Evaluation of 4% stabilized Sodium Hypochlorite activity in the repair of cutaneous excisional wounds in mice.

Wounds are conditions largely present in the clinical routine, and even though frequent, their complete resolution can be challenging. Several solutions can aid or stimulate the healing process, and for this reason, this work used a stabilized solution of 4% sodium hypochlorite for the treatment of excisional wounds in mice. This study was carried out in two distinct stages: in the first stage, the optimal concentration of the chlorinated solution was determined by using the sponge implantation technique in mouse subcutaneous tissue to evaluate the dose-response curve; and in the second phase, this concentration was tested in an experimental model of excisional skin wounds in mice. Soluble collagen, hemoglobin, myeloperoxidase (MPO) and N-acetyl-ß-D-glycosaminidase (NAG) activity were assessed, and total, type I and type III collagen deposition were quantified in both stages. Based on the results presented in the sponge implantation study, the chlorinated solution at 150 ppm (0.015%) was chosen for use in a preclinical trial of skin healing in mice. At 1, 3, 7 and 14 days of treatment, the % wound area repair in the group treated with 150 ppm chlorinated solution was higher when compared to the control group, with statistical differences at all time points (*p≤ 0.05 and **p≤ 0.01). 150 ppm chlorinated solution obtained from a stabilized 4% sodium hypochlorite solution was effective in accelerating cutaneous excision wound repair in mice, showing a positive influence on tissue repair.

Gene-environment interaction in molar-incisor hypomineralization.

Molar incisor hypomineralization (MIH) is an enamel condition characterized by lesions ranging in color from white to brown which present rapid caries progression, and mainly affects permanent first molars and incisors. These enamel defects usually occur when there are disturbances during the mineralization or maturation stage of amelogenesis. Both genetic and environmental factors have been suggested to play roles in MIH's development, but no conclusive risk factors have shown the source of the disease. During head and neck development, the interferon regulatory factor 6 (IRF6) gene is involved in the structure formation of the oral and maxillofacial regions, and the transforming growth factor alpha (TGFA) is an essential cell regulator, acting during proliferation, differentiation, migration and apoptosis. In this present study, it was hypothesized that these genes interact and contribute to predisposition of MIH. Environmental factors affecting children that were 3 years of age or older were also hypothesized to play a role in the disease etiology. Those factors included respiratory issues, malnutrition, food intolerance, infection of any sort and medication intake. A total of 1,065 salivary samples from four different cohorts were obtained, and DNA was extracted from each sample and genotyped for nine different single nucleotide polymorphisms. Association tests and logistic regression implemented in PLINK were used for analyses. A potential interaction between TGFA rs930655 with all markers tested in the cohort from Turkey was identified. These interactions were not identified in the remaining cohorts. Associations (p<0.05) between the use of medication after three years of age and MIH were also found, suggesting that conditions acquired at the age children start to socialize might contribute to the development of MIH.

IRF6, MSX1, TGFA, dental anomalies, and skeletal malocclusion.

OBJECTIVE: Verify the presence of association between four variables-transforming growth factor α (TGFA; C/T rs1523305), interferon regulatory factor 6 (IRF6; A/C rs2013162), muscle segment homeobox 1 (MSX1; A/G rs12532), and dental anomalies-with skeletal malocclusion by comparing these four variables with Angle Classes I, II, and III, and normal, hyperdivergent, and hypodivergent growth patterns. METHODS: A total of 505 orthodontic records of patients older than 8 years were evaluated. The sample consisted of 285 (56.4 per cent) females, 220 (43.6 per cent) males, 304 (60.2 per cent) Whites (the rest were mixed Blacks with Whites), with a mean age of 20.28 (±10.35) years (ranging from 8 to 25 years). Eight cephalometric points, which served as the anatomical framework for obtaining angles and cephalometric measurements, were used for skeletal characterization using the Dolphin Software. Samples of saliva were collected and the DNA was extracted, diluted and quantified. Markers in TGFA, IRF6, and MSX1 were used and genotypes were obtained using TaqMan chemistry. Odds ratio (OR) and 95 per cent confidence interval (CI) calculations, chi-square, Fisher's Exact, Mann-Whitney, and correlation coefficient tests (significance level: 95 per cent) were performed. Bonferroni correction was applied and an alpha of 0.0006 was considered statistically significant. RESULTS: There was no statistically significant associations between markers in TGFA or IRF6 with skeletal malocclusions. Tooth agenesis was associated with facial convexity (P < 0.001). MSX1 was associated with Class II skeletal malocclusion (P = 0.0001, OR = 0.6, CI = 0.46-0.78). CONCLUSION: Individuals with tooth agenesis were more likely to have a convex face. MSX1 was associated with Class II skeletal malocclusion.

Measuring the Microscopic Structures of Human Dental Enamel Can Predict Caries Experience.

OBJECTIVES: The hierarchical structure of enamel gives insight on the properties of enamel and can influence its strength and ultimately caries experience. Currently, past caries experience is quantified using the decayed, missing, filled teeth/decayed, missing, filled surface (DMFT/DMFS for permanent teeth; dmft/dmfs for primary teeth), or international caries detection and assessment system (ICDAS) scores. By analyzing the structure of enamel, a new measurement can be utilized clinically to predict susceptibility to future caries experience based on a patient's individual's biomarkers. The purpose of this study was to test the hypothesis that number of prisms by square millimeter in enamel and average gap distance between prisms and interprismatic areas, influence caries experience through genetic variation of the genes involved in enamel formation. MATERIALS AND METHODS: Scanning electron microscopy (SEM) images of enamel from primary teeth were used to measure (i) number of prisms by square millimeter and interprismatic spaces, (ii) prism density, and (iii) gap distances between prisms in the enamel samples. The measurements were tested to explore a genetic association with variants of selected genes and correlations with caries experience based on the individual's DMFT+ dmft score and enamel microhardness at baseline, after an artificial lesion was created and after the artificial lesion was treated with fluoride. RESULTS: Associations were found between variants of genes including ameloblastin, amelogenin, enamelin, tuftelin, tuftelin interactive protein 11, beta defensin 1, matrix metallopeptidase 20 and enamel structure variables measured (number of prisms by square millimeter in enamel and average gap distance between prisms and interprismatic areas). Significant correlations were found between caries experience and microhardness and enamel structure. Negative correlations were found between number of prisms by square millimeter and high caries experience (r value= -0.71), gap distance between prisms and the enamel microhardness after an artificial lesion was created (r value= -0.70), and gap distance between prisms and the enamel microhardness after an artificial lesion was created and then treated with fluoride (r value= -0.81). There was a positive correlation between number of prisms by square millimeter and prism density of the enamel (r value = 0.82). CONCLUSIONS: Our data support that genetic variation may impact enamel formation, and therefore influence susceptibility to dental caries and future caries experience. CLINICAL RELEVANCE: The evaluation of enamel structure that may impact caries experience allows for hypothesizing that the identification of individuals at higher risk for dental caries and implementation of personalized preventative treatments may one day become a reality.

Polymorphisms in Nonamelogenin Enamel Matrix Genes Are Associated with Dental Fluorosis.

The aim of this study was to evaluate whether genetic polymorphisms in AMELX, AMBN, ENAM, TFIP11, and TUFT1 genes are associated with dental fluorosis (DF). A total of 1,017 children from 2 Brazilian cohorts were evaluated. These populations lived in cities with fluoridation of public water supplies. DF was assessed in erupted permanent teeth using the modified Dean index. The polymorphisms rs946252, rs12640848, rs4694075, rs5997096, and rs4970957 were analyzed by real-time PCR from genomic DNA. Associations between DF, genotype, and allele distribution were evaluated using the χ2 test, with an alpha of 5%. The polymorphisms rs4694075, rs5997096, and rs4970957 in AMBN, TFIP11, and TUFT1 were associated with DF (p < 0.05). In conclusion, enamel matrix genes are associated with DF.

Diagnosis and Surgical Management of Nonsyndromic Nine Supernumerary Teeth and Leong's Tubercle.

Nonsyndromic multiple supernumerary teeth (ST) and Leong's tubercle are a condition with a very low prevalence and a multidisciplinary approach is required to restore function and aesthetics. So, this case report aimed at presenting a rare case of nonsyndromic nine supernumerary teeth and Leong's tubercle in a pediatric patient, without any evident familial history, showing its diagnosis and surgical management.

Genetic influences on dental enamel that impact caries differ between the primary and permanent dentitions.

Clinically, primary and permanent teeth are distinct anatomically and the presentation of caries lesions differs between the two dentitions. Hence, the possibility exists that genetic contributions to tooth formation of the two dentitions are different. The purpose of this study was to test the hypothesis that genetic associations with an artificial caries model will not be the same between primary and permanent dentitions. Enamel samples from primary and permanent teeth were tested for microhardness at baseline, after carious lesion creation, and after fluoride application to verify association with genetic variants of selected genes. Associations were found between genetic variants of ameloblastin, amelogenin, enamelin, tuftelin, tuftelin interactive protein 11, and matrix metallopeptidase 20 and enamel from permanent teeth but not with enamel from primary teeth. In conclusion, our data continue to support that genetic variation may impact enamel development and consequently individual caries susceptibility. These effects may be distinct between primary and permanent dentitions.

BMP2 Is Associated with Caries Experience in Primary Teeth.

Bone morphogenetic proteins (BMPs) play an important role during the initial process of enamel development and therefore may play a role in caries susceptibility. The purpose of this study was to evaluate the association between the polymorphisms in the BMP2, BMP4 and BMP7 genes and their association with caries experience and primary enamel microhardness characteristics. DNA from buccal cells as well as clinical and demographic information from 1,731 subjects from three different data sets from Brazil were included. Polymorphisms in BMP2, BMP4 and BMP7 were analyzed by real-time polymerase chain reaction from genomic DNA. Association between caries experience, genotype, and allele distribution in both cohorts was evaluated using χ(2) and logistic regression analyses. In the family-based set, the association between caries experience and alleles was tested using the transmission disequilibrium test. In the Rio de Janeiro cohort, microhardness data on 108 exfoliated primary teeth before and after demineralization and remineralization challenges was included. Associations between microhardness values and genotype and allele distribution were evaluated using χ(2) and logistic regression analyses. Differences between caries experience and some risk factors were statistically significant. In the cohort from Nova Friburgo, BMP2 was associated with caries experience in primary dentition during logistic regression analysis (p = 0.023; OR = 2.58; 95% CI 1.13-5.86). There was no association between genotype and allele distribution for BMP polymorphisms and primary enamel microhardness alterations. Our result suggests that BMP2 may be involved in caries experience in primary dentition from a Nova Friburgo cohort.

DLX1 and MMP3 contribute to oral clefts with and without positive family history of cancer.

OBJECTIVE: It has been suggested that oral clefts and cancer share a common genetic background. This study aimed to investigate the epidemiological and molecular association between oral clefts and cancer. METHODS: One hundred forty-eight nuclear families with oral clefts and 162 subjects with no birth defect were recruited. Data on self-reported family history of cancer among first, second, and third degree relatives of each patient were collected via a structured questionnaire. We also investigated the association between polymorphisms in the genes AXIN2, BMP2, BMP4, BMP7, DLX1, DLX2, and MMP3 and oral cleft with and without history of cancer. Markers in these genes were genotyped using real time PCR. Chi-square and t-test were used to assess the differences about self-reported family history of cancer between oral cleft and non-cleft individuals. The transmission disequilibrium test (TDT) was used to analyze the distortion of the inheritance of alleles from parents to their affected offspring. RESULTS: Families with oral clefts had an increased risk of having a family history of cancer (p=0.01; odds ratio=1.79; 95% confidence interval, 1.07-1.87). TDT results showed an association between DLX1 and cleft lip and palate, in which the A allele was undertransmited (p=0.022). For MMP3, G was undertransmited among affected progeny (p=0.019) in cleft palate subgroup. CONCLUSION: Oral clefts were associated with positive self-reported family history of cancer and with variants in DLX1 and MMP3. The association between oral clefts and cancer raises interesting possibilities to identify risk markers for cancer.

Calcium and magnesium levels in primary tooth enamel and genetic variation in enamel formation genes.

PURPOSE: Evidence exists that a genetic component in caries susceptibility is related to variation in enamel formation genes. The purpose of this study was to explore the trends of demineralization and remineralization of teeth from individuals whose genotypes for selected genes (ENAM, MMP20, TUFT, TFIP, and AMBN) are known. METHODS: In this study, primary baseline teeth (20) were exposed to an artificial caries solution, followed by a remineralizing solution. Biopsies of each tooth category (baseline, carious, and fluoridated) were completed via an acid wash solution. Concentrations of magnesium and calcium were measured using an optical emission spectrometer instrument. Allele and genotype frequencies for calcium and magnesium levels were compared between each tooth category. To help interpret the results, we also calculated odds ratios. RESULTS: Calcium levels exceeded magnesium levels in each sample. In addition, mineral concentration varied among samples. Associations could be seen between genetic variation in ENAM (P=.0003 baseline values for calcium, P<.001 baseline values for magnesium, P<.04 artificial caries values for magnesium) and AMBN (P<.02 artificial caries values for calcium) with mineral concentration. CONCLUSIONS: Our results suggest that genetic variation of enamel formation genes may influence calcium and magnesium concentrations of teeth and impact the development of caries.

Role of estrogen related receptor beta (ESRRB) in DFN35B hearing impairment and dental decay.

BACKGROUND: Congenital forms of hearing impairment can be caused by mutations in the estrogen related receptor beta (ESRRB) gene. Our initial linkage studies suggested the ESRRB locus is linked to high caries experience in humans. METHODS: We tested for association between the ESRRB locus and dental caries in 1,731 subjects, if ESRRB was expressed in whole saliva, if ESRRB was associated with the microhardness of the dental enamel, and if ESRRB was expressed during enamel development of mice. RESULTS: Two families with recessive ESRRB mutations and DFNB35 hearing impairment showed more extensive dental destruction by caries. Expression levels of ESRRB in whole saliva samples showed differences depending on sex and dental caries experience. CONCLUSIONS: The common etiology of dental caries and hearing impairment provides a venue to assist in the identification of individuals at risk to either condition and provides options for the development of new caries prevention strategies, if the associated ESRRB genetic variants are correlated with efficacy.

Studies of genes involved in craniofacial development and tumorigenesis: FGF3 contributes to isolated oral clefts and may interact with PAX9.

OBJECTIVE: Previous studies suggest individuals born with oral clefts and their families have a higher susceptibility for cancer, which raises the hypothesis that these two conditions share common molecular pathways. This study evaluated the association between oral clefts and polymorphisms in genes that play a role in craniofacial and tumor development. MATERIALS AND METHODS: Four hundred and ninety-seven subjects born with oral clefts and 823 unaffected subjects were recruited. Twenty-nine markers in 13 genes were genotyped by the Taqman method. Chi-square was used to compare allele and genotype frequencies. Bonferroni correction for multiple testing was used and the established alpha was 0.0003. This study also used logistic regression to test if genetic variants were associated with oral clefts using positive family history of cancer and age as covariates. RESULTS: There was no association between family history of cancer and oral clefts (p = 0.51). None of the 1320 study participants had a diagnosis of cancer at the time of participation in the study. The marker rs4980700 in FGF3 was associated with oral clefts (p = 0.0002). Logistic regression analysis also provided evidence for gene-gene interaction between FGF3 (rs4980700) and PAX9 (rs2073242), increasing the risk for isolated oral clefts (p = 0.0003). CONCLUSION: FGF3 is associated with oral clefts and may interact with PAX9.

Fine mapping of locus Xq25.1-27-2 for a low caries experience phenotype.

OBJECTIVE: The purpose of this study was to fine map the locus Xq25.1-27-2 in order to identify genetic contributors involved in low caries experience. DESIGN: Seventy-two families from the Philippines were studied. Caries experience was recorded and genomic DNA extracted from peripheral blood was obtained from all subjects. One hundred and twenty-eight polymorphisms in the locus Xq25.1-27-2, a region that contains 24 genes, were genotyped. Association between caries experience and alleles was tested using the transmission disequilibrium test (TDT). This initial analysis was followed by experiments with DNA samples from 1481 subjects from Pittsburgh, 918 children from Brazil, and 275 children from Turkey in order to follow up the results found in the Filipino families. Chi-square or Fisher's exact tests were used. Sequencing of the coding regions and exon-intron boundaries of MST4 and FGF13 were also performed on 91 women from Pittsburgh. RESULTS: Statistically significant association with low caries experience was found for 11 markers in Xq25.1-27-2 in the Filipino families. One marker was in MST4, another marker was in FGF13, and the remaining markers were in intergenic regions. Haplotype analysis also confirmed these results, but the follow up studies with DNA samples from Pittsburgh, Brazil, and Turkey showed associations for a subset of the 11 markers. No coding mutations were identified by sequencing. CONCLUSIONS: Our study failed to conclusively demonstrate that genetic factors in Xq25.1-27-2 contribute to caries experience in multiple populations.

Genetic variations in MMP9 and MMP13 contribute to tooth agenesis in a Brazilian population.

We investigated the association between polymorphisms in the MMP2 (rs243865), MMP9 (rs17576), and MMP13 (rs2252070) genes with tooth agenesis in humans. Two hundred eighty-five unrelated individuals (202 controls without tooth agenesis and 83 cases with tooth agenesis) were evaluated in a cross-sectional single-center study. The study participants were recruited through the Pediatric Dental Clinics of the Federal University of Rio de Janeiro, Brazil. Genotyping of the selected polymorphisms for MMPs was carried out by real-time PCR using the Taqman assay method from genomic DNA isolated from buccal epithelial cells of all the studied individuals. There was no significant association of MMP2 genotype or allele distribution with tooth agenesis or its absence. For MMP9, a significant difference in allele frequency was evident between the two groups (P = 0.05). With regard to the affected side, there was a significant difference between unilateral tooth agenesis and the control group in the distribution of MMP9 (P = 0.05). Also, there was a significant difference in MMP9 distribution between tooth agenesis in the maxilla and control individuals (P = 0.03). The genotype distribution of MMP13 differed significantly between the group with unilateral tooth agenesis and the controls (P = 0.01). Our findings provide evidence that MMP9 and MMP13 may be involved in tooth agenesis.

Genetic mapping of high caries experience on human chromosome 13.

BACKGROUND: Our previous genome-wide linkage scan mapped five loci for caries experience. The purpose of this study was to fine map one of these loci, the locus 13q31.1, in order to identify genetic contributors to caries. METHODS: Seventy-two pedigrees from the Philippines were studied. Caries experience was recorded and DNA was extracted from blood samples obtained from all subjects. Sixty-one single nucleotide polymorphisms (SNPs) in 13q31.1 were genotyped. Association between caries experience and alleles was tested. We also studied 1,481 DNA samples obtained from saliva of subjects from the USA, 918 children from Brazil, and 275 children from Turkey, in order to follow up the results found in the Filipino families. We used the AliBaba2.1 software to determine if the nucleotide changes of the associated SNPs changed the prediction of the presence of transcription-binding site sequences and we also analyzed the gene expression of the genes selected based on binding predictions. Mutation analysis was also performed in 33 Filipino individuals of a segment of 13q31.1 that is highly conserved in mammals. RESULTS: Statistically significant association with high caries experience was found for 11 markers in 13q31.1 in the Filipino families. Haplotype analysis also confirmed these results. In the populations used for follow-up purposes, associations were found between high caries experience and a subset of these markers. Regarding the prediction of the transcription-binding site, the base change of the SNP rs17074565 was found to change the predicted-binding of genes that could be involved in the pathogenesis of caries. When the sequence has the allele C of rs17074565, the potential transcription factors binding the sequence are GR and GATA1. When the subject carries the G allele of rs17074565, the potential transcription factor predicted to bind to the sequence is GATA3. The expression of GR in whole saliva was higher in individuals with low caries experience when compared to individuals with high caries experience (p = 0.046). No mutations were found in the highly conserved sequence. CONCLUSIONS: Genetic factors contributing to caries experience may exist in 13q31.1. The rs17074565 is located in an intergenic region and is predicted to disrupt the binding sites of two different transcription factors that might be involved with caries experience. GR expression in saliva may be a biomarker for caries risk and should be further explored.

Role of TRAV locus in low caries experience.

Caries is the most common chronic, multifactorial disease in the world today; and little is still known about the genetic factors influencing susceptibility. Our previous genome-wide linkage scan has identified five loci related to caries susceptibility: 5q13.3, 13q31.1, 14q11.2, 14q 24.3, and Xq27. In the present study, we fine mapped the 14q11.2 locus to identify genetic contributors to caries susceptibility. Four hundred seventy-seven subjects from 72 pedigrees with similar cultural and behavioral habits and limited access to dental care living in the Philippines were studied. An additional 387 DNA samples from unrelated individuals were used to determine allele frequencies. For replication purposes, a total of 1,446 independent subjects from four different populations were analyzed based on their caries experience (low versus high). Forty-eight markers in 14q11.2 were genotyped using TaqMan chemistry. Transmission disequilibrium test was used to detect over transmission of alleles in the Filipino families, and Chi-square, Fisher's exact and logistic regression were used to test for association between low caries experience and variant alleles in the replication data sets. We finally assessed the mRNA expression of TRAV4 in the saliva of 143 study subjects. In the Filipino families, statistically significant associations were found between low caries experience and markers in TRAV4. We were able to replicate these results in the populations studied that were characteristically from underserved areas. Direct sequencing of 22 subjects carrying the associated alleles detects one missense mutation (Y30R) that is predicted to be probably damaging. Finally, we observed higher expression in children and teenagers with low caries experience, correlating with specific alleles in TRAV4. Our results suggest that TRAV4 may have a role in protecting against caries.

Enamel formation genes influence enamel microhardness before and after cariogenic challenge.

There is evidence for a genetic component in caries susceptibility, and studies in humans have suggested that variation in enamel formation genes may contribute to caries. For the present study, we used DNA samples collected from 1,831 individuals from various population data sets. Single nucleotide polymorphism markers were genotyped in selected genes (ameloblastin, amelogenin, enamelin, tuftelin, and tuftelin interacting protein 11) that influence enamel formation. Allele and genotype frequencies were compared between groups with distinct caries experience. Associations with caries experience can be detected but they are not necessarily replicated in all population groups and the most expressive results was for a marker in AMELX (p=0.0007). To help interpret these results, we evaluated if enamel microhardness changes under simulated cariogenic challenges are associated with genetic variations in these same genes. After creating an artificial caries lesion, associations could be seen between genetic variation in TUFT1 (p=0.006) and TUIP11 (p=0.0006) with enamel microhardness. Our results suggest that the influence of genetic variation of enamel formation genes may influence the dynamic interactions between the enamel surface and the oral cavity.

MMP1 and MMP20 contribute to tooth agenesis in humans.

OBJECTIVE: Variations in genes that are critical for tooth formation may contribute to the tooth agenesis. MMPs are potential candidate genes for dental alterations based on the roles they play during embryogenesis. The aim of this study was to investigate the possible association between MMP1, MMP3, and MMP20 and tooth agenesis. METHODS: One hundred sixty-seven nuclear families from two different populations were analysed, 116 from Brazil and 51 from Turkey. Probands had at least one congenitally missing tooth. DNA samples were obtained from blood or saliva samples and genotyping was performed using TaqMan chemistry. In addition, Mmp20 was selected for quantitative real-time polymerase chain reaction analysis with SYBR Green I Dye in mouse tooth development. RESULTS: Associations between tooth agenesis and MMP1 (p=0.007), and MMP20 (p=0.03) were found in Brazilian families. In the total dataset, MMP20 continued to be associated with tooth agenesis (p=0.01). Mmp20 was not expressed during the initial stages of tooth development. CONCLUSION: Our findings provide evidence that MMP1 and MMP20 play a role in human tooth agenesis.