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hygiene tribune Dental Tribune Middle East & Africa Edition | January-February 20156B other, and also within the same channel. Structural heteroge- neity is a common feature of biofilms. Nevertheless, common features could be noted, as seen by the microscopic images (Ap- pendix Fig. 2): (i) the presence of individual bacteria and (ii) the presence of biofilm clusters of different sizes. When τw was increased from 0 to 2 Pa, there was a slight increase in the overall detachment rate of the biofilm (Appendix Fig. 2), seemingly caused by adhesive failure (von Fraunhofer, 2012). The bacterial cells as well as the biofilmaggregates appeared to slide along the surface before coming off. There was minimal detachment of the individual bacteria and the small aggre- gates over the applied elevated shear stress; but the larger bio- film clusters (with diameters over ~50 μm) detached when the shear stress ranged from 0.3 to 1.7 Pa. We extrapolated a con- servative “critical biofilm-aggre- gates detachment shear stress” (CDSSagg ) of 1.7 Pa for the com- putational modeling. Above 2 Pa, the smaller biofilm clusters still appeared to be firmly attached to the substrate, and remained attached even after the shear stress was increased to 3 Pa. Numerical Simulations Mesh Independence Study A mesh independence study was performed, and a cell size of 0.155 mm was selected for further numerical studies (Ap- pendix V). Quantification of Wall Shear Stress Distribution A representative contour plot of the fluid τw spatial distribution on the tooth surface is shown in Fig. 3B. This simulation corre- sponded to a velocity inlet of 60 m/sec, with the circular nozzle tip located at z/H = 0.5, gingi- vo-incisally, where z (mm) is a spatial coordinate from the su- pragingival base of the tooth per- pendicular to the tip of the tooth, and H (mm) is the supragingival height of the tooth. Thus, z/H = 0.5 equates to halfway up the tooth. The simulation showed the predicted fluid τw distribu- tion on the proximal surface of the tooth, starting from the labial side of the IP space close to the nozzle tip (τw ~2.7 kPa), to the midpoint of the palatal surface of the tooth (τw ~0.3 kPa). Computational Prediction of Shear Stress and Experimental Biofilm Removal The τw distribution obtained computationally was compared with experimentally measured removal of biofilms. A linear correlation of % removal as a function of τw was found accord- ing to: Percent removal = kτw (r2 = 0.94) (1) where τw is wall shear stress (in Pa), and k (in Pa-1 ) is the slope of the interpolating func- tion (Figs. 3C, 3D). Effect of the Nozzle z-position on Wall Shear Stress Distribu- tion Contours of fluid τw on the tooth surface at 5 nozzle tip zpositions were obtained to investigate the effect of tip positioning on the device’s hydrodynamic perfor- mance. Fig. 4 shows the tooth surface area where τw is lower than the critical value of 1.7 Pa. Computational results predicted that the maximum % of biofilm removal would take place when the nozzle tip is placed at z/H = 0.5 or z/H = 0.66, while the ef- ficacy of biofilm removal would be significantly reduced at ex- treme z/H positions, namely, z/H = 0.17 (i.e., close to the gum line) or z/H = 0.83 (i.e., close to the in- cisal edge). Discussion In the flow cell experiments, S. mutans biofilms were success- fully grown inside microchan- nels under gravitational flow conditions. Under transmitted light microscopy (Appendix Fig. 2), the biofilm size and mor- phology showed resemblance to previously reported data (Cos- terton et al., 1999; Heersink et al., 2003). When the biofilm was subjected to an increased shear stress from 0 to 2 Pa, the large aggregates resisted movement from the surface until the wall shear stress reached a critical value, or CDSSagg , which ranged between 0.3 and 1.7 Pa, at which they detached. However, even at this critical value, the smaller biofilm patches and individual bacterial cells remained at- tached. Generally, detachment of biofilm fragments (erosion), or even of the entire biofilm (sloughing), is caused by high flow shear stress levels that ex- ceed the adhesion strength of the biofilm (Ohashi and Harada, 1994, 1996). The detachment of the large aggregates occurred at a relatively low shear stress (~1 Pa), while the smaller patches remained firmly attached, even after the flow increased up to 3 Pa. The S. mutans biofilms were grown under static or low shear conditions, thus leading to the formation of large cell aggre- gates, which tend to be approxi- mately circular compared with the streamers that usually form under dynamic conditions. The streamlined shape has a signifi- cant effect on reducing the fluid drag on the elongated biofilms (Stoodley et al., 1998, 1999). Streamers develop viscoelastic flexible bodies which oscillate rapidlywhenexposedtotheflow forces, thus resisting detach- ment better than the large cir- cular biofilm-aggregates grown at low laminar flow conditions. The large circular aggregates show different behavior under flow, with less ability to flex, re- sulting in detachment at lower fluid shear stress. This explains the experimentally observed de- tachment of the large aggregates at a relatively low shear stress (~1 Pa). So, the CDSSagg estimat- ed here describes the detach- ment involving large aggregates only and not the total biofilm, which requires a higher criti- cal shear stress for detachment, which was beyond the range of the microfluidics system under our operating conditions. The critical shear stress value of 1.7 Pa is close to the range of previ- ously reported values of shear stress (5-12 Pa) required for detachment of non-dental bio- films (Ohashi and Harada, 1994; Stoodley et al., 2002). The exit velocity of the micro- drops from the prototype Air- Floss was 60 m/sec, and, based on earlier experiments, the flow was a steady stream (Rmaile et al., 2013). Even though the shearing force was applied over very short periods of 30 msec, the generated fluid τw proved to be effective in removing the at- tached biofilm by both adhesive and cohesive failure (Rmaile et al., 2013). However, fractions of the biofilm remained on the back of the teeth, due to tooth architecture and the fluid flow behavior in these regions, i.e., the inability of the fluid to flow around the anatomical curva- ture and undercuts associated with the palatal surface of the upper central incisors. These observations were predicted by the computational simulations in which τw on the proximal sur- face of the teeth was observed to decrease gradually in the labio- palatal direction. The simulations predicted τw distribution on the tooth surface caused by the microburst to be in the kPa range within the IP space, except in areas on the palatal side of the tooth, where τw became significantly lower (~200 Pa). The maximum com- putational values for the fluid τw were ~1,000 times higher than the CDSSagg obtained from the flow-cell experiments, and ~200 times higher than the estimated shear stress, reported in the literature, for biofilm detach- ment (Ohashi and Harada, 1994; Stoodley et al., 2002). Thus, the simulations predict that a sig- nificant percentage area of the tooth is subjected to τw values capable of removing the plaque from the IP spaces. The large difference in adhesive strength between the 2 systems illustrates the importance of the physi- cal growth conditions and sur- face type on adhesion strength. It was beyond the scope of this study to determine the influence of surface or hydrodynamics on adhesion strength. In mechani- cal testing, properties reports for the same species commonly vary by 3 orders of magnitude or greater (Shaw et al., 2004). Whether this variability is true at different locations in the mouth or between patients is unknown, but measurements of the adhe- sion strength of real oral biofilm plaques would be useful in de- veloping relevant in vitro mod- els which look at mechanically induced detachment. The 3D simulations for predict- ing τw were consistent with the experimental results obtained. As might be expected, the bio- film survived the burst at areas of low τw , but was flushed away at areas where τw was higher. A linear relationship was found between the predicted fluid τw and the amount of detached biofilm obtained experimentally (Eq. 1). This relationship could < Page 4B Figure 3. Biofilm removal as a function of shear stress and distance from the front of the tooth. (A) Percentage removal of the biofilm quantified from the CLSM images at 5 different locations on the tooth surface in the IP space. Three individual runs are shown by different symbols. The error bars repre- sent standard deviations of the mean from 5 CLSM images. Solid line and heavy bars are the mean of the individual means (n = 3), which have been slightly offset for clarity. The schematic inset shows the proximal view of the upper central incisor, where the black squares represent the different loca- tions where the CLSM images were taken. (B) Contour map showing the spatial distribution of τw on the tooth surface, as calculated from numerical simulations (circular nozzle tip; z/H = 0.5). The color bar is a linear scale showing the shear stress (Pa). (C) τw on the tooth surface (in kPa) at different y-positions along the tooth (i.e., from labial to palatal side), at a fixed z- position (gingivo-incisal), as also calculated from numerical simulations. Empty squares correspond to the measurement points (squares) in 3B. On the secondary y-axis, the mean percentage removal measured experimen- tally in 3A (i.e., solid line) is plotted, with the 5 empty circles (denoted as A, B, C, D, and E) corresponding to the same positions as in 3A. (D) Relationship between percentage removal (determined experimentally) and τw on the tooth surface (determined computationally). Datapoints were interpolated with a linear trend (red line). Figure 4. Effect of nozzle tip z-position (z/H) on fluid τw spatial distribution over the tooth surface. Tip cross-section is circu- lar, and z/H was varied between 0.17 and 0.83 (a-c). The blue arrow indicates the flow direction. The red area corresponds to the tooth surface area where the shear stress is lower than CDSSagg = 1.7 Pa. be used to predict the efficacy of oral health care devices that use shear forces to remove plaque. The computational model de- veloped allowed for prediction of the effect of changing the position of the nozzle tip in the z-direction (inciso-gingivally) on biofilm removal efficacy. The numerical simulations pre- dicted that placing the nozzle tip in or close to the middle of the inciso-gingival height (z/H = 0.5 or 0.67) provides more effective biofilm removal, in comparison with placing the tip closer to ei- ther the incisal edge or the gum line (Fig. 4). To the best of our knowledge, this is the first time that CFD has been used to cal- culate the wall shear stress dis- tribution, caused by water drops generated from an oral hygiene device, on the tooth surface. In this study, an experimental set-up was built and a methodol- ogy was developed to character- ize, visualize, and quantify the efficacy of biofilm detachment by high-velocity water droplets, which prevents the accumula- tion of biofilm and automatically translates into prevention of dental caries formation at these sites. Acknowledgments The use of both the IRIDIS High- Performance Computing Facil- ity, and μ-VIS (CT centre), and associated support services at the University of Southampton is sincerely acknowledged. The authors also acknowledge Dr. Phil Preshaw from Newcas- tle University for helping with the development of the typo- dont model, Dr. Suraj Patel from labtech for helping with the Bio- Flux experiments, Dr. Philipp Thurner from the University of Southhampton for advice with μ-CT, and the late Dr. Hansjür- gen Schuppe for helping with the CLSM images. This work was financially supported by Philips Oral Healthcare, Both- ell, WA, USA. M. Aspiras and M. Ward are employed by Philips Oral Healthcare, Bothell, WA, USA. The other authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article. A. Rmaile1 *, D. Carugo2 , L. Capret- to2 , M. Aspiras3 , M. De Jager4 , M. Ward3 , and P. Stoodley1,5 1 nCATS, Faculty of Engineer- ing and the Environment (FEE), University of Southampton, UK; 2 Bioengineering Group, Faculty of Engineering and the Environment (FEE), University of Southampton, UK; 3 Philips Oral Healthcare Inc. (POH), Bothell, WA, USA; 4 Philips Oral Healthcare, Philips Research, Eindhoven, The Netherlands; and 5 Center for Microbial Interface Biology, Departments of Micro- bial Infection and Immunity, and Orthopaedics, The Ohio State Uni- versity, Columbus, OH, USA; *cor- responding author, ar1a09@soton. ac.uk J Dent Res 93(1):68-73, 2014 About the Authors