RESUMO
Stem cells are maintained in specific niches that strictly regulate their proliferation and differentiation for proper tissue regeneration and renewal. Molecular oxygen (O2) is an important component of the niche microenvironment, but little is known about how O2 governs epithelial stem cell (ESC) behavior. Here, we demonstrate that O2 plays a crucial role in regulating the proliferation of ESCs using the continuously growing mouse incisors. We have revealed that slow-cycling cells in the niche are maintained under relatively hypoxic conditions compared with actively proliferating cells, based on the blood vessel distribution and metabolic status. Mechanistically, we have demonstrated that, during hypoxia, HIF1α upregulation activates the RhoA signal, thereby promoting cortical actomyosin and stabilizing the adherens junction complex, including merlin. This leads to the cytoplasmic retention of YAP/TAZ to attenuate cell proliferation. These results shed light on the biological significance of blood-vessel geometry and the signaling mechanism through microenvironmental O2 to orchestrate ESC behavior, providing a novel molecular basis for the microenvironmental O2-mediated stem cell regulation during tissue development and renewal.
Assuntos
Actomiosina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Epitélio/metabolismo , Incisivo/metabolismo , Oxigênio/metabolismo , Células-Tronco/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Proliferação de Células , Imunofluorescência , Hipóxia , Imuno-Histoquímica , Transdução de Sinais , Células-Tronco/citologia , Proteínas de Sinalização YAPRESUMO
Transient receptor potential melastatin 7 (TRPM7) is a unique ion channel connected to a kinase domain. We previously demonstrated that Trpm7 expression is high in mouse ameloblasts and odontoblasts, and that amelogenesis is impaired in TRPM7 kinase-dead mice. Here, we analyzed TRPM7 function during amelogenesis in Keratin 14-Cre;Trpm7fl/fl conditional knockout (cKO) mice and Trpm7 knockdown cell lines. cKO mice showed lesser tooth pigmentation than control mice and broken incisor tips. Enamel calcification and microhardness were lower in cKO mice. Electron probe microanalysis (EPMA) showed that the calcium and phosphorus contents in the enamel were lower in cKO mouse than in control mice. The ameloblast layer in cKO mice showed ameloblast dysplasia at the maturation stage. The morphological defects were observed in rat SF2 cells with Trpm7 knockdown. Compared with mock transfectants, the Trpm7 knockdown cell lines showed lower levels of calcification with Alizarin Red-positive staining and an impaired intercellular adhesion structures. These findings suggest that TRPM7 is a critical ion channel in enamel calcification for the effective morphogenesis of ameloblasts during amelogenesis.
Assuntos
Canais de Cátion TRPM , Camundongos , Ratos , Animais , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Camundongos Knockout , Esmalte Dentário/metabolismo , Ameloblastos/metabolismo , Epitélio , Amelogênese/genética , Proteínas de Transporte/metabolismo , IncisivoRESUMO
Various growth and transcription factors are involved in tooth development and developmental abnormalities; however, the protein dynamics do not always match the mRNA expression level. Using a proteomic approach, this study comprehensively analyzed protein expression in epithelial and mesenchymal tissues of the tooth germ during development. First molar tooth germs from embryonic day 14 and 16 Crlj:CD1 (ICR) mouse embryos were collected and separated into epithelial and mesenchymal tissues by laser microdissection. Mass spectrometry of the resulting proteins was carried out, and three types of highly expressed proteins [ATP synthase subunit beta (ATP5B), receptor of activated protein C kinase 1 (RACK1), and calreticulin (CALR)] were selected for immunohistochemical analysis. The expression profiles of these proteins were subsequently evaluated during all stages of amelogenesis using the continuously growing incisors of 3-week-old male ICR mice. Interestingly, these three proteins were specifically expressed depending on the stage of amelogenesis. RACK1 was highly expressed in dental epithelial and mesenchymal tissues during the proliferation and differentiation stages of odontogenesis, except for the pigmentation stage, whereas ATP5B and CALR immunoreactivity was weak in the enamel organ during the early stages, but became intense during the maturation and pigmentation stages, although the timing of the increased protein expression was different between the two. Overall, RACK1 plays an important role in maintaining the cell proliferation and differentiation in the apical end of incisors. In contrast, ATP5B and CALR are involved in the transport of minerals and the removal of organic materials as well as matrix deposition for CALR.
Assuntos
Proteômica , Dente , Camundongos , Animais , Masculino , Camundongos Endogâmicos ICR , Odontogênese/genética , Germe de Dente/metabolismo , Órgão do Esmalte/metabolismo , Proteínas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Dente/metabolismoRESUMO
In our previous study we established an animal model for immediately placed implants using mice and clarified that there were no significant differences in the chronological healing process at the bone-implant interface between immediately and delayed placed implants blasted with hydroxyapatite (HA)/ß-tricalcium phosphate (ß-TCP) (ratio 1:4). This study aimed to analyze the effects of HA/ß-TCP on osseointegration at the bone-implant interface after immediately placed implants in the maxillae of 4-week-old mice. Right maxillary first molars were extracted and cavities were prepared with a drill and titanium implants, blasted with or without HA/ß-TCP, were placed. The fixation was followed-up at 1, 5, 7, 14, and 28 days after implantation, and the decalcified samples were embedded in paraffin and prepared sections were processed for immunohistochemistry using anti-osteopontin (OPN) and Ki67 antibodies, and tartrate-resistant acid phosphatase histochemistry. The undecalcified sample elements were quantitatively analyzed by an electron probe microanalyzer. Bone formation occurred on the preexisting bone surface (indirect osteogenesis) and on the implant surface (direct osteogenesis), indicating that osseointegration was achieved until 4 weeks post-operation in both of the groups. In the non-blasted group, the OPN immunoreactivity at the bone-implant interface was significantly decreased compared with the blasted group at week 2 and 4, as well as the rate of direct osteogenesis at week 4. These results suggest that the lack of HA/ß-TCP on the implant surface affects the OPN immunoreactivity on the bone-implant interface, resulting in decreased direct osteogenesis following immediately placed titanium implants.
Assuntos
Implantes Dentários , Osseointegração , Camundongos , Animais , Maxila , Titânio/farmacologia , Osteogênese , Hidroxiapatitas/farmacologia , Propriedades de Superfície , Durapatita/farmacologiaRESUMO
Chondroitin sulfate proteoglycan (CSPG), one of the major extracellular matrices, plays an important part in organogenesis. Its core protein and chondroitin sulfate (CS) chain have a specific biological function. To elucidate the role of CS in the developmental and healing process of the dental pulp, we performed an experimental tooth replantation in CS N-acethylgalactosaminyltransferase-1 (T1) gene knockout (KO) mice. We also performed cell proliferation assay and qRT-PCR analysis for the WT and T1KO primary dental pulp cells using T1-siRNA technique and external CS. During tooth development, CS was diffusely expressed in the dental papilla, and with dental pulp maturation, CS disappeared from the differentiated areas, including the odontoblasts. In fully developed molars, CS was restricted to the root apex region colocalizing with Gli1-positive cells. In the healing process after tooth replantation, CD31-positive cells accumulated in the CS-positive stroma in WT molars. In T1KO molars, the appearance of Ki67- and Gli1-positive cells in the dental pulp was significantly fewer than in WT molars in the early healing stage, and collagen I-positive reparative dentin formation was not obvious in T1KO mice. In primary culture experiments, siRNA knockdown of T1 gene significantly suppressed cell proliferation in WT dental pulp cells, and the mRNA expression of cyclin D1 and CD31 was significantly upregulated by external CS in T1KO dental pulp cells. These results suggest that CS is involved in the cell proliferation and functional differentiation of dental pulp constituent cells, including vascular cells, in the healing process of dental pulp tissue after tooth injury.
Assuntos
Sulfatos de Condroitina , Polpa Dentária , Animais , Sulfatos de Condroitina/metabolismo , Polpa Dentária/metabolismo , Camundongos , Dente Molar/metabolismo , Odontoblastos , Reimplante DentárioRESUMO
The layered structures of facial muscles and their topographical relationship with facial fasciae are still not fully understood. This study aimed to clarify the layered structures of facial muscles and fasciae in the temporal-malar-mandible-neck region. Thirty-four human cadavers were examined during gross anatomy courses at Niigata University (2017-2020). The face was composed of 3-layered (deep, middle, and superficial) fasciae and 4-layered facial muscles (first superficial, second superficial, third, and fourth muscle layers) according to the attachment of muscles and their topographical relationship with the fasciae. The deep fascia covered the temporal and masseter muscles. The parotid gland and facial nerves were enveloped in the middle fascia. The superficial fascia was continuous with the second superficial muscle layer. The connection between fourth and superficial muscles was at the malar and buccal areas, where the platysma blended with the masseter and the plural muscles blended with the buccinator. Our findings suggest that cooperation between the 4-layered structure of the facial muscles surrounding the apertures of the eyes and mouth and the superficial fascia enables humans to produce complex facial expressions. Furthermore, the spread of inflammation in the face may be owing to the layered facial muscles and fasciae, as these layered structures separate tissues into multiple compartments.
Assuntos
Músculos Faciais , Sistema Musculoaponeurótico Superficial , Face/anatomia & histologia , Músculos Faciais/anatomia & histologia , Fáscia/anatomia & histologia , Humanos , Côndilo MandibularRESUMO
After dental implantation, osteopontin (OPN) is deposited on the hydroxyapatite (HA) blasted implant surface followed by direct osteogenesis, which is significantly disturbed in Opn-knockout (KO) mice. However, whether applying OPN on the implant surface promotes direct osteogenesis remains unclarified. This study analyzed the effects of various OPN modified protein/peptides coatings on the healing patterns of the bone-implant interface after immediately placed implantation in the maxilla of four-week-old Opn-KO and wild-type (WT) mice (n = 96). The decalcified samples were processed for immunohistochemistry for OPN and Ki67 and tartrate-resistant acid phosphatase histochemistry. In the WT mice, the proliferative activity in the HA binding peptide-OPN mimic peptide fusion coated group was significantly higher than that in the control group from day 3 to week 1, and the rates of OPN deposition and direct osteogenesis around the implant surface significantly increased in the recombinant-mouse-OPN (rOPN) group compared to the Gly-Arg-Gly-Asp-Ser peptide group in week 2. The rOPN group achieved the same rates of direct osteogenesis and osseointegration as those in the control group in a half period (week 2). None of the implant surfaces could rescue the direct osteogenesis in the healing process in the Opn-KO mice. These results suggest that the rOPN coated implant enhances direct osteogenesis during osseointegration following implantation.
Assuntos
Durapatita/química , Osseointegração/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteopontina/administração & dosagem , Fosfatase Ácida/metabolismo , Animais , Implantação Dentária , Implantes Dentários , Técnicas de Inativação de Genes , Camundongos , Modelos Animais , Osteopontina/química , Osteopontina/genética , Osteopontina/farmacologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologiaRESUMO
There is little anatomical evidence about the venous plexus in the floor of the oral cavity, although venous injury can elicit late postoperative bleeding after oral surgery and it is difficult to identify the exact location of such an injury. The aim of this study was to assess the relative risk for venous injury during surgery. We investigated the course patterns of the venous plexus in the floor of the oral cavity and analyzed their relationships to those of the arteries using 23 human cadavers (41 halves) in the anatomy course at Niigata University during 2016-2018. The venous plexus in the floor of the oral cavity comprised the perforating submental vein, the vena comitans of the hypoglossal nerve, the vena comitans of the submandibular duct, the vena comitans of the lingual nerve, the sublingual vein, and the deep lingual vein. Individual variations of this plexus include duplications or absences of some veins. There is a high incidence of a submental branch running above the mylohyoid or perforating submental artery in the sublingual fossa among individuals with the perforating submental vein piercing the mylohyoid muscle, whereas the sublingual artery has a high incidence there when there is no perforating submental vein. The course patterns of arteries in the floor of the oral cavity can be predicted by estimating the course patterns of the submental veins. The course patterns of the submental veins or veins associated with the nerves and submandibular duct need to be carefully considered during surgery.
Assuntos
Variação Anatômica , Boca/irrigação sanguínea , Boca/cirurgia , Veias/anatomia & histologia , Cadáver , Feminino , Hemorragia/prevenção & controle , Humanos , Masculino , Procedimentos Cirúrgicos BucaisRESUMO
BACKGROUND/AIM: Root length is a critical factor for dental pulp regeneration following tooth replantation. The aim of this study was to analyze the effects of reducing the root length by apicoectomy on the pulp healing process using a model for tooth replantation. MATERIAL AND METHODS: After extraction of the upper first molars (M1) of 3-week-old mice, the roots from the experimental group (EG) were shortened to half to two-thirds of their length before replantation, whereas in the control group (CG) the extracted teeth were immediately repositioned into their alveolar sockets. To determine the effects of root resection on the survival of inherent pulp cells, this study included tooth transplantation with root resection using wild-type (WT) and green fluorescent protein (GFP) transgenic mice. The M1 of GFP transgenic mice were transplanted into the alveolar socket of the M1 of WT mice. The roots of the right M1 were shortened (EG), whereas the left M1 remained untreated (CG). RESULTS: Apoptotic cells in the EG significantly decreased in number compared with the CG at day 3. Cell proliferative activity in the EG was significantly higher than that in the CG in the root pulp during days 3-5, and nestin-positive odontoblast-like cells began to arrange themselves along the pulp-dentin border in the cusp area at day 5 in the EG but not in the CG. At week 2, tertiary dentin had formed throughout the pulp in the EG, whereas the combined tissue of dentin and bone occupied the pulp space in 60% of the CG. Root resection also positively affected the survival of inherent pulp cells to differentiate into odontoblast-like cells as demonstrated by transplantation using GFP transgenic mice. CONCLUSIONS: Reducing the root length accelerated pulp regeneration following tooth replantation due to the better environment for revascularization.
Assuntos
Reimplante Dentário , Dente , Animais , Apicectomia , Polpa Dentária , Camundongos , RegeneraçãoRESUMO
BACKGROUND: Sulfated vizantin, a recently developed immunostimulant, has also been found to exert antibiofilm properties. It acts not as a bactericide, but as a detachment-promoting agent by reducing the biofilm structural stability. This study aimed to investigate the mechanism underlying this activity and its species specificity using two distinct ex vivo oral biofilm models derived from human saliva. RESULTS: The biofilm, composed mainly of the genus Streptococcus and containing 50 µM of sulfated vizantin, detached significantly from its basal surface with rotation at 500 rpm for only 15 s, even when 0.2% sucrose was supplied. Expression analyses for genes associated with biofilm formation and bacterial adhesion following identification of the Streptococcus species, revealed that a variety of Streptococcus species in a cariogenic biofilm showed downregulation of genes encoding glucosyltransferases involved in the biosynthesis of water-soluble glucan. The expression of some genes encoding surface proteins was also downregulated. Of the two quorum sensing systems involved in the genus Streptococcus, the expression of luxS in three species, Streptococcus oralis, Streptococcus gordonii, and Streptococcus mutans, was significantly downregulated in the presence of 50 µM sulfated vizantin. Biofilm detachment may be facilitated by the reduced structural stability due to these modulations. As a non-specific reaction, 50 µM sulfated vizantin decreased cell surface hydrophobicity by binding to the cell surface, resulting in reduced bacterial adherence. CONCLUSION: Sulfated vizantin may be a candidate for a new antibiofilm strategy targeting the biofilm matrix while preserving the resident microflora.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Glicolipídeos/farmacologia , Streptococcus/fisiologia , Trealose/análogos & derivados , Antibacterianos/química , Aderência Bacteriana/efeitos dos fármacos , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Cárie Dentária/microbiologia , Células Epiteliais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Gengivite/microbiologia , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Glicolipídeos/química , Humanos , Percepção de Quorum/efeitos dos fármacos , Percepção de Quorum/genética , Streptococcus/classificação , Streptococcus/efeitos dos fármacos , Streptococcus/crescimento & desenvolvimento , Sulfatos/química , Trealose/química , Trealose/farmacologiaRESUMO
OBJECTIVES: To determine glucose transporter 1 (GLUT1) and runt-related transcription factor 2 (RUNX2) expression during reparative dentinogenesis after pulpotomy with mineral trioxide aggregate (MTA) capping. SUBJECTS AND METHODS: Eight-week-old male Wistar rats were used. Pulp of the upper left first molar was exposed and capped with MTA. The upper right first molar of the same animal was used as a control. After collecting molars at various time points, GLUT1, RUNX2 and mammalian target of rapamycin (MTOR) were examined by immunohistochemistry. mRNA levels of Slc2a1 (encoding GLUT1), Runx2, Nestin and Mtor were determined by real-time PCR. RESULTS: Pulp exhibited progressive formation of reparative dentine lined with GLUT1- and MTOR-immunoreactive odontoblast-like cells at 5 days after pulpotomy. RUNX2 was detected in nuclei of most pulp tissue cells at day 5 after pulpotomy. Double immunofluorescence staining revealed GLUT1 immunoreactivity on odontoblast-like cells positive for Nestin or RUNX2, 5 days after pulpotomy. Slc2a1, Runx2, Nestin and Mtor mRNA levels were significantly upregulated on days 3-5 after pulpotomy. CONCLUSIONS: After rat molar pulpotomy, dental pulp induced formation of reparative dentine with colocalization of GLUT1 and Nestin or RUNX2. Moreover, mRNA levels of Slc2a1, Runx2, Nestin and Mtor were significantly upregulated in pulpotomized dental pulp.
Assuntos
Compostos de Alumínio/administração & dosagem , Compostos de Cálcio/administração & dosagem , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Capeamento da Polpa Dentária/métodos , Polpa Dentária/fisiologia , Dentinogênese/genética , Transportador de Glucose Tipo 1/genética , Óxidos/administração & dosagem , Pulpotomia , Silicatos/administração & dosagem , Serina-Treonina Quinases TOR/genética , Animais , Combinação de Medicamentos , Expressão Gênica , Imunoquímica , Masculino , Dente Molar/cirurgia , Nestina/genética , Odontoblastos/fisiologia , Ratos , Ratos WistarRESUMO
OBJECTIVES: This study is aimed at evaluating the effect of a new glass ionomer cement (GIC) containing fluoro-zinc-silicate fillers on biofilm formation and ion incorporation. MATERIALS AND METHODS: Streptococcus mutans biofilms were developed on two GIC materials: Caredyne Restore (CD) and Fuji VII (FJ); and hydroxyapatite (HA) for 24 h at 37 °C using a flow cell system. The morphological structure and bacterial viability were analyzed using a confocal laser scanning microscopy. Bacterial adhesion during the initial 2 h was also assessed by viable cell counting. To study the ion incorporation, restored cavities prepared on the root surfaces of human incisors were subjected to the elemental mapping of the zinc and fluoride ions in the GIC-dentin interface using a wavelength-dispersive X-ray spectroscopy electron probe microanalyzer. RESULTS: Morphological observations revealed that biofilm formation in the CD group was remarkably inhibited compared with the HA and FJ groups, exhibiting sparse, thinner biofilm clusters. The microorganisms adhering to the CD group were significantly inhibited, revealing 2.9 ± 0.4 for CD, 4.9 ± 0.2 for FJ, and 5.4 ± 0.4 log colony-forming units (CFU) for HA. The CD zinc ion incorporation depth was 72.2 ± 8.0 µm. The fluoride penetration of CD was three times deeper than that of FJ; this difference was statistically significant (p < 0.05). CONCLUSIONS: Enhanced by the incorporation of zinc and fluoride ions, the new GIC inhibited biofilm formation by interfering with bacterial adhesion. CLINICAL RELEVANCE: A novel GIC comprised of fluoro-zinc-silicate fillers may improve clinical outcomes, such as root caries and minimally invasive dentistry.
Assuntos
Biofilmes , Cimentos de Ionômeros de Vidro , Zinco , Dentina , Humanos , Teste de Materiais , SilicatosRESUMO
BACKGROUND: The aim of this in vitro study was to examine the possible enhancement of the biofilm peeling effect of a sonic toothbrush following the use of an antimicrobial mouth rinse. METHODS: The biofilm at a noncontact site in the interdental area was treated by sound wave convection with the test solution or by immersion in the solution. The biofilm peeling effect was evaluated by determining the bacterial counts and performing morphological observations. A Streptococcus mutans biofilm was allowed to develop on composite resin discs by cultivation with stirring at 50 rpm for 72 h. The specimens were then placed in recesses located between plastic teeth and divided into an immersion group and a combination group. The immersion group was treated with phosphate buffer, chlorhexidine digluconate Peridex™ (CHX) mouth rinse or Listerine® Fresh Mint (EO) mouth rinse. The combination group was treated with CHX or EO and a sonic toothbrush. RESULTS: The biofilm thickness was reduced by approximately one-half compared with the control group. The combination treatment produced a 1 log reduction in the number of bacteria compared to the EO immersion treatment. No significant difference was observed in the biofilm peeling effect of the immersion group compared to the control group. CONCLUSIONS: The combined use of a sonic toothbrush and a mouth rinse enhanced the peeling of the biofilm that proliferates in places that are difficult to reach using mechanical stress.
Assuntos
Esmalte Dentário/microbiologia , Antissépticos Bucais/farmacologia , Streptococcus mutans/efeitos dos fármacos , Escovação Dentária/instrumentação , Ultrassom/instrumentação , Aderência Bacteriana , Carga Bacteriana , Biofilmes/efeitos dos fármacos , Clorexidina , Humanos , Escovação Dentária/métodosRESUMO
The Nestin gene encodes type VI intermediate filament and is known to be expressed in undifferentiated cells during neurogenesis and myogenesis. To regulate Nestin expression, the first or second intron enhancer is activated in a tissue-dependent manner, for example, the former in mesodermal cells and the latter in neural stem cells. Although Nestin has also been used as a differentiation marker for odontoblasts during tooth development, how Nestin expression is regulated in odontoblasts remains unclear. Therefore, this study aimed to compare the expression patterns of Nestin-GFP (green fluorescent protein) with that of endogenous Nestin in developing teeth of Nestin-EGFP (enhanced GFP) transgenic mice, in which the second intron enhancer is connected with the EGFP domain, at postnatal 7d, 3w, and 8w. Immunohistochemical and in situ hybridization analyses revealed that endogenous Nestin protein and Nestin mRNA were intensely expressed in differentiated odontoblasts, while GFP immunoreactivity, which reflects the activity of Nestin second intron enhancer-mediated transcription, was mainly observed in the subodontoblastic layer. These results indicate that the first intron enhancer may be activated in differentiated odontoblasts. Intriguingly, Nestin-GFP expression in the subodontoblastic layer was found to be restricted to the coronal pulp of molars, which is susceptible to tooth injuries. Because the subodontoblastic layer serves as a reservoir of newly differentiated odontoblast-like cells upon exogenous stimuli to dentin, our findings suggest that the original odontoblasts and regenerated odontoblast-like cells may differently regulate Nestin expression.
Assuntos
Nestina/biossíntese , Odontoblastos/metabolismo , Animais , Diferenciação Celular , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nestina/genética , Odontoblastos/citologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
This study aimed to analyze the mRNA expression and protein localization of prostaglandin I2 (PGI2) synthase (PGIS), the PGI2 receptor (IP receptor) and transient receptor potential cation channel, subfamily V, member 1 (TRPV1) in force-stimulated rat molars, toward the elucidation of the PGI2-IP receptor-TRPV1 pathway that is in operation in the pulp and possibly associated with orthodontic pain and inflammation. Experimental force was applied to the maxillary first and second molars by inserting an elastic band between them for 6-72 h. PGIS, PTGIR (the IP receptor gene), and TRPV1 mRNA levels in the coronal pulp were analyzed with real-time PCR. PGIS, IP receptor, and TRPV1 proteins were immunostained. The force stimulation induced significant upregulation of PGIS at 6-24 h, and PTGIR and TRPV1 at 6 and 12 h in the pulp. PGIS was immunolocalized in odontoblasts and some fibroblasts in the force-stimulated pulp. The IP receptor and TRPV1 immunoreactivities were detected on odontoblasts and some nerve fibers. It was concluded that PGIS, PTGIR, and TRPV1 in rat molar pulp were significantly upregulated shortly after the force application, and that the IP receptor was co-expressed on TRPV1-expressing nerves and odontoblasts. These findings suggest that the PGI2-IP receptor-TRPV1 pathway is associated with the acute phase of force-induced pulp changes involving odontoblasts and nerves.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Polpa Dentária/metabolismo , Expressão Gênica , Oxirredutases Intramoleculares/genética , Receptores de Epoprostenol/genética , Canais de Cátion TRPV/genética , Técnicas de Movimentação Dentária , Animais , Técnicas Imunoenzimáticas , Masculino , Dente Molar , Odontoblastos/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Tempo , Regulação para CimaRESUMO
The mechanisms regulating the maintenance of quiescent adult stem cells in teeth remain to be fully elucidated. Our aim is to clarify the relationship between BrdU label-retaining cells (LRCs) and sonic hedgehog (Shh) signaling in murine teeth. After prenatal BrdU labeling, mouse pups were analyzed during postnatal day 1 (P1) to week 5 (P5W). Paraffin sections were processed for immunohistochemistry for BrdU, Sox2, Gli1, Shh, Patched1 (Ptch1) and Ki67 and for in situ hybridization for Shh and Ptch1. Dense LRCs, Gli1-(+) cells and Ptch1-(+) cells were co-localized in the outer enamel epithelium of the apical bud and apical dental papilla of incisors. In developing molars, dense LRCs were numerous at P1 but then decreased in number over the course of odontogenesis and were maintained in the center of pulp tissue. Gli1-(+) cells were maintained in the pulp horn during the examined stages, while they increased in number and were maintained in the center of pulp tissue during P2-5W. Ptch1-(+) cells were localized in the pulp horn at P1 and increased in number in the center of the pulp after P3W. Shh mRNA was first expressed in the enamel epithelium and then shifted to odontoblasts and other pulp cells. Shh protein was distributed in the epithelial and mesenchymal tissues of incisors and molars. These findings suggest that quiescent dental stem cells are regulated by Shh signaling, and that Shh signaling plays a crucial role in the differentiation and integrity of odontoblasts during epithelial-mesenchymal interactions and dentinogenesis.
Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Ciclo Celular , Proteínas Hedgehog/metabolismo , Dente/citologia , Animais , Animais Recém-Nascidos , Bromodesoxiuridina/metabolismo , Feminino , Proteínas Hedgehog/genética , Antígeno Ki-67/metabolismo , Camundongos Endogâmicos ICR , Mucosa Bucal/metabolismo , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Dente/crescimento & desenvolvimento , Proteína GLI1 em Dedos de Zinco/metabolismoRESUMO
Human molars exhibit a type of metameric variation, which is the difference in serially repeated morphology within an organism. Various theories have been proposed to explain how this variation is brought about in the molars. Actualistic data that support the theories, however, are still relatively scarce because of methodological limitations. Here we propose new methods to analyse detailed tooth crown morphologies. We applied morphometric mapping to the enamel-dentine junction of human maxillary molars and examined whether odontogenetic models were adaptable to human maxillary molars. Our results showed that the upper first molar is phenotypically distinct among the maxillary molars. The average shape of the upper first molar is characterized by four well-defined cusps and precipitous surface relief of the occlusal table. On the other hand, upper third molar is characterized by smooth surface relief of the occlusal table and shows greater shape variation and distinct distribution patterns in morphospace. The upper second molar represents an intermediate state between first and third molar. Size-related shape variation was investigated by the allometric vector analysis, and it appeared that human maxillary molars tend to converge toward the shape of the upper first molar as the size increases. Differences between the upper first molar and the upper second and third molar can thus be largely explained as an effect of allometry. Collectively, these results indicate that the observed pattern of metameric variation in human molars is consistent with odontogenetic models of molar row structure (inhibitory cascade model) and molar crown morphology (patterning cascade model). This study shows that morphometric mapping is a useful tool to visualize and quantify the morphological features of teeth, which can provide the basis for a better understanding of tooth evolution linking morphology and development.
Assuntos
Dente Molar/anatomia & histologia , Humanos , Microtomografia por Raio-XRESUMO
We have proposed the new hypothesis that dental pulp stem cells play crucial roles in the pulpal healing process following exogenous stimuli in cooperation with progenitors. This study aimed to establish an in vitro culture system for evaluating dentin-pulp complex regeneration with special reference to the differentiation capacity of slow-cycling long-term label-retaining cells (LRCs). Three intraperitoneal injections of BrdU were given to pregnant ICR mice to map LRCs in the mature tissues of born animals. The upper bilateral first molars of 3-week-old mice were extracted and divided into two pieces and cultured for 0, 1, 3, 5 and 7 days using the Trowel's method. We succeeded in establishing an in vitro culture system for evaluating dentin-pulp complex regeneration, where most odontoblasts were occasionally degenerated and lost nestin immunoreactivity because of the separation of cell bodies from cellular processes in the dentin matrix by the beginning of in vitro culture. Numerous dense LRCs mainly resided in the center of the dental pulp associating with blood vessels throughout the experimental periods. On postoperative days 1-3, the periphery of the pulp tissue including the odontoblast layer showed degenerative features. By Day 7, nestin-positive odontoblast-like cells were arranged along the pulp-dentin border and dense LRCs were committed in the odontoblast-like cells. These results suggest that dense LRCs in the center of the dental pulp associating with blood vessels were supposed to be dental pulp stem/progenitor cells possessing regenerative capacity for forming newly differentiated odontoblast-like cells.
Assuntos
Bromodesoxiuridina/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Polpa Dentária/citologia , Polpa Dentária/fisiologia , Dentina/fisiologia , Regeneração , Animais , Dentina/citologia , Camundongos , Camundongos Endogâmicos ICRRESUMO
Our recent study suggested that allogenic tooth transplantation may affect the maintenance of dental pulp stem/progenitor cells. This study aims to elucidate the influence of allograft on the maintenance of dental pulp stem/progenitor cells following tooth replantation and allo- or auto-genic tooth transplantation in mice using BrdU chasing, immunohistochemistry for BrdU, nestin and Ki67, in situ hybridization for Dspp, transmission electron microscopy and TUNEL assay. Following extraction of the maxillary first molar in BrdU-labeled animals, the tooth was immediately repositioned in the original socket, or the roots were resected and immediately allo- or auto-grafted into the sublingual region in non-labeled or the same animals. In the control group, two types of BrdU label-retaining cells (LRCs) were distributed throughout the dental pulp: those with dense or those with granular reaction for BrdU. In the replants and autogenic transplants, dense LRCs remained in the center of dental pulp associating with the perivascular environment throughout the experimental period and possessed a proliferative capacity and maintained the differentiation capacity into the odontoblast-like cells or fibroblasts. In contrast, LRCs disappeared in the center of the pulp tissue by postoperative week 4 in the allografts. The disappearance of LRCs was attributed to the extensive apoptosis occurring significantly in LRCs except for the newly-differentiated odontoblast-like cells even in cases without immunological rejection. The results suggest that the host and recipient interaction in the allografts disturbs the maintenance of dense LRCs, presumably stem/progenitor cells, resulting in the disappearance of these cell types.
Assuntos
Polpa Dentária/citologia , Dente Molar/transplante , Células-Tronco/citologia , Aloenxertos , Animais , Apoptose , Autoenxertos , Proliferação de Células , Polpa Dentária/metabolismo , Dentina/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Antígeno Ki-67/imunologia , Camundongos , Camundongos Endogâmicos ICR , Dente Molar/citologia , Nestina/imunologia , Odontoblastos/citologia , Fosfoproteínas/biossíntese , Sialoglicoproteínas/biossínteseRESUMO
Tooth crown patterning is governed by the growth and folding of the inner enamel epithelium (IEE) and the following enamel deposition forms outer enamel surface (OES). We hypothesized that overall dental crown shape and covariation structure are determined by processes that configurate shape at the enamel-dentine junction (EDJ), the developmental vestige of IEE. This this hypothesis was tested by comparing patterns of morphological variation between EDJ and OES in human permanent maxillary first molar (UM1) and deciduous second molar (um2). Using geometric morphometric methods, we described morphological variation and covariation between EDJ and OES, and evaluated the strength of two components of phenotypic variability, canalization and morphological integration, in addition to the relevant evolutionary flexibility, i.e. the ability to respond to selective pressure. The strength of covariation between EDJ and OES was greater in um2 than in UM1, and the way that multiple traits covary between EDJ and OES was different between these teeth. The variability analyses showed that EDJ had less shape variation and a higher level of morphological integration than OES, which indicated that canalization and morphological integration acted as developmental constraints. These tendencies were greater in UM1 than in um2. On the other hand, EDJ and OES had a comparable level of evolvability in these teeth. Amelogenesis could play a significant role in tooth shape and covariation structure, and its influence was not constant among teeth, which may be responsible for the differences in the rate and/or period of enamel formation.