In situ antibiofilm effect of glass-ionomer cement containing dimethylaminododecyl methacrylate

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In situ antibiofilm effect of glass-ionomer cement containing dimethylaminododecyl methacrylate Jin Feng a,d , Lei Cheng a , Xuedong Zhou a,∗∗ , Hockin H.K. Xu b , Michael D. Weir b , Markus Meyer c , Hans Maurer c , Qian Li d , Matthias Hannig d , Stefan Rupf d,∗ a

State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China Biomaterials & Tissue Engineering Division, Department of Endodontics, Prosthodontics and Operative Dentistry, University of Maryland Dental School, Baltimore, MD 21201, USA c Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Saarland University, 66421 Homburg/Saar, Germany d Clinic of Operative Dentistry, Periodontology and Preventive Dentistry, Saarland University Hospital, Homburg/Saar, Germany b

a r t i c l e

i n f o

a b s t r a c t

Article history:

Objective. The aim of this study was to investigate antibiofilm effects of a recently developed

Received 1 December 2014

glass ionomer cement (GIC) containing dimethylaminododecyl methacrylate (DMADDM)

Received in revised form

under oral conditions.

18 April 2015

Methods. Biofilms were allowed to form in situ on GIC specimens (n = 216) which contained

Accepted 15 May 2015

DMADDM (1.1 wt.% or 2.2 wt.%). Samples without DMADDM served as control (n = 108). GIC

Available online xxx

specimens were fixed on custom made splints and exposed to the oral cavity in six healthy volunteers for 24, 48 and 72 h, respectively. Biofilm viability and coverage were analyzed

Keywords:

by fluorescence microscopy (FM) and evaluated by red/green ratios and an established sco-

Biofilms

ring system. Bacterial morphology and biofilm accumulation were determined by scanning

Glass ionomer cements

electron microscopy (SEM). Additionally, material properties as surface charge density of

Dimethylaminododecyl

quaternary ammonium groups, surface roughness and DMADDM release were recorded.

methacrylate

Results. FM results showed a higher ratio (24 h: 0%: 0.5, 1.1%: 1.2, 2.2%: 2.5) of red/green

Anti-bacterial agents

fluorescence on GIC samples containing DMADDM. Biofilm coverage and viability scores were significantly reduced (24 h: q1/median/q3 for: 0%: 3/4/5, 1.1%: 2/3/3, 2.2%: 1/2/2) on DMADDM containing samples compared to controls after 24 h as well as 48 and 72 h in situ (p < 0.05). While surface charge density of quaternary ammonium groups and DMADDM release increased with the DMADDM concentration, surface roughness was lowest on specimens containing 2.2 wt.% DMADDM. Significance. An in situ dental biofilm model was used to evaluate the novel GIC containing DMADDM. This material strongly inhibited biofilms in situ and is promising to prevent bacterial colonization on the surface of restorations. © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

∗ Corresponding author at: Clinic of Operative Dentistry, Periodontology and Preventive Dentistry, Saarland University Hospital, Kirrberger Street, Building 73, D-66421 Homburg/Saar, Germany. Tel.: +49 6841 1624968; fax: +49 6841 1624954. ∗∗ Corresponding author at: West China School of Stomatology, Sichuan University, No. 14, Section 3rd, Renmin South Road, Chengdu, China. Tel.: +86 28 85501481; fax: +86 28 85501481. E-mail addresses: [email protected] (X. Zhou), [email protected] (S. Rupf).

http://dx.doi.org/10.1016/j.dental.2015.05.005 0109-5641/© 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Feng J, et al. In situ antibiofilm effect of glass-ionomer cement containing dimethylaminododecyl methacrylate. Dent Mater (2015), http://dx.doi.org/10.1016/j.dental.2015.05.005

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1.

Introduction

Glass ionomer cement (GIC) was invented by Wilson and Kent in 1971 [1]. It is widely used as a dental material, due to its ease of use, low coefficient of thermal expansion, good biocompatibility with dental pulp tissue, and long-term bonding to tooth surfaces and metals [2–4]. In addition, its unique fluoride ion release characteristics are supposed to have antimicrobial and remineralization effects [5,6]. However, clinical systematic review data were not supportive of an anti-caries effect of GICs [7], indicating that the fluoride-release from GICs is not potent enough to inhibit bacterial growth or combat bacterial destruction processes. One of the most common reasons for replacing a dental restoration is recurrent caries around the margins of the biomaterial [8,9]. Therefore, a dental biomaterial which creates a sustained antimicrobial environment around the restoration would be of considerable clinical benefit. Efforts were made to synthesize quaternary ammonium methacrylates (QAMs) for use in antibacterial dental materials [10–16]. Quaternary ammonium salts (QAS) can cause bacteria lysis by binding to cell membrane to cause cytoplasmic leakage [17,18]. When the negatively charged bacteria contact the positive quaternary amine charge (N+ ), the electric balance is disturbed and the integrity of the bacterial cell wall is damaged under the osmotic pressure [19]. Long cationic polymers can penetrate bacterial cells disrupting the membranes [20,21]. The primer incorporating 12-methacryloyloxydodecylpyridinium bromide (MDPB) demonstrated cavity-disinfecting effects, and the world’s first antibacterial adhesive system employing the MDPBcontaining primer was successfully commercialized [22]. Recently, a new quaternary ammonium monomer, dimethylaminododecyl methacrylate (DMADDM) has been synthesized. In vitro studies have shown a strong antibacterial effect on a DMADDM-containing adhesive without compromising its physical characteristics [23,24]. However, the potential of DMADDM for the prevention of biofilm formation and viability in vivo has not been proven, yet. Being an important factor in the occurrence of dental caries and periodontal diseases, dental biofilm comprises complex three-dimensional structures consisting of diverse communities of microbial multispecies complexes formed on oral tissue [25,26]. To evaluate the antibacterial activity of a material, an in situ model needs to be established in order to investigate the material properties under realistic conditions. The current study investigated antibacterial activities of a GIC containing DMADDM on biofilm formation in vivo. The null hypothesis tested was that biofilm formation on GIC surfaces under oral conditions is independent from the incorporation of DMADDM into the material.

2.

Materials and methods

2.1.

Study design and subjects

Biofilms were formed intra-orally on a total of 324 GIC specimens in a prospective, double-blind in situ trial. The study protocol was approved by the ethical committee of the

Saarland Medical Association (vote number: 193/08). Six healthy volunteers were involved after signing an informed consent form. Inclusion criteria were: full dentition, sufficient compliance, no periodontal or restorative treatment needs, no local or systemic hypersensitivity to the materials used (splints, silicone impression material, resin composite, antimicrobial agent), no systemic disease(s), no pregnancy, no smokers and, no antibiotic treatment in the last six months. The volunteers received detailed information on the handling of the intraoral splints containing the specimens (see below).

2.2.

Specimen preparation

Dimethylaminododecyl methacrylate (DMADDM) was synthesized via a modified Menschutkin reaction method. Briefly, 10 mmol of 1-(dimethylamino)docecane (DMAD) (Tokyo Chemical Industry, Tokyo, Japan) and 10 mmol of 2-bromoethyl methacrylate (BEMA) (Monomer-Polymer and Dajac Labs, Trevose, PA) were added in a 20 mL vial with a magnetic stir bar. The vial was capped and stirred at 70 ◦ C for 24 h. After the reaction was complete, the ethanol solvent was removed via evaporation, yielding DMADDM as a clear, colorless, and viscous liquid [24]. The glass ionomer cement chosen for the current study was a conventional GIC (Fuji IX GP, GC Corporation, Tokyo, Japan). The novel material was modified by adding 5%, 10% DMADDM (w/w) to the liquid of the GIC while keeping the original powder/liquid ratio of 3.6:1.0 g, thus achieving finial mass fractions of 1.1 wt.% and 2.2 wt.% DMADDM in GIC. GIC without DMADDM (0 wt.%) served as control. Specimens with nominal dimensions of 5 mm diameter and 1 mm thickness were formed by mixing the GIC according to the manufacturers’ instructions and packing into silicon molds covered by a mylar strip and glass plate under hand pressure. The mixing was carried out by one individual with extensive experience in GIC handling. Specimens were removed from the molds and coated with a thin layer of adhesive. They were placed for 1 day at 37 ◦ C in a chamber that contained wet tissue paper not in direct contact with specimen, to achieve an atmosphere of 100% humidity but to prevent the specimen from coming in contact with water which could result in dissolution during the critical early phases of setting [27,28]. After this, the specimens were polished by wet SiC paper (grit size 2500) at 300 rpm (Phoenix 3000, Buchler, Braunschweig, Germany) and disinfected in ethanol (70%) for 30 min and subsequently washed several times in distilled water.

2.3.

In situ formation of oral biofilms

Alginate impressions (Blueprint cremix® , Dentsply DeTrey, Konstanz, Germany) were made from the upper jaw of the six volunteers. Transparent custom made acrylic splints (Thermoforming foils® , Erkodent, Pfalzgrafenweiler, Germany) were fabricated as carrier of the GIC specimens. Six samples were fixed in the left and right buccal position in the molar and premolar regions with silicon impression material (President light body® , Colténe, Altstaetten, Switzerland) onto the splints [29] (Fig. 1). The splints were exposed intraorally for 24, 48 and 72 h, respectively. During meals or for tooth brushing, splints were removed and stored in a wet chamber. Tooth brushing

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interferometer (MicroProf WLI, FRT, Germany). Each specimen was individually fixed in a clamping apparatus and characterized by the roughness parameter Ra which means average surface roughness. The results were obtained by employing a scanning interferometry technique, a scan length of 20 ␮m, working distance of 5 mm. Each measurement was performed within a field-of-view of 90 ␮m × 90 ␮m. Five equidistant locations were measured on each disk, starting from its center and moving toward its periphery. Each experimental group comprised 6 samples.

Fig. 1 – Individual removable acrylic upper jaw splint in situ, which has been used for positioning the GIC specimens in the buccal region of the first premolar to the second molar. On each side 3 specimens were placed in every splint.

was performed twice daily just using tap water without tooth pastes. Neither were additional cleaning procedures applied, nor any agents for chemical plaque control. Splints with fixed specimens were not subjected to any cleaning measures. Volunteers were advised to maintain their normal eating habits. After intraoral exposure, specimens were rinsed for 10 s with sterile NaCl-solution (0.9%) and processed immediately for microscopic analysis. Half of the specimens were subjected to SEM analysis, the remaining half to FM analysis.

2.4.

Surface charge density measurement

The charge density of quaternary ammonium groups present on the GIC specimen’s surfaces was quantified using a fluorescein dye method [14,23,30]. Sample diameters (5 mm) and heights (1 mm) were measured using calipers. Samples were placed in a 48-well plate. Fluorescein sodium salt (200 ␮L of 10 mg/mL) in deionized (DI) water was added into each well, and specimens were left for 10 min at room temperature in the dark. After removing the fluorescein solution and rinsing extensively with DI water, each sample was placed in a new well, and 200 ␮L of 0.1% (by mass) of cetyltrimethylammonium chloride (CTMAC) in DI water was added. Samples were shaken for 20 min at room temperature in the dark to desorb the bound dye. The CTMAC solution was supplemented with 10% (by volume) of 100 mM phosphate buffer at pH 8.0. Samples were taken out and absorbance was read at 501 nm using a plate reader (Infinite® M200, Tecan, Switzerland). The fluorescein concentration was calculated using Beers Law and the molar extinction coefficient of 7.7 × 104 L mol−1 cm−1 . Using a ratio of 1:1 for fluorescein molecules to the accessible quaternary ammonium groups, the surface charge density was calculated as the total molecules of charge per exposed surface area (sum of top, bottom and side edge area, measured independently for each GIC disk due to slight variations in disk diameters). Six replicates were tested for each group.

2.6. In vitro/in situ LC–MSn measurement of DMADDM release Specimens were placed in 100 ␮L of LC–MS grade water (Fisher Scientific, Schwerte, Germany) for testing DMADDM release. Three sets (without DMADDM, 1.1 wt.% DMADDM, 2.2 wt.% DMADDM) of one specimen each were prepared and incubated at room temperature for 72 h. Water was replaced every hour. Accordingly, two specimens of each DMADDM concentration, mounted on the splints, were exposed to the oral cavity to present similar free surfaces in comparison to the in vitro experiments. 100 ␮L of saliva were collected after 1, 4, 8, 12 and, 24 h. All experiments were carried out threefold. Additionally, one set of 10 specimens containing 2.2 wt.% DMADDM was exposed to the oral cavity and saliva samples were taken as described above. All samples were analyzed using a ThermoFisher Scientific LXQ linear ion trap mass spectrometer (TF, Dreieich, Germany) equipped with a heated electrospray ionization source and coupled to a TF Accela ultra UHPLC system consisting of a degasser, a quaternary pump, and an autosampler. Gradient elution was performed on a TF Hypersil GOLD C18 column (100 mm × 2.1 mm, 1.9 ␮m) guarded by a TF Hypersil GOLD C18 Drop-in guard cartridge and a TF Javelin column filter with 10 mM aqueous ammonium formate plus 0.1% formic acid pH 3.4 (eluent A) and acetonitrile plus 0.1% formic acid (eluent B). The flow rate was set to 700 ␮L/min, and the gradient was programmed as follows: 0–1.0 min 98% A, 1.0–6.0 min to 2% A, and 6.0–8.0 hold 98% A. The injection volume for all samples was 10 ␮L each. The instrument was operated in positive electrospray ionization mode; sheath gas, nitrogen at flow rate of 34 arbitrary units (AU); auxiliary gas, nitrogen at flow rate of 11 AU; vaporizer temperature, 250 ◦ C; source voltage, 3.00 kV; ion transfer capillary temperature, 300 ◦ C; capillary voltage, 31 V; and tube lens voltage, 80 V. Automatic gain control was set to 15,000 ions for full scan and 5000 ions for MSn . Full MS2 product ion spectra of the predefined protonated molecule (at m/z 326) of the target analyte was recorded and a specific fragment ion (at m/z 113) was used as quantifier. Normalized wideband collision energies were 35.0% for MS2 . Other settings were as follows for MS2 : minimum signal threshold, 100 counts; isolation width, 1.5 u; activation Q, 0.25; activation time, 30 ms.

2.7. 2.5.

SEM-evaluation

Surface roughness evaluation

Surface roughness was determined on polished specimens (see specimen preparation above) using a white light

The specimens were fixed in a solution containing 2% glutaraldehyde and 0.1 M cacodylate buffer for 2 h at 4 ◦ C. This was followed by washing in 0.1 M cacodylate buffer, and

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Table 1 – SEM and FM analysis: scoring of pattern of biofilm formation.

Table 2 – Scoring system for the assessment of biofilm viability.

Score

Score

6 5 4 3 2 1

Description Established multilayer biofilm, multiple morphotypes, covering > 50% of the surface Established biofilm covering < 50% of the surface Multiple microbial aggregations or monolayer biofilm Few microbial aggregations, hundreds of microorganisms Few small microbial aggregations, dozens of microorganisms Distinct pellicle layer, none or scattered microorganisms

Scores 5 and 6 represent established biofilms with a distinct architecture in SEM and a high density of microorganisms (FM and SEM), scores 4, 3, 2 comprise reduced microbial colonization, and score 1 are pellicle layers with only scattered adherent microorganisms.

5 4 3 2 1

2.8.

Vital fluorescence microscopy (FM)

Biofilm coverage as well as the viability of the biofilms was assessed by fluorescence microscopy. The biofilms on the GIC specimens were stained using a live/dead staining kit (BacLight® Bacterial Viability Kit L7012, Molecular Probes, Carlsbad, USA). The live/dead stain was prepared by diluting 1 ␮L of SYTO 9 (green; living bacteria) and 1 ␮L of propidium iodide (red, dead bacteria) in 1 mL of distilled water. Specimens were placed in 24-well plates and 100 ␮L of the reagent mixture were added to each well followed by incubation at room temperature in the dark for 15 min. Each specimen was carefully positioned on a glass slide covered with mounting oil. Samples were evaluated under a reverse light fluorescence microscope (Axio Scope, Carl Zeiss AG, Oberkochen, Germany) in combination with the image processing software AxioVision 4.8 (Carl Zeiss Microimaging GmbH, Goettingen, Germany). One reading of biofilms on each quadrant and center area per specimen (magnification 1000×, oil immersion) was carried out (=5 FM-micrographs per specimen). Green and red FM-micrographs of the same section of the specimen were recorded separately and assembled hereafter using the AxioVision software. ImageJ 1.48 [National Institutes of Health (NIH), Bethesda, MD, USA; freeware from http://rsb.info.nih.gov/ij/] was used to quantify the coverage area and viability of the biofilm. The images for each color channel were assembled into image stacks. Total fluorescence area of each section was calculated as biofilm coverage. The images of green/red channel were calculated separately. Analogous to SEM investigation the biofilm coverage was also assessed using the scores shown in Table 1. For the assessment of biofilm vitality a 5-step scoring system [31] was

Mainly green fluorescence; ratio between red and green fluorescence 10:90 and lower More green fluorescence; ratio between red and green fluorescence 25:75 and lower Ratio between red and green fluorescence 50:50 More red florescence; ratio between red and green fluorescence 75:25 and higher Mainly red fluorescence; ratio between red and green fluorescence 90:10 and higher

used regarding ratios between red and green fluorescences (Table 2).

2.9. dehydration in an ascending series of 50–100% ethanol. After drying in 1, 1, 1, 3, 3, 3-hexamethyldisilazan, the samples were sputtered with carbon. SEM analysis was carried out using a FEI XL30 ESEM FEG (FEI Company, Eindhoven, NL). For each specimen, the biofilm coverage and its structure were assessed using the scores shown in Table 1. This scoring system was developed based on the experience of a previous study [31].

Description

Statistical analysis

A comprehensive explorative data analysis was performed for the results obtained from the SEM- and FM-analyses. Regarding biofilm coverage of the GIC samples, both methods were compared using Passing-Bablok regression analysis and McNemar test. Median values and interquartile ranges (25–75th percentiles) of the biofilm formation and viability (SEM-, FM-analysis) were calculated. The Kruskal–Wallis test and the Dunn’s Multiple Comparison test were used to test the influence of the DMADDM concentration. All statistical analyses were carried out at a significance level of 5% using the software SPSS (release 19, SPSS Inc., Chicago, IL, USA).

3.

Results

3.1.

Surface charge density

The surface charge density of GIC containing DMADDM (GICDMADDM) is plotted in Fig. 2A (mean ± sd; n = 6). Fluorescein binding to the cationic quaternary groups revealed statistically significant increases in the quaternary ammonium sites present on the surfaces of the specimens with increasing DMADDM concentration (p < 0.05). Samples with 0% DMADDM had slight nonspecific interaction with and absorption of the fluorescein salt.

3.2.

Surface roughness evaluation

Fig. 2B reports the quantitative topographical analysis of the experimental cements (mean ± sd; n = 6). According to the results of this study, the surface roughness of GICDMADDM decreases comparing with control group (p < 0.05). There were no significant differences between 1.1 wt.% and 2.2 wt.% DMADDM groups (p > 0.05).

3.3. In vitro/in situ LC–MSn measurement of DMADDM release In water, DMADDM concentrations decreased continuously; and after 12 h values were found near to the zero level for both 1.1 wt.% and 2.2 wt.% DMADDM containing specimens (Fig. 3). In saliva, no DMADDM release could be found for the

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Fig. 2 – Surface charge density (A) and surface roughness (B) of GIC containing different mass fractions of DMADDM. Charge density increased while roughness decreased with increasing DMADDM concentration. Each values is mean ± sd (n = 6) (*p < 0.05).

Fig. 3 – In vitro LC–MSn measurement of DMADDM release into water. Mean DMADDM release (␮L/L ± sd) from one specimen in vitro into water decreased within 12 h to values near zero line.

according sets of specimens for none of the DMADDM concentrations. Only one set presenting 10 specimens of 2.2 wt.% DMADDM revealed release of the substance near the detection level.

3.4.

SEM-evaluation

Representative SEM micrographs of GIC specimens not exposed intraorally are depicted in Fig. 4. Filler structure, size and arrangement within the GIC matrix were clearly detectable. Under the same polishing conditions, no general differences concerning the distribution of the glass filler particle and the morphology of the matrix could be detected when comparing the GIC specimens with and without DMADDM. However, the interface between the glass filler particles

and the surrounding matrix appears smoother in the GICDMADDM specimens compared to the controls. The SEM images of each group at different intraoral exposure times are shown in Fig. 5. After 24-h intraoral exposure early stage biofilms were formed (Fig. 5A), and after 48 h and 72 h, more and thicker established matured biofilms were present on the control specimens (Fig. 5D and G). The bacteria had a regular shape, extracellular matrix and intercellular links were even visible. In contrast, biofilms on GIC-DMADDM samples appeared thinner, displayed less microbial colonization and dispersed extracellular matrix structure; and the profile of bacteria was not regular. After 24-h intraoral exposure, the group with 1.1 wt.% DMADDM had less microbial colonization and dispersed extracellular matrix structure. On 2.2 wt.% DMADDM specimens, only scattered bacteria were apparent and the GIC surface pattern was clearly visible (Fig. 5B and C). After 48 h multiple microbial aggregations or monolayer biofilm were detectable on the GIC-DMADDM samples (Fig. 5E and F), predominantly damaged bacteria. After 72 h, multilayer biofilms were visible on the GIC-DMADDM samples, however, more damaged bacteria and a significant reduction in the thickness of biofilm were seen compared to controls (Fig. 5H and I). The Kruskal–Wallis test revealed a significant influence of DMADDM added to the GIC on biofilm formation for all three exposure times (24 h: p < 0.001, 48 h: p < 0.001, 72 h: p < 0.001). The scores for biofilm coverage were significantly lower (Dunn’s test: p < 0.05) in the groups of the GIC-DMADDM samples compared to the control (Fig. 5a–c). An increased concentration of the DMADDM was correlated with a higher reduction of biofilm coverage in 24 h biofilm. The scores for samples containing 2.2 wt.% DMADDM were significantly lower compared to 1.1 wt.% containing specimens (Dunn’s test: p < 0.05, Fig. 5a). After 48 h and 72 h, there were no statistically significant differences between the 2.2 wt.% DMADDM group and the 1.1 wt.% DMADDM group.

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Fig. 4 – Representative SEM micrographs of non-intraoral-exposure specimens containing 0 wt.% (A and D), 1.1 wt.% (B and E), 2.2 wt.% (C and F) DMADDM. Glass particle (G) was visible on the surface of specimens. The surface roughness decreased with the concentration of DMADDM. A–C: magnification: 500×, bar scale = 25 ␮m; D–F: magnification: 5000×, bar scale = 2.5 ␮m.

3.5.

FM-evaluation

The results obtained from FM were consistent with SEM. The images showed in general lower density of bacterial colonization on samples containing 1.1 wt.% or 2.2 wt.% DMADDM as compared to control samples without DMADDM (Fig. 6). Dense biofilms containing high proportions of living bacteria were visible on the control specimens (Fig. 6A, D and G). After 24-h intraoral exposure only scattered bacterial colonies or isolated bacteria were present on GIC-DMADDM samples (Fig. 6B and C). In addition, mainly dead bacteria (red fluorescence) were observed on 2.2 wt.% DMADDM specimens. After 48 h dispersed biofilms and increasing dead bacteria were observed on DMADDM containing specimens compared to controls (Fig. 6E and F). After 72 h, thinner biofilms were observed and living bacteria decreased compared to controls (Fig. 6H and I). Results of the FM analysis are presented in Fig. 7. The Kruskal–Wallis test revealed significant influences of DMADDM added to the GIC on biofilm viability and coverage for all three exposure times (24 h: p < 0.001, 48 h: p < 0.001, 72 h: p < 0.001). Red/green fluorescence intensity ratio analysis (Fig. 7A–C) showed that the intensity on DMADDM incorporated materials was significantly higher than that of non-DMADDM-incorporated materials after 24 h as well as 48 and 72 h (Dunn’s test: p < 0.05). The scores for biofilm viability (Fig. 7D–F) and biofilm formation (Fig. 7G–I) were significantly lower in the groups of DMADDM containing samples compared to the control (Dunn’s test: p < 0.05). Consistent with the results from SEM, there were significant differences in scores between 2.2 wt.% DMADDM containing specimens and 1.1 wt.% DMADDM specimens (Dunn’s test: p < 0.05) for 24-h intraoral biofilms.

3.6.

Method comparison SEM and FM

Passing-Bablok regression analysis (p = 0.15) and McNemar test (p = 0.20) did not reveal any statistically significant differences for results of biofilm coverage of the GIC-DMADDM samples analyzed by SEM and FM.

4.

Discussion

Among the bio-active functions proposed for restorative materials antibacterial activity seems the most promising [32]. Dental plaque is one of the most natural forms of biofilm growth, which represents a ‘micro-ecosystem’ of bacteria. The production of acid resulting from sugar metabolism by these bacteria and the subsequent decrease in environmental pH is responsible for demineralization of the tooth surface and formation of dental caries [33,34]. The dental biofilm could lead to secondary caries at the tooth-restoration margins, which has been suggested as a primary reason for restoration failure [35,36]. Research on various antibacterial materials containing novel monomer QAMs having antibacterial activity, has been conducted. DMADDM is one of QAMs with a carbon chain length of 12, which has a strong antibacterial effect in vitro [17,23,24]. Oral bacteria that bind to the tooth surface exhibit a behavioral pattern different from that of free-floating or planktonic bacteria. The most notable difference between the oral bacteria in dental biofilms and the same strain grown planktonically is the increased resistance of the former to antimicrobial agents in a mature biofilm. Accordingly, the present study was aimed to investigate the effect of conventional GIC containing DMADDM on biofilm

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Fig. 5 – Representative SEM micrographs of 24 h (A–C), 48 h (D–F) and 72 h (G–I) biofilms. Biofilms were strongly inhibited in 1.1 wt.% (B, E, H) and 2.2 wt.% (C, F, I) DMADDM containing samples compared to controls (A, D, G). Results of scoring of biofilm coverage (a, b, c) showed lower scores on DMADDM containing specimens compared to control (p < 0.05). On 24 h biofilms, the scores of 2.2 wt.% DMADDM group were significant lower than 1.1 wt.% (a). Magnification: 10,000x, bar scale = 1 ␮m.

formation under oral conditions. The results of this study indicate that the GIC-DMADDM affected quality as well as quantity of biofilm formation in situ over a period of 72 h, which requires rejection of the null-hypothesis. The application of in situ models in a biofilm study is an interim method between clinical trials and laboratory experiments, which provides many advantages [37]. The model used in this experiment provides the opportunity to non-invasively monitor biofilm formation under reproducible conditions at different exposure times within one individual. The removable intraoral splint does not cover the palate and therefore allows for unhindered salivary secretion from the minor palatine

glands [29]. It was the first time that non-destructive visualization of dental biofilm was combined with an assessment of the antibacterial effect of GIC incorporated with DMADDM in situ. Oral biofilm formation has been analyzed thoroughly using different approaches. SEM is considered the gold standard for ultrastructural exploration of bacterial biofilms [38]. However, quantification and characterization of viability of adherent microorganisms are difficult. Thus, FM was additionally used to assess the viability of the microorganisms within the biofilms in our study. The scoring system selected for biofilm coverage in the SEM-micrographs and FM-images considered

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Fig. 6 – Representative FM images of 24 h (A–C), 48 h (D–F) and 72 h (G–I) biofilms formed on GIC specimens without (A, D, G), with 1.1 wt.% (B, E, H) and 2.2 wt.% (C, F, I) DMADDM. Biofilm coverage and viability were inhibited by DMADDM and a shift from green to red fluorescence was observed. Bar scale = 10 ␮m.

both, the microbial colonization of the sample surface and the colonization density of the biofilm. Hence, this finally enabled qualitative and quantitative assessments [38,39]. The results of this in situ study confirm previous in vitro studies regarding the antimicrobial activity of primer or adhesive mixed with DMADDM [23,24]. In the present study three exposure times were chosen: 24, 48 and 72 h. The initial communities of bacteria found within dental plaque biofilm are of relatively low diversity and tolerance to antimicrobial agents in comparison with those present in more matured biofilms [25]. So it makes sense to evaluate the antibiofilm effect of GICDMADDM not only on 24-h, but also on 48-h and 72-h in situ biofilms. Fuji IX was selected as the base GIC material because it is one of the strongest commercially available conventional restorative GICs [40]. As demonstrated in the present study, incorporation of DMADDM significantly increased the antibiofilm function of Fuji IX. Four different mechanisms (isolated or in combination) might explain the biofilm inhibiting

properties observed in the present study on the GIC-DMADDM: (1) Decreased surface roughness of the experimental material. The surfaces of the GIC-DMADDM samples are smoother than controls, which could influence bacteria adhesion. (2) Release of DMADDM from the material’s surface, which devitalize planktonic organisms in the saliva, thus reducing the number of bacteria available for adherence. (3) Direct “contactkilling” effect on bacteria; The advantage of using QAMS is that they can kill the microorganism by touch or simple contact [41]. The positively charged N+ sites of QAMs could bind to the negatively charged bacterial cell, which causes cytoplasmic leakage and finally bacteria lysis [15]. The current study results suggest that the density of surface positive charges increased with the concentration of DMADDM in the GIC samples. Therefore, it might contribute to an increase of the ratio of dead bacteria. And (4): Inhibition of biofilm metabolism. Metabolic processes are constantly taking place in the dental biofilm as a result of microbial activity [33]. It is proposed that metabolism changes in plaque would produce an ecological

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Fig. 7 – Results of red/green fluorescence analysis (A–C) as well as scores of biofilm viability (D–F) and biofilm coverage (G–I) by FM. Red fluorescence ratio significantly increased in DMADDM containing specimens compared to the control (p < 0.05). Additionally, in 24 h biofilms, scores of 2.2 wt.% DMADDM specimens were significantly lower than 1.1 wt.% (p < 0.05). The scores for biofilm viability and coverage were significantly decreased on DMADDM containing specimens compared to controls (p < 0.05).

shift in the balance of the resident microflora [34]. A previous study showed that DMADDM could reduce metabolic activity and lactic acid production in S. mutans biofilms [23]. This has, however, to be addressed in subsequent in vivo studies. This in turn would inhibit bacterial growth and biofilm formation in vivo. Based on the present data, effect (1) might not be the dominating reason for biofilm inhibition. As shown in Fig. 2B, control group had Ra values below 0.15 ␮m and groups containing DMADDM had even lower Ra values. However, a smoothening below Ra = 0.2 ␮m showed no further significant

changes, either in the total amount or in the pathogenicity of adhering bacteria [42,43]. Therefore, the influence of decreased surface roughness of GIC-DMADDM on biofilm formation is very low. Results of DMADDM release measurements showed that the release was only detectable over a short period of time. Thus, effect (2) might influence the viability of bacteria in earlier biofilm, while in more matured biofilms it might not be the main reason. Effect (3) could kill early colonizing bacteria directly and influence the development of biofilm indirectly. As shown in the results of biofilm coverage scoring by SEM and FM, biofilm formation after 24 h was

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significantly inhibited on DMADDM containing samples compared to controls. On 1.1 wt.% DMADDM containing samples only insular bacterial aggregates were present, but no multilayered biofilm. On 2.2 wt.% DMADDM containing specimens only scattered bacteria were apparent. Hence, it is hypothesized that the effects observed can be related significantly to direct contact mechanisms: this hypothesis is corroborated by a high ratio of dead/live bacteria. After 48 h and 72 h biofilm formation and viability were also significantly inhibited on DMADDM containing material compared to controls, however, there were no significant differences between 1.1% and 2.2% DMADDM groups. Biofilm development is a process influenced by the physico-chemical properties of the underlying surface. After the early stage of biofilm formation, it is likely that metabolic communication and quorum-sensing are pivotal regulatory factors that determine the bacterial composition and metabolism [25,44]. Therefore, the metabolism inhibiting effect (4) might also contribute to explain the results observed in our study after 48 h and 72 h of biofilm formation. Summarizing of the results suggests beneficial short-term effects of GIC-DMADDM on biofilm formation under in vivo conditions. Future investigations have to clarify the long-term in vivo antibiofilm properties of GIC-DMADDM and the influence of DMADDM on biofilm metabolism, however, short term antibacterial effects might be beneficial also over the setting time of GIC.

Acknowledgments This study was supported by Forschungsgemeinschaft Dental e.V., 05/2013, International Science and Technology Cooperation Program of China, 2014DFE30180 (XZ) and Program for New Century Excellent Talents in University (LC). We thank Dr. Simone Grass, Dr. Natalia Umanskaya, Mr. Norbert Pütz, Ms. Stephanie Smolka and Ms. Kiriaki Papadopoulos for technical assistance.

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