RESUMO
BACKGROUND: Simulating a bacterial-induced pulpitis environment in vitro may contribute to exploring mechanisms and bioactive molecules to counteract these adverse effects. OBJECTIVE: To investigate the chronic exposure of human dental pulp cells (HDPCs) to lipopolysaccharides (LPS) aiming to establish a cell culture protocol to simulate the impaired odontogenic potential under pulpitis conditions. METHODOLOGY: HDPCs were isolated from four healthy molars of different donors and seeded in culture plates in a growth medium. After 24 h, the medium was changed to an odontogenic differentiation medium (DM) supplemented or not with E. coli LPS (0 - control, 0.1, 1, or 10 µg/mL) (n=8). The medium was renewed every two days for up to seven days, then replaced with LPS-free DM for up to 21 days. The activation of NF-κB and F-actin expression were assessed (immunofluorescence) after one and seven days. On day 7, cells were evaluated for both the gene expression (RT-qPCR) of odontogenic markers (COL1A1, ALPL, DSPP, and DMP1) and cytokines (TNF, IL1B, IL8, and IL6) and the production of reactive nitrogen (Griess) and oxygen species (Carboxy-H2DCFDA). Cell viability (alamarBlue) was evaluated weekly, and mineralization was assessed (Alizarin Red) at 14 and 21 days. Data were analyzed with ANOVA and post-hoc tests (α=5%). RESULTS: After one and seven days of exposure to LPS, NF-κB was activated in a dose-dependent fashion. LPS at 1 and 10 µg/mL concentrations down-regulated the gene expression of odontogenic markers and up-regulated cytokines. LPS at 10 µg/mL increased both the production of reactive nitrogen and oxygen species. LPS decreased cell viability seven days after the end of exposure. LPS at 1 and 10 µg/mL decreased hDPCs mineralization in a dose-dependent fashion. CONCLUSION: The exposure to 10 µg/mL LPS for seven days creates an inflammatory environment that is able to impair by more than half the odontogenic potential of HDPCs in vitro, simulating a pulpitis-like condition.
Assuntos
Pulpite , Humanos , Pulpite/metabolismo , NF-kappa B , Polpa Dentária , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Escherichia coli/metabolismo , Diferenciação Celular , Citocinas/metabolismo , Células CultivadasRESUMO
PURPOSE: To investigate the mechanism of polysaccharides from aloe vera (PAV), a main active ingredient of Aloe vera, treatment in pulpitis rats. METHODS: Pulpitis were modeled by drilling the occlusal central fossa with Sprague Dawley rats. Next, the rats were treated with 20, 40, and 80 mg/kg PAV for three weeks, respectively. Computed tomography scanning assay, hematoxylin and eosin staining, and tartrate-resistant acid phosphatase staining were used to detect the pathology change. Then, levels of tumor necrosis factor-α, interleukin-1ß, prostaglandin E2, and ciclooxigenase 2 were detected by enzyme-linked immunosorbent assay. The expressions of bone morphogenetic protein 2 human (BMP-2), osteocalcin, osterix, and runt-related transcription factor 2 (Runx2) were quantified by quantitative real-time polymerase chain reaction and Western blotting (WB). Finally, Wnt3a expression, p-GSK3ß/GSK3ß and p-ß-catenin/ß-catenin ratio were analyzed by WB. RESULTS: PAV up regulated the bone mineral density, and reduced the breakage of the crown and cervical structures, and the necrosis of the crown and root pulp of pulpitis rats. In addition, results indicated that PAV could inhibit osteoblast formation. While osteoblasts' number was decreased, proteins of BMP-2, osteocalcin, osterix, and Runx2 were up-regulated by PAV. Furthermore, PAV increased the Wnt3a expression and the p-ß-catenin/ß-catenin ratio, and decreased p-GSK3ß/GSK3ß ratio. Interestingly, these effects were all in dose dependence. CONCLUSIONS: PAV could inhibit pulp inflammation and promote osteoblasts differentiation via suppressing the activation of the Wnt/ß-catenin signaling, enhancing the dental bone density.
Assuntos
Aloe , Polissacarídeos , Pulpite , Via de Sinalização Wnt , Animais , Humanos , Ratos , Aloe/química , beta Catenina/metabolismo , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Osteoblastos , Osteocalcina/metabolismo , Osteogênese , Polissacarídeos/farmacologia , Pulpite/metabolismo , Ratos Sprague-DawleyRESUMO
BACKGROUND: Bone morphogenetic protein 9 (BMP9) tends to be associated with various inflammatory responses of diseases, but its relationship with pulpitis remains unknown. OBJECTIVE: This study aimed to evaluate the effects and mechanisms of BMP9 in pulpitis. METHODOLOGY: A rat model of pulpitis was used to evaluate the expression of BMP9, which was also analysed in Porphyromonas gingivalis lipopolysaccharide (Pg-LPS)-stimulated human dental pulp cells (hDPCs). The effects and mechanism of BMP9 on the regulation of inflammatory factors and matrix metalloproteinase-2 (MMP2) were evaluated using real-time quantitative PCR, western blotting, and immunocytofluorescence. Moreover, the migration ability of THP-1 monocyte-macrophages, treated with inflammatory supernate inhibited by BMP9, was previously tested by a transwell migration assay. Finally, a direct rat pulp capping model was used to evaluate in vivo the influence of the overexpression of BMP9 in pulpitis. RESULTS: The expression of BMP9 decreased after 24 h and increased after 3 and 7 d in rat pulpitis and inflammatory hDPCs. The overexpression of BMP9 inhibited the gene expression of inflammatory factors (IL-6, IL-8, and CCL2) and the secretion of IL-6 and MMP2 in Pg-LPS-stimulated hDPCs. The level of phosphorylated Smad1/5 was upregulated and the levels of phosphorylated ERK and JNK were downregulated. The inflammatory supernate of hDPCs inhibited by BMP9 reduced the migration of THP-1 cells. In rat pulp capping models, overexpressed BMP9 could partially restrain the development of dental pulp inflammation. CONCLUSION: This is the first study to confirm that BMP9 is involved in the occurrence and development of pulpitis and can partially inhibit its severity in the early stage. These findings provided a theoretical reference for future studies on the mechanism of pulpitis and application of bioactive molecules in vital pulp therapy.
Assuntos
Pulpite , Ratos , Humanos , Animais , Pulpite/metabolismo , Metaloproteinase 2 da Matriz , Fator 2 de Diferenciação de Crescimento/farmacologia , Fator 2 de Diferenciação de Crescimento/metabolismo , Polpa Dentária , Interleucina-6 , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Inflamação , Células CultivadasRESUMO
ABSTRACT: Researchers have reported false positive/negative results of the cold test in the diagnosis of pulpitis. Knowledge of the correlation between results of the cold test and proteins could aid in decreasing the frequency of incorrect diagnosis. To associate the levels of matrix metalloproteinase-8 (MMP-8) with the responses (in seconds) to the cold test in teeth diagnosed with reversible and irreversible pulpitis.A cross-sectional study was performed. A total of 150 subjects were evaluated, of which 60 subjects met the selection criteria. The participants were divided into 3 groups: Group 1, healthy pulps, 20 subjects with 20 posterior teeth (premolars) with clinically normal pulp tissue; Group 2, reversible pulpitis, 20 patients with 20 teeth diagnosed with reversible pulpitis; and Group 3, irreversible pulpitis, 20 subjects with 20 teeth diagnosed with irreversible pulpitis. All participants were evaluated based on the following variables: medical and dental history, cold test, and expression of MMP-8 by enzyme-linked immunosorbent assay in dentin samples.Responses to the cold test between 4 to 5âseconds (second evaluation; Pâ<â.0001) were associated with high levels of MMP-8 (mean, 0.36âng/mL) in the reversible pulpitis group. In the irreversible pulpitis group, the responses from 6 to ≥10âseconds (second evaluation; Pâ<â.0001) were associated with a higher average of MMP-8 levels (mean, 1.97âng/mL).We determined that an increase in the duration of response to the cold test was associated with an increase in MMP-8 levels (Rhoâ=â0.81, Pâ<â.0001) in teeth with pulpitis. The above correlations can be considered an adjunct to the clinical diagnosis of pulpitis.
Assuntos
Temperatura Baixa , Sensibilidade da Dentina/diagnóstico , Dentina , Metaloproteinase 8 da Matriz/análise , Pulpite , Adulto , Estudos Transversais , Dentina/metabolismo , Dentina/fisiopatologia , Erros de Diagnóstico/prevenção & controle , Feminino , Humanos , Masculino , México , Prognóstico , Pulpite/diagnóstico , Pulpite/metabolismoRESUMO
OBJECTIVES: To study the intensity of inflammatory infiltrate and production of interleukin-1ß (ll-1ß), tumor necrosis factor-ß (TNF-ß), fibroblast growth factor-2 (FGF-2), glutathione peroxidase (GPX), and osteocalcin in response to in-office tooth bleaching in rats. MATERIAL AND METHODS: Twenty male Wistar rats were randomized into four groups (n=5) according to the received treatment (tooth bleaching or no treatment - control) and the period of euthanasia after treatment (24 h or 10 days). We performed tooth bleaching using a 38% hydrogen peroxide gel on maxillary and mandibular incisors. After euthanasia, incisors (20 per group) were processed for histological analysis, immunohistochemistry staining of ll-1ß, TNF-ß, FGF-2 and GPX and osteocalcin by immunofluorescence. We analyzed data using the Mann-Whitney and Kruskal-Wallis/Dunn tests (p<0.05). RESULTS: The bleached groups presented statistically significant differences regarding the pulp inflammation stage compared with the control groups. Bleached teeth showed moderate/severe inflammatory infiltrate and control groups presented absent inflammatory cells or a negligible number of mononuclear cells (p<0.001) at two times (24 h and 10 days). There was strong staining for ll-1ß, TNF-ß, and GPX in bleached groups at 24 h and strong staining for ll-1ß, TNF-ß, GPX and FGF-2 at 10 days. After 10 days of tooth bleaching, the bleached group showed a statistically superior amount of osteocalcin than the other groups (p<0.01). CONCLUSIONS: Tooth bleaching with 38% hydrogen peroxide causes severe pulp inflammation, but characteristics of tissue repair after 10 days.
Assuntos
Peróxido de Hidrogênio/efeitos adversos , Pulpite/induzido quimicamente , Pulpite/patologia , Clareadores Dentários/administração & dosagem , Clareamento Dental/efeitos adversos , Animais , Fator 2 de Crescimento de Fibroblastos/biossíntese , Glutationa Peroxidase/biossíntese , Imuno-Histoquímica , Interleucina-1beta/biossíntese , Linfotoxina-alfa/biossíntese , Masculino , Microscopia de Fluorescência , Osteocalcina/biossíntese , Pulpite/metabolismo , Distribuição Aleatória , Ratos Wistar , Fatores de TempoRESUMO
Abstract Objectives: To study the intensity of inflammatory infiltrate and production of interleukin-1β (ll-1β), tumor necrosis factor-β (TNF-β), fibroblast growth factor-2 (FGF-2), glutathione peroxidase (GPX), and osteocalcin in response to in-office tooth bleaching in rats. Material and Methods: Twenty male Wistar rats were randomized into four groups (n=5) according to the received treatment (tooth bleaching or no treatment - control) and the period of euthanasia after treatment (24 h or 10 days). We performed tooth bleaching using a 38% hydrogen peroxide gel on maxillary and mandibular incisors. After euthanasia, incisors (20 per group) were processed for histological analysis, immunohistochemistry staining of ll-1β, TNF-β, FGF-2 and GPX and osteocalcin by immunofluorescence. We analyzed data using the Mann-Whitney and Kruskal-Wallis/Dunn tests (p<0.05). Results: The bleached groups presented statistically significant differences regarding the pulp inflammation stage compared with the control groups. Bleached teeth showed moderate/severe inflammatory infiltrate and control groups presented absent inflammatory cells or a negligible number of mononuclear cells (p<0.001) at two times (24 h and 10 days). There was strong staining for ll-1β, TNF-β, and GPX in bleached groups at 24 h and strong staining for ll-1β, TNF-β, GPX and FGF-2 at 10 days. After 10 days of tooth bleaching, the bleached group showed a statistically superior amount of osteocalcin than the other groups (p<0.01). Conclusions: Tooth bleaching with 38% hydrogen peroxide causes severe pulp inflammation, but characteristics of tissue repair after 10 days.
Assuntos
Animais , Masculino , Pulpite/induzido quimicamente , Pulpite/patologia , Clareamento Dental/efeitos adversos , Clareadores Dentários/administração & dosagem , Peróxido de Hidrogênio/efeitos adversos , Pulpite/metabolismo , Fatores de Tempo , Imuno-Histoquímica , Distribuição Aleatória , Osteocalcina/biossíntese , Fator 2 de Crescimento de Fibroblastos/biossíntese , Linfotoxina-alfa/biossíntese , Ratos Wistar , Interleucina-1beta/biossíntese , Glutationa Peroxidase/biossíntese , Microscopia de FluorescênciaRESUMO
INTRODUCTION: Pattern recognition receptors, such as toll-like receptor 2 (TLR-2) and TLR-4, participate in the activation of immune cells by microorganisms in dental pulp. However, the expression levels of pattern recognition receptors can be modulated by epigenetic factors, especially DNA methylation. In this study, the methylation status of the TLR-2 and CD14 (TLR4 co-receptor) genes in healthy and inflamed human dental pulp was examined. METHODS: The Methyl-Profiler DNA Methylation qPCR Assay was used to verify the DNA methylation patterns. RESULTS: No differences in the methylation patterns were observed between the 2 groups. Most DNA was unmethylated in both groups. CONCLUSIONS: The hypomethylation of TLR2 and CD14 genes is a usual feature in human dental pulp.
Assuntos
Metilação de DNA/genética , Polpa Dentária/metabolismo , Receptores de Lipopolissacarídeos/genética , Receptor 2 Toll-Like/genética , Adolescente , Adulto , Ilhas de CpG/genética , Metilação de DNA/imunologia , Cárie Dentária/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Pulpite/metabolismo , Adulto JovemRESUMO
The purpose of this study was to investigate whether the inflammation of rat dental pulp induces the muscarinic acetylcholine receptor (mAChR) constitutive receptor activity. Pulpitis was induced with bacterial lipolysaccharide in rat incisors dental pulp. Saturation assay with [(3)H]-quinuclidinyl benzilate ([(3)H] QNB), competitive binding with different mAChR antagonist subtypes, and nitric oxide synthase (NOS) activity were performed. A drastic change in expression and response to mAChR subtypes was observed in pulpitis. Inflamed pulp expressed high number of M(3) mAChR of high affinity, whereas the M(1) mAChR is the main subtype displayed in normal pulp. Consistent with the identification of the affinity constant (Ki) of M(3) and Ki of M(1) in both pulpitis and in normal pulps are the differences in the subtype functionality of these cells. In pulpitis, pilocarpine (1 × 10(-11) mol/L to 5 × 10(-9) mol/L) exerted an inhibitory action on NOS activity that was blocked by J 104129 fumarate (highest selective affinity to M(3) mAChR). In normal pulps, pilocarpine (1 × 10(-11) mol/L to 5 × 10(-9) mol/L) has no effect. NOS basal activity was 5.9 times as high in pulpitis as in the normal pulp as a result of the activation of inducible NOS. The irreversible pulpitis could induce a mAChR alteration, increasing the high-affinity receptor density and transduction-coupling efficiency of inducible NOS activity, leading to a spontaneously active conformation of the receptor. Pilocarpine acting as an inverse agonist might be useful therapeutically to prevent necrosis and subsequent loss of dental pulp.
Assuntos
Agonistas Muscarínicos/farmacologia , Óxido Nítrico Sintase/metabolismo , Pilocarpina/farmacologia , Pulpite/metabolismo , Receptor Muscarínico M3/efeitos dos fármacos , Análise de Variância , Animais , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/metabolismo , Modelos Animais de Doenças , Agonismo Inverso de Drogas , Inflamação/metabolismo , Masculino , Óxido Nítrico Sintase/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Ratos , Ratos Wistar , Receptor Muscarínico M3/agonistas , Receptor Muscarínico M3/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
INTRODUCTION: DNA methylation is characterized by the addition of methyl groups in cytosines within cytosine-phosphate-guanine (CpG) islands. Unmethylated islands are related with transcriptionally active structure, whereas methylated DNA recruits methyl-binding proteins that promotes chromatin compaction. Although epigenetic events can influence the expression of cytokines, such events have not been investigated in dental pulp yet. The purpose of the present study was to evaluate the methylation status of the interferon gamma (IFN-gamma) gene in human dental pulp affected by inflammation compared with pulp tissue of impacted molar teeth and to verify the impact of methylation status in the expression pattern of the gene. METHODS: Methylation-specific polymerase chain reaction (MSP) was used to verify the DNA methylation status of the IFN-gamma gene in 16 human dental pulps affected by inflammation and in 16 pulp samples of impacted molar teeth. Histologic sections stained by hematoxylin-eosin were used for histopathological evaluation, and the expression of IFN-gamma was assessed by quantitative real-time PCR (qPCR). RESULTS: Although total methylation was observed in 43.75% of the samples of normal dental pulp tissues, partial methylation or unmethylation was found in 93.75% of the samples of inflamed pulp tissues. All the samples with total methylation in MSP showed no transcription of IFN-gamma. The qPCR results showed expression of IFN-gamma in 5 of 10 samples with partial methylation. CONCLUSION: The present study gives the first evidence of the possible participation of epigenetic events in the development of dental pulp inflammation.
Assuntos
Metilação de DNA/fisiologia , Polpa Dentária/metabolismo , Interferon gama/genética , Pulpite/genética , Adolescente , Adulto , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Criança , Polpa Dentária/citologia , Feminino , Regulação da Expressão Gênica , Humanos , Interferon gama/biossíntese , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , Pulpite/metabolismo , Pulpite/patologia , Estatísticas não Paramétricas , Adulto JovemRESUMO
The purpose of this study was to quantify the percentage and the mean fluorescence intensity of viable alternatively activated monocytes/macrophages (AAMø) CD163+ positive for calcitonin gene-related peptide receptor (CGRPr) within the total AAMø population in human dental pulp. Pulp tissue samples were collected from teeth with a clinical diagnosis of irreversible pulpitis (n = 13), pulps with induced inflammation (n = 13), and normal pulps (n = 13). All samples were labeled to identify positive cells for CGRPr and CD163 using a flow cytometry assay. Results demonstrated that a high percentage of total viable AAMø CD163+ expressed CGRPr on their membranes (72.12% in healthy pulp, 62.20% in irreversible pulpitis, and 58.01% in induced pulpitis). Significant differences were found between mean AAMø CD163+ fluorescence for CGRPr according to pulp condition, being greater in irreversible pulpitis. It can be concluded that AAMø CD163+ are expressed during normal and inflammatory processes, supporting the hypothesis that they could exercise an anti-inflammatory action that could be controlled by CGRP signaling after its binding.