Evaluation of sealant retention and caries prevention of 2 % chitosan-based pit and fissure sealants in permanent 1st molars – A randomised trial

Abstract

Background

Dental caries is a significant public health concern, particularly in children, where occlusal surfaces are at high risk due to complex pit and fissure morphology. Pit and fissure sealants are a well-established preventive measure, with resin-based sealants offering superior retention compared to glass ionomer cement (GIC) sealants. Chitosan, a naturally derived biopolymer, may enhance resin-based sealants by improving their mechanical strength, antibacterial action, and adhesion, leading to better retention and reduced need for reapplication. This study evaluated the 6-month retention and caries-preventive effectiveness of a 2 % chitosan-modified resin-based sealant versus a conventional sealant.

Methodology

A double-blind, split-mouth randomised clinical trial (CTRI/2023/06/054321) was conducted in a pediatric dental setting. A total of 38 children aged 6–10 years, each with four fully erupted, caries-free permanent first molars, were enrolled, resulting in a total of 152 Molars out of which 32 children (128 teeth) completed the trial. Each participant received both a conventional resin-based sealant (Clinpro™) and a 2 % chitosan-modified Clinpro™ sealant on contralateral molars. Randomisation was performed using a SNOSE (Sequentially Numbered Opaque Sealed Envelope) to determine the allocation of sealants on each side. Teeth were prepared by professional prophylaxis using pumice slurry, followed by etching with 37 % phosphoric acid, rinsing, and drying per manufacturer's instructions before sealant application. Both sealants were light-cured for 20 s and evaluated for proper placement. Clinical assessments were conducted at baseline, 3 months, and 6 months. Primary outcomes included sealant retention, evaluated using modified retention criteria (complete, partial, or total loss), and caries incidence, assessed using the International Caries Detection and Assessment System-II (ICDAS-II). Data were analyzed using STATA 18 software, and statistical significance was determined using Chi-square test to compare categorical variables, Shapiro-Wilk test was used to assess normality. Friedman test was conducted for within-group comparisons over time, followed by the Durbin-Conover post-hoc test for pairwise comparisons. Between-group comparisons of ICDAS-II scores were conducted using the Wilcoxon signed rank test. Statistical significance was set at p < 0.05.

Results

At 3 months, complete retention was observed in 95.31 % of molars treated with the chitosan-modified sealant, compared to 81.25 % in the conventional sealant group. By 6 months, retention rates declined slightly to 92.19 % in the study group and 76.56 % in the control group, with the differences remaining statistically significant (p < 0.05). Regarding caries prevention, at 3 months, 100 % of teeth in the study group remained caries-free (ICDAS-II score 0), compared to 89.06 % in the control group. By 6 months, 95.31 % of teeth in the study group remained caries-free, whereas the percentage dropped to 84.38 % in the control group. The chitosan-modified sealant demonstrated significantly superior caries prevention compared to the conventional resin-based sealant.

Conclusions

The incorporation of 2 % chitosan into resin-based sealants significantly enhanced both retention and caries prevention over a six-month period. The bioadhesive and antimicrobial properties of chitosan likely contributed to these improved outcomes. Given its enhanced longevity and preventive benefits, chitosan-modified resin-based sealants may serve as a more effective alternative for pediatric dental care. Further studies with extended follow-ups and larger sample sizes are recommended to validate these findings.

Graphical abstract

1. Introduction

Recent advances in dental materials have focused on enhancing the preventive potential of pit and fissure sealants. Incorporation of bioactive agents such as chitosan into resin-based sealants has emerged as a promising strategy.¹ Considering these advancements, present study examines, over a six-month follow-up, whether a 2 % chitosan-modified sealant provides improved retention and protection against caries compared with the conventional Clinpro™ sealant on newly erupted permanent molars.

Dental caries remains one of the most prevalent chronic oral conditions worldwide, driven by cycles of demineralization and remineralization. No longer considered an infectious disease, its current understanding emphasizes the role of bacterial metabolism and host responses.^2,3 The condition primarily arises from acidic byproducts of bacterial metabolism,.⁴ with prevalence rates ranging from 49 % to 83 % globally according to the Global Oral Health Data Bank.⁵

Despite accounting for just 12.5 % of total tooth surfaces, occlusal surfaces of newly erupted permanent molars are highly vulnerable to caries because of deep pits, fissures, and partial eruption. More than half of carious lesions in schoolchildren were found on these occlusal sites (Ripa, 1973).^6,7 To protect these high-risk sites, pit and fissure sealants have been employed since the 1960s as effective barriers against bacterial penetration and plaque accumulation.⁸ Fluoride-releasing resin-based sealants such as Clinpro™ have shown better retention and caries-preventive outcomes than glass ionomer cements (GIC).^9,10 However, their long-term efficacy depends on durable retention, which may be reduced by microbial activity, mechanical wear, and chemical degradation, thereby limiting their protective effects. Hence, intending to improve the longevity and protective ability of sealants, recent research in dental material science has investigated the addition of bioactive and antimicrobial compounds. Chitosan, a natural biopolymer obtained from chitin, is recognized for its antimicrobial, biocompatible, and bioactive properties.^1,11, 12, 13 Proposed mechanisms include disruption of bacterial membranes through its cationic charge and improved fluoride retention via its mucoadhesive nature, which may in turn prolong release and enhance caries-preventive benefits.¹⁴ While these mechanisms are biologically plausible, to the best of our knowledge they have not yet been directly validated in vivo. Nonetheless, in vitro studies indicate that chitosan-modified sealants demonstrate superior antibacterial activity and more durable caries protection compared with conventional materials.^1,16 To address this gap, the present study evaluates the clinical performance of a 2 % chitosan-modified resin-based sealant using modified retention criteria¹⁷ and the International Caries Detection and Assessment System-II (ICDAS-II).¹⁸

2. Materials and methods

This study was designed as a randomized, double-blind, split-mouth clinical trial to evaluate the clinical retention and caries-preventive effect of a chitosan-modified pit and fissure sealant compared to a conventional resin-based sealant. Ethical clearance was obtained from the Institutional Ethical Committee of A.B. Shetty Memorial Institute of Dental Sciences, Nitte (Deemed to be University) (Approval No: ETHICS/ABSMIDS/336/2023). The trial was prospectively registered in the Clinical Trials Registry – India (CTRI/2023/06/054321). The study adhered to the CONSORT guidelines (2022) for randomized clinical trials and was conducted in accordance with the Declaration of Helsinki (2013), ensuring the ethical principles and guidelines for conducting human clinical research.

2.1. Participant selection and eligibility criteria

A total of 38 children aged 6–10 years were recruited from the outpatient department of Paediatric and Preventive Dentistry at A.B. Shetty Memorial Institute of Dental Sciences, Mangalore. Each participant contributed four caries-free permanent first molars, summing to 152 teeth evaluated in a split-mouth design. Before enrollment, written informed consent was obtained from parents/guardians, and assent was obtained from the children.

Inclusion criteria were as follows: healthy children within the 6–10 years age range, moderate to high risk for caries development based on the Caries Risk Assessment Form,¹⁹ and the presence of early non-cavitated lesions in permanent first molars.²⁰ Children with special healthcare needs displaying cooperative behavior were also included.²⁰

Exclusion criteria included uncooperative behavior, increased salivary flow, presence of molar incisor hypoplasia (MIH), dental fluorosis, or suspected carious lesions, participation in fluoride or mouthwash programs, presence of bruxism or parafunctional habits, a vegetarian diet or seafood allergy, and those undergoing orthodontic treatment at the time of the study.

2.2. Operator training and calibration

Prior to the study, all clinical operators and outcome assessors underwent standardized training and calibration sessions to ensure uniformity in clinical procedures and data recording. A total of 50 extracted human permanent molars were used for the pre-study calibration process. Two experienced paediatric dentists independently evaluated the sealant application, retention, and ICDAS-II scoring criteria. Inter and intra examiner reliability was assessed using Cohen's kappa statistics. Calibration showed good examiner agreement: inter-examiner kappa = 0.91; intra-examiner kappa = 0.85–0.88 based on duplicate exams of 10 % of teeth. Disagreements were resolved by consensus with a senior investigator.

2.3. Pilot study and feasibility assessment

A pilot study was conducted on 20 premolars extracted for orthodontic purposes to assess the feasibility of the modified chitosan-based sealant. The sealant was evaluated for polymerization stability, retention after thermocycling in artificial saliva (500 cycles, 4 °C, 37 °C, and 57 °C, with each temperature exposure lasting 30 s performed using a Prima-96 Himedia thermocycler, type LA949), and clinical handling properties.²¹ The pilot study confirmed 100 % retention in all cases and demonstrated no observable changes in the material's consistency or bonding ability. Based on these results, the main study proceeded without modifications.

2.4. Preparation and storage of chitosan-modified pit and fissure sealant

The chitosan-modified sealant was prepared in a dark room under red light to avoid premature polymerization. According to the Clinpro™ sealant (Clinpro, 3M ESPE, USA) datasheet, one 1.2 ml syringe provides approximately 70 applications. Considering potential dropouts and transfer losses, three syringes (total 3.6 ml, ∼3600 mg) were used. After dispensing into a pre-tared 25 ml sterile beaker, the total recovered weight was 3458 mg (slight wastage during disposal). Chitosan powder (2 wt/wt%, ∼69 mg; Product No. 448869, Sigma Aldrich, India; ≥75 % deacetylation, MW 50,000–190,000 Da, viscosity 20–300 cP in 1 % acetic acid) was added and mixed manually for 15 min, following study by Rajabnia et al.¹⁶ The modified sealant was transferred to insulin syringes (40 units), wrapped in aluminium foil, and placed in a black light-resistant container. Ambient conditions during both mixing and storage were continuously monitored, with an average temperature of 27 ± 1 °C and relative humidity of 70 % (range 66–75 %). The formulation retained its stability for six weeks, without evidence of premature polymerization or viscosity changes.

2.5. Sampling, randomisation, blinding, and trial design

This study followed a split-mouth, double-blind, parallel-group design with a 1:1 allocation ratio. Consecutive sampling was used to enrol eligible participants reporting to the outpatient department of A B Shetty Memorial Institute of Dental Sciences, Mangalore, between April 2024 and May 2024. All children meeting the inclusion criteria during the recruitment period were screened clinically, and eligibility was confirmed using predefined criteria. Written informed consent was obtained from parents/guardians.

The randomisation sequence was generated by an independent statistician using a computer-based random-number generator. Sequentially Numbered Opaque Sealed Envelopes (SNOSE) were prepared and safeguarded by a third party not involved in the study. Allocation concealment was ensured by opening the envelope only at the time of intervention. The side of the mouth (right/left) was randomised to receive either Clinpro (control) or chitosan-modified Clinpro (study group).

Blinding was maintained by masking the outcome assessor and the statistician from group allocation. The primary operator, who opened the envelope and applied the material, was not involved in outcome evaluation. Participants were also blinded to their allocation.

2.6. Clinical procedure and sealant application

At baseline, all participants underwent a comprehensive dental examination and case history recording. Sealant application was performed under optimal isolation using a rubber dam or cotton rolls with high-volume suction.²² The occlusal surface was first cleaned with a scaler tip and pumice slurry before applying 37 % phosphoric acid for 15–60 s. Following thorough water rinsing and air-drying, the sealant was applied using a 27-gauge modified insulin syringe tip to ensure precise placement. The material was light-cured (Woodpecker Mini S LED, calibrated at 1000 mW/cm², 420–480 nm using Woodpecker LED Light intensity meter, LM-1) for 20 s, maintaining the tip as close as possible to the sealant without contact.²³ The curing unit was calibrated at baseline and re-verified before every clinical session to ensure consistent output. Application varied slightly: cotton roll isolation in 88 %, rubber dam isolation in 12 %; etch duration was same.

Following polymerization, occlusal adjustment was performed using thin articulating paper, and excess material was removed using rotary instruments. Clinical photographs were taken for baseline documentation.

2.7. Outcome assessment and follow-up

Follow-up evaluations were conducted at three months (T1) and six months (T2) by a blinded outcome assessor using a mouth mirror and explorer. Sealant retention was assessed using the Modified Criteria¹⁷ for Sealant Retention: Occlusal surface (divided in three parts mesial-central-distal) retention was coded as: completely retained (all three sections = score 1), partially retained (two sections = score 1, third section = score 2/3), and completely lost (at least one section = score 4/5/6).

• •Score 1: Pits and fissures are completely covered with material.
• •Score 2: Pits and fissures are partly visible with a sharp fracture edge, creating a plaque retention site.
• •Score 3: Pits and fissures are partly visible with a crumbled fracture edge, not creating a plaque retention site.
• •Score 4: Pits and fissures are visible; if this score is assigned, further observation is conducted using compressed air.
• •Score 5: Pits and fissures are covered with remnants.
• •Score 6: Pits and fissures are partly covered with remnants.
• •Score 7: Other treatment performed.
• •Score 9: Unable to diagnose.

Caries preventive effects were assessed using the ICDAS-II scoring system. ICDAS-II scoring was applied as follows.

• •Score 0: Sound tooth surface.
• •Score 1: First visual change in enamel.
• •Score 2: Distinct visual change in enamel.
• •Score 3: Localized enamel breakdown without visible dentinal involvement.
• •Score 4: Underlying dark shadow from dentin.
• •Score 5: Distinct cavity with visible dentin.
• •Score 6: Extensive distinct cavity with visible dentin.

In cases where the sealant was partially or completely lost, the occlusal surface was re-evaluated using the ICDAS-II criteria. The presence of ICDAS codes 4, 5, and 6 indicated the development of a dentine caries lesion, necessitating appropriate restorative interventions.

2.8. Sample size calculation, dropouts and statistical analysis

The required sample size was determined using n-Master software (version 2), assuming a 75 % retention rate for conventional sealants (Kumaran P et al.),²⁴ with 15 % precision and 95 % confidence. This gave 32 participants (128 teeth). Allowing 20 % for dropouts, 38 children were enrolled (152 teeth). After exclusions (n = 3) and losses to follow-up (n = 3), 32 children (128 teeth) completed the trial. Analyses were conducted in Stata 18 using chi-square tests for categorical data, Friedman test with Durbin-Conover post-hoc for repeated ICDAS-II scores, and Wilcoxon signed rank test for between-group comparisons. Significance was set at p < 0.05.

CONSORT Flow Diagram

3. Results

3.1. Demographic characteristics of the study population

The study included children aged 7–10 years, with the highest proportion being 9-year-olds (43.75 %), followed by 8-year-olds and 10-year-olds (21.88 % each), while 7-year-olds had the lowest representation (12.5 %). A chi-square test (ꭓ² = 6.75, p = 0.0803) indicated no statistically significant difference in the distribution of age groups. Regarding gender distribution, 56.25 % of the participants were female and 43.76 % were male. The chi-square test revealed a statistically significant difference between genders (ꭓ² = 14.31, p = 0.0008). In terms of geographic location, 59.38 % of the children resided in semi-urban areas, while 40.63 % were from rural areas. This distribution was not statistically significant (ꭓ² = 1.125, p = 0.2888). For caries risk assessment, 59.38 % of the children fell into the moderate-risk category, whereas 40.63 % were classified as high-risk. However, the chi-square test (ꭓ² = 1.125, p = 0.2888) indicated no statistically significant difference in caries risk distribution (Table 1).

Table 1.

Distribution of age, gender, geographic location, and caries risk.

Categories	N (No. Of Children)	% (Percentage)	Chi-square Test (ꭓ2)	p-value
Age			6.75	0.0803
7	4	12.50
8	7	21.88
9	14	43.75
10	7	21.88
Gender			14.3125	0.0008∗
Female	18	56.25
Male	14	43.76
Geographic Location			1.125	0.2888
Rural	13	40.63
Semi-urban	19	59.38
Caries Risk			1.125	0.2888
High	13	40.63
Moderate	19	59.38

∗ Statistically significant p-value (p < 0.05).

At baseline (T0), both the control and study groups exhibited 100 % complete sealant retention, with no cases of partial retention or complete loss, confirming a uniform starting point for comparison. At 3 months (T1), a statistically significant difference in sealant retention emerged between the groups (ꭓ² = 6.1168, p = 0.0134). The study group showed higher complete retention (95.31 %) compared to the control group (81.25 %). Partial retention was more frequent in the control group (18.75 %) than in the study group (4.69 %), and no complete losses were reported in either group. By 6 months (T2), this trend persisted with another significant difference (ꭓ² = 8.0688, p = 0.0177). Complete retention dropped further in the control group (76.56 %) but remained relatively high in the study group (92.19 %). Partial retention was again higher in the control group (18.75 %) than in the study group (3.13 %). Both groups experienced the same proportion of complete sealant loss (4.69 %). These findings indicate superior long-term retention in the study group, suggesting the modified sealant is more durable (Table 2).

Table 2.

Comparison of Sealant Retention in Control group and Study group Across Three Time Intervals.

Categories	Control Group	Study Group	Chi-square value (ꭓ2)	p-value (T1)	p-value (T2)
T0 (Baseline)			–	–	–
Complete retention	64 (100 %)	64 (100 %)	–	–	–
Partial retention	0	0	–	–	–
Complete loss	0	0	–	–	–
T1 (3 months)			6.1168	0.0134∗	–
Complete retention	52 (81.25 %)	61 (95.31 %)
Partial retention	12 (18.75 %)	3 (4.69 %)
Complete loss	0	0
T2 (6 months)			8.0688	–	0.0177∗
Complete retention	49 (76.56 %)	59 (92.19 %)
Partial retention	12 (18.75 %)	2 (3.13 %)
Complete loss	3 (4.69 %)	3 (4.69 %)

∗ Statistically significant p-value (p < 0.05).

The Friedman test showed significant changes in caries scores over time in both groups. In the control group, ICDAS-II scores worsened from T0 to T2 (p = 0.0035), with a broader range (up to score 3) seen at T2. This indicates increased caries development over six months. In contrast, the study group also showed statistically significant but milder changes across time (p = 0.0498), with less progression in scores. Median scores remained at zero across all intervals, and the maximum score only reached 2 at T2. This suggests that the study group sealant helped maintain better caries prevention over time (Table 3).

Table 3.

Descriptive statistics of ICDAS-II scores.

Group	Time Point	Median	Min-Max	Shapiro-Wilk (p)	Friedman Test (ᵮf2, p)
Control Group	T0	0	0–0	–	11.29, 0.0035
	T1	0	0–1	<0.001
	T2	0	0–3	<0.001
Study Group	T0	0	0–0	–	6.0, 0.0498
	T1	0	0–0	–
	T2	0	0–2	<0.001

∗ Statistically significant p-value (p < 0.05).

At T0, both groups had 100 % of participants with a caries score of 0, confirming healthy baseline conditions. At T1, the control group showed early signs of demineralization with 10.94 % of children scoring 1. By T2, the deterioration progressed in the control group, with only 84.38 % retaining score 0 and the emergence of higher scores (score 2 in 6.25 % and score 3 in 1.56 %). Conversely, the study group maintained a higher percentage of caries-free teeth at both T1 (100 %) and T2 (95.31 %). Minimal progression to score 1 (3.13 %) and score 2 (1.56 %) was observed, and no children reached score 3. These trends demonstrate a better protective effect of the modified sealant in the study group (Table 4).

Table 4.

Distribution of ICDAS-II scores across time intervals.

Group	Time Point	ICDAS-II Score 0	ICDAS-II Score 1	ICDAS-II Score 2	ICDAS-II Score 3	ᵮf2	p-value
Control Group	T0	64 (100 %)	0	0	0	–	–
	T1	57 (89.06 %)	7 (10.94 %)	0	0	39.06	0.001
	T2	54 (84.38 %)	5 (7.81 %)	4 (6.25 %)	1 (1.56 %)	120.88	<0.001
Study Group II	T0	64 (100 %)	0	0	0	–	–
	T1	64 (100 %)	0	0	0	–	–
	T2	61 (95.31 %)	2 (3.13 %)	1 (1.56 %)	0	110.66	<0.001

∗ Statistically significant p-value (p < 0.05).

Within-group comparisons reveal significant caries progression in the control group over time (T0 vs. T1: p = 0.0188; T0 vs. T2: p = 0.0009), whereas the study group showed stability at 3 months (p = 1.0000) and only mild but significant change by 6 months (p = 0.0330). The control group did not show further significant worsening between T1 and T2 (p = 0.3095), indicating most damage occurred early, while the study group exhibited a delayed and milder progression (p = 0.0330). Between-group comparisons revealed significant differences at T1 (p = 0.011), with better outcomes in the study group. By T2, although the study group still performed better, the difference was not statistically significant (p = 0.053), suggesting some convergence but still clinically relevant trends favoring the study group (Table 5).

Table 5.

Comparison of ICDAS-II scores across different groups using statistical tests.

Comparison	Control Group p-value	Study Group p-value	Control Group vs. Study Group Effect size (p-value)
T0 vs. T1	0.0188∗	1.0000	–
T0 vs. T2	0.0009∗	0.0330∗	–
T1 vs. T2	0.3095	0.0330∗	–
Control Group T0 vs. Study Group T0	–	–	–
Control Group T1 vs. Study Group T1	–	–	1.0 (0.011)∗
Control Group T2 vs. Study Group T2	–	–	1.604 (0.053)

∗ Statistically significant p-value (p < 0.05).

ˆWithin-group comparisons (T0, T1, T2) using Durbin-Conover Test. Between-group comparisons (Control vs Study) using Wilcoxon signed rank test.

4. Discussion

Pit and fissure sealants have long been recognized as an effective preventive strategy against dental caries, particularly in pediatric patients, where the deep occlusal anatomy of posterior teeth predisposes them to plaque accumulation and subsequent decay.²⁵ Resin-based sealants, such as Clinpro™, have set the standard in this domain due to their ability to provide a physical barrier against cariogenic bacteria and their capability to release fluoride, which contributes to remineralization. Nevertheless, conventional resin-based sealants present certain limitations, such as polymerization shrinkage, microleakage, and a progressive decline in fluoride release over time.^26,27 These shortcomings have prompted ongoing research into modifying resin-based sealants with bioactive agents to enhance their mechanical properties, retention, antimicrobial efficacy, and sustained fluoride release. Chitosan, a natural polysaccharide, has emerged as a promising bioactive additive due to its biocompatibility, antimicrobial efficacy, and capacity to enhance the physicochemical properties of dental materials.^28,29 The present study was designed to evaluate the impact of incorporating 2 % chitosan microparticles into resin-based sealants, particularly focusing on retention, fluoride release, and caries-preventive efficacy over a 6-month period.

At the 3-month follow-up, the study group exhibited a high retention rate, with 95.31 % of sealed teeth demonstrating complete retention and only 4.69 % showing partial loss. These favorable retention outcomes were maintained at the 6-month follow-up, with minimal variation, suggesting that the incorporation of chitosan enhances the adhesive strength and durability of the resin-based sealant. In comparison, the control group treated with Clinpro™ showed a significantly lower retention rate at 6 months, with only 76.56 % of teeth exhibiting complete retention. This marked difference indicates that chitosan contributes to improved bonding integrity between the sealant and enamel surface.

The enhanced retention of chitosan-modified sealants can be attributed to several factors. Chitosan's inherent positive charge facilitates electrostatic interactions with the negatively charged enamel surface, thereby improving adhesive strength and reducing the risk of microleakage.³⁰ Previous studies have demonstrated that chitosan incorporation enhances the mechanical properties of composite resins, particularly in terms of wear resistance and flexural strength, which may contribute to the superior retention observed in this study.³¹ The findings align with prior research conducted by Krishnan C et al. (2022) and Hamilton et al. (2015), who reported that chitosan-based materials exhibited improved bond strength and resistance to degradation over time.^32,33 These results suggest that modifying resin-based sealants with chitosan may offer a viable solution for increasing their longevity in the oral environment.

Fluoride release is another crucial factor in the caries-preventive efficacy of dental sealants, as it aids in remineralizing early enamel lesions and inhibiting bacterial metabolism. While conventional resin-based sealants are formulated to release fluoride, the release is often limited to an initial burst phase, followed by a decline over time.^34,35 Although fluoride release was not measured in this study, it can be hypothesized that chitosan's ability to modulate ion exchange interactions may contribute to a more sustained fluoride release profile. Studies have suggested that chitosan is capable of binding fluoride ions and regulating their release in a controlled manner, thereby prolonging their therapeutic benefits. Keegan GM et al. (2016) demonstrated that chitosan-based formulations exhibited a more gradual fluoride release pattern, supporting the plausibility of this mechanism in our findings.¹⁵

Beyond retention and fluoride release, the caries-preventive efficacy of the sealants was also assessed using the ICDAS-II scoring system. At the 3-month follow-up, 89.06 % of the teeth in the chitosan-modified sealant group remained caries-free (ICDAS-II score 0), compared to 84.38 % in the control group. The difference became more pronounced at the 6-month follow-up, with only 1.56 % of teeth in the chitosan group developing caries (ICDAS-II score 3), whereas 6.25 % of teeth in the Clinpro™ group exhibited carious lesions. These findings strongly suggest that chitosan-modified sealants offer superior caries prevention, likely due to their combined benefits of higher retention, prolonged fluoride release, and inherent antimicrobial activity.

Chitosan disrupts bacterial cell walls and inhibits biofilm formation, thereby reducing bacterial adhesion on sealed surfaces.^1,36 Previous research has also indicated that chitosan-infused dental materials exhibit significantly lower bacterial colonization compared to conventional counterparts, further reinforcing the potential of chitosan as an antimicrobial agent in preventive dentistry.^16,33,37 The antibacterial activity of chitosan depends on its molecular weight (MW) and degree of deacetylation (DA). Lower MW chitosan shows stronger antibacterial activity due to better ionic interactions and binding with bacterial membranes.³⁸ The chitosan used in this study had a DA ≥ 75 %, similar to the chitosan in Mahapoka et al.'s study wherein it demonstrated superior antibacterial activity.³⁶

This study reinforces the WHO's (2022) call for cost-effective, minimally invasive strategies to combat dental caries by highlighting the potential of chitosan-modified sealants—effective in underserved communities (Griffin et al., 2016)³⁹ —to improve retention, reduce retreatment, and support early childhood caries (ECC) prevention in high-risk groups through their antimicrobial and fluoride-modulating properties (Li and Tanner, 2015)⁴⁰ while also promoting sustainable care via reduced chair time and costs, thereby advocating for the inclusion of chitosan-based materials in pediatric dental guidelines to advance equitable, prevention-focused public health care.

While the study offers promising results, limitations must be acknowledged. The 6-month follow-up captures only short-term performance, necessitating longer-term studies to assess durability and sustained efficacy. The relatively small sample size may limit generalizability, and variation in chitosan formulation—such as particle size and concentration—could affect outcomes. Future research should focus on optimizing these parameters, evaluating cost-effectiveness in broader populations, and directly assessing fluoride release and antimicrobial activity. These investigations will be critical to refining the clinical utility of chitosan-modified sealants and strengthening the case for their integration into mainstream pediatric dental care. Additionally, in a split-mouth design, each participant acts as their own control, reducing inter-individual variability. However, this approach is vulnerable to cross-over effects that may compromise internal validity. Behavioral cross-over—such as children's inability to distinguish sides during brushing—can lead to uniform plaque removal and fluoride exposure, masking differences between treatments like conventional and chitosan-modified sealants. Systemic factors like diet, saliva composition, and fluoride from toothpaste affect the entire oral cavity, further diminishing the distinction between interventions.

In conclusion, the results of this study highlight the significant advantages of incorporating 2 % chitosan microparticles into resin-based sealants. The findings suggest that chitosan-modified sealants exhibit superior retention and enhanced caries-preventive efficacy compared to conventional resin-based sealants. These attributes make chitosan a promising bioactive additive for improving the longevity and effectiveness of pit and fissure sealants in pediatric dentistry. With further research and clinical validation, chitosan-modified sealants may emerge as a viable alternative to traditional sealants, offering improved protection against dental caries while maintaining biocompatibility and ease of application. As the field of biomaterials advances, integrating bioactive components like chitosan into conventional dental materials may pave the way for more effective, long-lasting, and minimally invasive caries-prevention strategies.

Patient's/guardian's consent statement

Prior to enrollment in the study, written informed consent was obtained from the parents or guardians of all participating children. Additionally, assent was obtained from the children themselves, ensuring that they understood the nature of the study and agreed to participate. This process ensured that all participants and their guardians were fully informed about the study's procedures, potential risks, and benefits, and voluntarily agreed to take part in the research.

Ethical clearance statement

Ethical clearance for this study was obtained from the Institutional Ethical Committee of A.B. Shetty Memorial Institute of Dental Sciences, Nitte (Deemed to be University) (Approval No: ETHICS/ABSMIDS/336/2023). The trial was prospectively registered in the Clinical Trials Registry – India (CTRI/2023/06/054321). The study adhered to the CONSORT guidelines (2022) for randomized clinical trials and was conducted in accordance with the Declaration of Helsinki (2013), ensuring the ethical principles and guidelines for conducting human clinical research. Written informed consent was obtained from parents/guardians, and assent was obtained from the children before enrollment.

Funding

This study was financially supported by the NU Start up Research Grants (NUFR1) by NITTE, sanctioned under order number N/RG/NUFR1/ABSMIDS/2023/02.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.