Long-term bonding effectiveness of simplified etch-and-rinse adhesives to dentin after different surface pre-treatments.

OBJECTIVE: To evaluate the effect of 2% chlorhexidine (CHX) and 30% proanthocyanidin (PA) application on the immediate and long-term bond strength of simplified etch-and-rinse adhesives to dentin.
MATERIALS AND METHODS: One hundred twenty extracted human molar teeth were ground to expose the flat dentin surface. The teeth were equally divided into six groups according to the adhesives used, either Tetric N Bond or Solobond M and pretreatments given either none, CHX, or PA. Composite cylinder was bonded to each specimen using the respective adhesive technique. Half the samples from each group (n = 10) were then tested immediately. The remaining samples were tested after 6 month storage in distilled water.
RESULTS: The mean bond strength of samples was not significantly different upon immediate testing being in the range of 8.4(±0.7) MPa. The bond strength fell dramatically in the control specimens after 6 month storage to around 4.7(±0.33) MPa, while the bond strength was maintained in the samples treated with both CHX and PA.
CONCLUSION: Thirty percent PA was comparable to 2% CHX with respect to preservation of the resin dentin bond over 6 months.

INTRODUCTION

Modern adhesives have simplified application protocol with reduction in the number of clinical steps for ease of use.[1] Simplified etch-and-rinse systems have the hydrophilic primer and hydrophobic adhesive resin combined in a single bottle and a separate etchant which removes the debris, smear layer and demineralizes the dentin to expose the dentinal Type I collagen. The complete penetration of adhesives up till the entire depth of demineralized dentin surface, enveloping exposed collagen, and the subsequent conversion of resin are critically important for the success of adhesive resin-dentin bond. Although, incorporation of hydrophilic and acidic resin monomers has substantially improved the initial bond strength of contemporary etch-and-rinse and self-etch adhesives to intrinsically wet dental substrates, the problems with long-term bonding effectiveness continue to plague adhesive dentistry.[12]

Water sorption by hydrophilic and ionic resin monomers within both the hybrid layer and the adhesive layer may contribute to the degradation of resin-dentin bond strength over time. Furthermore, incompletely infiltrated zones along the bottom of hybrid layers contain denuded collagen fibrils, which are susceptible to degradation and also provide a pathway for host derived enzymes.[3] Auto-degradation of collagen matrices occurs in resin-infiltrated dentine by the slow action of host-derived matrix metalloproteinases (MMPs), which are a class of zinc- and calcium-dependent endopeptidases (collagenases, gelatinases, and co-elastinases) capable of degrading all extra-cellular matrix components.[4] Human dentin contains at least MMP-2, MMP-8, MMP-9, and MMP-20.[5] These host-derived proteases are trapped within the mineralized dentin matrix and are thought to play an important role in numerous physiological and pathological processes occurring in dentin and can degrade the unprotected Type I collagen fibrils that are exposed by sub-optimally infiltrated dental adhesive systems after acid etching.[14] This might lead to hydrolytic degradation, increased nanoleakage, and loss of bond strength over time due to thinning and disappearance of collagen fibrils from incompletely infiltrated hybrid layers in aged, bonded dentin. The activity of MMPs can be suppressed by protease inhibitors.

Specific and non-specific matrix metalloproteinase inhibitors (MMPIs) abound e.g., Chlorhexidine (CHX), Galardin (GL), chemically modified tetracycline (CMT), Gelatinase selective inhibitor SB-3CT [(4-phenoxyphenylsulfonyl) methylthirane], etc., Most are under development and have no proven biocompatibility to be used on human subjects despite being potent MMPIs. CHX has proven biocompatibility and good anti-bacterial action both in vivo and in vitro. CHX has been found to have MMP inhibitor and anti-enzyme properties (against MMP-2, -8, and -9) even at low concentrations. Several in vitro and in vivo resin-dentin bonding studies have confirmed the ability of 2% CHX to protect dentin collagen degradation.[567]

With the same objective i.e., in order to achieve dentin resin bond longevity through collagen preservation, the use of a collagen cross linking agent has also been considered. Cross-linking reagents have been used in medicine to ‘fix’ the tissues for study such as glutaraldehyde (GA), formaldehyde, and epoxy compounds. Han et al., found proanthocyanidins (PA) possess the same properties of tissue fixation due to collagen cross linking and are as potent as GA for the purpose but are less toxic.[8] The use of PAs on dentin blocks has been studied and the microtensile and microhardness of dentin were found to be enhanced in a time dependant manner.[9101112]

Although various studies have investigated the role of MMPIs and collagen cross-linkers in dentin bonding, still gaps remain in our knowledge regarding their effect on long-term bonding effectiveness of specific adhesive systems based on different solvents after clinically relevant applications time. Therefore, this study was designed to evaluate the effect of surface pre-treatment with CHX and PA on the immediate and long-term bond strength of simplified etch-and-rinse adhesives with different solvent compositions.

MATERIALS AND METHODS

One hundred and twenty freshly extracted non carious human molars were thoroughly cleaned and stored in 0.1% thymol until use. Depth cuts of 1.5 mm from the central fossa were drilled in each tooth with a round diamond bur to standardize the depth of the dentin. All samples were ground with a diamond disc, to expose a flat occlusal dentinal surface, which was polished with silicon carbide paper up to 1,000 grit. The samples were embedded in an auto-polymerizing acrylic resin placed perpendicular to the acrylic resin surface.

Clear tape with 3 mm punch hole was used to define the pre-treatment and bonding area. All the samples were acid etched with respective acid etchants for 15 seconds followed by rinsing and blot drying and were randomly divided equally into six groups according to the two simplified etch-and-rinse adhesive systems to be used [Table 1] and according to three different surface pre-treatments (none, CHX or PA). Thirty percent PA solution was prepared after dissolving the powder obtained from 95% purity tablets (Proanthodin, NexGen Pharma, India). The groups were as follows:

Table 1

Composition of the materials used

Group 1: Etched dentin surface was rinsed with distilled water for 1 min and gently blot dried with a cotton pellet prior to application of Solobond M adhesive.

Group 2: etched dentin surface treated with 2% CHX for 1 min and gently blot dried prior to application of Solobond M.

Group 3: etched dentin surface treated with 30% PA for 1 min and gently blot dried prior to application of Solobond M.

Group 4: etched dentin surface treated with distilled water for 1 min and gently blot dried prior to application of Tetric N Bond adhesive.

Group 5: etched dentin surface treated with 2% CHX for 1 min and gently blot dried prior to application of Tetric N bond.

Group 6: etched dentin surface treated with 30% PA for 1 min and gently blot dried prior to application of Tetric N bond.

Transparent plastic tubes of internal diameter 3 mm and 2 mm height with thickness 0.5 mm were pre-cut and placed perpendicular to etched and bonded dentinal surface. A hybrid resin composite (Flitek Z350 XT, Body Shade A1) was loaded into the pre-cut tubes and bonded to the adhesive by light curing at 600 mW/cm2 for 20 sec with the light tip in contact with plastic cylinder. The tubes were then removed. The bonded specimens were finally placed in distilled water at room temperature for 24 hrs.

Ten samples from each subgroup were subjected to immediate shear bond strength testing in a universal testing machine (Instron, ADMET, Enkay Enterprises, New Delhi). The specimens were placed and stabilized by the jig, while a straight knife-edge rod (2.0 mm) was applied at the tooth restoration interface at a cross-head speed of 0.5mm/min]. Load was applied until restoration failure. The remaining samples were subjected to shear bond strength evaluation after storage in distilled water for 6 months.

Statistical analysis

The shear bond strength values were statistically analyzed using Tukey's Honestly Significant Difference (HSD) test and three way analysis of variance (ANOVA) (Multivariate Assessment) at a significance level of P = 0.05.

RESULTS

The mean shear bond strength values obtained after immediate and delayed testing are depicted in Table 2. No significant difference in immediate shear bond strength of the subgroups was observed regardless of the surface pretreatment or the bonding agent used. However, after 6 months, water storage the magnitude of the shear bond strength fell significantly for both the control subgroups but no significant reduction in bond strength was observed in the subgroups with CHX or PA pretreatment.

Table 2

Comparison of immediate and delayed mean shear bond strength values (MPa) of all the groups

DISCUSSION

In the current study, application of 2% CHX to the acid etched dentin resulted in practically no reduction in bond strength over 6 months of ageing. Our results are supported by the study of Carhillo et al., who observed significant preservation of bond strength with CHX.[13] Zhou et al.,[1415] and Brackett et al.,[6] demonstrated considerable preservation of dentin bond in their respective in vitro studies over a 12 month period after incorporation of CHX in the bonding protocol.

CHX binding to dentin matrices represents a large reservoir of bound CHX that may provide long-term saturation of adjacent dentin, thereby, prolonging the durability of resin dentin bonds by inhibiting the MMPs in the hybrid layer from its Zn2+ cation-chelating property.[161718] When CHX solution is applied to demineralized dentin, it can diffuse into the 65% water-filled spaces of that matrix[1920] bind to exposed collagen fibrils and would also be trapped within the interfibrillar spaces between the collagen fibrils. The net result is a more than 8-fold increase in CHX uptake by demineralized matrices relative to CHX binding to mineralized dentin matrices.[19] CHX applied to dentin prior to the use of etch-and-rinse adhesives has been shown to markedly decrease the collagen degradation with time.[567]

Fixation of biological tissues can also preserve the integrity of the hybrid layer. Collagen stabilization can be achieved through cross-linking reagents like formaldehyde, GA, epoxy compounds, carbodiimide but all have drawbacks such as toxicity, difficult to regulate cross-linking rates, and stability.[8] Class of natural phytochemicals known as PA are bioflavinoids and collagen cross linking agents, used as natural antioxidants, free-radical scavengers and have proven to be safe in different clinical applications and as dietary supplements. PA contain a benzene-pyran- phenolic acid molecular nucleus (flavin) as part of their much larger molecular structure and are known by several names Oligomeric Phenolic Compound, pycno-genols, leukocyanidins, and condensed tannins.[8] PA is found to accelerate the conversion of soluble collagen to insoluble collagen.[8]

In the present study, although 1 minute application of 30% PA to acid etched dentin surface resulted in significant preservation of bond strength after 6 months of water storage, however, the difference between immediate shear bond strengths of all the groups was not significant, indicating that application of either CHX or PA did not interfere at all with the bonding procedures. Green et al., used model adhesives formulated with and without 5% PA and concluded that incorporation of PA in dental adhesives may inhibit the biodegradation of unprotected collagen fibrils within the hybrid layer.[12] Castellan et al., demonstrated significant increase in elastic modulus of dentin after PA treatment.[21]

Contrary to the present study, Al-Ammar et al.,[22] and Macedo et al.,[11] found that in vitro application of PA to etched dentin prior to bonding procedures significantly enhanced the immediate dentin bond strength and dentin collagen stability of caries affected and sound dentin. However, in their study, etched dentin surface was treated with 6.5% w/v grape seed extract (GSE) for 1 hour. But such application protocols are time consuming and have been judged clinically unrealistic to justify the use of PA. PA and collagen complex formation involves primarily hydrogen bonding between the protein amide carbonyl and the phenolic hydroxyl.[23] Proline-rich proteins like collagen have an extremely high affinity for PA especially at isoelectric pH.[823] When the collagen molecules are cross-linked, some of the sites that serve as substrate for the collagenase could be hidden or modified due to protein folding and the enzymatic digestion can also be significantly hindered.[16]

Increase in resin dentin bond longevity achieved by PA cannot be solely explained by augmentation in the cross-linking density. PA has a good and proven antioxidant effect.[24] PA is also a potential inhibitor of MMP 8 and MMP  9.

No significant difference was found upon comparing the specimen bonded using either Tetric N-Bond or Solobond M. Also the interaction between CHX and acetone or ethanol and between PA and acetone or ethanol, were essentially of the same extent. Various factors such as dentin origin, site and area of bonding affect the bond strength of adhesives.[25]

The results prove beyond doubt that PA does retard the rate of bond strength degradation and comparably to CHX. CHX only chelates and inactivates the MMPs and perhaps a minimal role of its antibacterial effect may also contribute to its success. In comparison, the effect of PA is many fold; stiffening or fixing the collagen thus decreasing susceptibility of the collagen to enzymatic decay, antioxidant properties reducing the oxygen inhibition layer and thus decreasing the osmotic blistering; supplemented by its own antibacterial effect.[924] The fact that all this was attained while keeping the application time clinically feasible indicates that this reagent has tremendous promise to be harnessed for clinical/commercial use in adhesive dentistry.

CONCLUSION

Surface pre-treatment with either 2% CHX or 30% PA had no significant effect on the immediate shear bond strength of both the etch-and-rinse simplified adhesives. However, it significantly reduced the fall in bond strength of both the adhesives after 6 months of water storage as compared to the groups without surface pre-treatment. Immediate bond strength of the surface pre-treated groups was maintained over 6 months period and did not differ significantly from the long-term bond strength.