Effectiveness of the DHMAI monomer in the development of an antibacterial dental composite

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Effectiveness of the DHMAI monomer in the development of an antibacterial dental composite Fatima Zohra Cherchali a,b,∗ , Mohamed Mouzali a , Jean Bernard Tommasino b , Dominique Decoret c , Nina Attik b,c , Hazem Aboulleil b,c , Dominique Seux b,c,d , Brigitte Grosgogeat b,c,d a

Laboratoire d’Etudes Physico-Chimiques des Matériaux, Application à l’Environnement (LEPCMAE), USTHB, Faculté de Chimie, Bab Ezzouar, Algérie b Laboratoire des Multimatériaux et Interfaces, UMR CNRS 5615, Université Lyon, Université Lyon1, Villeurbanne, France c UFR Odontologie, Université Lyon, Université Lyon1, Lyon, France d Service de Consultations et de Traitements Dentaires, Hospices Civils de Lyon, Lyon, France

a r t i c l e

i n f o

a b s t r a c t

Article history:

Objective. Development of antibacterial dental composites is the ultimate goal to decrease

Received 24 February 2017

carious disease occurrence and increase the restoration longevity. For this purpose, the qua-

Received in revised form

ternary ammonium dimethyl-hexadecyl-methacryloxyethyl-ammonium iodide (DHMAI)

28 July 2017

and the methacryloyloxyethylphosphorylcholine (MPC) have been incorporated in exper-

Accepted 11 September 2017

imental methacrylate-based composite resins. This aims to first investigate the effect of

Available online xxx

each alone and then their combined effect.

Keywords:

at different concentrations to experimental dental composite. Flexural strength (FS) and

Methods. Synthesized DHMAI and commercial MPC were added either alone or combined Dental composite

modulus (FM) were tested to select the optimal concentrations. Only selected composites

Quaternary ammonium salt

were evaluated for Vickers hardness (HV) and the degree of conversion (DC) using fourier

Streptococcus mutans

transform infrared spectroscopy analysis (FTIR-ATR). Antibacterial activity was assessed

DHMAI

using tests on colony-forming unit (CFU), scanning electron microscopy (SEM) and Alamar-

MPC

blue assay to measure the metabolic activity. Streptococcus mutans biofilm was chosen to be

Degree of conversion

grown on the composite surfaces during 96 h at 37 ◦ C.

Mechanical properties

Results. Incorporation of 7.5% DHMAI in composite improved the degree of conversion and

Antibacterial activity

gave a strong antibacterial effect with a reduction of (∼98%) in CFU and (∼50%) of metabolic activity with acceptable mechanical properties. Addition of MPC to DHMAI affects mechanical properties of composites without providing a better antibacterial activity. Significance. Composites with DHMAI greatly reduced S. mutans biofilm and improved the degree of conversion without scarifying the composites’ mechanical properties. DHMAI may have wide applicability to other dental materials in order to inhibit caries and improve the longevity of restorations. © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

∗ Corresponding author at: Équipe Biomatériaux et Interfaces (UMR CNRS 5615) UFR d’Odontologie, 11 rue Guillaume Paradin 69372 Lyon Cedex 08, France. Fax: +33 4 78 77 87 12. E-mail addresses: [email protected], [email protected] (F.Z. Cherchali). http://dx.doi.org/10.1016/j.dental.2017.09.004 0109-5641/© 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

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1.

Introduction

In dentistry, dental restorative composites are being used more often due to their aesthetics, practical handling and also their ability to adhere to the tooth structure. In the oral cavity, these materials are exposed to a complex environment of bacterial flora, saliva, gingival fluid, and food which lead to their degradation and limit their longevity. It is estimated that only about 60% of composite resin restorations are expected to survive more than 10 years [1]. While, regardless of clinical skills, 70% of dental restorations are being replaced because of restoration failure due to secondary caries in teeth restoration margins [2]. Oral microorganisms are known to play a crucial role in the biodegradation of dental composites [3,4]; probably due to the fact that dental composites clinically tend to accumulate more bacterial biofilms on their surface than the enamel or other restorative materials such as glass ionomer restorations [5,6]. Microorganisms are able to produce lactic acid responsible for secondary caries. Moreover they contain esterase that degrades the vinylic polymer matrix [7] by cleavage of the condensation bonds. On the other hand, volumetric shrinkage of dental resins leads to the creation of marginal gaps at the composite interface. Infiltration of saliva and microorganisms into these gaps can cause inflammation of the pulp and surrounding tissues, induces dental caries and reduces longevity of the material [4]. Resin composites consist of a vinyl organic matrix based on various monomers such as bisphenol A glycidyl methacrylate (BISGMA), urethane dimethacrylate (UDMA) and triethylene glycol dimethacrylate (TEGDMA). Inorganic fillers are added in order to reinforce the material and improve their physical and mechanical properties [8]. Despite the controversy about Bisphenol A (BPA) released by restorative composites based on BIS-GMA and its derivatives, the use of these materials remains widespread. Furthermore, new dental composites containing BPA free monomers claimed to be safer but are believed to be equally toxic [9]. In order to reduce biofilm formation on the resin composite surface and to resolve the problem of marginal degradation, a new strategy is being adopted to reduce bacterial adhesion. The local systemic antibiotic treatment is the most common way used to treat and prevent infections caused by bacteria. However, the increase of antibiotic resistance in different bacterial strains is the main concern related to this therapy. Development of composite with anti-bacterial properties is the ultimate aim to decrease recurrent caries occurrence and improve oral health. In this context, an effective method has been employed by incorporating soluble antibacterial agents such as fluoride and chlorhexidine into dental composites. The antibacterial activity is achieved through the release of these antibacterial agents in the humid oral environment but only until their final depletion [10]. On the contrary, new acrylic type monomers are being integrated into dental composite in an immobilized antibacterial state, thus monomethacrylate and dimethacrylate monomers associated with quaternary ammonium salts (QAM) are being developed [10–12]. Studies carried out on different alkyl chain lengths (CL) of QAM showed that those with 16 carbon chain lengths had the strongest antibacterial effect such

as N-dimethylamino-hexadecyl methacrylate (DMAHDM) [13], methacryloxylethylcetyl dimethyl ammonium chloride (DMAE-CB) [14,15], and methacryloxylethylhexadecyl methyl ammonium bromide (MAE-HB) [16]. These monomers have a bactericidal capacity against Streptococcuss mutans which are known to be the main oral bacteria involved in the formation of dental caries. Before photopolymerization, they have minimum inhibitory concentration (MIC) of 0.6–3.1 ␮g/ml comparable to that of chlorhexidine [16,17]. However, after photo-polymerization, their movements are limited within the resin matrix and they do not exhibit the same strong inhibitory effects. Each monomer has a different inhibitory effect depending on its chemical nature and according to the adequate amount that can be incorporated in the restoration without modifying its mechanical properties [10]. Moreover changing the type of halogen on the same antibacterial monomer chemical structure has an effect on the inhibiting force. Actually, DMAE-CB a chlorohexadecane-methacrylate showed a bacteriostatic effect of 3% in an adhesive resin [17,18]. While DMAHDM, a bromohexadecane-methacrylate showed a bactericidal effect of 10% in an adhesive resin [13]. We can hypothesis that a bacteriostatic molecule will have an inhibitory effect on the bacteria growth of the oral cavity but a less cytoxic effect on the eukaryotic oral cells. To our knowledge, the Dimethyl Hexadecyl Methacryloxyethyl Ammonium Iodide (DHMAI) has not been tested in any dental restorations. A study on a series of QAM monomers showed that DHMAI at CL = 16 had the lowest MIC on S. mutans (2.5 ␮g/ml) comparable to that of the DMAECB [19]. These cationic biomaterials tend to absorb the proteins of the physiological saliva fluid, which reduces the antibacterial effect. The presence of proteins is an important factor to consider in the fight against bacteria. The layers of adsorbed proteins may either reinforce or inhibit bacterial adhesion depending on the type of proteins and the bacterial strain [20]. Many studies have shown that hydrophilic surfaces are effective in minimizing protein adsorption and preventing bacterial biofilm. One of the most common biocompatible and hydrophilic biomedical polymers is the 2-methacryloyloxyethyl phosphorylcholine (MPC), a methacrylate with a phospholipid polar group in the side chain [20,21]. Due to its amphiphilic properties, MPC broadly repels proteins and inhibits their surface adsorption. This mechanism of protein repelling action has also been shown to be effective at preventing bacterial adhesion but without inducing a specific antiseptic action. MPC has been successfully introduced into dental restorations for two-fold benefit with antibacterial agents [22]. The aim of this work is to develop a dental composite with antibacterial properties without altering its mechanical properties. We hypothesized that DHMAI is an effective antibacterial agent and that the addition of MPC as a second agent may be beneficial to reduce bacterial growth and bacterial adhesion to the dental composites. Therefore, the adequate amounts of agents in dental resins have been optimized in order to obtain acceptable flexural strength and flexural modulus values. The hardness and the degree of conversion of the chosen formulations have been studied. The

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antibacterial activity has been confirmed in vitro by tests on colony-forming units (CFU), metabolic activity and scanning electron microscopy (SEM) images.

2.

The Menschutkin reaction was used for the synthesis of quaternary ammonium salt by addition of an alkyl halide on a tertiary amine [13,19,23]. All reagents were purchased from Sigma–Aldrich (Saint-Quentin Fallavier, France) and were used without further purification. DHMAI (C24 H48 O2 N+ I− ) was obtained by addition of 0.06 mol of 2-dimethylamino ethyl methacrylate (DMAEMA) to 0.05 mol of iodohexadecane and 0.05% of hydroquinone. The mixture was put in a 100 ml flask, equipped with a magnetic stirrer and a thermometer under an inert atmosphere at 50 ◦ C. After 12 h, the white paste formed was washed several times with diethyl ether, and then dried under vacuum (at 40 ◦ C for 7 h) until obtaining a white powder [19]. The latter was analyzed by 1 H-nuclear magnetic resonance spectra, 13 C NMR, 14 N NMR and mass spectra. The structure of the DHMAI and 1 H-nuclear magnetic resonance spectra are presented in Fig. 1.

Preparation of resin composites

Different formulations of light cured dental composites were synthesized. All experimental composites were composed of 29% of organic phase based on BISGMA and TEGDMA with mass ratio of (75/25%) respectively referred to as BT (BISGMA/TEGDMA). The comphoroquinone (CQ) and the DMAEMA, were mixed at mass ratio of 1 wt% each as priming system. Both the commercial MPC and the synthesized DHMAI were added into BT at different fractions separately at first and then combined. BT without MPC and DHMAI was prepared as a control group. All the constituents of the organic phase excepting DHMAI were purchased from Sigma–Aldrich (Saint-QuentinFallavier, France). Each resin was filled with 71 wt% of silanized glass particles ® (8235-SCHOTT Landshut, Germany) consisting of 10% Al2 O3 , 10% B2 O3 , 30% BaO and 50% SiO2 , (mean size 0.7 ␮m). All of the compounds were well blended with SpeedMixerTM (Hauschild, Hamm, Germany) for 4 × 3 min at 3000 rpm until obtaining a homogeneous paste and stored in sealed containers in darkness and at room temperature before use.

2.3.

to provide the mechanical properties of our experimental antibacterial composites according to ISO 4049 standard. According to the results of flexural strength and flexural modulus testing, four formulations were selected (F1–F4, see below).

Materials and methods

2.1. Procedure for the synthesis of quaternary ammonium salt monomers (DHMAI)

2.2.

3

Specimen preparation

According to the studies of other groups [22,24–26] on optimization of antibacterial monomer proportions for a compromise between antibacterial activity and good mechanical properties, three concentrations (5, 7.5, 10%) of DHMAI and two concentrations (7.5, 10%) of MPC were selected. Therefore, nine experimental composites (Table 1) were tested for their flexural and modulus strength. These composites were compared to three commercial composites (Table 2) in order

2.4.

Mechanical properties

2.4.1.

Flexural strength and flexural modulus

Flexural strength (FS) and flexural modulus (FM) were performed according to ISO 4049 standard [27]. Bar samples (n = 10) of each composites (Tables 1 and 2) were prepared in a rectangular Teflon mold (2 mm × 2 mm × 25 mm), covered on each side with a sheet of Mylar first, then glass slide. The samples were irradiated with an LED lamp (3 M ESPE Elipar TM S10, Seefeld, Germany) during 3 × 40 s at 1400 mw/cm2 on both sides. Bars were polished with 120-grit silicon carbide abrasive paper, stored in distilled water at 37 ◦ C for 24 h and then tested until fracture by three-point bending. The test was conducted under across head speed of 0.5 mm/min on a universal mechanical testing machine MTS-DY34 (Adamel Lhomargy, Roissy-en-Brie, France). FS (MPa) and FM (GPa) were calculated from the following formula: FS = 3FI/2bh2 FM = FI3 /4bh3 d where F is the load applied (N), I is the span length (20 mm), b is the specimen width, h the specimen thickness and d is the mid-span deflection corresponding to the load F.

2.4.2.

Vickers hardness test

Pellets samples (n = 6) of each composite (F1–F4) of 6 mm in diameter and 1 mm in thickness (weight 80–90 mg) were prepared. As described above, samples were irradiated for 40 s on both sides, polished and stored in distilled water at 37 ◦ C for 24 h before testing. Vickers hardness (HV) test was performed with Micromet 5104 (Buehler, Lake Bluff, Illinois, USA) with a pyramidal diamond indenter Vickers. A 100 g load was applied for 10 s on samples. Vickers hardness (kg/mm2 ) was calculated from the following formula: HV = 1854, 4 P/d2 where P is the load applied (g), and d the average length of both diagonals.

2.5.

Degree of conversion of resin composites

The degree of conversion (DC) was measured using FTIR spectroscopy (Safas Monaco IR 700) with an attenuated total reflectance (ATR) sampling accessory. The absorbance peaks of the uncured (F1–F4) and cured pellets samples (n = 6) prepared as described above were obtained using 4 scans at 4 cm−1 resolution. The ratio of absorbance intensities of the aliphatic (C C) peak at 1638 cm−1 and the internal aromatic (C C) at 1608 cm−1 was used and compared before and after photo-polymerization to calculate DC (%) from the following equation: DC (%) = [(P1/P2) − (P1 /P2 )/(P1/P2)] × 100

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Fig. 1 – Structure and 1 H NMR spectra of the quaternary ammonium salt DHMAI.

Table 1 – Composition of the organic phase of the experimental composites. Organic phase 29 wt%

Formulations BT (75/25) wt%

MPC wt%

DHMAI wt%

CQ wt%

DMAEMA wt%

F1

1

Control

28.42

0

0

0.29

0.29

F2

2 3 4

7.5% MPC 10% MPC 5% DHMAI

26.29 25.58 27.00

2.13 2.84 0

0 0 1.42

0.29 0.29 0.29

0.29 0.29 0.29

F3

5 6

7.5% DHMAI 10% DHMAI

26.29 25.58

0 0

2.13 2.84

0.29 0.29

0.29 0.29

F4

7

7.5% + 7.5% MPC + DHMAI 7.5% + 10% MPC + DHMAI 10% + 10% MPC + DHMAI

24.16

2.13

2.13

0.29

0.29

23.45

2.13

2.84

0.29

0.29

22.74

2.84

2.84

0.29

0.29

8 9

Abbréviations: BT = bisphenol A glycidyl methacrylate (BISGMA) mixed with triethylene glycol dimethacrylate (TEGDMA); MPC = methacryloyloxyethyl phosphorylcholine; DHMAI = dimethyl hexadecyl methacryloxyethyl ammonium iodide; CQ = comphoroquinone; DMAEMA = dimethylamino ethyl methacrylate.

Table 2 – Composition of commercial composites used in the study. Commercial composite

Resin

Fillers Barium, aluminium and fluoride glass Silica and zircona fillers

5 nm–20 ␮m

83%

0.6 ␮m–10 ␮m

78.5%

Barium glass, ytterbium trifluoride, mixed oxide and prepolymer.

40 nm–3000 nm

82–83%

VD

Venus Diamond

TCD-DI-HEA, UDMA

XTE

FiltekTM Supreme XTE Universel restorative 3M ESPE Tetric EvoCeram Ivoclar vivadent

BISGMA, UDMA TEGDMA BIS EMA PEGDMA BISGMA, UDMA TEGDMA

TEC

Fillers size

Fillers (wt%)

are provided by manufacturers; abbreviations: BISGMA = bisphenol A glycidyl methacrylate; UDMA = urethane Data dimethacrylate; TEGDMA = triethylene glycol dimethacrylate; TCD-DI-HEA = 2-propenoic acid, (octahydro-4,7 methano-1H-indene-5diyl)bis(methyleneiminocarbonyloxy-2,1-ethanediyl) ester; BISEMA = bisphenol-A-polyetheylene glycol dimethacrylate; PEGDMA = poly (ethylene glycol) dimethacrylate.

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where P1 and P1 are the absorbance peaks at 1638 cm−1 of (C C) aliphatic before and after photo-polymerization respectively. P2 and P2 are the absorbance peaks at 1608 cm−1 of (C C) aromatic before and after photo-polymerization respectively.

2.6.

Antibacterial activity

2.6.1.

Colony-forming unit (CFU) counts

S. mutans strain (ATCC 25175) (Institut Pasteur, Paris, France) were cultured in brain heart infusion medium (BHI). Pellet samples (n = 5) of each formulation (F1–F4) previously prepared as described above were selected. This sample preparation method is usually used for antibacterial activity assessment of resin-based composites and glass-ionomer cements [28,29]. Pellets were incubated for 24 h in distilled water, were placed in 24-well cell culture plates and sterilized by UV. Then, 1 ml of bacteria suspension prepared in BHI at the optical density of DO600 = (0.6–0.7) measured using Spectrometer (Helios Epsilon spectrophotometer, Thermo Spectronic, Rochester, NY, USA) was added to the tested samples’ surfaces. The plates were then incubated aerobically at 37 ◦ C for 96 h. The total number of viable bacteria was evaluated according to the number of colony forming units (CFU) on the samples’ surfaces. For this purpose, samples after incubation were well drained and transferred into sterile tubes with 1 ml fresh BHI. Biofilms attached onto the sample surfaces were collected by dispersing the bacteria with a vortex mixer (Mixer Uzusi VTX-3000L, Tokyo, Japan) for 1 min at 2400 rpm. Detached bacteria were serially diluted and inoculated on BHI agar plates then incubated for 96 h aerobically at 37 ◦ C. Finally, the colonies were counted manually and results were expressed as CFU/ml.

2.6.2.

Metabolic activity by Alamarblue assay

Pellet samples (n = 6) of each formulation (F1–F4) were prepared as described in CFU and aerobically incubated for 96 h at 37 ◦ C. Bacterial viability via the metabolic activity was performed using the Alamarblue assay. The Alamarblue Cell Viability reagent (R7017, Sigma Aldrich, St. Louis, MO, USA) works as colorimetric indicator through the conversion of blue resazurin (oxidized state) into pink resorufin via reduction reactions of metabolically active bacterial cells. The assay was carried out on the composite surfaces and in the culture mediums. Briefly, samples after incubation were drained and transferred into another new 24-well plate. Original medium samples were removed from the plates. Then, wells of original plates without samples and new plates with samples were delicately washed with 1 ml Dulbecco’s phosphate buffered saline (DPBS). Subsequently, 1 ml of Alamarblue solution at final concentration of 10% (V/V) in fresh BHI was added directly in wells and plates were incubated at 37 ◦ C. As a negative control, bacteria suspension without samples was incubated and treated in the same conditions. The absorbance was measured ® at 570 and 600 nm using a micro plate reader (Infinite M 200 PRO, NanoQuant plate, Tecan, France). Results were expressed as percentage of reduction compared to 100% of the negative control.

Fig. 2 – Flexural strength of commercial and experimental composites. Error bars represent standard deviation (n = 10). Groups sharing same label letters had statistically similar values (P > 0.05). Encircled columns represent the formulations selected for further evaluation.

2.6.3.

Scanning electron microscopy (SEM)

After 96 h of aerobic incubation as described previously, pellet samples of each formulation (F1–F4) were well drained and transferred into another new 24-well plate. To observe adhering biofim, samples were fixed in 2.5% glutaraldehyde (V/V) in DPBS for 24 h at 4 ◦ C, rinsed once with DPBS and dried in a graded series of ethanol solutions (25%–100%). Samples were sputter-coated to achieve a 10 nm copper layer then examined by field emission SEM at magnifications of 500× and 2000× using (FEI-Quanta 250, Thermo Fisher Scientific, France). Then, the obtained MEB images (2000×) were analyzed by Image J Software (plugin Cell Counter) of the samples was used to quantify adherent bacteria on the surface of representative images [30].

2.7.

Statistical analysis

All data were statistically analyzed using the SPSSTM Software version 23. The results of FS and FM were analyzed with t-test. Results of VH, DC, CFU and metabolic activity were performed with one-way analysis of variance (ANOVA) for multiple comparisons using Tukey’s and Games-Howell’s post hoc analysis. Statistical significance was accepted at P < 0.05.

3.

Results

3.1.

Mechanical properties

3.1.1.

FS and FM

Flexural strength and flexural modulus of commercial and experimental composites are shown in Figs. 2 and 3 respectively. The experimental control composite F1 had no statistically difference with commercials XTE (Filtek TM Supreme) in FS value and TEC (Tetric EvoCeram) in FM (P > 0.05). The experimental composites F2 (7.5% MPC) and F3 (7.5% DHMAI) had FS and FM values statistically similar to those of the commercial composites TEC and XTE (P > 0.05). The experimental composites 8 (7.5% MPC + 10% DHMAI) and 9 (10% MPS + 10% DHMAI) had the lowest FS (<80 MPa). While, F4 (7.5% MPC + 7.5% DHMAI) had FS and FM values sta-

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Fig. 3 – Flexural modulus of commercial and experimental composites. Error bars represent standard deviation (n = 10). Groups sharing same label letters had statistically similar values (P > 0.05). Encircled columns represent the formulations selected for further evaluation.

Fig. 4 – The Vickers hardness of selected experimental composites (F1–F4): error bars represent standard deviation (n = 10). Groups sharing same label letters had statistically similar values (P > 0.05).

Fig. 5 – The degree of conversion of experimental dental composites. Error bars represent standard deviation (n = 6). Groups sharing same label letters had statistically similar values (P > 0.05).

Fig. 6 – CFU counting of S. mutans on experimental composites’ surfaces after 96 h of aerobic growth in BHI medium. Error bars represent standard deviation (n = 5). Groups sharing same label letters statistically had similar values (P > 0.05).

tistically similar to those of the commercial composite TEC (P > 0.05).

3.1.2.

Vickers hardness

The Vickers hardness values of selected experimental composites (F1–F4) are shown in Fig. 4. Vickers hardness values revealed that there is no significant difference between F1 and F3. F2 and F4 exhibit a significantly reduced hardness (P < 0.05) compared to F1.

3.2.

Degree of conversion of resin composites

The degree of conversion of experimental dental composites is shown in Fig. 5. The DC revealed no significant difference between F1 and F2. F3 and mostly F4 exhibit a significant increase DC (P < 0.05).

Fig. 7 – Metabolic activity of S. mutans on experimental composites’ surfaces after 96 h of aerobic growth in BHI medium. Error bars represent standard deviation (n = 5). Groups sharing same label letters statistically had similar values (P > 0.05).

3.3.2. 3.3.

Antibacterial activity

3.3.1.

Colony-forming unit (CFU) counts

CFU counting of S. mutans on composites’ surfaces after 96 h of aerobic growth in BHI medium are shown in Fig. 6. No significant difference was found between F1 and F2 (no inhibitory function). Both the composite F3 and F4 had no significant difference and both drastically decreased the biofilm (∼98%) compared to F1 (P > 0.05).

Alamarblue assay

Metabolic activity of S. mutans after 96 h of aerobic growth in BHI medium on composites’ surfaces and in culture medium is shown in Figs. 7 and 8 respectively. For both tests composite F2 showed high reduction similar to the negative control and the composite control F1 with no statistical significant difference (P > 0.05). The test of metabolic activity showed that composite F3 greatly inhibited a reduction of Alamarblue than that of control F1 (∼−50%). The composite F4 had no significant difference with (P > 0.05)

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Fig. 8 – Metabolic activity of S. mutans in BHI culture medium after 96 h of aerobic growth. Error bars represent standard deviation (n = 5). Groups sharing same label letters statistically had similar values (P > 0.05).

in the test (A) (Biofilm on material surfaces) and (B) (Biofilm in culture medium) (P > 0.05).

3.3.3.

Scanning electron microscopy (SEM)

SEM images of S. mutans biofilms on composite surfaces after 96 h of aerobic growth in BHI medium are shown in Fig. 9 A thick biofilm was formed on the control F1 surface. On the surface of F2 the biofilm was more reduced than on F1, while on F3 and F4 it appeared much lesser and only dispersed bacterial colonies were observed. These changes were more clearly detected on higher magnitude (2000×). Segmentation by image J of bacterial clusters on F1 representative SEM image could not be achieved correctly due to dense biofilm formation. The approximate counting that was performed on one monolayer distribution gave a cell density greater than 475,000/mm2 . While, F2, F3, F4 representative SEM images were easily processed and bacterial cells were countable (251,000–162,000–163,000 cells/mm2 ) respectively. Thus, the adherent bacteria rate could be ranked as: F1 > F2 > F3 ≈ F4.

4.

Discussion

The incorporation of a QAM antibacterial monomer in dental resins was found to be an effective approach which may have durable antibacterial effect on bacteria responsible for tooth decay [31]. Our choice of QAM was based on the most effective alkyl chain length (CL). In fact, various studies carried out on several alkyl chains lengths have demonstrated that the antibacterial power increases considerably with the increase of chain lengths ranging from 2 to 18 carbon, but decreases beyond 16 Carbone. Thus, the QAM at 16 carbon chain lengths exhibit the best Antibacterial effects [13–16]. Another important parameter in the choice of the appropriate QAM is maintaining its inhibitory effect after photo-polymerization. The bacteriostatic or bactericidal effects depend on the nature of the antibacterial monomer and its maximum amount to be inserted in the dental resin without altering its mechanical properties [13,17]. Even, the type of halogen on the same chemical structure changes the monomer properties and influences its antibacterial effect. In this study, the monomethacrylate DHMAI with an iodohexadecane was chosen [19]. This later was incorporated at different concentrations in dental composite mixtures in order to evaluate its antibacte-

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rial effect after photo-polymerization, and in order to obtain a good compromise between the mechanical properties and the antibacterial efficiency. The bacterial adhesion to the composite surfaces is a complex process, comprising many factors, including bacterial properties, surface characteristics of the material and environmental factors such as the presence of proteins. Bacterial adhesion and biofilm formation begins by the phase of protein adsorption [32]. The addition of MPC as protein repellent should help reduce bacterial adhesion and minimize the formation of biofilm on the surface of the material [33]. MPC was first incorporated into our dental composites alone and then combined with the DHMAI for a further effect at different proportions. Through the Menschutkin reaction, the ammonium methacrylate monomer DHMAI was successfully synthesized for its incorporation in dental composites resin based BT matrix at (75/25) respectively. The results of the mechanical properties obtained in this study, showed that the combination of both DHMAI and MPC and their use separately in dental composites reduces the FS, the FM and the VH compared to the control. The only exception was the 7.5% DHMAI which showed FM and VH similar to those of the experimental control (P > 0.05). The alteration of the mechanical properties is attributed to the decrease in the level of the di-methacrylate monomers (BISGMA/TEGDMA) used; that were replaced with the monomethacrylate monomers (DHMAI/MPC) thus forming a less dense polymer structure. Moreover, decreasing the quantity of BISGMA from the resin matrix obviously reduces the rigidity and the mechanical properties of dental composite. In fact, BISGMA is a rigid molecule due to the two aromatic rings of its chemical structure [34]. This phenomenon which has already been reported in previous studies [25,26], prevents the use of high concentrations of antibacterial monomers. In the terms of FS, according to Fig. 2, it is quite clear that at 10% DHMAI, the composite presents properties above the limit of FS standard ISO 4049 (<80 Mpa) [27]. While, the 7.5% DHMAI is the optimal concentration to be incorporated into (75/25) BT respectively. The dental composite F3 (7.5% DHMAI) has acceptable FS comparable to that of the commercial composite TetricEvoCeram. In addition, it does not affect the FM and VH. Also in terms of FS, the 10% MPC seems acceptable but in combination with 7.5% DHMAI the limit of FS standard ISO 4049 [27] is exceeded. Whereas, the F4 (7.5% DHMAI + 7.5% MPC) appears to have the best combination with acceptable FS and FM comparable to those of the commercial composite TetricEvoCeram. In contrast, it has a low hardness compared to the control. The incorporation of the MPC had no effect on DC. While, DHMAI alone or mixed with MPC improves the degree of conversion. This phenomenon is due to the reduction of the concentration of BISGMA in the dental resin. It is known that DC is influenced by the viscosity of the photo-polymerizable mixture [35]. Due to the high viscosity and the rigidity of BISGMA, its reduction leads to an increase in the mobility of the reactive functional groups in the reaction medium. S. mutans are the human odontopathogenes most responsible for dental caries [36], hence they were chosen as a biofilm model to evaluate the antibacterial effect of our dental com-

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Fig. 9 – Representative SEM images of S. mutans biofilms on experimental composite surfaces after 96 h of aerobic growth in BHI medium. (A) Lower magnification micrographs (500×). (B) Higher magnification micrographs (2000×).

posites. The various tests on the antibacterial activity showed that dental composites F3 (7.5% DHMAI) gives a strong antibacterial effect with a reduction of (∼98%) in CFU and (∼50%) of metabolic activity on the surface of the materials. The SEM

imaging combined with image J quantification confirmed the strong reduction of biofilm formation on the surface of composites. It appears that the antibacterial activity at the surface of the composites is due to the monomers DHMAI immobi-

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lized in the dental resin. The mechanism of action is carried out by the interaction of the positive charge (N+ ) of the quaternary amine with the negative charge at the wall of the bacterial cell of S. mutans [12]. As the alkyl chain of DHMAI is 16-carbons long, it enters the S. mutans cell and destroys the cytoplasmic membrane [31], interrupts the activity of the proteins and damages the bacterial DNA [13]. It is disrupting the electron balance of S. mutans by inducing osmotic pressure leading to cell destruction [31]. This mode of action of the AMQ by (inhibition-contact) should ensure a durable antibacterial activity to the dental composites. Several studies confirmed this phenomenon for several AMQs modified dental resins [25,37] such as methacryloxylethyl dodecyl methyl ammonium bromide MAE-DB which keeps its effectiveness even after 6 months of aging process [38]. The dental composite F3 (7.5% DHMAI) also showed a significant reduction (∼50%) of the metabolic activity in the culture medium of the dental composites Fig. 8. This indicated that the planktonic S. mutans were damaged by the DHMAI which did not react during the photo-polymerization and was released by the dental composite. This mechanism of inhibition is an approach widely used in biomaterials using antimicrobial agents [39]. The combination of DHMAI with the MPC did not increase the inhibitory effect against bacterial adhesion. The various tests on the antibacterial activity showed that dental composites F4 (7.5% DHMAI + 7.5% MPC) gives similar effect to that of F3 (7.5% DHMI) on CFU, metabolic activity (P > 0.05) and SEM imaging. Nevertheless, the MPC has interesting biological properties. Various studies have confirmed its role in resisting protein adsorption to biomaterials [21,40,41], preventing bacterial adhesion [42] and its effectiveness against the formation of oral biofilm [43]. The MPC molecule consists of a side methacrylate function which ensures excellent polymerization in the dental resin and a polar phospholipid group which confers a high hydrophilicity. The adsorption of proteins is achieved through the binding of water molecules to the surface of biomaterials. The amphiphilic nature of the MPC should induce a weak interaction with the water molecules but an accumulation of free water around the material by forming a protein barrier [44]. One study showed that 10% MPC combined with 5% DMAHDM in a dental resin BT at (50/50) respectively did not alter the mechanical properties. The results demonstrated by CFU showed that the DMAHDM alone confers a strong antibacterial activity at two orders of magnitude lower than the control. While, when combined with the MPC it exhibited strong protein repulsion and conferred an antibacterial activity more than 3 orders of magnitude lower than the control [22]. In our study, the inefficient MPC in dental composites is probably due to the low incorporated amount. The alteration of the mechanical properties constrained us to be limited to 7.5% MPC in combination with 7.5% DHMAI. Based on the present data, the incorporation of the MPC at the 7.5% concentration did not provide any efficiency in reducing bacterial adhesion or biofilm formation. Moreover, it reduces hardness values below those recommended by ISO 4049 for dental composites (<40 Hv) [27]. These results call for further optimizations going beyond 7.5% MPC in dental resins. Incorporation of 7.5% DHMAI into a (75/25) dental resin BT considerably reduces bacterial growth, metabolic activity and thus biofilm formation. At this concentration, the mechanical prop-

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erties FS and FM are lower than the control but remain much higher than the ISO 4049 recommendations (>80 MPa) [27]. At 7.5%, DHMAI has no effect on hardness and improves DC. In the present study the optimal concentration of DHMAI in dental composite was determined and its antibacterial activity was evaluated. Further studies are needed to evaluate its long-term efficiency, its cytotocompatibility in the presence of human gingival fibroblasts, and its behavior in vivo. The use of DHMAI can also extend to other dental applications such as dental adhesives and flow composites.

5.

Conclusion

Our hypothesis on the efficiency of DHMAI as an antibacterial monomer has been confirmed. The results showed that the 7.5% DHMAI confers to the experimental composites a good compromise between: • Strong antibacterial activity against S. mutans. • Acceptable flexural strength and flexural modulus corresponding to those of the commercial composites. • Vickers hardness comparable to the experimental composite control without DHMAI. • Better degree of conversion compared to the experimental composite control without DHMAI. The DHMAI monomer gave promising results and thus can be a good candidate for various clinical applications such as antibacterial and anti-caries dental restorations. Our hypothesis on the efficiency of MPC addition to DHMAI in reducing bacterial growth was rejected. Within the limits of this study, (7.5% MPC + 7.5% DHMAI) affects the hardness of the dental composites without providing better antibacterial activity.

Acknowledgements The authors thank Dr. Maggy Hologne For NMR analyzes. We thank the laboratory Inserm UMR1033 Pathophysiology, Diagnosis and Treatments of Bone Diseases and especially Mr. Sébastien Rizzo for assistance with Vickers hardness study. We also wish to express our appreciation to the Microstructures Technology Center of University Claude Bernard Lyon1 for assistance with the SEM study, especially Mr. Xavier Jaurand. We are also grateful to Laiticia Scalone and Dr. Mark Cresswell for verification of English language.

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