Effect of resveratrol pretreatment of radicular dentin on immediate and delayed pushout bond strength of fiber post luted using self-adhesive resin cement: An in vitro study

Abstract

Aim:

To assess and compare the effect of resveratrol pretreatment of root dentin on immediate and delayed pushout bond strength (POBS) of fiber postluted using self-adhesive resin cement.

Methodology:

Postspace was prepared in endodontically treated roots and randomly divided into two groups based on pretreatment rinse before fiber postcementation: Group 1: Distilled water Group 2: 1 mL of 10 mg/mL resveratrol solution for 1 min. Fiber posts were luted using self-adhesive resin cement. Among, two 1 mm slices of the coronal and middle third of root, one slice was subjected to immediate POBS using the universal testing machine and the other slice was subjected to POBS after storing in distilled water for 12 months.

Results:

Resveratrol pretreatment showed significantly higher immediate POBS with more mixed failure than distilled water group. There was no significant difference in delayed POBS between the distilled water (mean: 7.07 MPa) and resveratrol pretreated group (mean: 8.20 MPa) with predominantly adhesive failure among them. There was significant difference between immediate and delayed POBS value of both the groups.

Conclusion:

Resveratrol pretreatment can be considered to improve bond strength stability of radicular dentin to self-adhesive resin cement.

INTRODUCTION

In endodontically treated teeth with reduced amount of coronal tooth structure, fiber posts are usually used which are luted with dual cure resin cements.[1-3] The resin cements used may be etch-and-rinse or self-etch adhesives or recently formulated self-adhesive cements.[4-6]

To simplify the clinical steps, self-adhesive resin cements were introduced. These cements allow simultaneous bonding between the fiber post and intra-radicular dentin by causing mild dentin demineralization that promotes resin infiltration and chemical bonding of the cement with hydroxyapatite. The durability of resin cement bond between the fiber post and radicular dentin is an important issue for long-term clinical success. Debonding of resin from dentin leads to failure of adhesively luted fiber post.[1,3] This may be due to hydrolytic degradation of resin component of the adhesive cement and collagen breakdown caused by matrix metalloproteinases (MMPs) released from dentin. The use of intracanal irrigants such as sodium hypochlorite and resin cements containing acidic monomers has shown to activate dentin MMPs, which might be responsible for the collagenolytic and gelatinolytic activity observed in the hybrid layer.[3,7,8]

In order to strengthen collagen fibrils at resin-dentin interface, the dentinal surface can be pretreated with natural crosslinking agents such as proanthocyanidine and riboflavin phosphate.[9] The use of these crosslinking agents has improved the bond strength; however, they cause discoloration of tooth.[8,10]

Resveratrol (3,5,4′-trihydroxystilbene, 228.25 g/mol), a polyphenolic compound of plant origin, has potent antioxidant, antimicrobial, cardioprotective, and neuroprotective properties extensively used in bio-medical field. In addition to these properties, it is known to have collagen crosslinking ability and MMP inhibition.[10,11]

Resveratrol has been tested on coronal dentin with favorable results and there is a lacunae in the literature regarding its application on the radicular dentin. Hence, this in vitro study was conducted to compare and assess the immediate and delayed effects of pretreating dentin with resveratrol on bond strength of self-adhesive resin cement used to lute the fiber posts to radicular dentin.

METHODOLOGY

Forty-two freshly intact single-rooted premolars extracted for orthodontic reasons were collected and stored in 0.5% chloramine solution (Hi-Media Labs, India) for 48 h and in distilled water until the preparation time at room temperature. Tooth samples were decoronated at the level of cementoenamel junction. Root canal preparation was done with Protaper rotary files (Dentsply, Maillefer Ballaigues, Switzerland) up to F3 size using 3% sodium hypochlorite, 17% ethylenediaminetetraacetic acid, and final irrigation with distilled water. Sectional obturation was done with F3 Protaper cone and AH plus sealer (Dentsply, De Trey, Germany). Postspace was prepared using peeso reamer (Mani Inc, Japan) till no.3 to accommodate the fiber post of 1.1 mm size. The prepared samples were randomly divided into two groups according to the pretreatment of radicular dentin.

• Group 1: (n = 21) Postspace debris was cleared with distilled water (no pretreatment)

• Group 2: (n = 21) Postspace debris was cleared with distilled water which was followed by pretreatment of radicular dentin with 1 mL of 10 mg/mL resveratrol solution for 1 min.

Preparation of resveratrol solution: 500 mg of resveratrol powder (Biotrex Nutraceuticals) was dissolved in 50 mL ethyl alcohol (100%) to produce 10 mg/mL experimental solution following the methods of Kang Li et al.[11]

Postspace was dried using absorbent paper points. Fiber post (Coltene/Whaledent, Switzerland) was luted using self-adhesive resin cement (Rely XU200 3MESPE) and light cured for 40s. The samples were then stored in distilled water for 24 h.

The samples were sectioned transversely into multiple slices obtaining two slices from the coronal third and second slices from the middle third from each sample. One slice from coronal and middle thirds of each sample was stored in distilled water for 24 h and subjected to immediate pushout bond strength (POBS). The other slice from coronal and middle thirds was stored in distilled water for 12 months and then subjected to delayed POBS. In delayed groups, the distilled water was changed every week. POBS (expressed in MPa) was determined using a universal testing machine by applying force at the crosshead speed of 1 mm/min using a plunger of 1 mm diameter in apico-coronal direction. The bond strength in MPa was calculated using the following formula: Force(n)/Area[2 ϖrh]; ϖ =3.14, r = radius of the postspace, h = thickness of the tooth slices (1 mm).

The failure pattern of debonded specimens was assessed using a stereomicroscope at ×50 magnification and classified as adhesive (between the dentin and resin cement), mixed, partially adhesive (fiber-post and resin cement) or cohesive (at the resin cement) according to Radovic et al.[12,13]

Statistical analysis

The comparison of POBS (MPa) among the groups was done by applying independent “t” test. Intragroup comparison was done using paired “t” test. Failure patterns between the two groups were compared using the Chi-square test. The level of significance was set at P < 0.05. The entire data were analyzed statistically using the Stastical Package for the Social Sciences version 22.0. Released 2013. Armonk, NY: IBM Corp.

RESULTS

Immediate POBS of resveratrol pretreated group (mean: 10.99 MPa) was significantly higher than distilled water (mean: 8.70 MPa). Delayed POBS of resveratrol pretreated group had higher POBS (mean: 8.20 MPa) as compared to distilled water (Mean: 7.07 MPa), but there was no statistically significant difference [Table 1]. Immediate POBS in both distilled water and resveratrol pretreated groups was higher than the delayed and this difference was statistically significant [Table 1]. The POBS was higher in the coronal third compared to the middle third [Graphs 1 and 2]. Predominantly adhesive failure, i.e. failure between dentin and resin cement was found in the distilled water group. Resveratrol group showed more of mixed failure compared to all the other groups [Table 2 and Figure 1].

Table 1.

Comparison of push-out bond strength between the two groups

Group	Mean	SD	Mean difference	t	P
Immediate
Distilled water (Group 1)	8.70	2.08	2.29	2.53	0.032*
Resveratrol (Group 2)	10.99	5.38
Delayed
Distilled water (Group 1)	7.07	1.51	1.13	9.45	0.59
Resveratrol (Group 2)	8.20	2.17
Distilled water (Group 1)
Immediate	8.70	2.08	0.33	1.63	<0.001*
Delayed	7.07	1.51
Resveratrol (Group 2)
Immediate	10.99	5.38	2.79	2.80	0.03*
Delayed	8.20	2.17

*Statistically significant. SD: Standard deviation

Graph 1.

Coronal versus middle immediate pushout bond strength

Graph 2.

Coronal versus middle delayed pushout bond strength

Table 2.

Comparison of mode of failure between groups at immediate and delayed duration

Groups	Adhesive (%)	Mixed (%)	χ2	P
Immediate
Distilled water	68	32	0.0674	0.845
Resveratrol	28	72
Delayed
Distilled water	65	35	0.357	0.547
Resveratrol	56	44

Figure 1.

Stereomicroscopic images. (a) Adhesive failure. (b) Mixed failure

DISCUSSION

Fiber post is indicated for endodontically treated teeth with loss of tooth structure due to previous caries, preexisting restorations, etc., to retain the core.[1,2] Long-term survival of such a tooth depends on the postretention as they are cemented with resin cement.[3] Among the resin cements, self-adhesive cement is currently used, as they simplify the clinical steps and are less technique sensitive.[4,5,14] Meta-analysis of laboratory studies by Sarkis-Onofre et al. 2014, indicated that the use of self-adhesive resin cement could improve the retention of fiberglass posts as compared to conventional resin cement.[15]

The bonding properties of these resin cements are related to their chemical composition which consists of multifunctional phosphoric acid methacrylates, which are proposed to be capable of simultaneously demineralizing and infiltrating the tooth surface. The phosphoric and carboxylic groups of polyalkenoic acid form ionic bonds with hydroxyapatite, thereby ensuring a second means of retention.[16] However, debonding of resin from dentin leads to failure of adhesively luted fiber post.[3,7,12]

The bond strength is affected by hydrolytic degradation and/or collagenolytic activity by MMPs. Two degradation patterns have been observed within the hybrid layer: Loss of resin from interfibrillar spaces and disorganization of collagen fibrils. MMPs get activated by the acidic nature of adhesive systems and resin cement. The bond between resin and dentin deteriorates over time due to hydrolytic and proteolytic action on collagen by activated endogenous enzymes (MMPs and cysteine cathepsins [CTs]). Hence, MMP inhibitors play a role in strengthening the dentin adhesive bond.[3,4,9]

Crosslinking agents form intra/inter-molecular and inter-microfibrillar crosslinks, hence, improving biomechanical characteristics and structural stability of dentin collagen. In addition, they also inactivate MMPs of dentin by crosslinking their peptide chains, after acid demineralization. This might contribute to a better bond strength between the radicular dentin and the resin cement.[7,9]

Polyphenols such as proanthocyanidin, riboflavin, and quercetin induce exogenous cross-linking with collagen mainly by hydrogen bonding between the protein amide carbonyl and the phenolic hydroxyl groups.[9,10] Resveratrol is a polyphenol, with antioxidant, anti-inflammatory, and collagen crosslinking properties.[10,11]

In this study, resveratrol was used to pretreat the dentin before bonding the fiber post with self-adhesive resin cement. On using resveratrol, there was an increase in the immediate POBS (mean =10.99 MPa) as compared to distilled water (mean = 8.70 MPa) which was statistically significant.

Resveratrol with its phenolic hydroxyl groups was believed to bio-modify collagen fibers through hydrogen bonding. Ethanol solvent may encourage the cross-linking of polyphenols and collagen by lowering the dielectric constant of the medium suggesting a synergistic effect.[11,17] The study by Porto et al. was the first to use resveratrol to improve dentin collagen resistance to degradation. In this study, resveratrol showed results similar to that of chlorhexidine.[18] Peng et al. evaluated the effects of resveratrol solution applied as a primer on the coronal-dentin to check the resin bond durability. Resveratrol pretreatment group presented significantly higher Microtensile bond strength (MTBS), showing the better inhibitory effect of MMP.[11]

With 12 months of storage in both groups, a decrease in the bond strength was obtained. This indicates deterioration at resin-dentin interfaces, due to hydrolytic degradation or activation of endogenous MMPs and CTs. Han et al. (2007) demonstrated that self-adhesive cement does not achieve a neutral pH, and thus maintaining a low initial pH for a long period can adversely affect the bonding of these types of cement.[16,19]

The samples were stored in a 100% humid environment for 12 months. This harsh environment might have led to the hydrolysis and leaching of resin adhesives, which is thought to be an additional reason for resin degradation, contributing to the deterioration of bond strength over time.[7,8]

In the delayed groups, resveratrol has shown higher mean POBS (Mean = 8.20 MPa) than the distilled water (mean =7.07 MPa) although not statistically significant. This implies that resveratrol has influenced the bond strength despite being subjected to moisture similar to the distilled water group. This may not be the exact clinical scenario as the resin dentin interface in the root is not exposed to such an extremely moist environment.

Self-adhesive resin cement (Rely XU 200) was used to bond the fiber post to the radicular dentin. Its mild acid concentration cannot etch the entire depth of the smear layer, preventing the collagen from being reached and exposed. The lack of difference between the groups after 12 months of storage could be explained by the fact that self-adhesive resin cements can only etch the most superficial layer of intact dentin beneath the smear layer and that collagen molecules with partially or fully mineralized dentin are inaccessible to collagen degradation pathways. Resveratrol might not have reached the collagen because no chelator was used to remove the smear layer.[6]

A statistically significant difference was found between the POBS of the coronal and middle third of the root in all the groups. This could be due to a relatively higher amount of debris in the middle third. The cavity configuration factor of the postspace and degree of conversion of dual-cure resin at the coronal and middle region may also have influenced the result.[7]

Clinically, adhesive failure at the interface was observed between resin cement and dentin making this the most critical area. The push-out test is considered to be the most suitable method for assessing postretention. As research suggests, thinner slices have lower frictional areas and fewer risks of results being overestimated, 1 mm sections were chosen to measure POBS.[7,20]

Following the push-out test, the debonded specimens were observed under a stereomicroscope at ×50 magnification for eliciting the failure pattern. The immediate distilled water group showed predominantly adhesive failure (68%), whereas resveratrol pretreatment showed predominantly mixed failure (72%). The delayed distilled water group showed predominantly adhesive failure (65%), whereas the resveratrol pretreatment group showed almost equal percentages of adhesive and mixed failure (56% - adhesive and 44% - mixed) implying a stronger resin dentin bond relatively. However, no statistically significant difference was found between the groups.

CONCLUSION

Within the scope of this study, resveratrol pretreatment on the POBS was found to be effective. Hence, resveratrol pretreatment can be considered to improve bond strength stability of radicular dentin to self-adhesive resin cement. Further research on testing this crosslinking agent following the use of chelators, its effect on biomechanical properties of radicular dentin need to be conducted before considering for its clinical application.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.