Milena Cadenaro

Use of a specific MMP-inhibitor (galardin) for preservation of hybrid layer.

OBJECTIVE Dentinal MMPs have been claimed to contribute to the auto-degradation of collagen fibrils within incompletely resin-infiltrated hybrid layers and their inhibition may, therefore, slow the degradation of hybrid layer. This study aimed to determine the contribution of a synthetic MMPs inhibitor (galardin) to the proteolytic activity of dentinal MMPs and to the morphological and mechanical features of hybrid layers after aging. METHODS Dentin powder obtained from human molars was treated with galardin or chlorhexidine digluconate and zymographically analyzed. Microtensile bond strength was also evaluated in extracted human teeth. Exposed dentin was etched with 35% phosphoric acid and specimens were assigned to (1) pre-treatment with galardin as additional primer for 30s and (2) no pre-treatment. A two-step etch-and-rinse adhesive (Adper Scotchbond 1XT, 3M ESPE) was then applied in accordance with manufacturers instructions and resin composite build-ups were created. Specimens were immediately tested for their microtensile bond strength or stored in artificial saliva for 12 months prior to being tested. Data were evaluated by two-way ANOVA and Tukeys tests (alpha=0.05). Additional specimens were prepared for interfacial nanoleakage analysis under light microscopy and TEM, quantified by two independent observers and statistically analyzed (chi(2) test, alpha=0.05). RESULTS The inhibitory effect of galardin on dentinal MMPs was confirmed by zymographic analysis, as complete inhibition of both MMP-2 and -9 was observed. The use of galardin had no effect on immediate bond strength, while it significantly decreased bond degradation after 1 year (p<0.05). Interfacial nanoleakage expression after aging revealed reduced silver deposits in galardin-treated specimens compared to controls (p<0.05). CONCLUSIONS This study confirmed that the proteolytic activity of dentinal MMPs was inhibited by the use of galardin in a therapeutic primer. Galardin also partially preserved the mechanical integrity of the hybrid layer created by a two-step etch-and-rinse adhesive after artificial aging.

The Inhibitory Effects of Quaternary Ammonium Methacrylates on Soluble and Matrix-bound MMPs

Matrix metalloproteinases (MMPs) bound to dentin contribute to the progressive degradation of collagen fibrils in hybrid layers created by dentin adhesives. This study evaluated the MMP-inhibiting potential of quaternary ammonium methacrylates (QAMs), with soluble rhMMP-9 and a matrix-bound endogenous MMP model. Six different QAMs were initially screened by a rhMMP-9 colorimetric assay. For the matrix-bound endogenous MMPs, we aged demineralized dentin beams for 30 days in calcium- and zinc-containing media (CM; control), chlorhexidine, or QAMs in CM to determine the changes in dry mass loss and solubilization of collagen peptides against baseline levels. The inhibitory effects of QAMs on soluble rhMMP-9 varied between 34 and 100%. Beams incubated in CM showed a 29% decrease in dry mass (p < 0.05), whereas beams incubated with QAMs showed only 0.2%-6% loss of dry mass. Significantly more solubilized collagen was detected from beams incubated in CM (p < 0.05). It is concluded that QAMs exhibited dentin MMP inhibition comparable with that of chlorhexidine, but required higher concentrations.

Effects of different exposure times and concentrations of sodium hypochlorite/ethylenediaminetetraacetic acid on the structural integrity of mineralized dentin.

INTRODUCTION This study tested the null hypothesis that there is no difference between the use of 1.3% NaOCl/17% ethylenediaminetetraacetic acid (EDTA) and 5.25% NaOCl/17% EDTA irrigation regimens on the collagen degradation and flexural strength reduction in mineralized dentin. METHODS Dentin powder and mineralized dentin sections were immersed in 1.3% or 5.25% NaOCl for 10-240 minutes and then rinsed with 17% EDTA as the final irrigant for 2 minutes. Untreated mineralized dentin powder/sections served as controls in the respective experiments. Dentin powders were examined by using Fourier transform infrared (FT-IR) spectroscopy to analyze their relative subsurface intact collagen content with the apatite/collagen ratio. Hydrated dentin sections were subjected to 3-point flexure under water for determining their flexural strengths. RESULTS Collagen degradation was significantly increased and the flexural strength of mineralized dentin was significantly reduced after the use of 5.25% NaOCl as the initial irrigant for more than 1 hour (P < .05). Conversely, changes were insignificant when 1.3% NaOCl was used as the initial irrigant for up to 4 hours (Kruskal-Wallis analysis of variance, n = 10, P < .05). CONCLUSIONS The null hypothesis was rejected. The deleterious effects attributed to the use of NaOCl on dentin are concentration-dependent and time-dependent and are not associated with the demineralization caused by the use of EDTA as the final active irrigant.

Contraction stress of low-shrinkage composite materials assessed with different testing systems

OBJECTIVES The contraction stress of a silorane-based material and a new low-shrinkage nanohybrid composite were compared to three conventional dimethacrylate-based resin composites using two different measuring systems. It was hypothesized that the silorane-based material and the low-shrinkage nanohybrid composite would exhibit lower contraction stress than dimethacrylate-based composites irrespective of measuring system. METHODS The materials tested were Filtek Silorane LS (3M ESPE), Venus Diamond (Heraeus Kulzer), Tetric EvoCeram (Ivoclar Vivadent), Quixfil (Dentsply DeTrey), and Filtek Z250 (3M ESPE). Shrinkage stress was assessed using a stress-strain analyzer consisting of two opposing attachments, one connected to a load sensor and the other fixed to the device, or a system fixed to a universal testing machine with an extensometer as a feedback system. All specimens were light-cured with 20 J/cm(2); the contraction force (N) generated during polymerization was continuously recorded for 300 s. Contraction stress (MPa) was calculated at both 40 s and 300 s. Data were statistically analyzed by three-way ANOVA and Tukeys post hoc test (alpha=0.05). RESULTS Venus Diamond exhibited the lowest stress under both experimental conditions. Stress values scored as follows: Venus Diamond<Tetric EvoCeram<Filtek Silorane LS<Quixfil<Filtek Z250 (p<0.05). Stress values measured with the stress-strain analyzer were significantly lower than those measured with the universal testing machine with feedback. SIGNIFICANCE The hypothesis was partially rejected because only Venus Diamond exhibited the lowest stress values among the tested materials. Contraction stress was higher for all composites when measured in a test system with a feedback. This study confirms that simply reducing the shrinkage does not ensure reduced stress development in composites.

Effects of residual ethanol on the rate and degree of conversion of five experimental resins

OBJECTIVES This study examined the extent of ethanol retention in five comonomer blends of experimental methacrylate-based dental adhesives, containing (10, 20, or 30 wt.%) ethanol, after solvent evaporation, as well as observing the effect of residual ethanol and exposure duration on degree of conversion (DC). The null hypothesis that was tested was that residual, unevaporated ethanol has no effect on the rate or extent of DC of polymerized adhesive resins. METHODS A known mass of each mixture was placed in glass wells and evaporated for 60s. The mass of the mixtures before and after evaporation was measured, allowing calculation of the gravimetric ethanol loss/retention. RESULTS The concentration of retained ethanol increased significantly with ethanol concentration (p<0.01): 1.1-1.9 mole/L for 10% ethanol/90% comonomers, 2.2-3.5 mole/L for 20% ethanol, and 2.6-3.7 mole/L for 30% ethanol/70% comonomers. As ethanol is evaporated from solvated comonomer mixtures, the molar concentration of comonomers increases, reducing the vapor pressure of the remaining ethanol. Thus, the fractional loss of ethanol solvent decreases as the comonomer concentration increases. The DC of 10, 20, and 30 wt.% ethanol blends increased with ethanol concentration in four of the five experimental resins (p<0.05), increasing by 30-45% when 10 or 20 wt.% ethanol was added to neat resins, regardless of exposure duration. Depending on the resin system, inclusion of 30% ethanol lowered DC at 20s but increased DC after 40-60s of light exposure. SIGNIFICANCE Since 10 and 20 wt.% ethanol-resin blends increased the DC of solvated resins by 30-45% over neat resins, the test null hypothesis is rejected. Even with prolonged evaporation, 4-9% residual ethanol concentration can remain in 90/10 (wt./wt.) comonomer-ethanol mixtures. This is thought to be because comonomers lower the vapor pressure of ethanol. This amount of residual ethanol facilitates DC but lowers the rate of polymerization.

Carbodiimide Cross-linking Inactivates Soluble and Matrix-bound MMPs, in vitro:

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

Adhesive performance of a multi-mode adhesive system: 1-Year in vitro study

OBJECTIVES The aim of this study was to investigate the adhesive stability over time of a multi-mode one-step adhesive applied using different bonding techniques on human coronal dentine. The hypotheses tested were that microtensile bond strength (μTBS), interfacial nanoleakage expression and matrix metalloproteinases (MMPs) activation are not affected by the adhesive application mode (following the use of self-etch technique or with the etch-and-rinse technique on dry or wet dentine) or by ageing for 24h, 6 months and 1year in artificial saliva. METHODS Human molars were cut to expose middle/deep dentine and assigned to one of the following bonding systems (N=15): (1) Scotchbond Universal (3M ESPE) self-etch mode, (2) Scotchbond Universal etch-and-rinse technique on wet dentine, (3) Scotchbond Universal etch-and-rinse technique on dry dentine, and (4) Prime&Bond NT (Dentsply De Trey) etch-and-rinse technique on wet dentine (control). Specimens were processed for μTBS test in accordance with the non-trimming technique and stressed to failure after 24h, 6 months or 1 year. Additional specimens were processed and examined to assay interfacial nanoleakage and MMP expression. RESULTS At baseline, no differences between groups were found. After 1 year of storage, Scotchbond Universal applied in the self-etch mode and Prime&Bond NT showed higher μTBS compared to the other groups. The lowest nanoleakage expression was found for Scotchbond Universal applied in the self-etch mode, both at baseline and after storage. MMPs activation was found after application of each tested adhesive. CONCLUSIONS The results of this study support the use of the self-etch approach for bonding the tested multi-mode adhesive system to dentine due to improved stability over time. CLINICAL SIGNIFICANCE Improved bonding effectiveness of the tested universal adhesive system on dentine may be obtained if the adhesive is applied with the self-etch approach.

Degree of conversion of Filtek Silorane Adhesive System and Clearfil SE Bond within the hybrid and adhesive layer: An in situ Raman analysis

OBJECTIVES To examine the degree of conversion (DC) of the adhesive interfaces created by Filtek Silorane Adhesive and Clearfil SE Bond using micro-Raman spectroscopy. METHODS The adhesives were applied on human dentin in accordance with manufacturers instructions. Specimens were cut to expose the bonded interfaces to the micro-Raman beam (Ranishaw InVia; laser wl 785 nm). Raman spectra were collected along the dentin/self-etching primer/adhesive interface at 1 microm intervals. The relative intensities of bands associated with mineral (P-O functional group at 960 cm(-1)) and adhesive (C-C-O group at 605 cm(-1)) components within the bonded interface were used to detect monomer penetration into the dentin matrix and to calculate the degree of conversion (C=C at 1640 cm(-1) as reaction peak, C-C-O at 605 cm(-1) as reference peak). Data were statistically analyzed with two-way ANOVA. RESULTS DC of Filtek Silorane Adhesive was 69+/-7% in the adhesive layer, increasing (p<0.05) to 93+/-5% in the primer and 92+/-9% in the hybrid layer. Clearfil SE Bond showed a DC of 83+/-3% in the hybrid and 85+/-3% in the adhesive layer. Thus, Filtek Silorane Adhesive showed a higher DC than Clearfil SE Bond in the hybrid layer (p<0.05), but a lower DC in the adhesive (p<0.05). SIGNIFICANCE As high DC is a fundamental pre-requisite for the stability of the bond over time, this study supports the hypothesis that optimal stability of Filtek Silorane Adhesive can be obtained. However, further research is needed to investigate the mechanical properties of the hybrid layer created by Filtek Silorane Adhesive and its long-term stability.

Polymerization efficiency and flexural strength of low-stress restorative composites.

OBJECTIVES To assess depth of cure (DOC), degree of conversion (DC), and flexural strength (FS) of several resin composites with low-stress behavior. METHODS SonicFill (Kerr), SureFil(®) SDR™ (Dentsply), everX Posterior (GC), Kalore (GC), and Filtek Silorane (3M ESPE) were tested. DOC was measured with the Acetone Shake test. DC was assessed with Fourier Transform Infra-Red spectroscopy on top and at the bottom of 4mm-thick disk-shaped specimens. Bottom to top ratios of DC percentages were calculated. FS was evaluated with the Three-Point Bending test. DOC, DC, and FS data were statistically analyzed. RESULTS SureFil(®) SDR™ and everX Posterior achieved significantly greater DOC than Kalore and Filtek Silorane. Also, SonicFill had significantly greater DOC than Filtek Silorane. Mean top DCs ranged between 71.46% and 52.44%. Kalore and everX Posterior had significantly lower top DCs than the other materials. Mean DC values at 4mm ranged largely from 57.95% to 6.82%. Kalore and Filtek Silorane had the lowest values of bottom DC and the difference was statistically significant. EverX Posterior and SonicFill recorded significantly higher FSs than the other materials. SIGNIFICANCE SureFil(®) SDR™ and everX Posterior exhibited DOC over 4mm, the maximum thickness recommended for bulk placement, while SonicFill recorded DOC values very close to the 4mm threshold. SonicFill achieved the highest DC at the irradiated surface, as well as at 4mm depth. SureFil(®) SDR™ demonstrated similarly uniform curing through the bulk increment. All the tested composites complied with the requirements of FS established by ISO 4049/2009.

Effects of ethanol addition on the water sorption/solubility and percent conversion of comonomers in model dental adhesives

OBJECTIVES This study evaluated the kinetics of water uptake and percent conversion in neat versus ethanol-solvated resins that were formulated to be used as dental bonding agents. METHODS Five methacrylate-based resins of known and increasing hydrophilicities (R1, R2, R3, R4 and R5) were used as reference materials. Resins were evaluated as neat bonding agents (100% resin) or they were solvated with absolute ethanol (95% resin/5% ethanol or 85% resin/15% ethanol). Specimens were prepared by dispensing the uncured resin into a circular mold (5.8 mm x 0.8 mm). Photo-activation was performed for 80s. The water sorption/diffusion/solubility was gravimetrically evaluated, while the degree of conversion (DC) was calculated by Fourier-transform infrared spectroscopy. RESULTS Water sorption increased with the hydrophilicity of the resin blends. In general, the solvated resins exhibited significantly higher water sorption, solubility and water diffusion coefficients when compared to their corresponding neat versions (p<0.05). The only exception was resin R1, the least hydrophilic resin, in which neat and solvated versions exhibited similar water sorption (p>0.05). Addition of ethanol increased the DC of all resins tested, especially of the least hydrophilic, R1 and R2 (p<0.05). Despite the increased DC of ethanol-solvated methacrylate-based resins, it occurs at the expense of an increase in their water sorption/diffusion and solubility values. SIGNIFICANCE Negative effects of residual ethanol on water sorption/solubility appeared to be greater as the hydrophilicity of the resin blends increased. That is, the use of less hydrophilic resins in dental adhesives may create more reliable and durable bonds to dentin.