In situantibiofilm effect of glass-ionomer cement containing dimethylaminododecyl methacrylate

Highlights

  • An in situ dental biofilm model was used to evaluate the novel glass ionomer cement (GIC) containing dimethylaminododecyl methacrylate (DMADDM).

  • DMADDM could increase surface charge density but reduce surface roughness.

  • The biofilm coverage and viability were significantly reduced on DMADDM containing samples compared to controls.

Abstract

Objective

The aim of this study was to investigate antibiofilm effects of a recently developed glass ionomer cement (GIC) containing dimethylaminododecyl methacrylate (DMADDM) under oral conditions.

Methods

Biofilms were allowed to form in situ on GIC specimens ( n = 216) which contained DMADDM (1.1 wt.% or 2.2 wt.%). Samples without DMADDM served as control ( n = 108). GIC 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 by fluorescence microscopy (FM) and evaluated by red/green ratios and an established scoring system. Bacterial morphology and biofilm accumulation were determined by scanning electron microscopy (SEM). Additionally, material properties as surface charge density of quaternary ammonium groups, surface roughness and DMADDM release were recorded.

Results

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

Introduction

Glass ionomer cement (GIC) was invented by Wilson and Kent in 1971 . 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 . In addition, its unique fluoride ion release characteristics are supposed to have antimicrobial and remineralization effects . However, clinical systematic review data were not supportive of an anti-caries effect of GICs , 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 . 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 . Quaternary ammonium salts (QAS) can cause bacteria lysis by binding to cell membrane to cause cytoplasmic leakage . 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 . Long cationic polymers can penetrate bacterial cells disrupting the membranes . The primer incorporating 12-methacryloyloxydodecylpyridinium bromide (MDPB) demonstrated cavity-disinfecting effects, and the world’s first antibacterial adhesive system employing the MDPB-containing primer was successfully commercialized . 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 . 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 . 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.

Materials and methods

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).

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 .

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 . 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.

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 ( 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 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.

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.

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.

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 . Sample diameters (5 mm) and heights (1 mm) were measured using calipers. Samples were placed in a 48-well plate. Fluorescein sodium salt (200 μL 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 μL 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 × 10 4 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.

Surface roughness evaluation

Surface roughness was determined on polished specimens (see specimen preparation above) using a white light 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 μm, working distance of 5 mm. Each measurement was performed within a field-of-view of 90 μm × 90 μm. Five equidistant locations were measured on each disk, starting from its center and moving toward its periphery. Each experimental group comprised 6 samples.

In vitro / in situ LC–MS n measurement of DMADDM release

Specimens were placed in 100 μL 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 μL 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 μm) 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 μL/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 μL 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 MS n . Full MS 2 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 MS 2 . Other settings were as follows for MS 2 : minimum signal threshold, 100 counts; isolation width, 1.5 u; activation Q, 0.25; activation time, 30 ms.

SEM-evaluation

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 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 .

Table 1
SEM and FM analysis: scoring of pattern of biofilm formation.
Score Description
6 Established multilayer biofilm, multiple morphotypes, covering > 50% of the surface
5 Established biofilm covering < 50% of the surface
4 Multiple microbial aggregations or monolayer biofilm
3 Few microbial aggregations, hundreds of microorganisms
2 Few small microbial aggregations, dozens of microorganisms
1 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.

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 μL of SYTO 9 (green; living bacteria) and 1 μL of propidium iodide (red, dead bacteria) in 1 mL of distilled water. Specimens were placed in 24-well plates and 100 μL 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 ] 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 was used regarding ratios between red and green fluorescences ( Table 2 ).

Table 2
Scoring system for the assessment of biofilm viability.
Score Description
5 Mainly green fluorescence; ratio between red and green fluorescence 10:90 and lower
4 More green fluorescence; ratio between red and green fluorescence 25:75 and lower
3 Ratio between red and green fluorescence 50:50
2 More red florescence; ratio between red and green fluorescence 75:25 and higher
1 Mainly red fluorescence; ratio between red and green fluorescence 90:10 and higher

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).

Materials and methods

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).

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 .

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 . 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.

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 ( 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 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.

Nov 23, 2017 | Posted by in Dental Materials | Comments Off on In situantibiofilm effect of glass-ionomer cement containing dimethylaminododecyl methacrylate
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