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Year : 2016  |  Volume : 19  |  Issue : 6  |  Page : 555-559
Resin bond strength to water versus ethanol-saturated human dentin pretreated with three different cross-linking agents

1 Department of Conservative Dentistry and Endodontics, Sri Sai College of Dental Surgery, Vikarabad, Telangana, India
2 Department of Conservative Dentistry and Endodontics, SVS Institute of Dental Sciences, Mahbubnagar, Telangana, India
3 Department of Conservative Dentistry and Endodontics, Guru Govind Singh College of Dental Sciences, Burhanpur, Madhya Pradesh, India

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Date of Submission07-Jul-2016
Date of Decision14-Sep-2016
Date of Acceptance22-Oct-2016
Date of Web Publication14-Nov-2016


Context: Resin-dentin bonds are unstable owing to hydrolytic and enzymatic degradation. Several approaches such as collagen cross-linking and ethanol-wet bonding (EWB) have been developed to overcome this problem. Collagen cross-linking improves the intrinsic properties of the collagen matrix. However, it leaves a water-rich collagen matrix with incomplete resin infiltration making it susceptible to fatigue degradation. Since EWB is expected to overcome the drawbacks of water-wet bonding (WWB), a combination of collagen cross-linking with EWB was tested.
Aim: The aim of this study was to compare the effect of pretreatment with different cross-linking agents such as ultraviolet A (UVA)-activated 0.1% riboflavin, 1 M carbodiimide, and 6.5 wt% proanthocyanidin on the immediate and long-term bond strengths of an etch and rinse adhesive system to water- versus ethanol-saturated dentin within clinically relevant application time periods.
Settings and Design: Long-term in vitro study evaluating the microtensile bond strength of adhesive-dentin interface after different surface pretreatments.
Subjects and Methods: Eighty freshly extracted human molars were prepared to expose dentin, etched with 37% phosphoric acid for 15 s rinsed, and grouped randomly. They were blot-dried and pretreated with different cross-linkers: 0.1% riboflavin for 2 min followed by UVA activation for 2 min; 1 M carbodiimide for 2 min; 6.5 wt% proanthocyanidin for 2 min and rinsed. They were then bonded with Adper Single Bond Adhesive (3M ESPE), by either WWB or EWB, followed by resin composite build-ups (Filtek Z350, 3M ESPE). Bonded specimens in each group were then sectioned and divided into two halves. Microtensile bond strength was tested in one half after 24 h and the other after 6 months storage in artificial saliva.
Statistical Analysis Used: Analysis was done using SPSS version 18 software (SPSS Inc., Chicago, IL, USA). Intergroup comparison of bond strength was done using ANOVA with post hoc Tukey's test, and intragroup comparison was done using paired t-test.
Results: The microtensile bond strength of cross-linked groups was higher compared to control group (P < 0.001). EWB showed much higher bond strength values on cross-linked dentin compared to noncross-linked dentin. UVA-activated riboflavin group exhibited highest bond strengths followed by carbodiimide and proanthocyanidin groups, respectively, on both water- as well as ethanol-saturated dentin. Even after 6 months storage, cross-linked groups showed significantly higher values compared to initial bond strength values of control group (P < 0.001).
Conclusions: 0.1% riboflavin pretreatment of dentin followed by UVA activation for 2 min exhibited highest increase in bond strength values at 24 h and least reduction in bond strength values after 6 months storage compared to other groups. Biomodification of dentin using collagen cross-linking followed by EWB exhibited a synergistic effect in improving the resin-dentin bond durability.

Keywords: Bond durability; carbodiimide; collagen cross-linking; ethanol-wet bonding; proanthocyanidin; ultraviolet A-activated riboflavin

How to cite this article:
Venigalla BS, Jyothi P, Kamishetty S, Reddy S, Cherukupalli RC, Reddy DA. Resin bond strength to water versus ethanol-saturated human dentin pretreated with three different cross-linking agents. J Conserv Dent 2016;19:555-9

How to cite this URL:
Venigalla BS, Jyothi P, Kamishetty S, Reddy S, Cherukupalli RC, Reddy DA. Resin bond strength to water versus ethanol-saturated human dentin pretreated with three different cross-linking agents. J Conserv Dent [serial online] 2016 [cited 2023 Oct 1];19:555-9. Available from:

   Introduction Top

Minimally invasive esthetic restorations are widely performed in clinical practice nowadays. However, the limited durability of resin-dentin bonds compromises the life span of these restorations.[1] Several strategies such as the use of matrix metalloproteinase (MMP) inhibitors, collagen cross-linking, and ethanol-wet bonding (EWB) have been attempted to overcome this problem.[2] Cross-linking agents improve the mechanical stability of dentin collagen as well as its resistance to thermal denaturation and enzymatic degradation.[3],[4] Although cross-linking makes the collagen matrix resistant to degradation, it does not remove residual water in dentinal tubules which hinders resin infiltration. EWB overcomes this problem by replacing water from the demineralized dentin collagen with ethanol resulting in better resin infiltration.[5]

The aim of the present study is to compare the immediate and long-term microtensile bond strengths of an etch and rinse adhesive to water- versus ethanol-saturated dentin, pretreated with three different cross-linking agents such as ultraviolet A-activated riboflavin (UVAR), a photo-oxidative cross-linker; 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), a synthetic chemical cross-linker; and proanthocyanidin, a natural cross-linker.

   Subjects and Methods Top

Eighty freshly extracted noncarious human molars were de-rooted, and occlusal crown was reduced to 2 mm above cementoenamel junction using slow-speed water-cooled diamond saw to achieve flat surface in dentin. Dentin was etched for 15 s with 37% phosphoric acid (Prime Dental Products, Pune, India), rinsed thoroughly with distilled water, and blot-dried.

Specimens were randomly allocated into eight groups, and respective treatments were performed [Table 1].
Table 1: Experimental groups and their treatment protocols

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Bonding procedures were performed using Adper Single Bond Adhesive (3M ESPE) according to manufacturer instructions. Then, composite build up was done on bonded specimens in four 1-mm thick increments (Filtek Z350, 3M ESPE). The restored specimens were then sectioned into 1 mm × 1 mm beams with IsoMet saw, according to microtensile nontrimming technique. Intact beams of all the groups were divided into two halves, of which one half was tested after 24 h, and the other was stored in artificial saliva at 37°C for testing after 6 months.

Bonded specimens were mounted on a custom-made jig and stressed to failure under tension at 1 mm/min crosshead speed in a Universal Testing Machine. Each specimen was tested for microtensile bond strength at both time periods for all the groups. The percentage reduction in microtensile bond strength values between 24 h and 6 months as well as failure mode percentages were calculated. Each specimen was examined for failure mode distribution under a stereomicroscope at ×50 magnification to determine the mode of failure classified as:

  • Adhesive (A)
  • Cohesive in Composite (CC)
  • Cohesive in Dentine (CD)
  • Mixed failure (M).

Statistical analysis

SPSS version 18 software (SPSS Inc., Chicago, IL, USA) was used and P < 0.05 was considered statistically significant. Intergroup bond strength comparison was done using ANOVA with post ho c Tukey's test. Intragroup comparison was done using paired t-test.

   Results Top

The mean and standard deviation of microtensile bond strength values of different groups at 24 h and 6 months are reported in [Table 2]. The results suggested that cross-linking treatment and EWB alone as well as their combination significantly improved the strength of resin-dentin bonds compared to control group (P < 0.001). All the experimental groups showed a significant increase in the bond strength values at 24 h. Even at 6 months, they showed significantly higher values compared to control group measured at 24 h (P < 0.05) [Graph 1].
Table 2: Mean and standard deviation of microtensile bond strength values of different groups at 24 h and 6 months

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Among all the groups tested, cross-linked EWB groups showed significantly higher values compared to their corresponding cross-linked water-wet bonded (WWB) groups at either of the time periods (P < 0.05). UVA-riboflavin cross-linked EWB group showed the highest microtensile bond strength values at 24 h as well as 6 months followed by carbodiimide cross-linked EWB group and the least values were seen in noncross-linked WWB group. Even without cross-linker treatment, EWB showed a significant rise in bond strength compared to noncross-linked WWB group (P < 0.05).

Among all the cross-linked groups, UVAR groups showed the highest values followed by carbodiimide group, while the least values were shown by proanthocyanidin group both with WWB as well as EWB at 24 h and 6 months. Considering the reduction in values of all the groups after 6 months storage, UVAR cross-linked EWB group showed an insignificant reduction (P > 0.05), followed by UVAR cross-linked WWB group, whereas the highest reduction in bond strength values was seen in noncross-linked WWB group [Graph 2].

Failure mode analysis [Table 2] showed that there were more adhesive failures in control group at 24 h and 6 months compared to experimental groups. In UVAR group, there were more of cohesive failures in resin composite after aging compared to other groups.

   Discussion Top

Resin-dentin bond degradation occurs by hydrolytic, enzymatic, and fatigue degradation processes.[5] Collagen cross-linking enhances the longevity of resin-dentin bonds by strengthening demineralized collagen with the introduction of native cross-links. The recent literature suggests that resistance to degradation may also be attributed to the silencing of endogenous collagenolytic enzymes such as MMPs and cysteine cathepsins through conformational changes in enzyme three-dimensional structure by the prevention of its recognition and complexing with Type I collagen.[6],[7] There was a significant increase in bond strength values of all the cross-linked groups over control group in the present study. Chiang et al. found that cross-linking agents also change the micromorphology of bonded dentin surfaces, allowing better resin infiltration.[8]

Another issue which compromises the bond durability is the increased hydrophilic components in adhesives, which improve adhesion to acid-etched dentin but in turn result in insufficient resin penetration, water sorption, and resin plasticization.[9],[10] In addition, they activate hydrolytic enzymes such as MMPs leading to adhesive failure.[8],[11] EWB is a concept of replacing water from interfibrillar and intrafibrillar spaces of demineralized dentin collagen with ethanol, thereby preventing phase separation due to its hydrophobic property.[12],[13],[14] Hosaka et al. found that EWB hybrid layers showed increased interfibrillar spacing and reduced collagen fibrillar diameter which improves resin uptake of the collagen matrix.[15],[16] Ethanol also has higher vapor pressure than water resulting in early evaporation facilitating better resin penetration. It permits the monomers to penetrate the surface of collagen molecules of the microfibrils resulting in complete water replacement, thereby blocking the action of MMPs.[2],[17] Sauro et al. partly attributed the success of EWB to a higher degree of conversion of resin monomers owing to ethanol's reduced viscosity, thus allowing radical propagation to continue longer.[18] These factors explain the significant increase in bond strength values of EWB groups irrespective of cross-linking agent used in our study when compared to control group. However, progressive EWB is time-consuming, technique sensitive, and clinically impractical.[19] Simplified EWB protocol was therefore carried out in the present study.

Riboflavin 5-phosphate (Vitamin B2), a photo-oxidative cross–linker, produces free radicals on UVA photo activation, thereby inducing covalent bonds to bridge amine groups (N-H) of one chain with carbonyl groups (C=O) of hydroxyproline in adjacent chains.[8],[20] In the present study, the highest bond strengths among UVAR groups at 24 h can be explained by the immediate production of free radicals through photo-oxidation resulting in faster cross-linking which effectively improves collagen matrix stiffness within a short period leading to better resin penetration.

Carbodiimide (EDC) is a zero-length synthetic cross-linking agent. It reacts with carboxyl groups in proteins to form an intermediate which further reacts with an amino group and neighboring protein chain to form a stable covalent bond.[21] The lower bond strengths in EDC group at either of the observation periods in this study, when compared to UVAR group, may be attributed to an intermediate product formed which slows down the reaction rate of carbodiimide.[22] Within a short time, the carboxyl and amino groups of collagen may not be as accessible as those in MMPs, thereby favoring more rapid cross-linking of MMPs over collagen. Therefore, EDC could behave as an effective MMP inactivator than a collagen cross-linker in a short time interval.[23]

Proanthocyanidins (grape seed extract) are natural polyphenols known for their cross-linking properties.[24],[25] The bond strength values of proanthocyanidin group were significantly lower than the other two cross-linker groups in this study at either of the time periods. Proanthocyanidins interact noncovalently, and their reaction rate is diffusion-dependent. Therefore, 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 within 2 min.[26]

Although cross-linking improves the intrinsic properties of conventional WWB dentin substrate,[4] it leaves a water-rich collagen matrix which might be susceptible to degradation over time. This problem may be solved by means of EWB.[1],[27] Collagen cross-linking results in a matrix which is more resistant to hydrolysis while EWB results in improved infiltration of resin monomers, improving the mechanical properties of the hybrid layer. Cross-linked collagen also shows increased stiffness and ultimate tensile strength, thereby reducing the chances of collagen network collapse which might occur with simplified EWB.[3],[28] Therefore, the combination of collagen cross-linking with EWB was tested, and it showed a synergistic effect on the preservation of resin-dentin bonds compared to individual strategies.

All the experimental groups showed a significant increase in bond strengths at 24 h with less reduction in bond strengths at 6 months compared to control group. The higher bond strength values among UVAR EWB group at 24 h and lesser reduction in their bond strengths at 6 months can be explained by the faster cross-linking rate of UVAR compared to other cross-linkers, resulting in immediate increase in the stiffness and strength of collagen matrix,[20] thereby preventing matrix collapse which might occur with simplified EWB, allowing better resin infiltration. The lower bond strengths of carbodiimide and proanthocyanidin EWB groups may be because of their slower reaction and diffusion rates as discussed previously.[21],[26]

The high amount of cohesive failures in composite resin in UVAR group may be attributed to the fact that one of the maximum absorption peaks of riboflavin lies in the visible blue light spectral range and, therefore, the competition between riboflavin and photo-activators in composite might have partly affected the polymerization reaction of resin monomers.

The collagen cross-linking in combination with EWB is a clinically feasible approach for improving the strength and durability of resin-dentin bonds. However, its clinical application protocol needs to be optimized and in vivo studies are required to further support this concept before recommending its routine clinical application.

   Conclusions Top

EWB on cross-linked dentin can be an effective strategy for improving the quality of resin-dentin bonds. UVA-riboflavin photo-oxidative cross-linking was more effective in enhancing resin-dentin bond strength and durability compared to other chemical cross-linking agents using either of the bonding techniques within clinically acceptable time limits.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

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Correspondence Address:
Dr. Bhuvan Shome Venigalla
Turquoise 1210, My Home Jewel, Madinaguda, Hyderabad, Telangana
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-0707.194019

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