High-Performance Dental Resins Containing a Starburst Monomer.

Dimethacrylate-based chemistries feature extensively as resin monomers in dental resin-based materials due to their distinguished overall performance. However, challenges endure, encompassing inadequate mechanical attributes, volumetric shrinkage, and estrogenicity. Herein, we first synthesized a novel resin monomer, 9-armed starburst polyurethane acrylate (NPUA), via the grafting-onto approach. Compared to the primary commercial dental monomer 2,2-bis [p-(2'-hydroxy-3'-methacryloxypropoxy) phenyl] propane (Bis-GMA) (with a viscosity of 1,174 ± 3 Pa·s and volumetric shrinkage of 4.7% ± 0.1%), the NPUA monomer achieves the lower viscosity (158 ± 1 Pa·s), volumetric shrinkage (2.5% ± 0.1%), and cytotoxicity (P < 0.05). The NPUA-based resins exhibit the higher flexural strength, flexural modulus, hardness, and hydrophobicity and lower volumetric shrinkage, water absorption, and solubility compared to the Bis-GMA (70 wt%)/TEGDMA (30 wt%) resins. The NPUA-based composites exhibit significantly higher flexural strength, flexural modulus, and hardness and lower volumetric shrinkage (171.4 ± 3.0 MPa, 12.6 ± 0.5 GPa, 2.0 ± 0.2 GPa, and 3.4% ± 0.2%, respectively) compared to the Bis-GMA group (120.3 ± 4.7 MPa, 9.4 ± 0.7 GPa, 1.5 ± 0.1 GPa, and 4.7% ± 0.2%, respectively; P < 0.05). This work presents a viable avenue for augmenting the physicochemical attributes of dental resins.

Enhanced Bonding to Caries-Affected Dentin Using an Isocyanate-Based Primer.

Dental caries is the most common oral disease and the most common cause of resin restorations. In minimally invasive dentistry, the principle behind cavity preparation is to remove external caries-infected dentin (CID) and preserve internal caries-affected dentin (CAD) and sound dentin (SD). The cavity floor is mainly composed of CAD, but the poor bonding performance of CAD has become a widespread concern. This study evaluated the performance of a new collagen-reactive monomer (ITCM) used as a primer to improve the bonding performance of CAD. The experimental specimens were grouped as follows: SD, CAD, and ITCM-pretreated CAD (CAD-ITCM). Dentin slices were obtained for attenuated total reflectance-Fourier transform infrared (ATR-FTIR) analysis. The bonded samples were subjected to microtensile bond strength analysis after 24 h of water storage or aging by thermocycling, and the bonding interface quality was evaluated by nanoleakage assessment, interfacial nanoindentation testing, and in situ zymography. Cytotoxicity experiments with ITCM were performed. ATR-FTIR showed that the isocyanate groups in ITCM can covalently bind and form hydrogen bonds with the collagen in CAD to mediate chemical bonding. ITCM pretreatment significantly improved the bond strength of CAD (P < 0.05), reduced interfacial nanoleakage, improved the sealing of the bonding interface, enhanced the homogeneity of the hybrid layer, and inhibited matrix metalloproteinase activity. In addition, ITCM presented acceptable biocompatibility for dental restorative application. Taken together, this study reported the application of ITCM to induce collagen-based chemical bonding in the CAD bonding system, which fills the gap in strategies to improve the bonding performance of CAD immediately and after aging and has important clinical application prospects.

Retention of Intrafibrillar Minerals Improves Resin-Dentin Bond Durability.

The concept of extrafibrillar demineralization involves selective removal of apatite crystallites from the extrafibrillar spaces of mineralized dentin without disturbing the intrafibrillar minerals within collagen. This helps avoiding activation of endogenous proteases and enables air-drying of partially demineralized dentin without causing collapse of completely demineralized collagen matrix that adversely affects resin infiltration. The objective of the present study was to evaluate the potential of quaternized carboxymethyl chitosan (QCMC)-based extrafibrillar demineralization in improving resin-dentin bond durability. Isothermal titration calorimetry indicated that QCMC synthesized by quaternization of O-carboxymethyl chitosan had moderate affinity for Ca2+ (binding constant: 8.9 × 104 M-1). Wet and dry bonding with the QCMC-based demineralization produced tensile bond strengths equivalent to the phosphoric acid (H3PO4)-based etch-and-rinse technique. Those bond strengths were maintained after thermocycling. Amide I and PO43- mappings of QCMC-conditioned dentin were performed with atomic force microscope-infrared spectroscopy (AFM-IR). Whereas H3PO4-etched dentin exhibited an extensive reduction in PO43- signals corresponding to apatite depletion, QCMC-conditioned dentin showed scattered dark areas and bright PO43- streak signals. The latter were consistent with areas identified as collagen fibrils in the amide I mapping and were suggestive of the presence of intrafibrillar minerals in QCMC-conditioned dentin. Young's modulus mapping of QCMC-demineralized dentin obtained by AFM-based amplitude modulation-frequency modulation recorded moduli that were the same order of magnitude as those in mineralized dentin and at least 1 order higher than H3PO4-etched dentin. In situ zymography of the gelatinolytic activity within hybrid layers created with QCMC conditioning revealed extremely low signals before and after thermocycling, compared with H3PO4-etched dentin for both wet and dry bonding. Confocal laser scanning microscopy identified the antibacterial potential of QCMC against Streptococcus mutans and Enterococcus faecalis biofilms. Taken together, the QCMC-based demineralization retains intrafibrillar minerals, preserves the elastic modulus of collagen fibrils, reduces endogenous proteolytic activity, and inhibits bacteria biofilms to extend dentin bond durability.

Applications of Cryogenic Electron Microscopy in Biomineralization Research.

Biological mineralization is a natural process manifested by living organisms in which inorganic minerals crystallize under the scrupulous control of biomolecules, producing hierarchical organic-inorganic composite structures with physical properties and design that galvanize even the most ardent structural engineer and architect. Understanding the mechanisms that control the formation of biominerals is challenging in the biomimetic engineering of hard tissues. In this regard, the contribution of cryogenic electron microscopy (cryo-EM) has been nothing short of phenomenal. By preserving materials in their native hydrated status and reducing damage caused by ion beam radiation, cryo-EM outperforms conventional transmission electron microscopy in its ability to directly observe the morphologic evolution of mineral precursor phases at different stages of biomineralization with nanoscale spatial resolution and subsecond temporal resolution in 2 or 3 dimensions. In the present review, the development and applications of cryo-EM are discussed to support the use of this powerful technique in dental research. Because of the rapid development of cryogenic sample preparation techniques, direct electron detection, and image-processing algorithms, the last decade has witnessed an exponential increase in the use of cryo-EM in structural biology and materials research. By amalgamating with other analytic techniques, cryo-EM may be used for qualitative and quantitative analyses of the kinetics and thermodynamic mechanisms in which organic macromolecules participate in the transformation of mineral precursors from their original liquid state to amorphous and ultimately crystalline phases. The present review concentrates on the biomineralization of calcium phosphate mineral phases, while that of calcium carbonate, silica, and magnetite is only briefly mentioned. Bioinspired organic matrix-mediated inorganic crystallization strategies are discussed from the perspective of tissue regeneration engineering.

Dentin Cross-linking Effect of Carbodiimide After 5 Years.

Carbodiimide (EDC)-based dentin primers preserve hybrid layer (HL) integrity. However, aging >1 y has not been investigated. The present study examined whether the cross-linking effect of EDC was reflected in dentin bond strength, endogenous enzymatic activity, and the chemical profile of the HL after 5-y aging in artificial saliva. Noncarious human third molars (N = 42) were cut to expose middle/deep coronal dentin and treated as follows: group 1, dentin etched with 35% H3PO4, pretreated with a 0.3M aqueous EDC primer for 1 min and restored with XP Bond (Dentsply Sirona); group 2, as in group 1 but without EDC pretreatment; group 3, Clearfil SE Bond (Kuraray-Noritake) primer applied to dentin surface, followed by EDC pretreatment as in group 1 and application of bond; group 4, as in group 3 without EDC pretreatment. After composite buildup, the specimens were cut into sticks or slabs, depending on the experiment. All tests were performed at baseline (T0) and after 5 y of aging (T5) in artificial saliva at 37 °C. Microtensile bond strength (µTBS) was tested at a crosshead speed of 1 mm/min until failure. Endogenous enzymatic activity was investigated with in situ zymography. The chemical profile of HL was determined via Raman spectroscopy. Three-way analysis of variance and post hoc Tukey test were used to analyze µTBS and in situ zymography data (α = 0.05). EDC pretreatment and aging significantly influenced µTBS and in situ zymography results (P < 0.05). Higher bond strength and lower gelatinolytic activity were identified in the EDC-treated groups at T5 (P < 0.05), especially in the etch-and-rinse groups. Raman spectra revealed less defined amide III peaks in control specimens at T5. The EDC cross-linking effect persisted in the HL for 5 y in terms of bond strength, collagen structure preservation, and dentinal enzyme silencing.

A novel dentin bonding scheme based on extrafibrillar demineralization combined with covalent adhesion using a dry-bonding technique.

Dentin bonding is a dynamic process that involves the penetration of adhesive resin monomers into the extrafibrillar and intrafibrillar demineralized collagen matrix using a wet-bonding technique. However, adhesive resin monomers lack the capacity to infiltrate the intrafibrillar space, and the excess water that is introduced by the wet-bonding technique remains at the bonding interface. This imperfectly bonded interface is inclined to hydrolytic degradation, severely jeopardizing the longevity of bonded clinical restorations. The present study introduces a dentin bonding scheme based on a dry-bonding technique, combined with the use of extrafibrillar demineralization and a collagen-reactive monomer (CRM)-based adhesive (CBA). Selective extrafibrillar demineralization was achieved using 1-wt% high-molecular weight (MW) carboxymethyl chitosan (CMCS) within a clinically acceptable timeframe to create a less aggressive bonding substance for dentin bonding due to its selectively extrafibrillar demineralization capacity. CMCS demineralization decreased the activation of in situ collagenase, improved the shrinking resistance of demineralized collagen, and thus provided stronger and more durable bonding than traditional phosphoric acid etching. The new dentin bonding scheme that contained CMCS and CBA and used a dry-bonding technique achieved an encouraging dentin bonding strength and durability with low technical sensitivity. This bonding scheme can be used to improve the stability of the resin-dentin interface and foster the longevity of bonded clinical restorations.

Application of Artificial Intelligence in Dentistry.

Artificial intelligence (AI) is a technology that utilizes machines to mimic intelligent human behavior. To appreciate human-technology interaction in the clinical setting, augmented intelligence has been proposed as a cognitive extension of AI in health care, emphasizing its assistive and supplementary role to medical professionals. While truly autonomous medical robotic systems are still beyond reach, the virtual component of AI, known as software-type algorithms, is the main component used in dentistry. Because of their powerful capabilities in data analysis, these virtual algorithms are expected to improve the accuracy and efficacy of dental diagnosis, provide visualized anatomic guidance for treatment, simulate and evaluate prospective results, and project the occurrence and prognosis of oral diseases. Potential obstacles in contemporary algorithms that prevent routine implementation of AI include the lack of data curation, sharing, and readability; the inability to illustrate the inner decision-making process; the insufficient power of classical computing; and the neglect of ethical principles in the design of AI frameworks. It is necessary to maintain a proactive attitude toward AI to ensure its affirmative development and promote human-technology rapport to revolutionize dental practice. The present review outlines the progress and potential dental applications of AI in medical-aided diagnosis, treatment, and disease prediction and discusses their data limitations, interpretability, computing power, and ethical considerations, as well as their impact on dentists, with the objective of creating a backdrop for future research in this rapidly expanding arena.

Oropharyngeal Secretion as Alternative for SARS-CoV-2 Detection.

This study aimed to determine if sampling of oropharyngeal secretions (OSs) helps improves detection of SARS-CoV-2 RNA by nucleic acid amplification testing of potential patients with COVID-19. The first prospective study consisted of 75 patients with COVID-19 who were ready for discharge and who had 2 consecutive negative results per nucleic acid amplification testing (NAAT) of viral samples retrieved with nasopharyngeal swabs (NPSs). Because of detection of potential false negatives in that cohort, the NAAT results of paired OS and NPS samples from 50 additional recruits with COVID-19 during their recovery stage were used in a second prospective study to compare the diagnostic values of the 2 viral RNA sampling methods. For identification of the frequency of inconsistency between the sampling methods, the McNemar's test was used for difference analysis and the kappa statistic for consistency analysis. OSs obtained from 2 of the 75 participants in the first study yielded positive results for SARS-CoV-2 nucleic acid. Both were male and aged >60 y. Subsequent chemiluminescence enzyme immunoassays indicated that they were positive for the SARS-CoV-2 IgM and IgG antibodies. For parallel NAAT of OS and NPS samples in the second study, McNemar's test indicated that the difference between the frequencies of inconsistent parts of OS and NPS was statistically significant (P = 0.021). Cohen's kappa coefficient for OS and NPS was 0.244, which is indicative of fair consistency. The NPS test has a risk of sending home more patients (59%) who still have the infection, while the OS test will make such an error in fewer patients (14%). Although OS sampling improves the accuracy of SARS-CoV-2 nucleic acid testing, it has to be emphasized that this conclusion is based on a very small sample size. Detection of viral RNA from a patient's secretions is not confirmative of viral infectivity.

Isocyanate-terminated urethane-based methacrylate for in situ collagen scaffold modification.

When damaged or fractured collagen-rich hard tissues are repaired by resin material, the collagen matrix may be used as a scaffold, after removal of the natural minerals, for resin monomers to penetrate and polymerize in-situ. Formation of a collagen-polymer hybrid biocomposite via mechanical hybridization provides a stable and strong link between endogenous tissue and the prosthesis for successful clinical integration. However, the heterogeneity between hydrophobic resin polymers and hydrophilic collagen presents a challenge to the quality of hybrid biocomposite. The objective of the present study was to evaluate the potential benefits of a collagen-reactive monomer (CRM, an isocyanate-terminated urethane-based methacrylate) with covalent affinity to collagen as "chemical link" to enhance in-situ resin hybridization within a collagen scaffold. Here, the CRM ligand with active isocyanate group may be chemically grafted onto the collagen receptor via covalent and hydrogen bonds. Dentin-derived collagen chemical modified by CRM shows improved mechanical property, thermostability and enzymatic stability. Moreover, CRM inhibited both exogenous and endogenous collagenase activities. The modification of collagen by chemical grafting of resin monomers improved its mechanical and physicochemical properties and demonstrated the potential of CRM for use in promoting chemical adhesion and creating a much stronger and durable bonding interface. Formation of a chemical bond between polymer and collagen scaffold in-situ improves the mechanical performance of collagen and may create a much stronger and durable collagen-polymer hybrid material. Addition of CRM into adhesives might effectively prolong the longevity of clinical resin-bonded restorations.

Evaluation of a Collagen-Reactive Monomer with Advanced Bonding Durability.

This study evaluated the use of a new collagen-reactive monomer (CRM), isocyanate-terminated urethane methacrylate precursor, which has covalent affinity to dental collagen, in the formation of dentin-resin bonds and compared it with 2 other dental adhesives. Dentin specimens were bonded with either the CRM-based adhesive (CBA), One-Step (OS; Bisco, Inc.), or a negative adhesive (NA) control and subjected to 24-h storage in water, thermocycling to simulate 1-y clinical function, or a matrix metalloproteinase-mediated aging process. We tested the microtensile bond strength (µTBS), characterized the bonding interface with an atomic force microscope, conducted micro-Raman analysis, and performed leakage tests and in situ zymography. CBA and OS exhibited comparable bonding strength after 24 h (P > 0.05); however, there was a sharp decrease in µTBS after aging for all except CBA (P < 0.001). Raman spectra results indicated increased collagen crosslinking and chemical reaction between the adhesive and collagen in the CBA group. CBA achieved high-quality hybridization with collagen, improving mechanical properties and integrity, and decreased the enzyme-mediated degradation of the bonding interface by inhibiting collagenolytic activity. With the promising bonding durability of coapplied CBA, CRM may be the first dental adhesive to provide strong and long-lasting resin-dental collagen bonding without the additional conditioning step. The use of CBA results in high-quality hybrid layers that protect the resin-dentin interface from harmful biological and chemical activities commonly occurring in the oral environment.

Improving the wear performance of feldspathic veneering porcelain by ion-exchange strengthening.

OBJECTIVES: The present study examined the effects of Na+→K+ ion-exchange on the wear performance of feldspathic veneering porcelain. METHODS: Bar and disk specimens were prepared using IPS classic as the feldspathic veneering porcelain. After ion-exchange by immersion of the specimens in melted KNO3 at two temperatures for different time-periods, the bars were tested for flexural strength and Vickers surface hardness. The disks were paired with zirconia antagonists and tested with a pin-on-disk tribometer with 10 N for 70☓104 wear cycles in artificial saliva. Wear analysis of the porcelain and zirconia was performed using 3D profilometer and analysed with one-way analysis of variance and Tukey's post-hoc pairwise comparison procedures. Worn surfaces were examined with scanning electron microscopy. RESULTS: The feldspathic veneering porcelain exhibited strong time-dependent wear behaviour, with typical running-in and steady wear stages. Ion-exchange treatments at 380 °C and 440 °C both enhanced the mechanical properties, decreased the wear rates of running-in wear and steady wear. The wear performance of porcelain treated by ion-exchange at lower temperature (380 °C) was improved significantly, especially reducing the wear rate of the running-in stage. CONCLUSION: A thicker ion-exchange layer with less stress relaxation may be obtained by ion-exchange at lower exchange temperature for a long processing time. Such a protocol improves the wear performance of the porcelain effectively. CLINICAL SIGNIFICANCE: Restorations with veneering porcelain may fail prematurely due to excessive wear. It important to improve the wear performance of the porcelain. Ion-exchange has the potential to strengthen dental veneering porcelain. Understanding the effect of ion-exchange on the wear performance of porcelain provides insight improving the wear performance of these restorations.

Contribution of biomimetic collagen-ligand interaction to intrafibrillar mineralization.

Contemporary models of intrafibrillar mineralization mechanisms are established using collagen fibrils as templates without considering the contribution from collagen-bound apatite nucleation inhibitors. However, collagen matrices destined for mineralization in vertebrates contain bound matrix proteins for intrafibrillar mineralization. Negatively charged, high-molecular weight polycarboxylic acid is cross-linked to reconstituted collagen to create a model for examining the contribution of collagen-ligand interaction to intrafibrillar mineralization. Cryogenic electron microscopy and molecular dynamics simulation show that, after cross-linking to collagen, the bound polyelectrolyte caches prenucleation cluster singlets into chain-like aggregates along the fibrillar surface to increase the pool of mineralization precursors available for intrafibrillar mineralization. Higher-quality mineralized scaffolds with better biomechanical properties are achieved compared with mineralization of unmodified scaffolds in polyelectrolyte-stabilized mineralization solution. Collagen-ligand interaction provides insights on the genesis of heterogeneously mineralized tissues and the potential causes of ectopic calcification in nonmineralized body tissues.

MMP-8-Responsive Polyethylene Glycol Hydrogel for Intraoral Drug Delivery.

Currently available drug delivery systems for oral diseases suffer from short retention time and poor local concentrations at the target site. A biodegradable stimulus-responsive hydrogel was synthesized in the present study to evaluate its application as an environmentally sensitive carrier for on-demand intraoral drug delivery. The hydrogel was synthesized from diacrylate-containing polyethylene glycol-based scaffolds and a cysteine-terminated peptide crosslinker (CGPQG↓IWGQC) via a Michael-type addition reaction. Because CGPQG↓IWGQC can be cleaved by matrix metalloproteinase 8 (MMP-8), minocycline hydrochloride, bovine serum albumin, or an antibacterial peptide (KSL) was incorporated into the scaffolds to evaluate the MMP-8-responsive release behavior of the on-demand drug delivery system. Hydrogel characterization and gelation kinetics were examined with gel time, Fourier-transform infrared spectroscopy, scanning electron microscopy, and measurements of rheologic parameters. Degradation behavior and MMP-8-responsive drug release were performed by high-performance liquid chromatography and protein-specific assay. Biocompatibility evaluation indicated that the hydrogels were noncytotoxic. Antibacterial testing demonstrated that the released drugs were able to maintain bioactivity. Taken together, these results suggest that the MMP-8-sensitive hydrogel is a promising candidate for on-demand intraoral localized drug delivery. Because MMP-8 is one of the most important biomarkers for periodontitis, the MMP-8-responsive hydrogel has potential to be used for in situ adaptive degradation in response to chronic periodontitis and peri-implantitis. This notion has to be tested in animal models of periodontal disease.

Evaluation of several instrumentation techniques and irrigation methods on the percentage of untouched canal wall and accumulated dentine debris in C-shaped canals.

AIM: To evaluate the effect of instrumentation using Reciproc Blue (RB; VDW, Munich, Germany) and XP-endo Shaper (XP-S; FKG Dentaire, La Chaux-deFonds, Switzerland) systems on the area of untouched canal wall (AUCW), accumulated hard-tissue debris (AHTD) and the efficacy of three irrigation protocols on percentage reductions (red%) of AHTD within C-shaped canals of mandibular molars. METHODOLOGY: Seventy mandibular molars with C-shaped canals were scanned, matched and assigned to two shaping groups (n = 35): RB and XP-S. Following instrumentation, specimens were triple-matched with respect to the amount of remaining debris and assigned to three irrigation subgroups (n = 10): syringe-and-needle irrigation (SNI), XP-endo Finisher (XP-F; FKG Dentaire) and passive ultrasonic irrigation (PUI). The AUCW% and AHTD% after instrumentation and the red% of AHTD after irrigation were calculated from micro-computed tomography. Data were analysed using comparisons for two groups (RB vs. XP-S) or multiple subgroups followed by pairwise comparison procedures (SNI vs. XP-F vs. PUI) at α = 0.05. RESULTS: For RB and XP-S, 33.04% and 30.45%, respectively, of the canal wall remained untouched (P > 0.05). For both groups, the apical third had larger AUCW% than the coronal third (P < 0.05). Instrumentation with RB left more debris (2.8%) than XP-S (1.1%) (P < 0.05). The PUI and XP-F subgroups had higher mean red% of AHTD than the SNI subgroup; the difference was significant for RB (P < 0.05) but not for XP-S. CONCLUSIONS: Both RB and XP-S systems were associated with similar AUCW after instrumenting C-shaped canals. RB left significantly greater levels of AHTD compared with XP-S. PUI and XP-F irrigation removed more debris than SNI when using the RB system.

Chitosan-Based Extrafibrillar Demineralization for Dentin Bonding.

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.

Zymography of Hybrid Layers Created Using Extrafibrillar Demineralization.

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.

Substantivity of Carbodiimide Inhibition on Dentinal Enzyme Activity over Time.

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.