Evaluation of tooth eruption rate of incisor teeth in rats with estrogen deficiency.

OBJECTIVES: To assess the influence of estrogen deficiency on tooth eruption rate (TER) and gene expression of estrogen receptor alpha and beta (ERα and ERß) in the odontogenic region of teeth with continuous formation in a rat model. MATERIALS AND METHODS: Ovariectomies (OVX; n = 25) and sham surgeries (SHAM; n = 25) were performed in female Wistar rats when animals were 25 days old. The TER of the lower incisors, both in impeded (hyperfunction condition) and unimpeded (trimmed incisal edge-hypofunction condition) conditions, was evaluated using standardized digital photographs acquired every 48-72 h for 3 weeks (35th-53rd day of life), using a camera coupled to a stereomicroscope. Quantitative real-time PCR was performed to evaluate the relative gene expression of ERα and ERß in the odontogenic region. RESULTS: The OVX group showed a significant reduction in TER when compared to the SHAM group, only in the impeded condition (p = 0.03). There was no statistically significant difference between the groups in ERα gene expression (p = 0.33). ERß showed a significantly higher gene expression in the OVX group (p ≤ 0.05). CONCLUSIONS: Estrogen deficiency decreases TER in teeth under impeded condition. Estrogen deficiency also increases ERß gene expression in the odontogenic region of teeth with continuous formation. CLINICAL RELEVANCE: Hormonal disturbances affecting estrogen levels can cause alterations in dental formation and teeth eruption.

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 ERα and ERß 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 ERα in the condyle of OVX group was observed (r = 0.90; p = 0.039 and it also between miRNA503 and ERß 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.

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.

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 µCT 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 µCT 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.

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. CLINICAL RELEVANCE: To understand the role of estrogen on the postnatal cranium development will impact the clinical diagnose and therapy during childhood and adolescence.

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 Ribeirão Preto, University of São 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 (ERα) and oestrogen receptor beta (ERß) 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). The ovariectomized rats showed significantly greater measures for all dimensions assessed. No differences in gene expression of ERα and ERß were identified at the different growth sites between the OVX and Sham groups (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.

Association between craniofacial morphological patterns and tooth agenesis-related genes.

BACKGROUND: The aim of the present study was to assess if genetic polymorphisms in tooth agenesis (TA)-related genes are associated with craniofacial morphological patterns. METHODS: This cross-sectional, multi-center, genetic study evaluated 594 orthodontic Brazilians patients. The presence or absence of TA was determined by analysis of panoramic radiography. The patients 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 buccal mucosa and genetic polymorphisms in MSX1 (rs1042484), PAX9 (rs8004560), TGF-α (rs2902345), FGF3 (rs1893047), FGF10 (rs900379), and FGF13 (rs12838463, rs5931572, and rs5974804) were genotyped by polymerase chain reaction using TaqMan chemistry and end-point analysis. RESULTS: Genotypes (p = 0.038) and allele (p = 0.037) distributions for the FGF3 rs1893047 were significantly different according to the skeletal malocclusion. Carrying at least one G allele increased in more than two times the chance of presenting skeletal class III malocclusion (OR = 2.21, CI 95% = 1.14-4.32; p = 0.017). There was no association between another skeletal craniofacial pattern and some polymorphism assessed in the present study. CONCLUSION: Our results suggest that the genetic polymorphism rs1893047 in FGF3 might contribute to variations in the craniofacial sagittal pattern.

Possible association between craniofacial dimensions and genetic markers in ESR1 and ESR2.

OBJECTIVE: To investigate the association of genetic markers in ESR1 and ESR2 with craniofacial measurements. DESIGN: Cross-sectional study. SETTING: School of Dentistry of Ribeirão Preto, University of São Paulo. PARTICIPANTS: A total of 146 biologically unrelated, self-reported Caucasian Brazilians with no syndromic conditions were included. METHODS: Sagittal and vertical measurements (ANB, S-N, Ptm'-A', Co-Gn, Go-Pg, N-Me, ANS-Me, S-Go and Co-Go) from lateral cephalograms were examined for craniofacial evaluation. DNA was extracted from saliva and genetic markers in ESR1 (rs2234693 and rs9340799) and in ESR2 (rs1256049 and rs4986938) were analysed by real-time polymerase chain reaction. Hardy-Weinberg equilibrium was evaluated using the Chi-square test within each marker. The associations between craniofacial dimensions and genotypes were analysed by linear regression and adjusted by sex and age. The established alpha was 5%. RESULTS: Individuals carrying CC in ESR1 rs2234693 had a decrease of -3.146 mm in ANS-Me (P = 0.044). In addition, rs4986938 in ESR2 was associated with S-N measurement (P = 0.009/ ß = -3.465). This marker was also associated with Go-Pg measurement, in which the CC genotype had a decrease of -3.925 mm in the length of the mandibular body (P = 0.043). CONCLUSION: The present study suggests that in ESR1 and ESR2 are markers for variations in the craniofacial dimensions. However, further research should confirm the results.

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 GLI2 (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). Subjects without TA showed significantly higher ANB angles (P < 0.05). Genotype and/or allele distributions of the SNPs in GLI2 (rs3738880, rs2278741) and GLI3 (rs846266) were associated with the presence of TA (P < 0.05). The SNPs rs3738880, rs2278741 and rs929387 were also associated with some type of skeletal malocclusion (P < 0.05), but not with the facial growth pattern (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 × 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 × 10-5). CONCLUSION: The present study suggests that SNPs in TA-associated GLI2 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.

Genetic variants in ACTN3 and MYO1H are associated with sagittal and vertical craniofacial skeletal patterns.

OBJECTIVE: This study aimed to evaluate the association of genetic variants inACTN3 and MYO1H with craniofacial skeletal patterns in Brazilians. DESIGN: This cross-sectional study enrolled orthodontic and orthognathic patients selected from 4 regions of Brazil. Lateral cephalograms were used and digital cephalometric tracings and analyzes were performed for craniofacial phenotype determination. Participants were classified according to the skeletal malocclusion in Class I, II or III; and according to the facial type in Mesofacial, Dolichofacial or Brachyfacial. Genomic DNA was extracted from saliva samples containing exfoliated buccal epithelial cells and analyzed for genetic variants inACTN3 (rs678397 and rs1815739) and MYO1H (rs10850110) by real-time PCR. Chi-square or Fisher's exact tests were used for statistical analysis (α = 5%). RESULTS: A total of 646 patients were included in the present study. There was statistically significant association of the genotypes and/or alleles distributions with the skeletal malocclusion (sagittal skeletal pattern) and facial type (vertical pattern) for the variants assessed inACTN3 (P < 0.05). For the genetic variant evaluated in MYO1H, there was statistically significant difference between the genotypes frequencies for skeletal Class I and Class II (P < 0.05). The reported associations were different depending on the region evaluated. CONCLUSION: ACTN3 and MYO1H are associated with sagittal and vertical craniofacial skeletal patterns in Brazilian populations.