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.

Abundance of MMPs and cysteine cathepsins in caries-affected dentin.

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.

Chlorhexidine inhibits the activity of dental cysteine cathepsins.

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.

The influence of time and cement type on push-out bond strength of fiber posts to root dentin.

The bond strength of fiber posts luted with resin cements was evaluated after two storage times in different regions of a post space. A total of 40 single-rooted human teeth were endodontically treated and prepared for cementation of fiber posts (White Post DC). In groups 1 and 3 (G1 and G3, respectively), posts were luted with RelyX ARC, whereas the posts in groups 2 and 4 (G2 and G4, respectively) were luted with RelyX Unicem. After one month of storage at 100% humidity, G1 and G2 were transversally sectioned in 1.7-mm slices of the cervical (C), middle (M), and apical (A) thirds of the post space and submitted to push-out testing at 1 mm/min. After nine months of storage, the roots of G3 and G4 underwent the same process. Mean values were analyzed using the Mann-Whitney and Kruskal-Wallis tests (α=0.05). The bond strengths in G2 (C=4.26±2.29; M=4.67±3.54; A=7.27±4.30) were statistically higher than in G1 (C=3.81±1.07; M=1.57±1.62; A=1.99±1.60) in the middle and apical thirds (p=0.001). Bond strengths in G4 (C=3.36±1.39; M=4.49±2.17; A=3.83±1.92) were higher than in G3 (C=2.13±0.47; M=0.94±1.05; A=0.95±1.02) in all evaluated regions (p=0.02, p<0.001, and p<0.001, respectively). When comparing the root regions for each group, G1 had higher values in the cervical third than the middle third (p=0.02). The self-adhesive resin cement showed better results than the conventional resin cement at both storage times. For both materials a similar performance among the three root regions was found. Storage time did not influence the shear bond strength.