2.1

Copolymer sample preparation

Cold cure acrylic resin (Wintercryl Rapid Repair; John Winter & Co., Halifax, UK) was used, and 40% of the MMA was replaced by HEMA (Sigma Aldrich, St. Louis, USA). For the fluoridated copolymer, 30% of the PMMA was replaced by an equal mass of NaF (Sigma Aldrich). The mixtures were transferred to custom-made plastic molds to produce either disc shapes for fluoride measurements under static conditions or stubs of dimensions to fit the Modified Robbins Device (MRD), and allowed to set by chemically-activated free radical polymerization for 48 h.

2.2

Fluoride release and recharge

Fluoride release from autoclaved samples was measured under static conditions or in some cases, from samples placed in the MRD under liquid flow. Fluoride was measured with an ion selective electrode (Orion Research, Thermo Scientific, MA, USA), after adding 500 μl acetate buffer (TISAB III, Orion Research, MA, USA) to 5 ml of the storage medium. For static conditions, five flat disc shaped samples of each copolymer were incubated independently in deionized water. Storage water was changed 24 h prior to measurements being taken and fluoride release over 24 h was determined by measuring the fluoride content in the water. For measurements of fluoride release under flowing conditions, a single stub was placed in the MRD system, and water was introduced at a rate of 40 ml h ⁻¹ . Fluoride was measured in the effluent periodically up to 48 h, and the experiment was repeated three times.

For fluoride recharging, freshly prepared fluoridated copolymer specimens were washed daily and placed into fresh water in order to take out as much fluoride as possible from the material. Before recharging, the daily fluoride release from all specimens was measured. Samples were sonicated in deionized water containing 0.2% w/v NaF for 3 min in an ultrasonic bath, rinsed with distilled water, dried and incubated at 37 °C in 5 ml of fresh deionized water. Fluoride release was measured daily for one week. Samples were incubated in deionized water for one further week and the storage water changed 24 h prior to a second recharge cycle, to facilitate measurement of daily fluoride release.

2.3

Growth media and microbial culture

Microbial strains used in this study were C. albicans VPSA1 , L. casei ATCC334 and S. mutans UA159. C. albicans was cultured aerobically in Yeast Extract-Peptone-Dextrose (YPD) medium (Sigma-Aldrich) at 37 °C. L. casei was cultured in Rogosa medium (Sigma-Aldrich) in a candle jar at 37 °C, and S. mutans was cultured in Tryptone Yeast Cystine (TYC) medium (Sigma-Aldrich) supplemented with 20% (w/v) sucrose in a candle jar at 37 °C. Solidified media were prepared by adding 15 g l ⁻¹ Bacto-Agar (Difco, Oxford, UK). Artificial saliva was employed for culturing organisms in the MRD, and sucrose was added as described below.

Standardised inocula were prepared for the MRD by culturing C. albicans , L. casei or S. mutans to mid-exponential phase, harvesting and re-suspending in one tenth volume of the appropriate growth medium supplemented with 25% (v/v) glycerol (Sigma-Aldrich). Cells were stored at −80 °C, and an aliquot was removed for measuring total viable counts (TVCs) by the method of Miles and Misra . Briefly, serial ten-fold dilutions were prepared in PBS (pH 7.4, Sigma-Aldrich) and triplicate 20 μl aliquots were added to solidified media. C. albicans was incubated aerobically at 37 °C for 24 h, while L. casei or S. mutans were incubated at 37 °C in a candle jar for 48 h. Colonies were counted from dilutions that contained between 10 and 100 colonies. For enumeration of individual species in mixed cultures, selective media were used as follows: YPD, counted at 24 h for C. albicans ; Rogosa Agar containing 21.6 μg ml ⁻¹ natamycin (Sigma-Aldrich) for L. casei ; and TYC Agar containing 21.6 μg ml ⁻¹ natamycin and 0.1 unit ml ⁻¹ bacitracin (Sigma-Aldrich) for S. mutans .

2.4

The Modified Robbins Device model biofilm system

Two 12-port polysulfone MRDs (Tyler Research, Alberta, Canada) were assembled with stubs of copolymer, sterilized by autoclaving for 15 min at 121 °C, and arranged in parallel at 37 °C ( Fig. 1 ). Fluoridated copolymer stubs were included in one device, and control stubs (non-fluoridated) in the other. To coat surfaces with human saliva, parafilm-stimulated saliva was collected from six different volunteers who had not consumed food or drink other than water in the previous hour and had not taken antibiotics in the previous three months. Dithiothreitol (Sigma-Aldrich, St. Louis, Mo.) was added to a final concentration of 2.5 mM and stirred gently on ice for 10 min. Samples were centrifuged at 15,000 g , 4 °C for 30 min and filter sterilized. The MRD was conditioned by the addition of 20 ml saliva at a rate of 40 ml h ^‐1 and inoculated with 1 × 10 ⁵ C. albicans cells, 1 × 10 ⁷ L. casei cells, and/or 1 × 10 ⁷ S. mutans cells. Artificial saliva was then flowed through the model at 40 ml h ^‐1 . At time points up to 48 h, stubs were removed and microbial biofilms were recovered by scraping the surfaces into 1 ml PBS (pH 7.4) or were prepared for microscopy as detailed below. Measurements of pH were made using a Mettler Toledo S20 SevenEasy pH meter.

Design of the MRD biofilm system. Biofilms are formed on copolymer pieces held within a plug and flush against the wall of the vessel. The samples were conditioned with natural saliva, and then inoculum was introduced. Artificial saliva and sucrose were added as required. The flow of liquid was controlled using clamps and a peristaltic pump.

2.5

Quantification of microorganisms by qPCR

For enumeration of microorganisms by qPCR, DNA was extracted using the ZR Fungal/Bacterial DNA extraction kit (Zymo Research Co., CA, USA). The following previously published oligonucleotide primers and TaqMan probes were used to quantify bacterial biofilm species: Smut3368-F, Smut3481-R, and Smut3423T (for S. mutans , targeting the gtfB gene) and L_case_IS, R_case_IS, and P_case_IS (for L. casei , targeting the 16S-23S intergenic region). For C. albicans VPSA1, the region surrounding the 5.8S rRNA gene was amplified by PCR using primers CaFP1 5′-GGAACCGAGAAGCTGGTCAA-3′ and CaRP1 5′-GTCATCTCATCGCACGGGAT-3′. The PCR product was cleaned using the EZ-10 Spin Column PCR Products Purification kit (NBS Biological, Huntingdon, UK), and sent for sequencing (Eurofins Genomics, Ebersberg, Germany). The sequence was deposited in GenBank (accession K J 39863). Based on this sequence, primers and TaqMan probe sequences for C. albicans were adapted from , and were: CaFP2 5′-GGGTTTGCTTGAAAGACGGTA-3′, CaRP2 5′-TGAAGATATACGTGGTAGACGTTAG-3′, Ca-probe 5′-ACCTAAGCCATTGTCAAAGCGATCCCG-3′). The probe was modified with FAM-6 fluorescence reporter at the 5′ end and MGBNFQ fluorescence quencher at the 3′ end.

Standards for qPCR were constructed by amplifying qPCR target regions from each microorganism. The amplified products were cloned in pCR2.1 (Invitrogen) to generate plasmids pTOPO- Ca , pTOPO- Lc and pTOPO- Sm , respectively, and used to transform Escherichia coli TOP10 cells (Invitrogen). Plasmids were extracted from E. coli using the Qiagen Plasmid Mini Kit, and concentrations were determined by staining with Quant-iT™ PicoGreen dsDNA Reagent and Kit (Invitrogen). Amplification and detection of DNA by qPCR were performed with an Opticon 2 DNA Engine (Bio-Rad Laboratories Ltd., Hertfordshire, UK) using optical grade 96-well plates (Eurogentec, Belgium). Quantitative PCR reactions were prepared, and contained the following reagents in each well: forward primer (stock concentration, 2.5 μM) 1.2 μl, reverse primer (2.5 μM) 1.2 μl, probe (2.5 μM) 0.6 μl, 2X Takara master mix 7.5 μl (Quantace, London, UK), H ₂ O 3.3 μl and template DNA 1.0 μl. The thermocycling conditions were as follows: initial denaturation at 95 °C for 2 min, denaturation at 95 °C for 5 s, annealing/extension at 60 °C for 30 s, plate read and cycle repeated 40 times. All the reactions were carried out in triplicate and the final analysis was based on the mean of the three reactions. The qPCR data analysis was carried out using Opticon Monitor 3 software (MJ Research). The quantity of template DNA in each sample well was calculated by reference to wells containing serial 10-fold dilutions of standards of known DNA concentration and corrections were made for target sequence copy number based on the assumption that individual cells contained 110 copies of C. albicans ITS2 , 5 copies of L. casei 16S-23S rRNA , or 1 copy of S. mutans gtfB .

2.6

Scanning electron microscopy (SEM)

For SEM, samples were fixed in 2% (v/v) glutaraldehyde at 4 °C for 16 h, rinsed with PBS (pH 7.4) and dehydrated through a series of ethanol washes as follows: 25% ethanol for 30 min, 50% ethanol for 30 min, 75% ethanol for 30 min, and two washes for 1 h in 100% ethanol. Samples were dried in a critical point dryer (Bal-tec, Reading, UK), mounted on aluminium stubs and sputter coated with gold at Electron Microscopy Research Services, Newcastle University. Biofilms were visualized using a Stereoscan 240 scanning electron microscope (Cambridge Instruments, Cambridge, UK).

2.7

Confocal scanning laser microscopy (CSLM)

For CSLM, the Live/Dead BacLight Bacterial Viability Kit (Life technologies Ltd., Paisley, UK) was used according to the manufacturer’s instructions to stain single-species biofilms. Mixed-species biofilms were stained with 50 μg ml ⁻¹ Alexa Fluor 488-conjugated Concanavalin A (ConA) (Life technologies Ltd., Paisley, UK), 5 μg ml ⁻¹ propidium iodide (Sigma Aldrich, St. Louis, USA) and 25 μg ml ⁻¹ 4′,6-diamidino-2-phenylindole (DAPI) (Life technologies Ltd., Paisley, UK). Biofilms were examined and image stacks collected using a Leica TCS SP2 confocal laser scanning microscope with a x63HCX PL APO oil immersion objective lens (numerical aperture 1.32) and an argon/neon laser for visualisation of SYTO 9 (excitation 488 nm, emission 530 nm), propidium iodide (excitation 530 nm, emission 630 nm), ConA (excitation 485 nm, emission 519 nm), and DAPI (excitation 358 nm, emission 461 nm). The collected image stacks of the biofilms were computationally rendered in three-dimensions using IMARIS Ver 7.3.1 software (Bitplane, Zurich, Switzerland) on a MSI PC computer (MSI computer corp, City of Industry, CA) equipped with a Radeon HD5850 graphics card (AMD, Sunnyvale, CA).

2.8

Statistical analyses

In each case, means and standard deviations were based on three independent biological experiments. Student’s unpaired two-sample T test was used for all statistical comparisons between means and p < 0.05 was considered statistically significant. Spearman’s rank correlation coefficients were calculated to assess correlations between qPCR and TVCs. All statistical tests were performed in Sigmaplot (Systat Software Inc., San Jose, CA).

2.1

Copolymer sample preparation

Cold cure acrylic resin (Wintercryl Rapid Repair; John Winter & Co., Halifax, UK) was used, and 40% of the MMA was replaced by HEMA (Sigma Aldrich, St. Louis, USA). For the fluoridated copolymer, 30% of the PMMA was replaced by an equal mass of NaF (Sigma Aldrich). The mixtures were transferred to custom-made plastic molds to produce either disc shapes for fluoride measurements under static conditions or stubs of dimensions to fit the Modified Robbins Device (MRD), and allowed to set by chemically-activated free radical polymerization for 48 h.

2.2

Fluoride release and recharge

Fluoride release from autoclaved samples was measured under static conditions or in some cases, from samples placed in the MRD under liquid flow. Fluoride was measured with an ion selective electrode (Orion Research, Thermo Scientific, MA, USA), after adding 500 μl acetate buffer (TISAB III, Orion Research, MA, USA) to 5 ml of the storage medium. For static conditions, five flat disc shaped samples of each copolymer were incubated independently in deionized water. Storage water was changed 24 h prior to measurements being taken and fluoride release over 24 h was determined by measuring the fluoride content in the water. For measurements of fluoride release under flowing conditions, a single stub was placed in the MRD system, and water was introduced at a rate of 40 ml h ⁻¹ . Fluoride was measured in the effluent periodically up to 48 h, and the experiment was repeated three times.

For fluoride recharging, freshly prepared fluoridated copolymer specimens were washed daily and placed into fresh water in order to take out as much fluoride as possible from the material. Before recharging, the daily fluoride release from all specimens was measured. Samples were sonicated in deionized water containing 0.2% w/v NaF for 3 min in an ultrasonic bath, rinsed with distilled water, dried and incubated at 37 °C in 5 ml of fresh deionized water. Fluoride release was measured daily for one week. Samples were incubated in deionized water for one further week and the storage water changed 24 h prior to a second recharge cycle, to facilitate measurement of daily fluoride release.

2.3

Growth media and microbial culture

Microbial strains used in this study were C. albicans VPSA1 , L. casei ATCC334 and S. mutans UA159. C. albicans was cultured aerobically in Yeast Extract-Peptone-Dextrose (YPD) medium (Sigma-Aldrich) at 37 °C. L. casei was cultured in Rogosa medium (Sigma-Aldrich) in a candle jar at 37 °C, and S. mutans was cultured in Tryptone Yeast Cystine (TYC) medium (Sigma-Aldrich) supplemented with 20% (w/v) sucrose in a candle jar at 37 °C. Solidified media were prepared by adding 15 g l ⁻¹ Bacto-Agar (Difco, Oxford, UK). Artificial saliva was employed for culturing organisms in the MRD, and sucrose was added as described below.

Standardised inocula were prepared for the MRD by culturing C. albicans , L. casei or S. mutans to mid-exponential phase, harvesting and re-suspending in one tenth volume of the appropriate growth medium supplemented with 25% (v/v) glycerol (Sigma-Aldrich). Cells were stored at −80 °C, and an aliquot was removed for measuring total viable counts (TVCs) by the method of Miles and Misra . Briefly, serial ten-fold dilutions were prepared in PBS (pH 7.4, Sigma-Aldrich) and triplicate 20 μl aliquots were added to solidified media. C. albicans was incubated aerobically at 37 °C for 24 h, while L. casei or S. mutans were incubated at 37 °C in a candle jar for 48 h. Colonies were counted from dilutions that contained between 10 and 100 colonies. For enumeration of individual species in mixed cultures, selective media were used as follows: YPD, counted at 24 h for C. albicans ; Rogosa Agar containing 21.6 μg ml ⁻¹ natamycin (Sigma-Aldrich) for L. casei ; and TYC Agar containing 21.6 μg ml ⁻¹ natamycin and 0.1 unit ml ⁻¹ bacitracin (Sigma-Aldrich) for S. mutans .

2.4

The Modified Robbins Device model biofilm system

Two 12-port polysulfone MRDs (Tyler Research, Alberta, Canada) were assembled with stubs of copolymer, sterilized by autoclaving for 15 min at 121 °C, and arranged in parallel at 37 °C ( Fig. 1 ). Fluoridated copolymer stubs were included in one device, and control stubs (non-fluoridated) in the other. To coat surfaces with human saliva, parafilm-stimulated saliva was collected from six different volunteers who had not consumed food or drink other than water in the previous hour and had not taken antibiotics in the previous three months. Dithiothreitol (Sigma-Aldrich, St. Louis, Mo.) was added to a final concentration of 2.5 mM and stirred gently on ice for 10 min. Samples were centrifuged at 15,000 g , 4 °C for 30 min and filter sterilized. The MRD was conditioned by the addition of 20 ml saliva at a rate of 40 ml h ^‐1 and inoculated with 1 × 10 ⁵ C. albicans cells, 1 × 10 ⁷ L. casei cells, and/or 1 × 10 ⁷ S. mutans cells. Artificial saliva was then flowed through the model at 40 ml h ^‐1 . At time points up to 48 h, stubs were removed and microbial biofilms were recovered by scraping the surfaces into 1 ml PBS (pH 7.4) or were prepared for microscopy as detailed below. Measurements of pH were made using a Mettler Toledo S20 SevenEasy pH meter.

Design of the MRD biofilm system. Biofilms are formed on copolymer pieces held within a plug and flush against the wall of the vessel. The samples were conditioned with natural saliva, and then inoculum was introduced. Artificial saliva and sucrose were added as required. The flow of liquid was controlled using clamps and a peristaltic pump.

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