Comparative evaluation of the effect of different disinfecting agents on bond strength of composite resin to dentin using two-step self-etch and etch and rinse bonding systems: An in-vitro study.

OBJECTIVE: This study was carried out to examine the effect of the application of different disinfecting agents on the shear bond strength (SBS) of an etch-and-rinse system.
MATERIALS AND METHODS: A total of 120 dentin surfaces of human molars were taken. Sixty samples were taken for immediate (testing was done after 24 h) and delayed (testing was done after 6 months) each. Further, they were divided into three subgroups (n = 20) according to the disinfectant used: Group I: control (no disinfectant); Group II: chlorhexidine based; and Group III: Aloe barbadensis miller (Aloe vera) based. Dentine bonding agent was applied, and resin composite build-ups were done for SBS testing. Samples were examined under scanning electron microscope for observing the gap formation for dentin-restoration interface. SBS results were statistically analyzed.
RESULTS: The results of the study showed that both chlorhexidine and aloe vera had improved bond strength as compared to the control group both for immediate and delayed SBSs.
CONCLUSION: Thus, aloe vera can be used as an alternative to chlorhexidine for cavity disinfection.

INTRODUCTION

Bacterial remnants after the cavity preparation have led to the use of different cavity disinfectants over the period of time.[1] These bacteria present in smear layer multiply and produce toxins which form degradation products that is a cause of irritation of pulp. Long-term studies have shown that the bond strength of resin-bonded dentin decreased over time due to collagen degradation within the hybrid layer.[2] The use of cavity disinfectants eliminates the residual bacteria, but a potent problem is that it may affect the bond strength of composite resins.[3]

Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes which are capable of degrading extracellular matrix proteins. Activated MMPs are not fully infiltrated with adhesive resin. They can slowly degrade the collagen fibrils at the resin-dentin bonded interface.[4] Thus, the use of such cavity disinfectants which are MMP inhibitors is a strategy to prevent degradation of dentin bonds and to increase the longevity of bonded restorations.

Chlorhexidine, a widely used antimicrobial, is a MMP inhibitor. Loss of hybrid layer integrity compromises resin-dentin bond stability.[5] CHX also minimizes the convective and evaporative water fluxes from the underlying dentin, thus enhancing the bonding capacity of certain adhesives.[5] Some of the common adverse reactions of chlorhexidine include contact dermatitis, damage and irritation to oral mucosa, altered taste sensations, and various other allergic reactions. One of the major drawbacks also includes discoloration of tooth surface.[67] Due to these disadvantages of chlorhexidine, various herbal alternatives are being tested which are of equal efficacy or better to be used as cavity disinfectants.

Aloe barbadensis miller (Aloe Vera) contains aloins and barbadoins as main chemical constituents.[8] Aloe Vera gel has inhibitory effects on microorganisms due to the action of anthraquinones.[9] Recently, a study by Kudalkar et al. exhibited that Aloe Vera has MMP-2 and MMP-9 inhibitory action.[10]

Thus, the objective of this study was to compare the effect of these two cavity disinfection materials, namely, chlorhexidine and Aloe Vera on bond strength of resin composite and to examine the null hypothesis; there are no differences in the shear bond strength (SBS) between etch and rinse and self-etch adhesives after application of different disinfecting agents.

MATERIALS AND METHODS

One twenty extracted intact caries, cracks and restorations free, permanent mandibular molars were collected, cleaned of debris, blood, and calculus, and then stored in normal saline at 4°C for no more than 2 weeks. Horizontal sections were cut using diamond disk (Markus Ink., Michigan, USA) in a high-speed handpiece under air and water spray; the long axes of the teeth were perpendicular to the surfaces cut. After removal of enamel, the midcoronal dentin was exposed. The sections of the teeth including the roots were embedded in autopolymerizing acrylic resin to form cylinders 2.5 cm in diameter and 3 cm high. Dentin surfaces were flattened up to 1000 grit silicon carbide paper under running water so that a very smooth surface and a homogenous smear layer were achieved. Sixty samples were taken for immediate shear testing and other sixty for delayed shear bond testing.[11]

Acid etching of the exposed dentin was performed for 15 s with 37% phosphoric acid gel (Scotchbond etchant, 3M ESPE, St. Paul, MN, USA). i.e:The teeth were divided into four groups (n=40) and then in Group 1, the specimens were not treated with any cavity disinfectant and served as control (n = 40).

Group 2: 2% CHX (n = 40) solution was prepared from dilution of 20% CHX Solution using distilled water (Basic pharma, Gujarat, India).

Group 3: Aloe barbadensis miller (Aloe Vera) (n = 40) solution was prepared using aloe vera powder of 99% purity and dissolving 20 mg of aloe vera powder in 10 ml of distilled water.[8]

The acid-etched dentin was pretreated with 2% CHX in Group 2 and Aloe barbadensis miller solution in Group 3 for 30 s, active application with a brush applicator (Microbrush International, WI, USA), and the excess was removed with cotton pellet before the application of bonding agent (Adper Single Bond 2). Adhesive tape with a 3 mm diameter hole in it was used to define the bonding agent. The dentin surfaces of the teeth were then dried with air for 10 s resin composite was applied in 5–6 increments (Filtek Z 350, 3M ESPE, St. Paul, MN, USA) with the aid of polyethylene tubes (3 mm diameter, 2 mm height, and 0.5 mm thickness) and individually light cured for 40 s using light-emitting diode (Dentsply), light-curing unit with an output of 600 milliwatts/cm2. The tubes were then removed. The teeth (20 samples from each group) were then stored in distilled water at room temperature for 24 h and tested for immediate SBS.

Sixty samples were stored in distilled water for 6 months and were then tested for delayed SBS. Each tooth was secured in a specially designed attachment jig to hold the specimens to the universal testing machine (Instron, ADMET, Enkay Enterprises, New Delhi, India). Load was applied by the testing machine through a wire loop adjusted to the bonded interface at a crosshead speed of 0.5 mm/min. SBS in MPa was calculated from the peak load at failure divided by the specimen surface area.

Two specimens from each group (1 for immediate and 1 for delayed) were sputter coated with gold after fracture and prepared for scanning electron microscope (LEO 435VF electron Microscope, England) examination. Coated specimens were then observed under scanning electron microscopy (SEM) for viewing the resin-dentin interface.

The SBS values were statistically analyzed using Tukey's honestly significant difference test and three-way analysis of variance (multivariate assessment) at a significance level of P > 0.05.

RESULTS

The mean SBS values obtained after immediate and delayed testing are depicted in Table 1 and Table 2. No significant difference in immediate SBS of the two test groups (CHX and Aloe Vera) was observed, but both the test groups exhibited bond strengths significantly higher than the control group. After 6months of water storage, the magnitude of SBS fell for all groups, but again the reduction in bond strength of the control group was more than that of test groups. Representative SEM images of the dentin/restoration interface for control and experimental groups are shown in Figures 1 (a-c) and 2(a-c).

Table 1

Shear bond strength (Mean±SE) of three groups at two different periods

Table 2

For each period, comparison of mean shear bond strength between the groups by Tukey test

Figure 1(a-c)

A representative Scanning electron microscopy image of bonded interface in control group (immediate bond strength tested specimen). Notice the gap between dentin and restoration (500X); A representative Scanning Electron Microscope image of bonded interface in Chlorhexidine group (immediate bond strength tested specimen) where gap free interface can be observed(500X); A representative SEM image of bonded interface in aloe vera group (immediate bond strength tested specimen) where gap free interface can be observed (500X)

Figure 2(a-c)

A representative Scanning Electron Microscope image of bonded interface in control group (delayed bond strength tested specimen). Notice the gap between dentin and restoration (500X); A representative Scanning Electron Microscope image of bonded interface in Chlorhexidine group (delayed bond strength tested specimen) where gap free interface can be observed (1000X); A representative Scanning Electron Microscope image of bonded interface in aloe vera group (delayed bond strength tested specimen) where gap free interface can be observed (1000X)

DISCUSSION

Degradation of the exposed collagen fibrils within the hybrid layer is the key factor which is primarily responsible for the deterioration of the adhesive dentin interface. This is mainly brought about by the action of MMP enzymes present in the dentin which get activated in the presence of zinc and calcium ions when low pH is created by the process of acid etching.[1213]

Certain mechanisms have been advocated to improve the bond strength and the durability of the resin-dentin bond. One of them is inhibition of MMPs. Chlorhexidine and Aloe Barbadensis Miller (Aloe Vera) both exhibit antimicrobial activities and are potent in inhibiting MMPs; especially MMPs 2, 8, and 9 for chlorhexidine[1415] and MMPs 2 and 9 for aloe vera.[10] The MMP inhibitory action of chlorhexidine is attributed to it chelating action whereby it scavenges calcium and zinc ions.[16] The MMP inhibitory action of aloe vera is attributed to the aloins which are effective inhibitors of stimulated granulocyte MMPs.[17]

Studies using chlorhexidine as cavity disinfectant were found to be effective in reducing the levels of Streptococcus mutans located in occlusal fissures and on exposed carious root surface. The use of chlorhexidine as cavity disinfectant after tooth preparation and before the application of bonding agent could help to reduce the potential for residual caries and postoperative sensitivity. However, some authors suggested that the increased amount of microleakage could hinder with bonding agent interaction to the dentin.[1518] On the other side, the antimicrobial effect of aloe vera has also been demonstrated in an in vitro study in which this phytotherapeutic agent inhibited the growth of diverse oral microorganisms such as S. mutans, Streptococcus Sanguis, and Candida albicans.[19] It was also found that aloe vera extract was able to inhibit the growth of S. mutans; thus, it is of great benefit as it prevents secondary caries.[20]

Chlorhexidine showed improved bond strength from that of the control group by blocking of the dentin MMPs activation;[2122] the results of our study are in support to the studies conducted by Sinha et al. and Boiter et al. They also suggested that application of 2% chlorhexidine prevents hybrid layer degradation and this procedure has a beneficial effect on maintaining bond strength.[1323]

Aloe barbadensis Miller (Aloe Vera) contains aloins, barbadoins, and anthraquinones as main chemical constituents which are responsible for its bactericidal activity.[89] It also possesses anti-MMP potential, especially against MMP 2 and 9.[10] The results of this study revealed that application of aloe vera to acid-etched dentin improves the longevity of the resin-dentin bond. This in support to the study conducted by Sinha et al.,[13] but in their study, only immediate bond strength was evaluated. Although the bond strength increased significantly from that of the control group, the difference between the bond strengths of the two test groups was not statistically significant (chlorhexidine≈aloe vera). The results of the study showed that both for immediate and delayed bond strengths; aloe vera was equally efficacious as chlorhexidine.

SEM analysis is important when one attempts to understand the mechanisms, leading to the degradation of the hybrid layer and the reduction in dentin bond strengths with time. Only cohesive failures within the resin and within the dentin are noted in SEM images of both the tested groups which are associated with higher bond strengths of these groups.

Based on the results of the study, the null hypothesis was rejected as statistically significant differences were obtained between both the adhesive groups.

CONCLUSION

Within the limitations of this in vitro study, it may be stated that the use of chlorhexidine and Aloe barbadensis Miller as cavity disinfectants has no adverse effect on the immediate and delayed SBSs of etch and rinse adhesive to dentin. Keeping in mind the several of drawbacks of chlorhexidine as cavity disinfectant and from the results of this study, the authors advocate the use of Aloe barbadensis Miller as an alternative to chlorhexidine for cavity disinfection. Still, long-term in vivo clinical trials are warranted in this regard.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.