Effect of chemical cross-linkers on surface topography and microtensile bond strength of sound dentin: An in vitro study.

AIM AND
OBJECTIVES: The aim of the study was to investigate the effect of two different collagen cross-linking agents proanthocyanidin (Grape seed extract [GSE] and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide) on the surface topography of etched dentin and microtensile bond strength (μTBS) of resin dentin bond.
MATERIALS AND METHODS: Fifty-two sound human 3rd molars were collected, and their occlusal surfaces were ground flat to expose dentin. Dentin surfaces were etched using phosphoric acid and then teeth were randomly divided into four groups, according to the dentin treatment: Group 1: wet bonding technique, Group 2: dry bonding technique, Group 3: 6.5% proanthocyanidin, and Group 4: 0.1M carbodiimide. Scanning electron microscope analysis was done for twenty specimens (n = 5 per group) at ×10,000 and ×30,000 magnification. Remaining 32 specimens were restored with TETRIC N-Bond adhesive systems and resin composite. After 24 h, teeth were sectioned to produce a cross-sectional surface area of 1.0 mm2 and tested for μTBS. STATISTICAL ANALYSIS: Data were statistically analyzed using ANOVA and post hoc least significant difference test (P < 0.05).
CONCLUSION: When acid-etched dentin is treated by 6.5% proanthocyanidin (GSE) and 0.1M carbodiimide, followed by application of adhesives, it results in increased μTBS due to cross-linking of collagen fibrils. Copyright:

INTRODUCTION

It has always been a challenge to achieve an optimal dentin-resin bond strength and maintain it over the period of time. There is a compelling need to explore the underlying mechanisms involved in preventing the degradation of resin–dentin bonds and extend their longevity.[1]

Conventional dry bonding technique causes collagen collapse and drying-induced shrinkage, resulting in reduced bond strength. Kanca developed the wet-bonding technique. This technique leaves far too much residual water in resin–dentin bonds and provides hydrolytic fuel for the endogenous proteases of dentin matrices such as matrix metalloproteinases (MMPs) and cysteine cathepsins. They are activated in mild acidic environment during restorative procedures and slowly hydrolyze collagen fibrils in resin-bonded dentin, resulting in poor durability of resin-dentin bonds.[2]

Over the past few years, the experimental use of cross-linking agents has been done to increase the longevity of resin–dentin bonds by cross-linking with collagen fibers of dentin. This will improve structural integrity of collagen fibers, so as to prevent collagen collapse when air drying is done after acid etching. These cross-linking agents also inhibit MMPs.[3] Mineralized dentin contains MMPs such as MMP-2, -3, -8, and -9. These host-derived proteases are a group of zinc- and calcium-dependent enzymes that contribute to the breakdown of collagen matrices.[2]

Most commonly used cross-linking agents are proanthocyanidin (Grape seed extracts [GSEs]), carbodiimide, riboflavin, glutaraldehyde, genipin, cinnamon, etc.[4]

In the present study, we used proanthocyanidin (GSEs), carbodiimide (EDC) as cross-linking agents. Proanthocyanidins, a class of bioflavonoids, are naturally occurring plant metabolites found in GSE, pine bark extract, cranberries, lemon tree bark, etc. It increases the density of the collagen network by inducing exogenous cross-links and decreases collagenase absorption, thereby enhancing the matrix resistance against enzymatic degradation. These cross-links involve covalent bonds that become stable over time, unlike reversible electrostatic binding seen in chlorhexidine. In addition to its cross-linking effect, Proanthocyanidin (PA) has also been shown to inhibit the production of MMPs. It can be incorporated in the current adhesive systems as an additive to the adhesives or as a primer.

Carbodiimide (EDC), a synthetic cross-linking agent causes cross-linking of dentin collagen. It is applied as primer on acid-etched dentin during bonding procedure.

Aims

Evaluation of surface topography of acid-etched dentin after application of collagen cross-linking agents using scanning electron microscope

Measurement of 24 h microtensile bond strength (μTBS) of resin-dentin bond using total etch-wet and dry bonding techniques and cross-linked dry bonding technique.

Objective

The objective of the study was to investigate the effect of two different collagen cross-linking agents proanthocyanidin (GSE/PA and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) on the μTBS of resin dentin bond.

Null hypothesis

There is no difference in mean μTBS in various groups compared in this study.

Materials and Method

Fifty-two extracted human 3rd molar teeth were stored in 0.01% sodium azide solution. Occlusal enamel and superficial dentin were removed using diamond disc under copious water cooling. The flat exposed mid-coronal dentin was abraded with wet 180-grit silicon carbide paper to create a standard smear layer. The flat occlusal dentin surface of all teeth was acid etched for 15 s with 37% phosphoric acid gel (N-ETCH– Ivoclar Vivadent) and rinsed with water. All acid-etched teeth divided into four groups:

Group 1 – wet bonding Technique – dentin surface left visibly moist after acid etching

Group 2 – Dry bonding technique – dentin is air dried for 30 s using 3-way syringe from 10 cm distance

Group 3 – 6.5% proanthocyanidin (GSE) – applied on etched dentin surface for 60 s and then rinsed with water for 10 s

Group 4 – 0.1M Carbodiimide (EDC) – applied on etched dentin surface for 60 s and then rinsed with water for 10 s.

In Groups 1 and 2, acid etching was followed by application of two coats of bonding agent (TETRIC N-Bond– Ivoclar Vivadent) on midcoronal dentin and light cured for 20 s

In Groups 3 and 4, acid etching was followed by application of cross-linking agents – GSE and EDC, respectively, rinsed for 10 s, followed by application of 2 coats of bonding agent (TETRIC N-Bond– Ivoclar Vivadent) applied on mid-coronal dentin and light-cured for 20 s.

Out of total 52 teeth taken, 20 (5 from each group) were sent for scanning electron microscope (SEM) analysis at ×10,000 and ×30,000 magnification.

The remaining 32 teeth (of all 4 groups) were further prepared for μTBS testing as follows:

In all the four groups, composite buildups were done using three 1.5 mm increments of resin composite (Tetric N – Ceram-Ivoclar Vivadent). Each increment was individually light cured for 40 s. Then, the specimens were stored at 37°C in water for 24 h. 120 resin–dentin sticks (n = 30 per group) were made from composite builtup specimens using a diamond disk under copious water cooling. 4–6 resin dentin sticks could be made from each specimen. To test the μTBS of these resin-dentin sticks, each stick was glued to custom-made acrylic jigs and placed on universal testing machine (UNITEST 10). The tensile force at failure was recorded and divided by the cross-sectional area of each stick and expressed in MPa.

RESULTS

All specimens were examined under SEM at ×10,000 and at ×30,000 magnification [Figure 1].

Figure 1

SEM images of specimen treated with WET bonding - group 1 (a) at 10,000x, and (b) at 30,000x. SEM images of specimen treated with DRY bonding - group 2 (c) at 10,000x, and (d) at 30,000x. SEM images of specimen treated with 6.5% Proanthocyanidin - group 3 (e) at 10,000x, and (f) at 30,000x. SEM images of specimen treated with 0.1M Carbodiimide - group 4 (g) at 10,000x, and (h) at 30,000x

Group 1: ×10,000 magnification shows the lack of peritubular dentin and the presence of individual collagen fibrils in the intertubular dentin and individual circumferential collagen fibrils within the dentinal tubules. At ×30,000, interfibrillar spaces could be identified between both the collagen fibrils in the intertubular dentin and within the circumferential collagen fibrils within the lining of the tubules

Group 2: ×10,000 magnification shows fine granular materials present on the surface of the collapsed intertubular dentin, which is silica gel used in etchant. At ×30,000 magnification, no individual collagen fibrils could be identified on the surface of the intertubular dentin or within the walls of the dentinal tubules because they have fused with each other by direct hydrogen bonding

Group 3 (6.5% Proanthocyanidin [GSE]) and Group 4 (0.1M Carbodiimide [EDC]): ×10,000 magnification shows the peritubular and intertubular dentin has a rough surface texture. At ×30,000 magnification, individual collagen fibrils can be recognized between the tubules and within dentinal tubules.

Evaluation of μTBS: [Graph 1 and Table 1]

Graph 1

Mean microtensile bond strength of all groups

Table 1

Intergroup comparison of mean microtensile bond strength (MPa) (one way ANOVA)

Groups	n	Minimum–maximum	Mean	SD	P
Group 1	30	10.00-41.50	22.55	7.10	<0.001**
Group 2	30	1.50-28.50	15.95	5.87
Group 3	30	13.00-51.00	27.09	9.04
Group 4	30	11.50-61.50	30.08	10.40

SD: Standard deviation. **- highly significant (P<0.001)

Group 1: Mean μTBS of samples was 22.55 MPa, with a maximum value of 41.50 and minimum value of 10.00. Standard deviation was 7.10, mean value was lesser than Groups 3 and 4 but greater than Group 2

Group 2: Mean μTBS of samples was 15.95 MPa, with a maximum value of 28.50 and minimum value of 1.50. Standard deviation was 5.87 for this group, and mean value was least among all groups

Group 3: Mean μTBS of samples treated with was 27.09MPa, with a maximum value of 51.00 and minimum value of 13.00. Standard deviation was 9.04 for this group, and mean value was lesser than Group 4 but greater than Groups 1 and 2

Group 4: Mean μTBS of samples treated was 30.08 MPa, with a maximum value of 61.50 and minimum value of 11.50. Standard deviation was 10.40 for this group. Mean value was highest among all groups. There is a statistically significant difference present in mean μTBS in various groups compared.

Intergroup comparison of μTBS was done by posthoc least significant difference test at 95% confidence interval. Intergroup comparison between Groups 1 and 2 and between Groups 1 and 4 shows mean difference of 6.59 and −7.53, respectively, which was statistically significant (P < 0.05), whereas comparison of Group 2 with Group 3 and comparison of Group 2 with Group 4 show mean difference −11.14 and −14.13 which was statistically highly significant (P < 0.05). Intergroup comparison of Group 1 with Group 3 and comparison between Group 3 and Group 4 were statistically not significant (P > 0.05) [Graph 2 and Table 2].

Graph 2

Mean differences in intergroup comparison

Table 2

Intergroup comparison of microtensile bond strength (MPa) (post hoc LSD)

Comparison between	Mean difference	SE	Significance
Group 1
Group 2	6.59	2.14002	0.013*
Group 3	−4.54	2.14002	0.152 (NS)
Group 4	−7.53	2.14002	0.003*
Group 2
Group 3	−11.14	2.14002	<0.001**
Group 4	−14.13	2.14002	<0.001**
Group 3
Group 4	−2.99	2.14002	0.503 (NS)

SE: Standard error, NS: Not significant. **-Highly significant (P<0.001), *-Significant (P<0.05), NS – Not significant (P>0.05)

μTBS of all groups was in the order: Group 4> Group 3≥ Group 1> Group 2.

DISCUSSION

Vital dentin contains a substantial proportion of mineral phase (50% by volume), water (20% by volume), and organic material, primarily Type I collagen (30% by volume). These collagen fibers have intrinsic tendency to form interpeptide hydrogen bond with each other in the absence of water. This interpeptide hydrogen bonding cannot occur in the presence of water.

During the acid-etching process in total etch strategy, the entire 50% volume of surface and subsurface mineral is solubilized, extracted, and is replaced by water. This, along with 20% intrinsic volume of water results in net water content of 70% by volume. The net water content surrounds the collagen fibrils that remain anchored into the underlying mineralized dentin.[35]

In resin–dentin bonding, the mineral phase of dentin is responsible for its high stiffness (20 GPa), is removed by acid-etching and partially to almost fully replaced with cross-linked resins with a much lower stiffness (3.4 GPa). Stress concentrations develop in resin-bonded assemblies where there is a mismatch in the modulus of elasticity of its constituents such as at the junction between the bottom of the hybrid layer (3-4 GPa) and the underlying mineralized dentin (20 GPa).[3]

To overcome this problem, newer strategies for the stabilization of hybrid layer are required that will prevent the degradation of resin–dentin bond over the period of time. Stabilization of hybrid layer is done by treating the demineralized dentin with synthetic or natural cross-linking agents that can inactivate the catalytic site of these enzymes[6] and improve mechanical properties of dentin collagen. This results in reduced susceptibility of cross-linked dentin collagen to enzymatic degradation by collagenases and increased stability of the resin-dentin interface.[2]

In SEM analysis, we observed the surface topography of mid-coronal dentin treated by wet and dry bonding and with 6.5% proanthocyanidin and 0.1M carbodiimide at ×10,000 and ×30,000 magnification. ×10,000 magnification shows that peritubular and intertubular dentin has a rough surface texture. At ×30000 magnification, individual collagen fibrils between the tubules and within dentinal tubules can be recognized. Non-cross-linked collagen fibrils collapsed completely when air-dried. There were no spaces between the collagen fibrils because, in the absence of water, they bonded to each other by hydrogen bonding. Interfibrillar spaces between collagen fibrils serve as diffusion channels for resin monomer infiltration. Without interfibrillar spaces, resin infiltration of surface collagen fibrils will be incomplete. In addition, lack of interfibrillar spaces in dentinal tubules result in lose resin tags that are not anchored to the surrounding intertubular dentin. The resulting μTBS of about 10 MPa is insufficient to oppose the forces of polymerization contraction.[7]

Evaluation of μTBS for all four groups was done. The highest bond strength was observed for 0.1M carbodiimide (Group 4)-treated groups (30.8MPa, P < 0.0001), which was statistically highly significant than all the other experimental groups. Improved resistance to collagenase challenge and increased mechanical properties of collagen-based materials have been reported following treatment with EDC.[3] Previously, studies done by Seseogullari-Dirihan et al.,[8] Scheffel et al.,[9] and Tezvergil-Mutluay et al.[10] show similar results as in our study.[11]

Treatment with 6.5% proanthocyanidin also resulted in a statistically increase in the μTBS when compared to dry bonding technique group. This can be attributed to the specificity of proanthocyanidin to facilitate the proline hydroxylase enzyme, which catalyzes proline hydroxylation, which is a step in collagen biosynthesis.[12] In the study by Walter et al. in 2008, they used 0.5% PA as collagen cross-linking agents. They concluded that PA could be used to modify dentin collagen and efficiently increase the resistance against enzymatic digestion.[13] In a similar study by Al-Ammar et al. in 2009 using 6.5% GSE., they concluded that GSE increased the bond strength of dentin.[14]

Since, proanthocyanidins interact noncovalently, and their reaction rate is diffusion dependent, an increase in their bond strength values may be limited by the diffusion of cross-linking agent in an adequate amount to the entire thickness of hybrid layer.[15]

The results of the present study show that pre-treatment of demineralized dentin with 6.5% GSE and 0.1M EDC for 60 s significantly increases μTBS of adhesive-dentin bond. Thus, the null hypothesis was rejected. Hence, in conclusion, the use of 6.5% proanthocyanidin and 0.1M carbodiimide as collagen cross-linking agents, increases the μTBS of adhesive-dentin bond through cross-linking of collagen fibrils. Demineralized dentin treated with cross-linking agents decreases collagenase degradation, reduces water absorption, and increases mechanical properties. Hence, it is probable that, during intraoral function, the dense collagen matrix would be potentially less susceptible to cyclic fatigue. Thus, it possibly increases the stability and biodegradation resistance of adhesive-dentin bond.

The present study was an in vitro study and could not simulate the actual clinical conditions and there is a lack of evidence for in vivo applications of this study. In the present study, 24 h μTBS was evaluated. Further studies over a longer period of time need to be done as resin-dentin bond strength may detoriate with time.

CONCLUSION

Within the limitations of this study, it can be concluded that acid-etched dentin treated by cross-linking agents such as 6.5% GSE and 0.1M EDC results in increased μTBS, with an application time of 1 min, which is clinically acceptable. Thus, the application of collagen cross-linkers during adhesive restorative procedures promises a new and practical approach to improve dentin bond strength properties in a clinical setting.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.