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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 (p0.05). 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.

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 (similar to 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.

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- 5 μM E-64, E-64-10- 10 μM E-64 and positive control (PC)- MP associated with activator/ catalyst. Then, slices were subjected to push-out test (0.5 mm/min) after 24 h/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 24 h, 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.

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 7 days, and treated for 10 min 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 220 μm) using a Knoop penetrator (10 g/10 s). Statistical analyses were performed with analysis of variance (ANOVA) and Tukey tests (p 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.

SUMMARY Objectives: The aim of this study was to evaluate the effect of proteolytic inhibitors on the bond strength of a universal adhesive system (etch-and-rinse mode) applied to artificial carious and eroded dentin. Methods: Ninety molars were prepared and randomly divided into three groups according to the substrate: N, no challenges; ACD, artificial carious dentin simulation and ERO, artificial erosion simulation with orange juice. All groups were redivided into three subgroups according to the dentin pretreatment: W, water; CHX, 2% digluconate chlorhexidine; and E-64 (trans-epoxysuccinyl-L-leucylamido-[4-guanidino] butane), 5 μM E-64 inhibitor. They constituted a total of nine 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/Filtek Z250. Beams (0.64 mm2) were obtained and subjected to the microtensile test (μTBS) in a universal testing machine at 0.5 mm/min. The failure mode of the interfaces was determined by optical microscopy (40× magnification). Data were statistically analyzed by three-way analysis of variance and Tukey tests (p

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.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) 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. Univ Estadual Campinas, Fac Dent Piracicaba, BR-13414903 Piracicaba, Brazil Univ Sao Paulo, Bauru Sch Dent, Dept Biol Sci, BR-17012901 Bauru, SP, Brazil Univ Bologna, Dept Biomed & Neuromotor Sci, I-40126 Bologna, Italy Univ Trieste, Dept Med Sci, I-34128 Trieste, Italy Univ Iowa, Coll Dent, Dept Operat Dent, 801 Newton Rd, Iowa City, IA 52246 USA CNR, IGM, Unit Bologna, IOR, I-40136 Bologna, Italy Univ Fed Ceara, Dept Pharm Dent & Nursing, BR-60020181 Fortaleza, Ceara, Brazil Univ Carlo Bo, Dept SteVA, I-61029 Urbino, Italy Georgia Hlth Sci Univ, Dent Coll Georgia, Oral Biol, Augusta, GA 30912 USA Univ Oulu, Inst Dent, FI-90014 Oulu, Finland Oulu Univ Hosp, Med Res Ctr Oulu, FI-90014 Oulu, Finland Univ Oulu, FI-90014 Oulu, Finland Univ Helsinki, Dept Oral & Maxillofacial Dis, FI-00014 Helsinki, Finland Helsinki Univ Hosp, FI-00014 Helsinki, Finland Univ Fed Sao Paulo, Dept Biochem, BR-04021001 Sao Paulo, Brazil Univ Mogi das Cruzes, Ctr Interdisciplinar Invest Bioquim, BR-08773520 Mogi Das Cruzes, Brazil Anhanguera Univ Sao Paulo UNIAN SP, Biomat Dent Program, BR-05145200 Sao Paulo, Brazil Anhanguera Univ Sao Paulo UNIAN SP, Biotechnol & Innovat Hlth Program, BR-05145200 Sao Paulo, Brazil Univ Fed Sao Paulo, Dept Biochem, BR-04021001 Sao Paulo, Brazil FAPESP: 2007/54618-4 FAPESP: 2009/14005-9 CNPq: 312768/2013-3 Web of Science

Case report: Even if directly veneering discolored anterior teeth with composite is quite widespread, proper reproduction of their natural appearance requires much from the professional. Comprehensive management, from planning to finishing and polishing, of a severely discolored upper central incisor through its directly veneering is critically reported. Relevance: Direct composite laminate veneers are, indeed, a viable treatment option to correct esthetic abnormalities involving most of the labial surface of anterior teeth. This relative conservative therapy, since accurately performed by the well qualified dentist, will assign the patient esthetic, functional and emotional rehabilitation, culminating in a harmonious and pleasant smile.

Auto-degradation of collagen matrices occurs within hybrid layers created by contemporary dentin bonding systems, by the slow action of host-derived matrix metalloproteinases (MMPs). This study tested the null hypothesis that there are no differences in the activities of MMP-2 and -9 after treatment with different etch-and-rinse or self-etch adhesives. Tested adhesives were: Adper Scotchbond 1XT (3M ESPE), PQ1 (Ultradent), Peak LC (Ultradent), Optibond Solo Plus (Kerr), Prime&Bond NT (Dentsply) (all 2-step etch-and-rinse adhesives), and Adper Easy Bond (3M ESPE), Tri-S (Kuraray), and Xeno-V (Dentsply) (1-step self-etch adhesives). MMP-2 and -9 activities were quantified in adhesive-treated dentin powder by means of an activity assay and gelatin zymography. MMP-2 and MMP-9 activities were found after treatment with all of the simplified etch-and-rinse and self-etch adhesives; however, the activation was adhesive-dependent. It is concluded that all two-step etch-and-rinse and the one-step self-etch adhesives tested can activate endogenous MMP-2 and MMP-9 in human dentin. These results support the role of endogenous MMPs in the degradation of hybrid layers created by these adhesives.

The co-expression of MMPs and cysteine cathepsins in the human dentin-pulp complex indicates that both classes of enzymes can contribute to the endogenous proteolytic activity of dentin. Chlorhexidine (CHX) is an efficient inhibitor of MMP activity. This study investigated whether CHX could also inhibit cysteine cathepsins present in dentin. The inhibitory profile of CHX on the activity of dentin-extracted and recombinant cysteine cathepsins (B, K, and L) was monitored in fluorogenic substrates. The rate of substrate hydrolysis was spectrofluorimetrically measured, and inhibitory constants were calculated. Molecular docking was performed to predict the binding affinity between CHX and cysteine cathepsins. The results showed that CHX inhibited the proteolytic activity of dentin-extracted cysteine cathepsins in a dose-dependent manner. The proteolytic activity of human recombinant cathepsins was also inhibited by CHX. Molecular docking analysis suggested that CHX strongly interacts with the subsites S2 to S2′ of cysteine cathepsins B, K, and L in a very similar manner. Taken together, these results clearly showed that CHX is a potent inhibitor of the cysteine cathepsins-proteolytic enzymes present in the dentin-pulp complex.

Orientador: Marcela Rocha de Oliveira Carrilho, Mario Fernando de Góes Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba Resumo: Para o entendimento do processo de adesão à dentina é fundamental conhecer a estrutura bioquímica e biomecânica deste substrato em condições normais ou quando submetido às diferentes etapas do procedimento restaurador adesivo. Evidências indicam que a reportada degradação da camada híbrida pode ocorrer pela ação de enzimas proteolíticas, pertencentes à família das metaloproteinases da matriz (MMPs) e das cisteíno-catepsinas (CTs). No entanto, é ainda necessário elucidar as funções biológicas dessas enzimas nesse processo, bem como, definir uma estratégia para prolongar a durabilidade das restaurações adesivas. O presente estudo teve como objetivo caracterizar o perfil proteolítico da dentina humana frente a sua exposição a diferentes concentrações de digluconato de clorexidina (CHX), um potente inibidor da atividade de MMPs. O efeito da CHX sobre a atividade proteolítica intrínseca da dentina foi avaliado a partir da análise do comportamento mecânico e bioquímico da matriz dentinária e da durabilidade de restaurações adesivas. No primeiro estudo, foi analisada a capacidade da CHX em inibir a atividade das CTs (B, K e L) por hidrólise de substratos fluorogênicos específicos, verificando a afinidade de ligação entre a CHX e as enzimas. No segundo estudo, o tratamento da matriz de dentina com diferentes concentrações de CHX foi avaliado pela análise do módulo de elasticidade e do grau de hidrólise do colágeno (liberação de hidroxiprolina) após armazenagem das amostras em solução fisiológica por 1 dia, 7 ou 30 dias. Finalmente, a função terapêutica da CHX como agente inibitório da atividade proteolítica da dentina foi investigada a partir de sua capacidade em preservar a integridade mecânica (resistência de união) e morfológica de interfaces adesivas tratadas com diferentes concentrações de CHX (0,2; 2,2 e 22 mM) e armazenadas por 6 a 18 meses. No terceiro estudo, a presença das CT-B e CT-K na dentina humana foram avaliadas por imunomarcação em MEV e MET. A atividade enzimática das MMPs e CTs na dentina e uma possível interação entre as duas famílias de enzimas foram verificadas por zimografia in situ e por espectrofluorimetria. Os resultados mostraram, de forma até então inédita, que a CHX é um potente inibidor das CTs presentes no complexo dentino-pulpar. No entanto, a CHX não foi capaz de preservar integralmente o módulo de elasticidade (E) da matriz dentinária após o período mais longo de armazenagem. De modo similar, maior grau de hidrólise do colágeno ocorreu após 30 dias de armazenamento para as amostras que não foram tratadas com CHX ou que foram tratadas com baixa concentração da mesma (0,2 mM) (p0,05). A CHX não afetou a resistência de união imediata da interface adesiva e preservou a resistência da união dentina/resina mesmo após 6 ou 18 meses de armazenamento. Similarmente, menor grau de nanoinfiltração com prata, significando maior integridade morfológica, foi observado para os espécimes tratados com CHX e envelhecidos por 6 ou 18 meses em comparação com as amostras do grupo controle. As imagens de imuno-histoquímica mostraram que as proteases CT-B e CT-K estão presentes na dentina humana, e não apenas na região de pré-dentina e interior dos túbulos dentinários como anteriormente antecipado. A zimografia in situ sugere que a atividade gelatinolítica das MMPs na dentina parece ser preponderante em relação à atividade das CTs, embora a espectrofluorimetria sugira que a atividade proteolítica de ambas as famílias de enzimas esteja presente no tecido dentinário. Dessa forma, concluiu-se que MMPs e CTs podem atuar sinergicamente na degradação da matriz orgânica dentinária, mas que parte dessa atividade proteolítica pode ser controlada pela presença de CHX, sobretudo, se essa estiver confinada no interior da camada híbrida Abstract: To better understand the process of adhesion to dentin is essential to understand the biochemical and biomechanical structures of this substrate under normal conditions or when subjected to the different steps of the adhesive restorative procedure. Evidences indicate that the hybrid layer degradation can result from the activity of proteolytic enzymes, belonging to the family of matrix metalloproteinases (MMPs) and cysteine-cathepsins (CTs). However, it is still necessary to comprehend the role of these enzymes in this degrading process as well as to determine the best way to extend the durability of adhesive restorations. The general purpose of the present study was to characterize the human dentin proteolytic profile when exposed to different concentration of chlorhexidine digluconate (CHX), a potent inhibitor of MMPs activity. The effect of CHX on the dentin endogenous proteolytic activity was evaluated by the analysis of dentin matrix mechanical and biochemical properties and adhesive restorations durability. The first study evaluated the CHX ability to inhibit CTs (B, K and L) activity by the hydrolysis of fluorogenic substrates, verifying the binding affinity between CHX and enzymes. The dentin matrix treatment with different CHX concentrations was evaluated in the second study by the elastic modulus (E) and degree of collagen hydrolysis (hydroxyproline release) after storage for 1, 7 or 30 days in saline solution. Finally, the CHX therapeutic action as an inhibitor of dentin proteolytic activity was investigated by its ability to maintain the mechanical (bond strength) and morphological (nanoleakage) properties of adhesive interfaces treated with different CHX concentrations (0.2, 2.2 and 22 mM) after aging for 6 to 18 months. The third study evaluated the presence of CT-B and CT-K in human dentin using immunolabeling in SEM and TEM. MMPs and CTs proteolytic activities and a possible interaction between these two families were verified by in situ zymography and by spectrofluorimetry. Results showed, for the first time, that the CHX is a potent inhibitor of CTs in the pulp-dentin complex. However, CHX was not able to preserve the integrity of the dentin matrix E after the longest storage period. Likewise, higher collagen hydrolysis occurred after 30 days of storage when the samples were not treated or treated with low CHX concentration (0.2 mM) (p0.05). CHX did not affect the immediate bond strength of adhesive interfaces and preserved the resin/dentin bond strength even after 6 or 18 months of storage. Similarly, less nanoleakage with silver particles, which means better morphological integrity, was observed for specimens treated with CHX and aged for 6 or 18 months in comparison with control samples. Immunohistochemistry images showed that the proteases CT-B and CT-K are present in human dentin matrix, not only in pre-dentin region and inside the dentin tubules as previous suggested. In situ zymography suggests that the MMPs gelatinolytic activity in dentin seems to be predominant when compared to CTs activities, although the spectrofluorimetry suggests that the proteolytic activity of both families of enzymes are present in dentin. In this way, it was concluded that MMPs and CTs may synergistically act in the dentin organic matrix degradation, but part of this proteolytic activity can be controlled by the presence of CHX, especially when it is restrained inside the hybrid layer Doutorado Materiais Dentários Doutora em Materiais Dentários

As ceramicas a base de zirconia (Y-TZP) apresentam propriedades mecanicas superiores as das demais ceramicas odontologicas. Entretanto, o aumento do conteudo cristalino modificou suas caracteristicas de adesao a cimentos resinosos, tornando necessario o desenvolvimento de metodos mais efetivos de uniao a estes materiais. Este trabalho in vitro pretende, portanto, avaliar a influencia de diferentes tratamentos de superficie e de diferentes agentes de uniao na resistencia ao cisalhamento entre uma zirconia estabilizada por itrio e um agente cimentante autoadesivo (RelyX U100®/3M ESPE). Para tal, cilindros de PROCERA® Allzircon receberam como tratamento de superficie jateamento com oxido de aluminio (Al2O3 - 50 m) ou jateamento com o sistema Rocatec. Sobre a superficie tratada dos cilindros foram aplicados, de acordo com os diferentes grupos experimentais, o sistema adesivo Clearfil SE Bond® (Kuraray Co.), e os primers Metal Zirconia Primer (Ivoclar Vivadent) e Alloy Primer (Kuraray Co.) e, finalmente, o cimento resinoso adotado. Cada especime foi, entao, submetido ao teste de cisalhamento (0,05 mm/min), por meio da acao de uma alca de fio ortodontico passando o mais próximo possivel da interface adesiva. A analise de variancia a um criterios (ANOVA) e o teste de Tukey (p