Effect of collagen cross-linkers on the shear bond strength of a self-etch adhesive system to deep dentin.

OBJECTIVES: To evaluate the shear bond strength of composite resin to deep dentin, bonded using a self-etch adhesive, after treatment with two collagen cross-linkers at varying time intervals.
MATERIALS AND METHODS: Thirty extracted human incisors were sectioned longitudinally into equal mesial and distal halves (n = 60). The proximal deep dentin was exposed and the specimens were divided based on the surface treatment of dentin prior to bonding as follows: Group I (n = 12, control): No prior dentin surface treatment; group II (n = 24): Dentin surface pretreated with 10% sodium ascorbate; and group III (n = 24): Dentin surface pretreated with 6.5% proanthocyanidin. Groups II and III were further divided into two subgroups based on the pre-treatment time of five and 10 min. Shear bond strength of the specimens was tested using universal testing machine and the data were statistically analyzed.
RESULTS: Significantly higher shear bond strength to deep dentin was observed in teeth treated with 10% sodium ascorbate and 6.5% proanthocyanidin compared to control group. No significant difference was observed between 5 min and 10 min pre-treatment times.
CONCLUSION: Dentin surface pre-treatment with both 10% sodium ascorbate and 6.5% proanthocyanidin resulted in significant improvement in bond strength of self-etch adhesive to deep dentin.

INTRODUCTION

The strength of adhesive joints formed by bonding resins to etched enamel and/or conditioned dentin has resulted in greater application of these systems in everyday dental practice. Dentin adhesives are classified as three step, two step and single step systems based on the manner in which the three fundamental steps of etching, priming and bonding to tooth substrate are accomplished. Self-etch adhesive systems use non-rinse acidic monomers that eliminate the need for a separate rinsing step thus reducing the clinical application time.[1] Although contemporary dentin bonding systems show improvement in better handling and bonding characteristics, the achievement of a strong and stable bond to deep dentin still remains a challenge in restorative dentistry.[23]

Dentin is a complex hydrated biological composite material with structural components and properties that vary with location. Studies have shown that a reduction in bond strength occurs when resin composite is bonded to deep dentin. This can be attributed to the complexities in structure of deep dentin, such as an increase in the number of tubules and their diameters with much less intertubular dentin matrix as compared with superficial dentin.[45]

The durability of the bond between dentin and adhesive system depends largely on the structural integrity and mechanical properties of acid-demineralized collagen fibers.[2] Mechanical properties of collagen and its resistance to enzymatic degradation can be improved by an increase in the formation of intra-and inter-molecular and intermicrofibrillar cross-links. This can be achieved by the use of various collagen cross-linkers, both synthetic and natural, on the dentin substrate prior to the bonding procedure.[67] Naturally occurring collagen cross-linkers such as sodium ascorbate and proanthocyanidin have been reported to increase the collagen cross-linking in sound and caries-affected dentin.[6-8] But, the effect of these collagen cross-linkers on the bond strength of a self-etch adhesive to deep dentin has not been reported in the literature. Hence, the aim of this in vitro study was to determine the shear bond strength of composite resin bonded to deep dentin using a self-etch adhesive, after treatment with two collagen cross-linking agents at varying time intervals.

MATERIALS AND METHODS

The materials used in this study and their composition are given in Table 1.

Table 1

Materials used in this study and their composition

Preparation of solutions

10% Sodium ascorbate solution

Ten grams of sodium ascorbate powder (sdfiNEcHEM Ltd., Mumbai, India) were dissolved in 100 mL of distilled water to make 10% sodium ascorbate solution.

6.5% Proanthocyanidin solution

6.5 g of grape seed extract in the form of powder (Puritans Pride Inc., Oakdale, NY, USA) was collected from the capsules and dissolved in 100 mL of distilled water to make 6.5% proanthocyanidin solu tion.

Specimen preparation

Thirty human maxillary central incisors extracted due to periodontal reasons were collected for the study. The teeth were cleaned of debris and stored in 0.2% thymol until use. Each tooth was sectioned longitudinally, parallel to the long axis of the tooth, into an equal mesial and a distal half by means of a low-speed diamond disc (HI – DI Diamond Precision Tools Ltd., London, UK) under copious water supply. The contents of the pulp chamber were then removed ultrasonically to determine its extent.

Using a diamond disc, the dentin in the proximal wall of each half of the crown incisal to the cemento-enamel junction was removed until the remaining dentin thickness was approximately 1 mm, as measured with a metal caliper (Iwanson Spring metal caliper, I.D-Tech, Sialkot, Pakistan), from the outer surface of the prepared proximal portion of the crown to the pulp chamber. All the specimens were immersed in an ultrasonic bath to remove the smear layer. The roots of the specimens were then mounted in self-cure acrylic resin. These specimens (n = 60) were randomly divided into three groups based on the surface treatment of dentin as follows:

Group I (n = 12, control): No pre-treatment was done on the exposed proximal dentin surface. Adhesive bonding and composite buildup was done according to the bonding protocol as described here.

Bonding protocol

The self-etch adhesive, Xeno III, was used according to the manufacturer's instructions. An equal amount of Liquid A and B (1:1) was mixed for 5 s and applied on the proximal dentin surface. The adhesive was left in place undisturbed for 20 s and then gently air dried and light cured (Astralis 3 light curing unit, IvoclarVivadent, Schaan, Liechtenstein) for 40 s. Composite buildup was done by placing two increments of 2-mm-thick composite resin (Ceram X Nano Ceramic Restorative, DentsplyDeTrey GmbH, Konstanz, Germany) using a 3 mm diameter plastic tube as a matrix, with each increment being light cured for 40 s.

Group II (n = 24): 10% sodium ascorbate pre-treatment. This group was further subdivided into two subgroups based on the pre-treatment time as follows:

Subgroup IIA (n = 12): The exposed proximal dentin surface was treated with 10% sodium ascorbate solution for 5 min and rinsed with water, followed by the bonding/buildup procedure as described above.

Subgroup IIB (n = 12): The exposed proximal dentin surface was treated with 10% sodium ascorbate solution for 10 min and rinsed with water. Adhesive bonding and composite buildup followed as described above.

Group III (n = 24): 6.5% proanthocyanidin pre-treatment. This group was further subdivided into two subgroups based on the pre-treatment time as follows:

Subgroup IIIA (n = 12): The exposed proximal dentin surface was treated with 6.5% proanthocyanidin solution for 5 min and rinsed with water, followed by the bonding/buildup procedure as described above.

Subgroup IIIB (n = 12): The exposed proximal dentin surface was treated with 6.5% proanthocyanidin solution for 10 min and rinsed with water. Adhesive bonding and composite buildup followed as described above.

Shear bond strength testing

All the specimens were then stored in distilled water at 37°C for 24 h. Shear bond strength was determined using a universal testing machine (LR 100 K, Lloyd Instruments, Largo, Florida, USA) at a crosshead speed of 1 mm/min. The results were tabulated and statistically analyzed using Statistical product and service solutions (SPSS) version 17.0 software (SPSS Inc., Chicago, IL, USA). Paired t-test was used to calculate the P value (P < 0.001).

RESULTS

The results of this study are shown in Table 2. The results showed that the mean shear bond strength values of subgroup IIA (21.43 ± 0.58), IIB (21.97 ± 0.48), IIIA (23.70 ± 0.58) and IIIB (23.82 ± 0.39) were significantly higher than the mean shear bond strength value in the control group (13.31 ± 0.87) (P < 0.001). Both 5 min and 10 min pre-treatment times of 6.5% proanthocyanidin (IIIA [23.70 ± 0.58] and IIIB [23.82 ± 0.39]) yielded significantly higher shear bond strength values than the corresponding pre-treatment times of 10% sodium ascorbate (IIA [21.43 ± 0.58] and IIB [21.97 ± 0.48]) (P < 0.001). No significant difference in shear bond strength values was found between the two pre-treatment times in both the 10% sodium ascorbate and the 6.5% proanthocyanidin groups (P > 0.05).

Table 2

Comparison of mean shear bond strength (MPa) of different study groups

DISCUSSION

The two main strategies that are currently in use for adhesive bonding to enamel and dentin are the total-etch technique and the self-etch technique. Although etch-and-rinse technique is still considered as the most effective approach to achieve efficient and stable bonding to enamel and dentin, the multiple application steps, the critical rinsing step and the frustratingly high incidence of post-operative sensitivity led to the evolution of more simplified, less technique-sensitive and user-friendly self-etch adhesives.[19]

The major shortcomings of dentin as a bonding substrate are its heterogeneous composition and hydrophilic nature. It presents an intricate composition comprised of 70% by weight of an inorganic phase of carbonate-rich hydroxyapatite crystals, 20% by weight of an organic phase of predominantly type I collagen and 10% water.[10-12]

Covalent inter-and intra-molecular cross-links are the basis for stability, tensile strength and viscoelasticity of the collagen fibrils. Various chemicals, both synthetic and natural, have the ability to increase these collagen cross-links in biological tissues.[61314]

Sodium ascorbate is an important component in the synthesis of hydroxyproline and hydroxylysine in collagen. Hydroxyproline serves to stabilize the collagen triple helix and hydroxylysine is necessary for the formation of intermolecular cross-links in collagen.[15]

Proanthocyanidins (PA) are naturally occurring bioflavonoids found in high concentrations in grape seed, pine bark, cranberries, lemon tree bark and hazel nut tree leaves.[16] Although PA from grape seed extract has been shown to effectively cross-link collagen in in vitro studies,[8141718] their effect on the bond strength of resin composite bonded using a self-etch adhesive to deep dentin remains an unexplored area in restorative dentistry.

In the present study, group I (control) recorded a mean shear bond strength value of 13.31 ± 0.87 Mega Pascals (MPa) to deep dentin, which is lesser than the optimal bond strength of self-etch adhesive resins to superficial dentin (20-24 MPa).[19-21] This is in accordance with previous studies done by Tagami et al.,[4] Pashley et al.,[22] Kaaden et al.[23] and Yazici et al.,[24] who showed that bond strength of self-etch adhesive resins to deep dentin can be as low as 10.3-16.7 MPa.

Results of this in vitro study showed that when deep dentin was treated with sodium ascorbate and proanthocyanidin, the bond strength reached values (21.43-23.82 MPa) comparable with the optimal bond strength values of superficial dentin. This can be attributed to improved dentin collagen stability obtained from an increase in the number of collagen cross-links, achieved by the use of these collagen cross-linkers.

Group III (6.5% proanthocyanidin) showed a significantly higher bond strength to deep dentin compared with group II (10% sodium ascorbate) in both the subgroups (P < 0.001). This is in accordance with the findings of Macedo et al.,[8] who showed that the application of 6.5% grape seed extract and 5% gluteraldehyde to caries-affected and sound dentin significantly improved the microtensile bond strength of composite to dentin. Similarly, Walter et al.[25] showed that 0.5% proanthocyanidin efficiently stabilized collagen and increased its resistance to caries compared with 0.625% genipin and 5% gluteraldehyde. Bedran-Russo, et al.[6] found that naturally occurring cross-linking agents such as 0.5% proanthocyanidin and 0.625% genipin are capable of stabilizing demineralized dentin collagen more effectively compared with 5% gluteraldehyde.

This could be attributed to the specificity of proanthocyanidin to facilitate the enzyme proline hydroxylase, which catalyzes the hydroxylation of proline, an essential step in collagen biosynthesis. PA and proteins have been shown to interact in four different ways: Covalent interactions, ionic interactions, hydrogen bonding interactions or hydrophobic interactions.[81718] Castellan et al.[14] showed that when demineralized, dentin is treated with PA, it results in improved mechanical properties and reduced water absorption as a result of the dense collagen network formed by the use of exogenous cross-linkers.

Unlike previous studies that employed longer application times,[68] application times of 5 and 10 min were chosen in the present study, and the results showed no statistically significant difference in the shear bond strength values between the two pre-treatment times in both 10% sodium ascorbate and 6.5% PA groups. Hence, dentin pre-treatment with collagen cross-linkers can be safely recommended as an effective chair side procedure to overcome the disadvantage of reduced bond strength of self-etch adhesive systems to deep dentin.

CONCLUSIONS

Under the limitations of this in vitro study, the following conclusions can be drawn:

A significant improvement in the bond strength of resin composite to deep dentin bonded using a self-etch adhesive was obtained when the dentin surface was pretreated with both 10% sodium ascorbate and 6.5% proanthocyanidin compared with the control group

The use of 6.5% proanthocyanidin as a collagen cross-linker on deep dentin significantly improved the shear bond strength values compared with the use of 10% sodium ascorbate

There was no significant difference in the shear bond strength values between the 5 min and 10 min pre-treatment times in both 10% sodium ascorbate and 6.5% proanthocyanidin groups.