Biofilm inhibition by an experimental dental resin composite containing octenidine dihydrochloride

Abstract

Objectives

The aim of the present study was to investigate an antimicrobial additive containing experimental resin composite with regards to its impact on biofilm formation under oral conditions.

Methods

Biofilms were established in situ on composite specimens ( n = 192) which contained octenidine dihydrochloride (ODH, 3 wt.% or 6 wt.%). Samples without antimicrobial additive served as control ( n = 96). Composite specimens were fixed on custom made splints and exposed to the oral cavity of six healthy volunteers for three or seven days. Biofilm formation was assessed by scanning electron microscopy (SEM) and fluorescence microscopy (FM).

Results

The biofilm formation was significantly reduced on ODH containing samples compared to controls after three as well as after seven days in situ . FM evaluation additionally showed a lower viability of the reduced biofilms for both ODH concentrations.

Significance

During this short term investigation, incorporation of ODH into resin based composite materials caused biofilm inhibiting effects in situ .

Introduction

Microbial biofilms play an important role for the development of caries and periodontal diseases . Biofilm formation on surfaces exposed in the oral cavity, e.g. teeth, restorative or implant materials, represents the ubiquitous phenomenon of microbial colonization on interfaces between solid surfaces and biological fluids.

This process is characterized by several stages. Adsorption of salivary proteins, glycoproteins and mucins form a so called “acquired pellicle” within minutes. Then, first bacterial colonizers adhere to the pellicle surface within minutes to a few hours . A multi-layer bacterial biofilm, the dental plaque, is formed by bacterial growth and co-adherence of further bacteria. The extracellular matrix of the plaque matures, which is characterized by network formation of water-soluble and -insoluble glucanes, synthesized by bacterial glycosyl transferases . The established oral biofilm is a three-dimensionally structured community of many microbial species. The microorganisms in the plaque interact and communicate by signal transduction processes.

An innovative approach to control biofilm formation, and in turn to prevent caries and periodontal disease, is to develop dental restorative materials with antimicrobial properties, inhibiting microbial colonization . Apart from fluoride containing materials , this approach has, however, hardly been investigated so far.

Pilot experiments have been carried out with restorative materials, supplemented with benzalconium chloride, chlorhexidine, triclosan, cetylpyridinium chloride, polyethyleneimine nanoparticles or silver. These substances are released from the materials and provide antimicrobial properties under in vitro conditions . Another concept is to bind the antimicrobial agent to the surface using e.g. 12-methacryloyl oxydodecyl pyridinium bromide . However, the efficiency of concepts for the prevention of biofilm formation and growth in vivo has not been proven yet.

Biofilm formation can be studied in situ on samples exposed to the oral environment; different methods are available for their assessment and analysis . Among others, scanning electron microscopy (SEM) and vital-fluorescence microscopy (FM) are widely accepted methods to investigate the quantity of biofilm formation and to estimate the share of viable and dead microorganisms, respectively .

To date there is, to the best knowledge of the authors, no report on the incorporation of bispyridines into resin based filling materials, even though this antimicrobial agent has been widely used for decades as mouth rinsing solution or for wound disinfection .

Hence, the present in situ study investigated the intraoral microbial adherence and biofilm formation on experimental resin composite filling materials, enriched with octenidine dihydrochloride (ODH), using SEM- and FM-analyses. The null-hypothesis tested was, that inhibitions effects on biofilm formation on resin composite surfaces under oral conditions is independent from the incorporation of ODH into the material.

Materials and methods

Study design and subjects

Biofilms were formed intra-orally on a total of 288 composite specimens in a prospective, volunteer blinded clinical trial. The study protocol was approved by the ethical board of the Saarland Medical Association (vote number 193/08).

Six healthy volunteers were involved after signing an informed consent form (three male, three female, aged 29–40 years). 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).

Experimental formulations, specimen preparation and biofilm formation

Two experimental resin based composite formulations were fabricated, containing 3 or 6 wt.% of N,N′-(1,10 decanediyldi-1[4H]-pyridinyl-4-ylidene) bis-(1-octanamine) dihydrochloride (ODH, C 36 H 62 N 4 ·2HCl, molar mass: 623.83 g/mol, Schuelke & Mayr, Norderstedt, Germany, Fig. 1 ). The composition of the resin composite was based on a commercially available light-curing composite filling material (Venus ® , Heraeus Kulzer, Hanau, Germany, Table 1 ).

Fig. 1
Chemical structure of octenidine dihydrochloride (Schuelke & Mayr, Norderstedt, Germany).

Table 1
Composition of the resin based composite filling materials.
Test group ODH a content [wt.%] Matrix Filler
Control (Venus ® ) 0 Bis-GMA, TEGDMA, campherchinone, stabilizer, pigments Ba–Al–B–Si glass ( D 50 = 0.7 μm; D max < 2.0 μm); SiO 2 (range: 0.01–0.07 μm; D 50 = 0.04 μm).
content: 78 wt.% (60 vol.%)
Exp.V 3% 3
Exp.V 6% 6
All information according to the manufacturer’s information. Exp.V: experimental composites (Venus ® containing 3 or 6 wt.% a octenidine dihydrochloride, ODH).

The dental monomers Bis-GMA and TEGDMA were mixed in a 70:30 ratio. After heating the Bis-GMA/TEGDMA matrix to 70 °C, ODH was added in small portions, facilitating to dissolve the antimicrobial agent. Afterwards, photoinitiators and stabilizers were added. At the same temperature, 75 g inorganic filler particles (dental glass, d 50 1 μm, and pyrogenic silica) were added consecutively in small portions to 25 g of the monomer mixture. The experimental composites were filled into molds (50 mm × 50 mm × 1.5 mm) after cooling down to room temperature and light cured with a Palatray light curing unit (Heraeus, Hanau, Germany) from both sides for 8 min each. The resulting plates were then cut into smaller specimens of 5 mm × 5 mm × 1.5 mm.

Ninety-six specimens were fabricated per experimental composition (3 or 6 wt.%) each, a further 96 specimens without antimicrobial additive served as control. The surfaces of the specimens were ground on wet SiC paper (grit size 2500) at 300 rpm (Gripo 2V, Metkon Instruments Ltd., Bursa, Turkey). Half of the specimens per test-group were then aged for two days by thermocycling (TC: 5–55 °C, dwell time 30 s, 2500 cycles) in deionized water (Thermocycler, Willitec, Munich, Germany). Control samples (no intraoral exposure) were aged over periods of two and seven days. All samples were disinfected in ethanol (70%) for 30 min and subsequently washed several times in distilled water. For reference purpose, resin composite specimens with and without antimicrobial additive were analyzed without intraoral exposure.

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 composite specimens. Eight composite 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. 2 ]. The splints were exposed intraorally for three and seven days, respectively. During meals or for tooth brushing, splints were removed and stored in tap water. Tooth brushing was performed twice daily using individual tooth pastes. Neither additional cleaning procedures nor agents for chemical plaque control were applied. Splints with fixed specimens were not subjected to any cleaning measures. Volunteers were advised to maintain their normal eating habits.

Fig. 2
Individual removable acrylic upper jaw splint in situ , which has been used for positioning the composite specimens in the buccal region of the first premolar to the second molar. On each side 4 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.

LC–MS/MS measurement of ODH release in vitro and in situ

In vitro specimens ( n = 18) were placed for testing ODH release in 300 μL of LC–MS grade water (Fisher Scientific, Schwerte, Germany). Three different sets (without ODH, 3% ODH, 6% ODH) of specimens were prepared for each storage condition (thermo-cycled vs. not thermo-cycled) and incubated at room temperature for one week each.

The in situ tests were performed by two volunteers. These collected 2.0 mL of saliva at different points in time (2 min, 1 h, 6 h, 12 h, 18 h, and 24 h) after intraoral exposure of 12 resin composite specimens containing 6% of ODH each.

Aliquots of all samples (100 μL water or saliva) were gently vortexed in an 1.5 mL Eppendorf tube after adding 100 μL of LC–MS grade methanol (Fisher Scientific, Schwerte, Germany) or methanolic calibration solutions and 50 μL of 0.01 mg/mL methanolic trimipramine-D3 solution (Promochem, Wesel, Germany). After centrifugation for 3 min at 10,000 × g , the supernatant was transferred into a glass vial and evaporated to dryness under a gentle stream of nitrogen at 50 °C. The residue was dissolved in 50 μL of a mixture of eluents A and B (1:1, V:V) to ensure sufficient wet ability.

All samples were analyzed by a TSQ Quantum Access (ThermoFisher Scientific, TF, Dreieich, Germany) mass spectrometer coupled to a TF Accela UHPLC system equipped with a TF Hypersil GOLD PFP column (100 mm × 2.1 mm, 1.9 μm). The mass spectrometer was operated in the multiple-reaction monitoring (MRM) using positive electrospray ionization. For unambiguous identification two MRM transitions were chosen for detection of the analyte ( m / z Q1 = 551, m / z Q3 = 345; m / z Q1 = 551, m / z Q3 = 207).

SEM-evaluation

The specimens were fixed in 2.5% glutaraldehyde solution for 2 h at 4 °C and dehydrated in an ascending series of 50–100% ethanol. After drying in 1,1,1,3,3,3-hexamethyl disilazan, the samples were sputtered with platinum. SEM analysis was carried out using a FEI XL30 ESEM FEG (FEI Company, Eindhoven, NL) at magnifications of 500×, 1600×, 5000×, and 20,000×.

For each specimen, the amount of biofilm coverage and its structure were assessed using the scores shown in Table 2A . This scoring system was developed based on the experience of a pilot experiment, carried out by 2 volunteers using specimens with or without ODH up to an intraoral exposure time of ten days.

Table 2A
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 were assessed by fluorescence microscopy. The biofilms on the composite 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 96-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 and stored dark at 4 °C until processing. 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 per specimen (magnification 1000×, oil immersion) was carried out (=4 FM-micrographs per specimen). Overview micrographs served as control (magnification: 10×, 100×). Green and red FM-micrographs of the same section of the specimen were recorded separately and assembled hereafter using the AxioVision software. According to SEM investigation the biofilm coverage was assessed using the scores shown in Table 2A .

For the assessment of biofilm viability a 5 step scoring system was used regarding ratios between red and green fluorescences ( Table 2B ).

Table 2B
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 fluorescence; ratio between red and green fluorescence 75:25 and higher
1 Mainly red fluorescence; ratio between red and green fluorescence 90:10 and higher

Each sample was assessed independently by two scientists. Disagreements were resolved through discussion and consensus. In some cases, no bacteria (and in turn no fluorescence) could be observed on the specimen surface. In this case, the FM-micrograph was rated n.a. (not applicable).

Statistics

A comprehensive explorative data analysis was performed for the results obtained from the SEM- and FM-analyses. Regarding biofilm coverage of the composite 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 (SEM-, FM-analysis) were calculated and frequency bars for biofilm viability compiled (FM-analysis). Specimens without biofilms were rated as “n.a.” during FM-evaluation and were excluded from the calculation of the biofilm viability frequencies.

The Kruskal–Wallis-test and the Mann–Whitney U -test were used to test the influence of the ODH concentration and TC, respectively. All statistical analyses were carried out at a significance level of 5% using the software SPSS (release 18.03, SPSS Inc., Chicago, IL, USA) and MedCalc (release 12.0.4.0, MedCalc Software bvba, Mariakerke, B).

Materials and methods

Study design and subjects

Biofilms were formed intra-orally on a total of 288 composite specimens in a prospective, volunteer blinded clinical trial. The study protocol was approved by the ethical board of the Saarland Medical Association (vote number 193/08).

Six healthy volunteers were involved after signing an informed consent form (three male, three female, aged 29–40 years). 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).

Experimental formulations, specimen preparation and biofilm formation

Two experimental resin based composite formulations were fabricated, containing 3 or 6 wt.% of N,N′-(1,10 decanediyldi-1[4H]-pyridinyl-4-ylidene) bis-(1-octanamine) dihydrochloride (ODH, C 36 H 62 N 4 ·2HCl, molar mass: 623.83 g/mol, Schuelke & Mayr, Norderstedt, Germany, Fig. 1 ). The composition of the resin composite was based on a commercially available light-curing composite filling material (Venus ® , Heraeus Kulzer, Hanau, Germany, Table 1 ).

Nov 28, 2017 | Posted by in Dental Materials | Comments Off on Biofilm inhibition by an experimental dental resin composite containing octenidine dihydrochloride
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