Tissue response to white mineral aggregate-based cement containing barium sulfate as alternative radiopacifier: A randomized controlled animal study.

The purpose of this study was to investigate the tissue reaction stimulated by BaSO4 - and Bi2 O3 -containing White MTA Angelus, in comparison with Bi2 O3 -containing white Portland cement, and white ProRoot MTA. Thirty-six adult male Wistar rats (Rattus norvegicus), weighing between 250 and 300 g, were distributed into three groups (n = 12) in accordance with the period of sacrifice (15, 30, and 60 days). Four polyethylene tubes filled with the tested cements were implanted into the dorsum of each rat. Lateral wall of the tubes served as the negative control. After the experimental periods, the animals were euthanized by overdose of pentobarbital anesthetic solution, and the specimens were prepared for microscopic analysis under ×50, ×100, and ×400 magnifications. Inflammatory scores (0-3) were used to grade the tissue reaction. Data were analyzed by the Kruskal-Wallis test and Dunn's test for individual comparisons (p < .05). A mild to moderate inflammatory tissue reaction was observed at the 15-day period, which decreased over the course of the periods for all cements, except for Portland cement. There was no significant difference among the tissue responses for ProRoot MTA, BaSO4 - and Bi2 O3 -containing White MTA Angelus at the 60-day period (p > .05). The Portland group had moderate inflammatory reaction at the final period of analysis, which was statistically different when compared to the other groups (p < .05). The microscopic findings of this animal study suggest that the addition of BaSO4 to White MTA Angelus does not hampers the biocompatibility of the cement.

In vivo and in vitro radiotherapy increased dentin enzymatic activity.

OBJECTIVES: The present study investigated the effects of in vitro and in-vivo radiotherapy on endogenous enzymatic activity in dentin using gelatin zymography and in-situ zymography. METHODS: Gelatin zymographic assays were performed on protein extracts obtained from dentin powder of sound non-irradiated (NRT), in vitro irradiated (VTRT) and in vivo irradiated (VIRT) human teeth. Their proteolytic activities were quantified using band densitometric evaluation. For in-situ zymography, dentin specimens from NRT, VIRT and VTRT were covered with fluorescein-conjugated gelatin and examined with confocal laser-scanning microscopy. Fluorescence intensity emitted by the hydrolyzed fluorescein-conjugated gelatin was quantified and statistically analyzed. In-situ zymography data were statistically analyzed using Kruskal-Wallis ANOVA and Dunn's multiple comparison procedures (α = 0.05). RESULTS: No difference between in vitro and in vivo radiotherapy treatment was found. Both VTRT and VIRT groups showed increase in MMP-9 expression when compared to NRT group. Significant increases (p < 0.05) in gelatinolytic activity (26 % for VTRT; 55 % for VIRT) were observed when compared to the NRT group. CONCLUSION: Radiotherapy increase endogenous enzymatic activity in non-restored dentin.

Insights into cathepsin-B activity in mature dentin matrix.

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.

Chlorhexidine preserves the hybrid layer in vitro after 10-years aging.

OBJECTIVE: The present study investigated the ability of a chlorhexidine (CHX)-containing primer (0.2% aqueous solution) to inhibit dentinal enzymes, preserve the hybrid layer (HL) and remain within the HL, after 10 years of aging in artificial saliva at 37°C. METHODS: Non-carious extracted molars were assigned to two groups, cut into slabs exposing middle/deep dentin, etched and bonded with Adper Scotchbond 1XT (SB1XT) with or without 0.2% CHX aqueous solution pretreatment. Composite build-ups were made, and the specimens were cut in 1-mm thick bonded sticks. In situ zymography was performed on freshly prepared specimens (T0) and specimens aged for 10 years (T10-yr) at 37°C in artificial saliva, to investigate endogenous gelatinolytic activity within the HL. At T10-yr, specimens were also decalcified and embedded in epoxy resin for TEM analysis. Micro-Raman spectroscopy was performed at T0 and T10-yr to evaluate the chemical profiles in intertubular dentin and the HL. RESULTS: In situ zymography showed less pronounced enzymatic activity in the CHX-pretreated group (p<0.05) regardless of aging, maintaining a similar level of fluorescence at T0 and T10-yr (p>0.05). TEM results showed that 98% of the HL had been degraded in the control group, while 95% of the HL was intact in the experimental group. Moreover, all the Raman spectra peaks assigned to CHX could be identified only in the CHX-pretreated group (T0 and T10-yr). SIGNIFICANCE: In vitro, CHX remains in the HL after 10 years with its inhibitory effect preserved. This may be the underlying factor for HL preservation after this long aging period.

Ultra-high-speed videography of resin-dentin interface failure dynamics under tensile load.

OBJECTIVES: Ultra-high-speed (UHS) videography was used to visualize the fracture phenomena at the resin-dentin interface during micro-tensile bond strength (µTBS) test. We also investigated whether UHS videography is applicable for failure-mode analysis. METHODS: Ten human mid-coronal dentin surfaces were bonded using Clearfil SE Bond either in self-etching (SE) or etch-and-rinse (ER) mode. After 24-h water storage, the samples were cut into beams for µTBS test and tested at a cross-head speed of 1 mm/min. The fracture phenomena at the bonded interface were captured using a complementary metal-oxide-semiconductor digital UHS camera at 299,166 frames per second. The failure modes were classified using UHS videography, followed by scanning electron microscopy (SEM) analysis. The failure-mode distributions determined by UHS videography and SEM analysis were statistically analyzed using Fisher's exact test with Bonferroni correction. RESULTS: The crack-propagation speed exceeded 1,500 km/h. No significant difference was found between the SEM and UHS videography failure-mode distributions in the SE mode. A significant difference appeared between them in the ER mode. Significant differences in the incidence of cohesive failures within the adhesive and at the adhesive-composite interface between the SE and ER modes were identified by both SEM and UHS videography. SIGNIFICANCE: UHS videography enabled visualization of the fracture dynamics at the resin- dentin interfaces under tensile load. However, the resolution at such high frame rate was insufficient to classify the failure mode as precisely as that of SEM. Nevertheless, UHS videography can provide more detailed information about the fracture origin and propagation.

Polymer conjugation optimizes EDTA as a calcium-chelating agent that exclusively removes extrafibrillar minerals from mineralized collagen.

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, 1H 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.

Carbodiimide inactivation of matrix metalloproteinases in radicular dentine.

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.

Biochemical and immunohistochemical identification of MMP-7 in human dentin.

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.

Optimizing resin-dentin bond stability using a bioactive adhesive with concomitant antibacterial properties and anti-proteolytic activities.

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 -C18H37 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.

Water-associated attributes in the contemporary dentin bonding milieu.

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.