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1.
Braz Oral Res ; 33: e117, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31939498

RESUMO

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-1ß 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.


Assuntos
Compostos de Alumínio/farmacologia , Anti-Inflamatórios/farmacologia , Compostos de Cálcio/farmacologia , Polpa Dentária/citologia , Polpa Dentária/efeitos dos fármacos , Óxidos/farmacologia , Própole/farmacologia , Silicatos/farmacologia , Antraquinonas , Brasil , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Combinação de Medicamentos , Humanos , Interleucina-1beta/análise , Interleucina-6/análise , Odontoblastos/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Estatísticas não Paramétricas , Fator de Necrose Tumoral alfa/análise
2.
J Appl Oral Sci ; 28: e20190023, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31800871

RESUMO

When exposure of the pulp to external environment occurs, reparative dentinogenesis can be induced by direct pulp capping to maintain pulp tissue vitality and function. These clinical situations require the use of materials that induce dentin repair and, subsequently, formation of a mineralized tissue. OBJECTIVE: This work aims to assess the effect of tricalcium silicate cements and mineral trioxide aggregate cements, including repairing dentin formation and inflammatory reactions over time after pulp exposure in Wistar rats. METHODOLOGY: These two biomaterials were compared with positive control groups (open cavity with pulp tissue exposure) and negative control groups (no intervention). The evaluations were performed in three stages; three, seven and twenty-one days, and consisted of an imaging (nuclear medicine) and histological evaluation (H&E staining, immunohistochemistry and Alizarin Red S). RESULTS: The therapeutic effect of these biomaterials was confirmed. Nuclear medicine evaluation demonstrated that the uptake of 99mTc-Hydroxymethylene diphosphonate (HMDP) showed no significant differences between the different experimental groups and the control, revealing the non-occurrence of differences in the phosphocalcium metabolism. The histological study demonstrated that in mineral trioxide aggregate therapies, the presence of moderate inflammatory infiltration was found after three days, decreasing during follow-ups. The formation of mineralized tissue was only verified at 21 days of follow-up. The tricalcium silicate therapies demonstrated the presence of a slight inflammatory infiltration on the third day, increasing throughout the follow-up. The formation of mineralized tissue was observed in the seventh follow-up day, increasing over time. CONCLUSIONS: The mineral trioxide aggregate (WhiteProRoot®MTA) and tricalcium silicate (Biodentine™) present slight and reversible inflammatory signs in the pulp tissue, with the formation of mineralized tissue. However, the exacerbated induction of mineralized tissue formation with the tricalcium silicate biomaterial may lead to the formation of pulp calcifications.


Assuntos
Compostos de Alumínio/farmacologia , Materiais Biocompatíveis/farmacologia , Compostos de Cálcio/farmacologia , Polpa Dentária/efeitos dos fármacos , Dentina/efeitos dos fármacos , Dentinogênese/efeitos dos fármacos , Óxidos/farmacologia , Silicatos/farmacologia , Animais , Polpa Dentária/patologia , Capeamento da Polpa Dentária/métodos , Exposição da Polpa Dentária/tratamento farmacológico , Exposição da Polpa Dentária/patologia , Combinação de Medicamentos , Proteínas da Matriz Extracelular/análise , Imuno-Histoquímica , Masculino , Imagem Molecular/métodos , Odontoblastos/efeitos dos fármacos , Fosfoproteínas/análise , Agentes de Capeamento da Polpa Dentária e Pulpectomia/farmacologia , Pulpite/tratamento farmacológico , Pulpite/patologia , Distribuição Aleatória , Ratos Wistar , Reprodutibilidade dos Testes , Sialoglicoproteínas/análise , Fatores de Tempo
3.
J Appl Oral Sci ; 28: e20190105, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31800873

RESUMO

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.


Assuntos
Compostos de Alumínio/química , Compostos de Alumínio/farmacologia , Compostos de Cálcio/química , Compostos de Cálcio/farmacologia , Cimentos Dentários/química , Cimentos Dentários/farmacologia , Polpa Dentária/citologia , Polpa Dentária/efeitos dos fármacos , Fosfatase Alcalina/análise , Fosfatase Alcalina/efeitos dos fármacos , Animais , Bismuto/química , Bismuto/farmacologia , Cloreto de Cálcio/química , Cloreto de Cálcio/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Combinação de Medicamentos , Expressão Gênica/efeitos dos fármacos , Teste de Materiais , Camundongos , Odontoblastos/efeitos dos fármacos , Óxidos/química , Óxidos/farmacologia , Reprodutibilidade dos Testes , Silicatos/química , Silicatos/farmacologia , Fatores de Tempo , Óxido de Zinco/química , Óxido de Zinco/farmacologia
4.
J Endod ; 45(11): 1332-1341, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31585735

RESUMO

INTRODUCTION: Leptin is secreted as a peptide hormone from adipose tissues. The aim of this study was to evaluate the effects of leptin on reparative dentin formation and angiogenesis in the pulp tissue of teeth in vivo. METHODS: Twenty-four 7-week-old male rats were anesthetized. Cavities were prepared in maxillary first molars. Pulp cappings were performed with collagen scaffold (Col) with a phosphate-buffered saline (PBS) vehicle (Col + PBS), leptin 1 µmol/L with Col (L1 + Col), or leptin 10 µmol/L with Col (L10 + Col). For the negative control group (no pulp capping), pulp capping was not performed. All cavities were sealed with resin-modified glass ionomer followed by a micro-computed tomographic scan, histologic examination, and immunohistochemical analysis. RESULTS: The volume of newly formed mineralized tissue in the leptin group was significantly (P < .01) higher than that in the control group based on micro-computed tomographic analysis. In histologic examination, hard tissue formation was rarely shown in the no pulp capping and Col + PBS groups. However, significantly (P < .01) larger amounts of newly mineralized tissue deposition were observed in the leptin groups. In immunohistochemical analysis, reparative dentin and new vessels formed in the pulp cavity of the leptin groups. Vascular endothelial growth factor, dentin sialoprotein, and dentin sialophosphoprotein were expressed around the newly formed mineralized tissue area. CONCLUSIONS: Leptin showed the ability to induce angiogenesis, odontogenic differentiation, and mineralization in exposed rat pulps. Leptin also exhibited favorable inflammatory responses in the pulp tissue. Not only osteodentin but also tubular dentin and new vessels were observed in the pulp cavity.


Assuntos
Polpa Dentária , Dentina Secundária , Leptina , Neovascularização Fisiológica , Odontoblastos , Animais , Diferenciação Celular , Polpa Dentária/efeitos dos fármacos , Capeamento da Polpa Dentária , Leptina/fisiologia , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Odontoblastos/efeitos dos fármacos , Ratos , Fator A de Crescimento do Endotélio Vascular
5.
J Appl Oral Sci ; 27: e20180453, 2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31411261

RESUMO

OBJECTIVE: This study was designed for the chemical activation of a 35% hydrogen peroxide (H2O2) bleaching gel to increase its whitening effectiveness and reduce its toxicity. METHODOLOGY: First, the bleaching gel - associated or not with ferrous sulfate (FS), manganese chloride (MC), peroxidase (PR), or catalase (CT) - was applied (3x 15 min) to enamel/dentin discs adapted to artificial pulp chambers. Then, odontoblast-like MDPC-23 cells were exposed for 1 h to the extracts (culture medium + components released from the product), for the assessment of viability (MTT assay) and oxidative stress (H2DCFDA). Residual H2O2 and bleaching effectiveness (DE) were also evaluated. Data were analyzed with one-way ANOVA complemented with Tukey's test (n=8. p<0.05). RESULTS: All chemically activated groups minimized MDPC-23 oxidative stress generation; however, significantly higher cell viability was detected for MC, PR, and CT than for plain 35% H2O2 gel. Nevertheless, FS, MC, PR, and CT reduced the amount of residual H2O2 and increased bleaching effectiveness. CONCLUSION: Chemical activation of 35% H2O2 gel with MC, PR, and CT minimized residual H2O2 and pulp cell toxicity; but PR duplicated the whitening potential of the bleaching gel after a single 45-minute session.


Assuntos
Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/toxicidade , Clareadores Dentários/química , Clareadores Dentários/toxicidade , Clareamento Dental/métodos , Análise de Variância , Catalase/química , Sobrevivência Celular , Células Cultivadas , Cloretos/química , Cor , Polpa Dentária/química , Polpa Dentária/diagnóstico por imagem , Dentina/química , Dentina/efeitos dos fármacos , Compostos Ferrosos/química , Compostos de Manganês/química , Odontoblastos/efeitos dos fármacos , Peroxidase/química , Valores de Referência , Reprodutibilidade dos Testes , Estatísticas não Paramétricas , Fatores de Tempo
6.
Cell Prolif ; 52(6): e12680, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31454111

RESUMO

OBJECTIVES: The odontoblastic differentiation of dental pulp stem cells (DPSCs) contributes to tertiary dentin formation. Our previous study indicated that epiregulin (EREG) enhanced odontogenesis potential of dental pulp. Here, we explored the effects of EREG during DPSC odontoblastic differentiation. METHODS: The changes in EREG were detected during tertiary dentin formation. DPSCs were treated with recombinant human EREG (rhEREG), EREG receptor inhibitor gefitinib and short hairpin RNAs. The odontoblastic differentiation was assessed with ALP staining, ALP activity assay, alizarin red S staining and real-time RT-PCR of DSPP, OCN, RUNX2 and OSX. Western blot was conducted to examine the levels of p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (Erk1/2). The expression of EREG and odontoblastic differentiation-related markers was investigated in human dental pulp from teeth with deep caries and healthy teeth. RESULTS: Epiregulin was upregulated during tertiary dentin formation. rhEREG enhanced the odontoblastic differentiation of DPSCs following upregulated p38 MAPK and Erk1/2 phosphorylation, but not JNK, whereas depletion of EREG suppressed DPSC differentiation. Gefitinib decreased odontoblastic differentiation with decreased phosphorylation of p38 MAPK and Erk1/2. And suppression of p38 MAPK and Erk1/2 pathways attenuated DPSC differentiation. In human dental pulp tissue, EREG upregulation in deep caries correlates with odontoblastic differentiation enhancement. CONCLUSION: Epiregulin is released during tertiary dentin formation. And EREG enhanced DPSC odontoblastic differentiation via MAPK pathways.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Polpa Dentária/efeitos dos fármacos , Epirregulina/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células-Tronco/citologia , Animais , Proliferação de Células/efeitos dos fármacos , Polpa Dentária/citologia , Proteínas da Matriz Extracelular/metabolismo , Masculino , Odontoblastos/citologia , Odontoblastos/efeitos dos fármacos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
7.
Cell Tissue Res ; 376(3): 413-423, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30707290

RESUMO

Dental caries is a chronic, infectious, and destructive disease that allows bacteria to break into the dental pulp tissue. As caries-related bacteria invade the human dentinal tubules, odontoblasts are the first line of dental pulp that trigger the initial inflammatory and immune responses. DNA methylation is a key epigenetic modification that plays a fundamental role in gene transcription, and its role in inflammation-related diseases has recently attracted attention. However, whether DNA methylation regulates the inflammatory response of human odontoblasts is still unknown. In the present study, we investigated the expression of DNA methyltransferase (DNMT)-1 in lipoteichoic acid (LTA)-stimulated human odontoblast-like cells (hOBs) and found that DNMT1 expression showed a decline that is contrary to the transcription of inflammatory cytokines. Knockdown of the DNMT1 gene increased the expression of several cytokines, including IL-6 and IL-8, in the LTA-induced inflammatory response. DNMT1 knockdown increased the phosphorylation of IKKα/ß, IκBα, and p65 in the NF-κB pathway and the phosphorylation of p38 and ERK in the MAPK pathway; however, only the NF-κB pathway inhibitor PDTC suppressed both IL-6 and IL-8 expression, whereas inhibitors of the MAPK pathway (U0126, SB2035580, and SP600125) did not. Furthermore, DNMT1 knockdown upregulated the expression of MyD88 and TRAF6 but only attenuated the MyD88 gene promoter methylation in LTA-treated hOBs. Taken together, these results demonstrated that DNMT1 depletion caused hypomethylation and upregulation of MyD88, which resulted in activation of the NF-κB pathway and the subsequent release of LTA-induced inflammatory cytokines in hOBs. This study emphasizes the critical role of DNA methylation in the immune defense of odontoblasts when dental pulp reacted to caries.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Cárie Dentária/imunologia , Fator 88 de Diferenciação Mieloide/genética , Odontoblastos/imunologia , Adolescente , Adulto , Citocinas/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , Humanos , Inflamação/induzido quimicamente , Inflamação/imunologia , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases , NF-kappa B/metabolismo , Odontoblastos/efeitos dos fármacos , Fosforilação , Transdução de Sinais , Ácidos Teicoicos/farmacologia
8.
Cell Reprogram ; 21(1): 18-25, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30735076

RESUMO

Rosuvastatin is a synthetic statin of 3-hydroxy-methyl-3-glutamyl coenzyme A reductase inhibitor. It has pleiotropic characteristics including hepatic selectivity, minimal metabolism, inhibition of inflammation, and induction of osteoblast differentiation. In this study, dental pulp stem cells (DPSCs) were treated with lipopolysaccharide alone or with rosuvastatin. Then, we examined the accelerative effects of rosuvastatin on odontoblast differentiation and mineralized nodule formation by real-time polymerase chain reaction (RT-PCR), western blot, alizarin red S staining, and alkaline phosphatase staining. The extent of anti-inflammation was determined by RT-PCR and analysis of the expression of tumor necrosis factor α, interleukin 1ß (IL-1ß), and IL-6. Furthermore, the activation of nuclear factor kappa B (NF-κB) was determined by western blot. This study demonstrates that rosuvastatin may speed up odontoblast differentiation and rescue inflammatory reaction by suppressing the NF-κB signaling pathway. It is believed that our findings provide novel perceptions on odontogenic differentiation of DPSCs.


Assuntos
NF-kappa B/antagonistas & inibidores , Odontoblastos/efeitos dos fármacos , Osteogênese , Rosuvastatina Cálcica/farmacologia , Adolescente , Adulto , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores/metabolismo , Western Blotting , Diferenciação Celular , Células Cultivadas , Polpa Dentária/citologia , Humanos , Inflamação/metabolismo , MicroRNAs/genética , NF-kappa B/metabolismo , Odontoblastos/citologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Células-Tronco/citologia , Adulto Jovem
9.
Braz Oral Res ; 33: e013, 2019 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-30758410

RESUMO

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.


Assuntos
Proliferação de Células/efeitos dos fármacos , Polpa Dentária/citologia , Polpa Dentária/efeitos dos fármacos , Extrato de Sementes de Uva/farmacologia , Odontoblastos/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Dentina/citologia , Dentina/efeitos dos fármacos , Camundongos , Odontogênese/efeitos dos fármacos , Valores de Referência , Reprodutibilidade dos Testes , Fatores de Tempo
10.
BMC Oral Health ; 18(1): 201, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30514371

RESUMO

BACKGROUND: Recombinant amelogenin protein (RAP) is reported to induce complete root apex formation in dog model when used as apexification therapy. It also induces pulp regeneration in 85% of the treated group. Thus, the aim of this study was to investigate the nature of the remaining regenerated calcified tissues of the RAP group that showed no pulp regeneration compared to the calcium hydroxide treated group (CH). METHODS: A total of 240 dogs' open apex root canals were used, after establishment of canals contamination. Canals were cleaned, irrigated, and filled with RAP as an apexification material and compared with CH. Treated teeth were assessed by H&E, trichrome staining, and/or immunohistochemistry technique, at 1, 3, and 6 months. RESULTS: A time-dependent increase in the calcified tissue barrier was observed in the apex of the RAP-treated group compared to the CH-treated group. The newly formed dentin in this RAP group was mainly tubular dentin and was functionally attached to the bone by periodontal ligament, while the CH group showed dentin-associated mineralized tissue (DAMT) associated with the newly formed apical barrier. CONCLUSIONS: Out results suggest that RAP can be used as novel apexification material, resulting in a thickening and strengthening of the canal walls, and achieving apical closure.


Assuntos
Amelogenina/farmacologia , Apexificação/métodos , Hidróxido de Cálcio/farmacologia , Polpa Dentária/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Ápice Dentário/fisiologia , Animais , Polpa Dentária/fisiologia , Cavidade Pulpar/efeitos dos fármacos , Cavidade Pulpar/fisiologia , Necrose da Polpa Dentária/patologia , Necrose da Polpa Dentária/terapia , Dentina/efeitos dos fármacos , Cães , Modelos Animais , Odontoblastos/efeitos dos fármacos , Ligamento Periodontal , Proteínas Recombinantes/farmacologia , Materiais Restauradores do Canal Radicular/farmacologia , Ápice Dentário/efeitos dos fármacos , Dente não Vital/patologia
11.
Arch Oral Biol ; 94: 54-61, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30168419

RESUMO

OBJECTIVE: To investigate the in vitro effects of CCN2 on odontoblast-like cells proliferation and differentiation. DESIGN: MDPC-23 cells were cultured in DMEM supplemented with 5% FBS. CCN2 was either added to culture media or coated onto culture polystyrene, addition or coating of dH2O was served as control. In the addition group, CCN2 (100 ng/mL) was added into culture media. In the coating group, CCN2 at the concentration of 1000 ng/mL was employed. Cell proliferation was performed using CCK-8 assay. Cell differentiation and mineralization were analyzed by ALPase activity assay, real time RT-PCR and alizarin red staining. One-way ANOVA with post-hoc tukey HSD test was used for statistical analysis. RESULTS: MDPC-23 cells exhibited robust proliferative activity upon exposure to either soluble or immobilized CCN2. ALP activity of cells cultured on CCN2-modified surface was continuously strengthened from day six (0.831 ±â€¯0.024 units/µg protein versus 0.563 ±â€¯0.006 units/µg protein of control) till day eight (1.035 ±â€¯0.139 units/µg protein versus 0.704 ±â€¯0.061 units/µg protein of control). Gene expression of BSP, OCN and OPN were promoted by soluble CCN2 after 48 h exposure. Moreover, gene expression of BSP, OCN, OPN, ALP, COL1 A1, Runx-2, DSPP and DMP-1 was significantly enhanced by immobilized CCN2. Finally, mineralization of MDPC-23 cells was accelerated by both soluble and immobilized CCN2 to different extent. CONCLUSIONS: The findings indicate that CCN2 promoted proliferation, odontogenic gene expression and mineralization of MDPC-23 cells. It is proposed that CCN2 may be a promising adjunctive formula for dentin regeneration.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento do Tecido Conjuntivo/farmacologia , Odontoblastos/efeitos dos fármacos , Actinas/genética , Actinas/metabolismo , Fosfatase Alcalina/análise , Análise de Variância , Regeneração Óssea/efeitos dos fármacos , Calcificação Fisiológica , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo/administração & dosagem , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Meios de Cultura , Citoesqueleto/efeitos dos fármacos , Dentina , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Perfilação da Expressão Gênica , Humanos , Técnicas In Vitro , Odontoblastos/citologia , RNA Mensageiro/metabolismo
12.
Dent Mater ; 34(11): e301-e308, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30236973

RESUMO

OBJECTIVE: This study investigated the effect of matrix metalloproteinase-8 inhibitor I (MMP8-I) and chlorhexidine (CHX) on the viability, oxidative stress and cytokine secretion of MDPC-23 under short-term (30min) and long-term (3 days) culture. METHODS: MDPC-23 were treated with MMP8-I or CHX for 30min, 1day, 2days and 3days to detect the proliferation by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. In the following assays, MDPC-23 treated with 0.0003% CHX were referred to CHX group, treated with 8µM MMP8-I were MMP8-I group. Cells without additional treatment were regarded as control group. The cell cycle, reactive oxygen species (ROS) level, and apoptosis were assessed by flow cytometry. The cytokine level was quantified by enzyme-linked immunosorbent assay (ELISA). RESULTS: In 30min, CHX at concentrations higher than 0.0003% dilution inhibited cell proliferation when compared to the control group. MMP8-I (0.1-500µM) showed no obvious cytotoxicity to MDPC-23, and MMP8-I (1000µM) inhibited cell proliferation. In 3 days, CHX (0.0003%) significantly inhibited cell growth, while MMP8-I (8µM) had no cytotoxicity. In the CHX group, the S phase population was decreased, and cellular ROS were increased in 3 days. In the MMP8-I group, the change of S phase population and cellular ROS was not significant compared with the control group. Cell apoptosis was not elevated in the MMP8-I group, while the apoptosis rate was increased in the CHX group both in 30min and 3 days. In 30min, CHX treatment significantly increased the secretion of interleukin (IL)-1ß and IL-8, but slightly increased the secretion of IL-10, while MMP8-I caused no change in cytokines. In 3 days, CHX treatment significantly increased the secretion of IL-1ß, IL-6, and IL-8, and inhibited the secretion of IL-10. MMP8-I treatment caused the increase of IL-6. SIGNIFICANCE: Compared with CHX, MMP8-I at low concentration did not result in cytotoxicity, oxidative stress, or the disorder of immune response.


Assuntos
Anti-Infecciosos Locais/farmacologia , Clorexidina/farmacologia , Metaloproteinase 8 da Matriz/química , Odontoblastos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Anti-Infecciosos Locais/toxicidade , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Clorexidina/toxicidade , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Propriedades de Superfície
13.
J Cell Physiol ; 234(1): 849-859, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-30078208

RESUMO

Cellular senescence has been suggested to be involved in physiological changes of cytokine production. Previous studies showed that the concentration of tumor necrosis factor-α (TNF-α) is higher in the blood of aged people compared with that of young people. So far, the precise effects of TNF-α on the odontoblastic differentiation of pulp cells have been controversial. Therefore, we aimed to clarify how this cytokine affected pulp cells during aging. Human dental pulp cells (HDPCs) were cultured until reaching the plateau of their growth, and the cells were isolated at actively (young HDPCs; yHDPCs) or inactively (senescent HDPCs; sHDPCs) proliferating stages. sHDPCs expressed senescence-related molecules while yHDPCs did not. When these HDPCs were cultured in an odontoblast-inductive medium, both young and senescent cells showed mineralization, but mineralization in sHDPCs was lower compared with yHDPCs. However, the administration of TNF-α to this culture medium altered these responses: yHDPCs showed downregulated mineralization, while sHDPCs exhibited significantly increased mineralization. Furthermore, the expression of tumor necrosis factor receptor 1 (TNFR1), a receptor of TNF-α, was significantly upregulated in sHDPCs compared with yHDPCs. Downregulation of TNFR1 expression led to decreased mineralization of TNF-α-treated sHDPCs, whereas restored the reduction in TNF-α-treated yHDPCs. These results suggested that sHDPCs preserved the odontoblastic differentiation capacity and TNF-α promoted odontoblastic differentiation of HDPCs with the progress of their population doublings through increased expression of TNFR1. Thus, TNF-α might exert a different effect on the odontoblastic differentiation of HDPCs depending on their proliferating activity. In addition, the calcification of pulp chamber with age may be related with increased reactivity of pulp cells to TNF-α.


Assuntos
Envelhecimento/genética , Polpa Dentária/citologia , Odontoblastos/citologia , Fator de Necrose Tumoral alfa/farmacologia , Calcificação Fisiológica/genética , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/crescimento & desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Odontoblastos/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/genética
14.
Cell Reprogram ; 20(4): 236-244, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30089027

RESUMO

Wedelolactone is a multitarget natural plant compound with many pharmacological activities, including anti-inflammatory, anticancer, and antiosteoporosis. In this study, dental pulp stem cells (DPSCs) were treated with or without wedelolactone. We found that wedelolactone stimulated odontoblast differentiation and mineralization. At the molecular level, wedelolactone directly promoted the nuclear accumulation of ß-catenin, and thereafter stimulated the expression of odontoblast-related marker genes containing dentin matrix protein-1 (DMP1), dentin sialophosphoprotein (DSPP), and runt-related transcription factor 2 (Runx2). Furthermore, wedelolactone upregulated the expression of IκBα and inhibited phosphonation and nuclear migration of p65. As a result, wedelolactone remarkably induced odontoblast differentiation through semaphorin 3A (Sema3A)/neuropilin-1 (NRP1) pathway-mediated ß-catenin activation and nuclear factor kappa B (NF-κB) pathway inhibition. Our findings provide novel perceptions on odontogenic differentiation of DPSCs.


Assuntos
Diferenciação Celular , Cumarínicos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , NF-kappa B/metabolismo , Odontoblastos/citologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Adulto , Células Cultivadas , Voluntários Saudáveis , Humanos , NF-kappa B/genética , Odontoblastos/efeitos dos fármacos , Odontoblastos/metabolismo , Proteínas Wnt/genética , Adulto Jovem , beta Catenina/genética
15.
J Endod ; 44(9): 1367-1375, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30144832

RESUMO

INTRODUCTION: iMatrix-511 is a novel integrin-binding fragment derived from laminin-511. Previous studies showed its superiority as a culture substrate for xeno-free culture and maintenance of pluripotency in stem cells. However, its effects in the dental field remain largely unknown. The aim of the present study was to unravel the in vitro effects of iMatrix-511 in comparison with vitronectin (VN). METHODS: Biochemical assays were performed in vitro in MDPC-23 cells. The optimal coating density for 2 proteins was determined using the cell counting kit-8. To evaluate cell proliferation to both proteins, MDPC-23 cells were directly seeded onto the iMatrix-511 or VN-modified polystyrene and analyzed by the cell counting kit-8. The phenotype of cells seeded on iMatrix-511 and VN was characterized. Phenotypic characterization included real-time reverse-transcription polymerase chain reaction and alizarin red staining. RESULTS: The optimal coating density for iMatrix-511 and VN was determined to be 1 µg/cm2 and 0.25 µg/cm2, respectively. Cells cultured on iMatrix-511 showed higher cell proliferative activity than the noncoated control and VN on days 1, 2, and 4. Cell morphology observation revealed MDPC-23 cells attach preferentially to iMatrix-511 and start to spread as early as 1 hour after inoculation. MDPC-23 cells exhibited more potent odontogenic differentiation on iMatrix-511 than the control and VN as shown by the marked enhancement of dentin matrix protein 1 and dentin sialophosphoprotein messenger RNA expression. Although both proteins showed more mineralized nodule formation than the control, iMatrix-511 remained to be the one that elicited stronger calcific deposition. CONCLUSIONS: iMatrix-511 supported the proliferation and acquisition of odontogenic cell phenotype in vitro, rendering this novel material a potential candidate for dentin regeneration.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteínas da Matriz Extracelular/farmacologia , Odontoblastos/efeitos dos fármacos , Odontoblastos/fisiologia , Odontogênese/efeitos dos fármacos , Fenótipo , Animais , Células Cultivadas , Dentina/fisiologia , Camundongos , Odontoblastos/citologia , Regeneração/efeitos dos fármacos , Estimulação Química
16.
J Endod ; 44(8): 1270-1275, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29935871

RESUMO

INTRODUCTION: In regenerative endodontic treatment (RET), practitioners favor the placement of bioceramics as sealing materials over blood clots. It is important to understand the interaction between sealing material and cells in the root canal. The purpose of this study was to compare the effectiveness of various bioceramic materials (ProRoot MTA [Dentsply, Tulsa, OK], Biodentine [Septodont, Saint-Maur-des-Fossés, France], and RetroMTA [BioMTA, Seoul, Korea]) as sealing materials in RET for the proliferation and differentiation of stem cells from the apical papilla (SCAPs). METHODS: SCAPs were seeded at 20,000 cells/well and cultured with soluble agents of testing materials through a transwell culture plate. The proliferation of SCAPs was investigated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on days 1, 3, 7, and 14 of testing. Alizarin red staining and quantitative real-time polymerase chain reaction were used for SCAP differentiation at different time points (1, 7, 14, and 21 days). The odontoblast genes expressed are dentin matrix acidic phosphoprotein 1, dentin sialophosphoprotein, osteocalcin, and matrix extracellular phosphoglycoprotein, which were used in this study. The SCAPs were cultured in odonto/osteogenic induction medium and also contacted soluble agents from the testing materials. RESULTS: All 3 tested biomaterials induced SCAP proliferation. The Biodentine, ProRootMTA, and RetroMTA groups showed significant SCAP proliferation on days 7 and 14 compared with the control. In regard to odontoblastic differentiation, only Biodentine showed positive alizarin red staining. The highest expressions of dentin matrix acidic phosphoprotein 1, dentin sialophosphoprotein, and matrix extracellular phosphoglycoprotein were found on day 21 in the Biodentine group. The expression of osteocalcin was found to be significant on day 7. CONCLUSIONS: Biodentine, ProRootMTA, and RetroMTA can induce SCAP proliferation. Biodentine induced significant SCAP differentiation among the 3 materials.


Assuntos
Materiais Biocompatíveis/farmacologia , Cerâmica/farmacologia , Papila Dentária/efeitos dos fármacos , Odontoblastos/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Ápice Dentário/citologia , Compostos de Alumínio/farmacologia , Compostos de Cálcio/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Papila Dentária/citologia , Papila Dentária/crescimento & desenvolvimento , Papila Dentária/fisiologia , Combinação de Medicamentos , Humanos , Odontoblastos/citologia , Odontoblastos/fisiologia , Óxidos/farmacologia , Endodontia Regenerativa/métodos , Materiais Restauradores do Canal Radicular/farmacologia , Silicatos/farmacologia , Células-Tronco/fisiologia , Ápice Dentário/efeitos dos fármacos , Ápice Dentário/crescimento & desenvolvimento , Ápice Dentário/fisiologia
17.
Biomed Res Int ; 2018: 9465383, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29854812

RESUMO

The aim for the present study was to evaluate the in vitro effects of iMatrix-411 in odontoblast-like cells. To that end, iMatrix-411 was coated to both nontissue culture treated- (Non-PS) and tissue culture treated-polystyrene (TCPS) multiwells. MDPC-23 cells were seeded into noncoated (control) or coated wells. Optimal coating density and cell proliferation were assessed by cell counting kit-8 (CCK-8) at day two, day three, and day five. Osteo/odontogenic differentiation was evaluated by real-time RT-PCR and alkaline phosphatase (ALP) activity at days seven and eight, respectively. Calcific deposition of cells was visualized by alizarin red staining. Data were analyzed with post hoc Tukey HSD test (p < 0.05). Optimal coating density for iMatrix-411 was 8 µg/cm2. Exposure of MDPC-23 cells to iMatrix-411 in either non-PS or TCPS significantly enhanced proliferative activity. iMatrix-411 elevated ALP activity in both types of culture plates. iMatrix-411 significantly increased the mRNA level of OCN, BSP, OPN, ALP, and DMP-1. Meanwhile, it enhanced the expression of several integrin subunits: ITGA1, ITGA5, ITGAV, ITGB1, and ITGB5. Finally, iMatrix-411 also accelerated the mineralization at day eight in Non-PS. The results indicated iMatrix-411 stimulates proliferation and favours differentiation of odontoblast-like cells.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Laminina/farmacologia , Odontoblastos/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Células Cultivadas , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/metabolismo , Camundongos , Odontoblastos/metabolismo , Odontogênese/efeitos dos fármacos , Osteogênese/efeitos dos fármacos
18.
J Endod ; 44(7): 1121-1125, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29884339

RESUMO

INTRODUCTION: The nuclear enzyme poly(adenosine phosphate ribose) polymerase 1 (PARP-1) has been implicated in the maintenance and differentiation of several stem cells. The role of PARP-1 in dental pulp stem cell (DPSC) differentiation, especially in the context of its ability to modulate nerve regeneration factors, has not been investigated. Regeneration of neuronal components in pulp tissue is important for the assessment of tooth vitality. Brain-derived neurotrophic factor (BDNF) is known to play an integral signaling factor during nerve regeneration. In this study, we identified the role of PARP-1 in the modulation of BDNF in DPSC differentiation into odontoblastlike cells. METHODS: Human DPSCs were prepared from healthy molars and cultured in regular and osteogenic media treated with PARP-1 antagonist and PARP-1 exogeneous protein. Polymerase chain reaction and immunohistochemistry analysis for BDNF and various differentiation markers were performed. RESULTS: Our polymerase chain reaction results showed that differentiated cells show odontoblastlike properties because they express odontogenic markers such as dentin sialophosphoprotein and dentin matrix protein 1. Both PARP-1 inhibitor and protein did not affect odontogenic differentiation and proliferation because the number of the differentiated cells was unaffected, and the expression of dentin sialophosphoprotein and dentin matrix protein 1 was not significantly changed. There is the possibility that PARP-1 treatment induces DPSCs into the unique cell lineage. Some differentiated cells show a very unique morphology with large irregular cytoplasm and an oval nucleus. Moreover, PARP-1 inhibition significantly increased BDNF secretion in DPSC-derived odontoblastlike cells. This observation was also confirmed by immunohistochemistry. CONCLUSIONS: Taken together, our results indicate PARP-1 as a negative regulator in BDNF secretion during odontogenic DPSC differentiation, showing its potential application for translational nerve regeneration strategies to improve dental pulp tissue vitality assessments.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Polpa Dentária/citologia , Odontoblastos/citologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Células-Tronco/metabolismo , Western Blotting , Diferenciação Celular , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/metabolismo , Humanos , Regeneração Nervosa , Odontoblastos/efeitos dos fármacos , Odontoblastos/metabolismo , Odontogênese , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Endodontia Regenerativa/métodos , Células-Tronco/efeitos dos fármacos
19.
J Dent Res ; 97(10): 1170-1177, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29649366

RESUMO

The goal of this study was to examine the effects of early and limited exposure of perivascular cells expressing α (αSMA) to fibroblast growth factor 2 (FGF2) in vivo. We performed in vivo fate mapping by inducible Cre-loxP and experimental pulp injury in molars to induce reparative dentinogenesis. Our results demonstrate that early delivery of exogenous FGF2 to exposed pulp led to proliferative expansion of αSMA-tdTomato+ cells and their accelerated differentiation into odontoblasts. In vivo lineage-tracing experiments showed that the calcified bridge/reparative dentin in FGF2-treated pulps were lined with an increased number of Dspp+ odontoblasts and devoid of BSP+ osteoblasts. The increased number of odontoblasts derived from αSMA-tdTomato+ cells and the formation of reparative dentin devoid of osteoblasts provide in vivo evidence for the stimulatory effects of FGF signaling on odontoblast differentiation from early progenitors in dental pulp.


Assuntos
Fator 2 de Crescimento de Fibroblastos/farmacologia , Odontoblastos/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/crescimento & desenvolvimento , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Hibridização In Situ , Camundongos , Odontoblastos/metabolismo , Odontoblastos/fisiologia
20.
Biomed Res Int ; 2018: 2370438, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29675422

RESUMO

Aim: To analyze the effect of three mitogen-activated protein kinase (MAPK) inhibitors, namely, SB202190 (p38 inhibitor), SP600125 (JNK inhibitor), and PD98059 (ERK inhibitor) in Dex-stimulated MDPC-23 cell differentiation and mineralization. Methods: Experiment was divided into five groups, control (cells without Dex and inhibitors treatment), Dex (cells with Dex treatment but without inhibitors), Dex + SB202190, Dex + SP600125, and Dex + PD98059. Cell differentiation was assessed by alkaline phosphatase (ALP) activity assay and real time RT-PCR. Cell mineralization was investigated by alizarin red staining. Results: Exposure to SB202190 (20 µM) significantly decreased the mineral deposition in Dex-treated cells as demonstrated by alizarin red staining. Treatment of SP600125 (20 µM) attenuated the mineralization as well, albeit at a lower degree as compared to SB202190 (20 µM). Similarly, SB202190 (20 µM) completely abrogated the ALP activity stimulated by Dex at six days in culture, while no changes were observed with regard to ALP activity in SP600125 (20 µM) and PD98059 (20 µM) treated cells. The upregulation of bone sialoprotein (BSP), ALP, and osteopontin (OPN) in Dex challenged cells was completely inhibited by SB202190. Conclusion: Blockade of p38-MAPK signaling pathway resulted in significant inhibition of ALP activity, mineralization, and downregulation of osteogenic markers. The data implicated that p38 signaling pathway plays a critical role in the regulation of MDPC-23 cells differentiation and mineralization.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Odontoblastos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Antracenos/farmacologia , Biomarcadores/metabolismo , Linhagem Celular , Dexametasona/farmacologia , Regulação para Baixo/efeitos dos fármacos , Flavonoides/farmacologia , Imidazóis/farmacologia , Sialoproteína de Ligação à Integrina/efeitos dos fármacos , Sialoproteína de Ligação à Integrina/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Odontoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteopontina/metabolismo , Piridinas/farmacologia , Ratos , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
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