P.M.C. Scaffa

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.

Phosphoric acid concentration affects dentinal MMPs activity

OBJECTIVES 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. METHODS 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. RESULTS 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 (p<0.05), 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 (p<0.05). CONCLUSIONS 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. CLINICAL SIGNIFICANCE 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.

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.

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.

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.