Antibacterial properties of copper iodide-doped glass ionomer-based materials and effect of copper iodide nanoparticles on collagen degradation.

OBJECTIVES: This study investigated the antibacterial properties and micro-hardness of polyacrylic acid (PAA)-coated copper iodide (CuI) nanoparticles incorporated into glass ionomer-based materials, and the effect of PAA-CuI on collagen degradation. MATERIALS AND METHODS: PAA-CuI nanoparticles were incorporated into glass ionomer (GI), Ionofil Molar AC, and resin-modified glass ionomer (RMGI), Vitrebond, at 0.263 wt%. The antibacterial properties against Streptococcus mutans (n = 6/group) and surface micro-hardness (n = 5/group) were evaluated. Twenty dentin beams were completely demineralized in 10 wt% phosphoric acid and equally divided in two groups (n = 10/group) for incubation in simulated body fluid (SBF) or SBF containing 1 mg/ml PAA-CuI. The amount of dry mass loss and hydroxyproline (HYP) released were quantified. Kruskal-Wallis, Student's t test, two-way ANOVA, and Mann-Whitney were used to analyze the antibacterial, micro-hardness, dry mass, and HYP release data, respectively (p < 0.05). RESULTS: Addition of PAA-CuI nanoparticles into the glass ionomer matrix yielded significant reduction (99.999 %) in the concentration of bacteria relative to the control groups. While micro-hardness values of PAA-CuI-doped GI were no different from its control, PAA-CuI-doped RMGI demonstrated significantly higher values than its control. A significant decrease in dry mass weight was shown only for the control beams (10.53 %, p = 0.04). Significantly less HYP was released from beams incubated in PAA-CuI relative to the control beams (p < 0.001). CONCLUSIONS: PAA-CuI nanoparticles are an effective additive to glass ionomer-based materials as they greatly enhance their antibacterial properties and reduce collagen degradation without an adverse effect on their mechanical properties. CLINICAL RELEVANCE: The use of copper-doped glass ionomer-based materials under composite restorations may contribute to an increased longevity of adhesive restorations, because of their enhanced antibacterial properties and reduced collagen degradation.

Cross-linked dry bonding: A new etch-and-rinse technique.

OBJECTIVE: To determine if acid-etched, cross-linked dentin can be dehydrated without lowering bond strength below that of cross-linked wet-bonded dentin in vitro. METHODS: Using extracted human third molars, control acid-etched dentin was bonded with Single Bond Plus, using either the wet- or dry-bonding technique. Experimental acid-etched dentin was treated with 5mass% grape seed extract (GSE) in different solvents for 1min before undergoing wet vs dry resin-dentin bonding with Single Bond Plus. Completely demineralized dentin beams were treated with 5% GSE for 0, 1 or 10min, before measuring stiffness by 3-point flexure. Other completely demineralized beams were treated similarly and then incubated in buffer for 1 week to measure the collagen solubilization by endogenous dentin proteases. RESULTS: 24h microtensile bond strengths (µTBS) in wet and dry controls were 53.5±3.6 and 9.4±1.8MPa, respectively (p<0.05). 5% GSE in water gave µTBS of 53.7±3.4 and 39.1±9.7MPa (p<0.05), respectively, while 5% GSE in ethanol gave µTBS of 51.2±2.3 and 35.3±2.0MPa (p<0.05). 5% GSE in 5% EtOH/95% water gave wet and dry µTBS of 53.0±2.3 and 55.7±5.1MPa (p>0.05). Cross-linking demineralized dentin with 5% GSE increased stiffness of dentin and decreased collagen degradation (p<0.05). SIGNIFICANCE: 5% GSE pretreatment of acid-etched dentin for 1min permits the dentin to be completely air-dried without lowering bond strength.

Effects of a Dicalcium and Tetracalcium Phosphate-Based Desensitizer on In Vitro Dentin Permeability.

The present study evaluated the effectiveness of a dicalcium and tetracalcium phosphate-based desensitizer in reducing dentin permeability in vitro. Dentin fluid flow was measured before and after treatment of dentin with patent dentinal tubules using 1 or 3 applications of the dicalcium and tetracalcium phosphate containing agent TeethmateTM (TM) and comparing the results with two sodium fluoride varnishes VellaTM (VLA) and VanishTM (VAN), after storage in artificial saliva for 24 h, 48 h and 7 days. Significant differences were observed among the 4 methods employed for reducing dentin permeability (p < 0.001) and the 3 post-treatment times (p < 0.001). VLA and VAN never achieved 50% permeability reductions consistently in any of the 3 time periods. Only the calcium phosphate-based desensitizer applied for 3 times consistently reduced dentin permeability by 50% after 24 h. When applied once, the permeability reduction of TM increased progressively over the 3 time periods. After 7 days, only one and three applications of the calcium phosphate-based desensitizer consistently reduced dentin permeability by more than 50%. Permeability reductions corresponded well with scanning electron microscopy examination of dentinal tubule orifice occlusion in dentin specimens treated with the agents. Overall, the dicalcium and tetracalcium phosphate-based desensitizer is effective in reducing dentin permeability via a tubule occlusion mechanism. The ability of the agent to reduce dentin permeability renders it to be potentially useful as a clinical dentin desensitizing agent, which has to be confirmed in future clinical studies. By contrast, the two sodium fluoride varnishes are not effective in dentin permeability reduction and should be considered as topical fluoride delivering agents rather than tubular orifice-blocking agents.

Is chlorhexidine-methacrylate as effective as chlorhexidine digluconate in preserving resin dentin interfaces?

OBJECTIVES: The aim of the current study was to evaluate the effect of 2% CHX and 2% CHX-methacrylate compared to the resin-dentin bonds created by a two-step etch-and-rinse adhesive system after 24h, 6min and 12min. METHODS: Microtensile bond strengths and interfacial nanoleakage within resin-dentin interfaces created by Adper Single Bond 2, with or without CHX or CHX-methacrylate pre-treatment for 30s on acid-etched dentin surfaces, were evaluated after 24h, 6min and 12min of storage in distilled water at 37°C. RESULTS: Twelve months of storage resulted in a significant decrease in microtensile bond strength in the control group, and significant increases in silver nanoleakage. In contrast, Single Bond 2+CHX, and to a greater extent CHX-methacrylate, significantly reduced the rate of deterioration of resin-dentin interfaces over the 12min water storage period, in terms of bond strength. CONCLUSIONS: Similar to Single Bond 2+CHX, Single Bond+CHX-methacrylates reduced the degradation of resin-bonded interfaces over a 12 month storage period. Thus it can be concluded that Single Bond 2+CHX-methacrylate may be important to improve durability of bonded interfaces and therefore, prolong the life span of adhesive restorations. CLINICAL SIGNIFICANCE: Although CHX primers have been shown to enhance the durability of etch-and-rinse adhesives, that protection is lost after 2h. The use of CHX-methacrylate should last much longer since it may copolymerize with adhesive monomers, unlike CHX.

Effect of Sodium Fluoride on the endogenous MMP Activity of Dentin Matrices.

OBJECTIVES: This study evaluated the effect of incorporating increasing concentrations of sodium fluoride in incubation media, on the loss of dry mass and solubilization of collagen from demineralized dentin beams incubated for up to 7 days. The effect of fluoride on the inhibition of matrix-bound metalloproteinases (MMPs) was also measured. METHODS: Dentin beams were completely demineralized in 10% phosphoric acid. After baseline measurements of dry mass, the beams were divided into six groups (n=10) and incubated at 37°C either in buffered media containing sodium fluoride (NaF) at 75, 150, 300, 450, 600 ppm or in fluoride-free media (control) for seven days. Following incubation, dry mass was re-measured. The incubation media was hydrolyzed with HCl for the quantitation of hydroxyproline (HYP) as an index of solubilization of collagen by endogenous dentin proteases. Increasing concentrations of fluoride were also evaluated for their ability to inhibit rhMMP-9. RESULTS: Addition of NaF to the incubation media produced a progressive significant reduction (p<0.05) in the loss of mass of dentin matrices, with all concentrations demonstrating significantly less mass loss than the control group. Significantly less HYP release from the dentin beams was found in the higher fluoride concentration groups, while fluoride concentrations of 75 and 150 ppm significantly reduced rhMMP-9 activity by 6.5% and 79.2%, respectively. CONCLUSIONS: The results of this study indicate that NaF inhibits matrix-bound MMPs and therefore may slow the degradation of dentin matrix by endogenous dentin MMPs.

Water distribution in dentin matrices: bound vs. unbound water.

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.

Can quaternary ammonium methacrylates inhibit matrix MMPs and cathepsins?

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.

Effects of quaternary ammonium-methacrylates on the mechanical properties of unfilled resins.

OBJECTIVE: Adding antimicrobial/anti-MMP quaternary ammonium methacrylates (QAMs) to comonomer blends should not weaken the mechanical properties of dental resins. This work evaluated the degree conversion and mechanical properties of BisGMA/TEGDMA/HEMA (60:30:10) containing 0-15 mass% QAMs A-E (A: 2-acryloxyethyltrimethyl ammonium chloride; B: [3-(methacryloylamino)propyl]trimethylammonium chloride; C: [2-(methacryloxy)ethyl] trimethyl ammonium chloride; D: diallyldimethyl ammonium chloride; E: 2-(methacryloyloxy) ethyltrimethyl ammonium methyl sulfate. METHODS: Unfilled resins with and without QAM were placed on ATR-FTIR and light-polymerized for 20s in a thin film at 30°C. Unfilled resin beams were casted from square hollow glass tubings. Half of the beams were tested after 3 days of drying (control); the other half were tested wet after 3 days of water storage. RESULTS: Addition of QAMs in control resins significantly increased conversion 600 s after light termination, with the exception of 5% MAPTAC (p<0.05). Increase of QAM content within a formulation significantly increased conversion. Control beams gave dry Young's moduli of ∼700 MPa. Addition of 5, 10 or 15 mass% QAMs produced significant reductions in dry Young's moduli except for 5% B or C. 15 mass% A, B and C lowered the wet Young's moduli of the resin beams by more than 30%. The ultimate tensile stress (UTS) of control dry resin was 89±11 MPa. Addition of 5-10 mass% QAMs had no adverse effect on the dry UTS. After water storage, the UTS of all resin blends fell significantly (p<0.05), especially when 15 wt% QAMs was added. Control dry beams gave fracture toughness (KIC) values of 0.88±0.1 MPa m(1/2). Wet values were significantly higher at 1.02±0.06 (p<0.05). KIC of dry beams varied from 0.85±0.08 at 5% QAMs to 0.49±0.05 at 15% QAMs. Wet beams gave KIC values of 1.02±0.06 MPa m(1/2) that fell to 0.23±0.01 at 15% QAMs. SIGNIFICANCE: Addition of 10% QAMs increased the degree of conversion of unfilled resins, but lowered wet toughness and UTS; addition of 15% QAMs lowered the mechanical properties of wet resins below acceptable levels.

Inhibition of endogenous human dentin MMPs by Gluma.

OBJECTIVE: The objective of this study was to determine if Gluma dentin desensitizer (5.0% glutaraldehyde and 35% HEMA in water) can inhibit the endogenous MMPs of dentin matrices in 60 s and to evaluate its effect on dentin matrix stiffness and dry mass weight. METHODS: Dentin beams of 2 mm×1 mm×6 mm were obtained from extracted human third molars coronal dentin. To measure the influence of Gluma treatment time on total MMP activity of dentin, beams were dipped in 37% phosphoric acid (PA) for 15 s and rinsed in water. The acid-etched beams were then dipped in Gluma for 5, 15, 30 or 60 s, rinsed in water and incubated into SensoLyte generic MMP substrate (AnaSpec, Inc.) for 60 min. Controls were dipped in water for 60 s. Additional beams of 1 mm×1 mm×6 mm were completely demineralized in 37% PA for 18 h, rinsed and used to evaluate changes on the dry weight and modulus of elasticity (E) after 60 s of Gluma treatment followed by incubation in simulated body fluid buffer for 0, 1 or 4 weeks. E was measured by 3-pt flexure. RESULTS: Gluma treatment inhibited total MMP activity of acid-etched dentin by 44, 50, 84, 86% after 5, 15, 30 or 60 s of exposure, respectively. All completely demineralized dentin beams lost stiffness after 1 and 4 weeks, with no significant differences between the control and Gluma-treated dentin. Gluma treatment for 60 s yielded significantly less dry mass loss than the control after 4 weeks. SIGNIFICANCE: The use of Gluma may contribute to the preservation of adhesive interfaces by its cross-linking and inhibitory properties of endogenous dentin MMPs.

Stabilization of dentin matrix after cross-linking treatments, in vitro.

OBJECTIVES: To evaluate the effect of EDC on elastic modulus (E), MMPs activity, hydroxyproline (HYP) release and thermal denaturation temperature of demineralized dentin collagen. METHODS: Dentin beams were obtained from human molars and completely demineralized in 10 wt% H3PO4 for 18 h. The initial E and MMP activity were determined with three-point bending and microcolorimetric assay, respectively. Extra demineralized beams were dehydrated and the initial dry mass (DM) was determined. All the beams were distributed into groups (n=10) and treated for 30 s or 60 s with: water, 0.5 M, 1 M or 2 M EDC or 10% glutaraldehyde (GA). After treatment, the new E and MMP activity were redetermined. The beams submitted to DM measurements were storage for 1 week in artificial saliva, after that the mass loss and HYP release were evaluated. The collagen thermal denaturation temperature (TDT) was determined by DSC analysis. Data for E, MMP activity and HYP release were submitted to Wilcoxon and Kruskal-Wallis or Mann-Whitney tests. Mass loss and TDT data were submitted to ANOVA and Tukey tests at the 5% of significance. RESULTS: EDC was able to significantly increase collagen stiffness in 60s. 10% GA groups obtained the highest E values after both 30 and 60s. All cross-linking agents decreased MMP activity and HYP release and increased TDT temperature. Significant differences were identified among EDC groups after 30 or 60 s of cross-linking, 1M or 2M EDC showed the lowest MMP activity. SIGNIFICANCE: Cross-linking agents are capable of preventing dentin collagen degradation. EDC treatment may be clinically useful to increase resin-dentin stability.

Effects of calcium silicate-based materials on collagen matrix integrity of mineralized dentin.

INTRODUCTION: Calcium silicate-based materials (CSMs) are used in various endodontic procedures. The present study examined whether prolonged contact of mineralized dentin with recently commercialized versions of these materials adversely affects dentin collagen matrix integrity. METHODS: Dentin slabs prepared from extracted human third molars (7 × 3 × 0.3 mm) were divided into 3 groups on the basis of the material to which dentin was exposed (MTA Plus, Biodentine, untreated control dentin slabs) and the time period of exposure (24 hours, 1, 2, and 3 months; n = 6). Hydroxyproline assay was performed on each group's supernatant to quantify the collagen extraction amounts of each group per time period. Data were analyzed with two-factor repeated-measures analysis of variance and Holm-Sidak pair-wise comparisons (α = 0.05) to determine the effects of material and aging time on collagen extraction. Dentin slabs from the 3 months of aging group were demineralized for transmission electron microscopy examination of collagen matrix ultrastructural changes. RESULTS: Material (P = .002), aging time (P < .001), and their interactions (P = .007) significantly affected the amount of hydroxyproline (pg/mg of mineralized dentin) extracted from mineralized dentin and were significantly correlated by power regression models. Collagen degradation was identified from the surface of dentin slabs that were in direct contact with CSMs. CONCLUSIONS: Prolonged contact of mineralized dentin with CSMs has an adverse effect on the integrity of the dentin collagen matrix. However, the amount of collagen extracted was limited to the contact surface. Clinicians can continue to apply CSMs in endodontic procedures; however, caution is advised when these materials are applied to thin dentinal walls.

Effects of calcium silicate-based materials on the flexural properties of dentin.

INTRODUCTION: Prolonged exposure of root dentin to calcium hydroxide alters the fracture resistance of dentin. Calcium silicate-based materials (CSMs) used in endodontics release calcium hydroxide on setting. This study examined whether prolonged contact of dentin with CSMs adversely affects its mechanical properties. METHODS: Dentin beams prepared from extracted human molars (7 × 3 × 0.3 mm) were divided into 3 groups on the basis of the material to which dentin was exposed (Biodentine, MTA Plus, and untreated control beams). Three-point flexure to failure was performed for each beam at designated exposure times (24 hours, 1, 2, and 3 months; n = 10). Data were analyzed with 2-factor repeated-measures analyses of variance to determine the effects of material and aging time on flexural modulus, flexural strength, and modulus of toughness (α = 0.05). RESULTS: For flexural modulus, there was no significant difference for material (P = .947) or aging time (P = .064) when compared with baseline control. For flexural strength, significant differences were associated with aging time (P < .001) but not with material (P = .349). Flexural strength of dentin exposed to Biodentine decreased significantly after 2 and 3 months, whereas that exposed to MTA Plus decreased significantly after 3 months of aging (P < .05). For modulus of toughness, significant declines were observed for both material (P < .004) and aging time (P < .001). CONCLUSIONS: Both CSMs alter material toughness more than the strength and stiffness of dentin after aging in 100% relative humidity. Because dentin toughness is attributed to its collagen matrix, the amount of collagen extracted from mineralized dentin and changes in collagen ultrastructure should be further examined after exposure of dentin to CSMs.

Inhibition of endogenous dentin matrix metalloproteinases by ethylenediaminetetraacetic acid.

INTRODUCTION: Endogenous dentin matrix metalloproteinases (MMPs) contribute to extracellular collagen matrix degradation in hybrid layers after adhesive dentin bonding procedures. Endodontic irrigants, including chlorhexidine and ethylenediaminetetraacetic acid (EDTA), might help protect the hybrid layer from this process. The objective of the present study was to determine the exposure time necessary for EDTA to inactivate endogenous MMP activity in human dentin. METHODS: Dentin beams (2 × 1 × 3 mm) were prepared from mid-coronal dentin of extracted third molars. The beams were demineralized in 10 wt% phosphoric acid, which also activated endogenous MMPs, and were divided into 4 experimental groups on the basis of exposure time to 17% EDTA (0, 1, 2, or 5 minutes). A generic colorimetric MMP assay measured MMP activity via absorbance at 412 nm. Data were evaluated by Kruskal-Wallis analysis of variance, followed by Dunn pair-wise comparisons at α = 0.05. RESULTS: All exposure times resulted in significant inhibition (P < .001) compared with unexposed controls. Specifically, percent inhibition for 1-, 2-, and 5-minute exposure times was 55.1% ± 21.5%, 72.8% ± 11.7%, and 74.7% ± 19.7%, respectively. CONCLUSIONS: Seventeen percent EDTA significantly inhibits endogenous MMP activity of human dentin within 1-2 minutes. This might minimize hybrid layer degradation after resin bonding procedures in the root canal space.

Inhibition of MMPs by alcohols.

OBJECTIVES: While screening the activity of potential inhibitors of matrix metalloproteinases (MMPs), due to the limited water solubility of some of the compounds, they had to be solubilized in ethanol. When ethanol solvent controls were run, they were found to partially inhibit MMPs. Thus, the purpose of this study was to compare the MMP-inhibitory activity of a series of alcohols. METHODS: The possible inhibitory activity of a series of alcohols was measured against soluble rhMMP-9 and insoluble matrix-bound endogenous MMPs of dentin in completely demineralized dentin. Increasing concentrations (0.17, 0.86, 1.71 and 4.28 mol/L) of a homologous series of alcohols (i.e. methanol, ethanol, propanols, butanols, pentanols, hexanols, the ethanol ester of methacrylic acid, heptanols and octanol) were compared to ethanediol, and propanediol by regression analysis to calculate the molar concentration required to inhibit MMPs by 50% (i.e. the IC(50)). RESULTS: Using two different MMP models, alcohols were shown to inhibit rhMMP-9 and the endogenous proteases of dentin matrix in a dose-dependent manner. The degree of MMP inhibition by alcohols increased with chain length up to 4 methylene groups. Based on the molar concentration required to inhibit rhMMP-9 fifty percent, 2-hydroxyethylmethacrylate (HEMA), 3-hexanol, 3-heptanol and 1-octanol gave the strongest inhibition. SIGNIFICANCE: The results indicate that alcohols with 4 methylene groups inhibit MMPs more effectively than methanol or ethanol. MMP inhibition was inversely related to the Hoy's solubility parameter for hydrogen bonding forces of the alcohols (i.e. to their hydrophilicity).

The anti-MMP activity of benzalkonium chloride.

OBJECTIVE: This study evaluated the ability of benzalkonium chloride (BAC) to bind to dentine and to inhibit soluble recombinant MMPs and bound dentine matrix metalloproteinases (MMPs). METHODS: Dentine powder was prepared from extracted human molars. Half was left mineralized; the other half was completely demineralized. The binding of BAC to dentine powder was followed by measuring changes in the supernatant concentration using UV spectrometry. The inhibitory effects of BAC on rhMMP-2, -8 and -9 were followed using a commercially available in vitro proteolytic assay. Matrix-bound endogenous MMP-activity was evaluated in completely demineralized beams. Each beam was either dipped into BAC and then dropped into 1 mL of a complete medium (CM) or they were placed in 1 mL of CM containing BAC for 30 days. After 30 days, changes in the dry mass of the beams or in the hydroxyproline (HYP) content of hydrolysates of the media were quantitated as indirect measures of matrix collagen hydrolysis by MMPs. RESULTS: Demineralized dentine powder took up 10-times more BAC than did mineralized powder. Water rinsing removed about 50% of the bound BAC, whilst rinsing with 0.5M NaCl removed more than 90% of the bound BAC. BAC concentrations 0.5wt% produced 100% inhibition of soluble recombinant MMP-2, -8 or -9, and inhibited matrix-bound MMPs between 55 and 66% when measured as mass loss or 76-81% when measured as solubilization of collagen peptide fragments. CONCLUSIONS: BAC is effective at inhibiting both soluble recombinant MMPs and matrix-bound dentine MMPs in the absence of resins.

The requirement of zinc and calcium ions for functional MMP activity in demineralized dentin matrices.

UNLABELLED: The progressive degradation of resin-dentin bonds is due, in part, to the slow degradation of collagen fibrils in the hybrid layer by endogenous matrix metalloproteinases (MMPs) of the dentin matrix. In in vitro durability studies, the storage medium composition might be important because the optimum activity of MMPs requires both zinc and calcium. OBJECTIVE: This study evaluated the effect of different storage media on changes in matrix stiffness, loss of dry weight or solubilization of collagen from demineralized dentin beams incubated in vitro for up to 60 days. METHODS: Dentin beams (1mm×2mm×6mm) were completely demineralized in 10% phosphoric acid. After baseline measurements of dry mass and elastic modulus (E) (3-point bending, 15% strain) the beams were divided into 5 groups (n=11/group) and incubated at 37°C in either media containing both zinc and calcium designated as complete medium (CM), calcium-free medium, zinc-free medium, a doubled-zinc medium or water. Beams were retested at 3, 7, 14, 30, and 60 days of incubation. The incubation media was hydrolyzed with HCl for the quantitation of hydroxyproline (HOP) as an index of solubilization of collagen by MMPs. Data were analyzed using repeated measures of ANOVA. RESULTS: Both the storage medium and the storage time showed significant effects on E, mass loss and HOP release (p<0.05). The incubation in CM resulted in relatively rapid and significant (p<0.05) decreases in stiffness, and increasing amounts of mass loss. The HOP content of the experimental media also increased with incubation time but was significantly lower (p<0.05) than in the control CM medium, the recommended storage medium. CONCLUSIONS: The storage solutions used to age resin-dentin bonds should be buffered solutions that contain both calcium and zinc. The common use of water as an aging medium may underestimate the hydrolytic activity of endogenous dentin MMPs.

The inhibitory effect of polyvinylphosphonic acid on functional matrix metalloproteinase activities in human demineralized dentin.

This study has examined the use of polyvinylphosphonic acid (PVPA) as a potential matrix metalloproteinase (MMP) inhibitor and how brief cross-linking of demineralized dentin matrix that did not affect its mechanical properties enhanced the anti-MMP activity of PVPA. The anti-MMP potential of five PVPA concentrations (100-3000 microgml(-1)) was initially screened using a rhMMP-9 colorimetic assay. Demineralized dentin beams were treated with the same five concentrations of PVPA to collagen and then aged for 30 days in a calcium- and zinc-containing medium. The changes in modulus of elasticity, loss of dry mass and dissolution of collagen peptides were measured via three-point bending, precision weighing and hydroxyproline assay, respectively. All tested PVPA concentrations were highly effective (P<0.05) in inhibiting MMP-9. Ageing in the incubation medium did not significantly alter the modulus of elasticity of the five PVPA treatment groups. Conversely, aged dentin beams from the control group exhibited a significant decline in their modulus of elasticity (P<0.05) over time. Mass loss from the dentin beams and the corresponding increase in hydroxyproline in the medium in the five PVPA treatment groups were significantly lower than for the control (P<0.05). PVPA is a potent inhibitor of endogenous MMP activities in demineralized dentin. It may be used as an alternative to chlorhexidine to prevent collagen degradation within hybrid layers to extend the longevity of resin-dentin bonds.

Chlorhexidine binding to mineralized versus demineralized dentin powder.

OBJECTIVES: The purposes of this work were to quantitate the affinity and binding capacity of chlorhexidine (CHX) digluconate to mineralized versus demineralized dentin powder and to determine how much debinding would result from rinsing with water, ethanol, hydroxyethylmethacrylate (HEMA) or 0.5M NaCl in water. METHODS: Dentin powder was made from coronal dentin of extracted human third molars. Standard amounts of dentin powder were tumbled with increasing concentrations of CHX (0-30 mM) for 30 min at 37 degrees C. After centrifuging the tubes, the supernatant was removed and the decrease in CHX concentration quantitated by UV-spectroscopy. CHX-treated dentin powder was resuspended in one of the four debinding solutions for 3 min. The amount of debound CHX in the solvents was also quantitated by UV-spectroscopy. RESULTS: As the CHX concentration in the medium increased, the CHX binding to mineralized dentin powder also increased up to 6.8 micromol/g of dry dentin powder. Demineralized dentin powder took up significantly (p<0.01) more CHX, reaching 30.1 micromol CHX/g of dry dentin powder. Debinding of CHX was in the order: HEMA

Substantivity of chlorhexidine to human dentin.

OBJECTIVES: To better comprehend the role of CHX in the preservation of resin-dentin bonds, this study investigated the substantivity of CHX to human dentin. MATERIAL AND METHODS: Dentin disks (n=45) were obtained from the mid-coronal portion of human third molars. One-third of dentin disks were kept mineralized (MD), while the other two-thirds had one of the surfaces partially demineralized with 37% phosphoric acid for 15 s (PDD) or they were totally demineralized with 10% phosphoric acid (TDD). Disks of hydroxyapatite (HA) were also prepared. Specimens were treated with: (1) 10 microL of distilled water (controls), (2) 10 microL of 0.2% chlorhexidine diacetate (0.2% CHX) or (3) 10 microL of 2% chlorhexidine diacetate (2% CHX). Then, they were incubated in 1 mL of PBS (pH 7.4, 37 degrees C). Substantivity was evaluated as a function of the CHX-applied dose after: 0.5 h, 1 h, 3 h, 6 h, 24 h, 168 h (1 week), 672 h (4 weeks) and 1344 h (8 weeks) of incubation. CHX concentration in eluates was spectrophotometrically analyzed at 260 nm. RESULTS: Significant amounts of CHX remained retained in dentin substrates (MD, PPD or TDD), independent on the CHX-applied dose or time of incubation (p<0.05). High amounts of retained CHX onto HA were observed only for specimens treated with the highest concentration of CHX (2%) (p<0.05). CONCLUSION: The outstanding substantivity of CHX to dentin and its reported effect on the inhibition of dentinal proteases may explain why CHX can prolong the durability of resin-dentin bonds.

Push-out strength of modified Portland cements and resins.

PURPOSE: Modified calcium-silicate cements derived from white Portland cement (PC) were formulated to test their push-out strength from radicular dentin after immersion for 1 month. METHODS: Slabs obtained from 42 single-rooted extracted teeth were prepared with 0.6 mm diameter holes, then enlarged with rotary instruments. After immersion in EDTA and NaOC1, the holes were filled with modified PCs or ProRoot MTA, Vitrebond and Clearfil SE. Different concentrations of phyllosilicate (montmorillonite-MMT) were added to experimental cements. ProRoot MTA was also included as reference material. Vitrebond and Clearfil SE were included as controls. Each group was tested after 1 month of immersion in water or PBS. A thin-slice push-out test on a universal testing machine served to test the push-out strength of materials. Results were statistically analyzed using the least squares means (LSM) method. RESULTS: The modified PCs had push-out strengths of 3-9.5 MPa after 1 month of immersion in water, while ProRoot MTA had 4.8 MPa. The push-out strength of PC fell after incubation in PBS for 1 month, while the push-out strength of ProRoot MTA increased. There were no significant changes in Clearfil SE Bond or Vitrebond after water or PBS storage.