2.1

Fabrication of composites

DMAHDM was synthesized using a modified Menschutkin reaction method, as previously described [ ]. Briefly, 10 mmol of 2-(dimethylamino) ethyl methacrylate (DMAEMA, Sigma-Aldrich, St. Louis, MO, USA), 10 mmol of 1-bromododecane (BDD, TCI America, Portland, OR, USA), and 3 g of ethanol were added into a vial, which was then capped and stirred at 70 °C to react for 24 h. After the reaction was complete, the ethanol solvent was removed by evaporation, yielding DMAHDM as a colorless and viscous liquid. FTIR spectra (Nicolet 6700, Thermo Scientific, Waltham, MA) of the starting materials and the viscous products were collected between two KBr windows in the 4000 cm ⁻¹ to 400 cm ⁻¹ region with 128 scans at 4 cm ⁻¹ resolution. Water and CO ₂ bands were removed from all spectra by subtraction. The results showed that the Menschutkin reaction was successful. The structure of DMAHDM was confirmed via ¹ HNMR (GSX 270, JEOL USA Inc., Peabody, MA) by assigning peaks to the alkyl group [ ].

NACP were synthesized via a spray-drying technique as described previously [ ]. Briefly, calcium carbonate and dicalcium phosphate anhydrous were dissolved into an acetic acid solution to achieve final Ca and P ionic concentrations of 8 mmol/L and 5.333 mmol/L. This solution was sprayed into a heated chamber, and the dried particles were collected via an electrostatic precipitator (AirQuality, Minneapolis, MN). Analysis with multipoint-BET (Brunauer, Emmet, and Teller) method (AUTOSORB-1, Quantachrome, Boynton Beach, FL) indicated that the NACP particles produced by this method had a mean particle size of 116 nm. X-ray diffractometry (XRD) pattern showed that the NACP particles were amorphous [ ].

Bisphenol glycidyl dimethacrylate (BisGMA, Esstech, Essington, PA, USA) and TEGDMA (Esstech, Essington, PA, USA) were mixed at a mass ratio of 1:1 [ , ]. Then 0.2% camphorquinone and 0.8% ethyl 4-N, N-dimethylaminobenzoate were added to render it light-curable (referred to as the BT resin) [ ]. DMAHDM was incorporated into BT at a DMAHDM/(BT + DMAHDM) mass fraction of 3%. Barium boroaluminosilicate glass particles with a median size of 1.4 μm (Caulk/Dentsply, Milford, DE, USA) were silanized with 4% 3-methacryloxypropyltrimethoxysilane [ , ]. A mass fraction of 65% of glass particles and 35% of BT resin were mixed by SpeedMixer (DAC 150.1 FVZ-K, Hauschild Engineering, Hamm, North Rhine-Westphalia, Germany) at a speed of 2800 rpm for 1 min. The paste was then mixed with a handheld device, and these two steps were repeated until a cohesive paste was obtained. The paste was kept in vacuum (2.7 kPa) overnight to eliminate any air bubbles [ ]. For the fabrication of composite with NACP, 35% glass particles and 35% BT resin were mixed with 30% NACP. This mass fraction was selected following a previous study [ ]. Heliomolar (Ivoclar, Amherst, NY, USA), a fluoride-releasing composite served as a control. It contained 40−200 nm nano-silica and ytterbium-trifluoride at a filler mass fraction of 66.7%. Four composites were tested:

• (1)

  Heliomolar nanocomposite (referred to as Commercial control);

• (2)

  Experimental composite control. 35% BT + 65% glass particles (referred to as 0% DMAHDM + 0% NACP);

• (3)

  Antibacterial composite. 32% BT + 65% glass particles + 3% DMAHDM + 0% NACP (referred to as 3% DMAHDM + 0% NACP);

• (4)

  Antibacterial and remineralizing composite. 32% BT + 35% glass particles + 3% DMAHDM + 30% NACP (referred to as 3% DMAHDM + 30% NACP).

2.2

Mechanical testing

Each composite paste was placed into molds of 2 × 2 × 25 mm for mechanical testing. Then each sample was covered in Mylar strips. Each specimen was placed on the turntable of a light-cure machine (Triad 2000; Dentsply, York, PA, USA) and light-cured for 1 min on each open side of the mold, using 270 watts and a distance of the light bulb to the specimen surface being approximately 6 cm [ , ]. After that, the specimens were stored at 37 °C in water for 24 h. The specimen edges were all polished to remove any edge defects. Specimen surfaces were covered with Mylar strips during curing, and were not further polished after curing.

The polished specimens were fractured in three-point flexure with a 10 mm span at a crosshead-speed of 1 mm/min on a computer-controlled Universal Testing Machine (5500R, MTS, Cary, NC) [ ]. Flexural strength was calculated as: S = 3Pmax/L(2bh ² ), where Pmax is the fracture load, L is span, b is specimen width and h is thickness. Elastic modulus was calculated as: E = (P/d)(L ³ /[4bh ³ ]), where load P divided by displacement d is the slope in the linear elastic region. Six specimens were tested for each group (n = 6).

2.3

Ca and P ion release

A sodium chloride (NaCl) solution (133 mmol/L) was prepared with deionized water and buffered to two different pH values: pH 4 with 50 mmol/L lactic acid, and pH 7 with 50 mmol/L HEPES. Following previous studies, three specimens of 2 × 2 × 12 mm were immersed in 50 mL of solution at each pH, yielding a specimen volume/solution of 2.9 mm ³ /mL, similar to a specimen volume per solution of approximately 3.0 mm ³ /mL in a previous study [ ]. The concentrations of Ca and P were measured at 1, 3, 7, 14, 21, and 28 days. At each time period, an aliquot of 0.5 mL was removed and replaced with a fresh solution. The aliquots were analyzed for Ca and P via a spectrophotometric method (DMS-80 UV-visible, Varian, Palo Alto, CA) using known standards and calibration curves [ ]. The released ions were reported in cumulative concentrations. In addition, the pH of the solution was regularly checked and adjusted to pH 4 using 50 mM lactic acid.

2.4

Bacteria culture and biofilm formation on composites

The use of S. mutans (ATCC 700610, UA159, American Type Culture, Manassas, VA, USA) was approved by University of Maryland Baltimore Institutional Review Board. S. mutans were incubated aerobically at 37 °C with 5% CO ₂ in brain heart infusion (BHI) broth (BD, Franklin Lakes, NJ, USA). The inoculum was adjusted to 10 ⁷ colony-forming unit counts (CFU/mL) based on the OD _600nm versus CFU/mL graph [ ]. For specimen preparation, composite disks were made using molds with a diameter of 9 mm and a thickness of 1 mm, and light-cured (Triad 2000; Dentsply, York, PA, USA) for 1 min on each side. In order to remove edge defects, the specimen edges were polished. Specimen surfaces were covered with Mylar strips during curing, and were not further polished after curing. The composite disks were immersed in 200 mL of deionized water and stirred with a magnetic bar at 100 rpm for 1 h to remove part of the initial burst of any uncured monomers [ ]. Subsequently, the disks were sterilized with ethylene oxide (AnproleneAN 74i, Andersen, HawRiver, NC, USA) and de-gassed for 1 week, following manufacturer’s instructions. The sterilized composite disks were placed into 24-well plates, and then S. mutans with a concentration of 10 ⁷ CFU/mL was inoculated in 1.5 mL of culture medium into each well onto a composite disk. The surface area to the volume of exposed solution ratio was 104 mm ² /mL. After 24 h of culture, the composite disks with adherent biofilms were transferred to new 24-well plates filled with fresh BHI, and incubated for another 24 h [ ].

2.5

Live/dead bacteria assay

For live/dead staining assay, each composite disk with 48 h biofilm was washed three time with cysteine peptone water (CPW) to remove the non-adherent bacteria. Three specimens of each group were stained with BacLight live/dead bacterial viability kit (Molecular Probes, Eugene, OR, USA) [ ]. Live bacteria cells were stained with SYTO 9 emitting green fluorescence, while dead cells were stained with propidium iodide emitting red fluorescence. The biofilms were imaged with an inverted epifluorescence microscope (TE2000-S, Nikon, Melville, NY).

2.6

Biofilm CFU counts

Six disks were made for each group. S. mutans with concentration of 10 ⁷ CFU/mL was inoculated in 1.5 mL of culture medium into each well onto a composite disk and cultured for 2 days as described in section 2.4 to form biofilms. After washing for three times in PBS, the disks with 2-day biofilms were transferred into tubes with 1 mL of cysteine peptone water (CPW). The biofilms were harvested by sonication (3510R-MTH, Branson, Danbury, CT) for 10 min, followed by vortexing at maximum speed for 30 s using a vortex mixer (Fisher, Pittsburgh, PA) [ ]. The suspensions were serially diluted and spread on BHI plates. After 48 h incubation at 37 °C in 5% CO ₂ , the colony number was counted and CFU counts were determined [ ].

2.7

MTT metabolic assay of biofilms

Disks with 48 h biofilms were washed twice with PBS and transferred into new 24-well plate (n = 6). One mL MTT dye (0.5 mg/mL MTT in PBS) was added into each well and incubated for 1 h at 37 °C in 5% CO ₂ [ ]. Then the disks were transferred into new 24-well plate with 1 mL dimethyl sulfoxide (DMSO) in each well and incubated at room temperature for 20 min to dissolve the formazan crystals. DMSO solution as then transferred into 96-well plate and OD _540nm was determined using a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA).

2.8

Lactic acid production by biofilms

After washed twice with PBS, the disk with biofilms were immersed in 1.5 mL buffered peptone water (BPW; Sigma-Aldrich) supplemented with 0.2% sucrose and incubated at 37 °C in 5% CO ₂ for 3 h (n = 6). The lactate concentrations in BPW were determined using a lactate dehydrogenase enzymatic method by measuring OD _340nm and compared to that of the lactic acid standard curves, as previously described [ ].

2.9

Polysaccharide production of biofilms

Water-insoluble polysaccharides of biofilms were measured using a phenol-sulfuric acid method [ ]. Six disks were tested for each group. Biofilms on disks were collected by scraping and sonication/vortexing in 2 mL PBS, followed by centrifugation. The precipitate was then washed twice with PBS and resuspended in 200 μL distilled water. 200 μL 5% phenol solution and 1 mL 95–97 % sulfuric acid were added, then incubation at room temperature for 30 min. 200 μL of the solution was transferred into a 96-well plate and OD _490nm was determined with the microplate reader. Six standard glucose concentrations of 0, 5, 10, 20, 50 and 100 mg/mL were used to plot the standard curve of OD _490nm versus polysaccharide concentrations.

2.10

S. mutans biofilm model for enamel demineralization

The use of freshly extracted bovine teeth was approved by University of Maryland Baltimore Institutional Review Board. 24 bovine incisors were used to prepare 24 enamel slabs with a diameter of 6 mm. Circular cavities with an approximate diameter of 4 mm and a depth of 1.5 mm were prepared. As shown in Fig. 1 A, a circle of 1 mm width of enamel surface area around the cavity was exposed. The rest of the enamel surfaces were covered with two layers of acid-resistant nail varnish. The 24 enamel slabs with cavities were randomly divided into four groups of 6 slabs each and restored with one of the four composites. In order to focus on the effects of the composite without interference from an adhesive, and to isolate and determine the effects of the composite alone, no adhesive was used in the present study [ ]. After 24 h, the specimens were polished with sandpapers using grit of # 600, 1200, 2400 and 4000 consecutively. Then, the enamel slabs were placed into a 24-well plate. S. mutans with a concentration of 10 ⁷ CFU/mL was inoculated in 1.5 mL of BHI into each well. The inoculum was first incubated aerobically at 37 °C with 5% CO ₂ for 8 h, then refreshed and incubated for another 16 h, for a total of 24 h ( Fig. 1 B). This method produced relatively mature S. mutans biofilm on the enamel slabs [ ]. Then, the samples were placed into a new 24-well plate. The biofilm on the slab was wiped off by soft sterilized paper every 24 h. After that, the slab was placed into a new well and inoculated to re-grow the biofilm, repeating the procedures as described above. As schematically shown in Fig. 1 C, the S. mutans biofilm acid attack was thus continued for 7 days.

Schematic of S. mutans biofilm model with restoration in enamel. (A) Cavity of 4 mm in diameter and 1.5 mm in depth was prepared and restored with composites. Varnish was applied to cover all surfaces except the 1 mm wide enamel region around the restoration. (B) Formation of S. mutans biofilm on the sample. (C) Culturing the biofilm to produce acid for 7 days.

2.11

Enamel hardness measurement

Vickers hardness was measured in enamel surface at different distances from the margin of the cavity: 50 μm, 150 μm, 250 μm. Vickers indentation (HMV II; Shimadzu Corporation, Kyoto, Japan) was performed at a load of 50 g [ ]. Five indentations were made at each distance for each enamel sample, with six samples for each group. Healthy enamel hardness was also measured as a control.

2.12

Statistical analysis

Data analyses were performed using one-way and two-way analyses of variance and Tukey’s honestly significant difference (HSD) multiple comparison tests. The statistical software SPSS 22.0 (SPSS Inc., Chicago, IL, USA) was used. P < 0.05 was considered to be significant.