Monica Yamauti

Zinc reduces collagen degradation in demineralized human dentin explants

OBJECTIVES Dentin matrix metalloproteinases are implicated in the pathogenesis of caries and contribute to collagen degradation in resin-dentin interfaces. The objective was to determine if collagen degradation may be modulated by an excess of zinc or zinc chelators. METHODS Mineralized and phosphoric acid demineralized human dentin specimens were tested. Chlorhexidine digluconate, doxycycline or ZnCl₂ were added to the media. In half of the groups, active exogenous metalloproteinase-2 was incorporated into the solution. C-terminal telopeptide determinations (radioimmunoassay) were performed after 24 h, 1 and 3 weeks. RESULTS Collagen degradation was prominent in demineralized dentin. Doxycycline fully blocked dentin proteolysis. Chlorhexidine digluconate reduced the degradation at the 24-h period. Zinc in excess strongly inhibits hydrolysis of collagen and its effect was maintained for 3 weeks. CONCLUSIONS Zinc in excess reduces MMP-mediated collagen degradation. The hypothesis that binding of zinc to collagen results in protection of sensitive cleavage sites of metalloproteinases requires further validation.

A ZnO-doped adhesive reduced collagen degradation favouring dentine remineralization

OBJECTIVES The objective of the study was to determine the efficacy of a ZnO-doped etch and rinse adhesive in decreasing MMPs-mediated collagen degradation at the resin-dentine hybrid layer, and increasing bonding stability. METHODS C-terminal telopeptide concentrations (ICTP) were determined after 24h, 1wk and 4wk in human dentine beams. Dentine was treated: (1) 37% phosphoric acid for 15s (PA), (2) PA-etched dentine infiltrated with Single Bond (SB), (3) PA-etched dentine infiltrated with ZnO doped SB (ZnO particles--10wt%--were added to the bonding resin) (ZnO-SB), and (4) Clearfil SE Bond primed-dentine was infiltrated with Clearfil SE bonding resin (CSE). Microtensile bond strength (MTBS) was assessed for the different groups at 24h and after 3months. Debonded dentine surfaces were studied by scanning electron microscopy. RESULTS MMPs-mediated collagen degradation occurred in demineralized dentine (PA). Resin infiltration decreased collagen degradation. The lowest collagen degradation was found for Zn-doped SB, followed by CSE. When these adhesives were applied, ICTP values did not change throughout the study period. At 24h, similar MTBS was attained for all adhesives. Only SB decreased MTBS after three months. CONCLUSIONS Addition of ZnO particles to SB produced a reduction in dentine collagen degradation and increased resin-dentine bonds durability. In Zn-doped adhesive interfaces, a calcium phosphate layer and tubular occlusion was encountered at the debonded interface. CLINICAL SIGNIFICANCE ZnO particles addition into the bonding resin of SB makes a breakthrough to prevent the hybrid layer degradation and to preserve its bonding efficacy overtime.

Zinc-doped dentin adhesive for collagen protection at the hybrid layer.

The aim of the study was to ascertain whether the addition of zinc to adhesives may decrease metalloproteinase-mediated collagen degradation without affecting bonding efficacy. Human dentin beams were treated with phosphoric acid, with Clearfil SE Bond Primer or with Clearfil SE Bond Primer plus ZnCl(2) (2 wt%). Acid-etched dentin was infiltrated with Single Bond, Single Bond plus ZnCl(2) (2 wt%), or Single Bond plus ZnO nanoparticles (10 wt%), and Clearfil SE Bond-primed dentin was infiltrated with Clearfil SE Bonding resin, Clearfil SE-Bonding resin with ZnCl(2) (2 wt%), or Clearfil SE-Bonding resin with ZnO nanoparticles (10 wt%). The C-terminal telopeptide concentrations were determined 24 h, and 1 and 4 wk after treatment. Microtensile bond strength to dentin was determined for the tested adhesives. Matrix metalloproteinases-mediated collagen degradation occurred in acid-etched and SE-primed dentin. Resin infiltration decreased collagen degradation. Lower collagen degradation was found for SE Bond than for Single Bond. Zinc-doped Single Bond resin always reduced collagen degradation, the ZnO particles being more effective than ZnCl(2) . Zinc-doped SE Bond reduced the liberation of C-terminal telopeptide only at 24 h. Bond strength to dentin was not decreased when Zn-doped resins were employed, except when ZnCl(2) was added to SE Primer. Zinc-doped resin reduced collagen degradation in Single Bond hybrid layers, but did not affect bond strength. The addition of zinc to SE Bond had no beneficial effects.

Zinc-Inhibited MMP-Mediated Collagen Degradation after Different Dentine Demineralization Procedures

Background: Dentine matrix metalloproteinases (MMPs) play an important role in the dentine caries process. Aims: To determine if MMP-mediated collagen degradation of acid-demineralized dentine may be inhibited by zinc or zinc chelators. Methods: Human dentine specimens were demineralized by phosphoric acid (PA), ethylenediaminetetraacetic acid (EDTA), Clearfil SE Bond primer (SE), or Xeno V (XE) and stored in artificial saliva. Chlorhexidine digluconate (CHX), doxycycline, EDTA, or ZnCl2 was added. C-terminal telopeptide determinations (ICTP) were performed by radioimmunoassay after 24 h and 4 weeks. Results: Collagen degradation was prominent in PA-demineralized (ICTP values from 74.01 µg/l at 24 h to 202.46 µg/l after 4 weeks) and EDTA-demineralized dentine (ICTP values from 83.93 µg/l at 24 h to 158.82 µg/l after 4 weeks) stored in artificial saliva. Doxycycline fully blocked proteolysis. CHX and EDTA reduced collagen degradation only at 24 h. Zinc in excess strongly inhibited hydrolysis of collagen in all tested groups (ICTP values were: PA, 13.56 µg/l; EDTA, 11.21 µg/l; SE, 1.52 µg/l, and XE, 2.37 µg/l) and its effect was maintained for up to 4 weeks, except for EDTA-treated dentine (ICTP values were: PA, 40.76 µg/l; EDTA, 79.15 µg/l; SE, 5.29 µg/l, and XE, 6.38 µg/l). Conclusion: EDTA and CHX exerted time-limited MMP inhibition, and excess zinc served as an effective inhibitor of MMP-mediated collagen degradation in strong or mildly demineralized dentine. MMP degradation of collagen was reduced in resin-infiltrated dentine; the presence of excess zinc chloride exerted an additional protective effect.

Experimental resin cements containing bioactive fillers reduce matrix metalloproteinase-mediated dentin collagen degradation.

INTRODUCTION Collagen dentin matrix may represent a suitable scaffold to be remineralized in the presence of bioactive materials. The purpose of this study was to determine if experimental resin cements containing bioactive fillers may modulate matrix metalloproteinase-mediated collagen degradation of etched dentin. METHODS Human dentin beams demineralized using 10% phosphoric acid or 0.5 mol/L EDTA were infiltrated with the following experimental resins: (1) unfilled resin, (2) resin with Bioglass 45S5 particles (Sylc; OSspray Ltd, London, UK), and (3) resin with β-tricalcium phosphate-modified calcium silicate cement (HCAT-β) particles. The filler/resin ratio was 40/60 wt%. The specimens were stored in artificial saliva, and the determination of C-terminal telopeptide (ICTP) was performed by radioimmunoassay after 24 hours, 1 week, and 4 weeks. Scanning electron microscopic analysis of dentin surfaces after 4 weeks of storage was also executed. RESULTS Collagen degradation was prominent both in phosphoric acid and EDTA-treated dentin. Resin infiltration strongly reduced the MMP activity in demineralized dentin. Resin-containing Bioglass 45S5 particles exerted higher and more stable protection of collagen at all tested dentin states and time points. HCAT-β induced collagen protection from MMPs only in EDTA-treated specimens. Dentin remineralization was achieved when dentin was infiltrated with the resin cements containing bioactive fillers. CONCLUSIONS MMP degradation of dentin collagen is strongly reduced in resin-infiltrated dentin. The inclusion of Bioglass 45S5 particles exerted an additional protection of collagen during dentin remineralization.

Bleaching agents increase metalloproteinases-mediated collagen degradation in dentin.

INTRODUCTION Tooth bleaching is based on hydrogen peroxide application. The Objective of this study was to determine whether dental bleaching agents affect metalloproteinases-mediated dentin collagen degradation. METHODS Human dentin specimens were subjected to different treatments: (1) untreated dentin; (2) demineralization by 37% phosphoric acid (PA); (3) demineralization by 37% PA, followed by application of Single Bond (SB); (4) 2 immersions of 7 days each in a nonvital bleaching agent, followed by PA; (5) 2 immersions of 7 days each in nonvital bleaching, followed by PA and SB application; (6) 3 immersions by using in-office bleaching gel for 20 minutes; (7) 3 immersions by using in-office bleaching gel for 20 minutes plus activation with a light source; and (8) immersion in home bleaching gel for 8 hours per day during 3 weeks. Specimens were stored in artificial saliva. C-terminal telopeptide determinations (radioimmunoassay) were performed after 24 hours, 1 week, and 4 weeks. RESULTS Bleaching agents increased collagen degradation, but C-terminal telopeptide of type I collagen (ICTP) values were higher when dentin was PA-demineralized. Nonvital bleaching plus PA promoted the highest collagenolytic activity, which was reduced after SB infiltration. Halogen light application did not influence ICTP values. At 24 hours, home bleaching exhibited high collagenolytic activity, which decreased up to 4 weeks. After 4 weeks of storage, all bleaching procedures showed similar values of collagen degradation, which were not different from those of PA-demineralized and resin-infiltrated dentin. CONCLUSIONS All tested bleaching agents increase matrix metalloproteinases-mediated collagen degradation in dentin. This effect was not completely reverted after 4 weeks. Home bleaching induced the highest collagen degradation.

In vitro load-induced dentin collagen-stabilization against MMPs degradation

INTRODUCTION Teeth are continuously subjected to stresses during mastication, swallowing and parafunctional habits, producing a significant reduction of the bonding efficacy in adhesive restorations. The purpose of this study was to evaluate the metalloproteinases (MMPs)-mediated dentin collagen degradation of hybrid layers created by using different demineralization processes, previous resin infiltration, and in vitro mechanical loading. METHODS Human dentin beams (0.75×0.75×5.0mm) were subjected to different treatments: (1) untreated dentin; (2) demineralization by 37% phosphoric acid (PA) or by 0.5% M ethylenediaminetetraacetic acid (EDTA); (3) demineralization by PA, followed by application of Adper(™) Single Bond (SB); (4) demineralization by EDTA, followed by application of SB. In half of the specimens, mechanical loadings (100,000 cycles, 2Hz, 49N) were applied to dentin beams. Specimens were stored in artificial saliva. C-terminal telopeptide (ICTP), determinations (which indicates the amount of collagen degradation) (radioimmunoassay) were performed after 24h, 1 week and 4 weeks. RESULTS Load cycling decreased collagen degradation when dentin was untreated or PA-demineralized and EDTA-treated. ICTP values increased when both PA-demineralized and EDTA-treated and infiltrated with SB dentin beams were loaded, except in samples that were subjected to EDTA treatment and SB infiltration after 4w of storage, which showed similar values of collagenolytic activity than the non loaded specimens. Load cycling preserved the initial (24h) ICTP determination at any time point, in all groups of the study, except in PA-demineralized and SB infiltrated dentin which showed an increased of collagen degradation values, over time. This same trend was observed in all groups without loading. INTERPRETATION Mechanical loading enhances collagens resistance to enzymatic degradation in natural and demineralized dentin. Mild acids (EDTA) lead to a lower volume of demineralized/unprotected collagen to be cleaved by MMPs. Load cycling produced an increase of collagen degradation when PA-demineralized dentin and EDTA-treated dentin were infiltrated with resin, but EDTA-treated dentin showed a constant collagenolytic degradation, over time.

Differential resin-dentin bonds created after caries removal with polymer burs.

The objective of this article was to investigate the effect of carbide and polymer burs caries removal methods on the bond strength of different adhesives to dentin. Resin restorations were performed in sound and caries-affected dentin, after using polymer or carbide burs and bonding with four different adhesive (Single bond, SB; Clearfil SE bond, SEB; FL-Bond II, FLB; and Fuji II-LC, FUJI). Microtensile bond strength (MTBS) was measured. Data were analyzed with ANOVA and Student-Newman-Keuls tests. Debonded surfaces were observed by scanning electron microscopy. Bonded interfaces were examined using light microscopy (Massons trichrome staining). In sound dentin, MTBS was similar for SEB and SB, and higher than that of FLB and FUJI. Bond strength to carbide bur prepared dentin was similar for SB, SEB, and FLB; FUJI presented the lowest. SB applied on polymer bur excavated dentin presented similar values to those of the carbide bur group; MTBS attained by SEB, FLB, and FUJI decreased when bonding to dentin treated with polymer burs; FUJI yielded pretesting failures in all specimens. Polymer burs created a thick smear layer that was not infiltrated by tested self-etching agents. The bonding effectiveness of self-etching and glass-ionomer-like adhesives to dentin decreased when polymer burs were used.

Resistance to degradation of resin-dentin bonds produced by one-step self-etch adhesives.

The objective of this article is to evaluate the resistance to degradation of resin-dentin bonds formed with three one-step adhesives. Flat, mid-coronal dentin surfaces were bonded with the self-etching adhesives [Tokuyama Bond Force (TBF), One Up Bond F Plus (OUB), and G-Bond (GB)]. The bonded teeth were subjected to fatigue loading, chemical degradation, and stored in distilled water for four time periods (up to 12 months). Specimens were tested for microtensile bond strength and microleakage. Fractographic analysis was performed by scanning electron microscopy. Bonded interfaces were examined by light microscopy using Massons trichrome staining. An atomic force microscope was employed to analyze phase separation and surface nanoroughness (Ra) at the polymers. Vickers microhardness and the degree of the conversion (DC) were also determined. ANOVA and multiple comparisons tests were performed. Bond strength significantly decreased after the chemical challenge, but not after load cycling. Aging decreased bond strength after 6 months in TBF and GB, in OUB after 12 months. An increase of the nonresin protected collagen zone occurred in all groups, after storing. TBF showed the highest roughness, microhardness, and DC values, and GB showed the lowest. Mild self-etch one-step adhesives (TBF/OUB) showed a higher degree of cure, lower hydrophilicity, and major resistance to degradation of resin-dentin bonds when compared to highly acidic self-etching adhesive (GB).

Characterization of Micro- and Nanophase Separation of Dentin Bonding Agents by Stereoscopy and Atomic Force Microscopy

The aim was to study the effect of solvents on the phase separation of four commercial dental adhesives. Four materials were tested: Clearfil™ SE Bond (CSE), Clearfil Protect Bond (CPB), Clearfil S3 Bond (CS3), and One-Up Bond F Plus (OUB). Distilled water or ethanol was used as a solvent (30 vol%) for microphase separation studies, by stereoscopy. For nanophase images, the mixtures were formulated with two different solvent concentrations (2.5 versus 5 vol%) and observed by atomic force microscopy. Images were analyzed by using MacBiophotonics ImageJ to measure the area of bright domains. Macrophase separations, identified as a loss of clarity, were only observed after mixing the adhesives with water. Nanophase separations were detected with all adhesive combinations. The area of bright domains ranged from 132 to 1,145 nm² for CSE, from 15 to 285 nm² for CPB, from 149 to 380 nm² for CS3, and from 26 to 157 nm² for OUB. In water-resins mixtures, CPB was the most homogeneous and OUB showed the most heterogeneous phase formation. In ethanol-resin mixtures, CSE attained the most homogeneous structure and OUB showed the most heterogeneous phase. Addition of 5 vol% ethanol to resins decreased the nanophase separation when compared with the control materials.