Changes in resin-infiltrated dentin stiffness after water storage.

Plasticization of polymers by water sorption lowers their mechanical properties in a manner that is predictable by the polarity of their component resins. This study tested the hypothesis that when adhesive resins were used to create resin-infiltrated dentin, the reductions in their flexural moduli after water storage would be lowered proportional to their hydrophilic characteristics. Three increasingly hydrophilic resin blends were used to fabricate polymer beams and macro-hybrid layer models of resin-infiltrated dentin for testing with a miniature three-point flexure device, before and after 1-4 weeks of water storage. Flexural modulus reductions in macro-hybrid layers were related to, and more extensive than, reductions in the corresponding polymer beams. Macro-hybrid layers that were more hydrophilic exhibited higher percent reductions in flexural modulus, with the rate of reduction proportional to the Hoy's solubility parameters for total intermolecular attraction forces (delta(t)) and polar forces (delta(p)) of the macro-hybrid layers.

Effects of resin hydrophilicity on dentin bond strength.

The purpose of this study was to determine if hydrophobic resins can be coaxed into dentin wet with ethanol instead of water. The test hypothesis was that dentin wet with ethanol would produce higher bond strengths for hydrophobic resins than would dentin wet with water. This study examined the microtensile bond strength of 5 experimental adhesives (50 wt% ethanol/50% comonomers) of various degrees of hydrophilicity to acid-etched dentin that was left moist with water, moist with ethanol, or air-dried. Following composite buildups, hourglass-shaped slabs were prepared from the bonded teeth for microtensile testing. For all 3 types of dentin surfaces, higher bond strengths were achieved with increased resin hydrophilicity. The lowest bond strengths were obtained on dried dentin, while the highest bond strengths were achieved when dentin was bonded moist with ethanol. Wet-bonding with ethanol achieved higher bond strengths with hydrophobic resins than were possible with water-saturated matrices.

The effects of ethanol on the size-exclusion characteristics of type I dentin collagen to adhesive resin monomers.

During dentin bonding with etch-and-rinse adhesive systems, phosphoric acid etching of mineralized dentin solubilizes the mineral crystallites and replaces them with bound and unbound water. During the infiltration phase of dentin bonding, solvated adhesive resin comonomers are supposed to replace all of the unbound collagen water and polymerize into copolymers. A recently published review suggested that dental monomers are too large to enter and displace water from tightly-packed collagen molecules. Conversely, recent work from the authors' laboratory demonstrated that HEMA and TEGDMA freely equilibrate with water-saturated dentin matrices. However, because adhesive blends are solvated in organic solvents, those solvents may remove enough free water to allow collagen molecules to come close enough to exclude adhesive monomer permeation. The present study analyzed the size-exclusion characteristics of dentin collagen, using a gel permeation-like column chromatography technique, filled with dentin powder instead of Sephadex beads as the stationary phase. The elution volumes of different sized test molecules, including adhesive resin monomers, studied in both water-saturated dentin, and again in ethanol-dehydrated dentin powder, showed that adhesive resin monomers can freely diffuse into both hydrated and dehydrated collagen molecules. Under these in vitro conditions, all free and some of the loosely-bound water seems to have been removed by ethanol. These results validate the concept that adhesive resin monomers can permeate tightly-bound water in ethanol-saturated collagen molecules during infiltration by etch-and-rinse adhesives. STATEMENT OF SIGNIFICANCE: It has been reported that collagen molecules in dentin matrices are packed too close together to allow permeation of adhesive monomers between them. Resin infiltration, in this view, would be limited to extrafibrillar spaces. Our work suggests that monomers equilibrate with collagen water in both water and ethanol-saturated dentin matrices.

Dimensional changes in acid-demineralized dentin matrices following the use of HEMA-water versus HEMA-alcohol primers.

Although dried acid-etched dentin can be reexpanded by hydroxyethyl methacrylate (HEMA)/water primers, the primed dentin collapses when the water is evaporated. Experimental HEMA/alcohol primers should stiffen the matrix and permit less shrinkage when the solvent is evaporated. The purpose of this study was to test the hypotheses that matrix shrinkage induced by solvent evaporation from HEMA primers is inversely related to solvent-induced matrix stiffness. Dentine discs were prepared from midcoronal dentine of unerupted human third molars. After demineralization in 37% phosphoric acid, the specimens were placed in the well of a linear variable differential transformer instrument, which measures changes in the matrix height and stiffness by load displacement after the application of weights. This was done in their hydrated state after water had been applied, after drying with dry nitrogen gas, and after the application of 35 vol % HEMA-water, HEMA-methanol, HEMA-ethanol, or HEMA-propanol primers. The degree of reexpansion after the application of the primers to the dentine in the dried state was found to be highest using the HEMA-water primer, followed by HEMA-methanol and HEMA-ethanol, with the HEMA-propanol primer producing no expansion. However, when the solvents were evaporated the HEMA-water-treated specimens shrank the most, the HEMA-ethanol-treated specimens shrank an intermediate amount, and the HEMA-methanol-treated specimens shrank the least. The net result of expansion minus shrinkage produced by evaporation determines how much HEMA remains in the hybrid layer just prior to polymerization.

Effects of sodium hypochlorite and RC-prep on bond strengths of resin cement to endodontic surfaces.

There is concern that the use of sodium hypochlorite (NaOCl) and RC-Prep may lower the bond strength of resin cements. The objective of this study was to evaluate the effect of 5% NaOCl and RC-Prep treatment on the bond strength of a resin cement, C&B Metabond. Control roots (group 1) were biomechanically prepared using 0.9% NaCl as an irrigant; group 2, roots with 5% NaOCl; group 3, roots with RC-Prep; group 4, roots with 0.9% NaCl followed by 10% ascorbic acid; group 5, roots with 5% NaOCl followed by 10% ascorbic acid (pH 4); group 6, roots with 5% NaOCl followed by 10% neutral sodium ascorbate; and group 7, roots with RC-Prep followed by 10% ascorbic acid. All roots were then filled with C&B Metabond, incubated in water for 24 h, and then cross-sectioned into six 1-mm thick slabs representing cervical and middle root dentin. The slabs were trimmed and tested for tensile bond strength. The results demonstrated that both 5% NaOCl and RC-Prep produced significantly (p < 0.05) large reductions in resin-dentin bond strengths, and the reductions could be completely reversed by the application of either 10% ascorbic acid or 10% sodium ascorbate.

Permeability of demineralized dentin to HEMA.

OBJECTIVES: The objectives of this study were to develop a macromodel of the demineralized layer of dentin that is formed after acid-etching, and to determine if the amount of HEMA uptake was responsive to air drying. The hypothesis that was tested was that HEMA uptake by demineralized dentin depends upon the degree of expansion of the collagen fibril network. METHODS: Dentin cubes (2 x 2 x 2 mm) were prepared in coronal dentin from extracted unerupted human third molars. They were incubated in 100% HEMA for up to 1000 min and then removed and blotted free of excess adherent HEMA. The HEMA taken up by each cube was extracted in 2 ml of water for 1 hr with shaking. This was repeated and the extracts combined. HEMA was quantitated spectrophometrically. The dentin cubes were then demineralized in 0.5 M EDTA for 10 days and the HEMA uptake remeasured at 1, 10, 100 and 1000 min. Then the cubes were air-dried and the HEMA uptake remeasured. After re-expanding the specimens in water, the cubes were stiffened in ascending concentrations of acetone of 100%, then allowed to air dry again in a more expanded state, and HEMA uptake was remeasured. Finally, after rehydration, the dentin cubes were "acid-etched" for 1 or 10 min with 37 wt% phosphoric acid and HEMA uptake remeasured. RESULTS: Before demineralization, the dentin took up little HEMA (ca. 4.8 x 10(-7) moles min-3) compared to after demineralization when the uptake for 10, 100 and 1000 min was 27.4, 43.8 and 51.4 x 10(-3) moles mm-3, respectively. Acid etching for 1 or 10 min had no effect on HEMA uptake. Air drying produced a 72% volumetric shrinkage but a 97% reduction in uptake. When the demineralized matrix was stiffened in acetone prior to air-drying, the volumetric shrinkage was only 27% and the HEMA uptake only fell 16% compared to the wet, fully expanded condition. SIGNIFICANCE: The results support the hypothesis that the uptake of HEMA by demineralized dentin depends on the degree of expansion of the dentin matrix.

Effect of phosphoric acid on the degradation of human dentin matrix.

This study determined if dentin proteases are denatured by phosphoric acid (PA) used in etch-and-rinse dentin adhesives. Dentin beams were completely demineralized with EDTA for 30 days. We "acid-etched" experimental groups by exposing the demineralized dentin beams to 1, 10, or 37 mass% PA for 15 sec or 15 min. Control beams were not exposed to PA but were incubated in simulated body fluid for 3 days to assay their total endogenous telopeptidase activity, by their ability to solubilize C-terminal crosslinked telopeptides ICTP and CTX from insoluble dentin collagen. Control beams released 6.1 ± 0.8 ng ICTP and 0.6 ± 0.1 ng CTX/mg dry-wt/3 days. Positive control beams pre-incubated in p-aminophenylmercuric acetate, a compound known to activate proMMPs, released about the same amount of ICTP peptides, but released significantly less CTX. Beams immersed in 1, 10, or 37 mass% PA for 15 sec or 15 min released amounts of ICTP and CTX similar to that released by the controls (p > 0.05). Beams incubated in galardin, an MMP inhibitor, or E-64, a cathepsin inhibitor, blocked most of the release of ICTP and CTX, respectively. It is concluded that PA does not denature endogenous MMP and cathepsin activities of dentin matrices.

The importance of size-exclusion characteristics of type I collagen in bonding to dentin matrices.

The mineral phase of dentin is located primarily within collagen fibrils. During development, bone or dentin collagen fibrils are formed first and then water within the fibril is replaced with apatite crystallites. Mineralized collagen contains very little water. During dentin bonding, acid-etching of mineralized dentin solubilizes the mineral crystallites and replaces them with water. During the infiltration phase of dentin bonding, adhesive comonomers are supposed to replace all of the collagen water with adhesive monomers that are then polymerized into copolymers. The authors of a recently published review suggested that dental monomers were too large to enter and displace water from collagen fibrils. If that were true, the endogenous proteases bound to dentin collagen could be responsible for unimpeded collagen degradation that is responsible for the poor durability of resin-dentin bonds. The current work studied the size-exclusion characteristics of dentin collagen, using a gel-filtration-like column chromatography technique, using dentin powder instead of Sephadex. The elution volumes of test molecules, including adhesive monomers, revealed that adhesive monomers smaller than ∼1000 Da can freely diffuse into collagen water, while molecules of 10,000 Da begin to be excluded, and bovine serum albumin (66,000 Da) was fully excluded. These results validate the concept that dental monomers can permeate between collagen molecules during infiltration by etch-and-rinse adhesives in water-saturated matrices.

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.

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.

Durability of resin-dentin bonds to water- vs. ethanol-saturated dentin.

Higher 24-hour resin-dentin bond strengths are created when ethanol is used to replace water during wet bonding. This in vitro study examined if ethanol-wet-bonding can increase the durability of resin-dentin bonds over longer times. Five increasingly hydrophilic experimental resin blends were bonded to acid-etched dentin saturated with water or ethanol. Following composite build-ups, the teeth were reduced into beams for 24-hour microtensile bond strength evaluation, and for water-aging at 37 degrees C for 3, 6, or 12 months before additional bond strength measurements. Although most bonds made to water-saturated dentin did not change over time, those made to ethanol-saturated dentin exhibited higher bond strengths, and none of them fell over time. Decreased collagen fibrillar diameter and increased interfibrillar spacing were seen in hybrid layers created with ethanol-wet-bonding. Increases in bond strength and durability in ethanol-wet-bonding may be due to higher resin uptake and better resin sealing of the collagen matrix, thereby minimizing endogenous collagenolytic activities.

Concentration dependencies of immunoturbidimetric dose-response curves: immunoturbidimetric titer and reactivity, and relevance to design of turbidimetric immunoassays.

To characterize polyclonal antisera for two-point immunoturbidimetric applications, we defined, as functions of antiserum concentration, two parameters derived from dose-response curves: the maximum bichromatic optical response, Tmax, and the antigen concentration in the region of excess antibody corresponding to one-half Tmax, or C50. We raised monospecific polyclonal antisera in goats against several human immunoglobulins, C-reactive protein, C3, C4, apolipoproteins A-I and B, and several other proteins. We could linearly relate the logarithm of the antiserum concentration to log C50 and to log Tmax. The concentration of polyethylene glycol affected not only C50 and Tmax but also their functional dependencies on antiserum concentration. We devised two definitions of immunoturbidimetric titer and related them to the titer obtained by the single radial immunodiffusion method of Becker (Immunochemistry 1969; 6:539-46).

Solvent-induced dimensional changes in EDTA-demineralized dentin matrix.

The purpose of this study was to test the null hypothesis that the re-expansion of dried matrix and the shrinkage of moist, demineralized dentin is not influenced by polar solvents. Dentin disks were prepared from midcoronal dentin of extracted human third molars. After complete demineralization in 0.5M of EDTA (pH 7), the specimens were placed in the well of a device that measures changes in matrix height in real time. Dry, collapsed matrices were created by blowing dry N(2) on the specimens until they shrank to a stable plateau. Polar solvents [water, methanol, ethanol, n-propanol, n-butanol, formamide, ethylene glycol, hydroxyethyl methacrylate (HEMA), or mixtures of water-HEMA] as model primers then were added and the degree of re-expansion measured. These same solvents also were applied to moist, expanded matrices and the solvent-induced shrinkages measured. Regression analysis was used to test the correlations between matrix height and Hansen's dispersive, polar, hydrogen bonding, and total solubility parameters (delta(d), delta(p), delta(h), delta(t)). The results indicate that water-free polar solvents of low hydrogen bonding (H-bond) ability (e.g., neat HEMA) do not re-expand dried matrices and that they shrink moist matrices. When HEMA was mixed with progressively higher water concentrations, the model water-HEMA primers expanded the dried matrix in proportion to their water concentrations and they produced less shrinkage of moist matrices. Solvents with higher H-bonding capacities (methanol, ethanol, ethylene glycol, formamide, and water) re-expanded the dried matrix in proportion to their solubility parameters for H-bonding (delta(h)). They also induced small transient shrinkages of moist matrices, which slowly re-expanded. The results require rejection of the null hypothesis.