2.1

Experimental design

DMAHDM concentration was tested at 3 levels (0%, 3%, and 5%) and incorporation of NACP at 2 levels [absence and presence of 20% NACP], as well as a desire to understand whether there is an interaction between these two factors on the dependent variable.

2.2

Synthesizing the DMAHDM monomer and NACP fillers

DMAHDM was synthesized via a modified Menschutkin reaction as described before . Briefly, 10 mmol of 2-(dimethylamino)ethyl methacrylate (DMAEMA, Sigma-Aldrich, St. Louis, MO, USA), 10 mmol of 1-bromohexadecane (BHD, TCI America, Portland, OR, USA), and 3 g of ethanol were mixed in a 20 mL-scintillation vial. Stirring was achieved at 70 °C for 24 h. Then, DMAHDM was extracted after evaporating the solvent .

NACP was synthesized via a spray-drying technique . Briefly, acetic acid was used to dissolve calcium carbonate and dicalcium phosphate anhydrous to construct calcium (Ca) and phosphate (P) with concentrations of 8 mmol/L and 5.333 mmol/L, respectively. The final molar ratio of Ca/P was 1.5, the same as that for ACP [Ca ₃ (PO ₄ ) ₂ ], which then was sprayed into a heated chamber of the spray-drying machine. Dry NACP with a particle size of 116 nm was collected using an electrostatic precipitator.

2.3

Formulation and optimizing of composites

Bisphenol glycidyl dimethacrylate (BisGMA, Esstech, Essington, PA, USA) and TEGDMA (Esstech, Essington, PA, USA) were mixed at a mass ratio of 1:1(referred to as BT resin). Then, 0.2% camphorquinone, and 0.8% ethyl 4-N, N- dimethylaminobenzoate photoinitiators were added. DMAHDM was introduced to achieve the final mass fractions of 3% and 5% in the dental composite. Barium boroaluminosilicate glass particles with a median size of 1.4 μm (Caulk/Dentsply, Milford, DE, USA) were silanized with 4% 3-methacryloxypropyltrimethoxysilane. All the groups had a filler mass fraction of 65%. Then, the following groups of dental composites were formulated:

• (1)

  Experimental composite control: 35% BT (bisphenol glycidyl dimethacrylate and TEGDMA at a mass ratio of 1:1(referred to as BT resin) + 65% glass particles (referred to as “control”);

• (2)

  32% BT + 3% DMAHDM + 65% glass particles (referred to as “3% DMAHDM”);

• (3)

  30% BT + 5% DMAHDM + 65% glass particles (referred to as “5% DMAHDM”);

• (4)

  32% BT + 3% DMAHDM + 20% NACP + 45% glass particles (referred to as “3% DMAHDM + 20% NACP”);

• (5)

  30% BT + 5% DMAHDM + 20% NACP (referred to as “5% DMAHDM + 20% NACP”).

2.4

Mechanical and physical properties

2.4.1

Flexural strength and elastic modulus

A stainless steel mold of 2 mm × 2 mm × 25 mm was used to fabricate the samples’ dimension. Mylar strips were used to assure the standardization of the surface smoothness and then light-cured (Labolight, DUO, GC, Tokyo, Japan) for 1 min at each side with the radiance emittance of 2330 mW/cm ² . Following the International Organization for Standardization (ISO) #4049:2000(E) , the samples were stored for 15 min in water at 37 °C before the molds were detached. Then, the samples were stored again in water for 24 h at 37 °C. Flexural strength and elastic modulus were recorded using a three-point flexural test with a 10 mm span at a crosshead speed of 1 mm/min on a computer-controlled Universal Testing Machine (5500R, MTS, Cary, NC, USA) by the following equations:

P _max = fracture load, L = the span, b = specimen width, and h = specimen thickness.

where P (the load) divided by d (displacement) is the slope in the linear elastic region.

2.4.2

Surface roughness

The surface roughness ( R _a , μm) was recorded from the surface that was subjected to the biofilm challenge, described below, using a surface roughness measurement instrument (Surftest SJ-310; Mitutoyo America, Aurora, IL). At a constant speed of 0.5 mm/s, a force of 4 mN, a 0.25-mm cutoff value, and 1.5-mm tracing length, each sample was traversed by the stylus tip (5 μm) . The average of five readings from each sample was taken to record the final R _a value of each sample.

2.5

Quantification of charge density of the quaternary ammonium groups

The cationic charge density of the composite surfaces was measured using fluorescein staining as described . Composite discs of each group were placed in 24-well plate, immersed in 1 mL of fluorescein disodium salt solution in demineralized water (10 mg/mL), and shaken at 60 rpm for 10 min in the dark at room temperature. Then, the samples were washed with demineralized water for three times. The composite disks were transferred to a new 24-well plate and immersed in 1 mL of a 0.1 wt% cetyltrimethylammonium chloride solution in demineralized water supplemented with 100 μL of 100 mM phosphate buffer, sonicated for 5 min, and then shaken in the dark for 20 min at 60 rpm to desorb complexed fluorescein dye. Composite disks absorbance values were read at 501 nm using a plate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA) to yield the concentration of fluorescein dye. The fluorescein concentration was calculated using Beers Law and an extinction coefficient of 77 mM ⁻¹ cm ⁻¹ by performing the following equation:

in which (Dye) is the extracted fluorescein concentration, Abs ₅₀₁ is the absorbance value at 501 nm, ε ₅₀₁ is the extinction coefficient that is equal to 77 mM ⁻¹ cm ⁻¹ for fluorescein, and L is the length of a polystyrene cuvette (1 cm) traversed by the spectrophotometer light. Then, The cationic charge density of composite samples was calculated by the following equation:

in which V is the volume of the extraction solution, N is Avogadro’s number = 6.023 × 10 ²³ , and A is the surface area of the composite sample.

2.6

Human saliva-based microcosm biofilm model

Composite disks were made using circular molds with a diameter of 8 mm and a thickness of 1 mm. Samples were cured (60 s; dual-mode wavelength 380–510 nm; 1200 mW/cm ² , Labolight, DUO, GC, Tokyo, Japan) and immersed in distilled water and stirred at 100 rpm using a magnetic bar for 1 h to remove uncured monomers . Then, sterilization of the composite samples was achieved with ethylene oxide (AnproleneAN 74i, Andersen, Haw River, NC, USA), and then de-gassed for 7 days to assure the complete release of entrapped ethylene oxide .

The University of Maryland Baltimore Institutional Review Board approved the use of human saliva for this study (HP-00050407). The biofilm model involved the use of human saliva as inoculum as it was described in previous studies . Briefly, ten healthy individuals with no active caries and no history of antibiotics in the last three months donated an equal volume of their saliva. The donors did not brush their teeth for 24 h and avoided food and drink intake 2 h before the collection. The collected saliva mixed to assure the complexity and heterogeneity of the microorganisms. Collected saliva was diluted in sterile glycerol to a concentration of 70%, and stored at −80 °C . The saliva-glycerol solution was added into a McBain artificial saliva growth medium with 1:50 final dilution. The components of the growth medium was prepared as the following: mucin (Type II, porcine, gastric), 2.5 g/L; bacteriological peptone, 2.0 g/L; tryptone, 2.0 g/L; yeast extract, 1.0 g/L; NaCl, 0.35 g/L, KCl, 0.2 g/L; CaCl ₂ , 0.2 g/L; 50 mM pipes, 15 g/L; hemin, 0.001 g/L; vitamin K ₁ , 0.0002 g/L, at pH 7 . 0.2% sucrose was added to this medium. Then, each sample was placed in the well of 24-well plates and immersed with 1.5 mL of the inoculum. The samples were incubated at 37 °C in 5% CO ₂ , and fresh medium was added after 8 and 24 h of the incubation. At a total of 48 h incubation, the samples were transferred to perform the live/dead assay, metabolic activity, lactic acid measurement, or colony-forming units (CFU). All experiments were performed with six to eight repetitions (composite samples) in each of three independent experiments .

2.6.1

Live/dead staining of biofilms

Composite samples subjected to 2-day biofilms were washed with phosphate-buffered saline (PBS) before they were stained with the BacLight live/dead kit (Molecular Probes, Eugene, OR, USA) . Samples were stained with an equal mixture of 2.5 μM SYTO 9 and 2.5 μM propidium iodide. Each sample was stained for 15 min. The green fluorescence indicated that the presence of live bacteria stained with SYTO9. The red fluorescence bacteria by propidium iodide indicated the presence of bacteria with defective and compromised membranes. An inverted epifluorescence microscope (Eclipse TE2000-S, Nikon, Melville, NY, USA) was used to examine the samples.

2.6.2

MTT assay for quantification of metabolic activity of biofilms

A colorimetric assay was performed to evaluate the enzymatic reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), a yellow tetrazole, to formazan . Composite samples subjected to 2-day biofilms were transferred to a new 24-well plate and immersed with 1 mL of tetrazolium dye and incubated at 37 °C in 5% CO ₂ for 1 h. Then, samples were transferred to another plate and immersed with 1 mL of dimethyl sulfoxide (DMSO) for 20 min in the dark to dissolve the formazan crystals. 200 μL of the DMSO solution of each sample was transferred to a 96-well plate, and the absorbance at 540 nm was measured via a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA). A higher absorbance is associated with a increased concentration of formazan, which indicates a higher metabolic activity.

2.6.3

Lactic acid production by biofilms

An enzymatic (lactate dehydrogenase) method was used to determine the lactate concentrations in the BPW solutions. Composite samples subjected to 2-day biofilms were transferred to a new 24-well plate and immersed with 1.5 mL of buffered-peptone water (BPW) plus 0.2% sucrose and incubated for 3 h to allow the biofilms to produce acid. The absorbance of the BPW solution of each well was measured at 340 nm (optical density OD ₃₄₀ ) using the microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA). A lactic acid standard (Sigma-Aldrich, St. Louis, MO, USA) was used to design the standard curves similar to previous studies .

2.6.4

Colony-forming unit (CFU) counts of biofilms on composites

Composite samples subjected to 2-day biofilms were transferred to a vial that had 1 mL CPW, and the biofilms were harvested by sonication and vortex . Four solid culture media were used to evaluate the growth of oral species .

• I.

  Tryptic soy blood agar culture plates to evaluate the biofilm growth of the total microorganisms.

• II.

  Mitis salivarius agar (MSA) culture plates having 15% sucrose to evaluate the biofilm growth of total Streptococci . This MSA agar contains agents such as crystal violet, Potassium tellurite was added to this MSA agar to inhibit most Gram-negative bacilli and most Gram-positive bacteria.

• III.

  MSA agar culture plates with 0.2 units of bacitracin per mL were used to evaluate the biofilm growth Mutans streptococci .

• IV.

  Rogosa agar culture plated to determine the growth of lactobacilli . In this rogosa agar, high levels of sodium acetate and ammonium citrate at a low pH were added to inhibit most oral microorganisms but not Lactobacilli.

The bacterial suspensions were serially diluted and transferred into each agar plate to determine the count of colonies. The agar plates were incubated at 37 °C in 5% CO ₂ for 48 h, except for rogosa plates, which were incubated for 72 h. CFU of each specimen was calculated by counting the colonies considering the dilution factor .

2.7

Statistical analysis

All experiments were performed with six repetitions in each of three independent experiments. The average of the three experiments per sample was considered as a statistical unit. The independent variables in this study were DMAHDM incorporation at three levels (0%, 3%, and 5%) and NACP incorporation at two levels (absence and presence of 20% NACP). Shapiro–Wilk test was performed to confirm the normality and equal variance of data. Two-way analyses of variance (ANOVA) and Bonferroni multiple comparison tests were performed to detect the significant effects of the dependent variables in the mechanical properties and antibacterial effects. A p -value <0.05 was considered statistically significant. All the statistical analyses were performed by SPSS statistics software (IBM version 26, Armonk, NY, USA).

3.1

Flexural strength and elastic modulus

The mechanical properties of the composites are plotted in ( Fig. 1 A) (mean ± standard deviation (SD); n = 6). 3% DMAHDM resin composite revealed the highest flexural strength among the experiment groups, 118.1 ± 6.2 MPa. The value attribitted to 3% DMAHDM was significantly higher than Heliomolar control resin composite and other experimental groups ( p ≤ 0.05), but close to experimental control ( p = 0.99). 5% DMAHDM resin composites revealed a flexural strength of 98.38 ± 6.8, which was higher but not statistically significant compared to Heliomolar control ( p = 0.28). However, the flexural strength of 5% DMAHDM resin composites was significantly higher compared to DMAHDM-NACP resin composites ( p ≤ 0.05). 3% and 5% DMAHDM-NACP demonstrated the lowest flexural strength, but the values were comparable to Heliomolar control ( p ≥ 0.05). Increasing the DMAHDM concentration, adding the NACP, and the interaction between them significantly reduced the flexural strength of the resin composite ( Table 1 ). The mean values of elastic modulus for all the groups ranged between 6 to 7.2 GPa ( Fig. 1 B). Heliomolar resin composites showed the highest elastic modulus score, which was significantly higher than the other groups ( p ≤ 0.05).

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