RESUMEN
Cleft palate (CP) is a common birth defect with a high incidence of occurrence in humans. The 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic halogenated aromatic hydrocarbon, with a strong CP effect on mice. Increasing recent evidences have shown that long-noncoding RNAs (lncRNAs) play an important role in several diseases, including CP. However, there is a paucity of studies on the role of lncRNA MEG3 in the occurrence and development of TCDD-induced CP. In this study, the relationship between MEG3 and the proliferation of palatal mesenchymal cells and the underlying molecular mechanism were studied by establishing fetal CP with TCDD (64 µg/kg) in C57BL/6N mice. The results revealed that MEG3 was highly expressed during the critical period of CP formation and that the fetal mesenchymal proliferation was significantly inhibited at certain critical periods in the mice receiving TCDD. In addition, we noted a possibility of a crosstalk between MEG3 and the TGF-ß/Smad pathway, such that the inhibition of the TGF-ß/Smad pathway was induced by TCDD. Cumulatively, our study suggests that TCDD-induced CP may be caused by MEG3 inhibition of the proliferation of palatal mesenchymal cells involving the TGFß/Smad pathway, which may provide a novel perspective to understand the pathogenesis of CP.
Asunto(s)
Proliferación Celular , Fisura del Paladar/metabolismo , Células Madre Mesenquimatosas/metabolismo , Paladar Duro/metabolismo , ARN Largo no Codificante/metabolismo , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Fisura del Paladar/inducido químicamente , Fisura del Paladar/genética , Fisura del Paladar/patología , Modelos Animales de Enfermedad , Femenino , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Células Madre Mesenquimatosas/patología , Ratones Endogámicos C57BL , Paladar Duro/anomalías , Fosforilación , Dibenzodioxinas Policloradas , Embarazo , ARN Largo no Codificante/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
OBJECTIVE: Patients with coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), exhibit not only respiratory symptoms but also symptoms of chemo-sensitive disorders. Cellular entry of SARS-CoV-2 depends on the binding of its spike protein to a cellular receptor named angiotensin-converting enzyme 2 (ACE2), and the subsequent spike protein-priming by host cell proteases, including transmembrane protease serine 2 (TMPRSS2). Thus, high expression of ACE2 and TMPRSS2 is considered to enhance the invading capacity of SARS-CoV-2. METHODS: To elucidate the underlying histological mechanisms of the aerodigestive disorders caused by SARS-CoV-2, we investigated the expression of ACE2 and TMPRSS2 proteins using immunohistochemistry, in the aerodigestive tracts of the tongue, hard palate with partial nasal tissue, larynx with hypopharynx, trachea, esophagus, and lung of rats. RESULTS: Co-expression of ACE2 and TMPRSS2 proteins was observed in the taste buds of the tongue, nasal epithelium, trachea, bronchioles, and alveoli with varying degrees of expression. Remarkably, TMPRSS2 expression was more distinct in the peripheral alveoli than in the central alveoli. These results coincide with the reported clinical symptoms of COVID-19, such as the loss of taste, loss of olfaction, and respiratory dysfunction. CONCLUSIONS: A wide range of organs have been speculated to be affected by SARS-CoV-2 depending on the expression levels of ACE2 and TMPRSS2. Differential distribution of TMPRSS2 in the lung indicated the COVID-19 symptoms to possibly be exacerbated by TMPRSS2 expression. This study might provide potential clues for further investigation of the pathogenesis of COVID-19. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E932-E939, 2021.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/patología , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/metabolismo , Enzima Convertidora de Angiotensina 2/análisis , Animales , COVID-19/virología , Esófago/metabolismo , Humanos , Inmunohistoquímica , Laringe/metabolismo , Pulmón/metabolismo , Masculino , Proteínas de la Membrana/análisis , Modelos Animales , Paladar Duro/metabolismo , Ratas , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Serina Endopeptidasas/análisis , Glicoproteína de la Espiga del Coronavirus/metabolismo , Lengua/metabolismo , Tráquea/metabolismo , Internalización del VirusRESUMEN
Although the matricellular protein periostin is prominently upregulated in skin and gingival healing, it plays contrasting roles in myofibroblast differentiation and matrix synthesis respectively. Palatal healing is associated with scarring that can alter or restrict maxilla growth, but the expression pattern and contribution of periostin in palatal healing is unknown. Using periostin-knockout (Postn-/-) and wild-type (WT) mice, the contribution of periostin to palatal healing was investigated through 1.5 mm full-thickness excisional wounds in the hard palate. In WT mice, periostin was upregulated 6 days post-wounding, with mRNA levels peaking at day 12. Genetic deletion of periostin significantly reduced wound closure rates compared to WT mice. Absence of periostin reduced mRNA levels of pivotal genes in wound repair, including α-SMA/acta2, fibronectin and ßigh3. Recruitment of fibroblasts and inflammatory cells, as visualized by immunofluorescent staining for fibroblast specific factor-1, vimentin, and macrophages markers Arginase-1 and iNOS was also impaired in Postn-/-, but not WT mice. Palatal fibroblasts isolated from the hard palate of mice were cultured on collagen gels and prefabricated silicon substrates with varying stiffness. Postn-/- fibroblasts showed a significantly reduced ability to contract a collagen gel, which was rescued by the exogenous addition of recombinant periostin. As the stiffness increased, Postn-/- fibroblasts increasingly differentiated into myofibroblasts, but not to the same degree as the WT. Pharmacological inhibition of Rac rescued the deficient myofibroblastic phenotype of Postn-/- cells. Low stiffness substrates (0.2 kPa) resulted in upregulation of fibronectin in WT cells, an effect which was significantly reduced in Postn-/- cells. Quantification of immunostaining for vinculin and integrinß1 adhesions revealed that Periostin is required for the formation of focal and fibrillar adhesions in mPFBs. Our results suggest that periostin modulates myofibroblast differentiation and contraction via integrinß1/RhoA pathway, and fibronectin synthesis in an ECM stiffness dependent manner in palatal healing.
Asunto(s)
Moléculas de Adhesión Celular/genética , Diferenciación Celular/genética , Fibronectinas/genética , Paladar Duro/crecimiento & desarrollo , Cicatrización de Heridas/genética , Actinas/genética , Animales , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Fibroblastos/patología , Fibronectinas/biosíntesis , Humanos , Integrina beta1/genética , Maxilar/crecimiento & desarrollo , Maxilar/metabolismo , Ratones , Ratones Noqueados , Miofibroblastos/metabolismo , Miofibroblastos/patología , Paladar Duro/metabolismo , Paladar Duro/fisiopatología , Transducción de Señal/genética , Proteína de Unión al GTP rhoA/genéticaRESUMEN
During mammalian palatogenesis, cranial neural crest-derived mesenchymal cells undergo osteogenic differentiation and form the hard palate, which is divided into palatine process of the maxilla and the palatine. However, it remains unknown whether these bony structures originate from the same cell lineage and how the hard palate is patterned at the molecular level. Using mice, here we report that deficiency in Shox2 (short stature homeobox 2), a transcriptional regulator whose expression is restricted to the anterior palatal mesenchyme, leads to a defective palatine process of the maxilla but does not affect the palatine. Shox2 overexpression in palatal mesenchyme resulted in a hyperplastic palatine process of the maxilla and a hypoplastic palatine. RNA sequencing and assay for transposase-accessible chromatin-sequencing analyses revealed that Shox2 controls the expression of pattern specification and skeletogenic genes associated with accessible chromatin in the anterior palate. This highlighted a lineage-autonomous function of Shox2 in patterning and osteogenesis of the hard palate. H3K27ac ChIP-Seq and transient transgenic enhancer assays revealed that Shox2 binds distal-acting cis-regulatory elements in an anterior palate-specific manner. Our results suggest that the palatine process of the maxilla and palatine arise from different cell lineages and differ in ossification mechanisms. Shox2 evidently controls osteogenesis of a cell lineage and contributes to the palatine process of the maxilla by interacting with distal cis-regulatory elements to regulate skeletogenic gene expression and to pattern the hard palate. Genome-wide Shox2 occupancy in the developing palate may provide a marker for identifying active anterior palate-specific gene enhancers.
Asunto(s)
Diferenciación Celular/genética , Proteínas de Homeodominio/genética , Osteogénesis/genética , Paladar Duro/metabolismo , Animales , Tipificación del Cuerpo/genética , Linaje de la Célula/genética , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Maxilar/citología , Maxilar/embriología , Maxilar/metabolismo , Ratones Noqueados , Ratones Transgénicos , Paladar Duro/citología , Paladar Duro/embriología , Transducción de Señal/genéticaRESUMEN
Buccal and palatal injections are required for administration of anesthetic agents before maxillary tooth extractions, but palatal injections are painful for patients. Studies suggest that the palatal injection can be eliminated when articaine is delivered as a local anesthetic agent via buccal injection, but the anatomical mechanism for this effectiveness remains unclear. The objective of this study was to explore the potential mechanism by which buccal infiltration results in palatal anesthesia. The study approach included examining cadaveric specimens and investigating the pharmacologic properties of articaine. Twenty-eight formalin-fixed cadaveric hemimaxillae were dissected and sectioned into anterior, premolar, and molar regions. The maxillary sections were measured in 3 planes: inferior, middle, and superior. Buccal cortical plate (BCP), palatal cortical plate (PCP), and total buccopalatal (TBP) thickness were independently evaluated by 2 measurers using standard digital calipers. Statistical analysis of regional maxillary thickness measurements was achieved via 2-way analysis of variance. Measurements of BCP and PCP thickness revealed no statistically significant differences along the maxillae (P > 0.05). Both the BCP and PCP mean values were significantly less than the TBP measurement (P < 0.0001). In all 3 regions, the mean TBP thickness in the superior plane was significantly greater than that of the inferior plane (P < 0.05). The mean TBP thickness was significantly greater in the molar and premolar regions than in the anterior region (P < 0.05). The mean BCP measurements were significantly lesser in the maxillary premolar and molar regions than in the corresponding mandibular regions (P < 0.0001). The pharmacologic properties of articaine, which is capable of diffusing greater distances than other local anesthetics, coupled with the uniformly thin, cancellous maxillary bone, provide a plausible explanation for the success of palatal anesthesia achieved through buccal infiltration of articaine, obviating the need for a palatal injection.
Asunto(s)
Anestesia Dental , Anestesia Local , Anestésicos Locales/administración & dosificación , Carticaína , Paladar Duro , Administración Bucal , Anestesia Dental/métodos , Anestesia Local/métodos , Carticaína/administración & dosificación , Humanos , Membrana Mucosa , Paladar Duro/metabolismoRESUMEN
Cleft palate is one of the most common craniofacial defects in newborn babies. The characteristics of this genetic disease produce soft and hard tissue defects on the lip and maxilla, which cause not only aesthetic but also functional problems with speech, eating, and breathing. Bone grafts using autologous cancellous bone have been a standard treatment to repair the hard tissue defect in cleft palates. However, such grafts do not fully integrate into host bone and undergo resorption. To overcome engraftment problems, it is common to engineer new tissues with a combination of multipotent cells and biomaterial frameworks. Here, we manufactured cell sheets for bone repair of cleft palates derived from two osteogenic cell sources, human mesenchymal stem cells (hMSCs) and stem cells from human exfoliated deciduous teeth (SHEDs). Cell sheets made from hMSCs and SHEDs gave rise to in vitro calcification, which indicated the osteogenic potential of these cells. The cell sheets of hMSCs and SHEDs expressed the bone-specific osteogenic markers, osterix, osteocalcin, and osteopontin, following insertion into ex vivo-cultured embryonic palatal shelves and in ovo culture. In conclusion, we showed that osteogenic stem cell sheets have mineralization potential and might represent a new alternative to autologous bone transplantation in the reconstruction of cleft palates.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Células Madre Mesenquimatosas/metabolismo , Paladar Duro/metabolismo , Ingeniería de Tejidos , Diente Primario/metabolismo , Animales , Embrión de Pollo , Niño , Fisura del Paladar/metabolismo , Fisura del Paladar/terapia , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Paladar Duro/citología , Diente Primario/citologíaRESUMEN
Synovial sarcoma is a high-grade soft tissue sarcoma that rarely arises in the head and neck region. It affects the parapharyngeal space and the hypopharynx most commonly and it has different presentations based on the affected site. In extremely rare occasions, it involves the hard palate such as in our case where a 24-year-old female patient presented with a mass lesion involving the left hard palate, which was identified clinically and by imaging studies. The histopathological assessment confirmed that it was a monophasic synovial sarcoma which was also confirmed with further molecular studies. The patient underwent surgical excision and postoperative radiotherapy. Her close follow up over a 6-year period that followed her curative treatment has demonstrated no evidence of disease recurrence or distant metastasis. Surgical excision is the mainstay of treatment for synovial sarcoma and adjuvant radiotherapy is advised. Long-term follow up is recommended because of the remote possibility of late recurrence of the tumor.
Asunto(s)
Neoplasias Maxilomandibulares , Paladar Duro , Sarcoma Sinovial , Adulto , Femenino , Humanos , Neoplasias Maxilomandibulares/diagnóstico por imagen , Neoplasias Maxilomandibulares/metabolismo , Neoplasias Maxilomandibulares/patología , Neoplasias Maxilomandibulares/terapia , Paladar Duro/metabolismo , Paladar Duro/patología , Paladar Duro/cirugía , Sarcoma Sinovial/diagnóstico por imagen , Sarcoma Sinovial/metabolismo , Sarcoma Sinovial/patología , Sarcoma Sinovial/terapiaRESUMEN
Periodic patterning of iterative structures is diverse across the animal kingdom. Clarifying the molecular mechanisms involved in the formation of these structure helps to elucidate the process of organogenesis. Turing-type reaction-diffusion mechanisms have been shown to play a critical role in regulating periodic patterning in organogenesis. Palatal rugae are periodically patterned ridges situated on the hard palate of mammals. We have previously shown that the palatal rugae develop by a Turing-type reaction-diffusion mechanism, which is reliant upon Shh (as an inhibitor) and Fgf (as an activator) signaling for appropriate organization of these structures. The disturbance of Shh and Fgf signaling lead to disorganized palatal rugae. However, the mechanism itself is not fully understood. Here we found that Lrp4 (transmembrane protein) was expressed in a complementary pattern to Wise (a secreted BMP antagonist and Wnt modulator) expression in palatal rugae development, representing Lrp4 expression in developing rugae and Wise in the inter-rugal epithelium. Highly disorganized palatal rugae was observed in both Wise and Lrp4 mutant mice, and these mutants also showed the downregulation of Shh signaling, which was accompanied with upregulation of Fgf signaling. Wise and Lrp4 are thus likely to control palatal rugae development by regulating reaction-diffusion mechanisms through Shh and Fgf signaling. We also found that Bmp and Wnt signaling were partially involved in this mechanism.
Asunto(s)
Tipificación del Cuerpo , Proteínas Morfogenéticas Óseas/metabolismo , Paladar Duro/embriología , Paladar Duro/metabolismo , Receptores de LDL/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Tipificación del Cuerpo/genética , Proteínas Morfogenéticas Óseas/genética , Difusión , Regulación del Desarrollo de la Expresión Génica , Proteínas Relacionadas con Receptor de LDL , Ratones , Ratones Mutantes , Paladar Duro/patología , Fenotipo , Receptores de LDL/genética , Transducción de SeñalRESUMEN
Fibroblast growth factor (FGF) signaling has been implicated in the regulation of osteogenesis in both intramembranous and endochondral ossifications. In the developing palate, the anterior bony palate forms by direct differentiation of cranial neural crest (CNC)-derived mesenchymal cells, but the signals that regulate the osteogenic cell fate in the developing palate remain unclear. In the present study, we investigated the potential role of FGF signaling in osteogenic fate determination of the palatal mesenchymal cells. We showed that locally activated FGF8 signaling in the anterior palate using a Shox2Cre knock-in allele and an R26RFgf8 allele leads to a unique palatal defect: a complete loss of the palatine process of the maxilla as well as formation of ectopic cartilaginous tissues in the anterior palate. This aberrant developmental process was accompanied by a significantly elevated level of cell proliferation, which contributes to an abnormally thickened palatal tissue, where the palatine process of the maxilla would normally form, and by a complete inhibition of Osterix expression, which accounts for the lack of bone formation. The coexpression of Runx2 initially with Sox9 and subsequently with Col II in the ectopic cartilaginous tissues indicates a conversion of osteogenic fate to a chondrogenic one. Consistent with the unique palatal phenotype, RNA-Sequencing analysis revealed that the augmented FGF8 signaling downregulated genes involved in ossification, biomineral tissue development, and bone mineralization but upregulated genes involved in cell proliferation, cartilage development, and cell fate commitment, which was further supported by quantitative real-time reverse transcription polymerase chain reaction validation of selected genes. Our results demonstrate that FGF8 signaling functions as a negative regulator of osteogenic fate and is sufficient to convert a subset of CNC cell-derived mesenchymal cells into cartilage in the anterior hard palate, which will have implications in future directed differentiation of CNC-derived precursor cells for clinical application.
Asunto(s)
Linaje de la Célula , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Paladar Duro/crecimiento & desarrollo , Animales , Diferenciación Celular , Ratones , Osteogénesis , Paladar Duro/citología , Paladar Duro/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
RNA interference (RNAi) is a powerful tool to silence or minimize gene expression, and palate culture in vitro is an important technique for study of the palate development. Our previous study demonstrated that the gene expression of glucose-regulated protein-78 (Grp78) was downregulation in the all-trans retinoic acid-induced mouse models of cleft palate (CP) during embryogenesis. To find the role of Grp78, the small interfering RNA (siRNA) of this gene carried by fluorescent vector was injected with a microinjector, through which about 30 pmol siRNA was injected into the Institute of Cancer Research (ICR) mouse palate explants. After 6, 12, 24, 48, and 72 h, these palate explants were removed from culture to observe their fluorescent and Alcian blue-staining phenotypes, and the expression of the unfolded protein response (UPR) key members (Grp78, Inositol-responsive enzyme 1, protein kinase RNA-like endoplasmic reticulum kinase, activating transcription factor-6 and X-box binding protein-1) was measured. After cultured for 72 h, the partially or completely fused bilateral palates were observed in the control siRNA group, while CPs were found in the Grp78 siRNA group. In the Grp78 siRNA group, the relatively mRNA abundance of the key genes belonged to UPR at each time point was lower than that of the control siRNA group, and their protein expression also displayed the same change. By the system of RNAi strategies with mouse palate culture, we found the siRNA of Grp78 disturbed the fusion of mouse palate cultured in vitro.
Asunto(s)
Fisura del Paladar/genética , Proteínas de Choque Térmico/genética , Paladar Duro/anomalías , Interferencia de ARN , Factor de Transcripción Activador 6/genética , Factor de Transcripción Activador 6/metabolismo , Animales , Fisura del Paladar/metabolismo , Chaperón BiP del Retículo Endoplásmico , Regulación del Desarrollo de la Expresión Génica , Proteínas de Choque Térmico/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos ICR , Morfogénesis , Paladar Duro/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Respuesta de Proteína Desplegada , Proteína 1 de Unión a la X-Box/genética , Proteína 1 de Unión a la X-Box/metabolismo , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
PURPOSE: Palatal local anesthetic injection is a painful procedure. Previous studies have reported successful extraction of maxillary teeth using only buccal infiltration of 4% articaine without palatal anesthesia. The aim of the present study was to determine levels of 4% articaine solution in palatal bone and mucosal tissues after buccal injection and compare those levels with 2% lidocaine solution in New Zealand white rabbits. MATERIALS AND METHODS: Eight rabbits received 2 different injections of 0.6 mL of 4% articaine with 1:100,000 epinephrine and 0.6 mL of 2% lidocaine with 1:100,000 epinephrine buccal to the right and left maxillary first molar, respectively, in a split-mouth study design using quantitative syringes. All injections were administered using the buccal infiltration technique without any palatal injection. Ten minutes later, palatal bone and mucosa specimens were collected for analysis. Levels of the 2 local anesthetic agents were measured in palatal tissues using high-performance liquid chromatography (HPLC). RESULTS: HPLC analysis showed markedly higher 4% articaine solution values (0.319 ± 0.037) in palatal mucosal tissues compared with palatal mucosal concentrations of 2% lidocaine solution (0.0839 ± 0.017). In palatal bone, the mean concentration of 2% lidocaine solution was markedly lower than the mean concentration of 4% articaine solution (0.085 ± 0.012 vs 0.155 ± 0.012, respectively). There was no relevant difference between levels of 2% lidocaine in the palatal bone and mucosal tissues. However, the mean concentration of 4% articaine in the palatal mucosa was markedly higher than its concentration in palatal bone. CONCLUSIONS: The buccal vestibule-palatal diffusion of 4% articaine solution with 1:100,000 epinephrine is greater than 2% lidocaine solution with 1:100,000 epinephrine in a rabbit model.
Asunto(s)
Anestesia Local/métodos , Anestésicos Locales/farmacocinética , Carticaína/farmacocinética , Lidocaína/farmacocinética , Paladar Duro/metabolismo , Administración Bucal , Anestésicos Locales/administración & dosificación , Animales , Carticaína/administración & dosificación , Cromatografía Líquida de Alta Presión , Lidocaína/administración & dosificación , Masculino , ConejosRESUMEN
Background: The role of microRNAs (miRNAs) in animal models of palatogenesis has been shown, but only limited research has been carried out in humans. To date, no miRNA expression study on tissues or cells from cleft palate patients has been published. We compared miRNA expression in palatal fibroblasts from cleft palate patients and age-matched controls. Material and Methods: Cultured palatal fibroblasts from 10 non-syndromic cleft lip and palate patients (nsCLP; mean age: 18 ± 2 months), 5 non-syndromic cleft palate only patients (nsCPO; mean age: 17 ± 2 months), and 10 controls (mean age: 24 ± 5 months) were analysed with next-generation small RNA sequencing. All subjects are from Western European descent. Sequence reads were bioinformatically processed and the differentially expressed miRNAs were technically validated using quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Results: Using RNA sequencing, three miRNAs (hsa-miR-93-5p, hsa-miR-18a-5p, and hsa-miR-92a-3p) were up-regulated and six (hsa-miR-29c-5p, hsa-miR-549a, hsa-miR-3182, hsa-miR-181a-5p, hsa-miR-451a, and hsa-miR-92b-5p) were down-regulated in nsCPO fibroblasts. One miRNA (hsa-miR-505-3p) was down-regulated in nsCLP fibroblasts. Of these, hsa-miR-505-3p, hsa-miR-92a, hsa-miR-181a, and hsa-miR-451a were also differentially expressed using RT-PCR with a higher fold change than in RNAseq. Limitations: The small sample size may limit the value of the data. In addition, interpretation of the data is complicated by the fact that biopsy samples are taken after birth, while the origin of the cleft lies in the embryonic period. This, together with possible effects of the culture medium, implies that only cell-autonomous genetic and epigenetic differences might be detected. Conclusions: For the first time, we have shown that several miRNAs appear to be dysregulated in palatal fibroblasts from patients with nsCLP and nsCPO. Furthermore, large-scale genomic and expression studies are needed to validate these findings.
Asunto(s)
Fisura del Paladar/genética , Fibroblastos/metabolismo , MicroARNs/genética , Paladar Duro/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Preescolar , Fisura del Paladar/patología , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Humanos , Lactante , Masculino , Paladar Duro/patología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodosRESUMEN
High mobility group box 1 (HMGB1) is tightly connected to the process of tissue organization upon tissue injury. Here we show that HMGB1 controls epithelium and connective tissue regeneration both in vivo and in vitro during palatal wound healing. Heterozygous HMGB1 (Hmgb1+/-) mice and Wild-type (WT) mice were subjected to palatal injury. Maxillary tissues were stained with Mallory Azan or immunostained with anti-HMGB1, anti-proliferating cell nuclear antigen (PCNA), anti-nuclear factor-κB (NF-κB) p50 and anti-vascular endothelial growth factor (VEGF) antibodies. Palatal gingival explants were cultured with recombinant HMGB1 (rHMGB1) co-treated with siRNA targeting receptor for advanced glycation end products (RAGEs) for cell migration and PCNA expression analysis. Measurement of the wound area showed differences between Hmgb1+/- and WT mice on Day 3 after wounding. Mallory Azan staining showed densely packed of collagen fibers in WT mice, whereas in Hmgb1+/- mice weave-like pattern of low density collagen bundles were present. At three and seven days post-surgery, PCNA, NF-κB p50 and VEGF positive keratinocytes of WT mice were greater than that of Hmgb1+/- mice. Knockdown of RAGE prevents the effect of rHMGB1-induced cell migration and PCNA expression in gingival cell cultures. The data suggest that HMGB1/RAGE axis has crucial roles in palatal wound healing.
Asunto(s)
Proteína HMGB1/genética , Queratinocitos/metabolismo , Paladar Duro/metabolismo , Receptor para Productos Finales de Glicación Avanzada/genética , Cicatrización de Heridas/genética , Animales , Anticuerpos Monoclonales/química , Regulación de la Expresión Génica , Encía/lesiones , Encía/metabolismo , Proteína HMGB1/metabolismo , Inmunohistoquímica , Queratinocitos/patología , Maxilar/lesiones , Maxilar/metabolismo , Ratones , Ratones Noqueados , Mucosa Bucal/lesiones , Mucosa Bucal/metabolismo , Subunidad p50 de NF-kappa B/genética , Subunidad p50 de NF-kappa B/metabolismo , Paladar Duro/lesiones , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor para Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Transducción de Señal , Técnicas de Cultivo de Tejidos , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Pulpal anesthesia success rates for ropivacaine following maxillary infiltration anesthesia seem to be low. We investigated the hypothesis that the addition of epinephrine would affect the pharmacokinetics of ropivacaine by retaining ropivacaine in the mucosa of the injected area through the time-dependent distribution of ropivacaine in the rat maxilla and serum following maxillary infiltration anesthesia using (3)H-labeled ropivacaine. We then examined the vasoactivity of ropivacaine with or without epinephrine on local peripheral blood flow. The addition of epinephrine to ropivacaine increased ropivacaine concentrations in the palatal mucosa and adjacent maxilla by more than 3 times that of plain ropivacaine at 20 minutes. By observing the autoradiogram of (3)H-ropivacaine, plain ropivacaine in the maxilla was remarkably reduced 20 minutes after injection. However, it was definitely retained in the palatal mucosa, hard palate, adjacent maxilla, and maxillary nerve after the administration with epinephrine. Ropivacaine with epinephrine significantly decreased labial blood flow. This study suggests that 10 µg/mL epinephrine added to 0.5% ropivacaine could improve anesthetic efficacy and duration for maxillary infiltration anesthesia over plain ropivacaine.
Asunto(s)
Amidas/farmacocinética , Anestésicos Locales/farmacocinética , Epinefrina/administración & dosificación , Hueso Paladar/metabolismo , Vasoconstrictores/administración & dosificación , Amidas/administración & dosificación , Amidas/sangre , Anestésicos Locales/administración & dosificación , Anestésicos Locales/sangre , Animales , Autorradiografía/métodos , Inyecciones , Labio/irrigación sanguínea , Masculino , Maxilar/irrigación sanguínea , Maxilar/efectos de los fármacos , Maxilar/metabolismo , Nervio Maxilar/irrigación sanguínea , Nervio Maxilar/metabolismo , Mucosa Bucal/irrigación sanguínea , Mucosa Bucal/efectos de los fármacos , Mucosa Bucal/metabolismo , Hueso Paladar/irrigación sanguínea , Hueso Paladar/efectos de los fármacos , Paladar Duro/efectos de los fármacos , Paladar Duro/metabolismo , Radiofármacos , Ratas , Ratas Wistar , Flujo Sanguíneo Regional/efectos de los fármacos , Ropivacaína , Organismos Libres de Patógenos Específicos , Factores de Tiempo , TritioRESUMEN
OBJECTIVE: To investigate the effects of social isolation on oral mucosal healing in rats, and to determine if wound-associated genes and microRNAs (miRNAs) may contribute to this response. METHODS: Rats were group housed or socially isolated for 4 weeks before a 3.5 mm wound was placed on the hard oral palate. Wound closure was assessed daily and tissues were collected for determination of gene expression levels and miRNAs (i.e., miR-29a,b,c and miR-203). The predicted target of these microRNAs (i.e., vascular endothelial growth factor A, VEGFA) was functionally validated. RESULTS: Social isolation stress delayed the healing process of oral palatal mucosal wounds in rats. Lower mRNA levels of interleukin-1ß (IL1ß), macrophage inflammatory protein-1α (MIP1α), fibroblast growth factor 7 (FGF7), and VEGFA were found in the biopsied tissues of isolated animals on days 1 and/or 3 post-wounding. Intriguingly, the isolated rats persistently exhibited higher levels of miR-29 family members and miR-203. Our results confirmed that VEGFA is a direct target of these miRNAs, as both miR-29a,c and miR-203 strongly and specifically suppressed endogenous VEGFA expression in vitro. CONCLUSIONS: This study in rats demonstrates for the first time that social isolation delays oral mucosal healing, and suggests a potential role for healing-associated gene and miRNA interactions during this process via modulation of VEGF expression.
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MicroARNs/genética , Mucosa Bucal/metabolismo , Paladar Duro/metabolismo , Aislamiento Social/psicología , Factor A de Crecimiento Endotelial Vascular/genética , Cicatrización de Heridas/genética , Animales , Secuencia de Bases , Quimiocina CCL3/genética , Quimiocina CCL3/metabolismo , Factor 7 de Crecimiento de Fibroblastos/genética , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Regulación de la Expresión Génica , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , MicroARNs/metabolismo , Datos de Secuencia Molecular , Mucosa Bucal/lesiones , Paladar Duro/lesiones , Unión Proteica , Ratas , Ratas Sprague-Dawley , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The tissue in the palatal region can be divided into the hard and the soft palates, each having a specialized function such as occlusion, speech, or swallowing. Therefore, an understanding of the mechanism of palatogenesis in relation to the function of each region is important. However, in comparison with the hard palate, there is still a lack of information about the mechanisms of soft palate development. In this study, the authors investigated the contribution of cranial neural crest (CNC) cells to development of both hard and soft palates. They also demonstrated a unique pattern of periostin expression during soft palate development, which was closely related to that of collagen type I (Col I) in palatine aponeurosis. Furthermore, organ culture analysis showed that exogenous transforming growth factor-ß (TGF-ß) induced the expression of both periostin and Col I. These novel patterns of expression in the extracellular matrix (ECM) induced by CNC cells suggest that these cells may help to determine the character of both the hard and soft palates through ECM induction. TGF-ß signaling appears to be one of the mediators of Col I and periostin expression in the formation of functional structures during soft palate development.
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Moléculas de Adhesión Celular/metabolismo , Colágeno Tipo I/metabolismo , Cresta Neural/metabolismo , Hueso Paladar/metabolismo , Animales , Animales Recién Nacidos , Matriz Extracelular/metabolismo , Técnica del Anticuerpo Fluorescente , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Ratones Transgénicos , Cresta Neural/citología , Técnicas de Cultivo de Órganos , Hueso Paladar/embriología , Hueso Paladar/crecimiento & desarrollo , Paladar Duro/embriología , Paladar Duro/crecimiento & desarrollo , Paladar Duro/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
BACKGROUND: Cleft lip-cleft palate is a common congenital disability and represents a large biomedical burden. Through the use of animal models, the molecular underpinnings of cleft palate are becoming increasingly clear. Indian hedgehog (Ihh) has been shown to be associated with craniofacial development and to be active in the palatine bone. The authors hypothesize that Indian hedgehog activity plays a role in osteogenesis within the secondary palate and that defects in this pathway may inhibit osteogenesis of the secondary palate. METHODS: Palates were isolated from wild-type mice during the period of palate development (embryonic days 9.5 to 17.5). Quantitative real-time polymerase chain reaction was used for detecting gene expression during osteogenic differentiation and cellular differentiation (Shh, Ihh, Ptc1, Gli1, Gli2, Gli3, Runx2, Alp, and Col1a1). Next, palates were analyzed by hematoxylin and eosin, aniline blue, pentachrome, and in situ hybridization to assess osteogenesis of the palatal shelf and expression of hedgehog pathway genes. Finally, the palates of Indian hedgehog-null mice were analyzed to determine the effect of genetic deficiency on palatal development osteogenesis. RESULTS: Increased Indian hedgehog and osteogenic signaling coincided with ossification and fusion of the palate in wild-type mice. This included a fivefold to 150-fold peak in expression of hedgehog elements, including Ihh, at embryonic day 15.5 as compared with embryonic day 9.5. Contrarily, loss of Indian hedgehog by genetic knockout (Ihh-/-) resulted in decreased secondary palate ossification. CONCLUSIONS: The authors' results suggest a role for hedgehog signaling during palatal ossification. The hedgehog pathway is activated during palatal fusion, and deletion of Indian hedgehog leads to diminished ossification of the secondary hard palate.
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Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Osteogénesis/fisiología , Paladar Duro/embriología , Preñez , ARN/genética , Transducción de Señal/fisiología , Animales , Femenino , Proteínas Hedgehog/biosíntesis , Ratones , Ratones Endogámicos C57BL , Paladar Duro/metabolismo , Reacción en Cadena de la Polimerasa , EmbarazoRESUMEN
Oral homeostasis depends largely on proteins and mucins present in saliva that coat all oral surfaces. The present study compared the protein composition of residual fluid on mucosal surfaces in subjects with normal salivary flow with that of patients with dry mouth caused by salivary hypofunction. Samples of residual mucosal fluid were collected using paper strips and then analysed by protein electrophoresis and immunoblotting. In both patients and controls, residual fluids on mucosal surfaces (except the anterior tongue in control subjects) had higher protein concentrations than unstimulated whole-mouth saliva. High-molecular-weight mucin (MUC5B) was present in greater amounts on the anterior tongue than on other surfaces in control subjects. In dry mouth patients who were unable to provide a measurable saliva sample, MUC5B was often still present on all mucosal surfaces but in reduced amounts on the anterior tongue. The membrane-bound mucin, MUC1, was prominent on buccal and labial surfaces in patients and controls. Statherin was still present on surfaces that were dried to remove salivary fluid, suggesting that it may be adsorbed as a protein pellicle. It is concluded that oral mucosal surfaces in dry mouth patients can retain MUC5B and other salivary proteins, although the functional integrity of these proteins is uncertain.
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Mucosa Bucal/metabolismo , Mucinas/metabolismo , Proteínas y Péptidos Salivales/metabolismo , Xerostomía/metabolismo , Adulto , Anciano , Amilasas/análisis , Artritis Reumatoide/metabolismo , Artritis Reumatoide/fisiopatología , Estudios de Casos y Controles , Inhibidores de Cisteína Proteinasa/análisis , Película Dental/metabolismo , Femenino , Humanos , Labio/metabolismo , Masculino , Persona de Mediana Edad , Mucina-1/análisis , Mucina 5B/análisis , Mucinas/análisis , Paladar Duro/metabolismo , Saliva/metabolismo , Cistatinas Salivales/análisis , Proteínas Salivales Ricas en Prolina/análisis , Proteínas y Péptidos Salivales/análisis , Tasa de Secreción/fisiología , Sialadenitis/metabolismo , Sialadenitis/fisiopatología , Síndrome de Sjögren/metabolismo , Síndrome de Sjögren/fisiopatología , Lengua/metabolismo , Viscosidad , Xerostomía/fisiopatologíaRESUMEN
Prenatal exposure to diazepam, a prototype sedative drug that belongs to Benzodiazepines, can lead to orofacial clefting in human newborns. By using real-time PCR, in the present study we investigated whether diazepam elicits gene expression alterations in extracellular matrix (ECM) components, growth factors and gamma-aminobutyric acid receptor (GABRB3), implicated in the coordinate regulation of palate development. Palate fibroblasts were treated with diazepam (Dz-N fibroblasts) and compared to cleft lip-palate (CLP) fibroblasts obtained from patients with no known exposure to diazepam or other teratogens. Untreated fibroblasts from non-CLP patients were used as control. The results showed significant convergences in gene expression pattern of collagens, fibromodulin, vitronectin, tenascin C, integrins and metalloprotease MMP13 between Dz-N and CLP fibroblasts. Among the growth factors, constitutive Fibroblast Growth Factor 2 (FGF2) was greatly enhanced in Dz-N and CLP fibroblasts and associated with a higher reduction of FGF receptor. Transforming Growth Factor beta 3 (TGFbeta(3)) resulted up-regulated in CLP fibroblasts and decreased in Dz-N fibroblasts. We found phenotypic differences exhibited by Dz-N and CLP fibroblasts in GABRB3 gene regulation, so further studies are necessary to determine whether GABAergic system could be involved in the development of diazepam mediated CLP phenotype. Taken together the results elucidate the molecular mechanisms underlying possible toxicology effects induced by diazepam. Counselling of women on the safety of diazepam exposure is clinically important, also for the forensic consequences.
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Ansiolíticos/toxicidad , Labio Leporino/inducido químicamente , Fisura del Paladar/inducido químicamente , Diazepam/toxicidad , Proteínas de la Matriz Extracelular/genética , Fibroblastos/efectos de los fármacos , Paladar Duro/efectos de los fármacos , Estudios de Casos y Controles , Proliferación Celular , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Niño , Labio Leporino/genética , Labio Leporino/patología , Fisura del Paladar/genética , Fisura del Paladar/patología , Proteínas de la Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Paladar Duro/crecimiento & desarrollo , Paladar Duro/metabolismo , ARN Mensajero/metabolismo , Receptores de GABA-A/genéticaRESUMEN
Cleft palate repair leaves full-thickness mucosal defects on the palate. Healing might be improved by implantation of a mucosal substitute. However, the genetic and phenotypic deviations of cleft palate cells may hamper tissue engineering. The aim of this study was to construct mucosal substitutes from cleft palate cells, and to compare these with substitutes from normal palatal cells, and with native palatal mucosa. Biopsies from the palatal mucosa of eight children with cleft palate and eight age-matched control individuals were taken. Three biopsies of both groups were processed for (immuno)histochemistry; 5 were used to culture mucosal substitutes. Histology showed that the substitutes from cleft-palate and non-cleft-palate cells were comparable, but the number of cell layers was less than in native palatal mucosa. All epithelial layers in native palatal mucosa and mucosal substitutes expressed the cytokeratins 5, 10, and 16, and the proliferation marker Ki67. Heparan sulphate and decorin were present in the basal membrane and the underlying connective tissue, respectively. We conclude that mucosal cells from children with cleft palate can regenerate an oral mucosa in vitro.