Abstract
Objectives
To characterize the properties of dentin matrix treated with two proanthocyanidin rich cross-linking agents and their effect on dentin bonded interfaces.
Methods
Sound human molars were cut into 0.5 mm thick dentin slabs, demineralized and either treated with one of two cross-linking agents (grape seed—GSE and cocoa seed—COE extracts) or left untreated. The modulus of elasticity of demineralized dentin was assessed after 10 or 60 min and the swelling ratio after 60 min treatment. Bacterial collagenase was also used to assess resistance to enzymatic degradation of samples subjected to ultimate tensile strength. The effect of GSE or COE on the resin–dentin bond strength was evaluated after 10 or 60 min of exposure time. Data were statistically analyzed at a 95% confidence interval.
Results
Both cross-linkers increased the elastic modulus of demineralized dentin as exposure time increased. Swelling ratio was lower for treated samples when compared to control groups. No statistically significant changes to the UTS indicate that collagenase had no effect on dentin matrix treated with either GSE or COE. Resin–dentin bonds significantly increased following treatment with GSE regardless of the application time or adhesive system used.
Significance
Increased mechanical properties and stability of dentin matrix can be achieved by the use of PA-rich collagen cross-linkers most likely due to the formation of a PA–collagen complex. The short term resin–dentin bonds can be improved after 10 min dentin treatment.
1
Introduction
Adhesive restorations are routinely used to replace carious dental tissue, fractured tooth, and replacement of defective restorations. Despite significant improvement of adhesives systems, the bonded interface formed by a mixture of dentin organic matrix, residual hydroxyapatite crystallites, resin monomers and solvents, still remains the weakest area of adhesive restorations . Moreover, failure at the bonded interface may lead to the formation of pathways in which oral fluid, bacterial products and endogenous proteolytic enzymes can degrade the components. Deterioration of the dentin collagen fibrils has been suggested as a possible mechanism responsible for adhesive bonds degradation .
Fibrillar type I collagen accounts for 90% of the dentin organic matrix while the remaining 10% consists of non-collagenous proteins such as phosphoproteins and proteoglycans . Lower biodegradation rates and high mechanical properties of collagen are desirable for many in vivo applications, such as restorative dentistry procedures. The induction of exogenous collagen cross-links has been proposed as a mechanism to improve the mechanical stability and reduce the biodegradation rates of collagen . Several synthetic (glutaraldehyde, carbodiimides and others) and natural occurring (genipin, proanthocyanidin from grape seed extract and others) agents can induce exogenous collagen cross-links .
Proanthocyanidin (PA) is a natural collagen cross-linker well known to readily precipitate proline rich proteins (such as collagen) due to hydrogen and covalent bonds . PAs are considered one of the most important classes of secondary metabolites in the plant kingdom available in fruits, vegetables, nut, seeds, flowers, and barks . It is well documented that cocoa and its products, along with grape seed are among the richest sources of PAs . Recent studies have shown that a PA-based cross-linker agent (grape seed extract) increased the mechanical properties of demineralized dentin matrix and enhanced the resin–dentin bond strength after 1 h treatment .
In an attempt to reduce treatment time to reproduce a more clinical relevant procedure, this study compared the effect of two different PA-based cross-linker (cocoa and grape seed extracts) on resin–dentin bonded interface using two different treatment times (10 and 60 min); and characterized the effect of different sources of PA on the mechanical properties and resistance against enzymatic degradation of demineralized dentin matrices. The null hypothesis tested was that the use of PA-based cross-linkers would not affect the dentin bond strength and properties of demineralized dentin when compared to a non-treated group.
2
Materials and methods
The use of seventy-eight sound molars was approved by the Institutional Review Board Committee from the University of Illinois at Chicago (protocol # 2009-0198). Cocoa seed extract ( Theobroma cacao–Foratero ) was obtained from Barry Callebaut (Lebbeke-Wieze, Belgium). Grape seed extract ( Vitis vinifera ) was obtained from Polyphenolics (Madera, CA, USA). Bacterial collagenase from Clostridium histolyticum (type I, ≥125 CDU/mg solid) was obtained from Sigma–Aldrich (St. Louis, MO, USA).
2.1
Modulus of elasticity of demineralized dentin
Seven teeth were sectioned into 0.5 ± 0.1 mm thick slabs ( n = 7 slabs per tooth) in the mesio-distal direction with a slow speed diamond wafering blade (Buehler-Series 15LC Diamond) under constant water irrigation. The sections were further trimmed to a final rectangular dimension of 0.5 mm thickness × 1.7 mm width × 7.0 mm length using a cylindrical high speed diamond bur (#557D, Brasseler, Savannah, GA). A dimple was made at one end of each sample to allow for repeated measurements to be performed on the same surface. Specimens were immersed in 10% phosphoric acid solution (LabChem, Pittsburgh, PA) for a period of 5 h and thoroughly rinsed with distilled water for 10 min. Demineralized specimens ( n = 12) were treated with one of two different cross-linkers, grape seed extract (GSE) or cocoa seed extract (COE), with the same concentration (6.5%) and dissolved in distilled water and acetone–water (30:70) respectively. All solutions had the pH adjusted to 7.4 using NaOH. Two control groups (no cross-linking agents) were tested, one in distilled water and other in acetone–water following the same protocol described for the experimental groups. Specimens were immersed in water for baseline measurements and then in their respective solutions for either 10 or 60 min.
An aluminum alloy fixture with 2.5 mm span where specimens were tested in 3-point bending while immersed in liquid using a 1 N load cell mounted on a universal testing machine (EZ Graph, Shimadzu, Kyoto, Japan) at crosshead speed of 0.5 mm/min. Displacement ( D ) during compression was displayed in millimeters and calculated at a maximum strain of 3%. The modulus of elasticity ( E ) was obtained as previously described and calculated as follows:
E = P L 3 4 D b T
where P is the maximum load, L is the support span, D is the displacement, b is the width of the specimen, and T is the thickness of the specimen. The data were collected and statistically analyzed using a General Linear Model SPSS program for ANOVA followed by Post Hoc Tukey’s test at a 95% confidence interval.
2.2
Swelling ratio
Dentin samples (0.5 × 1.7 × 7.0, n = 12) were treated for 1 h in either cross-linking solution (GSE and COE) or both control groups (distilled water and acetone) as described above. After treatment, samples were swollen in water and equilibrated overnight in PBS (pH 7.4) at room temperature. The specimens were removed quickly blotted with filter paper to remove excess surface water and weighed immediately. Dentin samples were then placed in a large volume of deionized water to remove the buffer salts and air dried to constant weight. The swelling ratio ( Q ) was calculated as the ratio of the weight of swollen sample to that of dry sample . Data was analyzed using a General Linear Model SPSS program for ANOVA followed by a Post Hoc Tukey’s test at a 95% confidence interval.
2.3
Resistance against enzymatic degradation: ultimate tensile strength (UTS)
Eleven teeth were sectioned into 0.5 mm thick slabs and trimmed to an hour-glass shaped sample with neck area of 0.5 ± 0.1 × 0.5 ± 0.1 mm at middle dentin using a cylindrical diamond bur (#557, Brasseler). Samples were fully demineralized using 10% phosphoric acid solution (LabChem, Inc.) for 5 h, thoroughly rinsed and immediately randomly divided into three treatments ( n = 12): control (distilled water), 6.5% GSE in distilled water and 6.5% COE in acetone–water (30:70). Specimens were kept in their respective solutions for 1 h, thoroughly rinsed and either subjected or not to enzymatic challenge for 24 h using bacterial collagenase (100 μg/ml) in 0.2 M ammonium bicarbonate buffer (pH = 7.5) or 24 h in buffer only. Enzymatic activity of bacterial collagenase has already been proved to successfully challenge dentin matrix . For UTS evaluation, the specimens were glued with a cyanoacrylate adhesive (Loctite Superbond, Henkel, Avon, OH, USA) to a jig, which was mounted on a microtensile tester machine (Bisco, Schaumburg, IL, USA) and subjected to a tension force at a crosshead speed of 1 mm/min. Means and standard deviations were calculated and expressed in MPa. Statistical analysis was performed using a General Linear Model SPSS program for ANOVA two-way (treatment and collagenase challenge) followed by a Post Hoc Tukey’s test at a 95% confidence interval.
2.4
Resin–dentin microtensile bond strength (μTBS)
The occlusal surfaces of 60 molars were ground flat using #180, 320 and 600 grit silicon carbide papers (Buehler, Lake Bluff, IL) under running water to expose middle coronal dentin. The prepared dentin surfaces were divided into six groups ( n = 20): control distilled water for 10 min, control distilled water for 60 min, 6.5% GSE for 10 min, GSE for 60 min and 6.5% COE for 10 min and COE for 60 min. Each group was either restored using Adper Single Bond Plus (SB, 3M ESPE, St. Paul, USA) or One Step Plus (OS, Bisco, Schaumburg, USA). Dentin surfaces were etched with the respective system’s etchants for 15 s, rinsed, treated with cross-linking solutions and then thoroughly rinsed. The bonding procedures were performed following manufacturers’ instructions. The control group followed the same protocol but distilled water was used instead of cross-linking solutions. Filtek supreme (3M ESPE) was used to build a 5 mm crown incrementally. Specimens were stored in distilled water at 37 °C for 24 h.
Teeth were sectioned into 0.7 × 0.7 ± 0.1 mm resin–dentin beans that had their edges glued with cyanoacrylate to a jig, and were then tested in a microtensile tester machine (Bisco, Schaumburg, IL, USA) at a crosshead speed of 1.0 mm/min. Five beams with at least 2 mm remaining dentin thickness were tested per tooth. Microtensile bond strength (MPa) was determined by dividing the fracture load by the cross-sectional area of the interface. Mean bond strength values for both adhesives systems were analyzed using a General Linear Model SPSS program for two-way ANOVA (treatment and exposure time) followed by a Post Hoc Tukey’s test at a 95% confidence interval.
2
Materials and methods
The use of seventy-eight sound molars was approved by the Institutional Review Board Committee from the University of Illinois at Chicago (protocol # 2009-0198). Cocoa seed extract ( Theobroma cacao–Foratero ) was obtained from Barry Callebaut (Lebbeke-Wieze, Belgium). Grape seed extract ( Vitis vinifera ) was obtained from Polyphenolics (Madera, CA, USA). Bacterial collagenase from Clostridium histolyticum (type I, ≥125 CDU/mg solid) was obtained from Sigma–Aldrich (St. Louis, MO, USA).
2.1
Modulus of elasticity of demineralized dentin
Seven teeth were sectioned into 0.5 ± 0.1 mm thick slabs ( n = 7 slabs per tooth) in the mesio-distal direction with a slow speed diamond wafering blade (Buehler-Series 15LC Diamond) under constant water irrigation. The sections were further trimmed to a final rectangular dimension of 0.5 mm thickness × 1.7 mm width × 7.0 mm length using a cylindrical high speed diamond bur (#557D, Brasseler, Savannah, GA). A dimple was made at one end of each sample to allow for repeated measurements to be performed on the same surface. Specimens were immersed in 10% phosphoric acid solution (LabChem, Pittsburgh, PA) for a period of 5 h and thoroughly rinsed with distilled water for 10 min. Demineralized specimens ( n = 12) were treated with one of two different cross-linkers, grape seed extract (GSE) or cocoa seed extract (COE), with the same concentration (6.5%) and dissolved in distilled water and acetone–water (30:70) respectively. All solutions had the pH adjusted to 7.4 using NaOH. Two control groups (no cross-linking agents) were tested, one in distilled water and other in acetone–water following the same protocol described for the experimental groups. Specimens were immersed in water for baseline measurements and then in their respective solutions for either 10 or 60 min.
An aluminum alloy fixture with 2.5 mm span where specimens were tested in 3-point bending while immersed in liquid using a 1 N load cell mounted on a universal testing machine (EZ Graph, Shimadzu, Kyoto, Japan) at crosshead speed of 0.5 mm/min. Displacement ( D ) during compression was displayed in millimeters and calculated at a maximum strain of 3%. The modulus of elasticity ( E ) was obtained as previously described and calculated as follows:
E = P L 3 4 D b T
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