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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.

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.

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.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).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 (p0.05). At 2-year evaluation, only OA exhibited significantly higher bond strength when cured for 20 and 40s (p0.05). 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).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.

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

Degradation of dentin matrix components within caries dentin has been correlated with the activity of host-derived proteases, such as matrix metalloproteases (MMPs) and cysteine cathepsins (CTs). Since this relationship has not been fully established, we hypothesized that the abundance of MMPs and CTs in caries-affected dentin must be higher than in intact dentin. To test this premise, we obtained 5 slices (200 µm) from 5 intact teeth and from 5 caries-affected teeth (1 slice/tooth) and individually incubated them with primary antibodies for CT-B, CT-K, MMP-2, or MMP-9. Negative controls were incubated with pre-immune serum. Specimens were washed and re-incubated with the respective fluorescent secondary antibody. Collagen identification, attained by the autofluorescence capture technique, and protease localization were evaluated by multi-photon confocal microscopy. The images were analyzed with ZEN software, which also quantitatively measured the percentages of collagen and protease distribution in dentin compartments. The abundance of the test enzymes was markedly higher in caries-affected than in intact dentin. CT-B exhibited the highest percentage of co-localization with collagen, followed by MMP-9, MMP-2, and CT-K. The high expression of CTs and MMPs in caries-affected teeth indicates that those host-derived enzymes are intensely involved with caries progression.

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.

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.

To evaluate whether the concentration of phosphoric acid (PA) has an effect on the proteolytic activity of sound human demineralized dentin. It is hypothesized that the activity of matrix-bound and extracted enzymes depends on the PA concentration used to demineralize dentin.One-gram aliquots of mid-coronal human dentin powder were demineralized with 1wt%, 10wt% and 37wt% PA. Concentrations of released calcium were measured for each set of demineralization. Extracted MMP-2 was immunologically identified by western blot and its activity was determined by conventional gelatin zymography. Analysis of released hydroxyproline (HYP) and in situ zymography were performed to evaluate the activity of insoluble, bound-matrix enzymes.The amount of released calcium from dentin powder treated with 37wt% PA was significantly higher (p≤0.05) than that obtained by dentin demineralization with 10wt% and 1wt% PA. Expression and activity of endogenous enzymes, extracted from or bound to dentin matrix, were detected for all samples regardless of the PA concentration. However, the expression and activity of extracted MMP-2 were significantly higher when dentin was treated with 10wt% PA (p0.05), followed by 1wt% and 37wt% PA. Similarly, the highest concentration of released HYP (i.e. meaning higher percentage of collagen degradation) and the highest activity in in situ zymography were observed when dentin samples were treated with 10wt% PA (p0.05).It was confirmed that PA does not denature endogenous enzymes of dentin matrices, but it may somehow modulate the expression and activity of these enzymes in a concentration-dependent manner.Endogenous proteases have been identified and suggested to be responsible for the digestion of dentin matrix when activated by the acidic components of dental adhesives. Proteolytic activity of dentinal MMPs showed to be dependent on phosphoric acid concentration. The clinically-used concentration (37%) does not inhibit MMPs activity, but slows it.

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 0.5 mM (0.004%) DMSO as additional primer for 30 s 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 0.5 mM 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 0.5 mM DMSO used for bonding.

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.