RESUMO
Dental pain arises from exposed dentin following bacterial, chemical, or mechanical erosion of enamel and/or recession of gingiva. Thus, dentin tissue and more specifically patent dentinal tubules represent the first structure involved in dentin sensitivity. Interestingly, the architecture of dentin could allow for the transfer of information to the underlying dental pulp via odontoblasts (dentin-forming cells), via their apical extension bathed in the dentinal fluid running in the tubules, or via a dense network of trigeminal sensory axons intimately related to odontoblasts. Therefore, external stimuli causing dentinal fluid movements and odontoblasts and/or nerve complex responses may represent a unique mechanosensory system bringing a new role for odontoblasts as sensor cells. How cells sense signals and how the latter are transmitted to axons represent the main questions to be resolved. However, several lines of evidence have demonstrated that odontoblasts express mechano- and/or thermosensitive transient receptor potential ion channels (TRPV1, TRPV2, TRPV3, TRPV4, TRPM3, KCa, TREK-1) that are likely to sense heat and/or cold or movements of dentinal fluid within tubules. Added to this, voltage-gated sodium channels confer excitable properties of odontoblasts in vitro in response to injection of depolarizing currents. In vivo, sodium channels co-localize with nerve terminals at the apical pole of odontoblasts and correlate with the spatial distribution of stretch-activated KCa channels. This highlights the terminal web as the pivotal zone of the pulp/dentin complex for sensing external stimuli. Crosstalk between odontoblasts and axons may take place by the release of mediators in the gap space between odontoblasts and axons in view of evidence for nociception-transducing receptors on trigeminal afferent fibers and expression of putative effectors by odontoblasts. Finally, how axons are guided to the target cells and which kind of signaling molecules are involved is extensively discussed in this review.
Assuntos
Sensibilidade da Dentina/fisiopatologia , Odontoblastos/fisiologia , Odontalgia/fisiopatologia , Axônios/fisiologia , Cílios/fisiologia , Polpa Dentária/inervação , Líquido Dentinal/fisiologia , Humanos , Canais de Potássio Cálcio-Ativados/fisiologia , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Pulpite/fisiopatologia , Transdução de Sinais , Canais de Sódio/fisiologia , Canais de Potencial de Receptor Transitório/fisiologia , Nervo Trigêmeo/citologiaRESUMO
Odontoblasts are organized as a single layer of specialized cells responsible for dentine formation and presumably for playing a role in tooth pain transmission. Each cell has an extension running into a dentinal tubule and bathing in the dentinal fluid. A dense network of sensory unmyelinated nerve fibers surrounds the cell bodies and processes. Thus, dentinal tubules subjected to external stimuli causing dentinal fluid movements and odontoblasts/nerve complex response may represent a unique mechano-sensory system giving to dentine-forming cells a pivotal role in signal transduction. Mediators of mechano-transduction identified in odontoblast include mechano-sensitive ion channels (high conductance calcium-activated potassium channel--K(Ca)--and a 2P domain potassium channel--TREK-1) and primary cilium. In many tissues, the latter is essential for microenvironment sensing but its role in the control of odontoblast behavior remains to be elucidated. Recent evidence for excitable properties and the concentration of key channels to the terminal web suggest that odontoblasts may operate as sensor cells.
Assuntos
Odontoblastos/fisiologia , Actinas/fisiologia , Encéfalo/fisiologia , Moléculas de Adesão Celular Neuronais/fisiologia , Células Cultivadas , Polpa Dentária/citologia , Polpa Dentária/inervação , Polpa Dentária/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Humanos , Proteínas do Tecido Nervoso/fisiologia , Odontoblastos/citologia , Proteína Reelina , Semaforinas/fisiologia , Serina Endopeptidases/fisiologia , Estresse Mecânico , Odontalgia/fisiopatologiaRESUMO
Members of the TGF-beta/BMP family of growth factors induce odontoblast differentiation and reparative dentin synthesis, and their use has been proposed to stimulate pulp healing during dental therapeutics in human. However, factors that modulate TGF-beta and/or BMP signalling during odontoblast differentiation and physiology remain largely unknown. To identify them, we compared expression profiles of TGF-beta/BMP-related genes in pulp fibroblast- and odontoblast-like cells cultured from human dental pulp explants using cDNA gene arrays. We evidenced that the gene encoding ecotropic viral integration site-1 (EVI1), a transcription factor that inhibits TGF-beta/BMP signalling, was under-expressed in odontoblast-like cells. This result was verified by real-time PCR and, at the protein level, by immunohistochemistry. In vivo, real-time PCR analysis revealed that EVI1 was expressed in the dental pulp, at a level similar to brain, but lower than in lung, kidney or trachea. The protein was localized in dental pulp samples in pulp core and subodontoblast cells. Staining intensity progressively decreased from the radicular to the coronal pulp where EVI1 staining was almost undetectable in odontoblasts. Our data suggest that fine regulation of the EVI1 level in the human dental pulp might be important in the TGF-beta/BMP-induced modulation of dental pulp cell kinetics and/or odontoblast differentiation.
Assuntos
Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas de Ligação a DNA/análise , Polpa Dentária/metabolismo , Fatores de Transcrição/análise , Fator de Crescimento Transformador beta/antagonistas & inibidores , Adolescente , Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Polpa Dentária/citologia , Regulação para Baixo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Imuno-Histoquímica , Proteína do Locus do Complexo MDS1 e EVI1 , Odontoblastos/citologia , Odontoblastos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Proto-Oncogenes/genética , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
Nerve growth factor (NGF) is a key regulator of the development and differentiation of neuronal and non-neuronal cells. In the present study we examined the distribution of NGF and its low and high-affinity receptors, p75NTR and TrkA respectively, in permanent human teeth under normal and pathological conditions. In intact functional teeth, NGF, p75NTR and TrkA are weakly expressed in dental pulp fibroblasts and odontoblasts that are responsible for dentine formation, while the NGF and p75NTR molecules are strongly expressed in nerve fibres innervating the dental pulp. In carious and injured teeth NGF and TrkA expression is upregulated in a selective manner in odontoblasts surrounding the injury sites, indicating a link between NGF signalling and dental tissue repair events. Accordingly, NGF and TrkA expression is strongly upregulated in cultured primary human dental mesenchymal cells during their differentiation into odontoblasts. Targeted release of NGF in cultured human tooth slices induced extensive axonal growth and migration of Schwann cells towards the NGF administration site. These results show that NGF signalling is strongly linked to pathological and regenerative processes in human teeth and suggest a potential role for this neurotrophic molecule in pulp regeneration.
Assuntos
Fator de Crescimento Neural/metabolismo , Regeneração , Dente/metabolismo , Dente/patologia , Adolescente , Adulto , Axônios/efeitos dos fármacos , Axônios/fisiologia , Células Cultivadas , Criança , Polpa Dentária/inervação , Polpa Dentária/metabolismo , Fibroblastos/metabolismo , Humanos , Pessoa de Meia-Idade , Fator de Crescimento Neural/administração & dosagem , Regeneração Nervosa , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/fisiologia , Odontoblastos/metabolismo , Receptor trkA/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Transdução de Sinais , CicatrizaçãoRESUMO
Semaphorin 7A (SEMA 7A) is a membrane-anchored member of the semaphorin family of guidance proteins, previously identified in the immune system. Expressed in central and peripheral nervous system during embryonic and post-natal stages, it can mediate neuronal functions by promoting axonal growth. We show here that SEMA 7A is expressed in human odontoblasts in vivo and in vitro and that its expression is correlated with the establishment of dentin-pulp complex terminal innervation . Co-cultures of trigeminal ganglion (TG) with COS cells overexpressing SEMA 7A demonstrate that SEMA 7A can promote the growth of trigeminal nerve fibers. Finally, by RT-PCR and immunochemistry, we show that beta1-integrin, a SEMA 7A putative receptor, is expressed in pulpal nerve fibers but we failed to detect a co-localization between nerves and odontoblasts through these molecules. On the basis of these data, we suggest that SEMA 7A might be a molecule involved in the terminal innervation of the dentin-pulp complex.
Assuntos
Antígenos CD/metabolismo , Dentina/inervação , Odontoblastos/metabolismo , Semaforinas/metabolismo , Adolescente , Animais , Animais Recém-Nascidos/metabolismo , Antígenos CD/genética , Células COS/metabolismo , Células Cultivadas , Chlorocebus aethiops , Técnicas de Cocultura , Polpa Dentária/citologia , Polpa Dentária/inervação , Embrião de Mamíferos/metabolismo , Proteínas Ligadas por GPI , Humanos , Integrina beta1/metabolismo , Fibras Nervosas/metabolismo , Fibras Nervosas/fisiologia , Fibras Nervosas/ultraestrutura , Sistema Nervoso/crescimento & desenvolvimento , Sistema Nervoso/metabolismo , Sistema Nervoso/ultraestrutura , Neuritos/fisiologia , Ratos , Ratos Sprague-Dawley , Semaforinas/genética , Distribuição Tecidual , Transfecção , Gânglio Trigeminal/citologia , Gânglio Trigeminal/fisiologiaRESUMO
Reelin is a large extracellular matrix (ECM) glycoprotein strongly expressed during embryonic development in the central nervous system and involved in architectonic brain development. It could participate in axon plasticity processes or adhesion-recognition between nerve fibers in adulthood. Previously identified from a subtractive cDNA library of fully differentiated human odontoblasts, reelin might be involved in the relationship between dental nerves and odontoblasts in as so far the latter are in close association with pulpal nerve fibers. Here, we show by in situ hybridization and immunohistochemistry that reelin is specifically expressed by human odontoblasts in vivo and in vitro and that an intense expression of the reelin gene is detected in odontoblasts in comparison with pulpal cells (PC). Co-cultures of rat trigeminal ganglion (TG) and odontoblasts allow to mimic odontoblast innervation and demonstrate that neurites contact these cells with reelin molecules as observed in vivo in human dental pulp. Moreover, by RT-PCR, we show that both reelin receptors (namely apolipoprotein E receptor [ApoER-2], very low density lipoprotein receptor [VLDLR] and cadherin-related neuronal receptor [CNR]) and the cytoplasmic adapter Disabled-1 implicated in the reelin signal transduction, were expressed by trigeminal ganglion. On the basis of these data, we suggest that reelin might be an extracellular matrix molecule involved in the terminal innervation of the dentin-pulp complex, promoting adhesion between dental nerve endings and odontoblasts.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Odontoblastos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adolescente , Animais , Células Cultivadas , Técnicas de Cocultura , Polpa Dentária/metabolismo , Humanos , Imuno-Histoquímica , Hibridização In Situ , Proteínas do Tecido Nervoso/metabolismo , Neuritos/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/metabolismo , Proteína Reelina , Serina Endopeptidases , Gânglio Trigeminal/fisiologiaRESUMO
OBJECTIVE: KLF4 and KLF5, members of the Krüppel-like factor (KLF) family, play key roles in proliferation, differentiation and apoptosis during development. In order to determine if these transcription factors are associated with tooth development, we examined the expression pattern of KLF4 and KLF5 during murine tooth development. DESIGN: In situ hybridization and immunohistochemistry were performed to detect the expression pattern of KLF4 and KLF5 from E12.5 to PN3 during murine tooth development. RESULTS: In situ hybridization analysis revealed that Klf4 was specifically expressed in polarizing odontoblasts from E16.5 (incisor) or E18.5 (first molar) to PN3. Immunohistochemistry staining showed that KLF4 was specifically expressed in both polarizing odontoblasts and ameloblasts at the same stages. KLF5 was mainly expressed from E18.5 to PN3 in secretory ameloblasts when enamel mineralization occurs and in secretory odontoblasts. However, an expression of KLF5 was also observed at earlier stages (E14.5 and E16.5) mainly in proliferating epithelial cells. CONCLUSIONS: These results suggest that the expression of KLF4 is closely correlated to the growth-arrest and the first step of odontoblast and ameloblast differentiation. Furthermore, KLF5 maybe involved in proliferation at the early stages of tooth development and related to mineralization of both enamel and dentin matrices at later stages.
Assuntos
Inibidores do Crescimento/análise , Fatores de Transcrição Kruppel-Like/análise , Odontogênese/genética , Dedos de Zinco/genética , Ameloblastos/fisiologia , Animais , Diferenciação Celular/genética , Proliferação de Células , Esmalte Dentário/citologia , Esmalte Dentário/embriologia , Polpa Dentária/citologia , Polpa Dentária/embriologia , Dentina/citologia , Dentina/embriologia , Células Epiteliais/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Inibidores do Crescimento/genética , Imuno-Histoquímica , Hibridização In Situ , Incisivo/citologia , Incisivo/embriologia , Antígeno Ki-67/análise , Antígeno Ki-67/genética , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Dente Molar/citologia , Dente Molar/embriologia , Odontoblastos/citologia , Fatores de Tempo , Calcificação de Dente/genética , Germe de Dente/embriologiaRESUMO
INTRODUCTION: Map-1B belongs to the family of proteins that govern the dynamic state and organization of microtubules within cells. MAP-1B is a microtubule-associated protein highly expressed during the development of the nervous system. Its expression, regulated by the fragile X mental retardation protein (FMRP), is essential to stabilize microtubules during the elongation of dendrites and neurites. Other microtubules-associated molecules such as tau or MAP2 seem to act similarly. The aim of this work was to identify the MAP-1B expression in in vitro and in vivo human odontoblasts during development and carious processes. The expression of MAP2 and tau was also studied. MATERIALS AND METHODS: In cultured cells, MAP-1B expression was analyzed by real-time polymerase chain reaction, flow cytometry, and Western blot. Its distribution was visualized by in situ hybridization and immunochemistry both in vitro and in vivo. The expression of FMRP, MAP2, and tau was identified by real-time polymerase chain reaction and immunochemistry. RESULTS: MAP-1B is specifically expressed in odontoblasts from adult third molars as well as incisor germs from human embryos. In adult carious teeth, it is also expressed in newly differentiated dentin-forming cells. In vitro, MAP-1B expression is related to the differentiation state of odontoblasts. MAP-1B clearly underlines the cellular architecture of cell bodies and processes of differentiated cells. FMRP, MAP2, and tau are also detected in vivo. CONCLUSION: On the basis of these data, MAP-1B could be considered as a new protein involved in the terminal differentiation of odontoblasts.
Assuntos
Cárie Dentária/metabolismo , Polpa Dentária/metabolismo , Proteínas Associadas aos Microtúbulos/biossíntese , Odontoblastos/citologia , Odontoblastos/metabolismo , Adolescente , Adulto , Biomarcadores , Diferenciação Celular , Células Cultivadas , Polpa Dentária/citologia , Dentina Secundária/metabolismo , Feto , Proteína do X Frágil da Deficiência Intelectual/biossíntese , Proteína do X Frágil da Deficiência Intelectual/fisiologia , Humanos , Imuno-Histoquímica , Dente Serotino/citologia , Dente Serotino/metabolismo , Neuritos/metabolismo , Odontogênese/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Germe de Dente/embriologia , Germe de Dente/metabolismo , Tubulina (Proteína)/biossíntese , Proteínas tau/biossínteseRESUMO
OBJECTIVES: Odontoblasts play a central role during the dentin formation by organic matrix production and mineralisation. Recently, suitable in vitro techniques for studying mature primary odontoblasts and the newly differentiated odontoblasts have been developed. Firstly, the gene expression profiles of native and cultured odontoblasts were compared at large-scale to investigate the similarities and differences between the samples. Secondly, differential expression levels of the genes encoding neuronal proteins were analyzed to study odontoblasts sensory function. DESIGN: Microarray analysis was performed to mature native and cultured pulp-derived odontoblast-like cells to compare their transcriptome. Then, the probes positive only in one sample were divided into gene ontology categories. Expression levels of selected neuronal proteins were further studied with quantitative PCR, and at the protein level by immunofluorescence of mature and newly differentiated odontoblasts in developing tooth. RESULTS: Remarkable similarities between the general and neuronal protein gene expression profiles were observed. Higher cortistatin, galanin, somatostatin receptor 1 (SSTR1) and tyrosine phosphatase receptor type Z1 (PTPRZ1) expression was detected in native than in cultured odontoblast at the mRNA level. Pronociceptin was more abundantly expressed in cultured than in native odontoblasts. Immunofluorescence of mature and newly differentiated odontoblasts on human tooth germs confirmed the results. CONCLUSIONS: Cultured odontoblasts used in this study have similar general gene expression pattern to native odontoblasts, and therefore offer a valuable tool for the in vitro odontoblast studies. The expression of PTPRZ1 and galanin, which participate in sensory signal transduction, supports the previously suggested role of odontoblasts as sensory cells.
Assuntos
Polpa Dentária/metabolismo , Neuropeptídeos/genética , Odontoblastos/metabolismo , Adolescente , Adulto , Células Cultivadas , Feminino , Galanina/genética , Perfilação da Expressão Gênica , Humanos , Masculino , Análise em Microsséries , Neuropeptídeos/metabolismo , Precursores de Proteínas/genética , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/genética , Receptores Opioides/genética , Receptores de Somatostatina/genética , Transdução de Sinais , Adulto JovemRESUMO
The Nav1.9 sodium channel is expressed in nociceptive DRG neurons where it contributes to spontaneous pain behavior after peripheral inflammation. Here, we used a newly developed antibody to investigate the distribution of Nav1.9 in rat and mouse trigeminal ganglion (TG) nerve endings and in enteric nervous system (ENS). In TGs, Nav1.9 was expressed in the soma of small- and medium-sized, peripherin-positive neurons. Nav1.9 was present along trigeminal afferent fibers and at terminals in lip skin and dental pulp. In the ENS, Nav1.9 was detected within the soma and proximal axons of sensory, Dogiel type II, myenteric and submucosal neurons. Immunological data were correlated with the detection of persistent TTX-resistant Na(+) currents sharing similar properties in DRG, TG and myenteric neurons. Collectively, our data support a potential role of Nav1.9 in the transmission of trigeminal pain and the regulation of intestinal reflexes. Nav1.9 might therefore constitute a molecular target for therapeutic treatments of orofacial pain and gastrointestinal syndromes.
Assuntos
Dor Facial/fisiopatologia , Plexo Mientérico/citologia , Neurônios Aferentes/fisiologia , Neuropeptídeos/genética , Canais de Sódio/genética , Plexo Submucoso/citologia , Gânglio Trigeminal/citologia , Sequência de Aminoácidos , Animais , Axônios/fisiologia , Polpa Dentária/inervação , Dor Facial/metabolismo , Lábio/inervação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.9 , Neurônios Aferentes/ultraestrutura , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Nociceptores/fisiologia , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Reflexo/fisiologia , Pele/inervação , Canais de Sódio/química , Canais de Sódio/metabolismoAssuntos
Canais de Cálcio/fisiologia , Sensibilidade da Dentina/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Odontoblastos/metabolismo , Canais de Cátion TRPM/fisiologia , Canais de Cátion TRPV/fisiologia , Sensação Térmica , Canais de Potencial de Receptor Transitório/fisiologia , Humanos , Canal de Cátion TRPA1RESUMO
Odontoblasts are responsible for the dentin formation. They are suspected to play a role in tooth pain transmission as sensor cells because of their close relationship with nerve, but this role has never been evidenced. We demonstrate here that human odontoblasts in vitro produce voltage-gated tetrodotoxin-sensitive Na(+) currents in response to depolarization under voltage clamp conditions and are able to generate action potentials. Odontoblasts express neuronal isoforms of alpha2 and beta2 subunits of sodium channels. Co-cultures of odontoblasts with trigeminal neurons indicate a clustering of alpha2 and beta2 sodium channel subunits and, at the sites of cell-cell contact, a co-localization of odontoblasts beta2 subunits with peripherin. In vivo, sodium channels are expressed in odontoblasts. Ankyrin(G) and beta2 co-localize, suggesting a link for signal transduction between axons and odontoblasts. Evidence for excitable properties of odontoblasts and clustering of key molecules at the site of odontoblast-nerve contact strongly suggest that odontoblasts may operate as sensor cells that initiate tooth pain transmission.
Assuntos
Odontoblastos/metabolismo , Dor , Odontalgia/metabolismo , Animais , Células Cultivadas , Técnicas de Cocultura , Eletrofisiologia , Cinética , Isoformas de Proteínas , Ratos , Ratos Sprague-Dawley , Sódio/metabolismo , Canais de Sódio/química , Tetrodotoxina/farmacologiaRESUMO
Gram-positive bacteria entering the dentinal tissue during the carious process are suspected to influence the immune response in human dental pulp. Odontoblasts situated at the pulp/dentin interface are the first cells encountered by these bacteria and therefore could play a crucial role in this response. In the present study, we found that in vitro-differentiated odontoblasts constitutively expressed the pattern recognition receptor TLR1-6 and 9 genes but not TLR7, 8, and 10. Furthermore, lipoteichoic acid (LTA), a wall component of Gram-positive bacteria, triggered the activation of the odontoblasts. LTA up-regulated the expression of its own receptor TLR2, as well as the production of several chemokines. In particular, an increased amount of CCL2 and CXCL10 was detected in supernatants from LTA-stimulated odontoblasts, and those supernatants augmented the migration of immature dendritic cells in vitro compared with controls. Clinical relevance of these observations came from immunohistochemical analysis showing that CCL2 was expressed in vivo by odontoblasts and blood vessels present under active carious lesions but not in healthy dental pulps. In contrast with this inflammatory response, gene expression of major dentin matrix components (type I collagen, dentin sialophosphoprotein) and TGF-beta1 was sharply down-regulated in odontoblasts by LTA. Taken together, these data suggest that odontoblasts activated through TLR2 by Gram-positive bacteria LTA are able to initiate an innate immune response by secreting chemokines that recruit immature dendritic cells while down-regulating their specialized functions of dentin matrix synthesis and mineralization.
Assuntos
Diferenciação Celular/imunologia , Quimiocinas/biossíntese , Dentina/metabolismo , Regulação para Baixo , Lipopolissacarídeos/farmacologia , Odontoblastos/metabolismo , Ácidos Teicoicos/farmacologia , Receptor 2 Toll-Like/biossíntese , Regulação para Cima , Células Cultivadas , Quimiocinas/genética , Quimiocinas/fisiologia , Células Dendríticas/imunologia , Dentina/imunologia , Regulação para Baixo/imunologia , Proteínas da Matriz Extracelular/metabolismo , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/imunologia , Humanos , Lipopolissacarídeos/metabolismo , Odontoblastos/citologia , Odontoblastos/imunologia , Técnicas de Cultura de Órgãos , Ácidos Teicoicos/metabolismo , Receptor 2 Toll-Like/genética , Regulação para Cima/imunologiaRESUMO
Previous reports have shown the expression of several mechanosensitive ionic channels on the plasma membrane in odontoblasts, which are the cells responsible for dentin formation. The membrane characteristics of odontoblasts imply that they could play critical roles in the mechano-transduction of fluid displacement within dentinal tubules into the electrical cell signals, to carry dentin sensation to the central nervous system. However, the direct ionic mechanism underlying such a dentin nociceptive function remains unclear. In the present study, we investigated the expression of the transient receptor potential vanilloid subfamily member 1 (TRPV1) channel--which essentially contributes to the detection of pain sensation--in rat odontoblasts by immunohistochemical and nystatin perforated patch-clamp techniques. Immunohistochemical observation showed the localization of TRPV1-immunoreactions on the distal regions of odontoblast membranes. In the patch-clamp experiments, we observed capsaicin-induced inward currents that were inhibited by capsazepine, a TRPV1 channel antagonist. Our results indicate a significant expression of TRPV1 channels in odontoblasts, suggesting that odontoblasts may directly respond to noxious stimuli such as a thermal-heat stimulus, and point to the necessity for a reconsideration of the cellular mechanisms of dentin sensation based on the transmembrane ionic signals in odontoblasts.
Assuntos
Odontoblastos/metabolismo , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/metabolismo , Canais de Cátion TRPV/biossíntese , Canais de Cátion TRPV/genética , Animais , Animais Recém-Nascidos , Dentina/fisiologia , Regulação da Expressão Gênica/fisiologia , Imuno-Histoquímica , Potenciais da Membrana/fisiologia , Nociceptores/química , Nociceptores/metabolismo , Nociceptores/fisiologia , Odontoblastos/química , Odontoblastos/fisiologia , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Células Receptoras Sensoriais/fisiologia , Canais de Cátion TRPV/fisiologiaRESUMO
Odontoblasts, the cells responsible for the dentine formation, are organized as a single layer of highly polarized and differentiated post-mitotic cells along the interface between the dental pulp and the mineralized tubules. They lay down the physiological secondary dentine throughout the life of the teeth. Odontoblasts play a central role in the transportation of calcium to the dentine and they possibly mediate early stages of sensory processing in teeth. A primary cilium, 9+0 configuration, have been regularly identified in a supra nuclear location. Calbindin D28k has been detected at the base of the cilium membrane. The cilium structure was positive with detyrosinated alpha tubulin antibodies in vivo and in cultured human odontoblasts. Transcripts of tektin, a protein involved in ciliogenesis, were expressed in vitro. The putative role of the primary cilium constituting a critical link between external teeth stimuli and odontoblast responses is extensively discussed.
Assuntos
Núcleo Celular/ultraestrutura , Cílios/ultraestrutura , Dentina/ultraestrutura , Odontoblastos/ultraestrutura , Calbindina 1 , Calbindinas , Células Cultivadas , Dentina/metabolismo , Humanos , Microscopia Eletrônica , Odontoblastos/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Dente/citologia , Dente/metabolismo , Calcificação de Dente/fisiologiaRESUMO
PURPOSE: The aim of this study was to investigate dental pulp reactions after a neodynium:yttrium aluminum perovskite laser pulse on the dentinal floor of occlusal cavities in an in vitro model. METHODS: A Lokki dt laser was used at 30 Hz, 5 W, and 160 mJ for 0.5 s. The pulp reactions were analyzed in a previously described human tooth slice cultured model. The following markers were identified by immunohistochemistry: collagens I, III, and IV and HLA-DR-positive cells. RESULTS: After 4 days of culture, under laser pulse, a concentration of type III collagen beneath the odontoblast layer, a higher level of vessels and an accumulation of HLA-DR-positive cells were routinely observed subjacent to the cavity. CONCLUSION: This laser treatment leads to the first step of rapid pulp repair under culture conditions.
Assuntos
Polpa Dentária/efeitos da radiação , Terapia a Laser , Odontoblastos/efeitos da radiação , Adolescente , Adulto , Células Dendríticas/imunologia , Preparo da Cavidade Dentária/métodos , Polpa Dentária/imunologia , Colágenos Fibrilares/efeitos da radiação , Antígenos HLA-DR/análise , Humanos , Técnicas Imunoenzimáticas , NeodímioRESUMO
Transforming growth factor beta 1 (TGF beta 1) is generally considered to be a potent inducer of dentin formation. In order to further assess this role, we studied the influence of this factor in human dental pulp cells on the expression of osteoadherin (OSAD), a newly described proteoglycan found in bone and dentin and suspected to play a role in mineralization events. We performed TGF beta 1 stimulation both in cultures of human tooth thick slices including mature odontoblasts and in pulp explant cultures giving rise to early secretory odontoblasts or pulpal fibroblasts. We first showed by immunohistochemistry that molecules involved in TGF beta 1 signal transduction, that is, membrane receptors T beta RI and T beta RII and intracellular proteins SMAD-2, SMAD-3, and SMAD-4, were present in human dental cells in vivo and were all maintained after culture of thick-sliced teeth in cells undergoing TGF beta 1 stimulation. In this culture system, OSAD synthesis was increased in mature odontoblasts close to the TGF beta 1 delivery system. In explant cultures, semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis indicated that the growth factor stimulated OSAD gene expression in early secretory odontoblasts and in pulpal fibroblasts. Taken together, these results indicate that OSAD expression is stimulated by TGF beta 1 in pulpal fibroblasts and in early secretory and mature odontoblasts. We suggest that TGF beta 1 in this way could control the organization and the mineralization of the extracellular matrix deposited by these cells during dentin formation.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Odontoblastos/metabolismo , Proteoglicanas/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/fisiologia , Técnicas de Cultura , Humanos , Odontoblastos/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1RESUMO
An antiserum was generated from synthetic peptides highly conserved between different mammalian species to immunolocalise the small leucine-rich proteoglycan osteoadherin (OSAD) in murine teeth. In 19-day-old embryos of rats and mice, a positive staining was found in incisor predentin and alveolar bone surrounding developing incisors and molars. In newborns, OSAD was detected at the tip of the first molar cusp where it accumulated in predentin concomitantly with odontoblast differentiation. In 2-day-old rats and mice, in the first molar, immunostaining revealed positive predentin, enamel matrix close to the apical pole of ameloblasts and a strong signal in dentin. At this stage, OSAD was detected in predentin in the second molar. Ultrastructural immunocytochemistry showed gold particles associated with collagen fibres in predentin and in foci at the dentin mineralisation front. Gold particles were also detected near the secretory pole of ameloblasts where enamel crystallites elongate. No staining was detected in pulp tissue and dental follicle. Restriction of OSAD expression to the extracellular matrix of bone, dentin and enamel suggests a role of this proteoglycan in the organisation of mineralised tissues.