nanog. PRP used as allogeneic medium is suitable for cultivation of hNDP-SCs.

Resumo Recentemente, células-tronco da polpa dental humana (hNDP-SCs) foram caracterizadas in vitro e foi demonstrado que elas satisfazem critérios que definem células mesenquimais do estroma humana (MSCs), tal como proposto pela Sociedade Internacional para Terapia Celular. No entanto, esses resultados foram alcançados na presença de meio de expansão xenogênico, que tem o potencial de alterar a capacidade funcional das células. Para determinar a validade das características das hNDP-SCs anteriormente relatadas para a terapia celular humana, cultivamos hNDP-SCs em meio de expansão alogênico. Duas linhagens hNDP-SC foram isoladas de dentes natais vitais, doadas por uma recém-nascida saudável, e cultivadas em plasma rico em plaquetas a 2% (PRP). Análises das expressões fenotípicas, taxas de proliferação, viabilidade, comprimento de telomerase e potenciais de diferenciação adipogênica, osteogênica e condrogênica in vitro das duas linhagens hNDP-SC (Zn001 e Zn002) foram realizadas. Ambas as linhagens apresentaram morfologia, taxas de proliferação, potencial de diferenciação adipogênico, condrogênico e osteogênico semelhantes. O encurtamento dos telômeros em 41,0% e 13,49% ocorreu da 3ª até a 14ª passagem para as linhagens Zn001 e Zn002, respectivamente. A viabilidade de ambas as linhagens foi superior a 90%. A citometria de fluxo demonstrou que ambas as linhagens foram positivas para a maioria dos marcadores testados, incluindo marcadores, que foram negativamente expressados quando hNDP-SCs foram previamente cultivadas em meio xenogênico. Usando análise imunocitoquímica, as células mostraram expressar a beta III-tubulina, nestina, neurofilamentos e Nanog. O PRP usado como meio alogênico mostrou-se adequado para o cultivo de hNDP-SCs.

Humans , Female , Infant, Newborn , Stem Cells/cytology , Cell Culture Techniques/methods , Dental Pulp/cytology , Natal Teeth/cytology , Phenotype , Cell Differentiation , Cell Survival , Cells, Cultured , Cell Proliferation , Platelet-Rich Plasma , Telomere Shortening
Braz. j. med. biol. res ; 51(5): e6754, 2018. tab, graf
Article in English | LILACS | ID: biblio-889074


Cell adhesion in three-dimensional scaffolds plays a key role in tissue development. However, stem cell behavior in electrospun scaffolds under perfusion is not fully understood. Thus, an investigation was made on the effect of flow rate and shear stress, adhesion time, and seeding density under direct perfusion in polycaprolactone electrospun scaffolds on human dental pulp stem cell detachment. Polycaprolactone scaffolds were electrospun using a solvent mixture of chloroform and methanol. The viable cell number was determined at each tested condition. Cell morphology was analyzed by confocal microscopy after various incubation times for static cell adhesion with a high seeding density. Scanning electron microscopy images were obtained before and after perfusion for the highest flow rate tested. The wall pore shear stress was calculated for all tested flow rates (0.005-3 mL/min). An inversely proportional relationship between adhesion time with cell detachment under perfusion was observed. Lower flow rates and lower seeding densities reduced the drag of cells by shear stress. However, there was an operational limit for the lowest flow rate that can be used without compromising cell viability, indicating that a flow rate of 0.05 mL/min might be more suitable for the tested cell culture in electrospun scaffolds under direct perfusion.

Humans , Dental Pulp/cytology , Perfusion , Polyesters , Stem Cells/cytology , Tissue Scaffolds , Cell Adhesion , Cell Culture Techniques
Braz. oral res. (Online) ; 32: e003, 2018. graf
Article in English | LILACS | ID: biblio-889476


Abstract The aim of this study was to investigate the viability of human dental pulp cells from extracted teeth kept at standard room temperature and atmospheric pressure for different periods of time. Twenty-one healthy permanent teeth were used. They were divided into five groups according to the expected time from extraction to processing. One group was tested immediately after extraction; the other groups were each tested at one of the following time points: 30 minutes, 1 hour, 2 hours, and 5 hours post-extraction. Cell morphology was analysed by light microscopy; cell proliferation was analysed using MTT assay and by counting the viable cells in a haemocytometer. Similar results were observed in all groups (p < 0.05). A delay of up to five hours for tooth processing and tissue collection does not preclude the establishment of dental pulp cell cultures, affect the morphology of these cells, or reduce their proliferative potential.

Humans , Cell Culture Techniques/methods , Dental Pulp/cytology , Tooth Extraction , Analysis of Variance , Cell Count , Cell Proliferation , Cell Survival , Cells, Cultured , Culture Media , Reproducibility of Results , Tetrazolium Salts , Thiazoles , Time Factors , Tissue Preservation/methods
Braz. oral res. (Online) ; 32: e48, 2018. tab, graf
Article in English | LILACS | ID: biblio-952159


Abstract The aim was to investigate the angiogenic effects of concentrated growth factors on human dental pulp cells and human umbilical vein endothelial cells. Cells were treated with concentrated growth factor extracts. The CCK-8 assay and cell cycle assay were conducted to evaluate cell growth. Cell migration was evaluated by the Transwell migration assay. Angiogenesis-associated mRNA and protein expression levels were determined using quantitative real-time PCR and Western blotting, respectively. A tube formation assay was conducted to evaluate the angiogenic capacity in vitro. The data showed that compared with the control, concentrated growth factor extracts significantly promoted dental pulp cell proliferation and differentiation and endothelial cell proliferation and migration in a dose-dependent manner (p < 0.05). Concentrated growth factor extracts also promoted the tube-like structure formation of endothelial cells in vitro. The RT-PCR and Western blot results showed that concentrated growth factor extracts upregulated the expression of angiogenesis-related genes - chemokine receptor-4, platelet-derived growth factor, and vascular endothelial growth factor - in dental pulp cells. In conclusion, concentrated growth factors showed proangiogenic effects on dental pulp cells and endothelial cells and have good application potential for dental pulp revascularization.

Humans , Male , Adult , Neovascularization, Physiologic/physiology , Intercellular Signaling Peptides and Proteins/physiology , Dental Pulp/cytology , Human Umbilical Vein Endothelial Cells/physiology , Reference Values , Time Factors , Platelet-Derived Growth Factor/analysis , Platelet-Derived Growth Factor/physiology , Cell Cycle/physiology , Cells, Cultured , Blotting, Western , Reproducibility of Results , Analysis of Variance , Receptors, CXCR4/analysis , Receptors, CXCR4/physiology , Intercellular Signaling Peptides and Proteins/analysis , Vascular Endothelial Growth Factor A/analysis , Vascular Endothelial Growth Factor A/physiology , Cell Proliferation/physiology , Cell Migration Assays , Real-Time Polymerase Chain Reaction
J. appl. oral sci ; 25(6): 631-640, Nov.-Dec. 2017. tab, graf
Article in English | LILACS, BBO | ID: biblio-893662


Abstract Objectives: The primary purpose of this study was to examine the effects of triethylene glycol dimethacrylate (TEGDMA) on odontoclastic differentiation in the dental pulp tissue. Material and Methods: The effects of different TEGDMA dosages on the odontoclastic differentiation capability of dental pulp cells were analyzed in vitro using the following methodologies: i) flow cytometry and tartrate-resistant acid phosphatase (TRAP) staining; ii) apoptotic effects using Annexin V staining; iii) mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor (NF)-kB ligand (RANKL) genes by quantitative Real-time PCR (qRT-PCR); and iv) OPG and RANKL protein expression by enzyme-linked immunosorbent assay (ELISA). Results: TEGDMA caused relatively less odontoclastic differentiation in comparison with the control group; however, odontoclastic differentiation augmented with increasing doses of TEGDMA (p<0.05). The mRNA and protein expression of OPG was lower in TEGDMA treated pulp cells than in the control group (p<0.05). While the mRNA expression of RANKL remained unchanged compared to the control group (p>0.05), its protein expression was higher than the control group (p<0.05). In addition, TEGDMA increased the apoptosis of dental pulp cells dose dependently. Conclusions: TEGDMA reduced the odontoclastic differentiation ability of human dental pulp cells. However, odontoclastic differentiation ratios increased proportionally with the increasing dose of TEGDMA.

Humans , Polyethylene Glycols/pharmacology , Polymethacrylic Acids/pharmacology , Cell Differentiation/drug effects , Dental Pulp/drug effects , Tartrate-Resistant Acid Phosphatase/drug effects , Enzyme-Linked Immunosorbent Assay , Lipopolysaccharide Receptors/metabolism , Dental Pulp/cytology , RANK Ligand/metabolism , Real-Time Polymerase Chain Reaction , Flow Cytometry
J. appl. oral sci ; 25(5): 515-522, Sept.-Oct. 2017. graf
Article in English | LILACS, BBO | ID: biblio-893656


Abstract Hypersensitivity, local irritative and cytotoxic effects are known for the chemical components of Syzygium aromaticum and Cinnamomum zeylanicum contained in dental materials. However, there is no intimate data in dentistry using the whole extracts of these plants and introducing new ones. Salvia triloba is a well-known anti-inflammatory plant that correspondingly could be used in several dental traumas. Objectives: We aimed to show and compare the effect of S. aromaticum, C. zeylanicum, and S. triloba extracts on dental pulp stem cells (DPSCs) proliferation, differentiation, and immune responses. Material and Methods: Using xCELLigence, a real time monitoring system, we obtained a growth curve of DPSCs with different concentrations of the Extracts. A dose of 10 μg/mL was the most efficient concentration for vitality. Osteogenic differentiation and anti-inflammatory activities were determined by using an ELISA Kit to detect early and late markers of differentiation. Results: The level of osteonectin (ON, early osteogenic marker) decreased, which indicated that the osteogenic differentiation may be accelerated with addition of extracts. However, the level of osteocalcin (OCN, late osteogenic marker and sign of calcium granulation) differed among the extracts, in which S. aromaticum presented the highest value, followed by S. triloba and C. zeylanicum. Surprisingly, the determined calcium granules were reduced in S. aromaticum and S. triloba. In response to tumor necrosis factor alpha (TNF-α), S. triloba-treated DPSCs showed the most reduced level of IL-6 cytokine level. We suggest C. zeylanicum as a promising osteogenic inducer and S. triloba as a potent anti-inflammatory agent, which could be used safely in biocomposite or scaffold fabrications for dentistry. Conclusions: Because calcium granule formation and cell viability play a critical role in hard tissue formation, S. aromaticum in dentistry should be strictly controlled, and the mechanism leading to reduced calcium granule formation should be identified.

Humans , Adolescent , Young Adult , Drugs, Chinese Herbal/pharmacology , Plant Extracts/pharmacology , Cinnamomum zeylanicum/chemistry , Syzygium/chemistry , Dental Pulp/cytology , Mesenchymal Stem Cells/drug effects , Anti-Inflammatory Agents/pharmacology , Osteogenesis/drug effects , Time Factors , Enzyme-Linked Immunosorbent Assay , Antigens, Differentiation/analysis , Osteocalcin/analysis , Osteonectin/analysis , Cell Differentiation/drug effects , Cells, Cultured , Calcium/analysis , Reproducibility of Results , Analysis of Variance , Cytokines/analysis , Dental Pulp/drug effects , Cell Proliferation/drug effects , Flow Cytometry
Acta cir. bras ; 32(7): 540-549, July 2017. tab, graf
Article in English | LILACS | ID: biblio-886216


Abstract Purpose: To investigate the therapeutic potential of human immature dental pulp stem cells in the treatment of chronic spinal cord injury in dogs. Methods: Three dogs of different breeds with chronic SCI were presented as animal clinical cases. Human immature dental pulp stem cells were injected at three points into the spinal cord, and the animals were evaluated by limb function and magnetic resonance imaging (MRI) pre and post-operative. Results: There was significant improvement from the limb function evaluated by Olby Scale, though it was not supported by the imaging data provided by MRI and clinical sign and evaluation. Conclusion: Human dental pulp stem cell therapy presents promising clinical results in dogs with chronic spinal cord injuries, if used in association with physical therapy.

Humans , Animals , Dogs , Spinal Cord Injuries/veterinary , Stem Cell Transplantation/veterinary , Dental Pulp/cytology , Dog Diseases/therapy , Spinal Cord Injuries/therapy , Magnetic Resonance Imaging , Chronic Disease , Treatment Outcome , Recovery of Function , Stem Cell Transplantation/methods
Cell Journal [Yakhteh]. 2017; 18 (4): 609-618
in English | IMEMR | ID: emr-185786


Objective: Pulp and periodontal tissues are well-known sources of mesenchymal stem cells [MSCs] that provide a promising place in tissue engineering and regenerative medicine. The molecular mechanisms underlying commitment and differentiation of dental stem cells that originate from different dental tissues are not fully understood. In this study, we have compared the expression levels of pluripotency factors along with immunological and developmentally-related markers in the culture of human dental pulp stem cells [hDPSCs], human dental follicle stem cells [hDFSCs], and human embryonic stem cells [hESCs]

Materials and Methods: In this experimental study, isolated human dental stem cells were investigated using quantitative polymerase chain reaction [qPCR], immunostaining, and fluorescence-activated cell sorting [FACS]. Additionally, we conducted gene ontology [GO] analysis of differentially expressed genes and compared them between dental stem cells and pluripotent stem cells

Results: The results demonstrated that pluripotency [OCT4 and SOX2] and immunological [IL-6 and TLR4] factors had higher expressions in hDFSCs, with the exception of the JAGGED-1/NOTCH1 ratio, c-MYC and NESTIN which expressed more in hDPSCs. Immunostaining of OCT4, SOX2 and c-MYC showed cytoplasmic and nucleus localization in both groups at similar passages. GO analysis showed that the majority of hDFSCs and hDPSCs populations were in the synthesis [S] and mitosis [M] phases of the cell cycle, respectively

Conclusion: This study showed different status of heterogeneous hDPSCs and hDFSCs in terms of stemness, differentiation fate, and cell cycle phases. Therefore, the different behaviors of dental stem cells should be considered based on clinical treatment variations

Humans , In Vitro Techniques , Dental Pulp/cytology , Dental Sac/cytology , Stem Cell Niche , Humans , Genetic Heterogeneity
Braz. oral res. (Online) ; 31: e112, 2017. graf
Article in English | LILACS | ID: biblio-952076


Abstract: Cellular retinoic acid-binding protein 2 (CRABP2) has been detected in several organs during embryonic development. Recent studies have demonstrated that CRABP2 plays important roles in the retinoic acid, β-catenin and Notch signaling pathways, as well as in the interaction between epithelial and mesenchymal cells, which are important for human dental pulp stem cells (hDPSCs) and tooth development. In the present study, the expression of CRABP2 during mouse molar development and the role of CRABP2 in hDPSC odontoblastic differentiation were evaluated. CRABP2 was gradually decreased during the development of the first maxillary molar, which exhibited the same trend as the expression of CRABP2 during the odontoblastic induction of hDPSCs. CRABP2 knockdown inhibited the proliferative ability of hDPSCs, while it enhanced odontoblastic differentiation via promoting mineralization nodule formation and upregulating the activity of alkaline phosphatase and the expression of mineralization-related genes. The present study uncovered a novel function of CRABP2 in hDPSCs. Our data suggest that CRABP2 may act as a regulator during the proliferation and differentiation of hDPSCs.

Humans , Animals , Male , Female , Stem Cells/physiology , Cell Differentiation/physiology , Receptors, Retinoic Acid/physiology , Dental Pulp/cytology , Cell Proliferation/physiology , Odontoblasts/physiology , Reference Values , Time Factors , Immunohistochemistry , Down-Regulation/physiology , Cell Communication , Cells, Cultured , Blotting, Western , Analysis of Variance , Anthraquinones , Receptors, Retinoic Acid/analysis , Reverse Transcriptase Polymerase Chain Reaction , Coloring Agents , Alkaline Phosphatase , Mice, Inbred C57BL
Braz. oral res. (Online) ; 31: e87, 2017. tab, graf
Article in English | LILACS | ID: biblio-952109


Abstract: The objective of this study was to realize a scoping review the literature in order to identify the profile of DPSCs isolation and analyze the possible risk factors that could change the native behavior of these cells. An initial search was conducted using the following MeSH terms: "(dental pulp stem cell [MeSH])"; "(dental pulp [MeSH])" AND "(stem cell [MeSH])"; "("dental pulp stem cell" [MeSH]")". The electronic search was done without date restriction up to and including April 2014, in PubMed, Scopus, Scielo and ISI Web of Knowledge databases. Studies were submitted to inclusion and exclusion criteria and 222 articles were included. Data showed that over the past 15 years many studies have been conducted using DPSCs. However this is the first systematic review regarding the isolation of stem cell, and more specifically of dental pulp stem cells. The isolation of dental pulp stem cells showed great variability, hampering the development of standard protocols to achieve in vitro dental pulp stem cells with similar characteristics. This scoping review combined, for the first time, the methodologies used for dental pulp stem isolation, highlighting the most frequently used.

Humans , Stem Cells/cytology , Dental Pulp/cytology , Risk Factors , Collagenases , Publication Bias , Cell Culture Techniques , Culture Media