Sergio Roberto Peres Line

A simple and cost-effective protocol for DNA isolation from buccal epithelial cells

Buccal cells provide a convenient source of DNA for epidemiological studies. The goal of this study was to develop a convenient method to obtain buccal cells from mouthwash samples to be used as a source of DNA, and to evaluate the stability of the DNA in mouthwash solution over time. The procedures used in the method described in this paper avoid the use of any organic solvents. This is achieved by salting out the cellular proteins by dehydration and precipitation with a saturated ammonium acetate solution. The protocol described here is fast, simple to perform, sensitive, economical and several samples can be processed at the same time. The analyses provide consistent evidence that DNA extracted by this methodology is sufficient for several PCR amplifications. The total DNA yield ranged from 5 to 93 microg (median 15 microg, mean 20.71 microg). DNA can be extracted and PCR amplified after storage of mouthwash solution at room temperature for periods of up to 30 days.

Inhibition of human gingival gelatinases (MMP-2 and MMP-9) by metal salts.

OBJECTIVES The interaction between metal ions and the oral environment is a major subject matter in dental research. Matrix metalloproteinases (MMPs) have been implicated in several pathologic oral processes such as periodontal tissue destruction, root caries, tumour invasion and temporomandibular joint disorders. The aim of this work was to test the effect of Zn, Cu, Sn and Hg ions on the activity of the major gingival gelatinolytic MMPs. METHODS Gingival explants were cultured overnight in DMEM and the activity of secreted enzymes was analyzed by gelatin zymography in buffers containing different metal ion concentrations. The major gelatinolytic proteinases present in the conditioned media were characterized as MMP-2 and MMP-9 by immunoprecipitation with specific antibodies. The eletrophoretic bands were scanned and the transmittance values were analyzed with the Sigmagel software (Sigma). RESULTS ZnSO4 was a strong inhibitor of MMP-2 (I50 = 15 microM) and MMP-9 (I50 = 40 microM), whereas CuSO4, HgSO4 and SnCl2 showed less efficient inhibition potential. SIGNIFICANCE Our findings show that the activity of oral tissue MMPs may be modulated by metal ions present in the oral environment. Therefore, the accumulation of metals in connective tissue may interfere with the formation and resorption of the extracellular matrix components.

Analysis of magnetic resonance imaging characteristics and pain in temporomandibular joints with and without degenerative changes of the condyle

The aim of this study was to investigate temporomandibular joint (TMJ) pain and magnetic resonance imaging characteristics in 104 TMJs with and 58 without degenerative changes of the condyle, such as osteophytes, erosion, avascular necrosis, subcondral cyst and intra-articular loose bodies. TMJ images were also assessed for flattening, retropositioning and hypomobility of condyle and disc displacement. Comparison of the TMJ side-related data showed a significant relationship between disc displacement without reduction (DDwoR) and the presence of degenerative bony changes (p=0.00). Flattening, retropositioning and hypomobility of condyle showed no significant difference in relation to the presence or absence of degenerative bony changes. Retropositioning of the condyle was significantly associated to disc displacement with reduction (DDwR) (p=0.00), while condylar hypomobility was significantly more frequent in TMJ with DDwoR (p<0.05). Independent of the presence or type of DD, TMJ pain was more frequent in the presence of degenerative bony changes. When considering only DDwR, TMJ pain was significantly associated to a degenerative condition (p=0.03). When there were no degenerative bony changes, TMJ pain was significantly more frequent in DDwoR (p=0.04). Despite the present findings, the absence of symptoms in some patients with condylar bony changes suggests that the diagnosis of osteoarthritis should be established by evaluation of magnetic resonance images in association with clinical examination.

Estrogen Receptor-α Polymorphisms and Predisposition to TMJ Disorder

UNLABELLED Temporomandibular joint disorders (TMJD) affect women with greater frequency than men, and sex hormones may contribute to this female predominance. Therefore, this study investigated whether estrogen receptor-alpha (XbaI/PvuII) single nucleotide polymorphisms (SNPs) are associated with TMJD in women. DNA was obtained from 200 women with TMJD (100 with chronic pain and 100 with signs of TMJD but no pain) diagnosed according to the Research Diagnostic Criteria for Temporomandibular Disorder (RDC/TMD) and 100 control women without TMJD. Restriction fragment length polymorphisms of polymerase chain reaction products were used to analyze XbaI and PvuII SNPs in DNA fragments. A model directly characterizing specific DNA sequence variants based on the risk haplotypic structure implemented with the EM algorithm was used to analyze the data. The [GC] haplotype of the XbaI locus was significantly more prevalent in both TMJD groups when compared with the control group (P = .0012). Specifically, the [GC] haplotype was more prevalent within the painful TMJD group versus the control group (OR = 3.203, 95% CI = 1.633, 6.284) and in the TMJD no pain versus the control group (OR = 2.51, 95% CI = 1.267, 4.97). In conclusion, the presence of [GC] haplotype in the XbaI locus may increase the susceptibility of women to develop TMJD. PERSPECTIVE This study suggests that a polymorphism in the estrogen receptor may increase the risk of women developing temporomandibular joint disorder. This finding may elucidate the interindividual differences in the contribution of estrogen to TMJD, the genetic influences on TMJD predisposition, and may serve as the basis for future treatment tailoring, which could enhance outcomes for these patients.

Variation of tooth number in mammalian dentition: connecting genetics, development, and evolution

SUMMARY A major question in modern biology is how gene mutations affect development and are translated into macroevolutionary changes in morphology. Variations in tooth number, a strategy used by many mammals to develop specialized dentitions, has been an important factor for species diversification. Changes in the number of teeth tend to occur in the reverse of the order teeth are formed during development, which also characterizes the general pattern of tooth loss observed during the evolution of placental mammals. To understand how changes at the molecular level affect the distinct stages of tooth development, we analyzed the ontogenesis of tooth growth arrest in sciurids and mice and in single and double knockout mutant mice. We show that the complexity of the genetic network that governs tooth development can change during ontogenetic trajectory, and these changes may be related to macroevolutionary changes. Furthermore, we show that the variation in tooth number in the affected members of human families bearing mutations in the MSX1 and PAX9 genes can help to understand how the genetic variations within a population can modulate evolutionary changes in dental patterning.

Structural analysis of bovine root dentin after use of different endodontics auxiliary chemical substances.

INTRODUCTION The use of chemical substances in endodontics during chemical-mechanical preparation may alter the structure of dentin, mainly collagen, which contributes considerably to the mechanical properties of this structure. This study evaluated the effect of different auxiliary chemical substances during endodontic treatment in root canal walls by implementing polarized light microscopy (PLM) and scanning electron microscopy (SEM). METHODS Sixty bovine incisors were randomly divided into six groups (n = 10): G1, 5.25% NaOCl + 17% EDTA; G2, 2% chlorhexidine (CHX) gel; G3, 5.25% NaOCl; G4, 17% EDTA; G5, 2% CHX + 17% EDTA; and G6, 0.9% sodium chloride solution (control). Teeth were chemomechanically prepared and fractured into two fragments. One fragment was used for PLM, and the other was used for SEM analysis. RESULTS Groups treated with 5.25% NaOCl, whether associated or not associated to 17% EDTA, presented a significantly different birefringence pattern compared with the control group. The results revealed that groups irrigated with 2% CHX and EDTA, independently or combined, did not present any significant differences compared with the control group. SEM analysis showed structural alterations of the root canal inorganic matrix for all groups that used 17% EDTA. CONCLUSIONS It is possible to conclude that 5.25% NaOCl, whether associated or not associated with 17% EDTA, causes alterations in the dentin collagen. The obtained SEM images revealed demineralization areas for groups that used 17% EDTA alone or associated with other substances.

BASEMENT MEMBRANE ASSOCIATED CHANGES IN THE RAT VENTRAL PROSTATE FOLLOWING CASTRATION

This study focuses on the basement membrane associated modifications that take place after androgen blockade, by studying some of its main components, through histochemical, immuno‐histochemical and Western blotting tests, and its ultrastructural aspects. It was demonstrated that laminin and collagen type IV remain associated with a thickened basement membrane and that there is an apparent increase in heparan sulfate content 21 days after castration. Ultrastructurally, basal lamina appeared extensively folded and pleated. It was also observed that detachment of epithelial cells is not dependent of basal lamina degradation and that the free basal lamina surfaces are folded by the action of adjacent cells. We have also observed some aspects of smooth muscle cell degeneration and death, that lead to modifications of the associated basal lamina. In this case, residual basal lamina also shows extensive folding. The results suggested that degradation of excess basement membrane does not occur or is a very slow process within the period examined, and that basement membrane is left re‐organized but ultrastructurally and compositionally unaffected.

Absence of mutations in the homeodomain of the MSX1 gene in patients with hypodontia

Hypodontia, the congenital absence of one or a few permanent teeth, is one of the most frequent alterations of the human dentition. Although hypodontia does not represent a public health problem, it may cause both speech and masticatory dysfunction and esthetic problems. A missense mutation in the homeodomain of MSX1 gene has been associated with hypodontia of second premolars and third molars in humans. However, another study excluded this gene as causative locus for hypodontia of incisors and premolars. To further investigate the role of the MSX1 gene in human hypodontia, we analyzed the homeobox region of the MSX1 gene in 20 individuals with different patterns of familial or isolated hypodontia. The direct sequencing of PCR products did not show any polymorphisms or mutations in the human MSX1 gene. Our results indicate that inactivation of MSX1 gene in humans must have a highly selective effect on dentition, and other genes must be involved in the cause of hypodontia in humans.

Interleukin-8 Gene Promoter Polymorphism (rs4073) May Contribute to Chronic Periodontitis

BACKGROUND The proinflammatory chemokine interleukin (IL)-8 is important in the regulation of the inflammatory response. Analyses of the single nucleotide polymorphism (SNP) reference sequence (rs) 4073 showed that the A allele upregulated IL-8 levels after stimulation with lipopolysaccharides. We investigated the association of the SNP rs4073 with chronic periodontitis. METHODS Genotyping was performed by a standard polymerase chain reaction-restriction fragment length polymorphism assay in 289 genomic DNA samples of healthy control subjects and patients with chronic periodontitis; analyses were adjusted by multivariate logistic regression modeling. A real-time polymerase chain reaction performance was used to detect levels of the IL-8 mRNA. RESULTS The analysis pointed to a statistically significant association of chronic periodontitis with the heterozygous TA genotype (P = 0.001); the results showed an increase in the frequency of the A allele in the diseased group (36% in the control group versus 48% in the periodontitis group). The higher levels of the IL-8 mRNA were found in the periodontitis group, mainly in individuals who presented the TA genotype (P = 0.03). CONCLUSION The SNP rs4073 was associated with chronic periodontitis in non-smoker Brazilian subjects because the frequency of the A allele was higher in the disease group than in the control group, and the TA genotype was associated with increased levels of IL-8 mRNA transcripts.

Effect of lead on dental enamel formation

In this work the effects of lead toxicity on dental enamel formation were studied. Epidemiological data and animal studies show an association between lead exposure and higher caries prevalence, but the mechanism underlying this association is still unknown. Here we present data on enamel formation in rats exposed to lead for 70 days in the drinking water. Enamel matrix was used for protein analysis and dry weight determination, while mature enamel was used for microhardness testing. Enamel matrix was scraped from the teeth and analyzed by electrophoresis in bulk or according to developing stage. Increased amounts of protein were observed in animals exposed to lead when the same weight of matrix was electrophoresed by protein electrophoresis. When extracts were prepared according to developing stages, no differences in the amount of protein or band pattern were observed. Upper incisors were cut longitudinally for Knoop enamel microhardness determination in four regions of the teeth. Microhardness analysis revealed statistically significant (P<0.05) decrease in the microhardness values of enamel from rats exposed to lead in regions of maturation but not of fully mature enamel. These results indicate a delay in enamel mineralization in incisor teeth from animals exposed to lead, highlighting a potentially important effect of lead toxicity not yet explored.