Revisiting gas-chromatography/mass-spectrometry molar response factors for quantitative analysis (FID or TIC) of glycosidic linkages in polysaccharides produced by oral bacterial biofilms.

Methylation analysis was performed on methylated alditol acetate standards and Streptococcus mutans extracellular polymeric substances (EPS) produced from wild-type and Gtf knockout strains (∆GtfB, ∆GtfB, and ∆GtfD). The methylated alditol acetate standards were representative of glycosidic linkages found in S. mutans EPS and were used to calibrate the GC-MS system for an FID detector and MS (TIC) and produce molar response factor, a necessary step in quantitative analysis. FID response factors were consistent with literature values (Sweet et al., 1975) and found to be the superior option for quantitative results, although the TIC response factors now give researchers without access to an FID detector a needed option for molar response factor correction. The GC-MS analysis is then used to deliver the ratio of the linkage types within a biofilm.

The potential use of glycosyl-transferase inhibitors for targeted reduction of S. mutans biofilms in dental materials.

Streptococcus mutans is the primary oral caries-forming bacteria, adept at producing "sticky" biofilms via the synthesis of insoluble extracellular polysaccharides (EPS), catalyzed by glucosyltransferases (GTFs). To circumvent the use of broad-spectrum antibiotics to combat these bacteria, this study sought to modify existing EPS-targeting small molecules with the ultimate goal of producing anti-biofilm polymer surfaces specifically targeting S. mutans. To achieve this, a known GTF inhibitor (G43) was modified with methoxy or tetraethyleneglycol substitutions in different positions (nine derivatives, tested at 50-µM) to pinpoint potential sites for future methacrylate functionalization, and then assessed against single-species S. mutans biofilms. As expected, the compounds did not diminish the bacterial viability. In general, the compounds with methoxy substitution were not effective in reducing EPS formation, whereas the tetraethyleneglycol substitution (G43-C3-TEG) led to a decrease in the concentration of insoluble EPS, although the effect is less pronounced than for the parent G43. This aligns with the reduced GTF-C activity observed at different concentrations of G43-C3-TEG, as well as the consequent decrease in EPS formation, and notable structural changes. In summary, this study determined that G43-C3-TEG is non-bactericidal and can selectively reduce the biofilm formation, by decreasing the production of EPS. This molecule will serve to functionalize surfaces of materials to be tested in future research.

Solvation role of dimethyl sulfoxide on the interaction with dentin bonding systems after 30 months.

OBJECTIVES: To determine whether DMSO could serve as an effective pretreatment to improve the mechanical properties and minimize the degradation of the adhesive interface, through the degree of conversion (DC) and bond strength to dentin of different categories of dentin bonding systems (DBSs) after 30 months. METHODS: DMSO (0, 0.5, 1, 2, 5, 10 vol%) were incorporated into four categories of DBSs: Adper Scotchbond Multipurpose (MP), Adper Single Bond 2 (SB), Clearfil SE Bond (CSE) and Adper Scotchbond Universal (SU). DC was evaluated by Fourier transform infrared spectroscopy (FTIR). For microtensile bond strength test (µTBS), 1 % DMSO were applied on dentin as pretreatment before DBSs. For SU, both strategies were tested. Specimens for µTBS were tested after 24 h, 6 and 30 months. DC and µTBS data were subjected to two-way ANOVA and Tukey test (α < 0.05). RESULTS: Incorporating 5 %/10 % DMSO increased the DC of CSE. Controversially, when combined with SU, 2 % and 10 % DMSO jeopardized the DC. Regarding µTBS, 1 % DMSO pre-treatment increased the bond strength for MP, SB, SU-ER and SU-SE. After 30 months, MP, SU-ER and SU-SE showed a decrease compared to baseline but remained higher than the control. CLINICAL SIGNIFICANCE: DMSO pretreatment may be a useful strategy to improve the bond interface over time. Its incorporation seems to favor the non-solvated systems regarding DC while it seems to show long-term benefits for bond strength using 1 % DMSO for MP and SU systems.

Differential analysis of the dentin soluble proteomic.

OBJECTIVES: To perform a differential analysis of the dentin soluble proteomic and assess the effects of tissue health state and protocol for protein extraction. We hypothesized the dentin soluble proteomic varies according to the tissue physiopathological state (intact vs. caries-affected) and protocol used to extract its proteins. METHODS: Dentin from freshly extracted non-carious and carious teeth were randomly assigned for protein extraction using either guanidine-HCl/ethylenediaminetetraacetic acid (EDTA) or acetic acid. Protein extracts from intact and caries-affected dentin were processed and digested with trypsin for shotgun label-free proteomic analysis (nLC-ESI-MS/MS). Peptides identification was performed on a nanoACQUITY UPLC-Xevo Q-Tof MS system. Peptides identified with scores of confidence greater than 95% were included in the quantitative statistical analysis embedded in the PLGS software. Differences between experimental conditions were calculated using Student test-t with significance pre-set at α=0.05. RESULTS: A total of 158 human proteins were identified. Approximately one-sixth of proteins (24/158) were present in at least two different extracts. Conversely, the greatest number of proteins (134/158) was identified uniquely in only one of the extracts. Overall, a larger number of soluble proteins was retrieved from caries-affected than intact dentin (86/158). Likewise, a greater number of proteins was extracted by the guanidine-HCl/EDTA (106/158) in comparison to acetic acid protocol. Several proteins detected in dentin extracts, mainly those from caries-affected teeth, are biological and/or metabolically involved with tissue turnover/remodeling. CONCLUSION: The identity/abundance of soluble proteins retrieved from and remained in dentin noticeably depend on this tissue physiopathological state and protocol used to remove its minerals. CLINICAL SIGNIFICANCE: The present findings brought new insight into the proteomic phenotype of human dentin and may provide targets for the development of novel caries disease-prevention therapies.

Gelatinolytic activity after dentin pretreatment with dimethyl sulfoxide (DMSO) combined to dental bonding systems: Perspectives for biological responses.

Endogeneous proteolytic responses in dentin bonding interface have addressing to strategies to preventive and therapeutic approaches of clinical use of dentin bonding systems (DBSs), but still present limitations. The aim of this study was to examine the gelatinolytic profile by means of in situ zymography regarding the use of 1% dimethyl sulfoxide (DMSO) as an aprotic solvent. Sound human third molars were prepared and randomized in 10 groups, following the factors 1- DBS: Adper™ Scotchbond Multipurpose [MP], Adper™ Single Bond 2 [SB], Clearfil™ SE Bond [CSE] and Adper™ Scotchbond Universal - Etch-and-rinse [SU-ER] mode and self-etch mode [SU-SE], 2- dentin pretreatment: Control - Water [C], 2% CHX and 1% DMSO and 3- time: Initial-24 h [I], 6 months [6M] and 30 months [30M]. Pretreatments were applied before primer application for 30s. After restoration, specimens were cut into slices, in which one third were incubated with fluorescein-conjugated gelatin for 24h at 37 °C and analyzed by confocal laser scanning microscopy. The other two-thirds were stored for 6 or 30 months at 37 °C. Fluorescence was quantified using Image J and data was subjected for two-way ANOVA followed by Tukey test (p<0.05). Neither DMSO nor CHX affected initial analyses for any tested conditions. After 6 months, it was observed increased fluorescence for MP using both pretreatments and for SB using only DMSO. Regardless time and pretreatment, CSE and SU-SE showed stabilized gelatinolytic pattern. For SU-ER, both CHX and DMSO were able to maintain a lower fluorescence compared to control group after 6 months. 30-month performance states the susceptibility of degradation for all etched-dentin systems. DMSO pretreatment can be promising to reduce gelatinolytic activity combined with an universal adhesive system under etch-and-rinse mode. For self-etching strategies, DMSO was successful to stabilize the gelatinolytic reactions.

Two-year randomized clinical trial of different restorative techniques in non-carious cervical lesions and MMP activity in gingival crevicular fluid.

OBJECTIVES: To evaluate different restorative techniques for non-carious cervical lesions (NCCLs) and the activity of matrix metalloproteinases (MMPs) in gingival crevicular fluid. MATERIALS AND METHODS: Two hundred restorations were performed in 50 patients using resin composite restorative system without (I) and with selective enamel conditioning (II) and resin-modified glass-ionomer cement without (III) and with EDTA pretreatment (IV). Gingival crevicular fluid samples were collected in 15 patients. Restorations were evaluated using USPHS criteria at baseline and after 2 years. Percentages of MMP activity were assessed by zymography as a surrogate outcome. Equality tests of two proportions, logistic regression analysis, survival analysis, ANOVA repeated measures, and Fisher tests were used. RESULTS: No differences in clinical performance were found among groups. Group I had lower retention at 2 years than at baseline. Decreased alpha scores for marginal integrity and marginal discoloration were observed for all groups after 2 years. MMP-2 decreased after 1 year, and its activity increased back to the initial level after 2 years, mainly for groups I, II, and III. MMP-9 increased after 1 year, and it was reduced to the initial level after 2 years, mainly for group I. CONCLUSIONS: All restorative techniques performed similarly in NCCLs after 2 years with initial marginal defect alterations. MMP-2 reestablished its initial levels after 2 years, and MMP-9 had few alterations over time in crevicular fluid. Clinical relevance The different restorative techniques are equally successful in NCCLs after 2 years of clinical functioning and have similar effects on MMPs present in crevicular fluid.

Sodium trimetaphosphate combined to calcium as a strategy to improve dentin-bonding interface / Trimetafosfato de sódio associado ao cálcio como uma estratégia para melhorar a interface adesiva da dentina

Objective: The aim of this study was to evaluate the effect of STMP as biomimetic analog of dentin matrix on the dentin bond strength submitted to artificial cariogenic challenge over time. Material and Methods: The total number of teeth used in the experiment was 60 teeth, which were divided into 6 groups (n = 10). Of these total amount, 10 teeth were not submitted to the artificial cariogenic challenge (ACC), serving as control group (Sound Dentin - SD) while the other 50 were submitted to an ACC (7d/37ºC), being treated with treatment solutions according to each group: SD- deionized water/sound dentin, CD- deionized water/ artificial caries dentin, GIII- STMP, GIV- STMP + Ca(OH)2, GV- STMP + NaF, and GVI- NaF. After treatments (24h), the specimens were restored (Adper Single Bond Universal + Filtek Z250), to obtain resin­dentin sticks with a cross sectional area of 0.8mm2, approximately. Two-third of these sticks were stored in artificial saliva (37°C) for analyzes after 6 and 12 months. The 1/3 remains were subjected to µTBS test (baseline). Data were analyzed by two-way ANOVA and Tukey tests (p<0.05). Results: In general, the highest µTBS values were obtained in sound condition (SD), while the artificial caries condition (CD) determined minimum values. Groups treated with NaF (with or without STMP- GV and GVI) were not able to improve adhesion over time. Only the use of STMP + Ca(OH)2(GIV) improved the µTBS compared to the others caries-challenged dentin after 1 year. The adhesive failure pattern was predominant in all time. Conclusion: The use of the STMP associated with Ca(OH)2 seems to be a viable therapeutic strategy conciliating the biomimetizing capacity to the adhesive process satisfactorily even its performance is not superior to initial condition (AU)

Objetivo: O objetivo deste estudo foi avaliar o efeito do STMP como análogo biomimético da matriz dentinária na resistência de união à dentina submetida a desafio cariogênico artificial ao longo do tempo. Material e Métodos:foram utilizados um total de 60 dentes neste experimento, os quais foram divididos em 6 grupos (n = 10). Desse total, 10 dentes não foram submetidos ao desafio cariogênico artificial (DCA), servindo como grupo controle (Dentina Hígida - DH) enquanto os outros 50 foram submetidos ao DCA (7d / 37ºC), sendo tratados com soluções de tratamento específicas para cada grupo: DH- água deionizada / dentina hígida, DC- água deionizada / dentina submetida ao DCA, GIII- STMP, GIV- STMP + Ca(OH)2, GV- STMP + NaF e GVI- NaF. Após os tratamentos (24h), os corpos-de-prova foram restaurados (Adper Single Bond Universal + Filtek Z250), para obtenção de palitos de resina-dentina com área transversal de aproximadamente 0,8mm2. Dois terços desses palitos foram armazenados em saliva artificial (37°C) para análises após 6 e 12 meses. Os outros 1/3 foram submetidos ao teste µTBS (baseline). Os dados foram analisados por ANOVA a dois fatores e testes de Tukey (p <0,05). Resultados:Em geral, os maiores valores de µTBS foram obtidos em condição hígidas (DH), enquanto a condição subtmetidas ao DCA determinou os menores valores. Os grupos tratados com NaF (com ou sem STMP associado -GV e GVI) não foram capazes de melhorar a resistência de união, ao longo do tempo. Somente o uso de STMP + Ca (OH)2(GIV) melhorou o µTBS em comparação com as outras condições desafiadas por cárie após 1 ano. O padrão de falha adesiva foi predominante em todos os tempos. Conclusão: O uso do STMP associado ao Ca (OH)2 parece ser uma estratégia terapêutica viável conciliando a capacidade biomimetizante ao processo adesivo de forma satisfatória mesmo que seu desempenho não seja superior à condição inicial.(AU)

Performance of MDP-based system in eroded and carious dentin associated with proteolytic inhibitors: 18-Month exploratory study.

The aim of this study was to explore the impact of the interaction between an MDP-based universal adhesive system in etch-and-rinse mode and two proteolytic inhibitors on the longevity of restorations bonded to artificially-affected-dentin substrates. 90 sound human third molars were randomly distributed into three groups according to the substrate: N-no challenges-control (stored in artificial saliva), ACD-artificial caries dentin (6 h DE + 18 h-RE/5 days + 48 h RE) and ERO-artificial erosion dentin (3 × 5 min/5 days with orange juice). They were further redistributed according to dentin pretreatment: W- water (control), CHX-2% digluconate chlorhexidine and E64- 5 µM E64-Trans-Epoxysuccinyl-L-Leucylamido-(4-guanidino) butane, which resulted in the following 9 groups (n = 10): N-W, N-CHX, N-E64, ACD-W, ACD-CHX, ACD-E64, ERO-W, ERO-CHX and ERO-E64. All specimens were restored with Adper Single Bond Universal (Etch-and-rinse mode)/Filtek Z250. Sticks (0.64 mm2) were obtained and subjected to microtensile test (µTBS) in a universal testing machine at 0.5 mm/min for 7-days, 6 and 18-month analyses. Failure modes were classified using optical microscopy (40X). Data were statistically analyzed by three-way ANOVA and Tukey tests (p < 0.05). All individual factors (p < 0.0001) and interaction between factors were statistically significant (substrate X pretreatment (p = 0.00093); substrate X time (p = 0.01035) and pretreatment X time (p = 0.0035). Caries-affected substrate was the most compromised one, disregarding the pretreatment. CHX was mostly affected compared with E64 up to 18 months, possibly due to its calcium-dependent mechanism.

Profile of a 10-MDP-based universal adhesive system associated with chlorhexidine: Dentin bond strength and in situ zymography performance.

The incorporation of functional monomers and proteolytic inhibitors into adhesive systems have shown to be promising strategies to improve the longevity of adhesive restorations. The aim of this study was to evaluate the long-term bonding performance and anti-gelatinolytic effect of a 10-MDP-based universal adhesive system applied in combination with 2% chlorhexidine digluconate (CHX). For that, this study assessed the resin-dentin bond strength and the in situ gelatinolytic activity profile at the adhesive interface at initial and after 6 month of storage. One hundred and two sound human third molars were prepared and randomly divided into 3 groups according to the adhesive strategy: SB (two-step etch-and-rinse adhesive, Adper Single Bond 2, 10-MDP-free control group); SU-ER (Adper Single Bond Universal, 10-MDP containing universal adhesive applied on etch-and-rinse mode); and SU-SE (SU applied on self-etching mode). The groups were subdivided into two according to the dentin pretreatment: W - water or CHX- 2% chlorhexidine digluconate aqueous solution (SB-W; SB-CHX; SU-ER-W; SU-ER-CHX; SU-SE-W; SU-SE-CHX) and subsequently restored according to the manufacturer's instructions. Bond strength (n = 12) was assessed by a microtensile test (µTBS) (500N/0.5 mm/min) after 24h or after 6 months of storage. In situ zymography was performed to evaluate anti-gelatinolytic activity (n = 5). Resin-dentin samples were incubated with fluorescein-conjugated gelatin for 24 h at 37 °C and analyzed by confocal laser scanning microscopy. Fluorescence indicating gelatinolytic activity at hybrid layer zone and adjacencies was quantified using Image J. Data was analyzed by three-way ANOVA and Tukey post-hoc tests (p < 0.05). Results: SU-SE showed the highest bond strength values, while similar results were observed for SU-ER and SB. No statistical significant differences were observed between pretreatment (CHX vs. W) or storage time (initial vs. 6 months of aging). For in situ zymography, fluorescence was detected in all groups and CHX pre-treatment was able to inhibit the gelatinolytic activity in all conditions. The 10-MDP-based universal adhesive system in self-etching mode was the strategy that showed the best bonding performance irrespective of its combination with chlorhexidine. Pre-treatment with CHX did not impair the bond strength when used in combination with 10-MDP and it may promote collagen stability overtime.

Insights into cathepsin-B activity in mature dentin matrix.

OBJECTIVE: Cysteine proteases are lysosomal enzymes that, under specific circumstances, may be secreted into the extracellular space and participate in protein turnover. This study investigated the involvement of cathepsin B in the gelatinolytic activity of mature dentin matrices at neutral pH. DESIGN: Human dentin fragments were made into powder and enzymes were extracted using guanidine-HCl/EDTA. Host-derived dentin proteases (cathepsin B, MMP-2 and MMP-9) were identified by immunoblotting, and their activities were evaluated spectrofluorimetrically using fluorogenic substrates. Proteases activities were monitored by measuring the rate of hydrolysis of substrates in the presence/absence of MMP- or cysteine cathepsin inhibitors, at neutral pH (7.4). Mass spectroscopy was used to determine the substrates' cleavage points. Reverse zymography was performed to examine the gelatinolytic activity of cathepsin B. RESULTS: Western-blots of dentin extracts yielded strong bands at 95, 72 and 30 kDa, corresponding respectively to MMP-9, MMP-2 and Cathepsin B. Greater fluorogenic substrates hydrolysis occurred in the absence of MMP and cysteine cathepsin inhibitors than in their presence. Cathepsin B exhibited significant gelatinolytic activity. CONCLUSIONS: Together with MMP-2 and MMP-9, cathepsin B also account for the host-derived gelatinolytic activity and matrix turnover of mature dentin at physiological, neutral pH.

Biochemical and immunohistochemical identification of MMP-7 in human dentin.

OBJECTIVES: Matrix metalloproteinases (MMPs) are dentinal endogenous enzymes claimed to have a vital role in dentin organic matrix breakdown. The aim of the study was to investigate presence, localization and distribution of MMP-7 in sound human dentin. METHODS: Dentin was powdered, demineralized and dissolved in isoelectric focusing buffer. Resolved proteins were transferred to nitrocellulose membranes for western blotting (WB) analyses. For the zymographic analysis, aliquots of dentin protein were electrophoresed in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing fluorescently labeled gelatin. Further, the concentrations of dentinal MMPs were measured using Fluorescent Microsphere Immunoassay with a human MMP-MAP multiplex kit. Pre- and post-embedding immunolabeling technique was used to investigate the localization and distribution of MMP-7 in dentin. Dentin was cryo-fractured, the fragments partially decalcified and labeled with a primary monoclonal anti-MMP-7 and a secondary antibody conjugated with gold nanoparticles. MMP-7 labelings were identified in the demineralized dentin matrix as highly electron-dense dispersed gold particles. RESULTS: WB and zymographic analysis of extracted dentin proteins showed presence of MMP-7 (∼20-28 KDa). Further, MMP-7 was found in the supernatants of the incubated dentin beams using Fluorescent Microsphere Immunoassay. FEI-SEM and TEM analyses established MMP-7 as an intrinsic constituent of the human dentin organic matrix. CONCLUSION: This study demonstrated that MMP-7 is an endogenous component of the human dentin fibrillar network. CLINICAL SIGNIFICANCE: It is pivotal to understand the underlying processes behind dentin matrix remodeling and degradation in order to develop the most optimal clinical protocols and ensure the longevity of dental restorations.

Sodium Trimetaphosphate as a Novel Strategy for Matrix Metalloproteinase Inhibition and Dentin Remineralization.

The effect of sodium trimetaphosphate (STMP) as an antiproteolytic and remineralizing agent on demineralized dentin was evaluated in vitro. The inhibitory potential of STMP at 0.5, 1.5, 3.5, and 5% against recombinant matrix metalloproteinases (MMPs) MMPs-2 and -9 was assessed by zymography. To investigate its remineralization potential, 40 bovine root specimens were obtained and subjected to a demineralization protocol to produce caries-like dentin lesions. After that, dentin surfaces were divided into 3 areas: (1) mineralized (no treatment); (2) demineralized; and (3) demineralized/treated with STMP and submitted to a pH-cycling associated or not with STMP (1.5, 3.5, or 5% STMP, 10 min of treatment). After that, superficial hardness (SH) and cross-sectional hardness (CSH) were determined. Polarized light microscopy (PLM) was used to qualitatively evaluate mineralization within the caries-like lesions. The zymographic analysis showed that STMP solution is a potent inhibitor of the gelatinolytic activity of MMPs-2 and -9 depending on the dose, since the lowest concentration (0.5%) partially inhibited the enzyme activity, while the higher concentrations completely inhibited enzyme activity. Regarding remineralization effect, only 1.5% STMP solution enhanced both the SH and CSH. PLM showed that the area treated with 1.5% STMP presented similar birefringence as mineralized sound dentin. In conclusion, 1.5% STMP solution is effective as an antiproteolytic agent against MMPs and promotes dentin remineralization.

How proteolytic inhibitors interact with dentin on glass-fiber post luting over 6 months.

OBJECTIVES: Enzyme inhibitors minimize the degradation of unprotected collagen of dentin promoted by matrix metalloproteinases (MMPs) and cysteine cathepsins (CCs). As the evidence of their effect on the root canal is limited, this study aimed to evaluate the role of EDTA, chlorhexidine and E-64 as antiproteolytic agents on the bond strength (BS) of glass-fiber posts in root canals. MATERIALS AND METHODS: Ninety-six bovine roots were distributed in groups for each time point (n = 8). Adper Scotchbond Multipurpose (MP)/ RelyX ARC system was used to lute the post according to the treatment: negative control (NC)- water, EDTA- 17% ethylenediaminetetraacetic acid, CHX- 2% digluconate chlorhexidine, E-64-5- 5µM E-64, E-64-10- 10µM E-64 and positive control (PC)- MP associated with activator/ catalyst. Then, slices were subjected to push-out test (0.5mm/min) after 24h/6 mons. Data were analyzed by three-way ANOVA/Tukey tests. Failure modes were analyzed (40×). RESULTS: The factors treatment, time, root canal third and the interaction between treatment and time were statistically significant. At 24h, no negative interactions were observed among the root dentin, bonding system and post. At 6 mons, CHX improved the BS for middle and apical root thirds. CONCLUSIONS: CHX was able to promote beneficial BS after 6 mons, which was not noted for any other tested enzyme inhibitors.

Dynamic Influence of pH on Metalloproteinase Activity in Human Coronal and Radicular Dentin.

The aim of this study was to evaluate the effect of pH on the activation of matrix metalloproteinases (MMPs) of human coronal (CD) and radicular dentin (RD). CD and RD were pulverized to powder, and proteins were extracted with 1% phosphoric acid. The extracted proteins and the demineralized powder were separately incubated in the following solutions: 4-aminophenylmercuric acetate (control) or a buffer solution at different pHs (2.5, 4.5, 5.0, 6.0, and 7.0). After incubation, proteins were separated by electrophoresis to measure MMP activities by zymography. To assess the solubilized dentin collagen, the demineralized dentin powder was sustained in incubation buffer, and the amount of hydroxyproline (HYP) released was measured. Zymography revealed MMP-2 gelatinolytic activities for CD and RD in all experimental groups. For both substrates, the lowest pH solutions (2.5, 4.5, and 5.0) yielded higher gelatinolytic activity than those obtained by the highest pH solutions (6.0 and 7.0). For HYP analysis, no detectable absorbance values were observed for pHs of 2.5 and 4.5. The amount of HYP was higher for pH 7.0 than those of all other groups (p < 0.05), except for pH 6.0. No statistical differences were found between pHs 6.0 and 5.0 and control (p > 0.05). The MMP-2 enzyme from human CD and RD is dynamically influenced by pH: at low pH, the extracted enzyme activates this latent form, whereas collagen degradation by the matrix-bound enzyme is only observed when pHs are close to neutral.

Use of sodium trimetaphosphate in the inhibition of dentin matrix metalloproteinases and as a remineralizing agent.

OBJECTIVES: Because of its ability to act as an antiproteolytic agent, the effect of sodium trimetaphosphate (STMP) against specific enzymes extracted from sound dentin and its performance under acidic challenge on demineralized dentin were investigated. METHODS: The antiproteolytic potential of STMP (0.5%, 1.0%, and 1.5%) was assessed in triplicate by zymography. For the evaluation of remineralization activity, 50 bovine-root dentin specimens were selected and randomly divided into 5 groups (n=10). Three areas were determined for each specimen: 1) control (no treatment); 2) demineralized (artificial caries-like challenge); 3) treated (demineralized and subjected to pH-cycling for 7days, and treated for 10min with 1.5% STMP, 1.5% STMP+calcium hydroxide (Ca[OH]2), 1.5% STMP+sodium fluoride (NaF), NaF, or deionized H2O). The dentin specimens were analyzed for superficial hardness (SH) and cross-sectional hardness (CSH) at different depths (10, 30, 50, 70, 90, 110, and 220µm) using a Knoop penetrator (10g/10s). Statistical analyses were performed with analysis of variance (ANOVA) and Tukey tests (p<0.05). RESULTS: The zymographic analysis showed that 1.5% STMP promoted complete inhibition of gelatinolytic activity. Therefore, 1.5% STMP was investigated in association with supplemented calcium or fluoride; a combination of 1.5% STMP and Ca(OH)2 significantly increased the mechanical properties of the treated dentin. CONCLUSION: 1.5% STMP serves as an antiproteolytic agent against matrix metalloproteinases extracted from human dentin. Furthermore, when supplemented with Ca(OH)2, 1.5% STMP may potentially induce remineralization. CLINICAL SIGNIFICANCE: STMP can be introduced as a novel strategy that combines enzymatic inhibition and remineralizing potential, which can serve to strengthen dentin and improve stability. STMP may have potential in the treatment of demineralized dentin lesions, especially when supplemented with calcium.

Comparative bonding ability to dentin of a universal adhesive system and monomer conversion as functions of extended light curing times and storage.

OBJECTIVES: The purpose of this in vitro study was to evaluate the bonding ability and monomer conversion of a universal adhesive system applied to dentin as functions of different curing times and storage. The results were compared among a variety of commercial adhesives. MATERIALS AND METHODS: Flat superficial dentin surfaces were exposed on human molars and assigned into one of the following adhesives (n = 15): total-etch Adper Single Bond 2 (SB) and Optibond Solo Plus (OS), self-etch Optibond All in One (OA) and Clearfil SE Bond (CSE), and Scotchbond Universal Adhesive in self-etch mode (SU). The adhesives were applied following the manufacturers' instructions and cured for 10, 20, or 40s. Specimens were processed for the microtensile bond strength (µTBS) test in accordance with the non-trimming technique and tested after 24h and 2 years. The fractured specimens were classified under scanning electron microscopy (SEM). Infrared (IR) spectra were obtained and monomer conversion (%) was calculated by comparing the aliphatic-to-aromatic IR absorption peak ratio before and after polymerization (n=5). Data were analyzed by 2-way ANOVA/Tukey's tests (α = 0.05). RESULTS: At 24-h evaluation, OA and CSE presented similar bond strength means irrespective of the curing time, whereas SB and SU exhibited significantly higher means when cured for 40s as did OS when cured for 20 or 40s (p < 0.05). At 2-year evaluation, only OA exhibited significantly higher bond strength when cured for 20 and 40s (p < 0.05). When the evaluation times were compared, OA also exhibited the same bonding ability when cured for longer periods of time (20 and 40s). All of the adhesives tested exhibited significantly lower monomer conversion when photoactivated according to the manufacturers' instructions (10s). CONCLUSIONS: Higher monomer conversions obtained with longer light exposure allow only higher immediate bond strength for most of the adhesives tested. After 2-year storage, only the self-etching adhesive Optibond All-In-One exhibited the same bonding ability when cured for longer periods of time.

Effect of a one-step self-etch adhesive on endogenous dentin matrix metalloproteinases.

Degradation of the hybrid layer created in dentin by dentin adhesives is caused by enzyme activities present within the dentin matrix that destroy unprotected collagen fibrils. The aim of the present study was to evaluate the effect of a one-step self-etch adhesive system on dentinal matrix metalloproteinases 2 and 4 (MMP-2 and MMP-9, respectively) using in situ zymography and an enzymatic activity assay. The null hypothesis tested was that there are no differences in the activities of dentinal MMPs before and after treatment with a one-step adhesive system. The MMP-2 and MMP-9 activities in dentin treated with the one-step adhesive, Adper Easy Bond, were quantified using an enzymatic activity assay system. The MMP activities within the hybrid layer created by the one-step adhesive tested were also evaluated using in situ zymography. The enzymatic assay revealed an increase in MMP-2 and MMP-9 activities after treatment with adhesive. In situ zymography indicated that gelatinolytic activity is present within the hybrid layer created with the one-step self-etch adhesive. The host-derived gelatinases were localized within the hybrid layer and remained active after the bonding procedure. It is concluded that the one-step self-etch adhesive investigated activates endogenous MMP-2 and MMP-9 with the dentin matrix, which may cause collagen degradation over time.

Co-distribution of cysteine cathepsins and matrix metalloproteases in human dentin.

It has been hypothesized that cysteine cathepsins (CTs) along with matrix metalloproteases (MMPs) may work in conjunction in the proteolysis of mature dentin matrix. The aim of this study was to verify simultaneously the distribution and presence of cathepsins B (CT-B) and K (CT-K) in partially demineralized dentin; and further to evaluate the activity of CTs and MMPs in the same tissue. The distribution of CT-B and CT-K in sound human dentin was assessed by immunohistochemistry. A double-immunolabeling technique was used to identify, at once, the occurrence of those enzymes in dentin. Activities of CTs and MMPs in dentin extracts were evaluated spectrofluorometrically. In addition, in situ gelatinolytic activity of dentin was assayed by zymography. The results revealed the distribution of CT-B and CT-K along the dentin organic matrix and also indicated co-occurrence of MMPs and CTs in that tissue. The enzyme kinetics studies showed proteolytic activity in dentin extracts for both classes of proteases. Furthermore, it was observed that, at least for sound human dentin matrices, the activity of MMPs seems to be predominant over the CTs one.

The effect of dimethyl sulfoxide (DMSO) on dentin bonding and nanoleakage of etch-and-rinse adhesives.

OBJECTIVE: The objective was to examine the effect of a solvent dimethyl sulfoxide (DMSO) on resin-dentin bond durability, as well as potential functional mechanisms behind the effect. METHODS: Microtensile bond strength (µTBS) was evaluated in extracted human teeth in two separate experiments. Dentin specimens were acid-etched and assigned to pre-treatment with 0.5mM (0.004%) DMSO as additional primer for 30s and to controls with water pre-treatment. Two-step etch-and-rinse adhesive (Scotchbond 1XT, 3M ESPE) was applied and resin composite build-ups were created. Specimens were immediately tested for µTBS or stored in artificial saliva for 6 and 12 months prior to testing. Additional immediate and 6-month specimens were examined for interfacial nanoleakage analysis under SEM. Matrix metalloproteinase (MMP) inhibition by DMSO was examined with gelatin zymography. Demineralized dentin disks were incubated in 100% DMSO to observe the optical clearing effect. RESULTS: The use of 0.5mM DMSO had no effect on immediate bond strength or nanoleakage. In controls, µTBS decreased significantly after storage, but increased significantly in DMSO-treated group. The control group had significantly lower µTBS than DMSO-group after 6 and 12 months. DMSO also eliminated the increase in nanoleakage seen in controls. 5% and higher DMSO concentrations significantly inhibited the gelatinases. DMSO induced optical clearing effect demonstrating collagen dissociation. SIGNIFICANCE: DMSO as a solvent may be useful in improving the preservation of long-term dentin-adhesive bond strength. The effect may relate to dentinal enzyme inhibition or improved wetting of collagen by adhesives. The collagen dissociation required much higher DMSO concentrations than the 0.5mM DMSO used for bonding.

Cavity instrumentation with chemical vapor deposition diamond-coated bur / Instrumentação cavitária com uso de ponta diamantada obtida pela tecnologia da Deposição Química por Vapor

High- and low-speed rotary dental handpieces have been used for a long time in restorative dentistry for cavity preparation. However, problems inherent to conventional burs, such as noise, heat and vibration, have led to the development of new dental burs, such as the chemical vapor deposition diamond-coated bur. Its advantages are many, such as less noise, less pain for the patient, precise cutting, conservative cavity preparation, longer lifetime, less injury to the dental structures, no cutting of soft tissues and easier access of the carious lesion. This case report uses a chemical vapor deposition diamond-coated bur to prepare a cavity by direct proximal access, preserving the marginal ridge. The cavity was then filled with glass ionomer cement. The clinical outcome was satisfactory. Direct access to the cavity was possible because of the chemical vapor deposition diamond-coated bur, resulting in comfort for the patient and dentist.

Os instrumentos rotatórios convencionais têm sido utilizados em alta e/ou baixa velocidade há tempos para a confecção de cavidades na odontologia restauradora. Contudo, problemas inerentes a estes equipamentos, como o ruído, calor e vibração mecânica levaram ao desenvolvimento de novos instrumentos, como a ponta de diamante pela tecnologia Deposição Química por Vapor. Esta apresenta uma série de vantagens como menor ruído, procedimento menos doloroso, desgaste preciso e preparo conservador, maior durabilidade da ponta, menor injúria à estrutura dentária, não corta os tecidos moles e acesso facilitado à lesão cariosa. Este trabalho apresenta um relato de caso clínico demonstrando o uso da ponta de diamante Deposição Química por Vapor para a confecção de um preparo cavitário proximal com acesso direto, com preservação da crista marginal, restauração com cimento de ionômero de vidro. O resultado clínico satisfatório foi conseguido, com acesso direto à cavidade em detrimento à configuração das pontas utilizadas, proporcionando conforto para o paciente e cirurgião dentista.