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 Table of Contents  
Year : 2021  |  Volume : 13  |  Issue : 5  |  Page : 470-477

Effect of remineralizing agents on resin-dentin bond durability of adhesive restorations: An in vitro study

Department of Conservative Dentistry and Endodontics, GITAM Dental College and Hospital, Visakhapatnam, Andhra Pradesh, India

Date of Submission17-Apr-2021
Date of Decision14-Jun-2021
Date of Acceptance16-Jul-2021
Date of Web Publication11-Oct-2021

Correspondence Address:
Jyothi Mandava
Department of Conservative Dentistry and Endodontics, GITAM Dental College and Hospital, Rushikonda, Visakhapatnam, Andhra Pradesh.
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JIOH.JIOH_96_21

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Aim: To prolong the clinical success and durability of bonded restorations, hybrid layer stabilization is crucial for better adhesion between the resin and tooth substrate. The aim of this in vitro study was to assess the ability of self-assembling peptide (curodont protect) and a remineralizing agent (MI paste plus) in promoting biomimetic remineralization for stabilization of the hybrid layer to improve the resin-dentin bond durability. Materials and Methods: Standardized mesio-occlusal (MO) and disto-occlusal (DO) cavities were prepared on 60 mandibular molars. Teeth were randomly allocated to different groups and after acid etching, half of the MO cavities (n = 30) of dentin were treated with curodont protect and another half (n = 30) with MI paste plus. Half of the DO cavities (n = 30) were pretreated with chlorhexidine, whereas the other half did not receive any dentin pretreatment before placing adhesive restoration. Half of the samples from each group (n = 15) were subjected to thermomechanical load cycles. Immediate and after aging bond strengths were estimated using a universal testing machine, and the type of bond failure was assessed under a scanning electron microscope. Data obtained in megapascals were analyzed by one-way analysis of variance and Tukey’s multiple post hoc test with the significance level set as P ≤ 0.05. Results: Immediate bond strength values were significantly high for the control group and low for the MI paste plus group (P = 0.002). Significant differences were not observed between the curodont protect and chlorhexidine (P = 0.0514) treated groups. In all groups, bond strength values were decreased significantly after thermomechanical cyclic (TMC) loading (P = 0.0001). Adhesive type fractures were reported more in failure mode analysis. Conclusion: All groups exhibited reduced microtensile bond strength values after aging. The dentin treated with chlorhexidine, curodont protect, and MI paste plus produced more durable bonding than the control group.

Keywords: Bond Strength, Chlorhexidine, Curodont Protect, Hybrid Layer, MI Paste Plus

How to cite this article:
Pulidindi H, Mandava J, Borugadda R, Ravi R, Angadala P, Penmatsa P. Effect of remineralizing agents on resin-dentin bond durability of adhesive restorations: An in vitro study. J Int Oral Health 2021;13:470-7

How to cite this URL:
Pulidindi H, Mandava J, Borugadda R, Ravi R, Angadala P, Penmatsa P. Effect of remineralizing agents on resin-dentin bond durability of adhesive restorations: An in vitro study. J Int Oral Health [serial online] 2021 [cited 2021 Dec 6];13:470-7. Available from:

  Introduction Top

Despite many improvements in adhesive dentistry, degradation of the resin-dentin interface still challenges the clinician with reduced longevity of composite restorations. With the currently recommended adhesive protocol, replacing 70 vol% of residual water from the etched dentin by resin monomers infiltration is hard to achieve, which, in turn, leads to hydrolytic degradation of the hybrid layer.[1],[2] In addition, exposed and unprotected collagen fibrils during the bonding procedure are susceptible to enzymatic degradation by the activation of dentin matrix metalloproteinases (MMPs).[3] Correspondingly, both etch-and-rinse and self-etch adhesives contribute to collagenolytic/gelatinolytic destruction of the hybrid layer by the activation of endogenous MMPs, mainly MMP-2, -8, and -9.[4] Other proteases such as cysteine cathepsin have also shown to participate in extracellular matrix degradation and failure of adhesive restoration over time.[5] Hence, preservation of hybrid layer integrity is crucial to furnish adhesive restorations with prolonged durability.

Various approaches have been suggested and tested to enhance bond stability either by increasing the resistance of dentin collagen to degradation or by inhibiting the activation of collagenolytic enzymes.[6] Among the enzyme inhibitors, the most tested nonspecific synthetic MMP inhibitor chlorhexidine (CHX) was able to suppress collagenolytic and gelatinolytic activity of dentin matrices.[7] However, both in vitro and in vivo studies demonstrated that the inhibitory action of CHX on MMPs was time-dependent (maximum for 18 months), as it leaches out slowly from the hybrid layer, making the resin-dentin interface prone to host-derived proteolytic enzyme activity.[8],[9] Other protease inhibitors such as gluteraldehyde, carbodiimide, proanthocyanidine, and riboflavin have been shown to increase the collagen stiffness and resistance to degradation by their cross-linking action.[10] However, their cytotoxicity, and need for prolonged application time (10 min to 1h) make them not suitable for clinical implementation.[11]

Biomimetic remineralization is a new strategy to backfill the water-containing voids in the resin-dentin interdiffusion zone with nano-size remineralizing apatite crystallite reagents.[6],[11] The use of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) nanoparticles can promote dentin biomineralization by infiltrating into intrafibrillar and interfibrillar collagen spaces, replacing water by mineralization and it consequently helps in the preservation of hybrid layer integrity.[12] The MI paste plus (GC America Inc, Alsip, IL) contains 0.20% w/w (900 PPM) fluoride and CPP-ACP. When applied on partially demineralized dentin, these agents can help in the epitaxial growth of the remaining hydroxyapatite crystals by localizing the bioavailability of calcium, fluoride, and phosphate, thereby inducing remineralization.[13]

The curodont protect (Credentis, Switzerland) tooth remineralizing gel developed for regeneration of enamel is based on patented CUROLOX technology. The active components of curodont are fluoride, sodium chloride, calcium phosphate, and oligopeptides.[14] Studies reported that P11-4, a self-assembled peptide matrix, can facilitate the growth and deposition of hydroxyapatite crystals on enamel.[15],[16] However, the influence of curodont protect on the durability of resin-dentin integrity has not been studied so far.

The aim of this study was to assess the influence of CPP-ACP and P11-4 self-assembling peptide dentin pretreatments on the microtensile bond strength (µTBS) of adhesive resin. The tested null hypothesis was that the restorative resin bond strength durability will not be influenced by dentin pretreatment with remineralizing agents.

  Materials and Methods Top

Setting and design

The Dr. NTR University of Health Sciences, Andhra Pradesh, India has provided the approval for conducting the research under the protocol no. D188601024. Ethical clearance was obtained from the Institutional research ethics committee. For this in vitro study, 60 extracted human mandibular molars having similar mesiodistal and buccolingual dimensions were selected after clinical and radiographic examination. Teeth with visible hypoplastic lesions, crack lines, or detectable carious defects were excluded from the study sample. Teeth were disinfected with 0.5% Chloramine-T (Viachem, TX) solution and stored in physiological saline at 4°C till the experimental period, for not more than three months after extraction. Considering the previous data published, sample size estimation was done using G* power software 20.0 version. A power analysis set at the level of 80% calculated the minimal sample size of 12 to test the variables and thus a total of 15 samples per group were taken. Teeth samples were randomly allocated into groups to test different dentin treatment protocols.

Sample preparation

Standardized class II mesio-occlusal (MO) and disto-occlusal (DO) cavities were prepared in all 60 teeth samples using # EX-41 pear-shaped diamond abrasive (Mani, Tochigi, Japan) and #245 carbide burs (SS White, NJ) with a high-speed handpiece. The prepared cavities were 3.5 mm deep and 3 mm wide occlusally with an axial wall depth of 1.5 mm by placing the gingival seat 0.5 mm coronal to the cemento-enamel junction. After every five teeth cavity preparations, burs were changed for standardization. The experimental design [Figure 1] and details of the test materials are presented in [Table 1].
Figure 1: Schematic diagram of investigation design

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Table 1: Details of materials used in the study

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Restorative procedure

The prepared mesial and distal class II cavities were cleaned with water to remove the debris and air-dried. N-etch (Ivoclarvivadent, Schaan, Europe) 37% phosphoric acid etchant was applied to all the prepared cavity walls with a syringe applicator tip. Application of etchant was done on enamel walls first and then on dentin, so that 20s of enamel etching time and 10s for dentin etching were maintained. The etchant was washed thoroughly with a jet of water for 10s, and excess moisture was removed with damp cotton pellets.

Pretreatment of dentin surfaces

After acid etching and rinsing, MO cavities in 30 teeth samples were treated with 0.1 mL of curodont protect remineralizing agent. All the prepared dentinal walls were coated with curodont, agitated with an applicator tip, and allowed to dry for 5 min. In another 30 samples, the MO cavities were treated with MI paste plus and left undisturbed for 3 min. DO cavities in 30 teeth samples were treated with 2% CHX after the etching procedure and allowed to dry for 3 min. DO cavities in the other 30 teeth (control) samples, after acid etching, did not encounter any dentin pretreatment before adhesive application.

Bonding procedure

In all mesial and distal class II cavities, two consecutive coats of Tetric N bond adhesive were applied on dentin surfaces and after the removal of an excess amount of solvent light-cured for 10s. Tetric N-Ceram restorative resin was inserted into the preparations, maintaining 2-mm-thick increments and light-cured for 20s with C8 light-emitting diode Bluephase light-curing unit (Ivoclar, Schaan, Austria) having 800 mW/cm² intensity. Finishing and polishing of all the restorations were done using Sof-Lex flexible fine-grit disks (3M ESPE, MN) and composite polishing rubber cups (Shofu Dental products, San Marcos, TX) at low speed.

The restored teeth were stored in saline for about 24h at room temperature and from each group half of the samples were tested for immediate µTBS.

Thermomechanical cyclic loading

Half of the samples from each group (n = 15) were subjected to thermomechanical cyclic (TMC) loading procedures to simulate intraoral conditions. Thermal changes of 5–55°C with 30s dwell time and 5s transit time were used for 10,000 thermal cycles. The teeth were then submitted to 1,00,000 mechanical load cycles by the application of 50N vertical occlusal load at 1 HZ frequency for 20 cycles/minute. It was proposed that in a day thermal changes might occur between 20 and 50 times and 10,000 cycles will represent one year of aging for restorations.[17],[18]

Microtensile bond strength evaluation

The restored teeth were sectioned at the furcation area to obtain mesial and distal halves with composite restorations. After mounting tooth halves in acrylic resin blocks, hard tissue microtome (Leisa SP 1600, Germany) was used for sectioning and 0.9 ± 0.1 mm² resin-dentin beams were prepared. A custom-made jig with cyanocrylate glue was used for fixing the specimen to the Instron testing machine (DAK Series 7200, India). A tensile load at a cross-head speed of 1 mm/min was applied to fracture the resin-dentin beams and for each sample the failure load was recorded in megapascals (MPa).

Failure mode analysis

To analyze the type of bond failure taking place, the fractured specimens were mounted on aluminum stubs and sputter coated with gold. The fracture patterns were examined under a scanning electron microscope (S-3700N, Hitachi, Japan) by taking photomicrographs at 200× magnification [Figure 2]. Failure modes were designated as: (1) Adhesive failures that represent the adhesive interface failures. They can be at the dentin-adhesive interface or the resin-adhesive interface; (2) cohesive failures in which the term “cohesive” represents failures within the adhesive resin or in dentin along with the non-specified cohesive failures; and (3) mixed failures are designated as the presence of a mixture of adhesive and cohesive failures in the same fractured surface.[19]
Figure 2: SEM bond failure images at 200× magnification. (A) curodont protect group-adhesive failure; (B) control group-adhesive failure; (C) MI paste plus group-cohesive failure; (D) MI paste plus group-cohesive failure; (E) control group-cohesive failure; (F) chlorhexidine group-cohesive failure; (G) chlorhexidine group-mixed failure; (H) curodont protect group-mixed failure. C = composite, D= dentin

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Statistical analysis

Recorded µTBS data were calculated using SPSS statistics for windows version 20.0 software (IBM, Armonk, NY). One-way analysis of variance (ANOVA) was applied to perform statistical comparisons of µTBS among the groups. Multiple pairwise comparisons between groups were performed by using dependent t-test and post hoc Tukey test. The bond failure comparisons were done with Fisher’s exact chi-square test. The entire evaluation was done at 95% confidence level, hence P < 0.05 was considered statistically significant.

  Results Top

The mean µTBS values and standard deviation of test and control groups are described in [Table 2] and [Figure 3]. The variables tested (dentin pretreatment with CHX or remineralizing agents and TMC) significantly influenced the µTBS (P < 0.05).
Table 2: Comparison of microtensile bond strength values (MPa) for all groups at different time intervals

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Figure 3: Box-plot graph representing immediate and after TMC microtensile bond strength values in all groups

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The immediate µTBS values for all groups were significantly higher (P = 0.0001) than the artificially aged samples with TMC. The CHX pretreated group has shown significantly higher immediate and after TMC bond strength values (P ≤ 0.05) among the test groups, whereas it yielded higher µTBS compared with the control group also after TMC. The immediate µTBS of curodont protect did not differ significantly from CHX (P = 0.0514). After TMC, bond strength values were not significantly different among control, curodont protect, and MI paste plus groups (P > 0.05)

The failure mode was not significantly different among the different dentin pretreated groups [Table 3] at the different time periods tested. Overall, failures at the adhesive interface were more in number and cohesive failures were observed to be less in number.
Table 3: Comparison of fracture pattern percentages using Fisher’s exact chi-square test

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  Discussion Top

Preservation of hybrid layer integrity is crucial to create adhesive restorations with prolonged durability. Either total-etch or self-etch adhesives containing acidic monomers can activate the latent pro-MMPs through a cysteine switch mechanism, exposing the polypeptide-covered MMPs. In addition, acidic monomers can inhibit tissue inhibitors of metalloproteinases-1 activating the MMPs and cysteine cathepsins, causing hybrid layer degradation over time.[20] The most investigated MMP inhibitor, CHX was able to effectively reduce MMP-2, -8, -9 and cysteine cathepsins activity only for a shorter duration.[8] Consequently, several studies reported that decalcified dentin mechanical properties can be improved by the application of remineralizing agents, which would favor the dentin-resin bond strengths.[4],[11],[21] Hence, this laboratory study investigated the influence of remineralizing agents on the µTBS durability.

Testing µTBS on flat dentinal surfaces may not predict clinically relevant material behavior and thus occluso-proximal preparations were done on mandibular molar teeth in the study to mimic intraoral conditions. Different teeth may have different mineral content, properties and may be subjected to varying types of age-related changes. Resin-dentin blocks of test and control group samples for performing µTBS test were taken from the same tooth to minimize the bias and to avoid variations in the results. The agents were applied on the etched dentinal walls and allowed to dry for 5 min in case of curodont protect, simultaneously 3 min for MI paste plus and CHX, according to the manufacturer’s directions.

The tested null hypothesis was rejected since a significant interaction between dentin pretreatment agents and bonding of adhesive resin composite was observed, though they were not capable of arresting bond degradation completely. The immediate mean µTBS of coronal dentin to composite resin was significantly more for all group samples compared with after TMC bond strength values.

The study results designate that the immediate µTBS was elevated significantly for control teeth in which after acid etching, no dentin pretreatment was done before adhesive application compared with the test groups in accordance with the other study results.[9],[22] The interfacial layer formed by CHX or the application of remineralizing agents might have caused incomplete resin penetration into the demineralized dentin, leading to decreased immediate bond strength values compared with control teeth samples. In contrast, another study reported no significant difference in control and 2% CHX-treated dentin immediate bond strength values for tested total-etch adhesives.[23] However, a decrease in µTBS was significantly more in the control group after TMC, suggesting maximum deterioration of integrity of the hybrid layer compared with dentin pretreated samples.

Among the test agents, CHX-treated teeth have shown maximum immediate and after aging bond strength values. Nevertheless, electrostatically bound CHX ions may get dissolved by the water remnants within the collagen matrix accompanying the depletion of its long-term efficacy. Although several in vitro investigations demonstrated higher aged µTBS with CHX treatment, a significant advantage was not observed in randomized clinical trials.[24]

Dentin pretreated samples with curodont protect exhibited immediate µTBS similar to CHX-treated teeth but after aging the bond strength values were inferior to the CHX group. Several in vitro and in vivo studies had demonstrated the potency of P11-4 in preventing demineralization and promoting remineralization of carious lesions.[14],[15],[16] The assembled scaffold of P11-4 acts as a hydroxyapatite nucleator and diffuses into the porous substrate, thereby reinforcing the demineralized collagen matrix.[25] Similar to earlier reported findings, immediate µTBS to dentin was less for P11-4 applied teeth than control teeth in this study. The rationale for this result could be the formation of a hydrophobic surface on application of the agent, which might have hampered the adhesion of resin to dentin.[26]

Lower µTBS values were observed for the MI paste plus group in the current study among the dentin pretreated groups. This finding was attributed to the presence of a residual CPP-ACP layer on the dentin surface, interfering with resin infiltration and micro-mechanical interlocking.[27] Further, it was reported that 3 min application time for CPP-ACP was not sufficient to induce adequate remineralization and longer treatment time was suggested.[28] However, among the test groups, dentin pretreated with MI paste plus has shown less amount of µTBS deterioration after TMC though the difference was not statistically significant. The CPP has the capacity of stabilizing nano-ACP and thus reinforces the collagen matrix demineralized structure, improving the mechanical properties of dentin.[12] In addition, this fluoride containing CPP-ACP having the benefit of anticariogenic activity reduces the secondary caries occurrence with improved bond strength to dentin.[29]

The type of bond failures reported in the study were more of an adhesive and mixed type, irrespective of the mode of dentin pretreatment used. The results of previous studies are in correlation with these findings, reporting more adhesive and mixed failures after thermocycling with reduced adhesive bond strengths.[21],[26] Pretreating dentin with therapeutic agents restricts the degradation of the resin-dentin interface after TMC, manifesting more mixed failures with some adhesive and some cohesive failures.[30]

From the current study results, we emphasize that treating dentin with P11-4 self-assembling peptide and CPP-ACP remineralizing agents could be a favorable approach for enhancing the stability of the hybrid layer. As an in vitro study, the current investigation has certain limitations of being not able to simulate the biological aspects of intraoral conditions such as intrapulpal pressure and the role of dentinal endogenous enzymes. Further clinical studies are essential to validate the current outcomes and to illuminate the combination of different strategies to attain durable dentin-resin bonding.

  Conclusion Top

It is concluded that

  • Dentin pretreatment with remineralizing agents P11-4 or CPP-ACP could be a viable option to improve resin-dentin bond durability.

  • CHX (2%) treated dentin yielded the highest µTBS values after TMC.

  • Overall, the adhesive failures observed were more in number and the cohesive failures were less in number.

  • Acknowledgment


    Financial support and sponsorship


    Conflict of interest

    There are no conflicts of interest.

    Author contributions

    HP contributed to concepts, design, definition of intellectual content, literature search, data acquisition, and article preparation. JM contributed to concepts, design, definition of intellectual content, literature search, data acquisition, article preparation, article editing, and article review. RB contributed to concepts, design, definition of intellectual content, literature search, data acquisition, statistical analysis, and article preparation. RR contributed to concepts, design, definition of intellectual content, and article preparation. PA contributed to design, statistical analysis, and article preparation. PP contributed to data analysis, statistical analysis, and article preparation.

    Ethical policy and Institutional Review board statement

    Not applicable as In-Vitro study.

    Patient declaration of consent

    Not applicable.

    Data availability statement

    Data are available on valid request by contacting the corresponding author via mail.

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      [Figure 1], [Figure 2], [Figure 3]

      [Table 1], [Table 2], [Table 3]


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