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Objective: Non-human teeth have been commonly used in research as replacements for human teeth, and potential dissimilarities between the dental tissues should be considered when interpreting the outcomes. To compare the proteolytic activity and degradation rate of bovine and human dentin matrices., Methodology: Dentin beam specimens were obtained from human molars (n=30) and bovine incisors (n=30). The beams were weighed hydrated and after complete dehydration to obtain the mineralized wet and dry masses. Then, the beams were demineralized in 10 wt% phosphoric acid. Next, 15 beams from each substrate were randomly selected and again dehydrated and weighed to obtain the initial demineralized dry mass (DM). Then, the beams were stored in saliva-like buffer solution (SLBS) for 7, 14 and 21 days. SLBS was used to evaluate hydroxyproline (HYP) release after each storage period. The remaining beams of each substrate (n=15) were tested for initial MMP activity using a colorimetric assay and then also stored in SLBS. DM and MMP activity were reassessed after 7, 14 and 21 days of incubation. The data were subjected to two-way ANOVA tests with repeated measures complemented by Bonferroni's tests. Unpaired two-tailed t-tests were also used (p<0.05)., Results: Similar water and inorganic fractions were found in human and bovine dentin, while human dentin had a higher protein content. The most intense proteolytic activity and matrix deterioration occurred short after dentin was demineralized. Both substrates exhibited a sharp reduction in MMP activity after seven days of incubation. Although human dentin had higher MMP activity levels, greater HYP release and DM loss after seven days than bovine dentin, after 14 and 21 days, the outcomes were not statistically different., Conclusion: Bovine dentin is a suitable substrate for long-term studies involving the degradation of dentin matrices.

Background: Primary and permanent teeth composition may influence dissolution and degradation rates., Aim: To compare the dissolution and degradation of primary and permanent teeth., Design: Enamel and dentin powders were obtained from primary molars and premolars and incubated within different pH buffers. Calcium and inorganic phosphate release was quantified in the buffers by atomic absorption and light spectrophotometry. A colorimetric assay was used to assess the MMP activity of primary dentin (PrD) and permanent dentin (PeD). Collagen degradation was assessed by dry mass loss, change in elastic modulus (E), and ICTP and CTX release. Data were submitted to ANOVA and Tukey's tests (α = 0.05)., Results: Similar dissolution was found between PrD and PeD after 256 hours. At pH 4.5, enamel released more minerals than dentin whereas at pH 5.5 the inverse result was observed. MMP activity was similar for both substrates. PrD showed higher dry mass loss after 1 week. In general, greater reduction in E was recorded for PrD. Higher quantities of ICTP and CTX were released from PrD after 1 week., Conclusions: Primary and permanent teeth presented similar demineralization rates. Collagen degradation, however, was faster and more substantial for PrD., (© 2020 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: Cysteine proteases are lysosomal enzymes that, under specific circumstances, may be secreted into the extracellular space and participate in protein turnover. This study investigated the involvement of cathepsin B in the gelatinolytic activity of mature dentin matrices at neutral pH., Design: Human dentin fragments were made into powder and enzymes were extracted using guanidine-HCl/EDTA. Host-derived dentin proteases (cathepsin B, MMP-2 and MMP-9) were identified by immunoblotting, and their activities were evaluated spectrofluorimetrically using fluorogenic substrates. Proteases activities were monitored by measuring the rate of hydrolysis of substrates in the presence/absence of MMP- or cysteine cathepsin inhibitors, at neutral pH (7.4). Mass spectroscopy was used to determine the substrates' cleavage points. Reverse zymography was performed to examine the gelatinolytic activity of cathepsin B., Results: Western-blots of dentin extracts yielded strong bands at 95, 72 and 30 kDa, corresponding respectively to MMP-9, MMP-2 and Cathepsin B. Greater fluorogenic substrates hydrolysis occurred in the absence of MMP and cysteine cathepsin inhibitors than in their presence. Cathepsin B exhibited significant gelatinolytic activity., Conclusions: Together with MMP-2 and MMP-9, cathepsin B also account for the host-derived gelatinolytic activity and matrix turnover of mature dentin at physiological, neutral pH., Competing Interests: Declaration of Competing Interest We, undersigned authors of the manuscript titled “Insights into Cathepsin-B Activity in Mature Dentin Matrix”, declare no conflict of interest of any nature and that the present manuscript has been submitted solely to the Achieves Oral Biology and is not under consideration for publication in any other journal. This paper has been compiled with the knowledge, input and approval of all the named authors., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

During development of mineralized collagenous tissues, intrafibrillar mineralization is achieved by preventing mineralization precursor inhibitors that are larger than 40 kDa from entering the collagen fibrils. Such a property is incorporated in the design of a calcium chelator for dentin bonding in the etch-and-rinse technique that selectively demineralizes extrafibrillar apatite while leaving the intrafibrillar minerals intact. This strategy prevents complete demineralization of collagen fibrils, avoids collapse of collagen that blocks resin infiltration after air-drying, and protects the completely demineralized fibrils from bacteria colonization and degradation by endogenous proteases after resin bonding. In the present study, a water-soluble glycol chitosan-EDTA (GCE) conditioner was synthesized by conjugation of EDTA, an effective calcium chelator, to high molecular weight glycol chitosan, which exhibits weak chelation property. The GCE conjugate was purified, characterized by FTIR, 1 H NMR, isothermal titration calorimetry and ICP-AES, and subjected to size exclusion dialysis to recover molecules that are >40 kDa. The optimal concentration and application time for etching dentin were determined by bond strength testing to ensure that the dentin bonding results were comparable to phosphoric acid etching, and maintained equivalent bond strength after air-drying of the conditioned collagen matrix. Extrafibrillar demineralization was validated with transmission electron microscopy. Inhibition of endogenous dentin proteases was confirmed using in-situ zymography. The water-soluble GCE dentin conditioner was non-cytotoxic and possessed antibacterial activities against planktonic and single-species biofilms, supporting its ongoing development as a dentin conditioner with air-drying, anti-proteolytic and antibacterial properties to enhance the durability of bonds created using the etch-and-rinse bonding technique. STATEMENT OF SIGNIFICANCE: The current state-of-the-art techniques for filling decayed teeth with plastic tooth-colored materials require conditioning the mineralized, biofilm-covered, decayed dentin with acids or acid resin monomers to create a surface layer of completely- or partially-demineralized collagen matrix for the infiltration of adhesive resin monomers. Nevertheless, fillings prepared using these strategies are not as durable as consumers have anticipated. Conjugation of polymeric glycol chitosan with EDTA produces a new conditioner for dentin bonding that demineralizes only extrafibrillar dentin, reduces endogenous protease activities and kills biofilm bacteria. The high molecular weight glycol chitosan-EDTA is non-cytotoxic to the key regenerative players within the dentin-pulp complex. This advance permits dry bonding and the use of hydrophobic resins., (Published by Elsevier Ltd.)

Objectives: The present in vitro study evaluated the effect of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), a cross-linking agent used as an additional therapeutic primer for luting fiber posts to radicular dentine to prevent hybrid layer degradation., Methods: Root canal treatment was performed on 80 extracted single-rooted human teeth. A 10-mm post space was prepared and pecimens were randomly assigned to four groups (n=20) according to the bonding system: 1) All Bond 3 (Bisco); 2) All Bond 3 + 0.3M EDC; 3) Prime&Bond XP (Dentsply Sirona); 4) Prime&Bond XP + 0.3M EDC. In groups 2 and 4, EDC was applied on phosphoric acid-etched dentine for 1 min. Fiber posts (RelyX Fiber Post, 3M ESPE) were luted with a dual-cured resin cement (Core-X flow, Dentsply Sirona). Slices were prepared for micro push-out test and interfacial nanoleakage evaluation of the coronal and apical region of the canal space after 24 h and 1 year storage in artificial saliva. In-situ zymography was performed to investigate endogenous matrix metalloproteinase activities within the hybrid layer. Results were statistically analysed with three-way ANOVA test or Chi Square test. Statistical significance was set at α=0.05., Results: No significant influence was identified between the two adhesives. The use of EDC significantly improved fiber post bond strength at 1 year but not at 24 h. Application of 0.3 M EDC prior to bonding significantly reduced gelatinolytic activities within the radicular hybrid layers., Conclusions: Carbodiimide was effective in preserving fibre post bond strength over time, through reducing the activities of intra-radicular endogenous proteases., Clinical Significance: Inhibition of matrix metalloproteinases using EDC over radicular dentin could play an important role in bond strength preservation. However, the clinical relevance of these findings needs to be proven., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Instability of resin-dentin bonds is the Achilles' heel of adhesive dentistry. To address this problem, a chelate-and-rinse extrafibrillar dentin demineralization strategy has been developed that keeps intrafibrillar minerals within collagen fibrils intact to prevent activation of endogenous proteases that are responsible for collagen degradation within hybrid layers. The objective of the present study was to evaluate the potential of using chitosan >40 kDa as an antimicrobial extrafibrillar dentin-chelating agent to enhance bond durability. Transmission electron microscopy provided evidence for retention of intrafibrillar minerals and smear plugs in dentin conditioned with 1 wt% chitosan. Analyzed by Kruskal-Wallis analysis of variance, Dunn's statistic, and separate Mann-Whitney tests, tensile bond strengths to wet- and dry-bonded dentin indicated that chelating dentin with chitosan for 60 s prior to bonding did not result in a significant decline in resin-dentin bond strength when compared with that of phosphoric acid etching ( P > 0.05). Gelatinolytic activity within the hybrid layers was examined via in situ zymography after 24-h storage or after thermomechanical cycling and analyzed with 3-factor analysis of variance. After 24 h, enzymatic activity was detected only within completely demineralized phosphoric acid-etched dentin, with values derived from dry bonding significantly higher than those derived from wet bonding ( P < 0.05). Negligible fluorescence was detected within hybrid layers when dentin was conditioned with chitosan, even after thermomechanical cycling, as compared with the controls. Reduction in water permeability in chitosan-conditioned dentin, attributed to smear plug retention, also fostered long-term bond stability. Antibacterial testing performed with live/dead staining indicated that the acetic acid-solubilized chitosan possessed antibacterial activities against 3 single-species biofilms: Streptococcus mutans, Actinomyces naeslundii, and Enterococcus faecalis. Taken together, the new chitosan-based extrafibrillar demineralization strategy retains intrafibrillar minerals, reduces endogenous protease-initiated collagen degradation, prevents water permeation within hybrid layers, and kills bacteria on dentin surfaces, which are crucial factors for enhancing resin-dentin bond durability.

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 (∼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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Secondary caries and hybrid layer degradation are two major challenges encountered in long-term resin-dentin bond stability. As a link between resin and dentin, adhesives that possess both antimicrobial and anti-proteolytic activities are in demand for eliminating bacteria-induced secondary caries and preventing hybrid layers from degradation. In the present study, a new quaternary ammonium methacryloxy silane (QAMS) prepared from sol-gel chemistry was incorporated into experimental adhesives to examine their antimicrobial effect and anti-proteolytic potential. This functional methacrylate resin monomer contains polymerizable methacryloxy functionalities as well as a positively-charged quaternary ammonium functionality with a long, lipophilic -C 18 H 37 alkyl chain for puncturing the cell wall/membrane of surface-colonizing organisms. Antibacterial testing performed using agar diffusion test, live/dead bacterial staining and colony-forming unit counts all indicated that the QAMS-containing adhesives killed Streptococcus mutans and Actinomyces naeslundii in a dose-dependent manner via a predominant contact-killing mechanism. Gelatinolytic activity within the hybrid layers created by these adhesives was examined using in-situ zymography. Hybrid layers created with 0% QAMS-containing adhesive exhibited intense green fluorescence emitted by the hydrolyzed fluorescein-conjugated gelatin, with 4-fold increase in enzymatic activity compared with an experimental adhesive containing 5% QAMS. Taken together, incorporation of 5% QAMS in the experimental adhesive provides simultaneous antimicrobial and anti-proteolytic activities that are crucial for the maintenance of long-term resin-dentin bond integrity., Statement of Significance: Durability of resin-dentin interfacial bond remains a clinically-significant challenge. Secondary caries caused by bacteria and the degradation of hybrid layers via endogenous dentin proteases are two important contributors to the poor resin-dentin bond durability. The present study developed a new 5% QAMS-containing adhesive that provides simultaneous antimicrobial and dentin protease inhibition functions to extend the longevity of resin-dentin bonds., (Published by Elsevier Ltd.)

Objectives: The water-associated attributes of resin-dentin interfaces created by contemporary adhesives are important determinants of bond integrity and stability. In the present work, these attributes were estimated from the perspectives of causality, to examine the behavior of the first and most-recently launched versions of universal adhesives when applied in either the etch-and-rinse mode or the self-etch mode., Methods: The immediate cause of interfacial permeability and the time-dependent cause of water sorption were investigated in conjunction with the intermediate effect of interface degradation and the more long-term effect of loss of mechanical strength, before and after thermomechanical cycling. The results were compared with control etch-and-rinse and self-etch adhesives., Results: Although the introduction of this new class of universal adhesives has brought forth significant changes to the dental adhesion arena, including more application options, reduced bonding armamentarium and increased user friendliness, the water-associated attributes that are critical for making resin-dentin bonds more durable to environmental challenges and less susceptible to degradation have remained unchanged at large, when compared with benchmarks established by former classes of adhesives., Conclusion: It appears that the current trend of adhesive development has brought forth significant changes but lacks the vigor that demarcates progress and technological sublimity., Clinical Significance: The advent of the user friendly universal adhesives has brought forth significant changes to the dental adhesion arena. However, the elements that are critical for making resin-dentin bonds more durable to environmental challenges and less susceptible to degradation have remained unchanged at large., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The aim of this study was to evaluate the effect of curcuminoids on the dentin endogenous protease activity. Demineralized dentin were pretreated with 50 or 100 µM of three different curcuminoids for 60 s and incubated up to 3 months. Untreated beams served as controls. Dry dentin mass was measured after incubation. Aliquots were analyzed for the quantity of ICTP and CTX releases for MMP and cathepsin-K mediated degradation, respectively. The effect of curcuminoids on matrix-bound MMP and soluble rhMMP-9 were measured using an activity assay. Data were subjected to repeated-measures-ANOVA (α=0.05). Gelatinolytic activity was analyzed using zymography. ICTP and CTX release and dry mass loss of curcuminoid-treated groups were significantly lower than the control. Inhibition of rhMMP-9 varied from 29-49% among curcumonoid-treated groups, whereas no inhibition was observed at untreated control (p>0.05). Results were confirmed by zymography. The study showed that the pretreatment of dentin matrices by curcuminoids decreases endogenous protease activity-mediated degradation in dentin.

A chelate-and-rinse extrafibrillar calcium chelation dentin bonding concept has recently been developed and investigated for its effectiveness in improving resin-dentin bonding by bridging the gap between wet and dry dentin bonding. The objective of the present study was to evaluate the gelatinolytic activity of hybrid layers (HLs) created using the chelate-and-rinse bonding technique. Gelatinolytic activity within the HL was examined using in situ zymography and confocal laser-scanning microscopy after 24-h storage or after thermomechanical cycling. Dentin specimens were bonded with Prime&Bond NT (Dentsply Sirona) after conditioning with 15 wt% phosphoric acid for 15 s (control) or 15 wt% polymeric chelators (sodium salt of polyacrylic acid; PAAN) of 2 different molecular weights for 60 s. For each reagent, bonding was performed using dry-bonding and wet-bonding techniques ( n = 10). Slices containing the adhesive-dentin interface were covered with fluorescein-conjugated gelatin and examined with a confocal laser-scanning microscope. Fluorescence intensity emitted by the hydrolyzed fluorescein-conjugated gelatin was quantified. Gelatinolytic activity was expressed as the percentage of green fluorescence emitted within the HL. After storage for 24 h, enzymatic activity was only detected within the completely demineralized phosphoric acid-etched dentin, with values derived from dry bonding higher than those from wet bonding ( P < 0.05). Almost no fluorescence signals were detected within the HL when dentin was conditioned with PAANs compared with the controls ( P < 0.05). After thermomechanical cycling, enzymatic activities significantly increased for the phosphoric acid-conditioned, drying-bonding group compared with 24-h storage ( P < 0.05). The present study showed that the use of the chelate-and-rinse bonding concept for both dry-bonding and wet-bonding approaches results in the near absence of matrix-bound collagenolytic activities in the HL even after aging. This may be attributed to fossilization of endogenous proteases via preservation of intrafibrillar minerals within the dentin collagen matrix.

Objective: The present study evaluated the influence of time, mass and surface area of demineralized dentin collagen matrices on telopeptides release. The hypotheses tested were that the rates of ICTP and CTX release by matrix bound endogenous proteases are 1) not time-dependent, 2) unrelated to specimen mass, 3) unrelated to specimen surface area., Methods: Non-carious human molars (N=24) were collected and randomly assigned to three groups. Dentin slabs with three different thicknesses: 0.37mm, 0.75mm, and 1.50mm were completely demineralized and stored in artificial saliva for one week. Collagen degradation was evaluated by sampling storage media for ICTP and CTX telopeptidases. Activity of MMPs in the aging medium was evaluated using fluorometric activity assay kit., Results: A statistically significant (p<0.05) decrease in the release of both ICTP and CTX fragments over time was observed irrespective of the specimen thickness. When data were normalized by the specimen mass, no significant differences were observed. Releases of ICTP and CTX were significantly related to the aging time as a function of surface area for the first 12h. Total MMP activity, mainly related to MMP-2 and -9, decreased with time (p<0.05)., Significance: Because the release of collagen fragments was influenced by specimen storage time and surface area, it is likely that cleaved collagen fragments closer to the specimen surface diffuse into the incubation medium; those further away from the exposed surface are still entrapped within the demineralized dentin matrix. Bound MMPs can only degrade the substrate within the limited zone of their molecular mobility., (Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Objectives: The objective of the present study was to elucidate the mechanism of bioactive molecule extraction from mineralized dentin by calcium hydroxide (Ca(OH) 2 ) and tricalcium silicate cements (TSC)., Methods and Results: Transmission electron microscopy was used to provide evidence for collagen degradation in dentin surfaces covered with Ca(OH) 2 or a set, hydrated TSC for 1-3 months. A one micron thick collagen degradation zone was observed on the dentin surface. Fourier transform-infrared spectroscopy was used to identify increases in apatite/collagen ratio in dentin exposed to Ca(OH) 2 . Using three-point bending, dentin exposed to Ca(OH) 2 exhibited significant reduction in flexural strength. Using size exclusion chromatography, it was found that the small size of the hydroxyl ions derived from Ca(OH) 2 enabled those ions to infiltrate the intrafibrillar compartment of mineralized collagen and degrade the collagen fibrils without affecting the apatite minerals. Using ELISA, TGF-β1 was found to be extracted from dentin covered with Ca(OH) 2 for 3 months. Unlike acids that dissolve the mineral component of dentin to release bioactive molecules, alkaline materials such as Ca(OH) 2 or TSC released growth factors such as TGF-β1 via collagen degradation., Significance: The bioactive molecule extraction capacities of Ca(OH) 2 and TSC render these dental materials excellent for pulp capping and endodontic regeneration. These highly desirable properties, however, appear to be intertwined with the untoward effect of degradation of the collagen matrix within mineralized dentin, resulting in reduced flexural strength., (Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Objective: The objectives of the study were to evaluate the ability of a 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC)-containing primer to improve immediate bond strength of either self-etch or etch-and-rinse adhesive systems and to stabilize the adhesive interfaces over time. A further objective was to investigate the effect of EDC on the dentinal MMPs activity using zymographic analysis., Methods: Freshly extracted molars (n=80, 20 for each group) were selected to conduct microtensile bond strength tests. The following groups were tested, immediately or after 1-year aging in artificial saliva: G1: Clearfil SE (CSE) primer applied on unetched dentin, pretreated with 0.3M EDC water-solution for 1min and bonded with CSE Bond; G2: as G1 but without EDC pre-treatment; G3: acid-etched (35% phosphoric-acid for 15s) dentin pretreated with 0.3M EDC, then bonded with XP Bond (XPB); Group 4 (G4): as G3 without EDC pre-treatment. Further, gelatinase activity in dentin powder treated with CSE and XPB with and without EDC pre-treatment, was analyzed using gelatin zymography., Results: The use of 0.3M EDC-containing conditioner did not affect the immediate bond strength of XPB or CSE adhesive systems (p>0.05), while it improved the bond strength after 1year of aging (p<0.05). Pre-treatment with EDC followed by the application of CSE resulted in an incomplete MMPs inactivation, while EDC pretreatment followed by the application of XPB resulted in an almost complete inactivation of dentinal gelatinases., Significance: The μTBS and zymography results support the efficacy of EDC over time and reveal that changes within the dentin matrix promoted by EDC are not adhesive-system-dependent., (Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Objectives: Efforts towards achieving durable resin-dentin bonds have been made for decades, including the understanding of the mechanisms underlying hybrid layer (HL) degradation, manufacturing of improved adhesive systems, as well as developing strategies for the preservation of the HL., Methods: This study critically discusses the available peer-reviewed research concerning the formation and preservation of the HL, the mechanisms that lead to the degradation of the HL as well as the strategies to prevent it., Results: The degradation of the HL occurs through two main mechanisms: the enzymatic degradation of its collagen fibrils, and the leaching of the resin from the HL. They are enabled by residual unbound water between the denuded collagen fibrils, trapped at the bottom of the HL. Consequently, endogenous dentinal enzymes, such as the matrix metalloproteinases (MMPs) and cysteine cathepsins are activated and can degrade the denuded collagen matrix. Strategies for the preservation of the HL over time have been developed, and they entail the removal of the unbound water from the gaps between the collagen fibrils as well as different modes of silencing endogenous enzymatic activity., Significance: Although there are many more hurdles to be crossed in the field of adhesive dentistry, impressive progress has been achieved so far, and the vast amount of available research on the topic is an indicator of the importance of this matter and of the great efforts of researchers and dental material companies to reach a new level in the quality and longevity of resin-dentin bonds., (Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

The medical literature is replete with the maxim 'primum non nocere', cautioning health care providers to avoid doing any harm to human subjects in their delivery of medical care. Sodium hypochlorite (NaOCl) is a well-established irrigant for root canal treatment because of its antimicrobial and organic tissue remnant dissolution capability. However, little is known about the deleterious effect of this strong oxidizing agent on the integrity of human mineralized dentin. Iatrogenically-induced loss of dentin integrity may precipitate post-treatment root fracture and has potential medico-legal complications. In the present work, transmission electron microscopy provided evidence for collagen destruction in the surface/subsurface of dentin treated with high NaOCl concentrations and long contact times. Size exclusion chromatography showed that the hypochlorite anion, because of its small size, penetrated the water compartments of apatite-encapsulated collagen fibrils, degraded the collagen molecules and produced a 25-35µm thick, non-uniform "ghost mineral layer" with enlarged, coalesced dentinal tubules and their lateral branches. Fourier transform-infrared spectroscopy identified increases in apatite/collagen ratio in NaOCl-treated dentin. The apatite-rich, collagen-sparse dentin matrix that remained after NaOCl treatment is more brittle, as shown by the reductions in flexural strength. Understanding the deleterious effects of NaOCl on mineralized dentin enables one to balance the risks and benefits in using high NaOCl concentrations for lengthy periods in root canal debridement. Delineating the mechanism responsible for such a phenomenon enables high molecular weight, polymeric antimicrobial and tissue dissolution irrigants to be designed that abides by the maxim of 'primum non nocere' in contemporary medical practices., Statement of Significance: The antimicrobial and tissue-dissolution capacities of NaOCl render it a well-accepted agent for root canal debridement. These highly desirable properties, however, appear to be intertwined with the untoward effect of collagen matrix degradation within mineralized dentin. Because of its small size, the hypochlorite anion is capable of infiltrating mineralized collagen and destroying the collagen fibrils, producing a mineral-rich, collagen sparse ghost mineral matrix with reduced flexural strength. Findings from the present work challenge the biosafety of NaOCl when it is used in high concentrations and for lengthy time periods during root canal treatment, and laid the background work for future biomaterials design in debridement of the canal space., (Published by Elsevier Ltd.)

Application of collagen cross-linkers to demineralized dentin improves bond durability. While the benefits of cross-linking treatments to bond strength and fatigue resistance have been explored, changes in hybrid layer stiffness with aging have not been examined., Objective: To examine the influence of a cross-linking treatment using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) on hybrid layer stiffness of resin-dentin adhesive bonds, using spatially-resolved nanoscopic Dynamic Mechanical Analysis (nanoDMA)., Methods: Bonded interface specimens were prepared using a two-step (SB) or three-step (SBMP) etch-and-rinse adhesive. Adhesive bonding of the treated groups was preceded by a 1min application of an experimental EDC conditioner to the acid-etched dentin. Control specimens did not receive EDC treatment. The bonded interfaces were evaluated using nanoDMA to determine the dynamic mechanical properties after storage in artificial saliva at 37°C for 0, 3 and 6 months., Results: The EDC treatment had no influence on the dynamic mechanical properties of the hybrid layer immediately after bonding. There was also no reduction in the hybrid layer stiffness after 3 and 6 months aging as defined by the complex modulus and storage modulus. However, there was a significant reduction in the loss modulus and tanδ components (i.e. viscous behavior) of the hybrid layers with aging. Degradation occurred to both adhesive systems with storage, but was greatest for SB. Without EDC treatment, the reduction in tanδ of the hybrid layer prepared with SB exceeded 80% in 6 months., Significance: The application of EDC to acid-etched dentin helps maintain the viscoelasticity of hybrid layers., (Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

A water-soluble crosslinking agent, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), has been used as a pretreatment of acid-etched dentin to inactivate matrix-bound endogenous dentin proteases. The aim of this study was to evaluate the effect of pH on the inactivation capacity of EDC. Demineralized dentin beams (1 × 2 × 6 mm) were divided into six groups (n = 8 per group). Then, EDC (0.3 M) was solubilized in distilled water with pH of 2, 4, 6, 7, 9, or 11. Control EDC was solubilized in 0.1 M 2-(N-morpholino) ethanesulfonic acid (MES) buffer and its pH was adjusted to 6. The dentin beams were pretreated for 1 min with EDC at each pH or with EDC in MES buffer at pH 6.0 and then incubated in 1 ml of simulated body fluid (pH 7.2) for 1, 3, 7, or 14 d. Untreated beams served as controls. At each study time-point, the dry mass of dentin beams was assessed and the incubation media were analyzed for carboxyterminal telopeptide of type-I collagen (ICTP) and C-terminal telopeptide of type I collagen (CTX) using specific ELISAs. Data were subjected to repeat-measures anova. The results of the study indicated that specimens pretreated with EDC in MES buffer showed the lowest collagen degradation in terms of mass loss and release of telopeptides, while specimens pretreated in alkaline media showed the highest collagen degradation. This study indicates that the pH of the EDC solution plays an important role in the stability of dentin protease inactivation., (© 2017 Eur J Oral Sci.)

Limitations associated with wet-bonding led to the recent development of a selective demineralization strategy in which dentin was etched with a reduced concentration of phosphoric acid to create exclusive extrafibrillar demineralization of the collagen matrix. However, the use of acidic conditioners removes calcium via diffusion of very small hydronium ions into the intrafibrillar collagen water compartments. This defeats the purpose of limiting the conditioner to the extrafibrillar space to create a collagen matrix containing only intrafibrillar minerals to prevent collapse of the collagen matrix. The present work examined the use of polymeric chelators (the sodium salt of polyacrylic acid) of different molecular weights to selectively demineralize extrafibrillar dentin. These polymeric chelators exhibit different affinities for calcium ions (isothermal titration calorimetry), penetrated intrafibrillar dentin collagen to different extents based on their molecular sizes (modified size-exclusion chromatography), and preserve the dynamic mechanical properties of mineralized dentin more favorably compared with completely demineralized phosphoric acid-etched dentin (nanoscopical dynamic mechanical analysis). Scanning and transmission electron microscopy provided evidence for retention of intrafibrillar minerals in dentin surfaces conditioned with polymeric chelators. Microtensile bond strengths to wet-bonded and dry-bonded dentin conditioned with these polymeric chelators showed that the use of sodium salts of polyacrylic acid for chelating dentin prior to bonding did not result in significant decline in resin-dentin bond strength. Taken together, the findings led to the conclusion that a chelate-and-rinse conditioning technique based on extrafibrillar collagen demineralization bridges the gap between wet and dry dentin bonding., Statement of Significance: The chelate-and-rinse dental adhesive bonding concept differentiates from previous research in that it is based on the size-exclusion characteristics of fibrillar collagen; molecules larger than 40kDa are prevented from accessing the intrafibrillar water compartments of the collagen fibrils. Using this chelate-and-rinse extrafibrillar calcium chelation concept, collagen fibrils with retained intrafibrillar minerals will not collapse upon air-drying. This enables adhesive infiltration into the mineral-depleted extrafibrillar spaces without relying on wet-bonding. By bridging the gap between wet and dry dentine bonding, the chelate-and-rinse concept introduces additional insight to the field by preventing exposure of endogenous proteases via preservation of the intrafibrillar minerals within a collagen matrix. If successfully validated, this should help prevent degradation of resin-dentine bonds by collagenolytic enzymes., (Published by Elsevier Ltd.)

The use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDC) has recently been investigated for its effectiveness in the prevention of collagen degradation over time and the improvement of resin-dentin bond durability. The objective of the present study was to evaluate the effects of a 0.3 M EDC-containing conditioner on endogenous enzymatic activities within the hybrid layer (HL) created by a self-etch or an etch-and-rinse adhesive after 1 y. The activity within the HL was examined using in situ zymography and confocal laser scanning microscopy after 24 h or 1-y storage in artificial saliva. Dentin specimens were bonded with Clearfil SE Bond (CSE) or XP Bond (XPB). For CSE, the self-etching primer was applied and treated with 0.3 M EDC for 1 min, and then the bonding agent was applied. For XPB, dentin was etched and treated with 0.3 M EDC for 1 min and then bonded with the primer-bonding agent. Control specimens were prepared without EDC treatment. Slices containing the adhesive-dentin interface were covered with fluorescein-conjugated gelatin and observed with a multiphoton confocal microscope. Fluorescence intensity emitted by hydrolyzed fluorescein-conjugated gelatin was quantified, and the amount of gelatinolytic activity was represented by the percentage of green fluorescence emitted within the HL. After 24 h of storage, enzymatic activity was detected by in situ zymography within the HLs of both tested adhesives, with XPB higher than CSE ( P < 0.05). Almost no fluorescence signal was detected when specimens were pretreated with EDC compared to controls ( P < 0.05). After 1 y of storage, enzymatic activities significantly increased for all groups (excluding XPB control) compared to 24-h storage ( P < 0.05), with EDC pretreated specimens exhibiting significantly lower activity than controls ( P < 0.05). The present study showed, for the first time, that the use of EDC for both the self-etch and the etch-and-rinse approaches results in the reduction but not complete inhibition of matrix-bound collagenolytic enzyme activities over time in the HL.

Objective: A recently-launched universal adhesive, G-Premio Bond, provides clinicians with the alternative to use the self-etch technique for bonding to dentine without waiting for the adhesive to interact with the bonding substrate (no-waiting self-etch; Japanese brochure), or after leaving the adhesive undisturbed for 10s (10-s self-etch; international brochure). The present study was performed to examine in vitro performance of this new universal adhesive bonded to human coronal dentine using the two alternative self-etch modes., Methods: One hundred and ten specimens were bonded using two self-etch application modes and examined with or without thermomechanical cycling (10,000 thermal cycles and 240,000 mechanical cycles) to simulate one year of intraoral functioning. The bonded specimens were sectioned for microtensile bond testing, ultrastructural and nanoleakage examination using transmission electron microscopy. Changes in the composition of mineralised dentine after adhesive application were examined using Fourier transform infrared spectroscopy., Results: Both reduced application time and thermomechanical cycling resulted in significantly lower bond strengths, thinner hybrid layers, and significantly more extensive nanoleakage after thermomechanical cycling. Using the conventional 10-s application time improved bonding performance when compared with the no-waiting self-etch technique. Nevertheless, nanoleakage was generally extensive under all testing parameters employed for examining the adhesive., Conclusion: Although sufficient bond strength to dentine may be achieved using the present universal adhesive in the no-waiting self-etch mode that does not require clinicians to wait prior to polymerisation of the adhesive, this self-etch concept requires further technological refinement before it can be recommended as a clinical technique., Clinical Significance: Although the surge for cutting application time to increase user friendliness remains the most frequently sought conduit for advancement of dentine bonding technology, the use of the present universal adhesive in the no-waiting self-etch mode may not represent the best use of the adhesive., (Published by Elsevier Ltd.)

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., (© 2017 Eur J Oral Sci.)

Objective: Inactivation of dentin proteases by crosslinkers has been suggested as a way to prevent the degradation of dentin collagen in the hybrid layer. However, it is not known if the inhibition is reversible. The aim of this study was to evaluate the inactivation effect of various crosslinkers on dentin protease activity over a period of 6 months., Methods: Demineralized dentin beams (1×2×6mm, n=10/group) were treated with (1) 1% glutaraldehyde (GA1), (2) 5% glutaraldehyde (GA5), (3) 1% grape seed extract (GS1), (4) 5% grape seed extract (GS5), (5) 10% sumac berry extract (S), (6) 20μM curcumin (CR20), and (7) 200μM curcumin (CR200) for 5min. Untreated beams served as control. The beams were incubated up to 6 months and incubation media were used to analyze solubilized telopeptide (ICTP and CTX) fragments as indicators of MMP- and cathepsin K-mediated degradation after 1, 3 and 6 months of incubation. The relative MMP activity of dentin beams was tested using a generic MMP assay. Data were analyzed using repeated-measures ANOVA, α=0.05., Results: All treated groups showed significant decrease in CTX release (32.2-469.5pg/mg dentin) and ICTP (1.8-47.6ng/mg dentin) fragments during the first month of incubation compared to control (1159pg/mg and 72.9ng/mg dentin, respectively). GA5, GS5 and CR200 maintained their inhibitory effect during 6-month incubation. The results were confirmed by dry mass loss and relative MMP activity following 6 months., Significance: The results of this study indicate that the long-term effect is both crosslinker and dose dependent., (Copyright © 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: The present study tested the central hypothesis that selective demineralisation of dentine extrafibrillar minerals by lowering the phosphoric acid concentration improves the quality of the resin-dentine interface., Methods: Dentine surfaces were etched with different concentrations of phosphoric acid (1, 5, 10, 20, 30 or 40wt%). Scanning electron microscopy was used to observe the micromorphology of the etched dentine surfaces. Energy dispersive X-ray analysis was performed to determine the residual Ca-content of the demineralised dentine matrix. Atomic force microscopy-based nanoindentation was used to analyse the nanomechanical properties of the treated dentine surfaces. The influence of H3PO4 concentration on resin-dentine bond strength was evaluated by microtensile bond strength testing. One-way ANOVA was used to compare the residual Ca-content ratio, reduced elastic modulus (Er) of the treated dentine surfaces and microtensile bond strength among groups., Results: Collagen fibrils appeared to be wider in diameter after etching with 5% and 10% H3PO4. The partially-demineralized collagen scaffold retained part of its rigidity to maintain an uncollapsed three-dimensional structure. Etching with 1% H3PO4 resulted in the highest residual Ca-content ratio and Er of demineralised dentine matrix, followed by 5% H3PO4. Those values were all significantly higher than values derived from the other groups. Etching with 30% H3PO4 resulted in the lowest Ca-content ratio and Er. Using 5% H3PO4 as etchant resulted in the highest resin-dentine bond strength., Conclusions: Selective demineralisation of the dentine matrix may be achieved by lowering the H3PO4 concentration to 5wt%, to achieve better bonding performance., Clinical Relevance: By retaining intrafibrillar minerals, more through air-drying of the partially demineralised collagen matrix may be accomplished without the need to worry about collapsing a mineral-free collagen matrix during air-drying. This may result in the elimination of water-wet bonding during the application of etch-and-rinse adhesives., (Published by Elsevier Ltd.)

Unlabelled: To investigate the contribution of nanolayering on resin-dentin bond durability, two phosphoric acid ester resin monomers, 10-methacryloyloxy-decyl-dihydrogen-phosphate (10-MDP) or its analog, methacryloyloxy-penta-propyleneglycol-dihydrogen-phosphate (MDA), were examined for their affinity for mineralized dentin powder in a column chromatography setup. Hydroxyapatite (HA) powder was dispersed in experimental primers consisting of 10-MDP or MDA solvated in ethanol/water and examined with FTIR, (31)P MAS-NMR and XPS. Light-curable 10-MDP or MDA primers were used for bonding to dentin, and examined after 24h or one-year of water-aging by TEM for evidence of nanolayering, and for microtensile bond strength evaluation. Primer-bonded dentin was examined by thin-film XRD to identify short-range order peaks characteristic of nanolayering of resin monomer-Ca salts. Although 10-MDP had better affinity for mineralized dentin than MDA, both monomers completely eluted from the mineralized dentin powder column using ethanol-water as mobile phase, indicating that the adsorption processes were reversible. This finding was supported by chemoanalytic data. XRD of 10-MDP-bonded dentin showed three diffraction peaks hat were absent from MDA-bonded dentin. Nanolayering was identified by TEM in 10-MDP-bonded dentin, but not in MDA-bonded dentin. Significant drop in bond strength (in MPa) was observed for both groups after one-year of water-aging compared with 24-h: 10-MDP group from 48.3±6.3 to 37.4±4.6; MDA group from 50.7±5.0 to 35.7±3.8 (P<0.05), with no significant difference between the two groups at the same time-point. Because both functional monomer-primed, resin-bonded dentin exhibited similar bond strength decline after water-aging, presence of nanolayering is unlikely to contribute to the overall resin-dentin bond durability., Statement of Significance: The durability of resin-dentin bonds in 10-MDP containing self-etching adhesives has been anecdotally attributed to the presence of nanolayering of 10-MDP-calcium salts in the resin-dentin interface. Results of the present work indicate that such a claim cannot be justified. Complete elution of the phosphoric acid ester monomer from mineralized dentin powder in the column chromatography experiments using ethanol-water mobile phase to simulate the solvent mixture employed in most 10-MDP-containing dentin adhesives further challenges the previously proposed adhesion-decalcification concept that utilizes chemical bonding of phosphoric acid ester monomers to apatite as a bonding mechanism in 10-MDP containing dentin adhesives., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The purpose of this study is to investigate the interaction of neat ethanol with bound and non-bound water in completely demineralized dentin that is fully hydrated, using molecular dynamics (MD) simulation method. The key to creating ideal resin-dentin bonds is the removal of residual free water layers and its replacement by ethanol solvent in which resin monomers are soluble, using the ethanol wet-bonding technique. The test null hypotheses were that ethanol cannot remove any collagen-bound water, and that ethanol cannot infiltrate into the spacing between collagen triple helix due to narrow interlayer spacing. Collagen fibrillar structures of overlap and gap regions were constructed by aligning the collagen triple helix of infinite length in hexagonal packing. Three layers of the water molecules were specified as the layers of 0.15-0.22nm, 0.22-0.43nm and 0.43-0.63nm from collagen atoms by investigating the water distribution surrounding collagen molecules. Our simulation results show that ethanol molecules infiltrated into the intermolecular spacing in the gap region, which increased due to the lateral shrinkage of the collagen structures in contact with ethanol solution, while there was no ethanol infiltration observed in the overlap region. Infiltrated ethanol molecules in the gap region removed residual water molecules via modifying mostly the third water layer (50% decrease), which would be considered as a loosely-bound water layer. The first and second hydration layers, which would be considered as tightly bound water layers, were not removed by the ethanol molecules, thus maintaining the helical structures of the collagen molecules., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Self-assembled nanolayering structures have been reported in resin-dentin interfaces created by adhesives that contain 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP). These structures have been hypothesized to contribute to bond durability. The objective of the present study was to determine the extent of nanolayering in resin-dentin interfaces after application of commercialized 10-MDP-containing self-etch and universal adhesives to human dentin. Seven commercialized adhesives were examined: Adhese Universal (Ivoclar-Vivadent), All-Bond Universal (Bisco, Inc.), Clearfil SE Bond 2, Clearfil S3 Bond Plus, Clearfil Universal Bond (all from Kuraray Noritake Dental Inc.), G-Premio Bond (GC Corp.), and Scotchbond Universal (3M ESPE). Each adhesive was applied in the self-etch mode on midcoronal dentin according to the respective manufacturer's instructions. Bonded specimens (n = 6) were covered with flowable resin composite, processed for transmission electron microscopy, and examined at 30 random sites without staining. Thin-film glancing angle X-ray diffraction (XRD) was used to detect the characteristic peaks exhibited by nanolayering (n = 4). The control consisted of 15%wt, 10%wt, and 5%wt 10-MDP (DM Healthcare Products, Inc.) dissolved in a mixed solvent (ethanol and water weight ratio 9:8, with photoinitiators). Experimental primers were applied to dentin for 20 s, covered with hydrophobic resin layer, and examined in the same manner. Profuse nanolayering with highly ordered periodicity (~3.7 nm wide) was observed adjacent to partially dissolved apatite crystallites in dentin treated with the 15% 10-MDP primer. Three peaks in the 2θ range of 2.40° (3.68 nm), 4.78° (1.85 nm), and 7.18° (1.23 nm) were identified from thin-film XRD. Reduction in the extent of nanolayering was observed in the 10% and 5% 10-MDP experimental primer-dentin interface along with lower intensity XRD peaks. Nanolayering and characteristic XRD peaks were rarely observed in specimens prepared from the commercialized adhesives. The sparsity of nanolayering in resin-dentin interfaces created by commercialized adhesives challenges its clinical effectiveness as a mechanism for improving bond longevity in dentin bonding., (© International & American Associations for Dental Research 2015.)

Objectives: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) has been shown to cross-link dentin type I collagen. Increased cross-linking usually elevates the glass transition temperature of polymers. The aim of this study was to evaluate the cross-linking reaction promoted by EDC in different aqueous concentrations by measuring the thermal denaturation temperature (Td) of human dentin collagen., Methods: The Td of dehydrated collagen and of insoluble dentin matrix collagen immersed in 0.5M or 1M EDC aqueous solution for different treatment times was obtained using a Differential Scanning Calorimeter (DSC). Specimens were also analyzed by Energy Dispersive X-Ray Spectroscopy., Results: EDC-treated dentin collagen showed a significantly higher Td than the untreated specimens when immersed in either 0.5M EDC or 1M EDC for 10min or longer (p<0.05). EDC-treated dentin collagen showed an increase of sulfur and chloride, not detectable in EDC-untreated dentin specimens. Conversely, the relative amount of carbon, nitrogen and oxygen was not modified by treatments., Significance: EDC-treated dentin collagen showed a higher Td than the untreated control at all tested concentrations and immersion times. A higher Td can be considered an indirect indicator of a more resistant and highly cross-linked collagen network. More data are needed to confirm these results., (Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

During dentin bonding with etch-and-rinse adhesive systems, phosphoric acid etching of mineralized dentin solubilizes the mineral crystallites and replaces them with bound and unbound water. During the infiltration phase of dentin bonding, solvated adhesive resin comonomers are supposed to replace all of the unbound collagen water and polymerize into copolymers. A recently published review suggested that dental monomers are too large to enter and displace water from tightly-packed collagen molecules. Conversely, recent work from the authors' laboratory demonstrated that HEMA and TEGDMA freely equilibrate with water-saturated dentin matrices. However, because adhesive blends are solvated in organic solvents, those solvents may remove enough free water to allow collagen molecules to come close enough to exclude adhesive monomer permeation. The present study analyzed the size-exclusion characteristics of dentin collagen, using a gel permeation-like column chromatography technique, filled with dentin powder instead of Sephadex beads as the stationary phase. The elution volumes of different sized test molecules, including adhesive resin monomers, studied in both water-saturated dentin, and again in ethanol-dehydrated dentin powder, showed that adhesive resin monomers can freely diffuse into both hydrated and dehydrated collagen molecules. Under these in vitro conditions, all free and some of the loosely-bound water seems to have been removed by ethanol. These results validate the concept that adhesive resin monomers can permeate tightly-bound water in ethanol-saturated collagen molecules during infiltration by etch-and-rinse adhesives., Statement of Significance: It has been reported that collagen molecules in dentin matrices are packed too close together to allow permeation of adhesive monomers between them. Resin infiltration, in this view, would be limited to extrafibrillar spaces. Our work suggests that monomers equilibrate with collagen water in both water and ethanol-saturated dentin matrices., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Objectives: The present study evaluated the long-term dentine bonding effectiveness of five universal adhesives in etch-and-rinse or self-etch mode after 12 months of water-ageing., Methods: The adhesives evaluated included All-Bond Universal, Clearfil Universal Bond, Futurabond U Prime&Bond Elect and Scotchbond Universal. Microtensile bond strength and transmission electron microscopy of the resin-dentine interfaces created in human coronal dentine were examined after 24h or 12 months., Results: Microtensile bond strength were significantly affected by bonding strategy (etch-and-rinse vs self-etch) and ageing (24h vs 12 months). All subgroups showed significantly decreased bond strength after ageing except for Prime&Bond Elect and Scotchbond Universal used in self-etch mode. All five adhesives employed in etch-and-rinse mode exhibited ultrastructural features characteristic of collagen degradation and resin hydrolysis. A previously-unobserved inside-out collagen degradation pattern was identified in hybrid layers created by 10-MDP containing adhesives (All-Bond Universal, Scotchbond Universal and Clearfil Universal Bond) in the etch-and-rinse mode, producing partially degraded collagen fibrils with intact periphery and a hollow core. In the self-etch mode, all adhesives except for Prime&Bond Elect exhibited degradation of the collagen fibrils along the thin hybrid layers. The three 10-MDP containing universal adhesives did not protect surface collagen fibrils from degradation when bonding was performed in the self-etch mode., Conclusions: Despite the adjunctive conclusion that bonds created by universal adhesives in the self-etch bonding mode are more resistant to decline in bond strength when compared with those bonds created using the etch-and-rinse mode, bonds created by universal adhesives are generally incapable of defying ageing., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: Success in adhesive dentistry means long lasting restorations. However, there is substantial evidence that this ideal objective is not always achieved. Current research in this field aims at increasing the durability of resin-dentin bonds. The objective of this paper is to examine the fundamental processes responsible for the aging mechanisms involved in the degradation of resin-bonded interfaces and the potential approaches to prevent and counteract this degradation., Methods: PubMed searches on the hybrid layer degradation were carried out. Keywords were chosen to assess hybrid layer degradation for providing up-dated information on the basis of scientific coherence with the research objective. Approaches to prevent and counteract this degradation were also reviewed., Results: 148 peer-review articles in the English language between 1982 and 2015 were reviewed. Literature shows that resin-dentin bond degradation is a complex process, involving the hydrolysis of both the resin and the collagen fibril phases contained within the hybrid layer. Collagen fibers become vulnerable to mechanical and hydraulic fatigue, as well as degradation by host-derived proteases with collagenolytic activity (matrix metalloproteinases and cysteine cathepsins). Inhibition of the collagenolytic activity and the use of cross-linking agents are the two main strategies to increase the resistance of the hybrid layer to enzymatic degradation., Significance: This review analyzes the issues regarding the durability of the adhesive interface, and the techniques to create stable resin-dentin bonds able to resist the collagenolytic hydrolysis that are currently studied., (Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Objective: This study evaluated the ability of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) to improve the stability of demineralized dentin collagen matrices when subjected to mechanical cycling by means of Chewing Simulation (CS)., Methods: Demineralized dentin disks were randomly assigned to four groups (N=4): (1) immersion in artificial saliva at 37°C for 30 days; (2) pre-treatment with 0.5 M EDC for 60 s, then stored as in Group 1; (3) CS challenge (50 N occlusal load, 30 s occlusal time plus 30 s with no load, for 30 days); (4) pre-treatment with 0.5 M EDC as in Group 2 and CS challenge as in Group 3. Collagen degradation was evaluated by sampling storage media for ICTP and CTX telopeptides., Results: EDC treated specimens showed no significant telopeptides release, irrespective of the aging method. Cyclic stressing of EDC-untreated specimens caused significantly higher ICTP release at day 1, compared to static storage, while by days 3 and 4, the ICTP release in the cyclic group fell significantly below the static group, and then remained undetectable from 5 to 30 days. CTX release in the cyclic groups, on EDC-untreated control specimens was always lower than in the static group in days 1-4, and then fell to undetectable for 30 days., Significance: This study showed that chewing stresses applied to control untreated demineralized dentin increased degradation of collagen in terms of CTX release, while collagen crosslinking agents may prevent dentin collagen degradation, irrespective of simulated occlusal function., (Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Unlabelled: Resin bonding to dentin requires the use of self-etching primers or acid etching to decalcify the surface and expose a layer of collagen fibrils of the dentin matrix. Acid-etching reduces the stiffness of demineralized dentin from approximately 19 GPa-1 MPa, requiring that it floats in water to prevent it from collapsing during bonding procedures. Several publications show that crosslinking agents like gluteraladehyde, carbodiimide or grape seed extract can stiffen collagen and improve resin-dentin bond strength., Objective: The objective was to assess a new approach for evaluating the changes in stiffness of decalcified dentin by polar solvents and a collagen cross-linker., Methods: Fully demineralized dentin beams and sections of etched coronal dentin were subjected to indentation loading using a cylindrical flat indenter in water, and after treatment with ethanol or ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The stiffness was measured as a function of strain and as a function of loading rate from 1 to 50 μm/s., Results: At a strain of 0.25% the elastic modulus of the fully demineralized dentin was approximately 0.20 MPa. It increased to over 0.90 MPa at strains of 1%. Exposure to ethanol caused an increase in elastic modulus of up to four times. Increasing the loading rate from 1 to 50 μm/s caused an increase in the apparent modulus of up to three times in both water and ethanol. EDC treatment caused increases in the stiffness in fully demineralized samples and in acid-etched demineralized dentin surfaces in situ., Significance: Changes in the mechanical behavior of demineralized collagen matrices can be measured effectively under hydration via indentation with cylindrical flat indenters. This approach can be used for quantifying the effects of bonding treatments on the properties of decalcified dentin after acid etching, as well as to follow the loss of stiffness over time due to enzymatic degradation., (Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Purpose: This review describes the evolution of the use of dental adhesives to form a tight seal of freshly prepared dentin to protect the pulp from bacterial products, during the time between crown preparation and final cementation of full crowns. The evolution of these "immediate dentin sealants" follows the evolution of dental adhesives, in general. That is, they began with multiple-step, etch-and-rinse adhesives, and then switched to the use of simplified adhesives., Methods: Literature was reviewed for evidence that bacteria or bacterial products diffusing across dentin can irritate pulpal tissues before and after smear layer removal. Smear layers can be solubilized by plaque organisms within 7-10 days if they are directly exposed to oral fluids. It is likely that smear layers covered by temporary restorations may last more than 1 month. As long as smear layers remain in place, they can partially seal dentin. Thus, many in vitro studies evaluating the sealing ability of adhesive resins use smear layer-covered dentin as a reference condition. Surprisingly, many adhesives do not seal dentin as well as do smear layers., Results: Both in vitro and in vivo studies show that resin- covered dentin allows dentin fluid to cross polymerized resins. The use of simplified single bottle adhesives to seal dentin was a step backwards. Currently, most authorities use either 3-step adhesives such as Scotchbond Multi-Purpose or OptiBond FL or two-step self-etching primer adhesives, such as Clearfil SE, Unifil Bond or AdheSE.

Objectives: The aim of this study was to evaluate the effect of using UVA-induced crosslinking with or without riboflavin as photosensitizers on degradation of dentin matrix by dentin proteases., Methods: Demineralized dentin specimens (0.4×3×6 mm(3), n=10/group) were subjected to: (RP1), 0.1% riboflavin-5 phosphate/UVA for 1 min; (RP5), 0.1% riboflavin-5 phosphate/UVA for 5 min; (R1), 0.1% riboflavin/UVA for 1 min; (R5), 0.1% riboflavin-UVA for 5 min; (UV1), UVA for 1 min; (UV5), UVA for 5 min. Specimens were incubated in 1 mL zinc and calcium containing media for 1 day and 1 week. An untreated group served as control (CM). After incubation, the loss of dry mass of samples was measured and aliquots of media were analyzed for the release of C-terminal fragment telopeptide (ICTP vs. CTX) of collagen to evaluate for cathepsin K (CA-K) and total matrix metalloproteinase (MMP)-mediated degradation. Data were analyzed using repeated measures ANOVA at α=0.05., Results: Although UVA radiation alone reduced dentin degradation, UVA-activated riboflavin or riboflavin-5 phosphate inhibited MMP and CA-K activities more than UVA alone. The effects of crosslinking were more pronounced in 7-day samples; only with CA-K were the effects of crosslinking with or without photosensitizer significantly different from controls in 1-day samples., Significance: The use of bioactive forms (RP) or longer treatment time did not result with better effect. The use of UVA crosslinking reduces dentin matrix degradation, especially with photosensitizers., (Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Objective: This study tested whether treatment of demineralized dentin with polyacrylic acid (PAA) has any activatory or inhibitory activity on dentin matrix metalloproteinases (MMP)s or cathepsin K (CAT-K)., Methods: Dentin beams (1mm×2mm×6mm; n=10) were completely demineralized with EDTA. After initial dry mass assessment, the beams were dipped into 37% phosphoric acid (PA), PA+2% benzalkonium chloride (BAC), PA+2% chlorhexidine digluconate (CHX), 10% PAA, PAA+BAC or PAA+CHX for 20s. Demineralized beams without treatment served as control. All beams were incubated in simulated body fluid (SBF) for 1 week and the dry mass loss was evaluated. Aliquots of SBF were used to analyze solubilized telopeptide fragments using ICTP as indicator of MMP-mediated collagen degradation and CTX for CAT-K-mediated degradation. Additional demineralized beams (n=10) were used to measure the influence of different chemical treatments on total MMP activity of EDTA-demineralized dentin using generic MMP assay. Data were analyzed by ANOVA (α=0.05)., Results: Dry mass loss ranged from 6% (PA) to 2% for (PA-BAC) or (PAA-BAC) (p<0.05). ICTP release of PAA-treated group was significantly higher (p<0.05) than the control, and not significantly different from the PA group (p>0.05). PA+CHX or PAA+CHX and PAA+BAC showed significantly lower ICTP than PA or PAA groups (p<0.05). CAT-K activity increased significantly after 10% PAA treatment compared to control (p<0.05) or to PA postreatment., Significance: Demineralized dentin treated with 10% polyacrylic acid activated CAT-K more than 37% phosphoric acid; 2% chlorhexidine digluconate seems to be a better inhibitor of MMPs and CAT-K than 2% benzalkonium chloride., (Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Objectives: This study evaluated the effect of dentin pretreatment with collagen crosslinkers on matrix metalloproteinase (MMP) and cathepsin K mediated collagen degradation., Methods: Dentin beams (1mm×2mm×6mm) were demineralized in 10% H3PO4 for 24h. After baseline measurements of dry mass, beams were divided into 11 groups (n=10/group) and, were pretreated for 5min with 1% glutaraldehyde (GA); 5% GA; 1% grape-seed extract (GS); 5% GS; 10% sumac (S); 20μM curcumin (CR); 200μM CR; 0.l% riboflavin/UV (R); 0.5% R; 0.1% riboflavin-5-phosphate/UV (RP); and control (no pretreatment). After pretreatment, the beams were blot-dried and incubated in 1mL calcium and zinc-containing medium (CM, pH 7.2) at 37°C for 3, 7 or 14 days. After incubation, dry mass was reassessed and aliquots of the incubation media were analyzed for collagen C-telopeptides, ICTP and CTX using specific ELISA kits. Data were analyzed by repeated-measures ANOVA., Results: The rate of dry mass loss was significantly different among test groups (p<0.05). The lowest 14 day mean dry mass loss was 6.98%±1.99 in the 200μM curcumin group compared to control loss of dry mass at 32.59%±5.62, p<0.05, at 14 days. The ICTP release over the incubation period (ng/mg dry dentin) ranged between 1.8±0.51 and 31.8±1.8. CTX release from demineralized beams pretreated with crosslinkers was significantly lower than CM (5.7±0.2ng/mg dry dentin)., Significance: The results of this study indicate that collagen crosslinkers tested in this study are good inhibitors of cathepsin K activity in dentin. However, their inhibitory effect on MMP activity was highly variable., (Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Objective: Multi-mode universal adhesives offer clinicians the choice of using the etch-and-rinse technique, selective enamel etch technique or self-etch technique to bond to tooth substrates. The present study examined the short-term in vitro performance of five universal adhesives bonded to human coronal dentine., Methods: Two hundred non-carious human third molars were assigned to five groups based on the type of the universal adhesives (Prime&Bond Elect, Scotchbond Universal, All-Bond Universal, Clearfil Universal Bond and Futurabond U). Two bonding modes (etch-and-rinse and self-etch) were employed for each adhesive group. Bonded specimens were stored in deionized water for 24h or underwent a 10,000-cycle thermocycling ageing process prior to testing (N=10). Microtensile bond testing (μTBS), transmission electron microscopy (TEM) of resin-dentine interfaces in non-thermocycled specimens and scanning electron microscopy (SEM) of tracer-infused water-rich zones within hybrid layers of thermocycled specimens were performed., Results: Both adhesive type and testing condition (with/without thermocycling) have significant influences on μTBS. The use of each adhesive in either the etch-and-rinse or self-etch application mode did not result in significantly different μTBS to dentine. Hybrid layers created by these adhesives in the etch-and-rinse bonding mode and self-etch bonding mode were ∼5μm and ≤0.5μm thick respectively. Tracer-infused regions could be identified within the resin-dentine interface from all the specimens prepared., Conclusion: The increase in versatility of universal adhesives is not accompanied by technological advances for overcoming the challenges associated with previous generations of adhesives. Therapeutic adhesives with bio-protective and bio-promoting effects are still lacking in commercialized adhesives., Clinical Significance: Universal adhesives represent manufacturers' attempt to introduce versatility in product design via adaptation of a single-bottle self-etch adhesive for other application modes without compromising its bonding effectiveness., (Published by Elsevier Ltd.)

Reducing collagen degradation within hybrid layers may contribute to the preservation of adhesive interfaces. This study evaluated the stability of resin-dentin interfaces treated with benzalkonium chloride (BAC)-modified adhesive blends and assessed collagen degradation in dentin matrices treated with BAC. The etch-and-rinse adhesive, Adper Single Bond Plus, modified with 0.5% and 1.0% BAC, was evaluated for microtensile bond strength (μTBS) and nanoleakage (NL) after 24 h and 1 yr. Thirty completely demineralized dentin beams from human molars were dipped for 60 s in deionized water (DW; control), or in 0.5% or 1.0% BAC, and then incubated in simulated body fluid (SBF). Collagen degradation was assessed by quantification of the dry mass loss and the amount of hydroxyproline (HYP) released from hydrolyzed specimens after 1 or 4 wk. Although all groups demonstrated a significant increase in NL after 1 yr, adhesive modified with 0.5% BAC showed stable bond strength after 1 yr (9% decrease) relative to the control (44% decrease). Significantly less HYP release and dry mass loss were observed for both 0.5% and 1.0% BAC relative to the control. This in vitro study demonstrates that BAC contributes to the preservation of resin-dentin bonds for up to 1 yr by reducing collagen degradation., (© 2015 Eur J Oral Sci.)

Objective: This work measured the amount of bound versus unbound water in completely-demineralized dentin., Methods: Dentin beams prepared from extracted human teeth were completely demineralized, rinsed and dried to constant mass. They were rehydrated in 41% relative humidity (RH), while gravimetrically measuring their mass increase until the first plateau was reached at 0.064 (vacuum) or 0.116 gH2O/g dry mass (Drierite). The specimens were then exposed to 60% RH until attaining the second plateau at 0.220 (vacuum) or 0.191 gH2O/g dry mass (Drierite), and subsequently exposed to 99% RH until attaining the third plateau at 0.493 (vacuum) or 0.401 gH2O/g dry mass (Drierite)., Results: Exposure of the first layer of bound water to 0% RH for 5 min produced a -0.3% loss of bound water; in the second layer of bound water it caused a -3.3% loss of bound water; in the third layer it caused a -6% loss of bound water. Immersion in 100% ethanol or acetone for 5 min produced a 2.8 and 1.9% loss of bound water from the first layer, respectively; it caused a -4 and -7% loss of bound water in the second layer, respectively; and a -17 and -23% loss of bound water in the third layer. Bound water represented 21-25% of total dentin water. Chemical dehydration of water-saturated dentin with ethanol/acetone for 1 min only removed between 25 and 35% of unbound water, respectively., Significance: Attempts to remove bound water by evaporation were not very successful. Chemical dehydration with 100% acetone was more successful than 100% ethanol especially the third layer of bound water. Since unbound water represents between 75 and 79% of total matrix water, the more such water can be removed, the more resin can be infiltrated., (Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Dentin can be described as a biological composite with collagen matrix embedded with nanosized hydroxyapatite mineral crystallites. Matrix metalloproteinases (MMPs) and cysteine cathepsins are families of endopeptidases. Enzymes of both families are present in dentin and collectively capable of degrading virtually all extracellular matrix components. This review describes these enzymes and their presence in dentin, mainly focusing on their role in dentin caries pathogenesis and loss of collagen in the adhesive hybrid layer under composite restorations. MMPs and cysteine cathepsins present in saliva, mineralized dentin, and/or dentinal fluid may affect the dentin caries process at the early phases of demineralization. Changes in collagen and noncollagenous protein structure may participate in observed decreases in mechanical properties of caries-affected dentin and reduce the ability of caries-affected dentin to remineralize. These endogenous enzymes also remain entrapped within the hybrid layer during the resin infiltration process, and the acidic bonding agents themselves (irrespective of whether they are etch-and-rinse or self-etch) can activate these endogenous protease proforms. Since resin impregnation is frequently incomplete, denuded collagen matrices associated with free water (which serves as a collagen cleavage reagent for these endogenous hydrolase enzymes) can be enzymatically disrupted, finally contributing to the degradation of the hybrid layer. There are multiple in vitro and in vivo reports showing that the longevity of the adhesive interface is increased when nonspecific enzyme-inhibiting strategies are used. Different chemicals (i.e., chlorhexidine, galardin, and benzalkonium chloride) or collagen cross-linker agents have been successfully employed as therapeutic primers in the bonding procedure. In addition, the incorporation of enzyme inhibitors (i.e., quaternary ammonium methacrylates) into the resin blends has been recently promoted. This review will describe MMP functions in caries and hybrid layer degradation and explore the potential therapeutic role of MMP inhibitors for the development of improved intervention strategies for MMP-related oral diseases., (© International & American Associations for Dental Research 2014.)

An experimental evaluation of human coronal dentin was performed using nanoscopic dynamic mechanical analysis (nanoDMA). The primary objectives were to quantify any unique changes in mechanical behavior of intertubular and peritubular dentin with age, and to evaluate the microstructure and mechanical behavior of the mineral deposited within the lumens. Specimens of coronal dentin were evaluated by nanoDMA using single indents and in scanning mode via scanning probe microscopy. Results showed that there were no significant differences in the storage modulus or complex modulus between the two age groups (18-25 versus 54-83 yrs) for either the intertubular or peritubular tissue. However, there were significant differences in the dampening behavior between the young and old dentin, as represented in the loss modulus and tanδ responses. For both the intertubular and peritubular components, the capacity for dampening was significantly lower in the old group. Scanning based nanoDMA showed that the tubules of old dentin exhibit a gradient in elastic behavior, with decrease in elastic modulus from the cuff to the center of tubules filled with newly deposited mineral., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: Dentin matrices release ICTP and CTX fragments during collagen degradation. ICTP fragments are known to be produced by MMPs. CTX fragments are thought to come from cathepsin K activity. The purpose of this study was to determine if quaternary methacrylates (QAMs) can inhibit matrix MMPs and cathepsins., Methods: Dentin beams were demineralizated, and dried to constant weight. Beams were incubated with rh-cathepsin B, K, L or S for 24h at pH 7.4 to identify which cathepsins release CTX at neutral pH. Beams were dipped in ATA, an antimicrobial QAM to determine if it can inhibit dentin matrix proteases. Other beams were dipped in another QAM (MDPB) to determine if it produced similar inhibition of dentin proteases., Results: Only beams incubated with cathepsin K lost more dry mass than the controls and released CTX. Dentin beams dipped in ATA and incubated for 1 week at pH 7.4, showed a concentration-dependent reduction in weight-loss. There was no change in ICTP release from control values, meaning that ATA did not inhibit MMPs. Media concentrations of CTX fell significantly at 15wt% ATA indicating that ATA inhibits capthesins. Beams dipped in increasing concentrations of MDPB lost progressively less mass, showing that MDPB is a protease-inhibitor. ICTP released from controls or beams exposed to low concentrations were the same, while 5 or 10% MDPB significantly lowered ICTP production. CTX levels were strongly inhibited by 2.5-10% MDPB, indicating that MDPB is a potent inhibitor of both MMPs and cathepsin K., Significance: CTX seems to be released from dentin matrix only by cathepsin K. MMPs and cathepsin K and B may all contribute to matrix degradation., (Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Objective: To evaluate the transdentinal cytotoxicity of three different concentrations of carbodiimide (EDC) or 5% glutaraldehyde (GA) on MDPC-23 cells., Methods: Seventy 0.4-mm-thick dentin disks obtained from human molars were adapted to artificial pulp chambers. MDPC-23 cells were seeded on the pulpal surface of the disks. After 48 hours, the occlusal dentin was acid-etched and treated for 60 seconds with one of the following solutions (n=10): no treatment (negative control); 0.1 M, 0.3 M, or 0.5 M EDC; 5% GA; Sorensen buffer; or 29% hydrogen peroxide (positive control). Cell viability and morphology were assessed by methyltetrazolium assay and scanning electron microscopy (SEM), respectively. The eluates were collected after the treatments and applied on MDPC-23 seeded in a 24-well plate to analyze cell death, total protein (TP), and collagen production. The last two tests were performed 24 hours and seven days after the challenge. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (p<0.05)., Results: EDC at all test concentrations did not reduce cell viability, while 5% GA did increase cell metabolism. Cell death by necrosis was not elicited by EDC or 5% GA. At the 24-hour period, 0.3 M and 0.5 M EDC reduced TP production by 18% and 36.8%, respectively. At seven days, increased TP production was observed in all groups. Collagen production at the 24-hour period was reduced when 0.5 M EDC was used. After seven days, no difference was observed among the groups. SEM showed no alteration in cell morphology or number, except in the hydrogen peroxide group., Conclusions: Treatment of acid-etched dentin with EDC or GA did not cause transdentinal cytotoxic effects on odontoblast-like cells.

Unlabelled: Spatial variations in the microstructure of dentine contribute to its mechanical behaviour., Objective: The objective of this investigation was to compare the microstructure and fatigue behaviour of dentine from donors of two different countries., Methods: Caries-free third molars were obtained from dental practices in Colombia, South America and the US to assemble two age-matched samples. The microstructure of the coronal dentine was evaluated at three characteristic depths (i.e. deep, middle and superficial dentine) using scanning electron microscopy and image processing techniques. The mechanical behaviour of dentine in these three regions was evaluated by the fatigue crack growth resistance. Cyclic crack growth was achieved in-plane with the dentine tubules and the fatigue crack growth behaviour was characterized in terms of the stress intensity threshold and the Paris Law parameters., Results: There was no difference in the tubule density between the dentine of patients from the two countries. However, there were significant differences (p≤0.05) in the tubule lumen diameters between the two groups in the deep and peripheral regions. In regards to the fatigue resistance, there was a significant increase (p≤0.05) in threshold stress intensity range, and a significant decrease in fatigue crack growth coefficient with increasing distance from the pulp in teeth from the US donors. In contrast, these properties were independent of location for the dentine of teeth from the Colombian donors., Conclusions: The microstructure of dentine and its mechanical behaviour appear to be a function of patient background, which may include environmental factors and/or ethnicity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: To directly test the effectiveness of ethanol-wet bonding (EW) in improving monomer infiltration into demineralized dentin through quantitative measurement of bis-GMA and TEG-DMA molar concentrations within hybrid layers, and to comprehensively evaluate the effect of EW and chlorhexidine on durability of resin-dentin bonds compared to conventional water-wet bonding (WW)., Materials and Methods: A three-step etch-and-rinse adhesive (70% bis-GMA/28.75%TEG-DMA) was applied to coronal dentin using a clinically relevant ethanol-wet bonding protocol (EW) or the conventional water-wet bonding (WW) technique. Bis-GMA and TEG-DMA molar concentrations at various positions across the resin/dentin interfaces formed by EW and WW were measured using micro-Raman spectroscopy. The experiment was repeated at the same positions after 7-month storage in phosphate buffer solution containing 0.1% sodium azide. The μTBS and hybrid layer morphology (TEM) of bonding groups with and without chlorhexidine application were compared immediately and after 1-year storage in terms of nanoleakage, collagen fibril diameter, collagen interfibrillar width, and hybrid layer thickness., Results: Specimens bonded with EW showed significantly higher monomer molar concentrations and μTBS throughout the hybrid layer immediately and after storage, providing direct evidence of superior infiltration of hydrophobic monomers in EW compared to WW. Microscopically, EW maintained interfibrillar width and hybrid layer thickness for resin infiltration and retention. The application of chlorhexidine further preserved collagen integrity and limited the degree of nanoleakage in EW after 1-year storage., Conclusion: EW enhances infiltration of hydrophobic monomers into demineralized dentin. The results suggest that a more durable resin-dentin bond may be achieved with combined usage of a clinically relevant EW and chlorhexidine.

This study analyzed the amounts of solubilized telopeptides cross-linked carboxyterminal telopeptide of type I collagen (ICTP) and C-terminal crosslinked telopeptide of type I collagen (CTX) derived from matrix-metalloproteinases (MMPs) and cysteine cathepsins (CTPs) subsequent to application of a filler-free (Res.A) or an ion-releasing resin (Res.B) to ethylenediaminetetraacetic acid (EDTA)-demineralized dentin with or without zoledronate-containing primer (Zol-primer) pre-treatment. The chemical modification induced following treatments and artificial saliva (AS) storage was also analyzed through attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). Totally EDTA-demineralized specimens were infiltrated with Res.A or Res.B with or without Zol-primer pre-treatment, light-cured, and immersed in AS for up to 4 wk. ICTP release was reduced following infiltration with Res.B and further reduced when Res.B was used with Zol-primer; remarkable phosphate mineral uptake was attained after AS storage. CTX release was increased in Res.A- and Res.B-treated dentin. However, when Zol-primer was used with Res.A, the CTX release fell significantly compared to the other tested resin-infiltration methods. In conclusion, zoledronate offers an additional inhibitory effect to the ion-releasing resins in MMP-mediated collagen degradation. However, Zol-primer induces a modest reduction in CTX release only when used with resin-based systems containing no ion-releasing fillers., (© International & American Associations for Dental Research.)

Purpose: This systematic review provides an overview of the different mechanisms proposed to regulate the degradation of dentin matrices by host-derived dentin proteases, particularly as it relates to their role in dental adhesion. Significant developments have taken place over the last few years that have contributed to a better understanding of all the factors affecting the durability of adhesive resin restorations. The complexity of dentin-resin interfaces mandates a thorough understanding of all the mechanical, physical and biochemical aspects that play a role in the formation of hybrid layers. The ionic and hydrophilic nature of current dental adhesives yields permeable, unstable hybrid layers susceptible to water sorption, hydrolytic degradation and resin leaching. The hydrolytic activity of host-derived proteases also contributes to the degradation of the resin-dentin bonds. Preservation of the collagen matrix is critical to the improvement of resin-dentin bond durability. Approaches to regulate collagenolytic activity of dentin proteases have been the subject of extensive research in the last few years. A shift has occurred from the use of proteases inhibitors to the use of collagen cross-linking agents. Data provided by 51 studies published in peer-reviewed journals between January 1999 and December 2013 were compiled in this systematic review., Results: Appraisal of the data provided by the studies included in the present review yielded a summary of the mechanisms which have already proven to be clinically successful and those which need further investigation before new clinical protocols can be adopted.

Objectives: To examine the effects of the combined use of chlorhexidine and ethanol on the durability of resin-dentin bonds., Methods: Forty-eight flat dentin surfaces were etched (32% phosphoric acid), rinsed (15 s) and kept wet until bonding procedures. Dentin surfaces were blot-dried with absorbent paper and re-wetted with water (water, control), 1% chlorhexidine diacetate in water (CHD/water), 100% ethanol (ethanol), or 1% chlorhexidine diacetate in ethanol (CHD/ethanol) solutions for 30 s. They were then bonded with All Bond 3 (AB3, Bisco) or Excite (EX, Ivoclar-Vivadent) using a smooth, continuous rubbing application (10 s), followed by 15 s gentle air stream to evaporate solvents. The adhesives were light-cured (20 s) and resin composite build-ups constructed for the microtensile method. Bonded beams were obtained and tested after 24-h, 6-months and 15-months of water storage at 37°C. Storage water was changed every month. Effects of treatment and testing periods were analyzed (ANOVA, Holm-Sidak, p<0.05) for each adhesive., Results: There were no interactions between factors for both etch-and-rinse adhesives. AB3 was significantly affected only by storage (p=0.003). Excite was significantly affected only by treatments (p=0.048). AB3 treated either with ethanol or CHD/ethanol resulted in reduced bond strengths after 15 months. The use of CHD/ethanol resulted in higher bond strengths values for Excite., Conclusions: Combined use of ethanol/1% chlorhexidine diacetate did not stabilize bond strengths after 15 months., (Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)