RESUMEN
Transforming growth factor-ß1 (TGF-ß1) regulates wound healing/regeneration and aging processes. Dental pulp stem cells from human exfoliated deciduous teeth (SHED) are cell sources for treatment of age-related disorders. We studied the effect of TGF-ß1 on SHED and related signaling. SHED were treated with TGF-ß1 with/without pretreatment/co-incubation by SB431542, U0126, 5Z-7-oxozeaenol or SB203580. Sircol collagen assay, 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) assay, alkaline phosphatase (ALP) assay, RT-PCR, western blotting and PathScan phospho-ELISA were used to measure the effects. We found that SHED expressed ALK1, ALK3, ALK5, TGF-RII, betaglycan and endoglin mRNA. TGF-ß1 stimulated p-Smad2, p-TAK1, p-ERK, p-p38 and cyclooxygenase-2 (COX-2) protein expression. It enhanced proliferation and collagen content of SHED that were attenuated by SB431542, 5Z-7-oxozeaenol and SB203580, but not U0126. TGF-ß1 (0.5-1 ng/ml) stimulated ALP of SHED, whereas 5-10 ng/ml TGF-ß1 suppressed ALP. SB431542 reversed the effects of TGF-ß1. However, 5Z-7-oxozeaenol, SB203580 and U0126 only reversed the stimulatory effect of TGF-ß1 on ALP. Four inhibitors attenuated TGF-ß1-induced COX-2 expression. TGF-ß1-stimulated TIMP-1 and N-cadherin was inhibited by SB431542 and 5Z-7-oxozeaenol. These results indicate that TGF-ß1 affects SHED by differential regulation of ALK5/Smad2/3, TAK1, p38 and MEK/ERK. TGF-ß1 and SHED could potentially be used for tissue engineering/regeneration and treatment of age-related diseases.
Asunto(s)
Pulpa Dental/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Regeneración/efectos de los fármacos , Proteína Smad2/metabolismo , Células Madre/efectos de los fármacos , Diente Primario/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Pulpa Dental/citología , Pulpa Dental/enzimología , Humanos , Fosforilación , Transducción de Señal , Proteína smad3/metabolismo , Células Madre/enzimología , Diente Primario/citología , Diente Primario/enzimologíaRESUMEN
Recent studies have demonstrated that IGF-1 modulates the pluripotent differentiation of dental pulp stem cells (DPSCs). Although mTOR pathway activation has been showed as responsible for IGF-1 induced pluripotent differentiation, the mechanism that the IGF-1-mTOR pathway induces the neural differentiation of DPSCs is still unclear. In our research, we have demonstrated that 0-10 ng/mL IGF-1 had no obvious effect on the proliferation of DPSCs, but IGF-1 nonetheless enhances the neural differentiation of DPSCs in a dose-dependent manner. Simultaneously, we found that phosphorylated mTOR was up-regulated, which indicated the involvement of mTOR in the process. Rapamycin, an inhibitor of mTOR activity, can reverse the effect of DPSCs stimulated by IGF-1. Next, we studied the role of mTORC1 and mTORC2, two known mTOR complexes, in the neural differentiation of DPSCs. We found that inhibition of mTORC1 can severely restricts the neural differentiation of DPSCs. However, inhibition of mTORC2 has the opposite effect. This latter effect disappears when both rictor and mTOR are inhibited, showing that the mTORC2 effect is mTORC1 dependent. This study has expanded the role of mTOR in DPSCs neural differentiation regulated by IGF-1.
Asunto(s)
Diferenciación Celular/genética , Pulpa Dental/enzimología , Factor I del Crecimiento Similar a la Insulina/genética , Células Madre/efectos de los fármacos , Adolescente , Adulto , Diferenciación Celular/efectos de los fármacos , Proliferación Celular , Pulpa Dental/citología , Pulpa Dental/efectos de los fármacos , Pulpa Dental/crecimiento & desarrollo , Femenino , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Diana Mecanicista del Complejo 2 de la Rapamicina/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/genética , Adulto JovenRESUMEN
MicroRNAs (miRNAs) play critical roles in various biological processes including cell differentiation. Some researchers suggested that the p38 mitogen-activated protein kinases (MAPK) signaling pathway had an effect on regulating the odontoblastic differentiation of human dental pulp stem cells (hDPSCs). This study focuses on the effects of miR-143-5p on hDPSCs by regulating the p38 MAPK signaling pathway. The targeting relationship of MAPK14 and miR-143-5p targets were verified by TargetScan and dual-luciferase reporter gene assay. Through overexpression of miR-143-5p or silencing of miR-143-5p, expressions of miR-143-5p, MAPK14, Ras, MAPK kinase (MKK) 3/6, dentin sialophosphoprotein (DSPP), alkaline phosphatase (ALP), and osteocalcin (OCN) were detected by reverse transcription quantitative polymerase chain reaction. Protein expressions of MAPK14, Ras, and MKK3/6 were determined by western blot analysis. ALP and alizarin red S staining were used to detect mineralization. Initially, MAPK14 was found to be negatively regulated by miR-143-5p. Meanwhile, the upregulated miR-143-5p decreased the p38 MAPK signaling pathway related genes (MAPK14, Ras, and MKK3/6) and odontoblastic differentiation markers (ALP, DSPP, and OCN) expression. On the contrary, the downregulated miR-143-5p increased the p38 MAPK signaling pathway related genes (MAPK14, Ras, and MKK3/6) and odontoblastic differentiation markers (ALP, DSPP, and OCN) expression. Furthermore, ALP activity and mineralized nodules increased after downregulation of miR-143-5p, and after its upregulation, ALP activity and mineralized nodules decreased. Our data suggest that poor expression of miR-143-5p promotes hDPSCs odontoblastic differentiation through the activation of the p38 MAPK signaling pathway by upregulating MAPK14.
Asunto(s)
Diferenciación Celular , Pulpa Dental/enzimología , MicroARNs/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Odontoblastos/enzimología , Células Madre/enzimología , Regiones no Traducidas 3' , Adulto , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Sitios de Unión , Pulpa Dental/citología , Regulación hacia Abajo , Activación Enzimática , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica , Células HEK293 , Humanos , Masculino , MicroARNs/genética , Proteína Quinasa 14 Activada por Mitógenos/genética , Osteocalcina/genética , Osteocalcina/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Transducción de Señal , Adulto JovenRESUMEN
Human dental pulp stem cells (hDPSCs) are primarily derived from the pulp tissues of permanent third molar teeth. They were widely used in human bone tissue engineering. It was previously indicated that microRNA (miR) expressions are closely associated with hDPSCs development. However, the specific effect of miR-488 on hDPSCs still remains unclear. In this study, we aimed to investigate effects of miR-488 on the differentiation of hDPSCs into odontoblast cells through the p38 mitogen-activated protein kinases (MAPK) signaling pathway by binding to MAPK1. The hDPSCs were isolated and cultured in vitro. Dual-luciferase reporter gene assay was performed to test the relationship between MAPK1 (p38) and miR-488. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to detect the mRNA and protein expressions of p38 MAPK signaling pathway-related genes (MAPK1, Ras, and Mitogen-activated protein kinase kinase 3/6 [MKK3/6]), along with expressions of dentin Sialophosphoprotein (DSPP), alkaline phosphatase (ALP), and osteonectin (OCN). ALP staining and alizarin red staining were conducted to detect ALP activity and degree of mineralization. Initially, we found that MAPK1 was the target gene of miR-488. Besides, downregulation of miR-488 was observed to stimulate the p38 MAPK signaling pathway and to increase the messenger RNA and protein expressions of DSPP, ALP, and OCN. Furthermore, ALP activity and formation of a mineralized nodule in hDPSCs were enhanced upon downregulation of miR-488. The aforementioned findings provided evidence supporting that downregulation of miR-488 promotes odontoblastic differentiation of hDPSCs through the p38 MAPK signaling pathway by targeting MAPK1, paving the basis for further study about hDPSCs.
Asunto(s)
Diferenciación Celular , Pulpa Dental/enzimología , MicroARNs/metabolismo , Odontoblastos/enzimología , Células Madre/enzimología , Calcificación de Dientes , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Pulpa Dental/citología , Regulación hacia Abajo , Activación Enzimática , Células HEK293 , Humanos , MicroARNs/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Transducción de SeñalRESUMEN
As one type of adult stem cells (ASCs), human dental pulp stem cells (HDPSCs) have several properties, including high proliferation rate, selfrenewal capability, and multilineage differentiation. However, the apoptotic mechanism underlying the development of dental pulp cells remains unclear. In the present study, a significant increase of apoptosis was observed in HDPSCs from the deciduous teeth compared with that from adult permanent teeth. In addition, the occurrence of cytochrome c expression and mitochondrialmediated apoptosis pathway activity in HDPSCs were confirmed by quantitative polymerase chain reaction, and western blotting. Although caspase8 and caspase9 showed higher expression in deciduous teeth than in adult permanent teeth, only the knockdown of caspase9 via RNA interference in HDPSC cells exhibited a significant reduction in apoptosis, and caspase3 expression and activity. All these results revealed that caspase9 and activated caspase3 predominantly regulates cell apoptosis in HDPSCs from deciduous teeth.
Asunto(s)
Células Madre Adultas/enzimología , Apoptosis , Caspasa 9/biosíntesis , Pulpa Dental/enzimología , Regulación Enzimológica de la Expresión Génica , Diente Primario/enzimología , Adolescente , Adulto , Células Madre Adultas/citología , Caspasa 3/biosíntesis , Niño , Pulpa Dental/citología , Femenino , Humanos , Masculino , Diente Primario/citologíaRESUMEN
AIM: To assess the effects of 2-hydroxyethyl methacrylate (HEMA) on proliferation and migration of human pulp cells, as well as on matrix metalloproteinase (MMP-2 and MMP-9) expression in human odontoblast-like cells, contributing to the goal of determining the relationship between resin materials and MMP activity in pulp-dentine complexes. METHODOLOGY: Dental pulp cell cultures were established from pulp tissue of human teeth extracted for orthodontic purposes. Pulp cell differentiation was characterized in the presence of dentine sialophosphoprotein, bone sialoprotein and alkaline phosphatase by reverse transcription polymerase chain reaction. MMP activity was assessed by gelatine zymography with media containing HEMA. Cell viability was evaluated using methyl thiazolyl tetrazolium assay for 24-72 h. Cell migration was tested using Transwell migration assay. Western blotting was used to visualize MMP expression with the nontoxic HEMA concentrations (0-400 µg mL-1 ) for 48 h. RESULTS: Pulp cell proliferation decreased with HEMA exposure in a time- and concentration-dependent manner. HEMA concentrations ≤400 µg mL-1 did not induce changes in cell viability at 48 h (P < 0.05). Pulp cells were induced to differentiate into odontoblast-like cells in media containing 5 mg mL-1 ascorbic acid and 10 mmol L-1 ß-sodium glycerophosphate for 3-4 weeks. After incubation with HEMA, dose-dependent inhibition was observed; HEMA had a strong inhibitory effect on MMP activity. Compared with the control group, cell migration and MMP expression were inhibited significantly with increasing HEMA concentration at noncytotoxic doses (P < 0.05). CONCLUSIONS: Cell viability was not affected at HEMA concentrations ≤400 µg mL-1 . Within this range, HEMA inhibited MMP-2 and MMP-9 expression and activity, which may protect against type I collagen degradation effectively during dentine adhesive procedures.
Asunto(s)
Cementos Dentales/farmacología , Pulpa Dental/citología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Metacrilatos/farmacología , Odontoblastos/citología , Adolescente , Adulto , Western Blotting , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Niño , Pulpa Dental/efectos de los fármacos , Pulpa Dental/enzimología , Humanos , Técnicas In Vitro , Metaloproteinasa 2 de la Matriz/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Odontoblastos/efectos de los fármacos , Odontoblastos/enzimología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
OBJECTIVES: Ten-eleven translocation 1 (TET1) is a DNA methylcytosine (mC) dioxygenase discovered recently that can convert 5-mC into 5-hydroxymethylcytosine (5hmC). We previously reported that TET1 promotes odontoblastic differentiation of human dental pulp cells (hDPCs). The gene encoding the family with sequence similarity 20, member C (FAM20C) protein, is a potential TET1 target and showed demethylation during odontoblastic differentiation of hDPCs in our previous study. This study aimed to explore whether TET1-mediated hydroxymethylation could activate the FAM20C gene, thereby regulating hDPC differentiation. MATERIALS AND METHODS: The expression pattern of FAM20C and its potential changes during odontoblastic induction of hDPCs were assessed by Western blotting. Lentivirus-mediated transduction with short hairpin RNA (shRNA) was used to knock down FAM20C and TET1 expression in hDPCs. The mineralization potential of hDPCs was evaluated with an ALPase activity assay and by observing the mineralized matrix deposition and the expression of odontoblast-related markers DSPP and DMP1. Recombinant human FAM20C protein (rhFAM20C) was reintroduced into shTET1 cells in a rescue experiment. The dynamic hydroxymethylation status of the FAM20C gene promoter was examined using hydroxymethylated DNA immunoprecipitation (IP)-PCR. Chromatin IP-PCR and agarose gel electrophoresis were utilized to validate the recruitment of TET1 to its target loci in the FAM20C promoter. RESULTS: FAM20C protein level was upregulated after the odontoblastic induction of hDPCs. shRNA-mediated FAM20C suppression reduced the expression of DSPP and DMP1 after odontoblastic induction for 7 and 14 days. ALPase activity was reduced on day 7, and the formation of mineralized nodules was attenuated on day 14 after odontoblastic induction in FAM20C-inhibited hDPCs. Genomic 5hmC levels significantly decreased, and total 5mC levels increased in TET1-deficient hDPCs. In addition, a significant reduction in FAM20C also emerged. The rhFAM20C treatment of shTET1 cells attenuated the mineralization abnormalities caused by TET1 depletion. TET1 depletion prompted a decline in 5hmC levels in several regions on the FAM20C promoter. Enhanced TET1 recruitment was detected at the corresponding loci in the FAM20C promoter during odontoblastic induction. CONCLUSION: TET1 knockdown suppressed odontoblastic differentiation by restraining its direct binding to FAM20C promoter, and hence inhibiting FAM20C hydroxymethylation and subsequent transcription. These results suggest that TET1 potentially promotes the cytodifferentiation potential of hDPCs through its DNA demethylation machinery and upregulation of FAM20C protein expression.
Asunto(s)
Calcificación Fisiológica , Quinasa de la Caseína I/biosíntesis , Diferenciación Celular , Pulpa Dental/enzimología , Proteínas de la Matriz Extracelular/biosíntesis , Regulación Enzimológica de la Expresión Génica , Oxigenasas de Función Mixta/biosíntesis , Odontoblastos/enzimología , Proteínas Proto-Oncogénicas/biosíntesis , Adolescente , Adulto , Quinasa de la Caseína I/genética , Pulpa Dental/citología , Proteínas de la Matriz Extracelular/genética , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Metilación , Oxigenasas de Función Mixta/genética , Odontoblastos/citología , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genéticaRESUMEN
OBJECTIVE: Recent studies suggested that head and neck radiotherapy increases active forms of matrix metalloproteinases (MMPs) in the dentin-enamel junction (DEJ), leading to enamel delamination and radiation-related caries. This study aimed to assess the expression and activity of the gelatinases MMP-2 and MMP-9 in the DEJ and dentin-pulp complex tissues of teeth irradiated in vivo. STUDY DESIGN: Thirty-six teeth were studied, including 19 irradiated and 17 non-irradiated controls. In situ zymography was used to investigate the gelatinolytic activity in the micromorphologic components of enamel, DEJ, dentin-pulp complex, and caries. Immunohistochemical analysis was conducted on the demineralized samples to assess MMP-2 and MMP-9 expression levels in the DEJ, dentin-pulp complex components, and caries. RESULTS: No statistically significant differences were detected between groups in gelatinolytic activity or in MMP-2 expression levels (P > .05). Odontoblast MMP-9 expression was reduced in the irradiated group (P = .02). CONCLUSIONS: The study rejected the hypothesis that MMP-2 and MMP-9 would be overexpressed or more activated in the DEJ and dentin-pulp complex of irradiated teeth. Direct effects of radiation should not be regarded as an independent factor for explaining radiation-related caries onset and progression.
Asunto(s)
Esmalte Dental/enzimología , Esmalte Dental/efectos de la radiación , Pulpa Dental/enzimología , Pulpa Dental/efectos de la radiación , Dentina/enzimología , Dentina/efectos de la radiación , Neoplasias de Cabeza y Cuello/radioterapia , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Diente/efectos de la radiación , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Purinergic signaling is involved in pain generation and modulation in the nociceptive sensory nervous system. Adenosine triphosphate (ATP) induces pain via activation of ionotropic P2X receptors while adenosine mediates analgesia via activation of metabotropic P1 receptors. These purinergic signaling are determined by ecto-nucleotidases that control ATP degradation and adenosine generation. Using enzymatic histochemistry, we detected ecto-AMPase activity in dental pulp, trigeminal ganglia (TG) neurons, and their nerve fibers. Using immunofluorescence staining, we confirmed the expression of ecto-5'-nucleotidase (CD73) in trigeminal nociceptive neurons and their axonal fibers, including the nociceptive nerve fibers projecting into the brainstem. In addition, we detected the existence of CD73 and ecto-AMPase activity in the nociceptive lamina of the trigeminal subnucleus caudalis (TSNC) in the brainstem. Furthermore, we demonstrated that incubation with specific anti-CD73 serum significantly reduced the ecto-AMPase activity in the nociceptive lamina in the brainstem. Our results indicate that CD73 might participate in nociceptive modulation by affecting extracellular adenosine generation in the trigeminal nociceptive pathway. Disruption of TG neuronal ecto-nucleotidase expression and axonal terminal localization under certain circumstances such as chronic inflammation, oxidant stress, local constriction, and injury in trigeminal nerves may contribute to the pathogenesis of orofacial neuropathic pain.
Asunto(s)
5'-Nucleotidasa/metabolismo , 5'-Nucleotidasa/fisiología , Adenosina Trifosfatasas/metabolismo , Axones/enzimología , Tronco Encefálico/enzimología , Pulpa Dental/enzimología , Fibras Nerviosas/enzimología , Núcleo Caudal del Trigémino/enzimología , Nervio Trigémino/enzimología , Adolescente , Animales , Pulpa Dental/citología , Dolor Facial/enzimología , Técnica del Anticuerpo Fluorescente , Humanos , Hidrólisis , Tercer Molar , Transducción de Señal , Coloración y Etiquetado , Adulto JovenRESUMEN
Recent evidence suggests that head-and-neck radiotherapy (HNRT) increases active forms of matrix metalloproteinase-20 (MMP-20) in human tooth crowns, degrading the dentin-enamel junction (DEJ) and leading to enamel delamination, which is a pivotal step in the formation of radiation-related caries (RRC). Additional participation of enzymatic degradation of organic matrix components in caries progression was attributed to MMP-20 in dentin. Therefore, the current study tested the hypothesis that MMP-20 is overexpressed in the DEJ, dentin-pulp complex components, and carious dentin of post-HNRT patients, leading to detectable micromorphological changes to the enamel and dentin. Thirty-six teeth were studied, including 19 post-HNRT specimens and 17 nonirradiated controls. Optical light microscopy was used to investigate the micromorphological components of the DEJ, dentin-pulp complex components, and carious dentin. The samples were divided into 2 subgroups: nondemineralized ground sections (n = 20) and demineralized histological sections (n = 16). In addition, immunohistochemical analysis using the immunoperoxidase technique was conducted to semiquantitatively assess MMP-20 expression in the DEJ, dentin-pulp complex components, and carious dentin. No apparent damage to the DEJ microstructure or other dentin-pulp complex components was observed and no statistically significant differences were detected in MMP-20 expression (p > 0.05) between the irradiated and control groups. This study rejected the hypothesis that MMP-20 is overexpressed in the DEJ, dentin-pulp complex components, and carious dentin of post-HNRT patients, leading to detectable micromorphological changes. Hence, direct effects of radiation may not be regarded as an independent factor to explain aggressive clinical patterns of RRC.
Asunto(s)
Caries Dental/etiología , Pulpa Dental/efectos de la radiación , Dentina/efectos de la radiación , Neoplasias de Cabeza y Cuello/radioterapia , Metaloproteinasa 20 de la Matriz/metabolismo , Cuello del Diente/efectos de la radiación , Adulto , Anciano , Caries Dental/enzimología , Pulpa Dental/enzimología , Dentina/enzimología , Progresión de la Enfermedad , Femenino , Humanos , Técnicas para Inmunoenzimas , Masculino , Persona de Mediana Edad , Factores de Riesgo , Cuello del Diente/enzimologíaRESUMEN
PIN1, which belongs to a family of prolyl isomerases, specifically binds to phosphorylated Ser/Thr-pro motifs to catalytically regulate the post-phosphorylation conformation of its substrates. This study aimed to investigate the importance of Pin1 expression in human dental pulp cells (hDPCs) to understand the involvement of Pin1 in the regulation of P2Y1 and the activation of ADP-mediated P2Y1 signaling. This study found that the protein levels of P2Y1 gradually decreased after the onset of cell recovery following heat stress. Interestedly, hDPC migration significantly decreased during the recovery period. An in vitro study revealed that the silencing of PIN1 by siRNA or the pharmacologic inhibition of its activity decreased the migration of P2Y1 and P2Y1 expression in these cells. In addition, we found that Pin1 directly interacts with S252 of P2Y1 and that its binding stabilizes the P2Y1 protein to increase migration activity. These results strongly suggest that Pin1 mediates cell migration by stabilizing P2Y1 and that the Pin1/P2Y1 signaling pathways might serve as a novel mechanism of cell migration progression in hDPCs.
Asunto(s)
Adenosina Difosfato/farmacología , Movimiento Celular/efectos de los fármacos , Pulpa Dental/efectos de los fármacos , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Agonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y1/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Pulpa Dental/citología , Pulpa Dental/enzimología , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Respuesta al Choque Térmico , Humanos , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Estabilidad Proteica , Interferencia de ARN , Receptores Purinérgicos P2Y1/genética , Receptores Purinérgicos P2Y1/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , TransfecciónRESUMEN
Nitric oxide (NO) possibly plays an important role in the events resulting in hyperalgesia. NO synthase (NOS) is a key enzyme in the production of NO. Changes in NOS expression in primary sensory neurons may be involved in the persistent sensory abnormalities that can be induced by inflammation. To assess the possible roles of NOS in trigeminal sensory system, we studied changes in the expression of NOS isoforms in the trigeminal ganglion (TG) following chronic inflammation after pulp exposure (PX) in rats. The neurons innervating injured tooth in the TG were labeled by fluoro-gold (FG). Immunohistochemical staining was used to reveal the presence of NOS. The results showed that within the FG-labeled population, neuron counts revealed a significant increase in the proportion of NOS neurons following PX, in which the frequency of iNOS and nNOS-positive neurons started to increase at 3 and 7day, respectively, and peaked at 28day. There was no eNOS expression observed in the control group and PX-treated groups. The results demonstrate that PX-induced chronic pulpal inflammation results in significant increase of nNOS and iNOS in the TG. It suggests that nNOS and iNOS could be involved in mediation of peripheral processing of nociceptive information following chronic tooth pulp inflammation.
Asunto(s)
Pulpa Dental/enzimología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Ganglio del Trigémino/enzimología , Animales , Enfermedad Crónica , Inflamación/enzimología , Isoenzimas/metabolismo , Masculino , Neuronas/enzimología , Ratas Sprague-DawleyRESUMEN
AIM: To evaluate catalase (CAT, EC 1.11.1.6) activity in healthy and inflamed dental pulp of young patient's teeth and to investigate if an active defense system oxidizing agents is present as a response to bacterial invasion. MATERIALS AND METHODS: Twenty young patients between 15 and 25 ages, who were diagnosed to be healthy, were the source of the pulp tissue. The situation of the dental pulps was evaluated using clinical and radiographic assessments. The patients were divided two groups from healthy, and inflamed pulp tissues were obtained; each participant provided one pulp tissue specimens. The specimens were collected during endodontic treatment or by longitudinally grooving and splitting the teeth (if extracted). Catalase activity was determined through spectrophotometric methods and an independent sample t-test assessed the significance of differences between the groups. RESULTS: There was statistically a difference between healthy pulp tissue and inflamed pulp tissue (P < 0.005, independent sample t-test). The catalase activity of healthy group was significantly lower than inflamed pulp groups. CONCLUSION: The present study has shown that a significant increase in catalase activity is determined in inflamed dental pulps, which is due to pulpitis in comparison to healthy dental pulp.
Asunto(s)
Catalasa , Pulpa Dental/enzimología , Pulpitis/enzimología , Adolescente , Adulto , Catalasa/análisis , Catalasa/metabolismo , Pulpa Dental/diagnóstico por imagen , Humanos , Pulpitis/diagnóstico por imagen , Adulto JovenRESUMEN
OBJECTIVE: The aim of the present study was to determine the influence of Notch ligands, Jagged-1 and Dll-1, on osteogenic differentiation by stem cells from human exfoliated deciduous teeth. DESIGN: Notch ligands were immobilized on tissue culture surface using an indirect affinity immobilization technique. Cells from the remaining of dental pulp tissues from human deciduous teeth were isolated and characterized using flow cytometry and differentiation assay. Alkaline phosphatase (ALP) enzymatic activity, osteogenic marker gene expression, and mineralization were determined using ALP assay, real-time polymerase chain reaction, and alizarin red staining, respectively. RESULTS: The isolated cells exhibited CD44, CD90, and CD105 expression but lack of CD45 expression. Further, these cells were able to differentiate toward osteogenic lineage. The upregulation of HES-1 and HEY-1 was observed in those cells on Dll-1 and Jagged-1 coated surface. The significant increase of ALP activity and mineralization was noted in those cells seeded on Jagged-1 surface and these results were attenuated when cells were pretreated with gamma secretase inhibitor. The significant upregulation of ALP and collagen type I gene expression was also observed in those cells seeded on Jagged-1 surface. The inconsistent Dll-1 induced osteogenic differentiation was found and high Dll-1 immobilized dose (50 nM) slightly enhanced alkaline phosphatase enzymatic activity. However, the statistical significant difference was not obtained as compared to the hFc control. CONCLUSION: The surface immobilization of Notch ligands, Jagged-1 and Dll-1, likely to enhance osteogenic differentiation of SHEDs. However, Jagged-1 had more ability in enhancing osteogenic differentiation than Dll-1 in our model.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/farmacología , Proteína Jagged-1/farmacología , Osteogénesis/efectos de los fármacos , Células Madre/citología , Células Madre/efectos de los fármacos , Diente Primario/citología , Diente Primario/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Calcificación Fisiológica , Proteínas de Ciclo Celular/genética , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colágeno Tipo I/genética , Pulpa Dental/citología , Pulpa Dental/efectos de los fármacos , Pulpa Dental/enzimología , Pulpa Dental/metabolismo , Humanos , Proteínas Inmovilizadas/farmacología , Péptidos y Proteínas de Señalización Intercelular/genética , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Osteogénesis/genética , Células Madre/enzimología , Células Madre/metabolismo , Técnicas de Cultivo de Tejidos/métodos , Diente Primario/enzimología , Factor de Transcripción HES-1/genética , Regulación hacia ArribaRESUMEN
INTRODUCTION: Differentiation between reversible pulpitis (savable pulp) and irreversible inflammation of the pulp tissue (nonsavable pulp) based only on clinical and radiographic diagnoses has proven to be difficult. Pulp exposure allows for the collection of pulpal blood to quantitatively determine the level of inflammation markers or proteolytic enzymes, even with small samples. Pulpitis is associated with the invasion of neutrophil granulocytes and their release of matrix metalloproteinase-9 (MMP-9). METHODS: Forty-four patients (aged 18-74 years, mean = 35 years), each with 1 tooth with carious pulp exposure presenting with different stages of pulpitis, were included in this prospective, 2-center clinical study; 26 patients presented with irreversible pulpitis (groups 3 and 4), 10 with reversible pulpitis (group 2), and 8 with completely asymptomatic teeth with deep carious lesions (group 1). Six of the 26 patients with teeth diagnosed with irreversible pulpitis had not taken any nonsteroidal anti-inflammatory drugs and were evaluated as a separate group (group 4). Partial pulpotomy and blood sample collection from the pulp chamber were performed. The total levels of MMP-9 and tissue inhibitor of metalloproteinase-1 were assessed by fluorometric and colorimetric enzyme-linked immunosorbent assays, respectively. The Mann-Whitney U test and Spearman rank correlations were used to compare the MMP-9 levels with different stages of pulpal inflammation; significance was set at .05. RESULTS: The MMP-9 levels in the asymptomatic teeth (group 1) were significantly different from those in the teeth with reversible pulpitis (group 2, P = .006) or irreversible pulpitis (group 4, P < .001). A statistically significant difference was also observed between the MMP-9 levels in group 1 and group 3 (P < .001) in which the patients had taken nonsteroidal anti-inflammatory drugs. CONCLUSIONS: These findings indicate that the MMP-9 levels in pulpal blood samples could be a useful ancillary diagnostic tool for distinguishing different stages of pulp tissue inflammation.
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Pulpa Dental/irrigación sanguínea , Metaloproteinasa 9 de la Matriz/sangre , Pulpitis/sangre , Adolescente , Adulto , Anciano , Biomarcadores/sangre , Pulpa Dental/enzimología , Pulpa Dental/patología , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Estudios Prospectivos , Pulpitis/tratamiento farmacológico , Pulpitis/patología , Inhibidor Tisular de Metaloproteinasa-1/sangre , Adulto JovenRESUMEN
Direct application of histone-deacetylase-inhibitors (HDACis) to dental pulp cells (DPCs) induces chromatin changes, promoting gene expression and cellular-reparative events. We have previously demonstrated that HDACis (valproic acid, trichostatin A) increase mineralization in dental papillae-derived cell-lines and primary DPCs by stimulation of dentinogenic gene expression. Here, we investigated novel genes regulated by the HDACi, suberoylanilide hydroxamic acid (SAHA), to identify new pathways contributing to DPC differentiation. SAHA significantly compromised DPC viability only at relatively high concentrations (5 µM); while low concentrations (1 µM) SAHA did not increase apoptosis. HDACi-exposure for 24 h induced mineralization-per-cell dose-dependently after 2 weeks; however, constant 14d SAHA-exposure inhibited mineralization. Microarray analysis (24 h and 14 days) of SAHA exposed cultures highlighted that 764 transcripts showed a significant >2.0-fold change at 24 h, which reduced to 36 genes at 14 days. 59% of genes were down-regulated at 24 h and 36% at 14 days, respectively. Pathway analysis indicated SAHA increased expression of members of the matrix metalloproteinase (MMP) family. Furthermore, SAHA-supplementation increased MMP-13 protein expression (7 d, 14 days) and enzyme activity (48 h, 14 days). Selective MMP-13-inhibition (MMP-13i) dose-dependently accelerated mineralization in both SAHA-treated and non-treated cultures. MMP-13i-supplementation promoted expression of several mineralization-associated markers, however, HDACi-induced cell migration and wound healing were impaired. Data demonstrate that short-term low-dose SAHA-exposure promotes mineralization in DPCs by modulating gene pathways and tissue proteases. MMP-13i further increased mineralization-associated events, but decreased HDACi cell migration indicating a specific role for MMP-13 in pulpal repair processes. Pharmacological inhibition of HDAC and MMP may provide novel insights into pulpal repair processes with significant translational benefit. J. Cell. Physiol. 231: 798-816, 2016. © 2015 Wiley Periodicals, Inc.
Asunto(s)
Pulpa Dental/enzimología , Pulpa Dental/patología , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Metaloproteinasa 13 de la Matriz/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Calcificación Fisiológica/efectos de los fármacos , Calcificación Fisiológica/genética , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , ADN Complementario/genética , Regulación de la Expresión Génica/efectos de los fármacos , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Factores de Tiempo , VorinostatRESUMEN
INTRODUCTION: Alkaline phosphatase (ALP) plays an important role in inducing mineralization events in the dental pulp. This study investigated and compared the ALP levels in healthy and inflamed pulp in young and old human pulp. METHODS: Tissue samples were collected from young (<30 years) and old (>60 years) donors. In both age groups, healthy human pulp (n = 18) were collected from extracted wisdom teeth. For reversible and irreversible pulpitis, pulp samples (n = 18 each) were obtained during endodontic treatment. ALP activity was assessed by spectrophotometry and immunhistochemistry. RESULTS: Regardless of age, reversible pulpitis group samples showed a slight elevation in ALP activity compared with normal healthy pulp. In elderly patients, ALP expression with irreversible pulpitis was significantly higher than those with a healthy pulp (P < .05). CONCLUSIONS: In the hyperemic state, both the young and old pulp shows a slight increase in ALP activity, whereas in irreversible pulpitis, only the old pulp shows significantly elevated ALP levels. Such an increase may trigger calcification events, which may eventually cause difficulties in endodontic treatment procedures in elderly individuals.
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Envejecimiento/metabolismo , Fosfatasa Alcalina/metabolismo , Pulpa Dental/enzimología , Pulpitis/enzimología , Adolescente , Adulto , Anciano , Humanos , Persona de Mediana Edad , Adulto JovenRESUMEN
Dental pulp is a dynamic tissue able to resist external irritation during tooth decay by using immunocompetent cells involved in innate and adaptive responses. To better understand the immune response of pulp toward gram-negative bacteria, we analyzed biological mediators and immunocompetent cells in rat incisor pulp experimentally inflamed by either lipopolysaccharide (LPS) or saline solution (phosphate-buffered saline [PBS]). Untreated teeth were used as control. Expression of pro- and anti-inflammatory cytokines, chemokine ligands, growth factors, and enzymes were evaluated at the transcript level, and the recruitment of the different leukocytes in pulp was measured by fluorescence-activated cell-sorting analysis after 3 h, 9 h, and 3 d post-PBS or post-LPS treatment. After 3 d, injured rat incisors showed pulp wound healing and production of reparative dentin in both LPS and PBS conditions, testifying to the reversible pulpitis status of this model. IL6, IL1-ß, TNF-α, CCL2, CXCL1, CXCL2, MMP9, and iNOS gene expression were significantly upregulated after 3 h of LPS stimulation as compared with PBS. The immunoregulatory cytokine IL10 was also upregulated after 3 h, suggesting that LPS stimulates not only inflammation but also immunoregulation. Fluorescence-activated cell-sorting analysis revealed a significant, rapid, and transient increase in leukocyte levels 9 h after PBS and LPS stimulation. The quantity of dendritic cells was significantly upregulated with LPS versus PBS. Interestingly, we identified a myeloid-derived suppressor cell-enriched cell population in noninjured rodent incisor dental pulp. The percentage of this population, known to regulate immune response, was higher 9 h after inflammation triggered with PBS and LPS as compared with the control. Taken together, these data offer a better understanding of the mechanisms involved in the regulation of dental pulp immunity that may be elicited by gram-negative bacteria.
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Pulpa Dental/inmunología , Pulpitis/inmunología , Linfocitos T/inmunología , Animales , Quimiocina CCL2/análisis , Quimiocina CXCL1/análisis , Quimiocinas/análisis , Citocinas/análisis , Células Dendríticas/patología , Pulpa Dental/enzimología , Dentina Secundaria/inmunología , Modelos Animales de Enfermedad , Femenino , Bacterias Gramnegativas/inmunología , Mediadores de Inflamación/análisis , Interleucina-10/análisis , Interleucina-1beta/análisis , Interleucina-6/análisis , Leucocitos/clasificación , Lipopolisacáridos/inmunología , Metaloproteinasa 9 de la Matriz/análisis , Óxido Nítrico Sintasa de Tipo II/análisis , Pulpitis/enzimología , Ratas , Ratas Sprague-Dawley , Linfocitos T Reguladores/patología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Camphorquinone (CQ) is a popularly-used photosensitizer in composite resin restoration. In this study, the effects of CQ on cytotoxicity and inflammation-related genes and proteins expression of pulp cells were investigated. The role of reactive oxygen species (ROS), ATM/Chk2/p53 and hemeoxygenase-1 (HO-1) and MEK/ERK signaling was also evaluated. We found that ROS and free radicals may play important role in CQ toxicity. CQ (1 and 2 mM) decreased the viability of pulp cells to about 70% and 50% of control, respectively. CQ also induced G2/M cell cycle arrest and apoptosis of pulp cells. The expression of type I collagen, cdc2, cyclin B, and cdc25C was inhibited, while p21, HO-1 and cyclooxygenase-2 (COX-2) were stimulated by CQ. CQ also activated ATM, Chk2, and p53 phosphorylation and GADD45α expression. Besides, exposure to CQ increased cellular ROS level and 8-isoprostane production. CQ also stimulated COX-2 expression and PGE2 production of pulp cells. The reduction of cell viability caused by CQ can be attenuated by N-acetyl-L-cysteine (NAC), catalase and superoxide dismutase (SOD), but can be promoted by Zinc protoporphyin (ZnPP). CQ stimulated ERK1/2 phosphorylation, and U0126 prevented the CQ-induced COX-2 expression and prostaglandin E2 (PGE2) production. These results indicate that CQ may cause cytotoxicity, cell cycle arrest, apoptosis, and PGE2 production of pulp cells. These events could be due to stimulation of ROS and 8-isoprostane production, ATM/Chk2/p53 signaling, HO-1, COX-2 and p21 expression, as well as the inhibition of cdc2, cdc25C and cyclin B1. These results are important for understanding the role of ROS in pathogenesis of pulp necrosis and pulpal inflammation after clinical composite resin filling.
Asunto(s)
Alcanfor/análogos & derivados , Pulpa Dental/efectos de los fármacos , Pulpa Dental/metabolismo , Dinoprostona/biosíntesis , Adulto , Apoptosis/efectos de los fármacos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Alcanfor/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Quinasa de Punto de Control 2/metabolismo , Pulpa Dental/citología , Pulpa Dental/enzimología , Dinoprost/análogos & derivados , Dinoprost/biosíntesis , Hemo-Oxigenasa 1/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Cultivo Primario de Células , Especies Reactivas de Oxígeno/metabolismo , Adulto JovenRESUMEN
AIMS: Dental pulp is the specialized tissue responsible for maintaining tooth viability. When tooth mineralized matrix is damaged, pulp is exposed to a plethora of environmental stimuli. In particular, in smokers, pulp become exposed to very high concentrations of nicotine. The aim of this study was to investigate the effect of direct nicotine stimulation on human dental pulp cell proliferation. Moreover, as it is known that nicotine could upregulate the expression of matrix metalloproteinases (MMPs), enzymes involved in pulpal inflammation, the effects of nicotine stimulation on MMP-2 and MMP-28 gene expression have also been investigated. MAIN METHODS: Human dental pulp cells were extracted from impacted third molars obtained from healthy patients undergoing routine orthodontic treatments. Such cells were treated with growing concentrations of nicotine in the presence or absence of a nicotine antagonist (hexamethonium chloride) or of a MEK signaling inhibitor (PD98059). Cell proliferation was evaluated by cell counting, while nicotine effects on MMP expression were evaluated by PCR. KEY FINDINGS: The data obtained indicate that nicotine is able to increase human dental pulp cell proliferation by acting through nicotinic cholinergic receptors and downstream MAPK signaling pathway. Moreover, it is also able to increase both MMP-2 and MMP-28 gene expression. SIGNIFICANCE: In summary these results highlight that direct exposure of human dental pulp cells to nicotine results in an inflammatory response, that could have a role in pulpal inflammation onset, a pathological condition that, when ignored, could eventually spread to the surrounding alveolar bone and progress to pulp necrosis.