2.1

Bacterial strains and culture conditions

Standard reference strains of S. oralis CECT 907T, V. parvula NCTC 11810, A. naeslundii ATCC 19039, Fusobacterium nucleatum DMSZ 20482, Aggregatibacter actinomycetemcomitans DSMZ 8324 and Porphyromonas gingivalis ATCC 33277 were used. These bacteria were grown on blood agar plates (Blood Agar Oxoid No 2; Oxoid, Basingstoke, UK), supplemented with 5% (v/v) sterile horse blood (Oxoid), 5.0 mg L ⁻¹ hemin (Sigma, St. Louis, MO, USA) and 1.0 mg L ⁻¹ menadione (Merck, Darmstadt, Germany) in anaerobic conditions (10% H ₂ , 10% CO ₂ , and balance N ₂ ) at 37 °C for 24–72 h.

2.2

Material specimens

Sterile discs of 5 mm of diameter made of two different surface materials were used: (1) titanium with a SLA grade 2 surface (Ti-SLA) (Sand-blasted, Large grit, Acid-etched; Straumann; Institut Straumann AG, Basel, Switzerland), and (2) sterile zirconium oxide (ZrO ₂ ), with a rough micro surface obtained after chemical treatment with a hot solution of hydrofluoric acid, according to a proprietary process of Institut Straumann AG (Institut Straumann AG, Basel, Switzerland). The resulting rough surface topography of ZrO ₂ discs has a S _a value of 0.55 mm (standard deviation, SD = 0.01) with a rough surface topography similar the Ti-SLA surface implants when evaluated with scanning electron microscopy (SEM), although zirconium surfaces seemed to have a flatter profile with less porosity [ S _a value of Ti-SLA surface of 1.17 mm (SD = 0.04)] .

2.3

Saliva preparation

Un-stimulated saliva was obtained from healthy volunteers in 10 mL aliquots at least 1.5 h after eating, drinking or tooth brushing. Each saliva sample was treated with 2.5 mmol L ⁻¹ DL–Dithiothreitol (Sigma) for 10 min with continuous stirring in order to reduce salivary protein aggregation. It was then centrifuged (10 min, 4 °C and 12,000 rpm) and the obtained supernatant was diluted (1:1) with phosphate buffered saline (PBS; pH = 7.4). The sample was then filtered and sterilized through a 0.22 μm pore size Millex GV low-protein-binding filter X50 (Millipore, Millipore Corporation Bedford, USA) and stored at −20 °C. The efficacy of this protocol was validated by plating processed saliva samples onto supplemented blood agar plates for 72 h at 37 °C, when confirmed by lack of any bacterial growth on either aerobically or anaerobically incubated plates.

2.4

Biofilm development

Biofilms were generated using the method described by Sánchez et al. with slightly different bacterial concentrations when preparing the bacterial suspension. Briefly, planktonic cultures of each bacteria were grown anaerobically in a protein-rich medium containing brain–heart infusion (BHI) (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) supplemented with 2.5 g L ⁻¹ mucin (Oxoid), 1.0 g L ⁻¹ yeast extract (Oxoid), 0.1 g L ⁻¹ cysteine (Sigma), 2.0 g L ⁻¹ sodium bicarbonate (Merck), 5.0 mg L ⁻¹ hemin (Sigma), 1.0 mg L ⁻¹ menadione (Merck) and 0.25% (v/v) glutamic acid (Sigma). Upon mid-exponential phase, the cells were mixed in modified BHI medium containing 10 ³ colony forming units (CFU) mL ⁻¹ for S. oralis , 10 ⁵ CFU mL ⁻¹ for V. parvula and A. naeslundii, and 10 ⁸ CFU mL ⁻¹ for F. nucleatum, A. actinomycetemcomitans and P. gingivalis . Ti-SLA and ZrO ₂ sterile discs were coated with treated saliva for 4 h at 37 °C in sterile plastic tubes, and then placed in the wells of a 24-well tissue culture plate (Greiner Bio-one, Frickenhausen, Germany). Each well was inoculated with 1.5 mL mixed bacteria suspension prepared and incubated in anaerobic conditions (10% H ₂ , 10% CO ₂ , and balance N ₂ ) at 37 °C for 72 h. Plates containing only culture medium were also incubated to check for sterility.

2.5

SEM analysis

Before SEM analysis, three discs of each material (Ti-SLA and ZrO ₂ ) covered with biofilms grown in vitro for 72 h were fixed in 4% paraformaldehyde and 2.5% glutaraldehyde for 4 h at 4 °C. After fixation, the discs were washed twice in PBS and again twice in sterile water (immersion time per washed, 10 min) and dehydrated through a series of graded ethanol solutions (50, 60, 70, 80, 90 and 100%; immersion time per series, 10 min). Then, the samples were critical point dried, sputter-coated with gold and analyzed by electron microscopy JSM 6400 (JSM6400; JEOL, Tokyo, Japan), with back-scattered electron detector and an image resolution of 25 KV. Analyses were carried out at ICTS National Centre of Electronic Microscopy (Campus of International Excellence Moncloa, University Complutense, Madrid, Spain).

2.6

Exposure to antimicrobial compounds

The following commercially available antiseptic mouth rinse formulations were used: (1) 0.12% CHX and 0.05% CPC without alcohol (CHX/CPC) (Perio-Aid tratameiento ^® ; Dentaid, Cerdanyola, Spain), (2) a combination of four EEOOs (thymol 0.06%, eucalyptol 0.09%, methyl salicylate 0.06% and menthol 0.01%) without alcohol (Listerine ^® Zero; Johnson & Johnson, Madrid, Spain), an (3) 0.05% CPC without alcohol (Oral B-ProExpert, Procter & Gamble, Weybridge, Surrey, UK). PBS was used as a negative control solution.

To monitor the bactericidal action of the three tested mouth rinses on 72 h biofilms, the discs were immersed during 1 min in the antiseptic solution and in PBS as control, with and without mechanical disruption by means of agitation through vortex at room temperature, which provided constant stirring at 90 rpm. After this exposure, the discs were sequentially rinsed in 2 mL of sterile PBS (immersion time per rinse, 10 s), three times, to remove the antiseptic solutions.

In each experiment, the three antimicrobial agents and the control solutions were tested with and without mechanical disruption (agitation). These experiments were repeated three times on different days using fresh bacterial cultures with trios of biofilms for each independent outcome variables.

2.7

Microbiological outcomes

After the antimicrobial treatment, biofilms were disrupted by vortex for 2 min in 1 mL of PBS. To discriminate between DNA from live and dead bacteria, propidium monoazide (PMA) (Biotium Inc., Hayword, CA, USA) was used. The use of this PMA dye has shown the ability to distinguish between viable and irreversibly damaged cells and hence when combined with quantitative polymerase chain reaction (qPCR) to detect the DNA from viable bacteria . PMA was added to sample tubes containing 250 μL of disaggregated biofilm cells, at a final concentration of 100 μM. Following an incubation period of 10 min at 4 °C in the dark, the samples were subjected to light-exposure for 20 min, using a 550 W halogen light source, placed 20 cm above the samples. During this exposure, the sample tubes were laid horizontally on ice to avoid excessive heating. After PMA photo-induced DNA cross-linking, the cells were centrifuged at 12,000 rpm for 3 min prior to DNA isolation. To control for any possible influence of the experimental process on bacterial viability, the same procedure (incubation at 4 °C and exposure to light source) but without the exposure to PMA, was used as negative control.

Bacterial DNA was isolated from all biofilms using a commercial kit (MolYsis Complete5; Molzym GmbH & CoKG, Bremen, Germany), following manufacturer’s instructions (the protocol for bacterial DNA extraction was followed from step 6, avoiding preliminary steps) and the hydrolysis 5′nuclease probe assay qPCR method was used for detecting and quantifying the bacterial DNA. The qPCR amplification was performed following a protocol previously optimized by our research group, using primers and probes targeted against 16S rRNA gene [obtained through Life Technologies Invitrogen (Carlsbad, CA, USA) and Applied Biosystems (Carlsbad, CA, USA) .

Each DNA sample was analyzed in duplicate. Quantification cycle (Cq) values, previously known as cycle threshold (Ct) values, describing the PCR cycle number at which fluorescence rises above the baseline, were determined using the provided software package (LC 480 Software 1.5; Roche Diagnostic GmbH; Mannheim, Germany). Quantification of viable cells by qPCR was based on standard curves . The correlation between Cq values and CFU/mL was automatically generated through the software (LC 480 Software 1.5; Roche).

All assays were developed with a linear quantitative detection range established by the slope range of 3.3–3.6 cycles/log decade, r ² > 0.997 and an efficiency range of 1.9–2.0.

Measures to avoid carryover DNA were established. In spite of this, when non-template control (NTC) was detectable, the limit of detection was established on the last value of the standard curve that holds five cycles of difference with NTC.

2.8

Statistical analyses

The following independent variables were considered: (1) the material surface (Ti-SLA and ZrO ₂ ), (2) the mechanical disruption applied by constant agitation (with or without), (3) type of antiseptic/control (PBS, CHX/CPC, EEOOs or CPC), and (4) their interaction. The number of viable bacteria present on in vitro biofilms formed on SLA titanium and zirconium oxide surfaces and measured as viable CFU/mL of A. actinomycetemcomitans , P. gingivalis and F. nucleatum was the primary dependent outcome variable.

An experiment-level analysis was performed for each study parameter (n = 9 or 72 for qPCR results). Shapiro–Wilk goodness-of-fit tests and distribution of data were used to assess normality. Data were expressed as means and 95% confidence intervals (CI).

In order to compare the effect of material surface, type of antiseptic with or without mechanical disruption, and their interaction on the main outcome variable (CFU/ml) a general lineal model was constructed for each bacterium ( A. actinomycetemcomitans , P. gingivalis and F. nucleatum ) using the method of maximum likelihood and Bonferroni corrections for multiple comparisons.

Results were considered statistically significant at p < 0.05. A software package (IBM SPSS Statistics 21.0; IBM Corporation, Armonk, NY, USA) was used for all data analysis.