Effect of proanthocyanidin and riboflavin on shear bond strength of a nanocomposite to deep dentin – An in vitro Study

Abstract

Aims:

The objective of this study was to evaluate the effect of two collagen crosslinking agents on shear bond strength of a nanocomposite using total-etch adhesive to deep dentin.

Materials and Methods:

Thirty maxillary central incisors were selected for the study and each tooth was divided into two equal halves with diamond disc (n = 60). Proximal surfaces were prepared to expose the deep dentin until the remaining dentin thickness was approximately 1 mm. The specimens were divided into three groups based upon the biomodification of dentin with collagen crosslinking agents. In Group I (n = 20), no collagen crosslinking agent was used before bonding system. In Group II, deep dentin biomodification was done with 6.5% proanthocyanidin (PA). In Group III, deep dentin biomodification was done with Riboflavin. Shear bond strength was evaluated using universal testing machine.

Statistical Analysis Used:

One-way analysis (ANNOVA). Pairwise comparison of groups was done with post hoc test.

Results:

Biomodification of deep dentin with PA showed the highest shear bond strength followed by riboflavin and control group.

Conclusion:

Biomodification of dentin surface with collagen cross-linking agents results in improvement of bond strength of total-etch adhesive to deep dentin.

INTRODUCTION

The rise in demand for esthetics has hugely inspired the evolution of many adhesive systems allowing fairly strong adhesion to tooth structures. Bonding to dentin has been considered more intricate and less predictable because of its varying structure. It includes the deposition of hydroxyapatite on the scaffold of collagen fibrils and a considerable proportion of water.[1] The content of collagen-rich inter-tubular dentin decreases to 12% at predentin, and the amount of water increases in deep dentin. From the dentin-enamel junction to the pulp, the number of tubules increases by more than threefold.[2] The factors mentioned above make bonding to deep dentin more demanding and strenuous.

For total-etch adhesives, the bonding mechanism is essentially based on the infiltration of adhesive resin inside the scaffold of exposed collagen fibrils, which should be as absolute as possible.[3] The most delicate portion of the bonded interface is the hybrid layer (a collagen-resin interface), where most failures take place due to stress concentration. The reliability of dentin bonding depends mostly upon this interface.[4,5] The enhancement of collagen stability within the hybrid layer has been a thought-provoking target for research.[6] The biomimetic strategy using collagen crosslinking agents has been reported to instigate the development of intermolecular as well as intramolecular cross-links.[4,5,6,7] This results in the protection of collagen against degradation and improvement of the properties of resin dentin interface, including the improved bond strength. Various agents that can be used for biomodification of dentin are genipin, glutaraldehyde, sodium ascorbate, riboflavin, and proanthocyanidin (PA).

PA is polyphenolic bioflavonoid extracted from grape seeds. PA is widely used as natural antioxidant, free-radical scavenger and it also induces the formation of exogenous collagen crosslinks.[7] Very few studies have evaluated the relation of crosslinking agents and bond strength of a total etch adhesive to deep dentin. Furthermore, the role of riboflavin as collagen crosslinking agent in dentistry is little known. Hence, this study was carried out in vitro to determine the influence of PA and riboflavin on shear bond strength of a nanocomposite to deep dentin utilizing total-etch adhesive system.

MATERIALS AND METHODS

After receiving approval from the institutional review board (GIDSR/2019/755), this research was carried out. Collection of 30 recently extracted maxillary central incisors devoid of any defects was done.

Preparation of solutions

The first solution (6.5% PA) was prepared using the capsules of 6.5 g of grape seed extract (Puritans Pride Inc., Oakdale, NY, USA) and dissolving the powder from capsules to 100 ml distilled water. For the second solution, Riboflavin-5-phosphate was dissolved in distilled water to prepare 1% riboflavin solution, and the pH was adjusted at approximately 7. Light-proof test tubes were used to preserve the prepared riboflavin solution to prevent any prior activation.

Preparation of specimens

Longitudinally, sectioning of the specimens was done with diamond disc. This resulted in the formation of equal mesial and distal halves thus making the number of specimens 60. Cementoenamel junction was marked on each specimen for the reference of extension for preparation of proximal wall. Proximal walls were prepared by stripping the dentin from one end until the remaining thickness of dentin (RDT) becomes 1 mm. Resistance Temperature Detector was measured with the help of digital caliper from the prepared proximal wall to the pulp chamber. The smear layer was removed by immersing all the samples in ultrasonic bath. Specimens were secured in place using epoxy resin. The prepared proximal walls were acid etched with 36% ortho-phosphoric acid (DeTrey Conditioner 36, Dentsply DeTrey GmbH, Konstanz, Germany) for 15 s. This was followed by water rinsing for 15 s and the specimens were air dried for 5 s. These specimens were randomly divided based on the method of surface pretreatment of dentin into three groups of 20 teeth each.

Group I

Total-etch adhesives were applied on the prepared proximal walls without any kind of dentin pretreatment. The cavities of the specimens were thoroughly applied with Prime and bond NT twice using fresh applicator tip for 20 s. It was made sure that all the cavity surfaces were completely wet. Prepared surfaces were light cured for 40 s. Then, composite resin (Filtek Z 350, 3M ESPE) layer was placed in two different increments of 2 mm thickness each. Composite was placed using a matrix prepared in the form of tube with 3 mm diameter, and both the increments were light cured for 40 s.

Group II

In this group, 6.5% PA solution was used for pretreatment on the etched dentin surface for 5 min, followed by rinsing with water. Bonding was done with total-etch adhesive and followed by build-up procedure as described in group I.

Group III

Pretreatment was done on the etched surface of dentin with 1% riboflavin for 5 min along with its activation with visible blue light for 20 s. The prepared surface was then treated with total etch adhesive system, and build-up procedure was done as described in group I.

For shear bond strength testing, universal testing machine was employed at crosshead speed of 1 mm/min. The specimens were loaded until they fractured. The forces were recorded and divided by surface area to obtain the shear bond strength.

Statistical analysis

SPSS Version 20.0 (Armonk, NY, USA: IBM Corp) was utilized, and data were analyzed by means of one-way analysis (ANNOVA). Pairwise comparison of groups was done with post hoc test. Statistical significance was set at P < 0.05.

RESULTS

Table 1 shows the mean value for each group. The mean value of shear bond strength was least for control group (15.598 ± 1.05) followed by Riboflavin group (22.29 ± 0.499) and most for PA group (27.05 ± 1.17) One way analysis of shear bond strength is PA > riboflavin > control group.

Table 1.

One-way analysis of shear bond strength

	n	Mean	SD	SE	95% CI for mean		Minimum	Maximum
					Lower bound	Upper bound
Control	20	15.5986	1.05181	0.23519	15.1063	16.0909	13.84	17.30
Riboflavin	20	22.2925	0.49935	0.11166	22.0588	22.5262	21.46	23.03
Proanthocyanidin	20	27.0522	1.17234	0.26214	26.5035	27.6009	25.52	28.81
Total	60	21.6478	4.82962	0.62350	20.4002	22.8954	13.84	28.81

SD: Standard deviation, SE: Standard error, CI: Confidence interval

The shear bond strength of all three groups is compared in Table 2. There were statistically significant differences between all of the groups.

Table 2.

Post hoc tests, Tukey HSD shows the pairwise comparisons of groups with the mean difference

Group (I)	Group (J)	Mean difference (I−J)	SE	Significant	95% CI
					Lower bound	Upper bound
Control	Riboflavin	−6.69392*	0.30166	0.000	−7.4199	−5.9680
	Proanthocyanidin	−11.45358*	0.30166	0.000	−12.1795	−10.7277
Riboflavin	Control	6.69392*	0.30166	0.000	5.9680	7.4199
	Proanthocyanidin	−4.75966*	0.30166	0.000	−5.4856	−4.0337
Proanthocyanidin	Control	11.45358*	0.30166	0.000	10.7277	12.1795
	Riboflavin	4.75966*	0.30166	0.000	4.0337	5.4856

SE: Standard error, CI: Confidence interval, Tukey HSD: Turkey Honest Significant Difference Test

DISCUSSION

Dentin is a delicate substrate for bonding because of its varying structure and properties. When dentin is treated with 37% phosphoric acid, it exposes a micro-porous network of hydroxyapatite-depleted collagen.[3,4,5,6,7,8] A rigid and space-filling biomaterial is formed by collagen, which provides viscoelasticity and can also act as a scaffold for hydroxyapatite deposition. A highly stable hybrid layer is created only after the thorough infiltration and encapsulation of exposed collagen fibrils with adhesive monomers.[4,5,9] Collagen fibrils fall down and collapse after drying resulting in the closing of the pores in inter-tubular collagen in vitro and impeding adhesive infiltration.[10] Two techniques can be utilized for increasing the properties of hybrid layer. One by developing the advanced bonding systems or can be done by utilizing biomodification approaches that improve the intrinsic properties of dentin.[11]

In order to increase the longevity of bond between deep dentin and nanocomposite, efforts are to be made to stabilize the acid demineralized collagen fibers. Various agents can improve the intrinsic properties of dentin by strengthening and reinforcing the collagen crosslinks. The properties of dentin can be enhanced using collagen crosslinking agents by two different courses of action. One is by preserving the expanded state of collagen fibers, thus improving the diffusion of solvents and resin. Second, these agents result in creating the prepared dentin surface to be stiffer, thus preventing it to be plasticized by water. In addition, dentin biomodification with collagen crosslinking agents can reduce the biodegradation rate of the hybrid layer and inhibits the collagenase activity.[10]

PA is a well-known natural collagen cross-linker. PA is believed to precipitate proline-rich proteins and cause inhibition of protease activity. The complexes formed by PA and collagen interactions are secured essentially by the interaction of the protein amide carbonyl and the phenolic hydroxyl, which forms the hydrogen bond. The covalent and hydrophobic bonds are also believed to be formed.[9,12,13] There is presence of four monomer molecules in the structure of PA.[14]

Riboflavin has the potential to cross-link dentin collagen. When Riboflavin is photoactivated with ultraviolet A light or visible light, it breaks down weak intrinsic crosslinks in collagen and generates free oxygen radicals forming covalent crosslinks between adjacent collagen molecules.[7,10,15] Although riboflavin results in collagen crosslinking but very few literature is available for its effect on bond strength of adhesive resin with relation to deep dentin.

In the present study, a flat surface was selected for bonding because of lower C-factor. When flat surfaces were restored, the forces of polymerization contraction were insignificant resulting in the formation of optimal bonds.[16] In the present study, acetone-based system Prime and Bond NT was used. When compared to ethanol based system, acetone-based yields deeper resin tags and wide hybrid layer.[9,17,18]

In the present study, Group I (control) attained a mean shear bond strength of 15.84 + 0.56 MPa. This value was less than as attained in the previous study carried out by Srinivasulu et al. which was 17 MPa.[19] This could be due to the different protocols followed in both the studies. In contrast to the prior study, this study employed air drying after rinsing instead of blot drying.

The results of the present study showed that collagen crosslinking agents are capable of enhancing the shear bond strength of a nanocomposite to deep dentin. Various studies stated that acid etching causes the increased formation of matrix mettaloproteinases (MMPs) resulting in disintegration of hybrid layer and degradation of collagen, which lead to loss of bond strength. PA and Riboflavin are capable of increasing the durability of resin dentin bond by inactivating MMPs.[20,21]

Group I (control) showed inferior bond strength when compared to Group II (PA). This is in accordance with the study of Epasinghe J, which stated that PA has the potency to stimulate the cross-links situated “within the fibril”, ”between the fibrils” and also intermicrofibrillar in the matrix of collagen.[22] Liu R showed the effect of PA on demineralized dentin. PA stabilizes the vulnerable adhesive demineralized dentin interface by retarding the proteolytic activity resulting in increased bond strength.[23] Han et al. suggested that dentin biomodification with PA resulted in rise in temperature at which denaturation of fixed tissue occurs which leads to enhancement of bond strength.[24] Bedran-Russo et al. stated that PA results in formation of stiffer dentin by reducing its biodegradation and reinforcing type 1 collagen.[12] AL-Ammar A stated that bond strength increased by 69% after pretreatment with PA.[4]

While group I was compared with group III, Group III revealed increased bond strength. Pretreatment with riboflavin enhances the mechanical properties of collagen as well as its structural integrity. This results in improving and maintaining the bond strength and interface integrity of the substrate.[25] Chiang et al. concluded that riboflavin helps to increase the stiffness of dentin and improved bond strength by maintaining the collagen matrix in expanded state.[10]

Group III showed with lower bond strength when compared to Group II. The lower bond strength of riboflavin could be due to the intensity of visible blue light used in the study being higher than Riboflavin's maximum peak value absorbed. According to Fawzy, riboflavin absorbs UV to visible light. It has a range with three maximum peak values at 270, 366, 445 nm. Moreover, the maximum emission of the blue light used in the Elipar Free Light curing light (3M ESPE) was approximately 465 nm. The other reasons could be the type of bond formation with collagen. Riboflavin results in the formation of only covalent crosslinking bonds between adjacent collagen.[17] PA can have four different interactions when applied on dentin surfaces. They can be hydrogen bonding, ionic bonding, covalent bonding, and/or hydrophobic bonding. Riboflavin and PA act on Type I collagen and as well as causes nonspecific inhibition of protease activity, but PA has interactions with proteoglycans also that might have influenced the results.[14]

In addition, a scanning electron microscope evaluation was conducted to check the type of fracture. For the control group, the type of fracture was mostly mixed and cohesive in nature. Failure is seen within the adhesive layer, which is due to the incomplete infiltration of monomer and dissolution of smear layer. In some of the specimens, there was the presence of cohesive failure within dentin which could be due to abnormal stress distribution during shear testing. PA group showed mixed type of fracture. Failure occurred through the top of the hybrid layer. Riboflavin group showed high frequency of mixed type of failure.

This study came up with the knowledge about the effect of few crosslinking agents on shear bond strength of teeth which could be the basis for continuing research in oral environment. An in vivo study would be required further for final evaluation of these agents.

Key learning points:

• Bonding of resin to deep dentin can be enhanced by two techniques. One is by the development of advanced bonding systems, other is biomimetic approach using collagen crosslinking agents

• When biomodification with collagen crosslinking agents is done, PA is more effective than riboflavin in increasing the bond strength of nanocomposite to deep dentin.

CONCLUSION

The present study concluded that collagen crosslinking agents when used before bonding composite resin to dentin result in enhancement of shear bond strength.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.