Effect of hesperidin incorporation into a self-etching primer on durability of dentin bond

Highlights

  • The use of natural cross-linkers incorporated primer is proposed.

  • The effect of hesperidin-incorporation primer on dentin bonding is demonstrated.

  • The immediate bond strength is increased by even at low concentration (i.e. 0.5%) of hesperidin.

  • Collagen matrix at bonded interface will be degradation over time even with self-etching adhesives.

  • To ensure long-term dentin bond durability, the higher concentration of hesperidin should be incorporated.

Abstract

Objective

Collagen degradation at the resin–dentin interface deteriorates dentin bond durability. The use of natural cross-linkers might offer a positive approach to stabilize the resin–dentin interface. This study evaluated the effects of incorporation of natural cross-linkers into a self-etch adhesive primer on the immediate and long-term micro-tensile bond strengths (μTBS) to dentin.

Methods

Experimental primers were prepared by incorporating either 0.5%, 1%, 2%, 5% of hesperidin (HPN) or 0.5% of proanthocyanidins (PA) into Clearfil SE primer. Extracted human molar teeth were restored using the experimental primers or the pure SE primer (control). The mechanical properties of the bonded interfaces were measured using the nano-indentation tests. Beam-shaped bonded specimens were sub-divided for one-day and one-year μTBS test. Interfacial collagen morphology was observed using transmission electron microscopy.

Result

The immediate μTBS significantly increased in 0.5%, 1% and 2% HPN-incorporated groups when compared with the control. The mechanical properties of bonded interface were improved with 1% and 2% HPN-incorporated primers. For the long-term μTBS, the 2% and 5% HPN-incorporated group were significantly higher than the control. The morphology of the collagen fibrils were preserved by 5% HPN-incorporation after one-year storage. The PA group, however, failed to improve the μTBS and the mechanical properties of the bonded interfaces.

Significance

The incorporation of 2% HPN into the self-etching primer had a positive effect on the immediate μTBS and mechanical properties of the resin–dentin interfaces. The 5% HPN group preserved the morphology of the collagen in the hybrid layer after one-year storage in artificial saliva.

Introduction

Adhesive restorations are widely distributed as the routine procedures in operative and restorative treatments. The use of self-etch adhesives became popular because it is less technique-sensitive, less aggressive to dentin, when compared with phosphoric-acid etching and shows less post-operative sensitivity . However, deterioration of dentin bond occurs due to degradation in resin–dentin interface . Unlike etch-and-rinse technique, self-etch technique does not completely expose the collagen matrix ; however, the stability of collagen fibrils within the hybrid layer is crucial for the maintenance of bond effectiveness over time . Past studies have focused on two major factors that can degrade resin–dentin interface. The first factor is the hydrophilic characteristic of the monomer that can be hydrolyzed in aqueous solution . The second factor is the uncured monomer that may remain at the bottom of the hybrid layer . These two phenomena consequently expose the collagen matrix that can be degraded by proteolytic enzyme . It has been reported that dentin collagenolytic and gelatinolytic activities can be suppressed by protease inhibitors . Therefore, matrix metalloproteinase (MMPs) inhibitors such as chlorhexidine have been reported to be beneficial to preserve the hybrid layer and improve bond strength over time . Recently, many researchers have reported that the use of collagen cross-linker to acid-etched dentin can prevent collagen degradation within the hybrid layer and maintain good dentin bond strength . To simplify the use of cross-linker in clinical situations, cross-linkers should be incorporated directly into the dental primer or adhesives . Since collagen matrices are partially denuded by acidity of the self-etching primer, the incorporation of cross-linker into the primer allows the cross-linking agents to interact with these denuded collagen substrate immediately upon removal of the mineral phase by the primer.

Hesperidin (HPN), hesperetin-7-O-rutinoside, is a flavonoid extracted from citrus fruits. The pharmacological properties and medicinal uses of HPN are associated with its wide range of benefits such as anti-inflammatory , analgesic , anti-microbial , and anti-oxidant effects . HPN is also capable of carcinogenesis inhibition , bone loss prevention and inhibition of MMPs’ proteolytic activities . We first attempted to apply HPN in root caries model in a pH cycle study, where HPN showed the potential to prevent collagen degradation against proteolytic enzyme . Considering its cross-linking effect on dentin bonding, we have incorporated HPN into a primer of self-etch adhesive system, which effectively increased the immediate resin–dentin bond strength .

In the present study, we aimed to evaluate the effect of incorporation of different concentrations of HPN into the primer of a self-etch adhesive on micro-mechanical properties of resin–dentin interfaces, immediate and long-term resin–dentin bond strength.

The null hypotheses tested were that the incorporation of HPN has no effects (i) on the mechanical properties of resin–dentin interfaces and (ii) on the immediate and long-term resin–dentin bond strength.

Materials and methods

Micro tensile bond strength testing

The teeth used in this study were collected after obtaining the patients’ informed consent. The Human Research Ethics Committee of Tokyo Medical and Dental University, Japan reviewed and approved this study under the protocol number 725. Forty-eight freshly extracted non-carious human molar teeth were used for bond strength testing. A flat dentin surface was created perpendicular to the tooth’s longitudinal axis using a slow-speed diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water cooling to remove occlusal dentin. Smear layer was produced on each surface using #600 Silicon Carbide paper under water irrigation. Eight teeth per group were allocated for the following self-etching primers. HPN (hesperetin-7-O-rutinoside, Wako Pure Chemical Industries, Ltd., Tokyo, Japan) or grape seed derived proanthocyanidins (PA) (proanthocyanidins, Kikkoman Biochemifa, Chiba, Japan) was added to Clearfil SE primer (Kuraray Noritake Dental Inc. Tokyo, Japan) to formulate the experimental primer groups (0.5%, 1%, 2%, 5% HPN and 0.5% PA ( Table 1 ). The pH value of each experimental primer was measured with a digital pH meter (Twin pH B-211, HORIBA, Ltd., Kyoto, Japan). The original SE primer served as control. The dentin surfaces were conditioned with the primers according to the manufacturer’s instructions, then Clearfil SE bond (Kuraray Noritake Dental Inc. Tokyo, Japan) was applied and light-cured for 10 s (OPTILUX 501, Kerr corporation, CA, USA. light intensity 650 mW/cm 2 ). Composite resin (Clearfil AP-X, Kuraray Noritake Dental Inc. Tokyo, Japan) was placed on dentin surfaces incrementally up to 5 mm of thickness. Each increment was light-cured for 30 s. After storage in de-ionized water at 37 °C for 24 h, the bonded teeth were sectioned longitudinally into serial slabs, and further sectioned to obtain (0.9 mm × 0.9 mm) composite-dentin beams. The beams were divided into two test groups. Half of the specimens from each group were used for immediate micro-tensile bond strength (μTBS) testing and the remaining half were stored in artificial saliva for one year at 37 °C. The artificial saliva contained (mM): CaCl 2 (0.7), MgCl 2 ·6H 2 O (0.2), KH 2 PO 4 (4.0), KCl (30), NaN 3 (0.3), and HEPES buffer (20).

Table 1
Study design and composition of SE primer and SE bond.
Group Material tested pH
1 Pure clearfil SE primer 2.0
2 Clearfil SE primer +0.5% HPN 2.0
3 Clearfil SE primer +1% HPN 2.0
4 Clearfil SE primer +2% HPN 2.0
5 Clearfil SE primer +5% HPN 2.0
6 Clearfil SE primer +0.5% PA 2.0
Clearfil SE Primer : MDP, HEMA, di-methyl-acrylate, monomer, water, catalyst.
Clearfil SE Bond : MDP, HEMA, di-methyl-acrylate, monomer, micro filler, catalyst.

Bond strength testing was performed for specimens after one day and one year storage. The exact dimension of each beam was measured using a pair of digital calipers. Any specimen close to pulp horns was discarded due to inadequate size of dentin for tensile testing. The number of beams used for bond strength testing ranged from 12 to 16 per tooth. Each beam was stressed to failure under tension using a universal testing machine (EZ Test, Shimadzu Co. Kyoto, Japan) at a crosshead speed of 1 mm/min.

Failure mode

The fractured dentin surfaces were air-dried, sputter-coated with gold/palladium and examined using a scanning electron microscope (SEM, JSM-5310LV scanning microscope, JEOL Ltd. Tokyo, Japan) operating at 5 kV. The failure modes were categorized into four groups according to the type and location, (A) mixed type of failure in resin composite and adhesive layer; (B) cohesive failure in adhesive layer; (C) mixed type of adhesive failure at the interface with retention of adhesive; (D) cohesive failure in dentin.

Examination of the etching effect of experimental primers on smear layer

To examine the effect of experimental SE primers on the smear layer, dentin discs of approximately 1 mm thickness were obtained from the mid-coronal dentin of another six extracted human third molars. The dentin surfaces were similarly treated with the pure primer or each of the experimental primers for 20 s. Immediately after treatment, the discs were soaked in 100% acetone for 5 min to remove the applied primer, followed by dehydration in ascending concentrations of ethanol, then immersion in hexamethyldisilazane (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) for 10 min and mounted on aluminum stubs and sputter-coated with gold/palladium and examined with the SEM operating at 5 kV.

Nano-indentation test

Dentin surfaces were prepared from 18 freshly extracted teeth. Three teeth in each group were bonded as previously described for tooth preparation. After storage in de-ionized water for 24 h at 37 °C, nano-indentation tests were conducted to measure Hardness ( H ) and Elastic Modulus ( EM ) of the adhesive layers and bonded interfaces. The test methodology was followed as described in our previous study . Three slabs of 2 mm thickness were sectioned perpendicular to the bonding surface and selected from the center of each bonded specimen. The specimens were embedded in epoxy resin. After polishing with Silicon Carbide papers from #600 to #2000 and diamond pastes of decreasing particle sizes down to 0.25 μm, nano-indentation test was performed at a constant temperature of 27.5 °C with a Berkovich indenter attached to a computer-controlled nano-indentation device (ENT-1100, Elionix. Tokyo, Japan). The positions of indentation points were programmed at an interface, 10 μm distance from the interface in the adhesive layer and in the dentin. Data of H were assigned to H 1 (hardness at the adhesive layer), H 2 (hardness at the interface) and H 3 (hardness at dentin). The relative hardness at the interface ( H 2/3 ) was calculated as the ratio of H 2 to H 3 ( H 2/3 = H 2 / H 3 ) to consider intrinsic difference in hardness of individual dentin substrate. The data of EM were determined in the same manner and assigned to EM 1 , EM 2 , EM 3 and EM 2/3 . The data were obtained from the average value of 10 points of each location. Statistical analysis was performed for 9 specimens (3 slabs each from 3 bonded teeth) in each group.

Transmission electron microscopy (TEM)

The deboned specimens near the mean μTBS from each group were allocated for TEM examination. The dentin sides of beams were demineralized in an aqueous solution 15% (w/v) EDTA a pH of 7.0 for 7 days at room temperature. The specimens were fixed in a solution 2.5% glutaraldehyde for 2 h followed by 2% paraformaldehyde in 0.1 mol/L cacodylate buffer (pH 7.3) for 1 h finally in osmium tetroxide for 2 h. The specimens were dehydrated in increasing concentrations of ethanol (50%, 60%, 70%, 80%, 90% for 25 min and 100% for 20 min each). Then the specimens were embedded in epoxy resin at 60 °C for 96 h. After resin embedding, ultra-thin transverse sections (ca.70 nm) were obtained with an ultra-microtome using a diamond knife and were collected onto 150 mesh copper grids under microscope (Sciences, Fort Washington, PA, USA). Specimens were double stained in 2% aqueous uranyl acetate for 20 min and Sato’s lead citrate for 5 min. After drying, the sections were observed with TEM (H-7100; Hitachi, Tokyo, Japan) operating at 75 kV.

Statistical analysis

The data were analyzed using a statistical software package (Sigma Stat Version 16.0, SPSS, Chicago, IL, USA). The μTBS data were analyzed using two-way ANOVA, followed by one-way ANOVA and Tukey’s post hoc for multiple comparisons. The mechanical properties ( H and EM ) were analyzed using one-way ANOVA and Tukey’s post hoc for multiple comparisons. Level of statistical significance was set at 5%.

Materials and methods

Micro tensile bond strength testing

The teeth used in this study were collected after obtaining the patients’ informed consent. The Human Research Ethics Committee of Tokyo Medical and Dental University, Japan reviewed and approved this study under the protocol number 725. Forty-eight freshly extracted non-carious human molar teeth were used for bond strength testing. A flat dentin surface was created perpendicular to the tooth’s longitudinal axis using a slow-speed diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water cooling to remove occlusal dentin. Smear layer was produced on each surface using #600 Silicon Carbide paper under water irrigation. Eight teeth per group were allocated for the following self-etching primers. HPN (hesperetin-7-O-rutinoside, Wako Pure Chemical Industries, Ltd., Tokyo, Japan) or grape seed derived proanthocyanidins (PA) (proanthocyanidins, Kikkoman Biochemifa, Chiba, Japan) was added to Clearfil SE primer (Kuraray Noritake Dental Inc. Tokyo, Japan) to formulate the experimental primer groups (0.5%, 1%, 2%, 5% HPN and 0.5% PA ( Table 1 ). The pH value of each experimental primer was measured with a digital pH meter (Twin pH B-211, HORIBA, Ltd., Kyoto, Japan). The original SE primer served as control. The dentin surfaces were conditioned with the primers according to the manufacturer’s instructions, then Clearfil SE bond (Kuraray Noritake Dental Inc. Tokyo, Japan) was applied and light-cured for 10 s (OPTILUX 501, Kerr corporation, CA, USA. light intensity 650 mW/cm 2 ). Composite resin (Clearfil AP-X, Kuraray Noritake Dental Inc. Tokyo, Japan) was placed on dentin surfaces incrementally up to 5 mm of thickness. Each increment was light-cured for 30 s. After storage in de-ionized water at 37 °C for 24 h, the bonded teeth were sectioned longitudinally into serial slabs, and further sectioned to obtain (0.9 mm × 0.9 mm) composite-dentin beams. The beams were divided into two test groups. Half of the specimens from each group were used for immediate micro-tensile bond strength (μTBS) testing and the remaining half were stored in artificial saliva for one year at 37 °C. The artificial saliva contained (mM): CaCl 2 (0.7), MgCl 2 ·6H 2 O (0.2), KH 2 PO 4 (4.0), KCl (30), NaN 3 (0.3), and HEPES buffer (20).

Nov 25, 2017 | Posted by in Dental Materials | Comments Off on Effect of hesperidin incorporation into a self-etching primer on durability of dentin bond
Premium Wordpress Themes by UFO Themes