RESUMO
Rupture of the basement membrane in fused palate tissue can cause the palate to separate after fusion in mice, leading to the development of cleft palate. Here, we further elucidate the mechanism of palatal separation after palatal fusion in 8-10-week-old ICR female mice. On day 12 of gestation, 40 µg/kg of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), sufficient to cause cleft palate in 100% of mice, was dissolved in 0.4 mL of olive oil containing toluene and administered as a single dose via a gastric tube. Fetal palatine frontal sections were observed by H&E staining, and epithelial cell adhesion factors, apoptosis, and cell proliferation were observed from the anterior to posterior palate. TUNEL-positive cells and Ki67-positive cells were observed around the posterior palatal dissection area of the TCDD-treated group. Moreover, in fetal mice exposed to TCDD, some fetuses exhibited cleft palate dehiscence during fusion. The results suggest that palatal dehiscence may be caused by abnormal cell proliferation in epithelial tissues, decreased intercellular adhesion, and inhibition of mesenchymal cell proliferation. By elucidating the mechanism of cleavage after palatal fusion, this research can contribute to establishing methods for the prevention of cleft palate development.
Assuntos
Fissura Palatina/induzido quimicamente , Fissura Palatina/metabolismo , Palato/efeitos dos fármacos , Palato/metabolismo , Dibenzodioxinas Policloradas/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Membrana Basal/efeitos dos fármacos , Membrana Basal/metabolismo , Membrana Basal/patologia , Proliferação de Células/efeitos dos fármacos , Fissura Palatina/patologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Epitélio/patologia , Feminino , Marcação In Situ das Extremidades Cortadas/métodos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Palato/patologiaRESUMO
PURPOSE: To observe, histologically, bone induced by recombinant human bone morphogenetic protein-2 (rhBMP-2) in onlay grafted and sinus lifted alveolaris. MATERIAL AND METHODS: Eighteen patients were treated with rhBMP-2 at concentration 1.5 mg/mL with an absorbable collagen sponge (ACS). The treated bone was harvested with small trephine bur at 5 or 7 months after surgery for the micro Computer Scanning (CT) and light microscopic observation. RESULTS: Micro CT showed clearly 3-dimensional trabecular bone structure. New bone formation and bone marrow structure were observed in the observed area. Osteoblastic cells existed along the new bone, and osteopontin was localized in the bone matrix weakly. In the connective tissue around the new bone, many CD34-positive blood vessel cells were present. Some tartrate-resistant acid phosphatase (TRAP)-positive osteoclastic cells were observed around bone at this stage. CONCLUSION: The application of rhBMP-2 with ACS induced a new bone accompanied by blood vessels in atrophied alveolaris. This suggests that rhBMP-2 is capable of osteoinductivity in human jaw.
Assuntos
Processo Alveolar/crescimento & desenvolvimento , Proteína Morfogenética Óssea 2/farmacologia , Processo Alveolar/anatomia & histologia , Processo Alveolar/química , Processo Alveolar/diagnóstico por imagem , Matriz Óssea/anatomia & histologia , Matriz Óssea/diagnóstico por imagem , Matriz Óssea/crescimento & desenvolvimento , Humanos , Osteopontina/análise , Proteínas Recombinantes/farmacologia , Levantamento do Assoalho do Seio Maxilar/métodos , Microtomografia por Raio-XRESUMO
Using the proliferating cell nuclear antigen (PCNA) immunostaining, we previously identified, after pulp exposure, three zones of proliferating cells in the rat molar pulp. Zones I and II were in the crown near the pulp. Zone III was near the apex revealing a recruitment of mitotic cells at distance from the lesion. To gain further insight into the spatio-temporal evolution of proliferating pulp cells of zone III, we performed a longitudinal study of PCNA staining in rat molar mesial root at 3, 8, and 15 d after pulp exposure associated to implantation of unloaded or amelogenin loaded agarose beads. At day 3 after implantation, PCNA-positive cells were located in the central part of the radicular pulp. At day 8, PCNA-labeled cells were aligned in the lateral part of the pulp beneath the odontoblast/sub-odontoblast layer. At day 15, PCNA labeling became undetectable in the root and was located in the coronal pulp. These results suggest that after pulp exposure, PCNA-positive cells may migrate from the central part of the radicular pulp to the sub-odontoblast cell layer and then from the apical root to the crown. Electron microscopy and immunostaining analysis showed that pulpal cells were linked by desmosome-like and gap-junctions. Extracellular matrix was composed of thin collagen fibrils associated with glycosaminoglycans favoring cell mobility. These data suggest that the syncytium-like structure formed by pulp radicular cells may be a pre-request for plithotaxis, a collective cell migration process. This emergent mechanism may govern pulp healing and regeneration after dental lesion.
Assuntos
Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Polpa Dentária/citologia , Odontoblastos/citologia , Regeneração/fisiologia , Animais , Polpa Dentária/fisiologia , Dente Molar , Ratos Sprague-DawleyRESUMO
This study involved a histologic, enzyme histologic, immunohistologic, and three-dimensional microstructure evaluating the extent of osteogenesis and repair in the human alveolar extraction socket achievable with an artificial bone substitute. After tooth extraction in 7 patients, extraction sockets were filled with Mastergraft (15% hydroxyapatite, 85% ß-tricalcium phosphate complex). Radiomicrographs and histologic examinations were performed on samples obtained during dental implant placement procedure. On micro-computed tomography, new bone was observed in all collected samples, and osteogenesis was observed to have taken place around the artificial bone substitute. Histologically, active osteogenesis was found throughout the region observed. Addition of new bone around the Mastergraft was observed, and osteoblast-like cells were present. Cells that had partially invaded the artificial bone included tartrate-resistant acid phosphate-positive and CD34-positive cells. These findings indicate that the Mastergraft artificial bone induced osteogenesis in the jawbone and seemed effective for repairing bone defects.
Assuntos
Substitutos Ósseos/uso terapêutico , Alvéolo Dental/cirurgia , Cicatrização/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteogênese , Técnicas de Sutura , Extração Dentária , Alvéolo Dental/diagnóstico por imagem , Resultado do Tratamento , Microtomografia por Raio-XRESUMO
Cementogenesis starts with the differentiation of cementoblasts. Mature cementoblasts secrete cementum matrix. Cementum components are similar to bone; moreover, cementoblasts possess many characteristics similar to those of osteoblasts. Runx2 and osterix, the transcriptional factors for osteoblast differentiation, participate in tooth formation. However, the characteristics of Runx2 and osterix during the differentiation process of cementoblasts remain unclear. In this study, we examined the immunolocalization patterns of Runx2, osterix, and osteopontin during rat molar tooth formation. Periodontal ligament cells and osteoblasts located on the alveolar bone surface showed immunoreactivity for Runx2. Colocalization of Runx2 and osterix was detected in cementoblasts, which penetrated the ruptured Hertwig's epithelial root sheath and attached to root dentin. Moreover, osteopontin was observed in Runx2-positive cementoblasts facing the root surface. However, the cells adjacent to cementoblasts showed only Runx2 reactivity. Neither Runx2 nor osterix was seen in cementocytes. These results suggest that both Runx2 and osterix are important for differentiation into cementoblasts. Additionally, osterix may be indispensable for transcription of osteopontin expression.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Dente Molar/metabolismo , Osteopontina/metabolismo , Raiz Dentária/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Cemento Dentário/citologia , Cemento Dentário/metabolismo , Imuno-Histoquímica , Dente Molar/citologia , Dente Molar/crescimento & desenvolvimento , Ratos , Ratos Wistar , Raiz Dentária/crescimento & desenvolvimentoRESUMO
Both periosteum and bone marrow have the potential to induce heterotopic bone when grafted. Whether the process of bone formation is controlled by the recipient environment where the donor graft is placed or by factors from the donor site is not well documented. The purpose of this study was to examine the histology of new bone induced by either autogenously grafted periosteum or autogenously grafted bone marrow using the rat calvarial defect model in Sprague-Dawley rats. Grafts of either bone marrow or periosteum obtained from tibias were placed in calvarial defects with beta-tricalcium phosphate. Ten days after grafting, active cell proliferation was observed in the defects of both types of grafts. After 20 days, cancellous bone formation was observed in the defects with bone marrow grafts, and intramembranous bone formation was observed in the defects with periosteal grafts. After 30 days, bone marrow grafts had developed bone with a bone marrow-like structure, and the periosteal grafts had produced cortical bone structure in the defects. The findings suggest that the type of bone formation is determined by characteristics of the donor site.
Assuntos
Transplante de Medula Óssea/métodos , Osteogênese/fisiologia , Periósteo/transplante , Animais , Materiais Biocompatíveis/farmacologia , Transplante Ósseo/métodos , Fosfatos de Cálcio/farmacologia , Técnicas Histológicas , Modelos Biológicos , Osteogênese/efeitos dos fármacos , Periósteo/citologia , Ratos , Ratos Sprague-Dawley , Crânio/cirurgia , Tíbia/cirurgia , Transplante AutólogoRESUMO
During cementogenesis, dental follicular cells penetrate the ruptured Hertwig's epithelial root sheath (HERS) and differentiate into cementoblasts. Mechanisms involved in basement membrane degradation during this process have not been clarified. Perlecan, a heparan sulfate (HS) proteoglycan, is a component of all basement membranes. Degradation of HS of perlecan by heparanase cleavage affects a variety of biological processes. We elucidated immunolocalization of perlecan and heparanase in developing murine molars to clarify their roles in cementoblast differentiation. At the initial stage of root formation, perlecan immunoreactivity was detected on the basement membrane of HERS. Weak heparanase immunoreactivity was detected in HERS cells. HERS showed intense staining for heparanase as root formation progressed. In contrast, labeling for perlecan disappeared from the basement membrane facing the dental follicle, and weak immunoreactivity for perlecan was detected on the inner side of the basement membrane of HERS. These findings suggest that perlecan removal is an important step for root and periodontal tissue formation. Heparanase secreted by the cells of HERS may contribute to root formation by degrading perlecan in the dental basement membrane.
Assuntos
Glucuronidase/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Dente Molar/metabolismo , Raiz Dentária/metabolismo , Animais , Sialoproteína de Ligação à Integrina , Queratinas/metabolismo , Laminina/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Dente Molar/crescimento & desenvolvimento , Osteopontina , Sialoglicoproteínas/metabolismo , Raiz Dentária/crescimento & desenvolvimentoRESUMO
Homeobox genes play important roles in craniofacial morphogenesis. However, the characteristics of the transcription factor Hoxc during palate formation remain unclear. We examined the immunolocalization patterns of Hoxc5, Hoxc4, and Hoxc6 in palatogenesis of cleft palate (Eh/Eh) mice. On the other hand, mutations in the FGF/FGFR pathway are exclusively associated with syndromic forms of cleft palate. We also examined the immunolocalization of Fgfr1 and Erk1/2 to clarify their relationships with Hoxc in palatogenesis. Some palatal epithelial cells showed Hoxc5 labeling, while almost no labeling of mesenchymal cells was observed in +/+ mice. As palate formation progressed in +/+ mice, Hoxc5, Hoxc4, and Hoxc6 were observed in medial epithelial seam cells. Hoxc5 and Hoxc6 were detected in the oral epithelium. The palatal mesenchyme also showed intense staining for Fgfr1 and Erk1/2 with progression of palate formation. In contrast, the palatal shelves of Eh/Eh mice exhibited impaired horizontal growth and failed to fuse, resulting in a cleft. Hoxc5 was observed in a few epithelial cells and diffusely in the mesenchyme of Eh/Eh palatal shelves. No or little labeling of Fgfr1 and Erk1/2 was detected in the cleft palate of Eh/Eh mice. These findings suggest that Hoxc genes are involved in palatogenesis. Furthermore, there may be the differences in the localization pattern between Hoxc5, Hoxc4, and Hoxc6. Additionally, Hoxc distribution in palatal cells during palate development may be correlated with FGF signaling. (228/250 words) © 2016 Japanese Teratology Society.
Assuntos
Proteínas de Homeodomínio/metabolismo , Organogênese , Palato/embriologia , Palato/metabolismo , Animais , Fissura Palatina/genética , Fissura Palatina/patologia , Modelos Animais de Doenças , Expressão Ectópica do Gene , Feminino , Expressão Gênica , Proteínas de Homeodomínio/genética , Imuno-Histoquímica , Masculino , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Família Multigênica , Organogênese/genética , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Transporte Proteico , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
CD44 is a multifunctional adhesion molecule that binds to hyaluronan (HA), type I collagen, and fibronectin. We investigated localization of CD44 and HA in mandibular condylar cartilage compared with the growth plate and the articular cartilage, to clarify the characteristics of chondrocytes. We also performed Western blotting using a lysate of mandibular condyle. In mandibular condyle, CD44-positive cells were seen in the surface region of the fibrous cell layer and in the proliferative cell layer. Western blotting revealed that the molecular weight of CD44 in condyle was 78 to 86 kD. Intense reactivity for HA was detected on the surface of the condyle and the lacunae of the hypertrophic cell layer. Moderate labeling was seen in cartilage matrix of the proliferative and maturative layer. Weak labeling was also seen in the fibrous cell layer. In growth plate and articular cartilage, HA was detected in all cell layers. However, chondrocytes of these cartilages did not exhibit reactivity for CD44. These results suggest that chondrocytes in the mandibular condylar cartilage differ in expression of CD44 from those in tibial growth plate and articular cartilage. Cell-matrix interaction between CD44 and HA may play an important role in the proliferation of chondrocytes in the mandibular condyle.
Assuntos
Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Côndilo Mandibular/metabolismo , Animais , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Cartilagem Articular/ultraestrutura , Diferenciação Celular , Condrócitos/citologia , Condrócitos/metabolismo , Condrócitos/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Lâmina de Crescimento/citologia , Lâmina de Crescimento/metabolismo , Lâmina de Crescimento/ultraestrutura , Receptores de Hialuronatos/química , Côndilo Mandibular/citologia , Côndilo Mandibular/ultraestrutura , Microscopia Imunoeletrônica , Peso Molecular , Ratos , Tíbia/citologia , Tíbia/metabolismo , Tíbia/ultraestruturaRESUMO
Ellis-van Creveld (EvC) syndrome is an autosomal recessive chondrodysplasia characterized by short limbs, postaxial polydactyly, natal teeth, and dysplastic nails. The Ellis-van Creveld (EVC) gene, which is mutated in patients with EvC syndrome, has been identified by positional cloning. However, the physiological roles of the EVC gene have not been elucidated. Histopathological analyses of EvC syndrome have shown disturbed chondrocytic phenotypes during cartilage development. We therefore postulated that the EVC gene is a critical factor for chondrocytes during endochondral ossification. The present study focuses on the relationship between the Evc gene and chondrocytes, and examines Evc gene expression in the rat tibial growth plate at the mRNA and protein levels. Evc mRNA in tibial epiphyseal cartilage was expressed at postnatal day (P) 1, P28, and P56 by RT-PCR. Immunohistochemical analyses localized the Evc protein mainly in prehypertrophic and hypertrophic chondrocytes of the epiphyseal growth plate in the tibia during the embryonic and postnatal periods. Evc mRNA was also detected in prehypertrophic and hypertrophic chondrocytes by in situ hybridization. These results indicate that the Evc gene functions mainly in the prehypertrophic and hypertrophic chondrocytes of the epiphyseal growth plate. The data presented here are important for future studies of the underlying mechanism of chondrodysplasia in EvC syndrome.
Assuntos
Lâmina de Crescimento/embriologia , Lâmina de Crescimento/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Tíbia/embriologia , Tíbia/metabolismo , Animais , Western Blotting , Embrião de Mamíferos/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Proteínas de Membrana/genética , Proteínas Nucleares/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Dioxins (e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) cause cleft palate at a high rate. A post-fusional split may contribute to the pathogenesis, and tissue fragility may be a concern. The objective of this study was to investigate the effects of TCDD on the palatal epithelium, bone and muscle, which contribute to tissue integrity. ICR mice (10-12 weeks old) were used. TCDD was administered on E12.5 at 40 mg/kg. Immunohistochemical staining for AhR, ER-α, laminin, collagen IV, osteopontin, Runx2, MyoD, and desmin were performed. Furthermore, western blot analysis for osteopontin, Runx2, MyoD, and desmin were performed to evaluate protein expression in the palatal tissue. Immunohistologically, there was little difference in the collagen IV and laminin localization in the palatal epithelium between control versus TCDD-treated mice. Runx2 and osteopontin immunoreactivity decreased in the TCDD-treated palatal bone, and MyoD and desmin decreased in the TCDD-treated palatal muscle. AhR and ER-α immunoreactivity were localized to the normal palatal bone, but ER-α was diminished in the TCDD-treated palate. On western blot analysis, Runx2, MyoD, and desmin were all downregulated in the TCDD-treated palate. TCDD may suppress palatal osteogenesis and myogenesis via AhR, and cause cleft palates via a post-fusional split mechanism, in addition to a failure of palatal fusion.
Assuntos
Fissura Palatina/induzido quimicamente , Palato/efeitos dos fármacos , Dibenzodioxinas Policloradas/efeitos adversos , Teratogênicos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos dos fármacos , Western Blotting , Fissura Palatina/embriologia , Colágeno Tipo IV/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/efeitos dos fármacos , Desmina/efeitos dos fármacos , Regulação para Baixo , Epitélio/efeitos dos fármacos , Epitélio/embriologia , Receptor alfa de Estrogênio/efeitos dos fármacos , Feminino , Idade Gestacional , Imuno-Histoquímica , Laminina/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Desenvolvimento Muscular/efeitos dos fármacos , Proteína MyoD/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteopontina/efeitos dos fármacos , Músculos Palatinos/efeitos dos fármacos , Músculos Palatinos/embriologia , Palato/embriologia , Palato Duro/efeitos dos fármacos , Palato Duro/embriologia , Gravidez , Receptores de Hidrocarboneto Arílico/efeitos dos fármacosRESUMO
Palatogenesis is directed by epithelial-mesenchymal interactions and results partly from remodeling of the extracellular matrix (ECM) of the palatal shelves. Here, we assessed heparanase distribution in developing mouse palates. No heparanase was observed in the vertically oriented palatal shelves in early stages of palate formation. As palate formation progressed, the palatal shelves were reorganized and arranged horizontally above the tongue, and heparanase localized to the epithelial cells of these shelves. When the palatal bilateral shelves first made contact, the heparanase localized to epithelial cells at the tips of shelves. Later in fusing palatal shelves, the cells of the medial epithelial seam (MES) were labeled with intense heparanase signal. In contrast, the basement membrane heparan sulfate (HS) was scarcely observed in the palatal shelves in contact. Moreover, perlecan labeling was sparse in the basement membrane of the MES, on which laminin and type IV collagen were observed. Moreover, we assessed the distribution of matrix metalloproteinase- (MMP-) 9, MMP-2, and MMP-3 in developing mouse palates and these MMPs were observed in the MES. Our findings indicated that heparanase was important for palate formation because it mediated degradation of the ECM of palatal shelves. Heparanase may, in concert with other proteases, participate in the regression of the MES.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Glucuronidase/biossíntese , Palato/embriologia , Animais , Membrana Basal/metabolismo , Colágeno Tipo IV/metabolismo , Matriz Extracelular/metabolismo , Feminino , Proteoglicanas de Heparan Sulfato/metabolismo , Heparitina Sulfato/metabolismo , Laminina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Fatores de TempoRESUMO
Mouse embryos exposed to 2,3,7,8-tetrachloridedibenzo-p-dioxin (TCDD) develop cleft palates and hydronephrosis. Cleft palates occur after TCDD exposure due to contact and/or fusion failure. We investigated whether cleft palate can be induced by dissociation of the palatine process after fusion. Pregnant mice on gestational day (GD) 12 were randomly divided into two groups: one group was administered through gastric tubes one dose of olive oil (control group) and the other group was administered one dose of TCDD diluted with olive oil, both at a dose of 40 microg/kg body weight. Embryos were removed by cesarean section from pregnant mice during the palatal formation stage (GD 13-18) and the palatal form was observed using a stereoscopic microscope. In TCDD-exposed embryos, palatal fusion was observed on GD 14, 15 and 16 and the incidence of cleft palate was 100% on GD 18. Fusion rates were 17.5 +/- 15.2% and 12.4 +/- 11.8% on GD 15 and 16, respectively. Some palates from the TCDD-exposed mouse embryos showed clearly developed cleft palate after fusion of the lateral palatine processes during palatal formation. A mass of cells, which were chiefly epithelial in the fused palates was observed in the TCDD-exposed mouse embryos. A decrease in E-cadherin expression was observed in this mass of cells, indicating its involvement in the development of cleft palate.
Assuntos
Fissura Palatina/embriologia , Palato/embriologia , Dibenzodioxinas Policloradas/toxicidade , Animais , Caderinas/metabolismo , Fissura Palatina/induzido quimicamente , Fissura Palatina/patologia , Feminino , Camundongos , Camundongos Endogâmicos ICR , GravidezRESUMO
In the present study, we evaluated the osteogenic potential of an autogenous bone marrow graft combined with beta-tricalcium phosphate (beta-TCP) in a rat calvarial bone defect model. The bone marrow harvested from the tibia of 7-week-old rats was grafted autogenously in a calvarial defect together with beta-TCP (=BTG group, n=16) or without beta-TCP (=BG group, n=16). Groups of animals were also treated with beta-TCP alone (=TG group, n=16) and control animals (n=8) received no graft implanted into the defect. We then observed the process of bone formation by histology, enzyme histochemistry and immunohistochemistry. Five days after grafting, in the BTG and BG groups, cell proliferation and osteogenic differentiation were observed. From 5 to 10 days after surgery, active Runx2, osteopontin (OPN), and TRAP- positive cells appeared in the BTG and BG groups. New bone formation started in the defect in both the BTG and BG groups. At 30 days after grafting, the BTG group showed new bone development and replacement of beta-TCP to fill the bone defect. New bone formation in the BTG group was significantly greater than in the BG group (P<0.01). The TG group showed no marked bone formation in the defect. The combination graft of bone marrow with beta-TCP showed marked bone formation in rat calvarial defects. Our results indicate that the combination grafts of bone marrow with beta-TCP may be an effective technique for repairing bone defects Beta-TCPgraft (TG) group.