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1.
Cell Prolif ; 53(1): e12697, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31713930

RESUMO

OBJECTIVES: miR-21 can promote osteoblast differentiation of periodontal ligament stem cells. However, the effect of miR-21 on bone remodelling in the midpalatal suture is unclear. This study aimed to elucidate the effects of miR-21 on the midpalatal suture bone remodelling by expanding the palatal sutures. MATERIALS AND METHODS: miR-21 deficient (miR-21-/- ) and wild-type (WT) mice were used to establish animal models by expanding the palatal sutures. Micro-CT, haematoxylin-eosin (HE) staining, tartrate-resistant acid phosphatase (TRAP) staining, fluorescence labelling and immunohistochemistry were used to investigate the function of miR-21 in midpalatal suture bone remodelling. Besides, bone mesenchymal stem cells (BMSCs) derived from both miR-21-/- and WT mice were cultured. The MTT, CCK8, EdU analysis, transwell and wound healing test were used to assess the effects of miR-21 on the characteristics of cells. RESULTS: The expression of ALP was suppressed in miR-21-/- mice after expansion except 28 days. The expression of Ocn in WT mice was much higher than that of miR-21-/-  mice. Besides, with mechanical force, miR-21 deficiency downregulated the expression of Opg, upregulated the expression of Rankl, and induced more osteoclasts as TRAP staining showed. After injecting agomir-21  to miR-21-/- mice, the expression of Alp, Ocn and Opg/Rankl were rescued. In vitro, the experiments suggested that miR-21 deficiency reduced proliferation and migration ability of BMSCs. CONCLUSIONS: The results showed that miR-21 deficiency reduced the rate of bone formation and prolonged the process of bone formation. miR-21 regulated the bone resorption and osteoclastogenesis by affecting the cell abilities of proliferation and migration.


Assuntos
Células da Medula Óssea/metabolismo , Remodelação Óssea , Suturas Cranianas/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Palato/metabolismo , Estresse Mecânico , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Proliferação de Células , Suturas Cranianas/citologia , Regulação da Expressão Gênica , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Knockout , MicroRNAs/genética , Osteoprotegerina/biossíntese , Osteoprotegerina/genética , Palato/citologia , Ligante RANK/biossíntese , Ligante RANK/genética , Fosfatase Ácida Resistente a Tartarato/biossíntese , Fosfatase Ácida Resistente a Tartarato/genética
2.
Toxicology ; 431: 152353, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31887333

RESUMO

Exposure to environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes cleft palate at high rates, but little is known about the underlying biological mechanisms. In the present study, we cultured osteoblasts from human fetal palate mesenchymal cells (hFPMCs) to explore the effects of TCDD on osteogenic differentiation. The results showed that TCDD significantly decreased cell proliferation, alkaline phosphatase (ALP) activity and calcium deposition. RNA analyses and protein detection demonstrated that TCDD downregulated a wide array of pro-osteogenic biomarkers. Further investigation of the underlying molecular mechanisms revealed that exposure to TCDD activated aryl hydrocarbon receptor (AhR) signaling and inhibited BMP-2/TGF-ß1/Smad pathway molecules. The inactivation of AhR signaling using CRISPR/Cas9-mediated AhR deletion or by genetic siRNA knockdown significantly blocked the effects induced by TCDD, suggesting a critical role of AhR activation in the TCDD-mediated inhibition of hFPMC osteogenic differentiation. The cotreatment with TGF-ß1 or BMP-2 and TCDD significantly relieved the activation of AhR and rescued the impairment of osteogenesis caused by TCDD. Taken together, our findings indicated that TCDD inhibited the osteogenic differentiation of hFPMCs via crosstalk between AhR and BMP-2/TGF-ß1/Smad signaling pathway.


Assuntos
Poluentes Ambientais/toxicidade , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Palato/citologia , Dibenzodioxinas Policloradas/toxicidade , Transdução de Sinais/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Palato/efeitos dos fármacos , Palato/embriologia , Gravidez , Receptores de Hidrocarboneto Arílico/efeitos dos fármacos , Proteínas Smad/efeitos dos fármacos , Fator de Crescimento Transformador beta/efeitos dos fármacos
3.
Cells ; 8(7)2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31336838

RESUMO

The presence of Candida albicans in the biofilm underlying the dental prosthesis is related to denture stomatitis (DS), an inflammatory reaction of the oral mucosa. The oral epithelium, a component of the innate immune response, has the ability to react to fungal invasion. In this study, we evaluated the in vitro effect of viable C. albicans on the apoptosis, nitric oxide (NO) production, and ß-defensin 2 (hBD-2) expression and production of human palate epithelial cells (HPECs). We further determined whether or not these effects were correlated with fungal invasion of epithelial cells. Interaction between HPEC primary culture and C. albicans was obtained through either direct or indirect cell-cell contact with a supernatant from a hyphal fungus. We found that the hyphae supernatants were sufficient to induce slight HPEC apoptosis, which occurred prior to the activation of the specific mechanisms of epithelial defense. The epithelial defense responses were found to occur via NO and antimicrobial peptide hBD-2 production only during direct contact between C. albicans and HPECs and coincided with the fungus's intraepithelial invasion. However, although the hBD-2 levels remained constant in the HPEC supernatants over time, the NO release and hBD-2 gene expression were reduced at a later time (10 h), indicating that the epithelial defense capacity against the fungal invasion was not maintained in later phases. This aspect of the immune response was associated with increased epithelial invasion and apoptosis maintenance.


Assuntos
Fibroblastos , Queratinócitos , Mucosa Bucal , Óxido Nítrico/metabolismo , Palato , beta-Defensinas/metabolismo , Biofilmes , Candida albicans/fisiologia , Candidíase/imunologia , Candidíase/microbiologia , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Queratinócitos/citologia , Queratinócitos/metabolismo , Mucosa Bucal/citologia , Mucosa Bucal/metabolismo , Palato/citologia , Palato/metabolismo
4.
Methods Mol Biol ; 1965: 93-105, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069670

RESUMO

The morphogenesis of the secondary palate provides an interesting model for many of the processes involved in embryonic development. A number of in vitro models have been used to study craniofacial development, including whole embryo culture, palatal mesenchymal and micromass cell cultures, and Trowell-like palatal cultures in which dissected palates are cultured individually or as pairs in contact on a support above medium. This chapter presents a detailed protocol for the culture of maxillary midfacial tissues, including the palatal shelves, in suspension culture. This method involves isolation of the midfacial tissues (maxillary arch and palatal shelves) and suspension of the tissues in medium in flasks. On a rocker in an incubator, the palatal shelves elevate, grow, make contact, and fuse in a time span analogous to that occurring in the intact embryo in utero.


Assuntos
Palato/citologia , Técnicas de Cultura de Tecidos/instrumentação , Animais , Proliferação de Células , Células Cultivadas , Incubadoras , Camundongos , Modelos Biológicos
5.
J Periodontal Res ; 54(1): 33-45, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30264516

RESUMO

BACKGROUND AND OBJECTIVE: The potential benefit of using hyaluronan (HA) in reconstructive periodontal surgery is still a matter of debate. The aim of the present study was to evaluate the effects of two HA formulations on human oral fibroblasts involved in soft tissue wound healing/regeneration. MATERIAL AND METHODS: Metabolic, proliferative and migratory abilities of primary human palatal and gingival fibroblasts were examined upon HA treatment. To uncover the mechanisms whereby HA influences cellular behavior, wound healing-related gene expression and activation of signaling kinases were analyzed by qRT-PCR and immunoblotting, respectively. RESULTS: The investigated HA formulations maintained the viability of oral fibroblasts and increased their proliferative and migratory abilities. They enhanced expression of genes encoding type III collagen and transforming growth factor-ß3, characteristic of scarless wound healing. The HAs upregulated the expression of genes encoding pro-proliferative, pro-migratory, and pro-inflammatory factors, with only a moderate effect on the latter in gingival fibroblasts. In palatal but not gingival fibroblasts, an indirect effect of HA on the expression of matrix metalloproteinases 2 and 3 was detected, potentially exerted through induction of pro-inflammatory cytokines. Finally, our data pointed on Akt, Erk1/2 and p38 as the signaling molecules whereby the HAs exert their effects on oral fibroblasts. CONCLUSION: Both investigated HA formulations are biocompatible and enhance the proliferative, migratory and wound healing properties of cell types involved in soft tissue wound healing following regenerative periodontal surgery. Our data further suggest that in gingival tissues, the HAs are not likely to impair the healing process by prolonging inflammation or causing excessive MMP expression at the repair site.


Assuntos
Tecido Conjuntivo/fisiologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Ácido Hialurônico/administração & dosagem , Ácido Hialurônico/farmacologia , Regeneração/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Composição de Medicamentos , Fibroblastos/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Gengiva/citologia , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Palato/citologia , Endodontia Regenerativa , Fator de Crescimento Transformador beta3/genética , Fator de Crescimento Transformador beta3/metabolismo , Cicatrização/genética
6.
Semin Cell Dev Biol ; 91: 75-83, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-28803895

RESUMO

Development of the secondary palate involves a complex series of embryonic events which, if disrupted, result in the common congenital anomaly cleft palate. The secondary palate forms from paired palatal shelves which grow initially vertically before elevating to a horizontal position above the tongue and fusing together in the midline via the medial edge epithelia. As the epithelia of the vertical palatal shelves are in contact with the mandibular and lingual epithelia, pathological fusions between the palate and the mandible and/or the tongue must be prevented. This function is mediated by the single cell layered periderm which forms in a distinct and reproducible pattern early in embryogenesis, exhibits highly polarised expression of adhesion complexes, and is shed from the outer surface as the epidermis acquires its barrier function. Disruption of periderm formation and/or function underlies a series of birth defects that exhibit multiple inter-epithelial adhesions including the autosomal dominant popliteal pterygium syndrome and the autosomal recessive cocoon syndrome and Bartsocas Papas syndrome. Genetic analyses of these conditions have shown that IRF6, IKKA, SFN, RIPK4 and GRHL3, all of which are under the transcriptional control of p63, play a key role in periderm formation. Despite these observations, the medial edge epithelia must rapidly acquire the capability to fuse if the palatal shelves are not to remain cleft. This process is driven by TGFß3-mediated, down-regulation of p63 in the medial edge epithelia which allows periderm migration out of the midline epithelial seam and reduces the proliferative potential of the midline epithelial seam thereby preventing cleft palate. Together, these findings indicate that periderm plays a transient but fundamental role during embryogenesis in preventing pathological adhesion between intimately apposed, adhesion-competent epithelia.


Assuntos
Fissura Palatina/embriologia , Epiderme/embriologia , Epitélio/embriologia , Palato/embriologia , Animais , Diferenciação Celular/genética , Fissura Palatina/genética , Epiderme/metabolismo , Epitélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Palato/citologia , Palato/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética
7.
Anat Rec (Hoboken) ; 301(11): 1861-1870, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30079585

RESUMO

The human soft palate plays an important role in respiration, swallowing, and speech. These motor activities depend on reflexes mediated by sensory nerve endings. To date, the details of human sensory innervation to the soft palate have not been demonstrated. In this study, eight adult human whole-mount (soft palate-tongue-pharynx-larynx-upper esophagus) specimens were obtained from autopsy. Each specimen was bisected in the midline, forming two equal and symmetrical halves. Eight hemi-specimens were processed with Sihler's stain, a whole-mount nerve staining technique. The remaining eight hemi-soft palates were used for immunohistochemical study. The soft palatal mucosa was dissected from the oral and nasal sides and prepared for neurofilament staining. Our results showed that the sensory nerve fibers formed a dense nerve plexus in the lamina propria of the soft palatal mucosa. There was a significant difference in the innervation density between both sides. Specifically, the oral side had higher density of sensory nerve fibers than the nasal side of the soft palate. The mean number and percent area of the sensory nerve fibers in the mucosa of the nasal side was 78% and 72% of those in the mucosa of the oral side, respectively (P < 0.0001). The data presented here could be helpful for further investigating the morphological and quantitative alterations in the sensory nerves in certain upper airway disorders involving the soft palate such as obstructive sleep apnea (OSA) and for designing effective therapeutic strategies to treat OSA. Anat Rec, 301:1861-1870, 2018. © 2018 Wiley Periodicals, Inc.


Assuntos
Palato Mole/citologia , Palato Mole/inervação , Idoso , Feminino , Humanos , Nervos Laríngeos/química , Nervos Laríngeos/citologia , Laringe/química , Laringe/citologia , Masculino , Pessoa de Meia-Idade , Mucosa Bucal/química , Mucosa Bucal/citologia , Mucosa Bucal/inervação , Palato/química , Palato/citologia , Palato/inervação , Palato Mole/química , Coloração e Rotulagem/métodos , Língua/química , Língua/citologia , Língua/inervação
8.
Dev Biol ; 441(1): 191-203, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29981310

RESUMO

Cleft palate is one of the most common craniofacial congenital defects in humans. It is associated with multiple genetic and environmental risk factors, including mutations in the genes encoding signaling molecules in the sonic hedgehog (Shh) pathway, which are risk factors for cleft palate in both humans and mice. However, the function of Shh signaling in the palatal epithelium during palatal fusion remains largely unknown. Although components of the Shh pathway are localized in the palatal epithelium, specific inhibition of Shh signaling in palatal epithelium does not affect palatogenesis. We therefore utilized a hedgehog (Hh) signaling gain-of-function mouse model, K14-Cre;R26SmoM2, to uncover the role of Shh signaling in the palatal epithelium during palatal fusion. In this study, we discovered that constitutive activation of Hh signaling in the palatal epithelium results in submucous cleft palate and persistence of the medial edge epithelium (MEE). Further investigation revealed that precise downregulation of Shh signaling is required at a specific time point in the MEE during palatal fusion. Upregulation of Hh signaling in the palatal epithelium maintains the proliferation of MEE cells. This may be due to a dysfunctional p63/Irf6 regulatory loop. The resistance of MEE cells to apoptosis is likely conferred by enhancement of a cell adhesion network through the maintenance of p63 expression. Collectively, our data illustrate that persistent Hh signaling in the palatal epithelium contributes to the etiology and pathogenesis of submucous cleft palate through its interaction with a p63/Irf6-dependent biological regulatory loop and through a p63-induced cell adhesion network.


Assuntos
Embrião de Mamíferos/metabolismo , Células Epiteliais/metabolismo , Proteínas Hedgehog/metabolismo , Palato/embriologia , Transdução de Sinais/fisiologia , Animais , Adesão Celular/fisiologia , Embrião de Mamíferos/citologia , Células Epiteliais/citologia , Proteínas Hedgehog/genética , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Camundongos , Camundongos Transgênicos , Palato/citologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transativadores/genética , Transativadores/metabolismo
9.
Sci Rep ; 8(1): 9975, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29967482

RESUMO

Oral mechanoreception is implicated in fundamental functions including speech, food intake and swallowing; yet, the neuroanatomical substrates that encode mechanical stimuli are not well understood. Tactile perception is initiated by intricate mechanosensitive machinery involving dedicated cells and neurons. This signal transduction setup is coupled with the topology and mechanical properties of surrounding epithelium, thereby providing a sensitive and accurate system to detect stress fluctuations from the external environment. We mapped the distribution of anatomically distinct neuronal endings in mouse oral cavity using transgenic reporters, molecular markers and quantitative histomorphometry. We found that the tongue is equipped with an array of putative mechanoreceptors that express the principal mechanosensory channel Piezo2, including end bulbs of Krause innervating individual filiform papillae and a novel class of neuronal fibers innervating the epithelium surrounding taste buds. The hard palate and gums are densely populated with three classes of sensory afferents organized in discrete patterns including Merkel cell-neurite complexes, Meissner's corpuscles and glomerular corpuscles. In aged mice, we find that palatal Merkel cells reduce in number at key time-points that correlate with impaired oral abilities, such as swallowing and mastication. Collectively, this work identifies the mechanosensory architecture of oral tissues involved in feeding.


Assuntos
Envelhecimento/fisiologia , Mucosa Bucal/citologia , Mucosa Bucal/inervação , Células Receptoras Sensoriais/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Gengiva/citologia , Gengiva/fisiologia , Imuno-Histoquímica , Células de Merkel/citologia , Células de Merkel/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mucosa Bucal/fisiologia , Palato/citologia , Palato/fisiologia , Compostos de Piridínio/farmacocinética , Compostos de Amônio Quaternário/farmacocinética , Células Receptoras Sensoriais/fisiologia , Língua/fisiologia
10.
Cells Tissues Organs ; 205(2): 93-104, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29734141

RESUMO

Immunohistochemistry for several neurochemical substances was performed on the human incisive papilla and other oral structures. Sodium channel alpha subunit 7 (SCN7A) protein-immunoreactive (IR) Schwann cells and protein gene product 9.5 (PGP 9.5)-IR nerve fibers made nerve plexuses beneath the epithelium of the palate, including the incisive papilla, tongue, and lip. SCN7A immunoreactivity could also be detected in lamellated and nonlamellated capsules of corpuscle endings. Lamellated SCN7A-IR corpuscle endings were mostly restricted to the mucous and cutaneous lips. These endings had thick and spiral-shaped PGP 9.5-IR axons without ramification. Nonlamellated SCN7A-IR corpuscle endings were most numerous in the incisive papilla among the oral regions. On the basis of axonal morphology, the nonlamellated endings were divided into simple and complex types. PGP 9.5-IR terminal axons in the simple type ran straight or meandered with slight ramification, whereas those in the complex type were densely entangled with abundant ramification. Substance P (SP)-, calcitonin gene-related peptide (CGRP)-, and transient receptor potential cation channel subfamily V member 2 (TRPV2)-IR varicose fibers were rarely seen beneath the epithelium of oral structures. The present study indicates that the human incisive papilla has many low-threshold mechanoreceptors with nonlamellated capsules. SP-, CGRP-, and TRPV2-containing nociceptors may be infrequent in the incisive papilla and other oral regions.


Assuntos
Boca/inervação , Palato/inervação , Idoso , Idoso de 80 Anos ou mais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Feminino , Humanos , Masculino , Palato/citologia , Palato/metabolismo , Canais de Cátion TRPV/metabolismo , Ubiquitina Tiolesterase/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo
11.
Reprod Toxicol ; 77: 137-142, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29526646

RESUMO

Numerous studies have been conducted to understand the molecular mechanisms controlling mammalian secondary palate development such as growth, reorientation and fusion. However, little is known about the signaling factors regulating palate initiation. Mouse fibroblast growth factor (FGF) receptor 2 gene (Fgfr2) is expressed on E11.5 in the palate outgrowth within the maxillary process, in a region that is responsible for palate cell specification and shelf initiation. Fgfr2 continues to express in palate on E12.5 and E13.5 in both epithelial and mesenchymal cells, and inactivation of Fgfr2 expression in mesenchymal cells using floxed Fgfr2 allele and Osr2-Cre leads to cleft palate at various stages including reorientation, horizontal growth and fusion. Notably, some mutant embryos displayed no sign of palate shelf formation suggesting that FGF receptor 2 mediated FGF signaling may play an important role in palate initiation.


Assuntos
Palato/crescimento & desenvolvimento , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Animais , Fissura Palatina/genética , Feminino , Mutação com Perda de Função , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Palato/citologia , Palato/metabolismo
12.
Hum Cell ; 31(2): 139-148, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29442285

RESUMO

Bone morphogenetic protein-2 (BMP-2), is a potential factor to enhance osseointegration of dental implants. However, the appropriate cellular system to investigate the osteogenic effect of BMP-2 in vitro in a standardized manner still needs to be defined. The aim of this study was to examine the effect of BMP-2 on the cell proliferation and osteogenic differentiation of human osteogenic progenitors of various origins: dental pulp stem cells (DPSC), human osteosarcoma cell line (Saos-2) and human embryonic palatal mesenchymal cell line (HEPM). For induction of osteogenic differentiation, cell culture medium was supplemented with BMP-2 homodimer alone or in combination with conventionally used differentiation inducing agents. Differentiation was monitored for 6-18 days. To assess differentiation, proliferation rate, alkaline phosphatase activity, calcium deposition and the expression level of osteogenic differentiation marker genes (Runx2, BMP-2) were measured. BMP-2 inhibited cell proliferation in a concentration and time-dependent manner. In a concentration which caused maximal cell proliferation, BMP-2 did not induce osteogenic differentiation in any of the tested systems. However, it had a synergistic effect with the osteoinductive medium in both DPSC and Saos-2, but not in HEPM cells. We also found that the differentiation process was faster in Saos-2 than in DPSCs. Osteogenic differentiation could not be induced in the osteoblast progenitor HEPM cells. Our data suggest that in a concentration that inhibits proliferation the differentiation inducing effect of BMP-2 is evident only in the presence of permissive osteoinductive components. ß-glycerophosphate, was identified interacting with BMP-2 in a synergistic manner.


Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Proteína Morfogenética Óssea 2/fisiologia , Proliferação de Células/efeitos dos fármacos , Polpa Dentária/citologia , Células-Tronco Mesenquimais/citologia , Osseointegração/efeitos dos fármacos , Células-Tronco/citologia , Fosfatase Alcalina/metabolismo , Cálcio/metabolismo , Células Cultivadas , Implantação Dentária Endo-Óssea , Sinergismo Farmacológico , Glicerofosfatos/farmacologia , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Osteossarcoma/patologia , Palato/citologia , Palato/embriologia , Multimerização Proteica , Células-Tronco/metabolismo
13.
Histochem Cell Biol ; 149(2): 143-152, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29209830

RESUMO

Epithelial differentiation is thought to be determined by mesenchymal components during embryogenesis. In mice, palatal mucosa showed the region-specific keratinization pattern along antero-posterior axis. However, developmental mechanisms involved in oral mucosa differentiation with fine tuning of keratinization are not elucidated yet. To reveal this developmental mechanism, first, we conducted tissue recombination assay of the palate at E16 for 2 days which revealed that epithelial differentiation with specific localization of CK10 is modulated by mesenchymal components. Based on the results, we propose that mesenchymal signaling would determine the presumptive fate of developing palatal epithelium in spatiotemporal manner. Genome-wide screening analysis using laser micro-dissection to collect spatiotemporal specific molecules between anterior and posterior palate suggested Meox2 in the posterior mesenchymal tissue to be a candidate regulator controlling epithelial differentiation. To examine the detailed spatiotemporal function of Meox2, we employed in vitro organ cultivation with the loss- and gain-of-function studies at E14.5 for 2 and 4 days, respectively. Our results suggest that posteriorly expressed Meox2 modulates non-keratinized epithelial differentiation through complex signaling regulations in mice palatogenesis.


Assuntos
Diferenciação Celular , Transição Epitelial-Mesenquimal , Mucosa Bucal/citologia , Mucosa Bucal/metabolismo , Palato/citologia , Palato/metabolismo , Transdução de Sinais , Animais , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Queratina-10/genética , Camundongos , Camundongos Endogâmicos ICR , Técnicas de Cultura de Tecidos
14.
Sci Rep ; 7(1): 9131, 2017 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-28831098

RESUMO

RNA-Seq is a powerful tool in transcriptomic profiling of cells and tissues. We recently identified many more taste buds than previously appreciated in chickens using molecular markers to stain oral epithelial sheets of the palate, base of oral cavity, and posterior tongue. In this study, RNA-Seq was performed to understand the transcriptomic architecture of chicken gustatory tissues. Interestingly, taste sensation related genes and many more differentially expressed genes (DEGs) were found between the epithelium and mesenchyme in the base of oral cavity as compared to the palate and posterior tongue. Further RNA-Seq using specifically defined tissues of the base of oral cavity demonstrated that DEGs between gustatory (GE) and non-gustatory epithelium (NGE), and between GE and the underlying mesenchyme (GM) were enriched in multiple GO terms and KEGG pathways, including many biological processes. Well-known genes for taste sensation were highly expressed in the GE. Moreover, genes of signaling components important in organogenesis (Wnt, TGFß/ BMP, FGF, Notch, SHH, Erbb) were differentially expressed between GE and GM. Combined with other features of chicken taste buds, e.g., uniquely patterned array and short turnover cycle, our data suggest that chicken gustatory tissue provides an ideal system for multidisciplinary studies, including organogenesis and regenerative medicine.


Assuntos
Galinhas/genética , Organogênese , Análise de Sequência de RNA/métodos , Papilas Gustativas/citologia , Animais , Embrião de Galinha , Perfilação da Expressão Gênica/métodos , Mesoderma/química , Mesoderma/citologia , Especificidade de Órgãos , Palato/química , Palato/citologia , Transdução de Sinais , Papilas Gustativas/química , Papilas Gustativas/embriologia , Língua/química , Língua/citologia
15.
J Dent Res ; 96(12): 1445-1450, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28759311

RESUMO

Previous studies demonstrated that chondroitin sulfate proteoglycans (CSPGs) on apical surfaces of palatal medial edge epithelial (MEE) cells were necessary for palatal adhesion. In this study, we identified 2 proteoglycans, biglycan and decorin, that were expressed in the palatal shelves prior to adhesion. In addition, we established that these proteoglycans were dependent on transforming growth factor ß (TGFß) signaling. Laser capture microdissection was used to collect selected palatal epithelial cells from embryonic mouse embryos at various palate development stages. The expression of specific messenger RNA (mRNA) for biglycan and decorin was determined with quantitative real-time polymerase chain reaction. The TGFßrI kinase inhibitor (SB431542) was used in palatal organ cultures to determine if blocking TFGß signaling changed biglycan and decorin distribution. Immunohistochemistry of both biglycan and decorin revealed expression on the apical and lateral surfaces of MEE cells. Biglycan protein and mRNA levels peaked as the palatal shelves adhered. Decorin was less abundant on the apical epithelial surface and also had reduced mRNA levels compared to biglycan. Their proteins were not expressed on MEE cells of palates treated with SB431542, an inhibitor of TGFß signaling. The temporal expression of biglycan and decorin on the apical surface of MEE, combined with the evidence that these proteins were regulated through the TGFß pathway, indicated that they may be important for adhesion.


Assuntos
Biglicano/metabolismo , Adesão Celular/fisiologia , Decorina/metabolismo , Palato/citologia , Animais , Benzamidas/farmacologia , Dioxóis/farmacologia , Imuno-Histoquímica , Microdissecção e Captura a Laser , Camundongos , Palato/embriologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Fator de Crescimento Transformador beta/farmacologia
16.
J Dent Res ; 96(12): 1451-1458, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28731788

RESUMO

Previous studies have identified the odd-skipped related 2 (Osr2) transcription factor as a key intrinsic regulator of palatal shelf growth and morphogenesis. However, little is known about the molecular program acting downstream of Osr2 in the regulation of palatogenesis. In this study, we isolated palatal mesenchyme cells from embryonic day 12.5 (E12.5) and E13.5 Osr2RFP/+ and Osr2RFP/- mutant mouse embryos and performed whole transcriptome RNA sequencing analyses. Differential expression analysis of the RNA sequencing datasets revealed that expression of 70 genes was upregulated and expression of 61 genes was downregulated by >1.5-fold at both E12.5 and E13.5 in the Osr2RFP/- palatal mesenchyme cells, in comparison with Osr2RFP/+ littermates. Gene ontology analysis revealed enrichment of signaling molecules and transcription factors crucial for skeletal development and osteoblast differentiation among those significantly upregulated in the Osr2 mutant palatal mesenchyme. Using quantitative real-time polymerase chain reaction (RT-PCR)and in situ hybridization assays, we validated that the Osr2-/- embryos exhibit significantly increased and expanded expression of many osteogenic pathway genes, including Bmp3, Bmp5, Bmp7, Mef2c, Sox6, and Sp7 in the developing palatal mesenchyme. Furthermore, we demonstrate that expression of Sema3a, Sema3d, and Sema3e, is ectopically activated in the developing palatal mesenchyme in Osr2-/- embryos. Through chromatin immunoprecipitation, followed by RT-PCR analysis, we demonstrate that endogenous Osr2 protein binds to the promoter regions of the Sema3a and Sema3d genes in the embryonic palatal mesenchyme. Moreover, Osr2 expression repressed the transcription from the Sema3a and Sema3d promoters in cotransfected cells. Since the Sema3 subfamily of signaling molecules plays diverse roles in the regulation of cell proliferation, migration, and differentiation, these data reveal a novel role for Osr2 in regulation of palatal morphogenesis through preventing aberrant activation of Sema3 signaling. Together, these data indicate that Osr2 controls multiple molecular pathways, including BMP and Sema3 signaling, in palate development.


Assuntos
Palato/embriologia , Fatores de Transcrição/genética , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Imunoprecipitação , Hibridização In Situ , Camundongos , Camundongos Endogâmicos , Morfogênese/genética , Palato/citologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética
17.
Sci Rep ; 7(1): 5148, 2017 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-28698574

RESUMO

Normal cell cycle progression and proliferation of palatal mesenchymal cells are important for palatal development. As targets of miR-17-92, E2F transcription factors family has been suggested to induce the transcription of miR-17-92 in several cell types. In the present study, we sought to investigate whether this negative feedback loop exists in mouse PMCs and what the function of this negative feedback loop would be in palatal mesenchymal cells. Using GeneMANIA, we revealed that the most important function of experimentally verified targets of miR-17-92 is cell cycle regulation. E2F1 and E2F3, but not E2F2, were extensively expressed in mouse palate. Over-expression of E2F1 significantly increased the expression of all the members of miR-17-92. After increased by E2F1, miR-17 and miR-20a may negatively target E2F1, and thereby prevent the cells from excessive proliferation. We suggest that the negative feedback loop between E2F1 and miR-17-92 may contribute to palatal development by regulating the proliferation and cell cycle of palatal mesenchymal cells.


Assuntos
Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , MicroRNAs/genética , Palato/crescimento & desenvolvimento , Animais , Ciclo Celular , Linhagem Celular , Proliferação de Células , Fator de Transcrição E2F2/genética , Fator de Transcrição E2F2/metabolismo , Fator de Transcrição E2F3/genética , Fator de Transcrição E2F3/metabolismo , Retroalimentação Fisiológica , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Família Multigênica , Palato/citologia , Palato/metabolismo
18.
Histochem Cell Biol ; 147(3): 377-388, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27600719

RESUMO

Current tissue engineering technology focuses on developing simple tissues, whereas multilayered structures comprising several tissue types have rarely been described. We developed a highly biomimetic multilayered palate substitute with bone and oral mucosa tissues using rabbit cells and biomaterials subjected to nanotechnological techniques based on plastic compression. This novel palate substitute was autologously grafted in vivo, and histological and histochemical analyses were used to evaluate biointegration, cell function, and cell differentiation in the multilayered palate substitute. The three-dimensional structure of the multilayered palate substitute was histologically similar to control tissues, but the ex vivo level of cell and tissue differentiation were low as determined by the absence of epithelial differentiation although cytokeratins 4 and 13 were expressed. In vivo grafting was associated with greater cell differentiation, epithelial stratification, and maturation, but the expression of cytokeratins 4, 13, 5, and 19 at did not reach control tissue levels. Histochemical analysis of the oral mucosa stroma and bone detected weak signals for proteoglycans, elastic and collagen fibers, mineralization deposits and osteocalcin in the multilayered palate substitute cultured ex vivo. However, in vivo grafting was able to induce cell and tissue differentiation, although the expression levels of these components were always significantly lower than those found in controls, except for collagen in the bone layer. These results suggest that generation of a full-thickness multilayered palate substitute is achievable and that tissues become partially differentiated upon in vivo grafting.


Assuntos
Órgãos Bioartificiais , Materiais Biocompatíveis , Palato/citologia , Engenharia Tecidual/métodos , Animais , Osso e Ossos/citologia , Diferenciação Celular , Células Cultivadas , Técnicas In Vitro , Mucosa Bucal/citologia , Mucosa Bucal/transplante , Palato/anatomia & histologia , Coelhos , Transplante Autólogo
19.
Am J Orthod Dentofacial Orthop ; 151(1): 132-142, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28024766

RESUMO

INTRODUCTION: The purpose of this study was to evaluate the effects of a newly designed magnetic palatal expansion appliance with a reactivation system. METHODS: A magnetic palatal expansion appliance was designed based on the repulsion forces of neodymium-iron-boron magnets combined with a reactivation system. Eighteen prepubertal male beagle dogs were assigned randomly to the magnetic expansion (ME) group, the mechanical screw expansion (SE) group, or the control group. Two pairs of nonmagnetic metal bone marker implants were inserted into palatal bone bilaterally 3 mm lateral to the midpalatal suture and in line with the first and fourth premolars, respectively, in each dog. The 6 animals in each group received (1) newly designed magnetic expanders, (2) jackscrew expanders, or (3) no expansion appliance. Expansion was stopped after 4 weeks when 6 mm of activation was achieved in the 2 treated groups. Three-dimensional evaluations of dental and skeletal effects were performed with cone-beam computed tomography. Histologic examinations were conducted using light microscopy to observe morphologic changes in the midpalatal suture after hematoxylin and eosin staining. RESULTS: The absolute transversal changes of both treated groups before and after expansion were significantly greater than those in the control group in all parameters (P <0.001). The differences of the distances of bilateral canines in the ME group were significantly greater than in the SE group (1.04 ± 0.16 mm; P <0.001); the differences of the distances between implants adjacent to the first premolars (0.77 ± 0.06 mm; P <0.001) and the distances between implants adjacent to the fourth premolars (0.37 ± 0.06 mm; P <0.001) in the SE group were significantly greater than in the ME group. Histologic observations of the palatal sutures in the ME and SE groups, when compared with the control group, showed widening of the sutures and many fibroblasts in an active, proliferative state. Counts of osteoblasts were increased in both expansion groups. Counts of osteoclasts were increased in the SE group. CONCLUSIONS: Both appliances expanded the maxilla effectively and induced processes of bone remodeling of the midpalatal sutures during expansion. The new magnetic palatal expansion appliances produced a smaller skeletal effect and a greater dental effect than did the mechanical screw palatal expansion appliances.


Assuntos
Técnica de Expansão Palatina/instrumentação , Animais , Contagem de Células , Tomografia Computadorizada de Feixe Cônico , Cães , Magnetismo , Masculino , Osteoblastos , Osteoclastos , Palato/anatomia & histologia , Palato/citologia , Palato/diagnóstico por imagem
20.
Toxicol Appl Pharmacol ; 305: 186-193, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27312872

RESUMO

Cleft palate is caused by the failure of palatal midline epithelial cells to disintegrate, which is necessary for palatal mesenchymal confluence. Although 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure is linked to cleft palate at a high rate, the mechanism remains to be elucidated. The present study was designed to determine the effects of TCDD on the fate of epithelial cell isolated from human fetal palatal shelves (hFPECs). We demonstrate that TCDD increased cell proliferation and promoted the progression of cells from G1 to S phase as well as increased the number of cells entering the G2/M phase. We found that TCDD has no measurable effect on apoptosis of hFPECs. The protein level assays revealed that TCDD increased cyclin-dependent kinases 4 (cdk4), cyclin D1, cyclin E and p21 (Waf1/Cip1) but not cdk2, bcl-2, cyclin B1 and cyclin A. Furthermore, TCDD activated PI3K/AKT signaling, and the PI3K inhibitor, LY294002, partially abrogated TCDD-induced cell proliferation and gene modulations. TCDD treatment increased CYP1A1 mRNA and protein levels, which indicated the activation of AhR signaling. Knockdown of the AhR with siRNA suppressed TCDD-induced cell proliferation and PI3K/AKT signaling activation. Taken together, these data demonstrated that TCDD is able to promote growth of hFPECs through AhR-dependent activation of the PI3K/AKT pathway, which may account for the underlying mechanism by which TCDD causes a failure of palatal fusion.


Assuntos
Células Epiteliais/efeitos dos fármacos , Palato/citologia , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Células Epiteliais/metabolismo , Feto , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética
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