Dental plaque build-up on the cervical area adjacent to gingival margins is a trigger factor for secondary caries around restored root caries lesions. Dimethylaminohexadecyl methacrylate (DMAHDM) and amorphous calcium phosphate nanoparticles (NACP) impart anti-caries effect by reducing the bacterial growth and releasing high concentrations of calcium and phosphate ions, respectively. The present study explored the optimization and formulation of dental composite with increased concentration of DMAHDM combined with NACP and its effect on mechanical behavior and antibacterial response.
DMAHDM was incorporated into dental composite formulation at 3% and 5% with 20% NACP fillers. Mechanical properties were assessed by flexural strength and elastic modulus. The cationic charge density of the samples was determined using fluorescein staining assay. A human saliva-derived microcosm biofilm model was used to assess antibacterial response via colony-forming units, metabolic activities, lactic acid production, and live/dead assay. Surface roughness was measured after 48 h-biofilm formation.
The viability of human saliva microcosm biofilms was DMAHDM concentration-dependent, where all the microbiological assays were substantially reduced in the presence of 5%DMAHDM. The increased DMAHDM concentration mirrors an increased surface charge density of composites by 8–12 folds and reduced the growth of cariogenic species by 2–5 log ( p ≤ 0.05). Metabolic activity and lactic acid were reduced by 70–90% and 48–99%, respectively. Increasing DMAHDM concentration up to 5% and its association with NACP fillers did not adversely affect the mechanical properties.
A highly potent antibiofilm bioactive composite for root caries restorations having DMAHDM-NACP could be flexibly tailored during formulation without detrimental outcome for mechanical function. The enhanced antibacterial performance of the novel bioactive composite has great potential to suppress the dental plaque build-up that triggers secondary caries around the restored root caries lesions.
With the population aging, the prevalence of root caries is rapidly growing, and without specific prevention and treatment approaches, it can lead to tooth loss . Currently, around 20% of senior individuals have untreated carious lesions . In aged individuals, decreased manual dexterity, physical or mental impairment are detrimental factors for satisfactory tooth brushing. These alterations, combined with dry mouth and low saliva flow, increases susceptibility to one of the most prevalent biofilm-driven oral diseases: root caries . In the root caries, the biofilm formation over the root surface is the most significant driver of the process . Cervical and root surfaces with exposed dentin or cementum are more susceptible to acid attack as the mineral content is less compared to enamel .
Current commercially available composite materials for root caries treatment do not address the specific challenges related to the development of root caries lesions. In contrast, composites accumulate more dental plaque (biofilm) compared to other types of dental restorations . The use of fluoride-releasing restorative materials has been suggested to prevent demineralization and secondary caries. However, the burst release of fluoride ions occurs only in the first 72 h, compromising the long-term impact of this material in the remineralization . With all these challenges, the class V-type restorations located in the roots with no long-term biointeractivity are at higher risk to develop secondary caries .
Many attempts were extensively conducted in the last two decades to impart biointeractivity into resin-based materials to introduce anti-caries functionalities . The two main approaches are (1) the presence of antibacterial functionalities in their polymer phase or filler particles, and (2) the calcium phosphate sources for restoring lost tooth mineral via the sustained release of calcium (Ca) and phosphate (PO 4 ) ions . Previous efforts to reach antibacterial response on dental materials have included the use of agents such as chlorhexidine, chitosan, triclosan, and different antibiotics. The resulting works have shown a deprived outcome due to their release rates from composites . Other studies also reported the ability to incorporate silver to induce antibacterial action with clinically stable mechanical properties .
The use of remineralizing micro-sized fillers has been attempted to enhance the buffering capacity and remineralize the tooth structure. Some limitations were reported related to the inability to neutralize the acid and restore the biofilm plaque pH . Therefore, nano-sized particles of amorphous calcium phosphate (NACP) (average size: 114 nm) have been suggested as improved remineralizing fillers. Due to the small size and high surface area of NACP fillers, reports have shown promising outcome toward to remineralization , which makes NACP as a potential filler for future biointeractive resin composites.
Following the antibacterial functionalities approach, resurgent interest has been focused on the use of an antibacterial monomer into the resin-based formulation as contact killing strategies . The antibacterial monomers that copolymerized with resin composite complex upon the photo-polymerization have shown promising results for long-lasting effects . As a result, 12-methacryloyloxydodecylpyridinium bromide (MDPB) was synthesized and incorporated into resin composites to enhance the antibacterial efficiency against caries-related pathogens . Bis(2-methacryloyloxyethyl) dimethylammonium bromide (QADM) also demonstrated the ability to reduce the growth and activities of S. mutans biofilm in situ . From a series of fine-tuning cationic bromide-containing antibacterial monomers with different alkyl chain lengths, Dimethylaminohexadecyl methacrylate (DMAHDM) has presented a potential effect on the reduction and modulation of cariogenic species . Although the exact mechanism of action of immobilized quaternary ammonium dimethacrylate (QAM) is still under debate, it has been proposed that the surface charge of the quaternary amine prompts unfavorable ion exchange with the bacterial membrane causing bacterial cells disturbance and lysis . Increasing the mass fraction of QAM was suggested to increase the antibacterial function of such material . Therefore, increasing the fraction of DMAHDM from 0.75 to 3% was found to increase the antibacterial function, which indicates a dose-dependent effect of this monomer . Previous reports demonstrated that the combination of DMAHDM and NACP fillers resulted in a 3-log reduction of caries related pathogens and great inhibition of metabolic activities and lactic acid production using saliva-derived biofilm .
Resin-based formulations with high concentrations of antibacterial monomer pose a great challenge for the clinical applications due to their impact on mechanical properties, in special when a combination with another approach is intended. Acid production and low pH environment are essential factors to establish the demineralization process . Limiting these factors by incorporating neutralizing fillers such as nano-sized amorphous calcium phosphate (NACP) particles may decrease the risk of secondary caries .
In view of the intended use for root caries restorations, the developing bioactive composite must present maximum anti-biofilm performance to confront high plaque build-up directly. Under this premise, it is highly demanded DMAHDM-NACP composite to acquire competence in resisting a wide range of mechanical forces, particularly under deflection forces pertinent to the cervical area of the tooth. Therefore, the aim of this study is to investigate the effect of DMAHDM concentration-dependence combined with NACP on mechanical behavior and the antibacterial response of biointeractive composite intent to root caries restorations. It was hypothesized that: (1) the new experimental bioactive composite would greatly reduce the biofilm viability, metabolic activity, and lactic acid production in a dental plaque microcosm biofilm model based on the present DMAHDM concentration; (2) incorporation of high concentration of DMAHDM would not decrease the mechanical properties; (3) increase DMAHDM concentration into the NACP composite will increase the surface charge density.
Material and methods
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.
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.
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:
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”);
32% BT + 3% DMAHDM + 65% glass particles (referred to as “3% DMAHDM”);
30% BT + 5% DMAHDM + 65% glass particles (referred to as “5% DMAHDM”);
32% BT + 3% DMAHDM + 20% NACP + 45% glass particles (referred to as “3% DMAHDM + 20% NACP”);
30% BT + 5% DMAHDM + 20% NACP (referred to as “5% DMAHDM + 20% NACP”).
Mechanical and physical properties
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.
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.
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.
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 .
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.
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.
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 .
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 .
Tryptic soy blood agar culture plates to evaluate the biofilm growth of the total microorganisms.
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.
MSA agar culture plates with 0.2 units of bacitracin per mL were used to evaluate the biofilm growth Mutans streptococci .
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 .
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).
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).