Abstract
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 5 mass% grape seed extract (GSE) in different solvents for 1 min 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 10 min, 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
24 h microtensile bond strengths (μTBS) in wet and dry controls were 53.5 ± 3.6 and 9.4 ± 1.8 MPa, respectively ( p < 0.05). 5% GSE in water gave μTBS of 53.7 ± 3.4 and 39.1 ± 9.7 MPa ( p < 0.05), respectively, while 5% GSE in ethanol gave μTBS of 51.2 ± 2.3 and 35.3 ± 2.0 MPa ( p < 0.05). 5% GSE in 5% EtOH/95% water gave wet and dry μTBS of 53.0 ± 2.3 and 55.7 ± 5.1 MPa ( 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 1 min permits the dentin to be completely air-dried without lowering bond strength.
1
Introduction
Water is one of the strongest hydrogen bonding solvents known, and has a Hoy’s solubility parameter for hydrogen bonding cohesive forces ( δ h ) of 40.4 (J/cm 2 ) ½ . The intrinsic tendency of collagen peptides to form interpeptide H-bonds with each other in the absence of water is 14.8 (J/cm 2 ) ½ . Such interpeptide hydrogen bonding cannot occur in the presence of water. That is, water molecules cluster around carbonyl oxygens and amide hydrogens in peptide bonds, which prevent direct hydrogen bonding between neighboring collagen peptides. The stiffness of demineralized dentin matrices is inversely related to the solubility parameter for hydrogen bonding cohesive forces of polar solvents . Water is not only a solvent, but participates in many protein-water-coupled phenomena .
During cavity preparations, dentists expose mineralized tooth dentin that has a modulus of elasticity of 20,000 MPa . To create microporosities in that dentin for resin-infiltration, they strip away the apatite crystallites in the mineralized matrix by acid-etching dentin, which solubilizes those crystallites to a depth of 10 μm . After water rinsing to extract the residual acid and solubilized minerals, the exposed demineralized collagen fibrils have a modulus of elasticity of only 3–5 MPa . As long as these collagen fibrils are suspended in water, they are very pliable. However, if that water is removed by evaporation or dehydrating solvents, the compliant collagen fibrils rapidly form interpeptide hydrogen bonds with their nearest neighbors. When this occurs, the 50–100 nm diameter collagen fibrils hydrogen bond to each other to form an impermeable membrane-like structure that prevents the permeation of solvated adhesive monomers around collagen fibrils . This results in resin-dentin bond values of only 10 MPa. To avoid drying-induced shrinkage, and to create higher resin-dentin bond strengths, Kanca developed what is called the “wet-bonding technique” , where demineralized dentin is allowed to float in 70% water during the monomer infiltration phase of dentin bonding. That bonding technique leaves far too much residual water in resin-dentin bonds , and provides hydrolytic fuel for the endogenous proteases of dentin matrices which slowly hydrolyze collagen fibrils in resin-bonded dentin, resulting in poor durability of resin-dentin bonds . The goal of resin infiltration during dentin bonding is to replace all of the 70 vol% rinse water with 70 vol% adhesive monomers . However, dimethacrylates such as triethylene glycol dimethacrylate are almost insoluble in water. They undergo phase changes from monomers in solution, to monomers in resin globules suspended in water . Because these resin globules are too large to permeate through the 20 nm wide interfibrillar spaces, this results in significant amounts of collagen fibrils in hybrid layers being surrounded by water instead of polymerized resin . To prevent phase changes, most manufacturers have added 30–50 vol% of water-soluble monomethacrylates such as 2-hydroxyethyl methacrylate (HEMA) to both scavenge residual water, and act as a solvent for dimethacrylates. However, monomethacrylates cannot produce strong cross-linked polymers. Rather, HEMA-rich polymers form elastomers that are not cross-linked. They are weak polymers which attract water to themselves that plasticizes their mechanical properties .
The authors propose to eliminate these problems by making the following modifications to the “wet-bonding technique”. After rinsing away the unreacted acid and solubilized minerals, collagen fibrils suspended in water would be cross-linked by grape seed extract (GSE) for 60 s. This agent is meant to be illustrative of cross-linking agents in general (i.e. carbodiimide, glutaraldehyde, etc.) . The excess, unreacted cross-linker would then be rinsed away with water and the stiffened collagen fibril matrix air dried. There is an inverse relationship between shrinkage and stiffness of demineralized dentin . That is, as stiffness increases, shrinkage decreases, allowing the individual collagen fibrils to be separated from each other by air.
The other problem is how to remove excess water. The vapor pressure of pure rinse water is much higher than it is after adding water-soluble adhesive monomers, which lower the vapor pressure of water (Raoult’s Law) . By evaporating the rinse water before adding primers or adhesives, it is possible to remove nearly all the rinse water added to dentin within 30 s using a strong, continuous air blast. In the absence of water, adhesive formulations free of HEMA and made entirely of dimethacrylates can be added to dry acid-etched, cross-linked dentin matrix . The end result should be a hybrid layer free of residual water and filled with dimethacrylates that absorb little water . Tay et al. reported that ethanol-solubilized BisGMA could infiltrate ethanol-rinsed, acid-etched dentin using a new bonding technique called “ethanol wet-bonding” . That bonding technique removed residual water by chemical dehydration with ethanol, an excellent solvent for dimethacrylates.
The purpose of the present work was to test three null hypotheses: (1) that there is no difference in the 24 h microtensile bond strengths (μTBS) of acid-etched dentin bonded to non-cross-linked wet vs dry specimens; (2) that there is no difference in the 24 h μTBS of acid-etched dentin bonded to GSE cross-linked dry vs GSE cross-linked wet specimens; (3) that there is no difference in the 24 h μTBS of acid-etched dentin bonded to non-crosslinked wet-bonded vs GSE cross-linked wet-bonded dentin.
2
Materials and methods
2.1
Teeth used for resin-dentin bonding
Thirty-two un-erupted human third molars were obtained from young (18–22 year old) patients in the Oral Surgery Clinics of The Dental College of Georgia at Augusta University with signed informed consent. They were stored in water containing 0.02% sodium azide as an antimicrobial, at 4 °C for less than 1 month before use.
2.2
Cross-linking agent
Proanthocyanidin was a gift from Dr. A. Bedran-Russo, who purchased it as Mega Natura-BP, from Polyphenolics, Madera, CA, USA. It was extracted from Vitis vinefera grapes and has been reported to contain 79.6 mass% total polyphenols . It was dissolved in water, ethanol or 5% ethanol/95% water at 5 mass%.
2.3
Bonding procedures
Occlusal enamel and superficial dentin were removed from the 32 extracted teeth using a Buehler diamond blade saw (Buehler Ltd., Lake Bluff, IL, USA) with copious water cooling. Then, the flat exposed mid-coronal dentin was sanded with wet 180-grit silicon carbide paper to create a standard smear layer . The flat occlusal dentin surface of all teeth were acid-etched for 15 s with 37% phosphoric acid gel (3M ESPE, St. Paul, MN, USA). All etched teeth were rinsed with water for 60 s to remove unreacted acid and to extract solubilized mineral. In experimental specimens, the acid-etched dentin surface was treated for 60 s with one of the following GSE experimental cross-linking primers: 5 mass% GSE in water (pH 3.26), 5 mass% GSE in ethanol (pH 4.17), or 5 mass% GSE in 5% ethanol/95% water (pH 3.48). Cross-linking was then terminated by rinsing dentin surface with air-water spray for 10 s.
Specimens in the wet-bonded control group were not pretreated with GSE, and bonded using the wet-bonding technique. They were left visibly moist when bonding with Single Bond Plus (3M ESPE). Bonding was accomplished by application of two separate layers of solvated adhesive, followed by evaporation of the solvent for 5 s and light-curing for 40 s at 600 mW/cm 2 using an Optilux 500 halogen light (Demetron/Kerr, Danbury, CT, USA). Creation of resin composite build-ups was made using three 1.5 mm increments of Z100 resin composite (3M ESPE) that were individually light-cured for 20 s each.
Specimens in the dry bonded control group were not pretreated with GSE and had their wet dentin surfaces completely dried for 30 s with a continuous air blast at a distance of 10 cm. They were then bonded with Single Bond Plus to dry dentin.
Specimens in the cross-linked wet bonded group were treated with various GSE primers for 60 s, and were then rinsed for 10 s with the appropriate solvent (water, ethanol, or 5% ethanol/95% water). They were then lightly blotted with a Kimwipe tissue (Fisher Scientific, Pittsburgh, PA, USA) moistened with the same solvent and immediately bonded as previously described.
Specimens in the cross-linked dry bonding group were treated with various GSE primers for 60 s, rinsed with air-water spray for 10 s and then air-dried using full strength air from a 3-way syringe at a distance of 10 cm for 30 s. They were then immediately dry bonded with Single Bond Plus and built up with resin composite as described above.
The resin-bonded teeth were immersed in labeled, separate containers in 37 °C water for 24 h. Then, using an Isomet saw with water cooling, the curved peripheries of each bonded tooth were cut away to yield a square bonded crown. The resulting “squared” crown was cut into 0.7 mm thick slabs. Each slab was, in turn, cut into 0.7 mm thick “sticks”. The μTBS of each stick was measured using Geraldeli testing jigs . The tensile force at failure was recorded and divided by the cross-sectioned area of each stick, and expressed in MPa.
2.4
Creation of dentin beams for 3-point flexure and hydroxyproline release
One 0.5 mm thick dentin disk was obtained from each tooth using a Buehler diamond blade saw (Buehler Ltd.). Sixty dentin beams were cut from these disks that were 3 mm wide × 6 mm long, using the same saw. These beams were then completely demineralized in 10% phosphoric acid at 4 °C by tumbling in sealed containers for 18 h. Complete demineralization was confirmed by measuring the modulus of elasticity of beams in water. A modulus of elasticity of 5 MPa was considered as completely demineralized .
2.5
Measurements of stiffness of completely demineralized dentin
Due to the excellent μTBS results obtained from using 5 mass% GSE in 5% ethanol/95% water, we decided to use this solvent alone for the remaining experimental procedures.
Thirty demineralized dentin beams were used to measure stiffness by 3-point flexure. The initial elastic modulus of each beam was determined by means of a testing machine (Vitrodyne 1000, Liveco Inc., Burlington, VA, USA) with a 1000 g load cell, at a crosshead speed of 1 mm/min. Load-displacement curves were converted to stress–strain curves, and modulus of elasticity ( E ) was calculated at the steepest, most linear portion of the curve, using the formula E = mL 3 /4 bd 3 , where m = slope (N/mm); L = support span (mm); d = thickness of beam (mm); b = width of beam (mm). After initial baseline testing in water, beams (10/group) were placed into 5% GSE in 5% ethanol/95% water for 1 min or 10 min. Immediately following incubation stiffening, the beams were rinsed in water and re-tested under the same parameters, and the new stiffness was measured. Each beam served as its own control.
2.6
Measurement of collagen solubilization by endogenous dentin proteases
Previous work from our laboratory showed that when completely demineralized dentin beams were incubated in buffer at 37 °C, they lost dry mass and stiffness . This loss of dry mass was associated with solubilization of hydroxyproline-containing collagen peptides. This hydrolytic activity is due to the presence of endogenous proteases in dentin matrices, including MMPs -2, -8, and -9 and cathepsin K . When these demineralized matrices were treated by cross-linking agents such as carbodiimide or glutaraldehyde, the loss of dry mass was significantly reduced .
Thirty 3 mm × 0.5 mm × 6 mm dentin beams were prepared from mid-coronal dentin as described above. After rinsing in water, the demineralized beams (10/group) were dipped in water for 10 min or in 5 wt% GSE in 5% ethanol/95% water for 1 min or 10 min, rinsed briefly and then dropped into 0.5 ml of 0.05 M HEPES buffer in sealed containers that were incubated at 37 °C for 1 week with shaking at 15 cycles/min. The HEPES buffer (pH 7.4) also contained 2.5 mM CaCl 2 ·2H 2 O and 0.02 mM ZnCl 2 (both from Sigma–Aldrich, St. Louis, MO, USA). At the end of one week, 100 μL of incubation media was mixed with an equal volume of 12 N HCl to create 6 N HCl, which was used to hydrolyze the soluble collagen fragments into their constituent amino acids in sealed glass ampoules at 118 °C for 16 h. After opening vials, the HCl was allowed to evaporate in a vacuum dessicator, the bottom of which was covered by NaOH pellets to neutralize the HCl. The dry residue was then analyzed for hydroxyproline using a colorimetric method .
2.7
Scanning electron microscopy
Control and experimental dentin specimens (3/group) were acid-etched with 37% phosphoric acid gel for 15 s, then rinsed with water for 15 s. The control specimens were then treated with water for 1 min. The experimental teeth were treated with 5 mass% GSE in water, ethanol or mixtures for 1 min. All surfaces were rinsed with water for 15 s and then air-dried for 30 s using continuous, full strength air for 30 s, 10 cm from the dentin surface. All specimens except those that were dry-bonded were critical-point dried (Samdri-790, Hummer Sputtering System, Anatech Corp., San Diego, CA, USA) prior to being coated with gold/palladium and examined in an SEM (Model XL-30 FEG, Philips Corp., Hillsboro, OR, USA) at 10 keV.
2.8
Statistics
The μTBS obtained from beams derived from each of the 4 teeth in each group (8 groups) were pooled together to obtain the mean bond strength value. Each tooth was treated as a statistical unit. The bond strength data were analyzed via two-way ANOVA (SigmaPlot 13, Systat Software Inc., San Jose, CA, USA) using cross-linking as one factor and bonding type (wet vs dry) as the second factor. There was a significant interaction between cross-linking and bond type ( p < 0.001). Thus, the data were re-analyzed by the least squares means test. Least squares means are the expected value of group or subgroup means that one expects for a balanced design involving the group variable with all covariates at their mean value.
The matrix stiffness and collagen solubilization data were logarithmically transformed to obtain normal distribution and equality of variance. They were then analyzed using separate, one-way ANOVAs and Holm-Sidak multiple comparison tests (SigmaPlot 13). Statistical significance was set in advance at the 0.05 level.
2
Materials and methods
2.1
Teeth used for resin-dentin bonding
Thirty-two un-erupted human third molars were obtained from young (18–22 year old) patients in the Oral Surgery Clinics of The Dental College of Georgia at Augusta University with signed informed consent. They were stored in water containing 0.02% sodium azide as an antimicrobial, at 4 °C for less than 1 month before use.
2.2
Cross-linking agent
Proanthocyanidin was a gift from Dr. A. Bedran-Russo, who purchased it as Mega Natura-BP, from Polyphenolics, Madera, CA, USA. It was extracted from Vitis vinefera grapes and has been reported to contain 79.6 mass% total polyphenols . It was dissolved in water, ethanol or 5% ethanol/95% water at 5 mass%.
2.3
Bonding procedures
Occlusal enamel and superficial dentin were removed from the 32 extracted teeth using a Buehler diamond blade saw (Buehler Ltd., Lake Bluff, IL, USA) with copious water cooling. Then, the flat exposed mid-coronal dentin was sanded with wet 180-grit silicon carbide paper to create a standard smear layer . The flat occlusal dentin surface of all teeth were acid-etched for 15 s with 37% phosphoric acid gel (3M ESPE, St. Paul, MN, USA). All etched teeth were rinsed with water for 60 s to remove unreacted acid and to extract solubilized mineral. In experimental specimens, the acid-etched dentin surface was treated for 60 s with one of the following GSE experimental cross-linking primers: 5 mass% GSE in water (pH 3.26), 5 mass% GSE in ethanol (pH 4.17), or 5 mass% GSE in 5% ethanol/95% water (pH 3.48). Cross-linking was then terminated by rinsing dentin surface with air-water spray for 10 s.
Specimens in the wet-bonded control group were not pretreated with GSE, and bonded using the wet-bonding technique. They were left visibly moist when bonding with Single Bond Plus (3M ESPE). Bonding was accomplished by application of two separate layers of solvated adhesive, followed by evaporation of the solvent for 5 s and light-curing for 40 s at 600 mW/cm 2 using an Optilux 500 halogen light (Demetron/Kerr, Danbury, CT, USA). Creation of resin composite build-ups was made using three 1.5 mm increments of Z100 resin composite (3M ESPE) that were individually light-cured for 20 s each.
Specimens in the dry bonded control group were not pretreated with GSE and had their wet dentin surfaces completely dried for 30 s with a continuous air blast at a distance of 10 cm. They were then bonded with Single Bond Plus to dry dentin.
Specimens in the cross-linked wet bonded group were treated with various GSE primers for 60 s, and were then rinsed for 10 s with the appropriate solvent (water, ethanol, or 5% ethanol/95% water). They were then lightly blotted with a Kimwipe tissue (Fisher Scientific, Pittsburgh, PA, USA) moistened with the same solvent and immediately bonded as previously described.
Specimens in the cross-linked dry bonding group were treated with various GSE primers for 60 s, rinsed with air-water spray for 10 s and then air-dried using full strength air from a 3-way syringe at a distance of 10 cm for 30 s. They were then immediately dry bonded with Single Bond Plus and built up with resin composite as described above.
The resin-bonded teeth were immersed in labeled, separate containers in 37 °C water for 24 h. Then, using an Isomet saw with water cooling, the curved peripheries of each bonded tooth were cut away to yield a square bonded crown. The resulting “squared” crown was cut into 0.7 mm thick slabs. Each slab was, in turn, cut into 0.7 mm thick “sticks”. The μTBS of each stick was measured using Geraldeli testing jigs . The tensile force at failure was recorded and divided by the cross-sectioned area of each stick, and expressed in MPa.
2.4
Creation of dentin beams for 3-point flexure and hydroxyproline release
One 0.5 mm thick dentin disk was obtained from each tooth using a Buehler diamond blade saw (Buehler Ltd.). Sixty dentin beams were cut from these disks that were 3 mm wide × 6 mm long, using the same saw. These beams were then completely demineralized in 10% phosphoric acid at 4 °C by tumbling in sealed containers for 18 h. Complete demineralization was confirmed by measuring the modulus of elasticity of beams in water. A modulus of elasticity of 5 MPa was considered as completely demineralized .
2.5
Measurements of stiffness of completely demineralized dentin
Due to the excellent μTBS results obtained from using 5 mass% GSE in 5% ethanol/95% water, we decided to use this solvent alone for the remaining experimental procedures.
Thirty demineralized dentin beams were used to measure stiffness by 3-point flexure. The initial elastic modulus of each beam was determined by means of a testing machine (Vitrodyne 1000, Liveco Inc., Burlington, VA, USA) with a 1000 g load cell, at a crosshead speed of 1 mm/min. Load-displacement curves were converted to stress–strain curves, and modulus of elasticity ( E ) was calculated at the steepest, most linear portion of the curve, using the formula E = mL 3 /4 bd 3 , where m = slope (N/mm); L = support span (mm); d = thickness of beam (mm); b = width of beam (mm). After initial baseline testing in water, beams (10/group) were placed into 5% GSE in 5% ethanol/95% water for 1 min or 10 min. Immediately following incubation stiffening, the beams were rinsed in water and re-tested under the same parameters, and the new stiffness was measured. Each beam served as its own control.
2.6
Measurement of collagen solubilization by endogenous dentin proteases
Previous work from our laboratory showed that when completely demineralized dentin beams were incubated in buffer at 37 °C, they lost dry mass and stiffness . This loss of dry mass was associated with solubilization of hydroxyproline-containing collagen peptides. This hydrolytic activity is due to the presence of endogenous proteases in dentin matrices, including MMPs -2, -8, and -9 and cathepsin K . When these demineralized matrices were treated by cross-linking agents such as carbodiimide or glutaraldehyde, the loss of dry mass was significantly reduced .
Thirty 3 mm × 0.5 mm × 6 mm dentin beams were prepared from mid-coronal dentin as described above. After rinsing in water, the demineralized beams (10/group) were dipped in water for 10 min or in 5 wt% GSE in 5% ethanol/95% water for 1 min or 10 min, rinsed briefly and then dropped into 0.5 ml of 0.05 M HEPES buffer in sealed containers that were incubated at 37 °C for 1 week with shaking at 15 cycles/min. The HEPES buffer (pH 7.4) also contained 2.5 mM CaCl 2 ·2H 2 O and 0.02 mM ZnCl 2 (both from Sigma–Aldrich, St. Louis, MO, USA). At the end of one week, 100 μL of incubation media was mixed with an equal volume of 12 N HCl to create 6 N HCl, which was used to hydrolyze the soluble collagen fragments into their constituent amino acids in sealed glass ampoules at 118 °C for 16 h. After opening vials, the HCl was allowed to evaporate in a vacuum dessicator, the bottom of which was covered by NaOH pellets to neutralize the HCl. The dry residue was then analyzed for hydroxyproline using a colorimetric method .
2.7
Scanning electron microscopy
Control and experimental dentin specimens (3/group) were acid-etched with 37% phosphoric acid gel for 15 s, then rinsed with water for 15 s. The control specimens were then treated with water for 1 min. The experimental teeth were treated with 5 mass% GSE in water, ethanol or mixtures for 1 min. All surfaces were rinsed with water for 15 s and then air-dried for 30 s using continuous, full strength air for 30 s, 10 cm from the dentin surface. All specimens except those that were dry-bonded were critical-point dried (Samdri-790, Hummer Sputtering System, Anatech Corp., San Diego, CA, USA) prior to being coated with gold/palladium and examined in an SEM (Model XL-30 FEG, Philips Corp., Hillsboro, OR, USA) at 10 keV.
2.8
Statistics
The μTBS obtained from beams derived from each of the 4 teeth in each group (8 groups) were pooled together to obtain the mean bond strength value. Each tooth was treated as a statistical unit. The bond strength data were analyzed via two-way ANOVA (SigmaPlot 13, Systat Software Inc., San Jose, CA, USA) using cross-linking as one factor and bonding type (wet vs dry) as the second factor. There was a significant interaction between cross-linking and bond type ( p < 0.001). Thus, the data were re-analyzed by the least squares means test. Least squares means are the expected value of group or subgroup means that one expects for a balanced design involving the group variable with all covariates at their mean value.
The matrix stiffness and collagen solubilization data were logarithmically transformed to obtain normal distribution and equality of variance. They were then analyzed using separate, one-way ANOVAs and Holm-Sidak multiple comparison tests (SigmaPlot 13). Statistical significance was set in advance at the 0.05 level.
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