Interproximal biofilm removal by intervallic use of a sonic toothbrush compared to an oral irrigation system

Bacterial strains were obtained from the Institute for Oral Biology, Section for Oral Microbiology and General Immunology, University of Zürich, Zürich, Switzerland. Before biofilm formation, the strains (Streptococcus mutans OMZ 918, Streptococcus oralis OMZ 607, Actinomyces naeslundii OMZ 745) were gained from precultures streaked on Columbia sheep’s blood agar (CSBA) plates (bioMérieux, Marcy l’Etoile, France). Colonies were propagated planktonic in a substrate composed of 30 % saliva solution and 70 % modified fluid universal medium (mFUM) [14] separately on a rocker at 37 °C in jars using gas-paks to create anaerobic conditions (GENbox anaer and GENbag anaer, bioMérieux, Marcy l’Etoile, France). Therefore, fresh saliva was gained by one healthy donor and centrifuged two times for 30 min by 13400 rpm. Following the opinion of the Ethics Committee of the Canton of Zurich, Switzerland, no ethical approval is needed for the donation of saliva as explained above (no. 0324/2013 and no. 50/14). The pellet was removed each time and the remaining supernatant was diluted 1:2 in sodium chloride (0.9 % NaCl) prior to sterile filtration (TPP syrenge filters with 0.2 μm pores, Faust, Schaffhausen, Switzerland). The resulting saliva solution was used in all experimentations. FUM, a well-established tryptone-yeast based broth medium was described by Loesche et al. [15]. FUM contained (per liter of distilled water): 10 g of tryptone, 5 g of yeast extract, 3 g of glucose, 2 mg of hemin, 1 mg of menadione, 0.5 g of cysteine hydrochloride, 0.1 g of dithiothreitol, 2.9 g of 0.9 % NaCl, 0.5 g of Na₂CO₃, 1 g of KNO₃, 0.45 g of K₂HPO₄, 0.45 g of KH₂PO₄, 0.9 g of (NH₄)₂SO₄, and 0.188 g of MgSO₄ * 7H20. It was modified by supplementing 67 mmol/l Sørensen’s buffer to a final pH of 7.2. Glucose was replaced by 3 g of a 1:1 mixture of glucose and sucrose. The modification used in this study was adopted from the Zurich biofilm protocols [14]. After approximately 6 - 7 h the bacterial solutions were adjusted to the optical density (OD550) of 1 and mixed in a tube as inoculum. To quantify the inocula per ml, colony forming units (CFU) were plated out on CSBA plates and incubated anaerobically in jars using gas-paks (t = 2 d). In the meantime, sterile sintered hydroxyapatite discs (Ø 5 mm, Clarkson Chromatography Products, South Williamsport, USA) were incubated in 800 μl of non-stimulated saliva solution for 4 h at gentle agitation to form a pellicle (100 rpm at room temperature). For biofilm formation, pellicle-coated discs were then placed in new 24-well polystyrene cell culture plates and incubated with 1 ml of the prepared inocula during gentle agitation for 24 h in jars at 37 °C using gas-paks (GENbox anaer and GENbags anaer, bioMérieux, Marcy l’Etoile, France). Media was refreshed daily prior to treatment procedures and directly after treatment by transferring the specimens in new plates filled with fresh media (30 % saliva solution + 70 % mFUM). pH was controlled daily in the overnight medium directly after the first media change using a pH meter (Mettler-Toledo Easy Five, Mettler-Toledo AG, Schwerzenbach, Switzerland).

Specimens were divided into four groups. Three independent experiments were performed to obtain n = 18 specimens per group (first experiment n = 4, second n = 6, last experiment n = 8 specimens per group). Each experiment consisted of three treatment days (d1, d2, d3). Prior to each treatment, measurements of the metabolic activity were performed to obtain baseline values for each specimen. Then, specimens were placed carefully into an interproximal device with 2 specimens in a distance of 0.5 mm face to face (Fig. 1). The brushing device for electric toothbrushes was build in a co-operation between the Institute of Fluid Dynamics, ETH Zürich and the Department of Preventive Dentistry, Periodontology and Cariology of the University of Zurich, Switzerland. For experimentation, 25 ml water of 36 °C was pipetted into the device to cover the interproximal regions and the specimens. For the WF-group, the oral irrigator (Waterfloss, Waterpik® Sensonic WP-100E) was adjusted using the JT-100E Classic Jet Tip at a 90° angle towards the interproximal region as described in the manufacturer’s information. The pressure control was positioned at level 10 (highest water pressure) and activated for 10 s. Afterwards, the specimens were carefully taken from the interproximal device and restored in plates with 0.9 % NaCl. For the WPa-group, the sonic toothbrush (Waterpik® Sensonic SR-3000E) was adjusted onto the device using the respective standard brush head with a load of the brush head onto the interproximal region of < 0.9 N as measured for sonic toothbrushes (total load 70 ± 5 g) [2, 16]. The brushing was performed for 10 s under static conditions. For the specimens of the WPi-group, the procedures were repeated for the inactivated brushes (power off). Specimens without treatment were used as control group (CO).

Prior to experimentation, the alamarBlue assay was validated in preliminary tests [see Additional file 1]. The above-described inoculum was diluted 1:2 in phosphate buffered saline in seven series. Samples of each dilution series were determined by colony forming units, resulting in dilution series from 5.5 to 350 × 10⁶ CFU/ml. Ten samples of each series were then used for kinetic measurements. Therefore, samples were incubated in wells containing 10 vol.% alamarBlue Cell Viability Assay Reagent (Life technologies, Zug, Switzerland) and 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). Measurements were conducted every 15 min for 2 h. The measured relative fluorescence units of each dilution were plotted against the incubation time. The findings of these preliminary tests showed constant initial rates of the enzymatic reaction within a range of approximately 30 % of the total substrate conversion for each dilution curve [see Additional file 1]. To gain measurements within the range of linear increase for the current experimentations, the incubation time in the alamarBlue solution was set to 15 min.

For experimentation, biofilm coated hydroxyapatite discs were first stored in a new well plate with 0.9 % NaCl to avoid further growing during treatment. Then, specimens were transferred carefully into 96-well plates and incubated in 300 μl alamarBlue solution containing fresh media (30 % saliva solution + 70 % mFUM) with 10 vol.% alamarBlue under anaerobic conditions. Additionally, two wells were filled with blank alamarBlue solution (without specimens) and one well was filled with centrifuged bacteria from the planktonic inocula in alamarBlue solution to gain values for the maximal metabolic activity. After 15 min, 200 μl of each alamarBlue solution was pipetted into new 96-well plates and metabolic activity was measured in a Spectrophotometer with plate-reader at 560 nm excitation/585 nm emission. The resulting relative fluorescence units (rfu) were defined as baseline values or pre-treatment rfu. After measurements, specimens were stored in 0.9 % NaCl and further experiments were conducted (treatments using the different oral devices). Then, post-treatment values were obtained by using the alamarBlue assay as described before. After rfu-measurements, specimens were transferred to fresh substrate to enable regrowth and retreated with the identical procedures 24 h later. Therefore, specimens were distributed into the same groups. Altogether, each specimen underwent three treatment cycles (d1, d2, d3).

After the last treatment and measurement using alamarBlue (d3), three specimens of each group were analyzed additionally using CFU. Therefore, specimens were vortexed in 1 ml 0.9 % NaCl for 2 min and sonified for 5 s. Each bacterial suspension was diluted in 0.9 % NaCl and plated out on Columbia sheep’s blood agar (CSBA) plates (bioMérieux, Marcy l’Etoile, France). CSBA plates were then incubated in jars at 37 °C, using gas -paks (GENbag anaer, bioMeriux, Marcy l’Etoile, France) for 2 days.

Metabolic activity was analyzed for each disc separately, and the ratio of post-treatment to baseline values were compared within the treatment days (d1, d2, d3). Analysis of the data was performed using ANOVA with the post-hoc Scheffe test, or Kruskal-Wallis with post-hoc Mann–Whitney test.