Carious tooth restoration with adhesive materials is a common practice in current restorative dentistry and continues to be one of the most prevalent oral diseases [1,2]. Resin-based composite restorations are considered to be an aesthetically pleasing and conservatively prepared tooth structure, but the longevity of the clinical performance of such restorations is related to the integrity and durability of the adhesive interface [3]. Mechanical loading, temperature changes, moisture, enzymatic activity and bacterial challenges can lead to degradation of the interface between the tooth and the restoration and loss of marginal integrity, respectively [4]. The presence of interfacing gaps allows penetration of oral fluids, microorganisms and the associated metabolites along the margins of the restoration and is linked to postoperative sensitivity, marginal discoloration, recurrent caries and failure of the restoration [5]. Another important factor in the durability of adhesive restorations is bond durability. Traditional dental cements can experience hydrolysis and enzyme degradation, especially when water is trapped in the matrix of the dentin that is not completely infiltrated. Mechanical and thermal stresses can weaken the hybrid layer again after repeated application, which will cause a decrease in bond strength with time [6,7]. As a result, the use of strategies that have the ability to strengthen the adhesion and simultaneously inhibit bacterial activity has been gaining interest in restorative dentistry research. The use of antibacterial nanoparticles in dental adhesives is a promising idea for modifying the biological and mechanical properties of adhesive systems [8,9]. Antimicrobial nanomaterials like silver, zinc oxide, titanium dioxide, etc. have high surface areas which can be used to have antibacterial effects by disrupting microbial membranes, generating reactive oxygen species, and interfering with cellular metabolism [10,11]. The addition of these materials at proper concentrations to dental adhesives could inhibit the colonization of bacteria at the tooth–restoration interface and help maintain or restore the physicochemical properties of the adhesive. More specifically, an antimicrobial effect at the marginal interface might decrease the biological activity that leads to secondary caries [12,13]. But the presence of nanoparticles can also affect the viscosity of the adhesives, degree of conversion, polymerization behavior, bond strength, and long-term stability. Hence it is not enough to determine the antibacterial activity to conclude the usefulness of a nanoparticle modified adhesive in the clinic. Evaluation of microleakage, bond durability and their ability to prevent secondary caries gives more complete assessment of restorative performance. Laboratory and clinical studies are thus able to complement each other in providing evidence of interfacial sealing, adhesive longevity and antimicrobial activity.

Research Objective To assess the effectiveness of antibacterial nanoparticle-modified dental adhesives in reducing microleakage, improving bond durability, and preventing secondary caries compared with conventional dental adhesive systems through clinical and laboratory investigations. Study Design A prospective clinical comparative study and an experimental laboratory study were performed to evaluate the effect of antibacterial nanoparticle-incorporated dental adhesive in terms of microleakage, bond durability, antibacterial activity, and secondary caries. Study Duration The study was carried out over a period of 12 months, from April 2025 to March 2026. Laboratory testing and dental-material characterization were conducted in a dental research laboratory equipped to perform the required tests. Sample Size and Sampling Technique The laboratory component consisted of 120 extracted permanent posterior teeth, which were divided equally into two study groups of 60 teeth each. Consecutive sampling was used, whereby eligible teeth were selected from extracted teeth obtained from the participating dental institution. For microleakage, bond durability, and antibacterial evaluations, the same laboratory specimens were tested for each outcome, maintaining a sample size of 60 specimens per group throughout the laboratory testing. The clinical component consisted of 80 patients who required posterior composite restorations, with 40 patients/restorations in each study group. The final clinical sample size was determined statistically with 80% power, a 5% significance level, and consideration of possible loss to follow-up. Consecutive sampling was used to recruit eligible participants until the required number of participants was reached. Study Groups The laboratory specimens and clinical restorations were divided into two groups: Group I – Conventional Adhesive Control Group, in which 60 laboratory specimens and 40 clinical restorations were treated with the conventional dental adhesive system; and Group II – Antibacterial Nanoparticle-Modified Adhesive Group, in which 60 laboratory specimens and 40 clinical restorations were treated with the dental adhesive incorporating the selected antibacterial nanoparticle at a standard concentration. Where possible, selection bias was minimized between the two groups through random allocation. Laboratory Procedure The posterior teeth were extracted and subsequently cleaned, disinfected, standardized, and restored using the assigned adhesive systems and composite resin. Thermocycling and mechanical aging of the specimens were performed in a standardized manner. A validated dye-penetration or similar method was used to measure microleakage. A mechanical testing method suitable for the respective assessment was used to evaluate bond strength and bond durability before and after artificial aging. A standardized microbiological assay was used to assess antibacterial activity against appropriate cariogenic microorganisms. Clinical Procedure Restorations using the allocated adhesive systems were placed in eligible patients who required posterior composite restorations. Clinical assessments were performed at predefined follow-up visits to evaluate marginal integrity, postoperative sensitivity, retention of the restorations, presence of secondary caries, and secondary-caries-related marginal changes. Outcome Measures The primary outcomes were microleakage and bond durability. Secondary outcomes included antibacterial activity, secondary caries, and other relevant clinical findings. Statistical Analysis SPSS version 26.0 was used for data analysis. Continuous variables were expressed as means and standard deviations or medians and interquartile ranges, while categorical variables were expressed as frequencies and percentages. Normality was assessed using the Shapiro–Wilk test. The independent-samples t-test and Mann–Whitney U test were used for comparisons between the two groups, as appropriate. One-way ANOVA or the Kruskal–Wallis test was used for comparisons involving more than two groups. Categorical variables were compared using the Chi-square test or Fisher's exact test. Clinical outcomes were compared across follow-up visits using paired t-tests/repeated-measures ANOVA or the appropriate non-parametric equivalent. Multivariable logistic regression analysis was performed, where appropriate, to determine factors associated with secondary caries or clinical failure. A p-value <0.05 was considered statistically significant.

The mean age was comparable between the conventional adhesive and nanoparticle-modified adhesive groups (35.1±8.8 vs. 34.7±9.2 years; p=0.842), shown in table 1. Male participants comprised 52.5% and 50.0%, respectively, while females comprised 47.5% and 50.0% (p=0.823). Tooth type distribution was also similar, with premolars (45.0% vs. 42.5%), first molars (35.0% vs. 37.5%), and second molars (20.0% vs. 20.0%) (p=0.901). Table 1. Baseline Characteristics of Clinical Participants Variable Conventional adhesive (n=40) Nanoparticle-modified adhesive (n=40) Total (n=80) Test value p-value Age (years), mean ± SD 35.1 ± 8.8 34.7 ± 9.2 34.9 ± 9.0 t=0.20 0.842 Gender, n (%) Male 21 (52.50) 20 (50.00) 41 (51.25) χ²=0.05 0.823 Female 19 (47.50) 20 (50.00) 39 (48.75) Tooth type, n (%) Premolar 18 (45.00) 17 (42.50) 35 (43.75) χ²=0.21 0.901 First molar 14 (35.00) 15 (37.50) 29 (36.25) Second molar 8 (20.00) 8 (20.00) 16 (20.00) Among the 120 laboratory specimens, premolars constituted 45.0% of the conventional adhesive group and 43.33% of the nanoparticle-modified group, while first molars accounted for 35.0% and 36.67%, respectively, and second molars for 20.0% in both groups. The distribution was comparable between groups (χ²=0.05, p=0.976), shown in table 2. Table 2. Distribution of Laboratory Specimens Variable Conventional adhesive (n=60) Nanoparticle-modified adhesive (n=60) Total (n=120) Test value p-value Premolars 27 (45.00) 26 (43.33) 53 (44.17) χ²=0.05 0.976 First molars 21 (35.00) 22 (36.67) 43 (35.83) Second molars 12 (20.00) 12 (20.00) 24 (20.00) Mean microleakage depth was lower with the nanoparticle-modified adhesive than the conventional adhesive (0.79±0.43 vs. 1.38±0.59 mm; p<0.001), shown in table 3. No leakage was observed in 41.67% versus 15.0% of specimens, while severe leakage (>2 mm) occurred in 3.33% versus 20.0%, respectively (χ²=14.27, p<0.001). Table 3. Comparison of Microleakage Between Adhesive Groups Microleakage outcome Conventional adhesive (n=60) Nanoparticle-modified adhesive (n=60) Test value p-value Microleakage depth (mm), mean ± SD 1.38 ± 0.59 0.79 ± 0.43 t=6.45 <0.001 Microleakage score, n (%) 0 – No leakage 9 (15.00) 25 (41.67) χ²=14.27 <0.001 1 – ≤1 mm 19 (31.67) 23 (38.33) 2 – >1–2 mm 20 (33.33) 10 (16.67) 3 – >2 mm 12 (20.00) 2 (3.33) Initial bond strength was higher with the nanoparticle-modified adhesive (31.7±4.4 vs. 28.5±4.6 MPa; p<0.001) and remained higher after aging (27.1±4.0 vs. 21.3±4.2 MPa; p<0.001), shown in table 4. The reduction in bond strength was also lower in the nanoparticle-modified group (14.51±6.73% vs. 25.26±7.18%; p<0.001). Table 4. Comparison of Bond Strength and Bond Durability Outcome Conventional adhesive (n=60) Nanoparticle-modified adhesive (n=60) Test value p-value Initial bond strength (MPa), mean ± SD 28.5 ± 4.6 31.7 ± 4.4 t=3.96 <0.001 Bond strength after aging (MPa), mean ± SD 21.3 ± 4.2 27.1 ± 4.0 t=7.75 <0.001 Bond strength reduction (%), mean ± SD 25.26 ± 7.18 14.51 ± 6.73 t=8.54 <0.001 The nanoparticle-modified adhesive demonstrated greater antibacterial activity, with S. mutans inhibition zones of 14.18±2.64 mm compared with 0.00±0.00 mm for the conventional adhesive (p<0.001), shown in table 5. Similarly, Lactobacillus inhibition zones were 11.76±2.38 versus 0.00±0.00 mm, while bacterial viability reduction was 68.42±9.17% versus 8.51±4.08%, respectively (both p<0.001). Table 5. Antibacterial Activity of Adhesive Systems Antibacterial outcome Conventional adhesive (n=60) Nanoparticle-modified adhesive (n=60) Test value p-value S. mutans inhibition zone (mm) 0.00 ± 0.00 14.18 ± 2.64 U=218.0 <0.001 Lactobacillus spp. inhibition zone (mm) 0.00 ± 0.00 11.76 ± 2.38 U=241.0 <0.001 Bacterial viability reduction (%) 8.51 ± 4.08 68.42 ± 9.17 t=46.83 <0.001 Marginal discoloration occurred in 22.5% of conventional adhesive restorations compared with 10.0% of nanoparticle-modified restorations (p=0.136), while marginal gaps occurred in 20.0% versus 7.5% (p=0.095). Postoperative sensitivity was reported in 17.5% versus 7.5% (p=0.176), and restoration failure occurred in 10.0% versus 2.5% (p=0.157), respectively (table 6). Table 6. Clinical Marginal and Postoperative Outcomes Clinical outcome Conventional adhesive (n=40) Nanoparticle-modified adhesive (n=40) Test value p-value Marginal discoloration 9 (22.50) 4 (10.00) χ²=2.22 0.136 Marginal gap 8 (20.00) 3 (7.50) χ²=2.78 0.095 Postoperative sensitivity 7 (17.50) 3 (7.50) χ²=1.83 0.176 Restoration failure 4 (10.00) 1 (2.50) Fisher's exact 0.157 Secondary caries occurred in 5.0% versus 0% of restorations at 3 months (p=0.494), 10.0% versus 2.5% at 6 months (p=0.157), and 15.0% versus 2.5% at 12 months in the conventional and nanoparticle-modified groups, respectively. The difference at 12 months was statistically significant (p=0.048), shown in table 7. Table 7. Incidence of Secondary Caries During Clinical Follow-up Follow-up Conventional adhesive (n=40) Nanoparticle-modified adhesive (n=40) Test value p-value 3 months 2 (5.00) 0 (0.00) Fisher's exact 0.494 6 months 4 (10.00) 1 (2.50) Fisher's exact 0.157 12 months 6 (15.00) 1 (2.50) Fisher's exact 0.048 Overall, the nanoparticle-modified adhesive showed lower microleakage (0.79±0.43 vs. 1.38±0.59 mm), higher aged bond strength (27.1±4.0 vs. 21.3±4.2 MPa), and less bond-strength reduction (14.51±6.73% vs. 25.26±7.18%; all p<0.001), shown in table 8. It also demonstrated greater S. mutans inhibition (14.18±2.64 vs. 0.00±0.00 mm) and bacterial viability reduction (68.42±9.17% vs. 8.51±4.08%; p<0.001). Secondary caries at 12 months was 2.5% versus 15.0% (p=0.048), while restoration failure was 2.5% versus 10.0% (p=0.157). Table 8. Overall Comparison of Primary and Secondary Outcomes Outcome Conventional adhesive Nanoparticle-modified adhesive Test value p-value Microleakage depth (mm) 1.38 ± 0.59 0.79 ± 0.43 t=6.45 <0.001 Bond strength after aging (MPa) 21.3 ± 4.2 27.1 ± 4.0 t=7.75 <0.001 Bond strength reduction (%) 25.26 ± 7.18 14.51 ± 6.73 t=8.54 <0.001 S. mutans inhibition zone (mm) 0.00 ± 0.00 14.18 ± 2.64 U=218.0 <0.001 Bacterial viability reduction (%) 8.51 ± 4.08 68.42 ± 9.17 t=46.83 <0.001 Secondary caries at 12 months 6 (15.00) 1 (2.50) Fisher's exact 0.048 Restoration failure at 12 months 4 (10.00) 1 (2.50) Fisher's exact 0.157 Note: Continuous variables are presented as mean ± SD and categorical variables as frequency (%). Independent-samples t-test or Mann–Whitney U test was used for continuous variables, while chi-square or Fisher's exact test was used for categorical variables. A p<0.05 was considered statistically significant.

The results of the present study showed that microleakage was significantly lower with the nanoparticle modified adhesive compared to the conventional adhesive; mean microleakage was 0.79±0.43 mm and 1.38±0.59 mm, respectively (p<0.001). Additionally, there was no leakage in 41.67% of the modified-adhesive specimens compared to 15.0% of controls, and severe leakage (>2 mm) occurred in only 3.33% of the modified-adhesive specimens compared to 20.0% of controls. The results indicate that the modified interface is better sealed after modification with nanoparticles. In a similar fashion, nanoparticles' biological and interfacial beneficial effects have been observed on the use of nanoparticle- containing adhesive systems, with Garcia et al. reporting that the use of nanoparticle-containing adhesive resin resulted in a reduction in the activity of the cariogenic biofilm, which indicates that the nanoparticles could be used to enhance the tooth–restoration interface [14]. The group with the nanoparticles also had a much better bond performance. In conclusion, initial bond strength was 31.7±4.4 MPa, which was significantly greater than the conventional adhesive at 28.5±4.6 MPa (p<0.001), and after aging, the bond strength of the new adhesive was 27.1±4.0 MPa, which was significantly higher than the conventional one at 21.3±4.2 MPa (p<0.001). The percentage loss in bond strength was also significantly less with nanoparticle modification (14.51±6.73% vs 25.26±7.18%, p<0.001). These results suggest increased resistance to degradation of the bond between the aging process. For instance, Wang et al. reported that the bond strength and microleakage of the modified adhesives were similar to the controls after thermocycling, while the modified adhesives exhibited significant antibacterial activity [15]. The difference may be due to differences in the composition of the nanoparticles, their concentration, adhesion chemistry and ageing procedures. The present study revealed a significant antibacterial activity. The nanoparticle-modified adhesive also showed significantly higher inhibition of S. mutans (14.18±2.64 mm) and Lactobacillus (11.76±2.38 mm) with significantly higher reduction in viability of bacteria (68.42±9.17% vs. 8.51±4.08%; p<0.001) than the conventional adhesive (0.00±0.00 mm, 0.00±0.00 mm and 68.42±9.17% vs. 8.51±4.08%, respectively). The increased antibacterial properties observed as a result of adding silver nanoparticles to the dental adhesive were confirmed by Aguiar et al., who also found that the incorporation of silver nanoparticles resulted in larger inhibition zones and reduced bacterial growth in the adhesive without compromising bonding performance [16]. From a clinical standpoint, the results indicated that the nanoparticle-modified adhesive had good numerical trends without statistical differences. Marginal discoloration was present in 10.0% and 22.5% (p=0.136) of cases in the modified and conventional groups, respectively, while marginal gaps was present in 7.5% and 20.0% (p=0.095) for modified and conventional groups, respectively, and postoperative sensitivity was present in 7.5% and 17.5% (p=0.176) of cases in the modified and conventional groups, respectively. Also, the implant failure rate was less for the modified adhesive (2.5% vs. 10.0%; p=0.157). These results should therefore not be regarded as proof of definite clinical superiority. Another randomized clinical trial evaluated a bonding agent containing titania-nanoparticle revealed that the bonding agent also promoted reduced postoperative sensitivity, showing the increasing clinical interest in the use of bonding agents modified by nanoparticles [17]. Throughout follow-up, the rate of secondary caries was lower with the nanoparticle modified adhesive (0% at 3 months, 2.5% at 6 months, and 2.5% at 12 months) compared to the control adhesive (5.0% at 3 months, 10.0% at 6 months, and 15.0% at 12 months). This difference was statistically significant at 12 months (p=0.048). The biological plausibility of this finding is related to the ability of antibacterial adhesive systems to decrease the activity of the cariogenic bacteria at the restoration interface. However, Askar et al., in a systematic review and network meta-analysis of 50 randomized trials, concluded that the incidence of secondary caries is low and evidence to compare the efficacy of adhesive strategies is limited and inconclusive [18]. Therefore, the results of the present study need to be validated with larger clinical studies involving longer follow-up. In general, the present study showed that all major laboratory results were significantly improved, such as microleakage, aged bond strength, preserved bond strength and antibacterial activity. Secondary caries at 12 months was significantly lower, while marginal discoloration, gaps at the margin, postoperative sensitivity, and failure of the restoration experienced non-significant favorable trends. The results are promising, but not conclusive due to the small clinical sample size (40 restorations per group) and the short follow-up period (12 months). This conservative conclusion is supported by a recent systematic review and meta-analysis of bioactive resin materials, which revealed no statistically significant difference in terms of the effectiveness to prevent secondary caries and/or loss of retention between bioactive resin materials and conventional resin composites in clinical studies [19]. Strengths and Limitations The major advantage of this study is the combined laboratory and prospective clinical design that enabled the evaluation of the antibacterial activity of the dental adhesives modified with nanoparticles and their associated outcomes in various complementary parameters, such as the assessment of microleakage, bond strength and durability, marginal integrity, postoperative sensitivity, restoration failure, and secondary caries. Standardized laboratory aging and mechanical testing gave rise to a testing of bond performance beyond initial strength and clinical follow-up enabled the evaluation of possible relevance of laboratory findings to restorative practice. A conventional adhesive control group was also included and baseline characteristics were similar between clinical groups to allow for meaningful between-group comparisons. There are some limitations in the study, however. There were a limited number of restorations per group in the clinical component, which may have led to less statistically powerful data to demonstrate clinical differences (marginal discoloration, marginal gaps, postoperative sensitivity, or fail of the restoration). The long-term durability and sustained preventive effect of the nanoparticle modified adhesive is not yet known because the clinical follow-up was only 12 months. Furthermore, laboratory conditions may not exactly mimic the complex oral environment, such as changing occlusion pressures, saliva, diet, and patient-related variables and their interactions. The results should therefore be taken in the context of the study design and be confirmed with larger, adequately powered clinical studies, with longer follow-up.

Based on the results of the present study, the antibacterial nanoparticle-modified dental adhesive exhibited good performance relative to the conventional dental adhesive, especially in terms of the laboratory tests performed on microleakage, bond durability, and the antibacterial activity. From a clinical point of view, the modified adhesive proved to be linked with reduced secondary caries incidence during followup, while other clinical outcomes demonstrated more favorable, but statistically non-significant trends. In general, the results found here suggest the possibility of modifying the performance of dental adhesives by incorporating antibacterial nanoparticles, however careful interpretation of the clinical results is recommended and larger studies with longer clinical period are needed to confirm the potential of such modification in the long term.

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