2.1

Synthesis of nCaF ₂

The nCaF ₂ was synthesized via spray-drying as described previously [ , ]. Briefly, a two-liquid nozzle (Visco Mist, Lechler, St. Charles, IL) was employed to allow two solutions to be mixed at the time of atomization. A calcium solution (calcium hydroxide, Ca(OH) ₂ at 2 mmol/L) and a fluoride solution (ammonium fluoride, NH ₄ F at 4 mmol/L) were prepared using reagent grade chemicals and simultaneously fed to the nozzle. The feed rate was 10 mL/min and the solution was atomized into a heated air stream of approximately 70 ℃ in a glass column (VM770-48, VM Glass, Vineland, NJ). The reaction of Ca(OH) ₂ and NH ₄ F led to the formation of CaF ₂ and NH ₄ OH: Ca(OH) ₂ + NH ₄ F → CaF ₂ + NH ₃ ↑ + H ₂ O↑. The nCaF ₂ suspended in the flow were trapped in an electrostatic precipitator (Mist Buster, Air Quality Engineering, Minneapolis, MN). The NH ₄ OH was removed as NH ₃ and H ₂ O vapors with the air flow. The collected nanoparticles were verified to be CaF ₂ in a previous study, with particles sizes ranging from 15 nm to 335 nm and a median particle size of 56 nm [ ].

2.2

Synthesis of DMAHDM

DMAHDM was synthesized using a Menschutkin reaction where a tertiary amine group was react with an organohalide. The reaction products were generated at virtually quantitative amounts and required minimal purification [ ]. Briefly, 10 mmol of 2-(dimethylamino) ethyl methacrylate (DMAEMA, Sigma-Aldrich, MO, USA) and 10 mmol of 1-bromohexadecane (BHD, TCI America, Portland, OR, USA) were combined with 3 g of ethanol in a 20 mL scintillation vial. The vial was stirred at 70 ℃ for 24 h for the reaction to occur. The solvent was then evaporated, yielding DMAHDM as a clear, colorless, and viscous liquid [ ].

2.3

Preparation of pit and fissure sealant containing nCaF ₂ and DMAHDM

BisGMA and triethylene glycol dimethacrylate (TEGDMA) (Esstech, Essington, PA) were mixed at a mass ratio = 1:1, and rendered light-curable with 0.2% camphorquinone (CQ) and 0.8% ethyl 4- N,N -dimethylaminobenzoate (4E) (all mass %) [ ]. For mechanical reinforcement, the resin was filled with barium boroaluminosilicate glass particles which were silanized with 4% 3-methacryloxypropyltrimethoxysilane and 2% n -propylamine (Caulk/Dentsply, Milford, DE) [ ]. The total mass fraction of nCaF ₂ plus glass fillers was 50% to yield a flowable paste; the specific filler levels for nCaF ₂ and glass were listed in the next section. DMAHDM was incorporated into the BisGMA-TEGDMA resin (referred to as BT) at DMAHDM/(BT + DMAHDM) mass fractions of 10%. Since the resin mass fraction in the final sealant was 50%, the DMAHDM mass fraction in the sealant was 5%. This DMAHDM mass fraction was selected following previous studies [ , , ].

2.4

Flow test

The following seven sealants were tested for the paste flow property.

• (1)

  Helioseal F (Commercial sealant control);

• (2)

  50% BT + 0% DMAHDM + 0% nCaF ₂ + 50% glass (Experimental sealant control);

• (3)

  45% BT + 5% DMAHDM + 0% nCaF ₂ + 50% glass (referred to as 5%DMAHDM+0%nCaF ₂ );

• (4)

  45% BT + 5% DMAHDM + 10% nCaF ₂ + 40% glass (referred to as 5%DMAHDM+10%nCaF ₂ );

• (5)

  45% BT + 5% DMAHDM + 15% nCaF ₂ + 35% glass (referred to as 5%DMAHDM+15%nCaF ₂ );

• (6)

  45% BT + 5% DMAHDM + 20% nCaF ₂ + 30% glass (referred to as 5%DMAHDM+20%nCaF ₂ );

• (7)

  45% BT + 5% DMAHDM + 25% nCaF ₂ + 25% glass (referred to as 5%DMAHDM+25%nCaF ₂ ).

A commercial pit and fissure sealant with fluoride release (Helioseal F, Ivoclar Vivadent, Liechtenstein) served as a control (referred to as “Commercial sealant control”). The fillers in Helioseal F consisted of fluorosilicate glass and silicon dioxide, with a filler lever of 40.5%. Helioseal F is photo-cured and indicated to seal pits, fissures, and foramina caeca.

The paste flow was measured following the International Organization for Standardization (ISO) Specification 6876/2012 and ANSI/ADA2000 [ ]. A volume of 0.05 mL of the sealant paste was placed on a glass plate of 40 mm × 40 mm. A second glass plate weighing approximately 20 g was then placed on the top of the sealant paste, followed by a weight of 100 g to make a total mass of 120 g. Ten minutes after the commencement of mixing the paste, the weight was removed and the maximum and minimum diameters of the compressed sealant paste disk were measured using a digital caliper, and the average diameter was calculated. The flow test was used to help choosing the optimal mass fractions of nCaF ₂ and glass fillers.

2.5

Enamel shear bond strength testing

Twenty-five healthy, caries-free, extracted human third molars were collected with donor consent, under a protocol approved by the Institutional Review Board (IRB) of the University of Maryland. All teeth were cleaned to remove adherent tissue and debris, and the crowns were sectioned into four surfaces (mesial, distal, oral, buccal) with a diamond disk (REF 5000484U0, Brasseler, USA). This process resulted in 100 tooth surfaces, and 70 tooth surfaces with good quality were randomly assigned to seven groups (n = 10/group). All tooth surfaces were embedded in self-curing methyl methacrylate resin (REF 1845, Lang, IL, USA). Each enamel surface was polished with 600-grit SiC paper under running water for 10 s to create a standardized smear layer. All enamel surfaces were rinsed with a water spray and dried with water and oil-free air, then were etched with 37% phosphoric acid gel (Total Etch, Ivoclar Vivadent, Schaan, Liechtenstein) for 30 s [ ]. The tooth surfaces were rinsed with a water spray and dried with pressurized air for 5 s until a chalky-white enamel surface was visible. The teeth were then inserted into the sample jig (ISO 29022, Ultradent Products, South Jordan, UT, USA), and the cylindrical plastic mold (Button Mold Insert, ISO 29022, Ultradent Products, Inc., South Jordan, UT, USA) was lowered to achieve a gap-free fit on the etched enamel surface. The sealant material was applied in two steps. Each layer was light cured for 20 s with a light-curing unit, then the plastic mold was carefully removed. The procedure rendered a sealant cylinder with a 2.37 mm diameter perpendicular to the enamel surface, as required by ISO 29022 (2013). The specimens were stored in distilled water at 37 ℃ for 24 h and then tested for shear bond strength using a Universial Testing Machine (MTS, Eden Prairie, MN, USA). The notched-edge fixture with the shear blade was placed over the composite cylinder on the aligned specimen. After precise positioning of the shear blade over the cylinder, the load was applied at a constant crosshead speed of 1 mm/min until the bond failed. The enamel shear bond strength = 4 P/(πd ² ), where P is the load at failure, and d is the diameter of the bonded area of the pit and fissure sealant on the tooth surface [ ].

2.6

Measurement of hardness

The sealant with 25% nCaF ₂ was not included in hardness test due to a decrease in enamel shear bond strength. Three pit and fissure sealants were selected for hardness measurement to determine the influence of nCaF ₂ and DMAHDM on hardness: Helioseal F control, 0%DMAHDM+20%nCaF ₂ , and 5%DMAHDM+20%nCaF ₂ . This was because the 20% nCaF ₂ yielded a good enamel bond strength while having a high level of F ion release.

Disks were made using molds with a diameter of 10 mm and thickness of 0.8 mm. Sealant was light-cured for 1 min and stored in a NaCl solution (133 mmol/L) buffered with 50 mmol/L HEPES (pH = 7.4; 37 ℃) for 24 h. Vickers hardness was measured. A Vickers diamond indenter was used at a load of 20 g and a dwell time of 10 s.

2.7

Measurement of fluoride ion release

The aforementioned three sealants were tested for fluoride release. A NaCl solution (133 mmol/L) buffered with 50 mmol/L HEPES (pH = 7.4; 37 ℃) was used to immerse the specimens. As in previous studies, three specimens of approximately 2 mm × 2 mm × 12 mm were immersed in 50 mL of solution to yield a specimen volume/solution of 2.9 mm ³ /mL [ , ]. The concentrations of fluoride ion released from the specimens were measured at 1, 2, 4, 7, 14, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, and 98 days. At each time point, aliquots of 0.5 mL were removed and replaced by fresh solution. The amount of fluoride release was measured with a combination of a fluoride ion selective electrode and a reference electrode (Orion, Cambridge, MA). The collected solutions were combined with equal volume of total ionic strength adjustment buffer (TASAB) solution (Fisher, Fair lawn, NJ). Fluoride standard solutions ranging from 1 × 10 ⁻⁶ to 1 × 10 ⁻³ mol/L were measured to form a calibration curve, which was used to determine the fluoride ion concentration.

2.8

S. mutans biofilm formation

The following five groups were tested for biofilm experiments.

• (1)

  Helioseal F (Commercial sealant control);

• (2)

  50% BT + 0% DMAHDM + 0% nCaF ₂ + 50% glass (Experimental sealant control);

• (3)

  50% BT + 0% DMAHDM + 20% nCaF ₂ + 30% glass (The purpose of including this group was to test the effect of F ion release on biofilm growth);

• (4)

  45% BT + 5% DMAHDM + 0% nCaF ₂ + 50% glass (The purpose of including this group was to test the effect of DMAHDM on biofilm growth);

• (5)

  45% BT + 5% DMAHDM + 20% nCaF ₂ + 30% glass (The purpose of including this group was to test the effect of DMAHDM + F ion release on biofilm growth).

The mold with a diameter of 10 mm and thickness of 1 mm was used to fabricate disks. The sealant was light-cured for 1 min between transparent Mylar strips. The cured disks were immersed in 200 mL of distilled water and magnetically-stirred with a bar at a speed of 100 rpm for 1 h to remove any uncured monomers. The disks were then sterilized with ethylene oxide (AnproleneAN 74i, Andersen, Haw River, NC) and de-gassed for 3 days, following the manufacturer’s instructions [ ].

The use of S. mutans (ATCC 700610, UA159) (American Type Culture, Manassas, VA) was approved by the University of Maryland IRB. S. mutans was chosen because it is a cariogenic, aero-tolerant anaerobic bacterium and it is the primary causative agent of dental caries. Brain heart infusion broth (BHI) (Becton, Sparks, MD) supplemented with 1% sucrose (pH = 7.4) was used as growth medium. 10 μL of stock bacteria was added into 10 mL of BHI broth (pH = 7.3) and incubated at 37 ℃ with 5% CO ₂ for 16 h. Then, the S. mutans culture was diluted by 10-fold in the growth medium to form the inoculation medium for subsequent experiments. Each pit and fissure sealant disk was placed in a well of a 24-well plate, and 1.5 mL of the inoculation medium was added to each well. The samples were incubated at 5% CO ₂ and 37 ℃ for 24 h. Then each disk with biofilm was transferred to a new 24-well plate containing 1.5 mL of fresh growth medium, and cultured for another 24 h. This totaled 48 h of incubation, which was shown to form relatively mature biofilms on resins, as shown in previous studies [ , ].

2.9

Live/dead assay of S. mutans biofilms on sealant resins

Pit and fissure sealant specimens with 2-day biofilms were washed with phosphate buffered saline (PBS). Then the biofilms on disks were stained using a live/dead kit (Molecular probes, Eugene, OR). Live bacteria were stained with Syto 9 to produce green fluorescence, and bacteria with compromised membranes were stained with propidium iodide to produce red fluorescence. The stained disks were imaged using an inverted fluorescence microscope (Eclipse TE2000-S, Nikon, Melville, NY) [ ]. Three disks were evaluated for each group. Five images were collected at random locations on each disk, yielding 15 images per group.

2.10

MTT metabolic activity of S. mutans biofilms on sealant resins

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole to formazan [ ]. Pit and fissure sealant discs with 2-day biofilms were transferred to a new 24-well plate. 1 mL of 0.5 mg/mL MTT was added to each well and incubated at 37 ℃ in 5% CO ₂ for 1 h. After 1 h, the biofilm specimens were transferred to a new 24-well plate. An aliquot of 1 mL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals. The discs were incubated for 20 min in the dark at room temperature. After 20 min, 200 μL of the DMSO solution in each well was transferred to a new 96-well plate, and the absorbance at 540 nm was measured via a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA). A higher absorbance is related to a higher formazan concentration, which indicates a higher metabolic activity in the biofilm.

2.11

Lactic acid production of S. mutans biofilms on sealant resins

Pit and fissure sealant disc with 2-day biofilms were rinsed in cysteine peptone water (CPW) to remove loose bacteria. Then, the discs were transferred to a new 24-well plate with 1.5 mL of buffered peptone water (BPW) supplemented with 0.2% sucrose in each well. The sealant discs were incubated at 37 °C in 5% CO ₂ for 3 h to allow the biofilms to produce acid. After 3 h, the BPW solutions were stored for lactate analysis. Lactate concentrations were determined using an enzymatic (lactate dehydrogenase) method [ ]. The microplate reader was used to measure the absorbance at 340 nm. Standard curves were prepared using a lactic acid standard (Supelco Analytical, Bellefonte, PA).

2.12

Colony-forming units (CFU) of S. mutans biofilms on sealant resins

Biofilms were cultured for 2-days as described above. The biofilms on the disks were harvested in PBS by scraping and sonication (3510R-MTH, Branson, Danbury, CT) for 3 min and vortexing (Fisher, Pittsburgh, PA) for 20 s. The bacterial suspensions were serially diluted and spread onto BHI ager plates for CFU analysis (n = 6). After 48 h incubation at 37 ℃ in 5% CO ₂ , the colony number was counted and CFU counts were determined [ ].

2.13

The pH of S. mutans biofilm culture medium

Each sterilized sealant disk was placed in a well of a 24-well plate with 1.5 mL of S. mutans inoculation medium. The samples were incubated at 5% CO ₂ and 37 °C for 24 h to form biofilms on the sealant disk. After 24 h, the disk with biofilm was transferred to a new 24-well plate containing 1.5 mL of fresh growth medium and incubated at 5% CO ₂ and 37 ℃ for another 24 h. This totaled two days of culture, which could form relatively mature biofilms on resins. The pH of the first 24 h of culture was not measured, because the non-adherent planktonic bacteria in the medium could contribute to the pH changes, while the purpose of the experiment was to determine the pH caused by the biofilms. Therefore, the pH of the biofilm medium was measured at 48 h of culture using a pH meter (Accumet Excel XL25, Fisher, Pittsburgh, PA). In this way, the measured pH was solely related to the relatively mature 2-day biofilms on the resins [ ].

2.14

Statistical analyses

Statistical analysis was conducted with one-way analysis of variance to detect the significant effects of the variables. Post-hoc pair-wise comparisons were performed using the Tukey’s statistic. Parametric statistical methods were employed with the confirmation that the normality and equal variance assumptions of the data sets were not violated. Statistical analyses were performed by SPSS 18.0 software (SPSS, Chicago, IL) at p < 0.05.