Hygiene Tribune Middle East & Africa Edition

hygiene tribune Dental Tribune Middle East & Africa Edition | January-February 20154B Removal of interproximal dental biofilms by high-velocity Water Microdrops By A. Rmaile, D. Carugo, L. Capretto, M. Aspiras, M. De Jager, M. Ward and P. Stoodley A bstract The influence of the im- pact of a high-velocity water microdrop on the detach- ment of Streptococcus mutans UA159 biofilms from the inter- proximal (IP) space of teeth in a training typodont was studied experimentally and computa- tionally. Twelve-day-old S. mu- tans biofilms in the IP space were exposed to a prototype AirFloss delivering 115 μL wa- ter at a maximum exit velocity of 60 m/sec in a 30-msec burst. Using confocal microscopy and image analysis, we obtained quantitative measurements of the percentage removal of bio- films from different locations in the IP space. The3Dgeometryofthe typodont and the IP spaces was obtained by micro-computed tomography (μ-CT) imaging. We performed computational fluid dynamics (CFD) simulations to calculate the wall shear stress (τw ) distri- bution caused by the drops on the tooth surface. A qualitative agreement and a quantitative re- lationship between experiments and simulations were achieved. The wall shear stress (τw ) gen- erated by the prototype AirFloss and its spatial distribution on the teeth surface played a key role in dictating the efficacy of biofilm removal in the IP space. Key words: oral hygiene, Strep- tococcus mutans, micro-com- puted tomography, microscopy, interproximal cleaning, dental plaque. Introduction Good oral hygiene practice maintains a healthy oral cavity, controls the progress of dental plaque biofilms (ten Cate, 2006) and calculus, and prevents fur- ther complications such as gum diseases and tooth decay (Cos- terton et al., 1999; Jakubovics and Kolenbrander, 2010; Bjarn- sholt et al., 2011; Marsh et al., 2011). The challenge of dental care products is to efficiently and quickly remove plaque from the interproximal (IP) space. Me- chanical removal of IP plaque by traditional dental flossing products has been accompanied with bleeding, stuck or shred- ded floss, and prolonged flossing time (Darby, 2003). Fluid shear stress is an alternative mechani- cal approach for controlling biofilm build-up (Stewart, 2012). Previous studies have demon- strated that if sufficiently high fluid shear stress can be gener- ated, this alone can stimulate biofilm detachment (Rutter and Vincent, 1988; Hope et al., 2003; Sharma et al., 2005a; Paramon- ova et al., 2009). High-velocity water droplets (Cense et al., 2006) and entrained air bubbles (Parini et al., 2005; Sharma et al., 2005b) have also been shown to be able to remove bacteria and biofilms from surfaces utilizing the additional effect of gener- ating a “surface-tension force” way from the surface by the pas- sage of an air/water interface (Gómez-Suárez et al., 2001). An advantage of using fluid forces to remove biofilms is that me- chanical forces can be projected beyond the device itself, by gen- erating currents in the fluid sur- rounding the teeth by powered brushing (Adams et al., 2002) or through the generation of wa- ter jets by oral irrigation (Lyle, 2011). However, continuous wa- ter jets have a disadvantage of requiring large reservoirs and can be messy to use because of the large volumes of water involved. More recently, the Sonicare™ AirFloss device has been introduced for removing IP plaque. The AirFloss shoots a microdrop volume of water and entrained air at a high velocity into the IP space in a discrete burst, thus creating high wall shear stress (τw ) and high-im- pact pressure over short periods of time, minimizing water vol- ume and cleaning times. We previously reported the in- fluence of high-velocity water microdrop impact on the de- tachment of artificial plaque from the IP spaces, to demon- strate how a real biofilm might detach (Rmaile et al., 2013). Here, we go on to use the same in vitro model to look at bacterial biofilm removal and apply com- putational fluid dynamics (CFD) numerical techniques to model and predict the spatial distribu- tion of fluid wall shear stress (τw ) required to remove the biofilm. This paper reports the results of an experimental and numerical study on the influence of a high- velocity water microdrop impact on the detachment of Strepto- coccus mutans biofilms from the IP spaces of a typodont model. Materials & Methods Bacteria and Growth Media Biofilms were grown from S. mutans UA159 (ATCC 700610). Stock cultures of S. mutans were stored at -80o C in 10% glycerol in physiological buffered saline (PBS). Biofilms were cultured with sucrose (2% w/v) supple- mented brain heart infusion (BHI+S) medium (Sigma-Al- drich, Dorset, UK) and incubat- ed at 37o C and 5% CO2 . Typodont Model and Micro- burst To recreate a realistic geom- etry associated with the IP space, we grew biofilms on the 2 upper central incisors (teeth 8 and 9) removed from a train- ing typodont (A-PZ periodontal model 4030025, Frasaco GmbH, Tettnang, Germany) (Fig. 1A). A prototype AirFloss was used to generate a microburst of 115 μL (±50; n = 30) over a time period of approximately 0.033 sec (Ap- pendix I). CLSM Microscopy and Image Analysis The amount of biofilm on the IP surfaces of the typodont teeth was measured with a Leica TCS SP2 AOBS (Leica Microsystems, Nanterre, France) confocal laser scanning microscope (Fig. 2; Ap- pendix II). Micro-computed Tomography ( μ-CT) Geometry Reconstruction of the Typo- dont Model μ-CT was used to image the ty- podont in 3D and construct a model of the IP space to be used in subsequent CFD modeling (Fig. 1B; Appendix III). Streptococcus mutans Bio- films inside Microfluidic Channels To estimate a critical hydrody- namic shear stress required for S. mutans biofilm detachment, which could be used as a model input parameter for predicting the spatial distribution of biofilm removal, we used a BioFluxTM 1000 device (Fluxion Bioscienc- es, South San Francisco, CA, USA) (Appendix IV). Computational Fluid Dynam- ics Simulations To model the dynamic behavior of the microburst created within the IP space, the tomography ob- tained from μ-CT was converted to a 3D computer-aided design (CAD) file geometry with Amira software (Mercury Computer Systems, Fürth, Germany). The computational domain, repre- sented by the IP space, was dis- cretized with software Gambit 2.4.6 (Symetrix Inc., Mountlake Terrace, WA, USA) and using a tetrahedral meshing scheme. A cell size of ~155 μm was chosen, which led to a total number of 143,985 mesh tetrahedral cells. Since the IP space was symmet- rical, only half of it was modeled, reducing computational cost and time. CFD simulations were per- formedwithANSYSFluent12.1.4 software (ANSYS Inc., Canons- burg, PA, USA), which allowed for the determination of the flow field within the IP space and τw Figure 1. Digitization process of the training typodont. (A) Photograph showing the typodont used in the study. (B) Micro-CT image of the typodont (maxillary dental arch). (C) CAD-based 3D rendering of the IP space used in the study. (D) The 3D meshwork showing the geometry of the tooth sur- face that was used for the computational simulations. The sketch (right) shows the mesial view of a maxillary left central incisor, and the dashed square shows the region of interest used in the study. Figure 2. Representative CLSM images of S. mutans biofilm of 5 different lo- cations (A, B, C, D, E) across the IP space at the level of the prototype AirFloss tip from the proximo-labial to the proximo-palatal side of a maxillary cen- tral incisor (the 5 locations are identified clearly in Fig. 3). A1, B1, C1, D1, and E1 are the images of the biofilm before the burst (on the untreated tooth), and A2, B2, C2, D2, and E2 are the corresponding images after threshold- ing with ImageJ (the biofilm is in black in these images, while the white areas are biofilm-free regions). Meanwhile, A3, B3, C3, D3, and E3 are the images of the biofilm on the treated tooth after the burst, and A4, B4, C4, D4, and E4 are the corresponding thresholded images. The untreated samples (columns 1 and 2) and treated samples (columns 3 and 4) are not from the same specimens. We calculated the % removal by subtracting the amount of biofilm that remained from the original amount of biofilm. generated on the tooth surface (Appendix V). Statistical Analysis Statistical comparisons were made by one-way analysis of variance (ANOVA) (Excel 2003, Microsoft). Differences were re- ported as statistically significant for p < .05. Results 3DImagingofTypodontModel High-resolution 3D images de- tailing the micro-architecture of the typodont were obtained by μ-CT (Fig. 1B). This allowed us to computationally disassemble the typodont, maintaining the relevant juxtaposition between the individual teeth, and to cre- ate computational meshes of the teeth without interference from the other typodont materials. Quantification of Biofilm Re- moval With confocal microscopy, S. mutans biofilms grown in the IP space showed bacterial cells ag- gregating and forming complex cell cluster colonies consisting of ‘tower’-, ‘mushroom’-, and ‘mound’-shaped structures. The thickness of the resulting bio- film on each tooth surface was approximately 200 to 300 μm. After the microburst, the images taken for the proximal surface of the teeth showed almost no bio- film close to the nozzle tip of the prototype AirFloss. Image analy- sis showed 95% removal close to the tip, 62% removal at approxi- mately half the labio-palatal dis- tance from the tip to the back of the teeth, and 8% removal at the back of the teeth (Fig. 3). The percentage removal values were plotted vs. the distance from the nozzle tip to the midpoint of the palatal surface of the teeth (Fig. 3A). The resulting curve was compared with the values obtained from the numerical simulations for τw at the same locations (Figs. 3C, 3D). Critical Shear Stress for Biofil- maggregate Detachment The morphology of the biofilms in the BioFluxTM 1000 microflu- idic channels varied markedly between one channel and the > Page 6B

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