A bio-based superhydrophobic pit and fissure sealant for enhanced stability and anticaries application

Abstract

Dental caries is one of the most prevalent chronic diseases worldwide, with most lesions occurring in occlusal pits and fissures. To prevent occlusal caries, pit and fissure sealants are considered one of the most effective measures. However, their anticaries effect are often compromised by secondary caries resulting from biofilm accumulation. Furthermore, traditional sealants based on bisphenol A glycidyl methacrylate (Bis-GMA) may release bisphenol A (BPA), raising concerns about potential endocrine disruption. In this study, we developed a bio-based superhydrophobic pit and fissure sealant by utilizing isosorbide dimethacrylate (IM) as a bio-based resin matrix, fluorinated methacrylate (FMA) as a low-surface-energy component, and a combination of micro- and nano-sized SiO₂ to construct a micro-nano hierarchical surface architecture, after light curing treatment. The bio-based micro-nano structured superhydrophobic pit and fissure sealant (m-n SUP) retained superhydrophobicity after immersion in artificial saliva for 5 days, thermocycling for 5000 cycles, pH cycling for 5000 cycles, tape peeling for 100 cycles, and sandpaper abrasion for 40 cycles. m-n SUP also exhibited favorable mechanical properties and enamel bonding comparable to those of the commercial sealant, together with favorable marginal sealing performance. Moreover, m-n SUP exhibited good cytocompatibility, and markedly reduced salivary mucin adsorption and provided sustained inhibition of the initial adhesion and biofilm formation for five representative oral cariogenic bacteria over 72 h. In a rat model, m-n SUP effectively reduced the incidence and severity of occlusal carie. Overall, this bio-based superhydrophobic pit and fissure sealant can provide a promising bio-based material strategy for pit and fissure caries prevention.

Graphical abstract

1. Introduction

Dental caries is one of the most prevalent chronic diseases worldwide [1,2]. Numerous studies have shown that most carious lesions occur in the occlusal pits and fissures of molars [3]. Pits and fissures are irregular enamel depressions and grooves formed during tooth development. Their complex anatomy facilitates the accumulation of dental plaque and the formation of cariogenic biofilms, making these sites particularly susceptible to dental caries [4,5]. Currently, pit and fissure sealants have become one of the most effective clinical strategies to prevent occlusal caries [6,7]. Nevertheless, the premature failure of these sealants is frequently observed in clinical practice. A substantial proportion of these failures are attributed to secondary caries caused by biofilm accumulation at the sealant tooth interface [8]. To improve the stability of caries-preventive efficacy of pit and fissure sealants, researchers have attempted to incorporate antimicrobial agents such as fluorides, antibiotics, and inorganic antibacterial materials into resin matrices to inhibit bacterial growth and biofilm formation [[9], [10], [11], [12], [13]]. However, these antibacterial agents are simply dispersed within the matrix, so it is difficult to control their release kinetics. Additionally, the sustained or excessive release of antimicrobial agents may disrupt the ecological balance of the oral microbiota [14,15]. Therefore, there is an urgent need to develop new anticaries strategies that prevent bacterial adhesion and biofilm formation without disrupting oral microbial homeostasis.

Light-curable bisphenol A glycidyl methacrylate (Bis-GMA) composite resins are widely used in pit and fissure sealants because of their suitable flowability, favorable wettability, and excellent mechanical properties. However, BPA release from these materials has been proved [[16], [17], [18], [19], [20]]. BPA is an endocrine disruptor that binds estrogen receptors and exerts estrogen-like activity owing to its structural similarity to estrogen. Long-term BPA exposure is associated with reproductive dysfunction, developmental abnormalities, and other health risks [21,22]. Therefore, developing BPA-free alternatives to replace conventional dental resin monomers is important to reduce potential BPA-related health risks. In recent years, bio-based monomers have emerged as promising alternatives for developing safer and more sustainable dental resin materials. Sun et al. [23] synthesized BPA-free dimethacrylate monomers from bio-based lignin-derived creosol and developed dental resin composites with favorable mechanical properties and cytocompatibility. Zhang et al. [24] synthesized the isosorbide-derived monomers IBMEDS and IBM to develop a Bis-GMA- and TEGDMA-free pit and fissure sealant, which exhibited reduced polymerization shrinkage, improved biocompatibility and enamel bonding as well as marginal adaptation. Bragaglia et al. [25] developed a light-curable bio-based resin based on acrylated epoxidized soybean oil for DLP-printed customized guided bone regeneration meshes with favorable biocompatibility and mechanical performance. Among these bio-based alternatives, isosorbide-derived monomers, which are derived from starch or cellulose and possess a unique rigid bicyclic structure, are promising candidates for the development of bio-based pit and fissure sealants.

Superhydrophobic surfaces with water contact angle (WCA) more than 150° and water sliding angle (WSA) less than 10° have attracted increasing attention in dental applications due to their antifouling and self-cleaning capabilities [[26], [27], [28]]. In recent years, various strategies have been developed to prepare superhydrophobic interfaces in the oral field. Chen et al. [28] developed amphiphilic Janus SiO₂ nanoparticles by introducing fluorinated low-surface-energy components and an enamel-binding peptide, enabling the nanoparticles to adhere to the enamel surface and form a superhydrophobic coating, thereby reducing bacterial adhesion and biofilm formation. Zhao et al. [29] developed a sprayable superhydrophobic dental protectant (ZFP) composed of ZnO, fluorine-modified nano-silica, and PDMS, which can reduce the adhesion of bacteria, proteins, and food residues on tooth surfaces, exhibiting antibacterial, acid-resistant, and self-cleaning properties. Tao et al. [27] constructed microscale structures on denture bases and orthodontic retainers, followed by spraying superhydrophobic nanoparticles. These microstructures served as both mechanical protection and reservoirs for the nanostructures, thereby improving the resistance of the superhydrophobic interfaces to salivary flushing and mechanical brushing. However, the oral cavity presents a complex environment, with continuous salivary flow and frequent mechanical abrasion. Under such conditions, current strategies to prepare superhydrophobic materials are susceptible to wear and gradual physical degradation, which may trigger the loss of superhydrophobicity/hydrophobicity or even a localized hydrophobic-to-hydrophilic transition, ultimately weakening their antibiofouling function [30,31]. Moreover, the ability of previously developed superhydrophobic dental materials to maintain superhydrophobicity under complex oral conditions and sustained mechanical challenges remains insufficiently investigated. In addition, evidence regarding their caries-preventive effects through the inhibition of initial bacterial adhesion and subsequent biofilm formation remains limited.

In this study, a bio-based superhydrophobic pit-and-fissure sealant was developed using isosorbide dimethacrylate (IM) as the resin matrix, fluorinated methacrylate (FMA) as a low-surface-energy component, and micro- and nano-sized SiO₂ to construct a hierarchical surface architecture. The resulting micro-nano-structured bio-based superhydrophobic pit and fissure sealant (m-n SUP) exhibited enhanced superhydrophobic stability and retained its superhydrophobicity after immersion in artificial saliva for 5 days, thermocycling for 5000 cycles, pH cycling for 5000 cycles, tape peeling for 100 cycles, and sandpaper abrasion for 40 cycles. In vitro results showed that the isosorbide-based monomer did not significantly promote MCF-7 cell proliferation, suggesting limited estrogen-like activity. The m-n SUP prepared from this monomer reduced protein adsorption through a stable superhydrophobic interface and provided inhibition of initial bacterial adhesion, colonization, and biofilm formation on the material surface, thereby achieving sustained antibiofouling performance. In a rat model, application of m-n SUP effectively reduced the incidence and severity of carious lesions without causing adverse effects for 60 days. In summary, m-n SUP can be applied onto pits and fissures, and cured in situ through a simple sealing procedure, directly forming a stable superhydrophobic interface with sustained antibiofouling performance. This study provides a promising material design strategy for superhydrophobic antibiofouling approaches for stability and caries prevention in dental field.

2. Materials and methods

2.1. Materials

Nano-sized SiO₂ particles (15 nm; n-SiO₂; ≥99.5%), 2-(perfluorooctyl)ethyl methacrylate (FMA; ≥98%), 3-(trimethoxysilyl)propyl methacrylate (KH570; ≥97%), (±)-camphorquinone (CQ; ≥98%), and ethyl 4-dimethylaminobenzoate (4-EDMAB; ≥98%) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Micro-sized SiO₂ particles (5 μm; m-SiO₂; 99.99%) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Isosorbide dimethacrylate (IM) was obtained from Weijite New Material Co., Ltd. (Taizhou, Jiangsu, China). The commercial adhesive (3M™ Single Bond Universal Adhesive) was purchased from 3M ESPE (USA). The commercial pit and fissure sealant (MEDENTAL Light Cure Pit & Fissure Sealant) was purchased from MEDENTAL International (USA). MCF-7 cells were obtained from Suzhou Haixing Biosciences Co., Ltd. (TCH-C247; Suzhou, China).

2.2. Preparation of bio-based superhydrophobic pit and fissure sealant (m-n SUP)

IM and FMA were mixed at a mass ratio of 5:2 and magnetically stirred for 0.5 h. m-SiO₂ and n-SiO₂ were then incorporated to construct a hierarchical rough structure, with m-SiO₂/n-SiO₂ mass ratios of 0:1, 1:1, 2:1, 3:1, 4:1, and 5:1. An in situ silanization approach was employed to promote interfacial compatibility between SiO₂ and the resin matrix [32]. The silane coupling agent KH570 was added dropwise to the SiO₂-containing resin mixture under magnetic stirring at a SiO₂/KH570 mass ratio of 2:1, followed by continuous stirring for another 0.5 h. Subsequently, 0.3 wt% CQ and 0.7 wt% 4-EDMAB were added and stirred for 1 h. The resulting mixture was poured into molds, light-cured with an LED curing light for 20 s, then cured under oxygen-free conditions for an additional 60 s and polymerized in a light-curing unit for 1 h to prepare disc-shaped specimens with a diameter of 10 mm and a thickness of 2 mm for in vitro material property testing. Because the specimens used for in vitro material property testing had a certain thickness, more extensive light curing was required. Therefore, additional curing time was employed for the in vitro specimens to ensure sufficient and uniform polymerization throughout the specimens. The preparation process and reaction mechanism are shown in Fig. 1A and D.

Fig. 1.

(A) Schematic illustration of the preparation process of m-n SUP. (B) Effect of different m-SiO₂/n-SiO₂ ratios on the WCA. (C) FTIR spectra of the hydrophilic resin and m-n SUP before and after curing. (D) Reaction mechanism of m-n SUP. Data are presented as the mean ± standard deviation (SD; n = 3).

To evaluate the performance of the dental resin composites prepared, three control groups were included:

• (1)Hydrophilic: the hydrophilic resin was prepared without SiO₂ fillers, KH570 and FMA.
• (2)Bio-based nano structured superhydrophobic pit and fissure sealant (nano SUP): the nano SUP contained only 1.5 g of n-SiO₂ as the filler with all other components which are identical to those of the m-n SUP.
• (3)Commercial sealant: a commercial pit and fissure sealant (Medental International Inc., USA).

2.3. MCF-7 cell proliferation assay

MCF-7 cells were used to evaluate estrogenic activity. The cells were seeded in 96-well plates at a density of 0.6 × 10⁴ cells per well. After 6 h of incubation, 10 μL of various concentrations of IM, estradiol, or BPA solution in dimethyl sulfoxide (DMSO) was added to each well. The treated cells were then incubated for 24 h at 37 °C and 5% CO₂. Cell proliferation was quantified using a CCK-8 assay, and live/dead fluorescence images were acquired to visualize cell viability.

2.4. Material characterization and performance testing

2.4.1. Chemical composition analysis and degree of conversion determination

The chemical composition of the samples was analyzed by Fourier transform infrared (FTIR) spectroscopy (Nicolet iS50; Thermo Fisher Scientific, USA). The degree of conversion of the resin was evaluated based on the conversion of carbon-carbon double bonds (C=C), and the degree of conversion (DC) was calculated as follows:

$$DC\left(t\right)=\frac{{\left(A_{C=C}/A_{C=O}\right)}_0‐{\left(A_{C=C}/A_{C=O}\right)}_t}{{\left(A_{C=C}/A_{C=O}\right)}_0}\times 100\%$$ (1)

where A_C=C and A_C=O are the characteristic peak areas of C=C and C=O, respectively; the subscripts 0 and t refer to the spectra obtained before and after curing, respectively.

2.4.2. Surface wettability, morphology, and roughness measurements

Water contact angle (WCA) and water sliding angle (WSA) were measured using a contact angle goniometer (OCA 15 EC, DataPhysics, Germany) at an ambient temperature of 25 ± 2 °C. For WCA measurements, a 5 μL droplet of deionized water was deposited onto the sample surface, and the contact angle was recorded. For WSA measurements, a 5 μL droplet of deionized water was placed on the sample surface, and the sample stage was gradually tilted until the droplet began to slide. The corresponding inclination angle was recorded as the WSA. The surface morphology of the samples was examined using scanning electron microscopy (SEM; Gemini 300, Carl Zeiss, Germany), and the surface roughness was measured using atomic force microscopy (AFM; NanoWizard 4XP, Bruker, Germany).

2.5. Stability tests

2.5.1. pH cycling and thermocycling tests

The samples were subjected to either pH cycling between pH = 3 and pH = 7 using HCl and NaCl solutions, or thermocycling between 5 °C and 55 °C in a pH = 7 NaCl solution. A total of 5000 cycles were performed, with a dwell time of 15 s in each condition, using a thermocycling unit (SD Mechatronik, Germany). Changes in WCA were measured using a contact angle goniometer (OCA 15 EC, DataPhysics, Germany).

2.5.2. Artificial saliva immersion test

The samples were immersed in artificial saliva for different days and changes in WCA were measured using a contact angle goniometer.

2.5.3. Tape-peeling test

The tape-peeling test was performed using 3M™ VHB™ 5925 tape with a thickness of 0.6 mm and a width of 20 mm. A 2 kg weight was applied to provide uniform pressure, and WCA was measured every 20 cycles.

2.5.4. Sandpaper abrasion test

The sandpaper abrasion test was conducted using 1000-grit sandpaper under a 200 g weight. One reciprocating abrasion path of 10 cm was defined as one cycle, and WCA was measured every 10 cycles.

2.6. Evaluation of mechanical properties

2.6.1. Microhardness test

The microhardness of each group was measured using a Vickers microhardness tester (MHV-2000Z, Shimadzu, Japan). A diamond indenter was used under a load of 100.0 g with a dwell time of 15 s, after which the hardness value was calculated.

2.6.2. Friction and wear test

The coefficient of friction (COF) of each group was measured using a friction and wear tester (MFT-5000, Rtec, USA). The test was performed in the artificial saliva using a 6.33-mm-diameter ball as the counterbody under a normal load of 30 N, with a sliding distance of 2 mm and a linear speed of 1 mm/s. Reciprocating sliding was carried out for 30 min, and the COF was recorded.

2.6.3. Diametral tensile strength and compressive strength tests

Cylindrical specimens with a diameter of 6 mm and a height of 3 mm were prepared. Diametral tensile strength (DTS) was measured using a universal testing machine (AGS-X, Shimadzu, Japan) at a crosshead speed of 0.5 mm/min and calculated as follows:

$$DTS=\frac{2F}{\mathrm{\pi }dt}$$ (2)

where (F) is the maximum load (N), (d) is the specimen diameter (mm), and (t) is the specimen height (mm).

For compressive strength (CS) testing, cylindrical specimens with a diameter of 4 mm and a height of 8 mm were prepared. The test was performed using the same universal testing machine at a crosshead speed of 1 mm/min and calculated as follows:

$$CS=\frac{4F}{\mathrm{\pi }{d}^2}$$ (3)

where (F) is the maximum load (N), and (d) is the specimen diameter (mm).

2.6.4. Microtensile bond strength (μTBS) test

The microtensile bond strength (μTBS) test was performed with modifications based on a previously reported method [33]. Bovine teeth were ultrasonically cleaned and air-dried. The cleaned bovine teeth enamel surfaces were etched with 37% phosphoric acid for 30 s, thoroughly rinsed, and air-dried. Subsequently, commercial adhesive was applied to the etched enamel surfaces according to the manufacturer's instructions, air-thinned to form a uniform thin layer, and light-cured. According to the experimental groups, the hydrophilic resin, commercial sealant, nano SUP, and m-n SUP were respectively coated on the treated enamel surfaces and light-cured for 80 s. After bonding, the specimens were stored in the distilled water at 37 °C for 24 h and then sectioned using a standard procedure into resin-bovine enamel sticks with a cross-sectional area of approximately 1 mm². The dimensions of each stick were measured for subsequent calculation of the bond strength.

For the μTBS test, each specimen was fixed to the testing jig using cyanoacrylate adhesive and subjected to tensile loading in a universal testing machine at a crosshead speed of 1 mm/min until failure.

$$\mu TBS=\frac{F}{A}$$ (4)

where (F) is the maximum load at failure (N), and (A) is the actual cross-sectional area of the bonding interface of each stick (mm²).

2.6.5. Microleakage test

Microleakage was evaluated using a modified dye penetration method based on a previously reported protocol [34]. Tooth blocks of approximately 5 × 5 × 5 mm³ were obtained from the bovine tooth crowns. Standardized cubic cavities with a side length of 2 mm were prepared on the bovine enamel surface. The cavity walls and margins were etched with 37% phosphoric acid for 30 s, thoroughly rinsed, and air-dried. Subsequently, commercial adhesive was applied according to the manufacturer's instructions. According to the experimental group, the cavities were filled with hydrophilic resin, commercial sealant, nano SUP, and m-n SUP, and then light-cured with an LED curing light for 80 s.

The restored specimens were subsequently subjected to 5000 thermocycles and then immersed in 2% methylene blue solution at 37 °C for 24 h. After staining, the specimens were thoroughly rinsed with distilled water and sectioned vertically through the center of the restoration using a low-speed precision cutting machine to expose the restoration-tooth interface.

Dye penetration was examined under a stereomicroscope at ×25 magnification and scored as follows: 0 = no dye penetration; 1 = dye penetration not exceeding one-half of the tooth-material interface; 2 = dye penetration exceeding one-half of the tooth-material interface but not reaching the cavity floor; and 3 = dye penetration reaching the cavity floor.

2.7. Cytotoxicity assay

Human oral keratinocytes (HOK) were used to evaluate cytotoxicity. HOK were seeded in 12-well plates at a density of 0.7 × 10⁴ cells per well and then co-cultured with extracts from the different resin materials. After 1, 3, and 5 days of incubation at 37 °C with 5% CO₂, cell proliferation and viability were assessed using CCK-8 assay and Calcein-AM/PI cell staining assay. The cytoskeletal structure was evaluated by FITC-phalloidin/DAPI staining. Cell morphology was observed using a fluorescence microscope and a two-photon laser scanning confocal microscope (LSM 980 NLO, Carl Zeiss, Germany).

2.8. Antibiofouling performance

2.8.1. Inhibition of salivary mucin adsorption

Samples from each group were incubated with 10 mg/mL bovine submaxillary mucin at 37 °C for 12 and 72 h. After incubation, the samples were rinsed with PBS and stained with Alcian blue solution for 30 min. The samples were then rinsed three times with PBS and transferred to 1 mL of PBS, followed by ultrasonication for 10 min. The supernatant was collected, and the optical density (OD) was measured at 595 nm using a microplate reader (Infinite 200Pro Nano, Tecan, Switzerland).

2.8.2. Inhibition of bacterial adhesion and biofilm formation

Streptococcus mutans (S. mutans, ATCC 35668), Streptococcus sanguinis (S. sanguinis, ATCC 10556), Streptococcus gordonii (S. gordonii, CGMCC 1.2496), Actinomyces viscosus (A. viscosus, CGMCC 2.2411), and Lactobacillus casei (L. casei, BNCC 364409) were selected to evaluate the anti-adhesion properties of the materials. Sterilized samples were placed in well plates with prepared microbial suspensions (OD₆₀₀ = 0.5) and incubated at 37 °C for 24 and 72 h. After incubation, non-adherent bacteria were removed by rinsing with sterile PBS, and the retained microorganisms were subjected to MTT analysis. For fluorescence observation, the samples were stained with SYTO 9 green-fluorescent nucleic acid stain (Thermo Fisher Scientific, USA) for 15 min. CFU counting was performed by transferring each sample to 1 mL PBS, ultrasonication for 15 min, and plating after a 10⁴-fold dilution. For SEM observation, the samples were fixed overnight at 4 °C with 2.5% glutaraldehyde (Solarbio, China), dried using a critical point dryer (K850, Quorum, UK), sputter-coated with gold (108Auto, Cressington, UK), and imaged by SEM.

The multispecies biofilm model consisted of the five microorganisms described above. The microbial suspensions were mixed to prepare an inoculum containing each species at 10⁷ CFU/mL in brain heart infusion (BHI) medium supplemented with 1% sucrose. Sterile samples were placed in culture plates containing 1 mL of the microbial suspension and incubated at 37 °C for 3 days. The biofilms were then fluorescently stained followed by the description above and observed using a two-photon laser scanning confocal microscope (LSM 980 NLO, Carl Zeiss, Germany). Z-stack images were analyzed using ZEN 3.13 software (Carl Zeiss, Germany). The fluorescence images were quantitatively analyzed using ImageJ software, and the integrated optical density (IOD) was calculated.

2.9. In vivo evaluation

All animal experimental procedures were reviewed and approved by the Ethics Committee of Anhui Medical University (Approval No. LLSC20261936) and were performed in strict compliance with relevant national and international guidelines to minimize animal suffering.

Twenty-four 6-week-old male Sprague-Dawley (SD) rats were obtained from the Laboratory Animal Center of Anhui Medical University. To deplete the endogenous oral microbiota, the rats were supplied with drinking water containing 0.05% (w/v) streptomycin for 3 consecutive days and then randomly assigned to four groups (n = 6):

• (1)Control: the tooth surfaces were cleaned without further treatment.
• (2)Sealant: after cleaning, etching, rinsing, and drying, a commercial adhesive was applied and light-cured for 10 s. The commercial sealant was then applied to the tooth surface and light-cured for 80 s.
• (3)nano SUP: after cleaning, etching, rinsing, and drying procedures, a commercial adhesive was applied and light-cured for 10 s. The nano SUP was then applied to the tooth surface and light-cured for 80 s.
• (4)m-n SUP: after cleaning, etching, rinsing, and drying procedures, a commercial adhesive was applied and light-cured for 10 s. The m-n SUP was then applied to the tooth surface and light-cured for 80 s.

After the treatments, all rats were fed a cariogenic Keyes 2000 diet (Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd., China) and inoculated orally once daily with 100 μL of the mixed bacterial suspension (10⁷ CFU/mL each strain) for 60 days.

2.10. Statistical analysis

All data were analyzed using GraphPad Prism software and are presented as the mean ± standard deviation. For multiple comparisons, one-way analysis of variance (one-way ANOVA) followed by Tukey's honestly significant difference (Tukey HSD) post hoc test was performed. For experiments involving multiple time points, each time point was analyzed independently to compare differences among material groups at the same time point. In the figures, **** denotes p < 0.0001, *** denotes p < 0.001, ** denotes p < 0.01, * denotes p < 0.05, and ns denotes no significant difference.

3. Results and discussion

3.1. Estrogenic activity assay

The estrogenic activity of the materials was evaluated based on estrogen-induced proliferation of MCF-7 cells [35]. The estrogenic activity assay results are shown in Fig. S1. Within the concentration range of 10⁻¹²-10⁻⁶ M, IM exhibited the lowest proliferative effect on MCF-7 cells. Within the tested concentration range, 10⁻⁸ M estradiol and 10⁻⁹ M BPA induced the highest proliferation rates of MCF-7 cells, reaching 167.02% and 137.19%, respectively. In contrast, the maximum proliferation rate induced by IM was only 105.41%, indicating that IM did not exhibit an obvious proliferative effect on MCF-7 cells. Subsequently, the morphology of MCF-7 cells was evaluated by Calcein-AM/PI staining. As shown in Fig. S2, the number of cells in the IM was comparable to that in the DMSO group, with intact morphology, uniform distribution, and no obvious cell proliferation. In contrast, the estradiol and BPA groups exhibited a higher number of cells and a greater cell density, consistent with the CCK-8 results.

This result may be related to the molecular structure of IM. Unlike BPA and estradiol, IM lacks the phenolic hydroxyl groups and key structural features required for binding to estrogen receptors. As a result, estrogen receptor-mediated signaling may be less readily activated, explaining the limited estrogen-like proliferative response observed in the MCF-7 assay under the tested conditions [36,37]. However, it should be noted that the MCF-7 cell proliferation assay provides only a preliminary assessment of the estrogen-like proliferative activity of IM and the findings mentioned above indicate that IM has no significant estrogen-like proliferative activity under the experimental conditions in this study.

3.2. Preparation of the m-n SUP

In this study, m-n SUP was prepared using the bio-based monomer IM, with m-SiO₂, n-SiO₂, and FMA incorporated to construct a micro-nano-structured superhydrophobic resin. As shown in Table 1 and Fig. 1B, the WCA of the resin first increased and then decreased as the proportion of m-SiO₂ increased. The highest WCA (156.70 ± 1.41°) was obtained when the mass ratio of m-SiO₂ to n-SiO₂ was 3:1. The WSA (6.24 ± 0.51°) and surface energy followed a similar trend, indicating that the 3:1 formulation provided the most favorable surface wettability.

Table 1.

WCA, WSA, surface energy and f₁ of different groups.

Samples			WCA (°)	WSA (°)	Surface energyb (mJ/m2)	f1c
m-SiO2 (g)	n-SiO2 (g)	FMA (g)
0	0	0	81.65 ± 1.22	> 90a	44.95 ± 5.16	-
0	0	1	112.46 ± 2.61	> 90a	16.58 ± 3.10	-
0	1	1	136.47 ± 1.72	> 90a	17.05 ± 3.19	44.50%
1	1	1	143.59 ± 2.33	> 90a	3.20 ± 0.69	31.59%
2	1	1	150.63 ± 1.37	13.13 ± 2.04	1.43 ± 0.26	20.80%
3	1	1	156.70 ± 1.41	6.24 ± 0.51	0.62 ± 0.16	13.20%
4	1	1	152.30 ± 1.25	9.92 ± 1.46	1.81 ± 1.09	18.55%
5	1	1	150.83 ± 0.47	11.35 ± 1.73	2.55 ± 1.29	20.52%

^a> 90 represents the 5 μL water droplet cannot slide off from the test surface even if the surface is inclined to 90°.

^bSurface energy was calculated from the WCA of water and diiodomethane used as probe liquids.

^cf₁ represents the apparent solid-liquid contact fraction calculated using the Cassie-Baxter equation.

The surface morphology and roughness of the samples were further examined by SEM and AFM. SEM images (Fig. S3) revealed that the gaps within the micro-nano-structured surface gradually narrowed as the m-SiO₂ content increased. However, when the amount of m-SiO₂ exceeded an appropriate range, n-SiO₂ particles became less discernible, thereby disrupting the formation of the desired surface microstructure and reducing hydrophobicity. The AFM results (Fig. S4) were consistent with the SEM images. At a m-SiO₂/n-SiO₂ mass ratio of 3:1, the surface root-mean-square roughness (Rq) reached its maximum value (436.9 ± 68.6 nm).

Collectively, the SEM, AFM, and wettability results suggest that an appropriate ratio of m-SiO₂ to n-SiO₂ allows the two fillers to work together in constructing a multiscale hierarchical rough structure, while FMA provides a low-surface-energy chemical environment. However, excessive m-SiO₂ may disturb the hierarchical organization and spatial distribution of the micro-nano structure, thereby weakening the superhydrophobic performance [31].

As shown in Fig. 1C, compared with the hydrophilic resin, m-n SUP displayed additional absorption bands at 1100 and 1245 cm⁻¹, which were assigned to Si-O-Si and -CF₂ stretching vibrations, respectively. The characteristic -CF₃ band at 1199 cm⁻¹ was not clearly resolved because it overlapped with the broad and intense Si-O-Si absorption band; however, the distinct -CF₂ peak at 1245 cm⁻¹ verified the incorporation of fluorinated groups and SiO₂ into m-n SUP. In addition, after curing, the intensity ratio of the C=C absorption band at 1636 cm⁻¹ to the C=O absorption band at 1720 cm⁻¹ decreased markedly compared with that in the uncured samples. This decrease indicates a reduction in C=C double bonds and confirms that photopolymerization occurred under light irradiation.

The DC of m-n SUP was 80.81%, slightly lower than the hydrophilic resin (85.75%). Furthermore, the DC decreased gradually as the SiO₂ content increased (Table S1). This decrease was probably associated with the elevated inorganic filler loading. Specifically, the dispersion state of the fillers and the filler/matrix interfacial interactions interfere with light transmittance and the free-radical polymerization process, thereby diminishing the photopolymerization efficiency and the final DC of the resin system [38].

Despite this reduction, m-n SUP still achieved a DC exceeding 80%, suggesting adequate photopolymerization. Such a degree of conversion is suitable for a pit and fissure sealant and provides a basis for its subsequent mechanical performance [39].

3.3. Wettability

Surface wettability is a key physicochemical parameter that directly affects protein adsorption and bacterial colonization [40]. As presented in Fig. 2A, B and Table 1, the hydrophilic resin and commercial sealant exhibited WCAs of 81.65 ± 1.22° and 90.06 ± 6.38°, respectively, and water droplets remained stably attached to the material surfaces with a hemispherical shape. By contrast, the WCA of m-n SUP increased to 156.70 ± 1.41°, with droplets forming an almost spherical shape on the resin surface and rolling off readily (Movie S1). The WSA was only 6.24 ± 0.51°. In addition, m-n SUP also had a very low surface energy of 0.62 ± 0.16 mJ/m², consistent with the WCA and WSA results. These findings indicate that m-n SUP possessed excellent superhydrophobicity.

Fig. 2.

Wettability, surface morphology, stability, and mechanical properties of the resin samples. (A) WCA and SEM images of the hydrophilic resin, sealant, nano SUP, and m-n SUP. (B) Dynamic behavior of water droplets on the four resin surfaces. (C) Silver mirror reflection phenomenon observed on m-n SUP after immersion in water. (D) AFM images of the four resin samples. (E) Schematic illustration of the Cassie-Baxter wetting state. (F) WCA of the resin samples before treatment and after pH cycling, thermocycling, artificial saliva immersion for 3 days, and artificial saliva immersion for 5 days. (G) WCA of nano SUP and m-n SUP after tape-peeling cycles. (H) WCA of nano SUP and m-n SUP after sandpaper abrasion cycles. (I-L) Vickers hardness, COF, DTS, and CS of the four resin samples, respectively. Data are presented as the mean ± standard deviation (n = 3) and were analyzed by one-way ANOVA. *p < 0.05, **p < 0.01, ****p < 0.0001; ns, not significant.

When m-n SUP was immersed in water, its surface displayed a silver mirror reflection phenomenon at specific viewing angles (Fig. 2C). This phenomenon was attributed to the stable air layer trapped within the micro-nano-structured superhydrophobic surface, which partially transformed the underwater solid-liquid interface into a solid-air-liquid composite interface. The difference in refractive index between the trapped air and water produced strong light reflection, giving rise to the visible mirror-like appearance. This behavior is consistent with the Cassie-Baxter wetting state (Fig. 2E) [41].

The wetting behavior of m-n SUP can be described by the Cassie-Baxter model, in which a rough heterogeneous surface is composed of solid regions and trapped air pockets. The Cassie-Baxter equation is expressed as follows:

$$\cos {\theta }_{\mathrm{A}}=f_1\cos {\theta }_1+f_2\cos {\theta }_2$$ (5)

where θ_A is the apparent contact angle; f₁ and f₂ are the area fractions of the solid-liquid and air-liquid interfaces, respectively (f₁ + f₂ = 1); and θ₁ and θ₂ are the intrinsic contact angles on the solid and air phases, respectively. Because θ₂ = 180°, the equation can be simplified as:

$$\cos {\theta }_{\mathrm{A}}=f_1\cos {\theta }_1‐\left(1‐f_1\right)$$ (6)

As shown in Table 1, the solid fraction (f₁) of m-n SUP was 13.2%, indicating that 86.8% of the apparent interface consisted of trapped air. This high air fraction, together with the hierarchical rough structure and fluorinated low-surface-energy modification, enabled the formation of a stable superhydrophobic surface.

The SEM results (Fig. 2A) indicated that the surface of m-n SUP contained distributed pores with sizes of approximately 1-5 μm, and these pores were filled with nanoparticles. This multiscale architecture allows air to be trapped at the solid–liquid interface [42], thereby contributing to the superhydrophobicity. On the surface of nano SUP, which was constructed using only n-SiO₂, a large number of n-SiO₂ particles were evenly distributed. By contrast, the hydrophilic resin and commercial sealant had much smoother and flatter surfaces.

AFM analysis further supported these observations. Fig. 2D presents the 3D surface morphology and Rq of the four samples. The hydrophilic resin and commercial sealant displayed relatively flat surfaces, with Rq values of 8.3 ± 0.74 nm and 24.61 ± 0.84 nm, respectively. In contrast, both m-n SUP and nano SUP had pronounced height variations and isolated protrusions, with Rq values far higher than that of the hydrophilic resin. These findings are consistent with the SEM observations.

Overall, these results indicate that the combination of m-SiO₂ and n-SiO₂ particles generated a hierarchical surface with micro-nano-scale roughness. Combined with the fluorinated low-surface-energy modification, this architecture trapped air at the solid-liquid interface, thereby stabilizing the Cassie-Baxter wetting state and accounting for the superhydrophobicity of m-n SUP. This finding is consistent with the study by Huang et al. [31], which showed that constructing hierarchical micro-nano roughness in combination with low-surface-energy modification was an effective strategy for enhancing surface water repellency and achieving superhydrophobicity.

3.4. Stability and mechanical properties

The stability of the materials was evaluated by artificial saliva immersion, thermocycling, pH cycling, tape peeling, and sandpaper abrasion. As shown in Fig. 2F, the initial WCA of m-n SUP and nano SUP were 157.64 ± 0.97° and 160.04 ± 0.90°, respectively. nano SUP exhibited a marked loss of superhydrophobicity under simulated oral and mechanical challenges. After immersion in artificial saliva for 3 days and after 5000 thermocycles or pH cycles, its WCA decreased to 136.93 ± 0.91°, 126.20 ± 0.33°, and 124.48 ± 2.02°, respectively. In the tape-peeling test, the WCA decreased to 139.62 ± 0.42° after 10 cycles and further dropped to 118.04 ± 1.62° after 100 cycles. Similarly, in the sandpaper abrasion test, the WCA decreased to 138.14 ± 3.57° after 10 cycles and further declined to 89.63 ± 1.35° after 50 cycles. In contrast, m-n SUP maintained a WCA above 150° after immersion in artificial saliva for 5 days, 5000 thermocycles, 5000 pH cycles, 100 tape-peeling cycles, and 40 sandpaper abrasion cycles, demonstrating markedly improved superhydrophobic stability. As shown in the SEM images (Fig. S5), after 50 abrasion cycles, the nanostructures on nano SUP were largely destroyed, whereas m-n SUP retained a recognizable hierarchical structure. The microscale particles appeared to help preserve the underlying nanoscale roughness, thereby contributing to the maintenance of the superhydrophobic state.

These results indicate that m-n SUP retained its superhydrophobicity under simulated oral conditions and showed greater resistance to the loss of superhydrophobicity induced by mechanical abrasion than nano SUP.

The mechanical properties of the materials were evaluated by microhardness testing, friction and wear testing, CS testing, and DTS testing. As presented in Fig. 2I–m-n SUP reached a Vickers hardness of 140.07 HV, which was significantly higher than that of nano SUP (114.3 HV, p < 0.01) and much higher than those of the sealant (52.33 HV, p < 0.001) and the hydrophilic resin (67.37 HV, p < 0.0001).

The COF of m-n SUP was 0.22 (Fig. 2J), which was comparable to that of nano SUP (p > 0.05). However, it was significantly lower than that of the sealant (0.25, p < 0.05) and was much higher than that of the hydrophilic resin (0.15, p < 0.001).

CS is an important mechanical property for dental restorative materials, as it reflects the ability of a material to withstand masticatory stress and resist fracture [43]. The CS of m-n SUP reached 130.35 MPa (Fig. 2K), with no significant difference from that of the sealant (140.56 MPa, p > 0.05). It was much higher than those of the hydrophilic resin (65.33 MPa, p < 0.0001) and nano SUP (80.99 MPa, p < 0.001).

DTS, which reflects the toughness of a material, was measured by diametral compression testing [44]. The DTS of m-n SUP was 38.41 MPa (Fig. 2L), with no significant difference from that of the sealant (42.99 MPa, p > 0.05). It was significantly higher than those of the hydrophilic resin (29.52 MPa, p < 0.05) and nano SUP (27.69 MPa, p < 0.001).

To evaluate the bonding performance of the materials to adhesive-treated enamel, μTBS testing was performed. The μTBS of m-n SUP was 58.55 MPa (Fig. S6A), with no significant difference from that of the commercial sealant (62.86 MPa, p > 0.05), indicating that m-n SUP exhibited bonding performance comparable to that of the commercial sealant.

In addition, the marginal sealing performance of the materials was evaluated using a dye penetration method. As shown in Fig. S6B, the distributions of microleakage scores were generally similar among different the groups and were predominantly distributed concentrated in the lower score categories. Corresponding representative stereomicroscopic images were shown in Fig. S6C. Overall, m-n SUP exhibited a favorable marginal sealing performance.

The stability of superhydrophobic surfaces generally depends on both the surface chemical composition and the integrity of the micro/nanoscale roughness [45]. Changes in the low-surface-energy components or mechanical damage to the hierarchical roughness required for superhydrophobicity may increase the solid-liquid contact area and destabilize the Cassie-Baxter wetting state, resulting in the loss of superhydrophobicity. In this study, the superhydrophobic stability and mechanical durability of m-n SUP may arise from the synergistic effects of its low-surface-energy chemistry and multiscale surface architecture. FMA provides a low-surface-energy modification, while the combination of m-SiO₂ and n-SiO₂ generates hierarchical micro/nanoscale roughness that favors air entrapment at the solid-liquid interface to form a trapped air layer, reducing the actual solid-liquid contact area, and facilitating maintenance of the Cassie-Baxter wetting state. Meanwhile, the cured resin matrix forms a crosslinked polymer network with mechanical stability that embeds and anchors the SiO₂ particles, thereby providing structural support and protection for the surface micro/nanostructures [46]. The larger-scale structures formed by m-SiO₂, together with their embedding and encapsulation within the resin matrix, may provide a supporting and protective framework, reducing direct mechanical damage to the nanoscale surface features and thereby facilitating the retention of the interfacial air layer and the superhydrophobic state [31,45]. However, increasing the m-SiO₂ content in the present study was accompanied by changes in the total SiO₂ loading. Therefore, the differences in wettability and durability among the formulations cannot be attributed solely to the micro/nano hierarchical structure and may also be influenced by the total SiO₂ loading, micro/nano particle composition, and the resulting surface architecture. Future studies should maintain a constant total filler content while varying only the micro/nano-SiO₂ ratio to further clarify the independent contribution of the micro/nano hierarchical structure to superhydrophobic stability. Similar findings were reported by Tao et al. [27], who showed that surfaces constructed solely from exposed nanoparticles were readily damaged by mechanical abrasion, whereas microscale supporting or protective structures reduced direct wear of the nanoscale features and improved superhydrophobic stability. This proposed mechanism is consistent with the stability tests in the present study. m-n SUP maintained a WCA >150° after immersion in artificial saliva for 5 days, thermocycling for 5000 cycles (which, based on previous empirical estimates, may approximate 6 months of intraoral thermal aging based on the previous empirical estimates [47]), pH cycling for 5000 cycles, tape peeling for 100 cycles, and sandpaper abrasion for 40 cycles, indicating its resistance to multiple simulated oral environments and mechanical challenges.

Taken the results obtained above, m-n SUP can achieve stable superhydrophobicity with good CS, DTS, enamel bonding performance and marginal sealing performance comparable to those of the commercial sealant.

3.5. Evaluation of cytocompatibility

Good cytocompatibility is a prerequisite for materials intended for use in the human body. In this study, human oral keratinocytes (HOK) were used to evaluate cytotoxicity, and the experimental workflow is illustrated in Fig. 3A.

Fig. 3.

In vitro cytocompatibility evaluation. (A) Schematic illustration of the cytotoxicity assay procedure. (B) Cell viability evaluated using the CCK-8 assay. (C) Live/dead staining images of cells after 1, 3, and 5 days, captured by fluorescence microscopy. Green signals (Calcein-AM) indicate live cells, and red signals (PI) indicate dead cells. (D) Cytoskeletal staining images captured by CLSM. F-actin was labeled with FITC-phalloidin (green), and nuclei were labeled with DAPI (blue). Data are presented as the mean ± standard deviation (n = 3).

CCK-8 assay results showed that cell viability gradually increased with culture time in all groups, and the values in each material group were close to Blank (Fig. 3B). Cell viability was further evaluated by Calcein-AM/PI staining after coculture of HOK with the extracts from each material. As shown in Fig. 3C, Calcein-AM-positive cells predominated in all groups on days 1, 3, and 5, whereas only a few PI-positive cells were observed, with no extensive red fluorescence or obvious cellular debris. F-actin cytoskeleton and nuclei were subsequently stained with FITC-phalloidin and DAPI, respectively. As shown in Fig. 3D, all groups exhibited intact nuclear morphology and well-organized actin cytoskeletons. These observations were consistent with the CCK-8 and live/dead staining results.

These results indicate that HOK maintained normal proliferation after coculture with extracts of the m-n SUP. The m-n SUP exhibited good cytocompatibility.

3.6. Sustained antibiofouling performance of the materials

Salivary mucin adsorption can alter the physicochemical properties of the tooth surface, provide specific attachment receptors for bacteria, and accelerate plaque colonization and caries development [48]. In this study, bovine submaxillary mucin was selected as a model protein to evaluate the antibiofouling performance of the materials. A schematic illustration of the proposed antibiofouling mechanism is presented in Fig. 4C.

Fig. 4.

Inhibition of salivary mucin adsorption on the resin surfaces. (A) Photographs of the surfaces of the four resin samples after Alcian blue staining. (B) Quantitative absorbance of mucin adsorption on each resin. (C) Schematic of the inhibition of salivary mucin and bacterial adhesion on the superhydrophobic surface due to the trapped air layer. Data are presented as the mean ± standard deviation (n = 3) and were analyzed by one-way ANOVA. ****p < 0.0001; ns, not significant.

Alcian blue staining indicated that, within 12 h, only minimal salivary mucin adsorption occurred on the surfaces of m-n SUP and nano SUP (Fig. 4A). Quantitative analysis (Fig. 4B) further revealed that the OD values for mucin adsorption on m-n SUP and nano SUP were only 0.12 and 0.14, respectively, with no significant difference between the two groups (p > 0.05). These values were significantly lower than those of the hydrophilic resin (0.36, p < 0.0001) and the sealant (0.59, p < 0.0001).

After 72 h, however, salivary mucin accumulation on the surface of nano SUP increased markedly, accompanied by a marked increase in Alcian blue staining. In contrast, the OD value for mucin adsorption on m-n SUP was 0.19, which was significantly lower than those of nano SUP (0.65, p < 0.0001), the hydrophilic resin (0.77, p < 0.0001), and the sealant (0.94, p < 0.0001). These results suggest that m-n SUP effectively inhibited salivary mucin adsorption. By comparison, nano SUP exhibited some short-term resistance to mucin adsorption, salivary mucin gradually accumulated on its surface as the immersion time increased, indicating a clear decline in anti-protein-adsorption performance.

To further evaluate the antibiofouling performance of the resin materials, S. mutans, S. sanguinis, S. gordonii, A. viscosus, and L. casei were selected as representative oral bacteria. S. mutans is an acidogenic and aciduric bacterium and has long been regarded as one of the major cariogenic species. S. sanguinis and S. gordonii are important early colonizers in oral tooth-surface biofilms and contribute to plaque microbial homeostasis through alkali-generating metabolism [49,50]. A. viscosus is a Gram-positive bacterium in oral biofilms, with strong tooth-surface adhesion and coaggregation capacities, and participates in early plaque colonization and the assembly of multispecies communities [51]. L. casei is mainly found in saliva, the dorsum of the tongue, and mucosal surfaces. It is also highly enriched in carious lesions and is among the most acid-tolerant bacteria in the oral cavity [52].

The overall metabolic activity of the five bacterial species attached to the surfaces of different samples within 24 and 72 h was assessed using the MTT assay (Fig. 5A–C and Fig. S7A–B). At 24 h, the OD values of the m-n SUP and nano SUP were significantly lower than those of the hydrophilic resin and sealant (p < 0.0001). However, after 72 h of incubation, the OD value of the nano SUP increased markedly and was significantly higher than that of the m-n SUP (p < 0.0001).

Fig. 5.

Inhibition of S. mutans, S. sanguinis and A. viscosus adhesion on the material surfaces. (A-C) MTT assay results for bacterial adhesion on the surfaces of the hydrophilic resin, sealant, nano SUP, and m-n SUP after 24 and 72 h. (E-G) Photographs of agar plates and fluorescence staining images of bacteria attached to the surfaces of different samples. (I-K) SEM images of bacteria attached to the surfaces of different samples. (D) Quantitative analysis of CLSM images of mixed bacteria. (H) CLSM images of mixed bacteria. S. m: S. mutans; S. s: S. sanguinis; A. v: A. viscosus. Data are presented as the mean ± standard deviation (n = 3) and were analyzed by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.

CFU counting was then performed to evaluate bacterial adhesion on the material surfaces. As shown in Fig. 5E–G and Fig. S7C–D, after 24 h, the colony numbers of the five bacterial species on m-n SUP and nano SUP were similar, and both were significantly lower than those on the hydrophilic resin and sealant (Fig. S8A, p < 0.0001). After 72 h of incubation, the colony numbers on nano SUP increased markedly, whereas those on m-n SUP remained at a low level (Fig. S8B). Staining with SYTO 9 was further performed to observe microbial attachment on the surfaces of the different samples after 72 h. Almost no fluorescence signal from the five microorganisms was observed on the surface of m-n SUP. By contrast, a certain amount of green fluorescence was detected on nano SUP, whereas only scattered fluorescence signals were observed on m-n SUP. Continuous and intense green fluorescence was observed on the surfaces of the hydrophilic resin and sealant, suggesting a large number of bacteria adhering to their surfaces.

The SEM images (Fig. 5I–K and Fig. S7E–F) showed that after 72 h of incubation, only a few scattered bacteria were observed on the surface of m-n SUP, with no continuous bacterial aggregates or dense biofilm structure observed. By contrast, more bacteria were visible on the surface of nano SUP. The surfaces of the hydrophilic resin and the sealant were extensively covered with bacteria, with dense bacterial colonization. This result was consistent with previous findings.

The ability of m-n SUP to inhibit multispecies biofilm formation was further evaluated. The CLSM results are shown in Fig. 5D. The surfaces of hydrophilic resin and sealant were covered with strong green fluorescence, indicating extensive biofilm coverage. On the surface of nano SUP, moderate green fluorescence was observed. By contrast, only very few scattered green fluorescence signals were observed on the surface of m-n SUP. Quantitative analysis of the CLSM images (Fig. 5H) showed that m-n SUP had the lowest integrated optical density (53.04), significantly lower than nano SUP (173.05, p < 0.05), the hydrophilic resin (333.27, p < 0.001), and the sealant (270.96, p < 0.001). The biofilm thickness in the m-n SUP group was only 5.39 μm, markedly lower than those in the nano SUP (12.75 μm, p < 0.0001), hydrophilic resin (21.48 μm, p < 0.0001), and sealant (21.45 μm, p < 0.0001). These results indicate that m-n SUP provided sustained inhibition of biofilm formation and exhibited favorable antibiofouling performance.

In summary, m-n SUP significantly reduced salivary mucin adsorption. The MTT assay, CFU counting, fluorescence staining, and SEM observations further showed that m-n SUP reduced the adhesion for 5 tested oral bacterial species on the material surface for 72 h. Further multispecies biofilm analysis by CLSM showed that m-n SUP exhibited the lowest biofilm-associated fluorescence intensity and biofilm thickness. These findings are consistent with those reported by Chen et al. [28], who showed that superhydrophobic surfaces can reduce bacterial adhesion, which can further support that m-n SUP can reduce protein adsorption and bacterial adhesion, thereby inhibiting subsequent biofilm formation. However, the maximum observation period of the in vitro antibiofouling experiments in the present study was 72 h. Therefore, longer-term dynamic multispecies biofilm models are still needed to further evaluate the stability and durability of the antibiofouling performance of m-n SUP.

It is noted that the antibiofouling effect of m-n SUP was primarily associated with its surface anti-adhesion properties rather than direct bacterial killing. In this study, the micro-nano hierarchical structure, together with the low-surface-energy components, promoted the formation of a Cassie-Baxter wetting state, allowing air to be trapped within the surface microstructures. This can reduce the effective solid-liquid contact area and limited protein adsorption and effective contact between bacteria and the material surface. It can be calculated that m-n SUP s owns solid-liquid contact fraction of only 13.2%, indicating that a large proportion of the apparent interface was occupied by trapped air. It should be noted that increased surface roughness alone does not necessarily inhibit bacterial adhesion; under certain conditions, rough surfaces may instead provide additional attachment sites for bacteria.

Moreover, unlike conventional direct bactericidal strategies, m-n SUP primarily reduces initial bacterial adhesion through its physicochemical surface properties rather than relying on the sustained release of antibacterial agents, thereby interfering with subsequent biofilm formation. Therefore, this passive antibiofouling approach mainly acts at the early adhesion stage of biofilm development without requiring the continuous release of bactericidal components, thereby reducing dependence on sustained antibacterial agent release and providing an alternative anticaries strategy for inhibiting early bacterial colonization and subsequent biofilm formation.

3.7. In vivo application of anticaries efficacy and biosafety evaluation

To evaluate the in vivo anticaries performance of m-n SUP, SD rats were used to establish an experimental caries model. After m-n SUP was applied to pits and fissures of the molar in SD rats, a mixed bacterial suspension was introduced into the oral cavity. The experimental protocol is shown in Fig. 6A.

Fig. 6.

In vivo anticaries efficacy and biosafety of m-n SUP. (A) Schematic illustration of the animal experimental procedure. (B) 2D sagittal micro-CT images, pseudocolor images, and 3D reconstructed images of the mandibular molars; red arrows indicate carious lesion sites. (C) Quantitative analysis of enamel volume based on the 3D micro-CT reconstructions. (D) H&E images for major organs and tongue of the rats. Data are presented as the mean ± standard deviation (n = 6 rats per group) and were analyzed by one-way ANOVA. **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.

Images of the occlusal surfaces (Fig. S9) showed dark-brown carious lesions on the molars in the control group, as indicated by the red arrows. In contrast, the teeth in the m-n SUP group retained the most intact appearance, with only a few superficial lesions and a milder degree of caries compared with the other groups. To further assess the severity of carious lesions, samples from each group were scanned by micro-CT and subjected to 3D reconstruction. In the control group, the results (Fig. 6B) showed extensive low-density defect areas in the pit and fissure regions, with lesions extending into the dentin, as indicated by the red arrows. In contrast, the m-n SUP group showed intact dentin with almost no low-density areas. The pseudocolor images showed only mild enamel demineralization, represented by the yellow areas, in the m-n SUP group, whereas the other three groups exhibited extensive low-density defect areas. 3D reconstructed images and quantitative analysis (Fig. 6C) showed that m-n SUP exhibited higher mineral density and a more intact tooth surface structure than the other three groups. These results indicate that, under the 60-day experimental conditions of the present study, m-n SUP sealing treatment reduced enamel demineralization and the severity of occlusal carious lesions in rat molars. However, the animal experiment evaluated the caries-preventive effect of the complete m-n SUP sealing system, which involved the combined effects of acid etching and adhesive pretreatment, the physical barrier provided by the sealing material, and the antibiofouling effect of the superhydrophobic surface. Therefore, the independent contribution of each factor to the in vivo caries-preventive effect should be distinguished reasonably.

The in vivo biosafety of m-n SUP was further evaluated. At the end of the experiment, peripheral blood was collected from SD rats in each group for routine hematological and serum biochemical analysis. The heart, liver, spleen, lungs, kidneys and tongue were harvested for histological evaluation using H&E staining.

Routine hematological and serum biochemical analyses (Figs. S10 and S11) showed that all measured parameters remained within normal physiological ranges. These findings indicated that m-n SUP did not induce detectable systemic inflammation, hematological abnormalities, or liver and kidney dysfunction, supporting its good in vivo biosafety. H&E staining results are presented in Fig. 6D. In the m-n SUP group, the lingual mucosal epithelium remained intact, with no evidence of inflammatory cell infiltration, tissue necrosis, or epithelial disruption. The heart, liver, spleen, lungs, and kidneys also maintained normal histological architecture with regularly arranged cells. No pathological abnormalities, including congestion, edema, inflammatory infiltration, or tissue necrosis, were observed. These findings were comparable to those in the control, nano SUP, and sealant.

Despite the favorable anticaries performance observed within the 60-day experimental period, several limitations should be acknowledged. Although daily inoculation with a mixed-species bacterial suspension was used to establish sustained and high-intensity cariogenic challenges, this artificially induced environment cannot fully recapitulate the naturally formed and dynamically evolving oral microbiota under clinical conditions. Therefore, in vivo studies using animal models that better mimic native oral microbial communities and clinically relevant conditions are still needed.

Collectively, these findings demonstrate that m-n SUP effectively reduced enamel demineralization and suppressed the development of occlusal caries in rat molars. In addition, no detectable systemic toxicity or histopathological damage was observed during the 60-day experimental period, supporting the in vivo biosafety of m-n SUP and its potential application as a pit and fissure sealant for caries prevention.

4. Conclusions

In this study, a bio-based micro/nano-structured superhydrophobic pit and fissure sealant based on isosorbide dimethacrylate (IM) was developed. By incorporating fluorinated low-surface-energy components and hierarchical structures formed by micro- and nano-sized SiO₂, m-n SUP exhibited favorable superhydrophobic properties and maintained superhydrophobic stability after multiple simulated oral challenges, including artificial saliva immersion, thermocycling, pH cycling, and mechanical disturbances. In addition, m-n SUP exhibited favorable mechanical properties, maintained bonding performance to adhesive-treated enamel comparable to that of the commercial sealant, and showed favorable marginal sealing performance and cytocompatibility. In vitro biocompatibility and in vivo evaluations revealed no obvious cytotoxicity or histopathological damage to major organs.

m-n SUP significantly reduced salivary mucin adsorption and the initial adhesion of multiple oral bacterial species and suppressed subsequent multispecies biofilm formation. The in vivo results further showed that m-n SUP reduced enamel demineralization and occlusal caries severity in rat molars.

Overall, this study integrates a bio-based resin system with a superhydrophobic antibiofouling strategy for pit and fissure caries prevention, providing experimental support for the development of novel pit and fissure sealants combining physical sealing with surface-mediated antibiofouling properties. However, material retention, maintenance of superhydrophobicity under long-term oral conditions, and potential effects on the overall oral microbial community require further investigation.

CRediT authorship contribution statement

Xu Chen: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Xiaolei Zhang: Writing – original draft, Methodology, Investigation. Xin Wang: Writing – review & editing, Investigation, Formal analysis. Chen Zhang: Investigation, Data curation. Yanping Lv: Data curation. Ruilu Zhu: Formal analysis. Yuhan Cao: Investigation. Bingqin Li: Formal analysis. Shunli Zheng: Writing – review & editing, Supervision, Methodology, Funding acquisition, Formal analysis, Conceptualization.

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.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.