Effect of genetic polymorphisms rs2301113 and rs2057482 in the expression of HIF-1α protein in periodontal ligament fibroblasts subjected to compressive force.

OBJECTIVE: Many genes and signaling molecules are involved in orthodontic tooth movement, with mechanically and hypoxically stabilized HIF-1α having been shown to play a decisive role in periodontal ligament signaling during orthodontic tooth movement. Thus, this in vitro study aimed to investigate if genetic polymorphisms in HIF1A (Hypoxia-inducible factor α-subunits) influence the expression pattern of HIF-1α protein during simulated orthodontic compressive pressure. METHODOLOGY: Samples from human periodontal ligament fibroblasts were used and their DNA was genotyped using real time Polymerase chain reaction for the genetic polymorphisms rs2301113 and rs2057482 in HIF1A . For cell culture and protein expression experiments, six human periodontal ligament fibroblast cell lines were selected based on the patients' genotype. To simulate orthodontic compressive pressure in fibroblasts, a 2 g/cm2 force was applied under cell culture conditions for 48 hours. Protein expression was evaluated by Western Blot. Paired t-tests were used to compare HIF-1α expression with and without compressive pressure application and unpaired t-tests were used to compare expression between the genotypes in rs2057482 and rs2301113 (p<0.05). RESULTS: The expression of HIF-1α protein was significantly enhanced by compressive pressure application regardless of the genotype (p<0.0001). The genotypes in the genetic polymorphisms rs2301113 and rs2057482 were not associated with HIF-1α protein expression (p>0.05). CONCLUSIONS: Our study confirms that compressive pressure application enhances HIF-1α protein expression. We could not prove that the genetic polymorphisms in HIF1A affect HIF-1α protein expression by periodontal ligament fibroblasts during simulated orthodontic compressive force.

Single Nucleotides Polymorphisms in COX2 Gene and their Association with Signs and Symptoms of Teething - A Pilot Study

ABSTRACT Objective: To investigate the association between single nucleotide polymorphisms in the COX2 gene (rs689466 and rs5275) and local and systemic signs and symptoms of teething. Material and Methods: Forty-four pairs of mothers-babies/toddlers were included. Erupted primary teeth were evaluated during clinical examination. Local and systemic signs and symptoms of teething were obtained from mothers' reporting via anamnesis. Samples of buccal cells were retrieved for DNA genotyping using real-time PCR. The T-test, Chi-square test, logistic regression, and haplotype analyses were applied. Results: Almost all mothers (95.5%) reported at least one local or systemic sign and symptom of teething. The most common was increased salivation (79.5%), diarrhea (72.3 %), and fever (70.5 %). The mean number of signs and symptoms per child was higher in boys than girls (mean = 5.1; SD= 1.5; p=0.008). Sleep disturbance (p=0.03) and loss of appetite (p=0.05) were more reported in boys. The rs689466 and rs5275 were not associated with signs and symptoms of teething (p>0.05). Conclusion: The single nucleotide polymorphisms in the COX2 gene (rs689466 and rs5275) were not associated with local and systemic signs and symptoms of teething.

Effect of genetic polymorphisms rs2301113 and rs2057482 in the expression of HIF-1α protein in periodontal ligament fibroblasts subjected to compressive force

Abstract Objective Many genes and signaling molecules are involved in orthodontic tooth movement, with mechanically and hypoxically stabilized HIF-1α having been shown to play a decisive role in periodontal ligament signaling during orthodontic tooth movement. Thus, this in vitro study aimed to investigate if genetic polymorphisms in HIF1A (Hypoxia-inducible factor α-subunits) influence the expression pattern of HIF-1α protein during simulated orthodontic compressive pressure. Methodology Samples from human periodontal ligament fibroblasts were used and their DNA was genotyped using real time Polymerase chain reaction for the genetic polymorphisms rs2301113 and rs2057482 in HIF1A . For cell culture and protein expression experiments, six human periodontal ligament fibroblast cell lines were selected based on the patients' genotype. To simulate orthodontic compressive pressure in fibroblasts, a 2 g/cm2 force was applied under cell culture conditions for 48 hours. Protein expression was evaluated by Western Blot. Paired t-tests were used to compare HIF-1α expression with and without compressive pressure application and unpaired t-tests were used to compare expression between the genotypes in rs2057482 and rs2301113 (p<0.05). Results The expression of HIF-1α protein was significantly enhanced by compressive pressure application regardless of the genotype (p<0.0001). The genotypes in the genetic polymorphisms rs2301113 and rs2057482 were not associated with HIF-1α protein expression (p>0.05). Conclusions Our study confirms that compressive pressure application enhances HIF-1α protein expression. We could not prove that the genetic polymorphisms in HIF1A affect HIF-1α protein expression by periodontal ligament fibroblasts during simulated orthodontic compressive force.

The role of 25-hydroxyvitamin-D3 and vitamin D receptor gene in human periodontal ligament fibroblasts as response to orthodontic compressive strain: an in vitro study.

BACKGROUND: This study aimed to investigate, if different physiological concentrations of vitamin D (25(OH)D3) and single nucleotide polymorphisms in vitamin D receptor (VDR) gene have an impact on gene expression in human periodontal ligament (hPDL) fibroblasts induced by simulated orthodontic compressive strain. METHODS: A pool of hPDL fibroblasts was treated in absence or presence of 25(OH)D3 in 3 different concentrations (10, 40 and 60 ng/ml). In order to evaluate the role of single nucleotide polymorphisms in the VDR gene, hPDL fibroblasts from 9 patients were used and treated in absence or presence of 40 ng/ml 25(OH)D3. Each experiment was performed with and without simulated orthodontic compressive strain. Real-time PCR was used for gene expression and allelic discrimination analysis. Relative expression of dehydrocholesterol reductase (DHCR7), Sec23 homolog A, amidohydrolase domain containing 1 (AMDHD1), vitamin D 25-hydroxylase (CYP2R1), Hydroxyvitamin D-1-α hydroxylase, receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), cyclooxygenase-2 (COX-2) and interleukin-6 (IL6) was assessed. Three single nucleotide polymorphisms in VDR were genotyped. Parametric or non-parametric tests were used with an alpha of 5%. RESULTS: RANKL, RANKL:OPG ratio, COX-2, IL-6, DHCR7, CYP2R1 and AMDHD1 were differentially expressed during simulated orthodontic compressive strain (p < 0.05). The RANKL:OPG ratio was downregulated by all concentrations (10 ng/ml, 40 ng/ml and 60 ng/ml) of 25(OH)D3 (mean = 0.96 ± 0.68, mean = 1.61 ± 0.66 and mean = 1.86 ± 0.78, respectively) in comparison to the control (mean 2.58 ± 1.16) (p < 0.05). CYP2R1 gene expression was statistically modulated by the different 25(OH)D3 concentrations applied (p = 0.008). Samples from individuals carrying the GG genotype in rs739837 presented lower VDR mRNA expression and samples from individuals carrying the CC genotype in rs7975232 presented higher VDR mRNA expression (p < 0.05). CONCLUSIONS: Simulated orthodontic compressive strain and physiological concentrations of 25(OH)D3 seem to regulate the expression of orthodontic tooth movement and vitamin-D-related genes in periodontal ligament fibroblasts in the context of orthodontic compressive strain. Our study also suggests that single nucleotide polymorphisms in the VDR gene regulate VDR expression in periodontal ligament fibroblasts in the context of orthodontic compressive strain.

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.