Influence of Single-Nucleotide Polymorphisms on Vitamin D Receptor Expression in Periodontal Ligament Fibroblasts as a Response to Orthodontic Compression.

This study aimed to evaluate if single-nucleotide polymorphisms (SNPs) in the vitamin D receptor (VDR) gene are associated with gene expression in human periodontal ligament (hPDL) fibroblasts under simulated orthodontic compressive force. hPDL samples from 57 patients were used. A physiological compressive strain was performed to simulate orthodontic tooth movement in pressure areas under cell culture conditions. The RNA from hPDL fibroblasts was isolated to determine the relative gene expression (mRNA) of the VDR. The DNA was also isolated for the genotyping analysis of five SNPs in the VDR gene: BglI (rs739837, G/T), BsmI (rs1544410, T/C), ApaI (rs7975232, A/C), FokI (rs2228570, A/G), and TaqI (rs731236, A/G). Real-time polymerase chain reaction was used for both analyses. Kruskal−Wallis tests were used to compare VDR expression among genotypes of each SNP. A linear regression analysis was performed to evaluate SNP−SNP interaction. An established alpha of 5% was used. The relative mRNA VDR expression according to the genotypes in the SNPs BglI, BsmI, ApaI, FokI, and TaqI was not statistically significantly different (p > 0.05). The SNP−SNP interaction evaluated by regression analysis did not demonstrate any statistically significant association. No association was observed (p > 0.05). In conclusion, the SNPs BglI (rs739837), BsmI (rs1544410), ApaI (rs7975232), FokI (rs2228570), and TaqI (rs731236) did not show an impact on VDR gene expression in hPDL fibroblasts under simulated orthodontic compressive force.

Tooth bleaching effects on the adhesive interface of composite restorations.

The objective of this study was to evaluate the effects of different bleaching techniques on the tooth-restoration interface of composite restorations. Cavities (3 x 3 x 2 mm) were prepared in 100 bovine incisor fragments, which were etched with a conventional adhesive system and restored with a nanocomposite. The fragments were randomly divided into five groups (n = 20): Control (no bleaching), At-home bleaching (HB) (10% hydrogen peroxide [HP]), In-office bleaching (OB) (35% HP), LED-activated bleaching (LB) (35% HP activated by LED), and Laser-activated bleaching (LaB) (35% HP activated by diode laser, λ = 880 nm). After bleaching, 10 samples per group were thermocycled (500 cycles, 5°C to 55°C), immersed in 50% silver nitrate solution, sectioned, evaluated under a stereomicroscope, and scored for microleakage. The other samples were pH cycled for 14 consecutive days, sectioned, and the enamel adjacent to the adhesive interface assessed by cross-sectional Knoop hardness. The data were compared using the one-way ANOVA (α = 0.05). No differences between the microleakage indexes found for the control and experimental groups were observed. The enamel of the bleached groups located near the adhesive interface presented the same Knoop hardness numbers as the samples of the control group. Tooth bleaching does not damage the tooth-restoration interface of composite restorations.

Light-Emitting Diode at 460 ± 20 nm Increases the Production of IL-12 and IL-6 in Murine Dendritic Cells.

BACKGROUND DATA: Light emitting diode (LED) therapy has been proposed as an option for the treatment of many skin inflammatory processes. Dendritic cells (DCs) are important cells of skin that participate in the initiation and activation of skin immunity. The modulation of these cells by LED could explain much of its effects. OBJECTIVE: Thus, the aim of this study was to examine the effects of LED at 460 ± 20 nm on cytokine production and the expression of surface markers on DCs. MATERIALS AND METHODS: DCs were obtained from mouse bone marrow-derived dendritic cells (BMDCs). The LED was applied giving a fluence of 3.3, 8.2, or 16.5 J/cm2 on BMDCs or lipopolysaccharide (LPS)-matured BMDCs. The production of cytokine was analyzed by enzyme linked immunosorbant assay (ELISA) and the expression of DC co- and stimulatory was analyzed markers by cytometry. RESULTS: LED increases IL-12p40 and IL-6 production in both nonstimulated BMDCs and LPS-matured BMDCs. The expression of MHC-II molecule was inhibited and the expression of the CD86 molecule was increased in nonstimulated BMDCs but not in LPS-matured BMDCs. The production of tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) and the expression of CD40 were not altered. CONCLUSIONS: These results demonstrate that LED stimulated cytokine production in BMDCs, suggesting a proinflammatory role in the tested conditions and maybe it can increase DC maturation.

Use of hand held photopolymerizer to photoinactivate Streptococcus mutans.

OBJECTIVES: The main focus of this research was to investigate the photodynamic therapy (PDT), in vitro, acting on Streptococcus mutans and fibroblasts. A hand held photopolymerizer (HHP) and a classical photosensitizer (Rose Bengal) were used to induce photodynamic response. METHODS: S. mutans and fibroblast were treated with different concentrations of Rose Bengal (0-50 microM) irradiated with light (400-500 nm) for different time periods (0-40s) and then cell viability was evaluated. RESULTS: It was observed that the light (per se) is not toxic and in the dark Rose Bengal is toxic to the cells tested only at concentrations above 2.5 microM. Under light exposure concentrations of Rose Bengal above 0.5 microM all S. mutans were killed with no cytotoxic effects to fibroblasts. CONCLUSIONS: For the purpose of this work, the photoactivation of Rose Bengal, using the HHP, inactivated the bacteria without affecting the fibroblast viability.

Kinetic characterization of P-type membrane ATPase from Streptococcus mutans.

The proton translocating membrane ATPase of oral streptococci has been implicated in cytoplasmatic pH regulation, acidurance and cariogenicity. Studies have confirmed that Streptococcus mutans is the most frequently detected species in dental caries. A P-type ATPase that can act together with F(1)F(o)-ATPase in S. mutans membrane has been recently described. The main objective of this work is to characterize the kinetic of ATP hydrolysis of this P-type ATPase. The optimum pH for ATP hydrolysis is around 6.0. The dependence of P-type ATPase activity on ATP concentration reveals high (K(0.5)=0.27 mM) and low (K(0.5)=3.31 mM) affinity sites for ATP, exhibiting positive cooperativity and a specific activity of about 74 U/mg. Equimolar concentrations of ATP and magnesium ions display a behavior similar to that described for ATP concentration in Mg(2+) saturating condition (high affinity site, K(0.5)=0.10 mM, and low affinity site, K(0.5)=2.12 mM), exhibiting positive cooperativity and a specific activity of about 68 U/mg. Sodium, potassium, ammonium, calcium and magnesium ions stimulate the enzyme, showing a single saturation curve, all exhibiting positive cooperativities, whereas inhibition of ATPase activity is observed for zinc ions and EDTA. The kinetic characteristics reveal that this ATPase belongs to type IIIA, like the ones found in yeast and plants.

Use of visible light-based photodynamic therapy to bacterial photoinactivation.

The main focus of this laboratory exercise was to investigate the photodynamic therapy (PDT) acting over Streptococcus mutans. A handheld photopolymerizer and a classical photosensitizer (Rose Bengal) were used to induce photodynamic response. In this way, a suspension of S. mutans was treated with different concentrations of Rose Bengal (0-10 µmol/liter), irradiated with a light (400-600 nm) for 20 s, and then cell viability was evaluated. It was observed that the light (per se) is not toxic, and in the dark, Rose Bengal is toxic only to the cells tested at concentrations above 5.0 µmol/liter. Under light exposure, concentrations of Rose Bengal above 0.5 µmol/liter killed all S. mutans. Therefore, for the purpose of our work, the photoactivation of Rose Bengal using the handheld photopolymerizer was efficient in bacteria inactivation.

A 100 kDa vanadate and lanzoprazole-sensitive ATPase from Streptococcus mutans membrane.

The cariogenic potential of Streptococcus mutans is due to the production of organic acids derived from energy metabolism, which implies the need of mechanisms for the organism to tolerate this acidic environment. The F(1)F(o)-ATPase is generally considered as the main enzyme responsible for cytoplasmic proton extrusion, but mutations that resulted in a 50% reduction in F(1)F(o)-ATPase activity in S. mutans still allowed the micro-organism to grow and extrude acid, keeping the intracellular pH one pH unit above the extracellular ambient. This finding suggests the existence of other enzymatic (or cellular) mechanisms that keep the cytosolic pH neutral during micro-organism growth. This paper describes a membrane protein in S. mutans, with a molecular weight of 100 kDa, which exhibits ATPase activity inhibited by classic inhibitors of P-type ATPases (orthovanadate) and H(+),K(+)-ATPase (lanzoprazole), has an optimum pH comparable to other H(+)-ATPases and undergoes phosphorylation during the catalytic reaction, like that of H(+)-ATPases described in yeast and plant plasma membrane. Together, these results strongly suggest that the enzyme we describe here is a P-type H(+)-ATPase or H(+),ion-ATPase that can act in association with F(1)F(o)-ATPase during the growth of the S. mutans.

Lipid composition-dependent incorporation of multiple membrane proteins into liposomes.

Membrane proteins from bacteria Pasteurella multocida were used as a model for studying its incorporation into liposomes. An important step to achieve efficient high yield protein incorporation in proteoliposomes is the study of the more suitable lipid composition. To this end, we compared the amount of total protein, reconstituted by co-solubilization methods, into liposomes of phospholipids with different polar head groups and acyl chain lengths. The liposomes and proteoliposomes were characterised by isopycnic centrifugation in sucrose gradient and by dynamic light scattering. Experimental and theoretical results were compared considering the effects exerted through the hydrocarbon chain length, volume, and optimal cross-sectional area of the phospholipid (combined in the geometrical critical packing parameter, lipid-protein matching), critical spontaneous radius of curvature of the bilayer vesicle, phase transition temperature of the lipid and ratio of lipid-protein molecules present in the vesicles. The highest incorporation of multiple proteins was found with dipalmitoylphosphatidylcholine (DPPC), reaching a yield of 93% compared to the lower relative amounts incorporated in proteoliposomes of the other lipids. The incorporation of multiple proteins induces a proportional enhancement of vesicular dimension, since DPPC-proteoliposomes have an average diameter of 1850A, compared to the 1430A for pure DPPC vesicles.

The effect of carbon source and fluoride concentrations in the streptococcus mutans biofilm formation*.

The main objective of this class experiment is to show the influence of carbon source and of different fluoride concentrations on the biofilm formation by the bacterium Streptococcus mutans. The observation of different biofilm morphology as a function of carbon source and fluoride concentration allows an interesting discussion regarding the metabolic pathways that lead to cavity development, about the role of fluoride on this disease prevention, and also on the importance of biofilm formation to the cariogenic potential of this bacterium, one of the main responsible for this multifatorial disease appearance. On addition to that, the low execution cost and the simple technical apparatus makes this experiment easy to perform.

Photodynamic therapy reduces bone resorption and decreases inflammatory response in an experimental rat periodontal disease model.

OBJECTIVE: The purpose of the present study was to analyze inflammatory conditions after application of photodynamic therapy (PDT) in the gingival tissues of Wistar rats with ligature-induced periodontal disease. BACKGROUND DATA: The work investigated the effectiveness of PDT in decreasing the inflammatory response, in order to avoid bone loss. METHODS: Ligatures were positioned at the mandibular first molar of rats (n=6). After 7 days the ligatures were removed and the animals were divided into two groups, one of which received eosin, with both groups subsequently being treated by light-emitting diode irradiation. The animals were killed 7 days after the treatments, and the mandibles were histologically processed (hematoxylin and eosin stain) to assess bone loss, while gingival tissues were removed for quantification of neutrophil infiltration (using the myeloperoxidase assay) and tumor necrosis factor (TNF)-? expression (by ELISA). RESULTS: Histomorphological analysis of periodontal tissues demonstrated that PDT-treated animals presented decreased bone resorption, as well as reduced neutrophil migration and lower TNF-? expression, compared to ligatured animals treated with eosin alone. CONCLUSIONS: Within the limitations of this study, it can be concluded that PDT may be useful in the treatment of periodontal disease, because of immunomodulatory effects that decrease the inflammatory response and consequently the bone resorption.

Photodynamic therapy with rose bengal induces GroEL expression in Streptococcus mutans.

UNLABELLED: Heat-shock proteins (HSPs) are indicative of stressing conditions that may affect cell viability. In Streptococcus mutans, acid stress induces high levels of GroEL, an HSP, in addition to metabolic alterations, as shown by proteomic analysis. OBJECTIVE: We tested whether the expression of GroEL by S. mutans was enhanced after photodynamic therapy (PDT) with rose bengal. METHODS: S. mutans was grown in complete medium supplemented with 50 mmol/L glucose. The test conditions used were as follows: Rose bengal (0.1 micromol/L) with and without light treatment (500 mJ/cm(2)), light treatment alone, and 1 mol/L NaCl (as a stress condition). The extracellular pH of bacteria was monitored; HSP expression was assayed with Western blot, and possible DNA damage analyzed. RESULTS: Higher HSP expression was detected in bacteria after PDT treatment as compared with light or dye alone (negative controls). The expression of HSP after PDT was similar to that induced by osmotic stress. No DNA degradation was observed after PDT of S. mutans. CONCLUSIONS: PDT may cause effects similar to those of other stressing conditions in S. mutans, and cell death induced by this treatment reflects its incapacity to protect itself sufficiently against the deleterious effects of PDT with Rose bengal.

Comparative study of methylene blue and erythrosine dyes employed in photodynamic therapy for inactivation of planktonic and biofilm-cultivated Aggregatibacter actinomycetemcomitans.

AIM: The proposal of this work was to test the efficacy of photodynamic therapy (PDT) by using methylene blue (MB) and erythrosine (ERY) to inactivate Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), the main pathogen in aggressive periodontitis. METHODS: A. actinomycetemcomitans was cultivated in planktonic cultures and biofilm by using Tryptic Soy Broth medium. The sensibility (dark toxicity) to MB and ERY was determined, and its ideal concentration for PDT protocols was established. An odontologic resin photopolymerizer was used as the light source. The bacterial viability was determined by CFU (planktonic cultures) and microscopic observation (biofilms). RESULTS: The results show that ERY is more efficient at killing bacterial cells of A. actinomycetemcomitans in planktonic (75%) and biofilm (77%) culture compared with MB (50% and 54%, respectively). CONCLUSION: PDT using MB or ERY as a photosensitizing agent and odontologic resin photopolymerizer as a light source could be an efficient option for pocket decontamination in aggressive periodontal disease.

Photodynamic therapy in planktonic and biofilm cultures of Aggregatibacter actinomycetemcomitans.

OBJECTIVE: To evaluate the inactivation of Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), responsible for causing aggressive periodontitis, using photodynamic therapy (PDT) by rose bengal (RB) as a model of a reactive oxygen species (ROS) generator, in planktonic and biofilm cultures. MATERIALS AND METHODS: A. actinomycetemcomitans was grown in planktonic and biofilm cultures using tryptic soy broth medium. The sensibility (dark toxicity) to RB was determined, and its ideal concentration for PDT was established. Concentrations in the range from 0.01 to 50.0 micromol L(-1) RB, with different light potencies and incubation times, were used. An odontological resin photopolymerizer that emits the adequate wavelength for absorption of the RB dye was applied. Bacterial viability was determined by colony- forming units (CFU). RESULTS: RB photosensitizer dye in concentrations up to 0.1 micromol L(-1) did not show toxicity per se toward A. actinomycetemcomitans cells. In a PDT study with photoirradiation (1 min) at 0.1 micromol L(-1), a 55% reduction of A. actinomycetemcomitans viability was obtained in planktonic cultures. Preincubation (30 min) of the bacteria with the dye resulted in a 90% reduction of its viability. It is important to note that, for dye concentrations up to 1 micromol L(-1), in the same experimental conditions, no death effect on gingival fibroblasts was observed. The A. actinomycetemcomitans biofilm was not affected by RB or light alone. After PDT, the reduction in the biofilm (about 45%) is significantly dependant on RB concentration and irradiation time when this dye was used as a ROS generator. CONCLUSION: Photodynamic therapy-generated ROS inactivates A. actinomycetemcomitans both in planktonic and biofilm cultures, even in small concentrations of the photosensitizing agent, and it does not cause damage to fibroblast cells under the same conditions.

Local delivery of EGF-liposome mediated bone modeling in orthodontic tooth movement by increasing RANKL expression.

AIMS: It has long been demonstrated that epidermal growth factor (EGF) has catabolic effects on bone. Thus, we examined the role of EGF in regulating mechanically induced bone modeling in a rat model of orthodontic tooth movement. MAIN METHODS: The maxillary first molars of rats were moved mesially using an orthodontic appliance attached to the maxillary incisor teeth. Rats were randomly divided into 4 groups: (G1) administration of PBS (phosphate buffer saline) solution (n=24); (G2) administration of empty liposomes (n=24); (G3) administration 20ng of EGF solution (n=24); and (G4) 20ng of EGF-liposomes solution (n=24). Each solution was injected in the mucosa of the left first molar adjacent to the appliance. At days 5, 10, 14 and 21 after drug administration, 6 animals of each group were sacrificed. Histomorphometric analysis was used to quantify osteoclasts (Tartrate-resistant acid phosphatase (TRAP)+cells) and tooth movement. Using immunohistochemistry assay we evaluated the RANKL (receptor activator of nuclear factor kappaB ligand) and epidermal growth factor receptor (EGFR) expression. KEY FINDINGS: The EGF-liposome administration showed an increased tooth movement and osteoclast numbers compared to controls (p<0.05). This was correlated with intense RANKL expression. Both osteoblasts and osteoclasts expressed EGFR. SIGNIFICANCE: Local delivery of EGF-liposome stimulates osteoclastogenesis and tooth movement.

Purification and properties of pi-repressible acid phosphatases from Aspergillus nidulans.

Two forms of the pacA-encoded acid phosphatase (designated acid phosphatases I and II) secreted by the mold Aspergillus nidulans grown in low-Pi medium at 37 degrees, pH5.0, were purified to apparent homogeneity by PAGE. The M(r) of the purified enzyme forms were ca 115000 (60000) and 113000 (62000) respectively for forms I and II secreted by strain biA1 and ca 118000 (60000) and 121000 (61000) respectively for forms I and II secreted by strain biA1 pacA1, as determined by exclusion chromatography (number between brackets are the M(r) as determined by SDS-PAGE). All of these purified enzyme forms showed an apparent optimum pH ranging from 6.0 to 6.5 and no deviation from Michaelis kinetics for the hydrolysis of both p-nitrophenylphosphate and alpha-naphthylphosphate. Heat inactivation at 60 degrees and at pH6.0 showed half-lives of 14min (k=0.033min(-1)) and 10min (k=0.069min(-1)), respectively, for the purified acid phosphatases I and II secreted by biA1 strain and half-lives of 0.8min (k=0.92min(-1)) and 0.6min (k=0.95min(-1)), respectively, for the purified forms I and II secreted by the biA1 pacA1 strain. The neutral sugar content of purified acid phosphatases I and II secreted by strain biA1 was 48% and 37% (w/w), respectively, whereas the content of forms I and II secreted by strain biA1 pacA1 was 18% and 11%, respectively.