Oral biofilm and caries-infiltrant interactions on enamel

Graphical abstract

Abstract

Objectives

This study aimed to analyze interactions between oral biofilms and a dental triethylene glycol dimethacrylate (TEGDMA)-based resin infiltration material on enamel.

Methods

Demineralized enamel specimens (14 days, acidic buffer, pH 5.0) were either infiltrated with a commercial TEGDMA resin and subjected to a three-species biofilm ( Streptococcus mutans UAB 159 , Streptococcus oralis OMZ 607 and Actinomyces oris OMZ 745) (group 1), applied to the biofilm (group 2), or merely resin infiltrated (group 3). A control group received no treatment (4). Biofilm formation and metabolic activity of biofilms were measured for group (1) and (2) after 24 h CFU and a resazurin assay. Resin biodegradation was measured for group (1) and (3) by high performance liquid chromatography (HPLC) coupled with mass spectrometry after 6 and 24 h incubation. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) images were taken to study the biofilm and material’s autofluorescence in groups (1–4) after 24 h.

Results

SEM and CLSM images showed reduced biofilm formation on resin-infiltrated specimens (group 1) compared to group 2, while no biofilm was detectable on groups 3 and 4. CFU data (log 10 CFU per mL) of group 1 showed significantly reduced bacterial numbers (p < 0.05) compared to group 2. However, HPLC analysis of TEGDMA leakage after 6 h and 24 h revealed no differences between group 1 and group 3.

Conclusions

The results of the current study indicate that freshly resin-infiltrated enamel surfaces show a biofilm reducing effect, while monomer leakage was not affected by bacterial presence.

Clinical significance

Resin infiltrated enamel surfaces are constantly exposed to the oral microflora. Yet, it is not known how biofilms interact with enamel-penetrated resins and if and to which extent accessory alignments in oral hygiene are needed.

Introduction

New approaches for prevention and so-called non-invasive or micro-invasive treatment are increasingly studied to avoid restorative treatment . Resin infiltration is a micro-invasive treatment option for demineralized, non-cavitated enamel, which is not expected to remineralize anymore. Further caries progression is suppressed by the penetration of low viscous and light curable resins into porous enamel . Positive side effects were found in masking white spot lesions by modifying the refractive index of demineralized enamel . Although several studies provided insight into the mechanical and chemical properties of resin-infiltrated enamel , only very little is known about the interaction of oral microflora with resin-infiltrated enamel.

It is known that biofilms develop on all orally exposed surfaces and consist of different cross-linked bacteria and extracellular polymeric substances . Bacteria in biofilms show a higher pathogenicity compared to their planktonic counterparts . External parameters, such as surface properties, nutrition supply and pH in the surrounding media were shown to be relevant for the biofilm formation and composition . Yet, especially the role of different surface characteristics on biofilm growth is controversially discussed . Some studies revealed no differences of biofilm formation on varying surfaces as on different saliva-coated composites and glass ionomer cements . In contrast, other studies found significantly different levels of bacterial adherence on different restorative materials, irrespective of the initial saliva coating .

The resin infiltrant mentioned above is mainly composed of triethylene glycol dimethacrylate (TEGDMA). Besides its high penetration capability and wettability , TEGDMA has been reported to influence growth patterns of certain bacterial strains and biomass formation . However, the impact of TEGDMA on bacterial growth patterns was investigated only under very controlled in vitro conditions e.g. with regard to selective bacterial species, showing contradictory effects depending on the concentration of TEGDMA and the pH of the surrounding media . Leakage of TEGDMA monomers was only investigated in set-ups with cured plain resin material in molds. TEGDMA leakage of demineralized enamel specimens after resin infiltration and their interaction with complex biofilms was, to the authors’ knowledge, not investigated yet. Thus, it is still unknown whether caries lesions infiltrated with TEGDMA are a preferential site for biofilm formation or if the resin infiltrant exhibits some antibacterial effects on multispecies biofilms as found in the oral cavity. Furthermore, it is still unknown, how the enamel mesh affects TEGDMA leakage after resin infiltration.

The aim of this study was to investigate the initial formation of oral biofilms on resin-infiltrated demineralized enamel surfaces in comparison to mere demineralized enamel and the TEGDMA leakage of resin-infiltrated enamel specimens with and without biofilms. The null hypothesis was that the groups are not significantly different with regard to bacterial adhesion or TEGDMA leakage.

Materials & Methods

Specimen preparation and biofilm formation

Demineralized bovine enamel specimens (n = 48) were allocated into four groups: (1) resin-infiltrated enamel with biofilm, (2) enamel with biofilm, (3) resin-infiltrated enamel, (4) no treatment (control). Enamel specimens were produced using bovine incisors. In brief, crowns were cut off from the roots and stored in a 0.1% thymol solution (VWR International, Dietikon, Switzerland) for no longer than 6 month. Cylindrical specimens (4 mm in diameter) were punched out from each crown and ground stepwise from 1200 to 4000 Fepa P (1200, 2400, 4000 grit, Water Proof silicon carbide Paper, Struers, Erkath, Germany).

Initial carious lesions with intact surface layer were created in all enamel specimens by the following demineralization procedure. Briefly, specimens were immersed for 14 days in an acidic buffer with traces of thymol at a pH of 5 and 37 °C . The solution was renewed every second day to maintain a constant pH. After demineralization, control specimens (n = 6) were cut in 100 μm slices and demineralization depth and intact surface layer were controlled using transverse microradiography. In this study, lesions of about 200 μm were used. Resin infiltration with Icon (DMG, Hamburg, Germany), a caries infiltrant system, was performed for specimens of group (1) and (3) according to the manufacturer’s instructions. In brief, 15% hydrochloric acid was used to etch the surfaces for 2 min (Icon Etch, DMG) and removed by 30 s water rinsing and subsequent air-drying. Specimens were then rinsed with 99% ethanol (Icon Dry, DMG) for 30 s and air-dried. 0.75 μL resin (Icon Infiltrant, DMG) was applied for 3 min. Excess material was removed with a cotton roll prior to light-curing for 40 s. This was followed by a second application of 0.75 μL of resin for 60 s and gentle cotton roll application. Light curing followed again for 40 s (800 W/cm 2 bluephase, Ivoclar Vivadent, Schaan, Liechtenstein). Subsequently, specimens were fine grinded using a holder with an integrated spring and silicon carbide discs (4000 Fepa P) for 10 s to remove the oxygen inhibition layer under constant pressure. Specimens of all groups underwent gas sterilization for 16 h (Ethylene oxide, 3 M™ Steri-Gas™ Cartridges, Healthcare, Rüschlikon, Switzerland) and were stored sterile in a moist chamber until further treatment.

For biofilm formation in groups (1) and (2), three-species biofilms were grown on specimens for 24 h. The bacterial strains ( Streptococcus mutans OMZ 918, Streptococcus oralis OMZ 60, Actinomyces oris OMZ 745) were provided by the Institute for Oral Biology, Section for Oral Microbiology and General Immunology, University of Zurich, Zurich, Switzerland. Prior to bacterial incubation, specimens were immersed in diluted saliva supernatant to form a pellicle. One healthy subject donated fresh whole mouth saliva, which was used in all experiments. Donated non-stimulated saliva was centrifuged twice for 30 min (12′100 g). The supernatant was diluted 1:2 in 0.9% NaCl (Braun, Melsungen, Germany) and subsequently sterile filtrated (TPP syringe filters with 0.2 μm pores, Faust, Schaffhausen, Switzerland). For pellicle formation, specimens were incubated with gentle agitation in 800 μL diluted saliva supernatant for 4 h. Bacterial strains in a mixture of 30% diluted saliva supernatant and 70% modified fluid universal medium (mFUM) were adjusted to an OD 550 of 1 and mixed as inoculum. Pellicle-coated specimens were incubated during gentle agitation in 2 mL inoculum. Incubation was performed anaerobically in jars using gas-paks for 24 h at 37 °C (GENbox anaer and GENbag anaer, bioMérieux, Marcy l’Etoile, France). After 6 h, specimens were transferred to new wells with fresh medium and analyzed after 24 h incubation, whereas expended media (after 6 and 24 h incubation) were subjected to high performance liquid chromatography (HPLC).

HPLC

Analysis of uncured Icon Infiltrant (DMG) was carried out using HPLC on an Agilent 1100 LC/MS (Agilent Technologies, Basel, Switzerland). An Agilent column (ZORBAX Eclipse XDB-C8, 4.6 × 150 mm, 5 μm) with a 50/50 mixture of acetonitrile and water at a flowrate of 0.75 mL/min and a run time of 10 min was used. Identification of uncured Icon Infiltrant, namely TEGDMA, was performed with single ion detection at 309 m / z after diluting the experimental media (n = 12) with 50 vol% of a methanol-water (Millipore) mixture (80/20). To quantify the amount, the chromatogram peak area was compared with a calibration curve (linear range, R 2 = 0.998) of mere TEGDMA (95%). All chemicals for HPLC analysis were purchased from Sigma Aldrich (Buchs, Switzerland) and were of HPLC analytical grade, except otherwise stated.

Bacteria counts and metabolic activity

Plate counts were performed for specimens w/wo resin infiltration and subjected to biofilm (groups 1 and 2, n = 8) after 24 h. Specimens were sonified in 1 mL 0.9% NaCl and vortexed. 50 μL of different bacterial dilutions (in 0.9% NaCl) were plated out on Columbia sheep blood agar plates (CSBA, bioMérieux, Geneve, Switzerland) and incubated under anaerobic conditions using gas-paks. Plate counting followed after 2 days of incubation using a light microscope with 10-fold magnification (Wild Stereoskop, Heerbrugg, Switzerland).

Bacterial metabolic activity of groups (1) and (2) was measured after 24 h (n = 10). Specimens were transferred into 96-well plates and incubated in 300 μL resazurin solution consisting of 10 vol% alamarBlue Cell Viability Assay Reagent (Life Technologies, Zug, Switzerland) and fresh media (30% saliva solution + 70% mFUM) under anaerobic conditions. Two wells were additionally filled with blank resazurin solution (without specimens for background detection) and one well with bacteria from the planktonic inocula. After 15 min, 200 μL of each solution was pipetted into new 96-well plates and fluorescence was measured in a spectrophotometer with plate-reader at 560 nm excitation/585 nm emission at 37 °C (Spectramax M2, Molecular Devices, Bucher Biotec, Basel, Switzerland). Results were presented as relative fluorescence units (rfu) after background subtraction.

Bacteria imaging

Two specimens of each group were analyzed by scanning electron microscopy (SEM) after 24 h of incubation (SUPRA 50VP and Genesis, Carl Zeiss, Oberkochen, Germany). Briefly, specimens were washed with 0.9% NaCl solution and fixed for at least 24 h in 4% glutaraldehyde solution (in 0.1 M sodium potassium phosphate buffer, pH 7). A dehydration procedure followed and subsequent critical point drying was performed. Specimens were coated by gold sputtering for 60 s. Images were taken to show the detailed surface characteristics’.

Biofilms and resin-infiltrated surfaces were examined using confocal laser scanning microscopy (CLSM) after 24 h of incubation. Two specimens of each group were washed in 0.9% NaCl to remove loosely bound bacteria, fixed with 4% paraformaldehyde in the dark at room temperature for 60 min and washed again with 0.9% NaCl. Fluorescence staining was performed using Syto 59 (Life Technologies) to visualize all bacteria. The detailed staining procedure has been described elsewhere . In brief, each specimen was incubated in 500 μL staining solution consisting of 5 μM Syto 59 for 15 min at room temperature in the dark. After staining, specimens were washed in phosphate buffered saline (PBS, Invitrogen, Carlsbad, CA, USA) and mounted onto chamber slides (Fisher Scientific, Schwerte, Germany) using Mowiol 4-88 (Sigma Aldrich). Images were taken with a CLSM (SP5, Leica Microsystems, Heidelberg, Germany) using a 20× objective (numerical aperture: 1.25) and a helium laser (561 nm). Emission was detected with a photomultiplier between 630 and 660 nm. Three random areas of each specimen were examined with a z step size of 1 μm (512 × 512 pixels). Image processing was performed using Imaris Software 7.7.2 (Bitplane, Zurich, Switzerland).

Data presentation analysis

Leakage of TEGDMA monomers was compared between the resin-infiltrated specimens with biofilm (group 1) and without biofilm (group 3) after 6 h and after 24 h. Plate counts and relative fluorescence units were separately analyzed after 24 h between group (1) and group (2). All values were not normally distributed and Wilcoxon/Kruskal-Wallis test was applied for non-parametrical analysis. JMP (version 10, SAS, Cary, NC, USA) was used for statistical analysis. The overall level of significance was set at p ≤ 0.05.

Materials & Methods

Specimen preparation and biofilm formation

Demineralized bovine enamel specimens (n = 48) were allocated into four groups: (1) resin-infiltrated enamel with biofilm, (2) enamel with biofilm, (3) resin-infiltrated enamel, (4) no treatment (control). Enamel specimens were produced using bovine incisors. In brief, crowns were cut off from the roots and stored in a 0.1% thymol solution (VWR International, Dietikon, Switzerland) for no longer than 6 month. Cylindrical specimens (4 mm in diameter) were punched out from each crown and ground stepwise from 1200 to 4000 Fepa P (1200, 2400, 4000 grit, Water Proof silicon carbide Paper, Struers, Erkath, Germany).

Initial carious lesions with intact surface layer were created in all enamel specimens by the following demineralization procedure. Briefly, specimens were immersed for 14 days in an acidic buffer with traces of thymol at a pH of 5 and 37 °C . The solution was renewed every second day to maintain a constant pH. After demineralization, control specimens (n = 6) were cut in 100 μm slices and demineralization depth and intact surface layer were controlled using transverse microradiography. In this study, lesions of about 200 μm were used. Resin infiltration with Icon (DMG, Hamburg, Germany), a caries infiltrant system, was performed for specimens of group (1) and (3) according to the manufacturer’s instructions. In brief, 15% hydrochloric acid was used to etch the surfaces for 2 min (Icon Etch, DMG) and removed by 30 s water rinsing and subsequent air-drying. Specimens were then rinsed with 99% ethanol (Icon Dry, DMG) for 30 s and air-dried. 0.75 μL resin (Icon Infiltrant, DMG) was applied for 3 min. Excess material was removed with a cotton roll prior to light-curing for 40 s. This was followed by a second application of 0.75 μL of resin for 60 s and gentle cotton roll application. Light curing followed again for 40 s (800 W/cm 2 bluephase, Ivoclar Vivadent, Schaan, Liechtenstein). Subsequently, specimens were fine grinded using a holder with an integrated spring and silicon carbide discs (4000 Fepa P) for 10 s to remove the oxygen inhibition layer under constant pressure. Specimens of all groups underwent gas sterilization for 16 h (Ethylene oxide, 3 M™ Steri-Gas™ Cartridges, Healthcare, Rüschlikon, Switzerland) and were stored sterile in a moist chamber until further treatment.

For biofilm formation in groups (1) and (2), three-species biofilms were grown on specimens for 24 h. The bacterial strains ( Streptococcus mutans OMZ 918, Streptococcus oralis OMZ 60, Actinomyces oris OMZ 745) were provided by the Institute for Oral Biology, Section for Oral Microbiology and General Immunology, University of Zurich, Zurich, Switzerland. Prior to bacterial incubation, specimens were immersed in diluted saliva supernatant to form a pellicle. One healthy subject donated fresh whole mouth saliva, which was used in all experiments. Donated non-stimulated saliva was centrifuged twice for 30 min (12′100 g). The supernatant was diluted 1:2 in 0.9% NaCl (Braun, Melsungen, Germany) and subsequently sterile filtrated (TPP syringe filters with 0.2 μm pores, Faust, Schaffhausen, Switzerland). For pellicle formation, specimens were incubated with gentle agitation in 800 μL diluted saliva supernatant for 4 h. Bacterial strains in a mixture of 30% diluted saliva supernatant and 70% modified fluid universal medium (mFUM) were adjusted to an OD 550 of 1 and mixed as inoculum. Pellicle-coated specimens were incubated during gentle agitation in 2 mL inoculum. Incubation was performed anaerobically in jars using gas-paks for 24 h at 37 °C (GENbox anaer and GENbag anaer, bioMérieux, Marcy l’Etoile, France). After 6 h, specimens were transferred to new wells with fresh medium and analyzed after 24 h incubation, whereas expended media (after 6 and 24 h incubation) were subjected to high performance liquid chromatography (HPLC).

HPLC

Analysis of uncured Icon Infiltrant (DMG) was carried out using HPLC on an Agilent 1100 LC/MS (Agilent Technologies, Basel, Switzerland). An Agilent column (ZORBAX Eclipse XDB-C8, 4.6 × 150 mm, 5 μm) with a 50/50 mixture of acetonitrile and water at a flowrate of 0.75 mL/min and a run time of 10 min was used. Identification of uncured Icon Infiltrant, namely TEGDMA, was performed with single ion detection at 309 m / z after diluting the experimental media (n = 12) with 50 vol% of a methanol-water (Millipore) mixture (80/20). To quantify the amount, the chromatogram peak area was compared with a calibration curve (linear range, R 2 = 0.998) of mere TEGDMA (95%). All chemicals for HPLC analysis were purchased from Sigma Aldrich (Buchs, Switzerland) and were of HPLC analytical grade, except otherwise stated.

Bacteria counts and metabolic activity

Plate counts were performed for specimens w/wo resin infiltration and subjected to biofilm (groups 1 and 2, n = 8) after 24 h. Specimens were sonified in 1 mL 0.9% NaCl and vortexed. 50 μL of different bacterial dilutions (in 0.9% NaCl) were plated out on Columbia sheep blood agar plates (CSBA, bioMérieux, Geneve, Switzerland) and incubated under anaerobic conditions using gas-paks. Plate counting followed after 2 days of incubation using a light microscope with 10-fold magnification (Wild Stereoskop, Heerbrugg, Switzerland).

Bacterial metabolic activity of groups (1) and (2) was measured after 24 h (n = 10). Specimens were transferred into 96-well plates and incubated in 300 μL resazurin solution consisting of 10 vol% alamarBlue Cell Viability Assay Reagent (Life Technologies, Zug, Switzerland) and fresh media (30% saliva solution + 70% mFUM) under anaerobic conditions. Two wells were additionally filled with blank resazurin solution (without specimens for background detection) and one well with bacteria from the planktonic inocula. After 15 min, 200 μL of each solution was pipetted into new 96-well plates and fluorescence was measured in a spectrophotometer with plate-reader at 560 nm excitation/585 nm emission at 37 °C (Spectramax M2, Molecular Devices, Bucher Biotec, Basel, Switzerland). Results were presented as relative fluorescence units (rfu) after background subtraction.

Bacteria imaging

Two specimens of each group were analyzed by scanning electron microscopy (SEM) after 24 h of incubation (SUPRA 50VP and Genesis, Carl Zeiss, Oberkochen, Germany). Briefly, specimens were washed with 0.9% NaCl solution and fixed for at least 24 h in 4% glutaraldehyde solution (in 0.1 M sodium potassium phosphate buffer, pH 7). A dehydration procedure followed and subsequent critical point drying was performed. Specimens were coated by gold sputtering for 60 s. Images were taken to show the detailed surface characteristics’.

Biofilms and resin-infiltrated surfaces were examined using confocal laser scanning microscopy (CLSM) after 24 h of incubation. Two specimens of each group were washed in 0.9% NaCl to remove loosely bound bacteria, fixed with 4% paraformaldehyde in the dark at room temperature for 60 min and washed again with 0.9% NaCl. Fluorescence staining was performed using Syto 59 (Life Technologies) to visualize all bacteria. The detailed staining procedure has been described elsewhere . In brief, each specimen was incubated in 500 μL staining solution consisting of 5 μM Syto 59 for 15 min at room temperature in the dark. After staining, specimens were washed in phosphate buffered saline (PBS, Invitrogen, Carlsbad, CA, USA) and mounted onto chamber slides (Fisher Scientific, Schwerte, Germany) using Mowiol 4-88 (Sigma Aldrich). Images were taken with a CLSM (SP5, Leica Microsystems, Heidelberg, Germany) using a 20× objective (numerical aperture: 1.25) and a helium laser (561 nm). Emission was detected with a photomultiplier between 630 and 660 nm. Three random areas of each specimen were examined with a z step size of 1 μm (512 × 512 pixels). Image processing was performed using Imaris Software 7.7.2 (Bitplane, Zurich, Switzerland).

Data presentation analysis

Leakage of TEGDMA monomers was compared between the resin-infiltrated specimens with biofilm (group 1) and without biofilm (group 3) after 6 h and after 24 h. Plate counts and relative fluorescence units were separately analyzed after 24 h between group (1) and group (2). All values were not normally distributed and Wilcoxon/Kruskal-Wallis test was applied for non-parametrical analysis. JMP (version 10, SAS, Cary, NC, USA) was used for statistical analysis. The overall level of significance was set at p ≤ 0.05.

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Jun 19, 2018 | Posted by in General Dentistry | Comments Off on Oral biofilm and caries-infiltrant interactions on enamel
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