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In dentistry, the wet-bonding approach relies on water to maintain demineralized collagen expanded for proper resin infiltration; nevertheless, hydrolytic instability of the resin-dentin interface is inevitable with current bonding techniques. Considering dimethyl sulfoxide's (DMSO) ability to "biomodify" collagen and precipitate enzymes, the aim was to test whether the use of DMSO would permit adequate resin bonding to H3PO4-etched dehydrated dentin and assess its impact on collagen degradation by host-derived enzymes. Etched dentin surfaces from extracted sound human molars were randomly bonded in wet or dry conditions using aqueous or ethanolic DMSO solutions as pretreatments and bonding resins with or without DMSO. Bonded teeth were sectioned into resin-dentin slabs for confocal in situ zymography and beams for microtensile bond strength test. Demineralized powdered dentin was incubated in the tested DMSO -media and a hydroxyproline assay evaluated dissolution of collagen peptides. Zymography was performed on protein extracts obtained from dry and wet H3PO4-ecthed dentin powder treated with the DMSO- media. The correlative biochemical analysis demonstrated that reduction of water content during dentin hybridization by the innovative dry-bonding approaches with DMSO is effective to inactivate host-derived MMP-2 and MMP-9 and thus reduce collagen degradation while simultaneously optimizing resin-dentin bonding.

The aim of this study was to evaluate the degradation of completely demineralized dentin specimens in contact with a filler-free or 2 ion-releasing resins containing micrometer-sized particles of Bioglass 45S5 (BAG) or fluoride-containing phosphate-rich bioactive glass (BAG-F). Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) were also used to evaluate the remineralization induced by the experimental ion-releasing resin-based materials. Dentin beams were totally demineralized in H 3 PO 4 (10%) and placed in direct contact with a filler-free (RESIN) or 2 experimental ion-releasing resins (BAG or BAG-F) and immersed in artificial saliva (AS) up to 30 d. Further specimens were also processed and submitted to FTIR and SEM analysis to evaluate the remineralization induced by such ion-releasing resins before and after AS immersion. BAG and BAG-F alkalinized the incubation media. A significant decrease of the dry mass was observed between the specimens of all groups stored for 3 and 30 d in AS. However, the fluoride-containing phosphate-rich bioactive glass incorporated into a resin-based material (BAG-F) showed greater ability in reducing the solubilization of C-terminal cross-linked telopeptide (ICTP) and C-terminal telopeptide (CTX) after prolonged AS storage. Moreover, after 30 d of AS storage, BAG-F showed the greatest remineralizing effect on the stiffness of the completely demineralized dentin matrices. In conclusion, fluoride-containing phosphate-rich bioactive glass incorporated as micrometer-sized filler in dental composites may offer greater beneficial effects than Bioglass 45S5 in reducing the enzyme-mediated degradation and remineralization of demineralized dentin

This study determined if dentin proteases are denatured by phosphoric acid (PA) used in etch-and-rinse dentin adhesives. Dentin beams were completely demineralized with EDTA for 30 days. We “acid-etched” experimental groups by exposing the demineralized dentin beams to 1, 10, or 37 mass% PA for 15 sec or 15 min. Control beams were not exposed to PA but were incubated in simulated body fluid for 3 days to assay their total endogenous telopeptidase activity, by their ability to solubilize C-terminal crosslinked telopeptides ICTP and CTX from insoluble dentin collagen. Control beams released 6.1 ± 0.8 ng ICTP and 0.6 ± 0.1 ng CTX/mg dry-wt/3 days. Positive control beams pre-incubated in p-aminophenylmercuric acetate, a compound known to activate proMMPs, released about the same amount of ICTP peptides, but released significantly less CTX. Beams immersed in 1, 10, or 37 mass% PA for 15 sec or 15 min released amounts of ICTP and CTX similar to that released by the controls (p > 0.05). Beams incubated in galardin, an MMP inhibitor, or E-64, a cathepsin inhibitor, blocked most of the release of ICTP and CTX, respectively. It is concluded that PA does not denature endogenous MMP and cathepsin activities of dentin matrices.

Matrix metalloproteinases (MMPs) cause collagen degradation in hybrid layers created by dentin adhesives. This in vitro study evaluated the feasibility of using a cross-linking agent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), to inactivate soluble rhMMP-9, as an example of dentin MMPs, and matrix-bound dentin proteases. The inhibitory effects of 5 EDC concentrations (0.01-0.3 M) and 5 incubation times (1-30 min) on soluble rhMMP-9 were screened with an MMP assay kit. The same EDC concentrations were used to evaluate their inhibitory effects on endogenous proteinases from completely demineralized dentin beams that were incubated in simulated body fluid for 30 days. Decreases in modulus of elasticity (E) and dry mass of the beams, and increases in hydroxyproline content of hydrolysates derived from the incubation medium were used as indirect measures of matrix collagen hydrolysis. All EDC concentrations and pre-treatment times inactivated MMP-9 by 98% to 100% (p < 0.05) compared with non-cross-linked controls. Dentin beams incubated in 0.3 M EDC showed only a 9% decrease in E (45% decrease in control), a 3.6% to 5% loss of dry mass (18% loss in control), and significantly less solubilized hydroxyproline when compared with the control without EDC cross-linking (p < 0.05). It is concluded that EDC application for 1 min may be a clinically relevant and effective means for inactivating soluble rhMMP-9 and matrix-bound dentin proteinases if further studies demonstrate that EDC is not toxic to pulpal tissues.

Objectives. This study evaluated the endogenous matrix metalloproteinase (MMP) activity of demineralized dentin matrix following 1 or 5 min pretreatment by various collagen crosslinkers. Generic MMP activity assay, total protein analysis, in situ zymography, gelatin zymography and multiplex bead technology were used to evaluate matrix-bound MMP activity. Methods. Six different crosslinkers; glutaraldehyde, riboflavin/UVA, riboflavin-5-monophospate/UVA, sumac berry extract, grape seed extract, and curcumin were used. Demineralized dentin beams were pretreated with respective crosslinkers for 1 or 5 min. Demineralized dentin beams with no crosslinker pretreatment served as control. The reduction in the total activity of dentin matrices were measured using generic MMP activity assay. Dentin slabs were used for in situ zymography and evaluated by using hydrolysis of self-quenched fluorescein-conjugated gelatin under confocal microscopy. Dentin beam extracts were used for total protein assay and multiplex analysis and powder extracts were used for gelatin zymography. Results. MMP activity in crosslinker pretreated samples decreased significantly between 21% and 70%, whereas untreated control samples' activity increased up to 84%. Zymograms confirmed a decrease in the gelatinolytic activity and in the amount of extractable total protein content. Multiplex analysis of extracts of crosslinker-treated dentin showed a reduction in the MMP-8, MMP-2 and MMP-9 release. Significance. The result of this work suggests that the effect of the crosslinkers is source dependent. The use of crosslinkers for as little as 1 min on demineralized dentin can inactivate the endogenous protease activity of dentin matrices. (C) 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.