Effects of estrogen deficiency during puberty on maxillary and mandibular growth and associated gene expression – an μCT study on rats

Background Estrogen is a well-known and important hormone involved in skeletal homeostasis, which regulates genes involved in bone biology. Some studies support that estrogen is important for craniofacial growth and development. Therefore this in vivo animal study aimed to investigate, whether and in which way low estrogen levels in the prepubertal period affect craniofacial development in the postpubertal stage and to quantify the gene expression of RANK, RANKL and OPG in cranial growth sites in ovariectomized estrogen-deficient rats during puberty. Methods Control (sham-operated, n = 18) and ovariectomy (OVX, n = 18) surgeries were performed on 21-days-old female Wistar rats. Animals euthanized at an age of 45 days (pubertal stage) were used for gene expression analyses (n = 6 per group) and immunohistochemistry of RANK, RANKL and OPG. Animals euthanized at 63 days of age (post-pubertal stage) were used for craniofacial two-dimensional and three-dimensional craniofacial measurements using μCT imaging (n = 12 per group). Results In the μCT analysis of the mandible and maxilla many statistically significant differences between sham-operated and OVX groups were observed, such as increased maxillary and mandibular bone length in OVX animals (p < 0.05). Condylar volume was also significantly different between groups (p < 0.05). The sham-operated group showed a higher level of RANK expression in the midpalatal suture (p = 0.036) and the RANKL:OPG ratio levels were higher in the OVX group (p = 0.015). Conclusions Our results suggest that estrogen deficiency during the prepubertal period is associated with alterations in the maxillary and mandibular bone length and condylar growth

Evaluation of the hole of estrogen in the maxilla and mandible growth and development

Os hormônios sexuais desempenham um importante papel no metabolismo ósseo. Além disso, polimorfismos genéticos em genes expressos durante o desenvolvimento craniofacial estão associados com alterações dimensionais na maxila e na mandíbula. Desta forma, o objetivo deste trabalho foi avaliar, por meio de modelo animal e de genética humana, o papel do estrógeno e dos polimorfismos genéticos nos seus receptores (ER&alpha; e ER&beta; ), codificados pelos genes ESR1 e ESR2, nas alterações dimensionais da maxila e da mandíbula. Um total de 40 ratas Wistar foram divididas em 2 grupos: Ovariectomizadas (para estimular deficiência de estrógeno) e Sham. Foram realizadas 3 tomadas radiográficas da região craniofacial das ratas, sendo a primeira aos 21 dias, a segunda aos 45 dias e a última aos 63 dias de vida. Medidas cefalométricas da maxila e da mandíbula foram obtidas para avaliação das alterações verticais e sagitais. Após a eutanásia, peças anatômicas das regiões de interesse foram utilizadas para realização de imuno-histoquímica e PCR em tempo real para os genes ESR1 e ESR2. Paralelamente, na área da genética humana, um total de 143 amostras de DNA genômico e dados relativos à análise cefalométrica destes indivíduos foram avaliados quanto aos polimorfismos genéticos nos genes de interesse pelo método de discriminação alélica por PCR em tempo real. Os resultados foram agrupados de acordo com os diferentes testes; as variáveis contínuas foram avaliadas com testes paramétricos e/ou não paramétricos para comparação das médias entre os grupos. Os testes do qui-quadrado e exato de Fisher foram usados para avaliar a associação do padrão esquelético facial com as distribuições genotípica e alélica na população humana (alfa de 5%). No modelo animal, foi possível observar um aumento nas dimensões maxilares e mandibulares do grupo sob condição de deficiência de estrógeno (p0,05), onde não houve diferença na expressão do RNAm de ambos os genes (p>0,05). Para mais, a presença de ER&beta; foi confirmada nos sítios de crescimento mandibulares de ambos os grupos. Na população humana, foi possível identificar associação estatisticamente significante entre medidas cefalométricas e polimorfismos em ESR1 e ESR2 (p0,05). Desta forma, conclui-se que o estrógeno tem um papel importante no crescimento e desenvolvimento da maxila e da mandíbula em modelo animal e que polimorfismos genéticos nos seus receptores estão associados com o padrão esquelético facialSex hormones play an important role on bone metabolism. In addition, genetic polymorphisms in genes expressed during craniofacial development are associated with dimensional changes in maxilla and mandible. Thus, the aim of this study was to evaluate, using animal models and human genetics, the role of estrogen and genetic polymorphisms in its receptors (ER&alpha; and ER&beta; ), codified by ESR1 and ESR2, in the dimensional alterations of the maxilla and the mandible. A total of 40 Wistar rats were divided into 2 groups: Ovariectomized (to stimulate estrogen deficiency) and Sham. Radiographic exams of rats craniofacial region were performed, the first at 21 days, the second at 45 days and the last at 90 days old. Cephalometric measurements of maxilla and mandible were obtained to evaluate the vertical and sagittal skeletal alterations. After euthanasia, anatomical parts of interesting regions were used to performed immunohistochemistry and real time PCR for ESR1 and ESR2. In parallel, a total of 143 genomic DNA samples and cephalometric data of these individuals were evaluated for genetic polymorphisms by real time PCR (allele discrimination method). The results were grouped according to the different tests; continuous variables were evaluated with parametric and/or non-parametric test to compare means between groups. Chi-square and Fishers exact tests were used to evaluate the association of facial skeletal pattern with genotypic and allelic distributions in the human population (5% alpha). In the animal model, it was possible to detect an increase in the maxillary and mandibular dimensions of ovariectomized group (p0,05). There was no difference in the mRNA expression of both genes (p>0.05). Moreover, the ER&beta; presence was confirmed at mandibles growth sites of both groups. In the human population, it was possible to identify statistically significant association in cephalometric measures and polymorphisms in ESR1 and ESR2 (p0.05). Therefore, in conclusion, estrogen has an important role in the mandible and maxilla growth and development in the animal model, and genetic polymorphisms in estrogen receptors are associated with facial skeletal pattern

Evaluation of the hole of estrogen in the maxilla and mandible growth and development

Os hormônios sexuais desempenham um importante papel no metabolismo ósseo. Além disso, polimorfismos genéticos em genes expressos durante o desenvolvimento craniofacial estão associados com alterações dimensionais na maxila e na mandíbula. Desta forma, o objetivo deste trabalho foi avaliar, por meio de modelo animal e de genética humana, o papel do estrógeno e dos polimorfismos genéticos nos seus receptores (ER&alpha; e ER&beta; ), codificados pelos genes ESR1 e ESR2, nas alterações dimensionais da maxila e da mandíbula. Um total de 40 ratas Wistar foram divididas em 2 grupos: Ovariectomizadas (para estimular deficiência de estrógeno) e Sham. Foram realizadas 3 tomadas radiográficas da região craniofacial das ratas, sendo a primeira aos 21 dias, a segunda aos 45 dias e a última aos 63 dias de vida. Medidas cefalométricas da maxila e da mandíbula foram obtidas para avaliação das alterações verticais e sagitais. Após a eutanásia, peças anatômicas das regiões de interesse foram utilizadas para realização de imuno-histoquímica e PCR em tempo real para os genes ESR1 e ESR2. Paralelamente, na área da genética humana, um total de 143 amostras de DNA genômico e dados relativos à análise cefalométrica destes indivíduos foram avaliados quanto aos polimorfismos genéticos nos genes de interesse pelo método de discriminação alélica por PCR em tempo real. Os resultados foram agrupados de acordo com os diferentes testes; as variáveis contínuas foram avaliadas com testes paramétricos e/ou não paramétricos para comparação das médias entre os grupos. Os testes do qui-quadrado e exato de Fisher foram usados para avaliar a associação do padrão esquelético facial com as distribuições genotípica e alélica na população humana (alfa de 5%). No modelo animal, foi possível observar um aumento nas dimensões maxilares e mandibulares do grupo sob condição de deficiência de estrógeno (p0,05), onde não houve diferença na expressão do RNAm de ambos os genes (p>0,05). Para mais, a presença de ER&beta; foi confirmada nos sítios de crescimento mandibulares de ambos os grupos. Na população humana, foi possível identificar associação estatisticamente significante entre medidas cefalométricas e polimorfismos em ESR1 e ESR2 (p0,05). Desta forma, conclui-se que o estrógeno tem um papel importante no crescimento e desenvolvimento da maxila e da mandíbula em modelo animal e que polimorfismos genéticos nos seus receptores estão associados com o padrão esquelético facialSex hormones play an important role on bone metabolism. In addition, genetic polymorphisms in genes expressed during craniofacial development are associated with dimensional changes in maxilla and mandible. Thus, the aim of this study was to evaluate, using animal models and human genetics, the role of estrogen and genetic polymorphisms in its receptors (ER&alpha; and ER&beta; ), codified by ESR1 and ESR2, in the dimensional alterations of the maxilla and the mandible. A total of 40 Wistar rats were divided into 2 groups: Ovariectomized (to stimulate estrogen deficiency) and Sham. Radiographic exams of rats craniofacial region were performed, the first at 21 days, the second at 45 days and the last at 90 days old. Cephalometric measurements of maxilla and mandible were obtained to evaluate the vertical and sagittal skeletal alterations. After euthanasia, anatomical parts of interesting regions were used to performed immunohistochemistry and real time PCR for ESR1 and ESR2. In parallel, a total of 143 genomic DNA samples and cephalometric data of these individuals were evaluated for genetic polymorphisms by real time PCR (allele discrimination method). The results were grouped according to the different tests; continuous variables were evaluated with parametric and/or non-parametric test to compare means between groups. Chi-square and Fishers exact tests were used to evaluate the association of facial skeletal pattern with genotypic and allelic distributions in the human population (5% alpha). In the animal model, it was possible to detect an increase in the maxillary and mandibular dimensions of ovariectomized group (p0,05). There was no difference in the mRNA expression of both genes (p>0.05). Moreover, the ER&beta; presence was confirmed at mandibles growth sites of both groups. In the human population, it was possible to identify statistically significant association in cephalometric measures and polymorphisms in ESR1 and ESR2 (p0.05). Therefore, in conclusion, estrogen has an important role in the mandible and maxilla growth and development in the animal model, and genetic polymorphisms in estrogen receptors are associated with facial skeletal pattern

Estrogen deficiency during puberty affects the expression of microRNA30a and microRNA503 in the mandibular condyle

Background: The aim of this study was investigated if estrogen deficiency during puberty affects the expression of miRNA30a and miRNA503 in maxillary and mandibular growth centers, and also evaluated if ER alpha and ER beta are correlated with miRNA30a and miRNA503 expressions. Methods: Samples from 12 female Wistar rats randomized into experimental group (OVX) and control group (SHAM). At an age of 45 days animals were euthanized for miRNA expression analyses. RT-qPCR was performed to determine miRNA30a and miRNA503 expression in growth sites: midpalatal suture, condyle, mandibular angle, symphysis/parasymphysis and coronoid process. The data was carried out using the parametric tests at 5% of significance level. Results: miRNA 30a and miRNA503 presented higher levels in the condylar site in SHAM group when compared with OVX (p = 0.002 and p = 0.020, respectively). In the growth centers, a statistical significant difference was observed only for miRNA30a (p = 0.004), when compared mandibular angle with condyle the in OVX group (p = 0.001). A strong positive correlation between miRNA503 and ER alpha in the condyle of OVX group was observed (r = 0.90; p = 0.039 and it also between miRNA503 and ER beta in the coronoid process of the OVX group (r = 0.88; p = 0.05). Conclusion: The results suggested that estrogen regulates specific miRNAs in maxillary and mandibular growth centers, which may participate in posttranscriptional regulation of estrogen-regulated genes. (c) 2021 Published by Elsevier GmbH

Estrogen deficiency affects tooth formation and gene expression in the odontogenic region of female rats

Background: There is some evidence that estrogen regulates the expression of several genes in different cells, including dental cells. Therefore, the aim of this study was to investigate the role of estrogen deficiency during tooth development regarding tooth structure morphology and its impact on the expression of odontogenesis-related genes. Methods: A total of 40 female Wistar rats was divided into OVX (estrogen deficiency) and Sham (control) groups. Bilateral ovariectomy was performed in the OVX group, while Sham surgery was performed in the control group at the age of 21 days. At an age of 56 days, 16 rats were euthanized for gene expression analyses of Bmp4, Smad6, Tgfb1 and Runx2. At the age of 63 days, the remaining rats were euthanized for histological and morphometric analyses of teeth. The mandibles of the rats were submitted to mu CT analysis. Tooth structures (enamel, dentin and dental pulp) were analyzed. T test was used to compare the mean differences between groups (p < 0.05). Results: In the mu CT analysis, enamel and dentin thickness were significantly increased in the control group (p < 0.0001). Pulp dimensions were significantly larger in the OVX group (p < 0.0001). A reduction of tooth structures in the OVX group was confirmed in HE staining. Smad6 was differentially expressed in the OVX group (p = 0.04). Conclusion: Estrogen deficiency affects gene expression in the odontogenic region and tooth structure morphology. (C) 2021 Elsevier GmbH. All rights reserved

The role of postnatal estrogen deficiency on cranium dimensions

Objectives The aim of this study was investigate the cranium dimensions of adult female rats, who suffered estrogen deficiency during the prepubertal stage, to assess the impact of estrogen deficiency on craniofacial morphology. Material and methods Twenty-two female Wistar rats were divided into ovariectomy (OVX) (n = 11) and sham-operated control (n = 11) groups. Bilateral ovariectomy were performed in both groups at 21 days old (prepubertal stage), and rats were euthanized at an age of 63 days (post-pubertal stage). Micro-CT scans were performed with rat skulls, and the cranium morphometric landmark measurements were taken in the dorsal, lateral, and ventral view positions. Differences in measurements between the OVX and sham control groups were assessed using t test with an established alpha error of 5%. Results The measures of the rats' skull showed that the inter-zygomatic arch width and anterior cranial base length were significantly larger in OVX group (p = 0.020 and p = 0.050, respectively), whereas the length of parietal bone was significantly higher in the sham group (p = 0.026). For the remaining measurements no significant differences between groups were detected (p > 0.05). Conclusion This study provides evidence that ovariectomized rats had alterations in cranial bone dimensions, demonstrating that estrogens during puberty are important for skull morphology

Effect of ovariectomy on maxilla and mandible dimensions of female rats

Objective The role of oestrogen in craniofacial growth still remains unclear. Therefore, the present study aimed to assess the effect of oestrogen deficiency on maxilla and mandible dimensions. Setting and Sample Population The study was conducted at the Department of Pediatric Dentistry at the School of Dentistry of Ribeirao Preto, University of Sao Paulo, and used forty female Wistar rats. Material and Methods Ovariectomy (OVX) and placebo surgery (Sham) were performed when animals were twenty-one days old (prepubertal stage). Dimensions of the maxilla and mandible were assessed by craniometric analysis using radiographs, during and after puberty of the animals (45 and 63 days old, respectively). Quantitative real-time PCR and immunohistochemical analyses were performed to determine the expression and localization, respectively, of oestrogen receptor alpha (ER alpha) and oestrogen receptor beta (ER beta) in different growth sites of the evaluated structures at puberty. The differences between the groups for each outcome were evaluated using the t test with an established alpha error of 5%. Results There were significant differences between the OVX and Sham groups for horizontal and vertical linear measurements in the maxilla and the mandible at both pubertal and post-pubertal stages (P .05). Immunohistochemical analyses revealed the presence of both oestrogen receptors in osteoblasts and chondrocytes in the midpalatal suture and mandibular condyle, respectively, in the OVX and Sham groups. Conclusion Our results suggest that oestrogen deficiency from the prepubertal stage might increase the growth of the maxilla and mandible in female rats

Effect of ovariectomy on maxilla and mandible dimensions of female rats

Objective The role of oestrogen in craniofacial growth still remains unclear. Therefore, the present study aimed to assess the effect of oestrogen deficiency on maxilla and mandible dimensions. Setting and Sample Population The study was conducted at the Department of Pediatric Dentistry at the School of Dentistry of Ribeirao Preto, University of Sao Paulo, and used forty female Wistar rats. Material and Methods Ovariectomy (OVX) and placebo surgery (Sham) were performed when animals were twenty-one days old (prepubertal stage). Dimensions of the maxilla and mandible were assessed by craniometric analysis using radiographs, during and after puberty of the animals (45 and 63 days old, respectively). Quantitative real-time PCR and immunohistochemical analyses were performed to determine the expression and localization, respectively, of oestrogen receptor alpha (ER alpha) and oestrogen receptor beta (ER beta) in different growth sites of the evaluated structures at puberty. The differences between the groups for each outcome were evaluated using the t test with an established alpha error of 5%. Results There were significant differences between the OVX and Sham groups for horizontal and vertical linear measurements in the maxilla and the mandible at both pubertal and post-pubertal stages (P .05). Immunohistochemical analyses revealed the presence of both oestrogen receptors in osteoblasts and chondrocytes in the midpalatal suture and mandibular condyle, respectively, in the OVX and Sham groups. Conclusion Our results suggest that oestrogen deficiency from the prepubertal stage might increase the growth of the maxilla and mandible in female rats

Tooth agenesis-related GLI2 and GLI3 genes may contribute to craniofacial skeletal morphology in humans

Objective: The present cross-sectional, multi-centre, genetic study aimed to determine, whether single nucleotide polymorphisms (SNPs) in tooth agenesis (TA)-associated GLI2 and GLI3 genes contribute to the development of craniofacial skeletal morphology in humans. Design: Orthodontic patients from an ethnically heterogeneous population were selected for the present study (n = 594). The presence or absence of TA was determined by analysis of panoramic radiography and dental records. The subjects were classified according to their skeletal malocclusion and facial growth pattern by means of digital cephalometric analysis. Genomic DNA was extracted from squamous epithelial cells of the buccal mucosa and SNPs in GL12 (rs3738880, rs2278741) and GLI3 (rs929387, rs846266) were analysed by polymerase chain reaction using TaqMan chemistry and end-point analysis. Results: Class II skeletal malocclusion presented a significantly lower frequency of TA (P 0.05). The G allele for TA-related GLI2 rs3738880 was strongly linked to the presence of Class III skeletal malocclusion (OR = 2.03; 95% CI = 1.37-3.03; P < 3125 x 10(-6)). GLI2 rs2278741 C allele was overrepresented in individuals without TA, suggesting it as a protective factor for this dental phenotype (OR = 0.43; 95% CI = 0.24-0.78; P < 625 x 10(-5)). Conclusion: The present study suggests that SNPs in TA-associated GL12 and GLI3 genes may also play a role in the development of skeletal malocclusions. rs3738880 and rs2278741 in GLI2 seems to contribute to the genetic background for skeletal Class III and TA, respectively. TA could be an additional predictor of craniofacial morphology in some cases. Further research replicating the reported associations should be performed