Mechanical properties and disruption of dental biofilms

A literature review of dental plaque biofilms formation, progression and detachment mechanisms is presented in this thesis. Various strategies that have been employed to reduce or eliminate dental biofilms are discussed. The focus of the thesis was on the mechanical properties and disruption of dental biofilms, especially from hard-to-access areas of the oral cavity, such as the interproximal (IP) sites between the teeth. Various methods to measure mechanical properties of dental biofilms were investigated, and physical and chemical strategies to disrupt these biofilms were employed. Streptococcus mutans, the bacterium responsible for initiation of dental plaque biofilms, was used in our studies.A uniaxial compressive test was utilized to characterize the mechanical behaviour of biofilms, while manipulating the chemical microenvironment. Initially, the mechanical properties of a dextran gel were characterized. The gel was used as an artificial dental plaque biofilm (Chapter 5). The elastic modulus of the gel was 17 kPa (± 12; n = 3), and the stress relaxation time was 25 seconds (± 18; n = 3), demonstrating a viscoelastic behaviour similar to that reported for real biofilms. After optimizing the technique with the gel, the mechanical properties of S. mutans biofilms were studied, the elastic modulus was 380 Pa (± 350; n = 30), and the stress relaxation time was 12 seconds (± 11; n = 10). The elastic modulus increased by increasing the sucrose percentage in the media, and decreased when the biofilms were treated with increasing concentrations of ethylene di-amine tetra acetic acid, EDTA. Treating the biofilms with different solutions of poly (ethylene glycol), PEG, resulted in behaviour similar to that previously observed for synthetic polymers.The flow field and local hydrodynamics of high velocity water microdrops impacting the interproximal (IP) space of typodont teeth, and their influence on the structure and detachment of both surrogate dental plaque and Streptococcus mutans biofilms, were studied experimentally and computationally. Water droplets of 115 ?L were produced by a prototype AirFloss (PT-AirFloss) device provided by Philips Oral Healthcare, bursting water at a velocity of 60 m/s into the IP space between the maxillary central incisors. High-speed imaging, was used to characterise the PT-AirFloss microburst of pressurized air and water micodrops, and demonstrated the removal mechanism of adental plaque biofilm substitute and the S. mutans biofilms. Using various microscopy and image analysis techniques, quantitative measurements of the removal rate and the percentage removal of biofilms from different locations in the IP space were obtained. Microcomputed Tomography (?-CT) imaging was used to obtain 3D images of the typodont and the IP spaces. The shear stress distribution generated by the drop impacting the tooth surface was calculated by Computational Fluid Dynamics (CFD) simulations based on the finite element method (FEM). There was good agreement between experimentally measured biofilm removal and the pattern of predicted wall shear stress (?w) generated in the IP space by the microburst. High velocity water microdrops, with minimal fluid volume and time, effectively removed both the surrogate and the biofilm. The shear stress generated by the PT-AirFloss and its spatial distribution on the teeth surface played a key role in dictating the efficacy of biofilm removal. In addition, CFD models were used to predict optimal water drop or burst design with respect to more effective biofilm removal performance. Furthermore, the influence of fluid shear flow on the detachment of Streptococcus mutans biofilms inside microfluidic channels was studied using a commercially available flow-cell system. A critical biofilm detachment shear stress was estimated for the large biofilm-aggregates (CDSSagg). The CDSSagg value was used in the CFD model to predict the spatial distribution of biofilm aggregates detachment from the IP surface caused by the PT-AirFloss microburst.Next the effect of three biofilm matrix-degrading enzymes on the structure and detachment of Streptococcus mutans biofilms inside microtiter plates and on typodont teeth was studied experimentally. The enzymes used were: Bromelain (a protease), DNase, and RNase. The biofilms were treated with different enzymatic preparations, stained with Live/Dead and Crystal Violet, and the corresponding optical density (OD) and fluorescence intensity (FI) were measured by a microplate reader. The results detailed the degradation effect of each enzyme, separately and in combination. The three enzymes demonstrated different efficacies in degrading the biofilm in 6, 24 and 96 well-plates, as well as on the typodont teeth. Also, there was a large variability which could be explained by the heterogeneity of the biofilm. Using epifluorescence microscopy and image analysis, quantitative measurements of the percentage surface area coverage were obtained, and the preliminary results were consistent with the datafrom the plate reader. Furthermore, pre-coating the plates with the three enzymes did not inhibit biofilm from formation and accumulation. Lastly the use of a biocompatible copolymer of methylvinyl ether and maleic anhydride, with excellent mucosal adhesive properties and biocompatibility to improve enzymatic digestion by offering a prolonged contact of the enzymes with the teeth and oral tissues, was investigated. The rationale was to eliminate a major obstacle facing the efficacy of the enzymatic therapy which was the relatively short residence time of the enzymes at the site of administration. The adhesive copolymer could possibly enhance enzyme biofilm degradation. Combining the adhesive copolymer with the enzymes could potentially allow near total degradation of the laboratory-grown S. mutans biofilms

High-velocity microsprays enhance antimicrobial activity in S. mutans biofilms

Streptococcus mutans in dental plaque biofilms play a role in caries development. The biofilm’s complex structure enhances the resistance to antimicrobial agents by limiting the transport of active agents inside the biofilm. We assessed the ability of high-velocity water microsprays to enhance delivery of antimicrobials into 3-days old S. mutans biofilms. Biofilms were exposed to a 90° or 30° impact, firstly using a 1-?m tracer beads solution (109 beads/mL) and secondly, a 0.2% Chlorhexidine (CHX) or 0.085% Cetylpyridinium chloride (CPC) solution. For comparison, a 30-sec diffusive transport and simulated mouthwash were also performed. Confocal microscopy was used to determine number and relative bead penetration depth (RD) into the biofilm. Assessment of antimicrobial penetration was determined by calculating the killing depth (KD) detected by live/dead viability staining. We firstly demonstrated that the microspray was able to deliver significantly more microbeads deeper in the biofilm compared to diffusion and mouthwashing exposures. Next our experiments revealed that the microspray yielded better antimicrobial penetration evidenced by deeper killing inside the biofilm and a wider killing zone around the zone of clearance than a diffusion transport with the same antimicrobials. Interestingly the 30° impact in the distal position delivered approximately 16 times more microbeads and yielded approximately 20% more bacteria killing (for both CHX and CPC) than the 90o impact. These data suggest that high-velocity water microsprays can be used as an effective mechanism to deliver micro-particles and antimicrobials inside S. mutans biofilms. High shear stresses generated at the biofilm/burst interface might have enhanced beads and antimicrobials delivery inside the remaining biofilm by combining forced advection into the biofilm matrix and physical restructuring of the biofilm itself. Further, the impact angle has potential to be optimized both for biofilm removal and active agents’ delivery inside biofilm in those protected areas where some biofilm might remai

Streptococcus mutans biofilm transient viscoelastic fluid behaviour during high-velocity microsprays

Using high-speed imaging we assessed Streptococcus mutans biofilm–fluid interactions during exposure to a 60-ms microspray burst with a maximum exit velocity of 51 m/s. S. mutans UA159 biofilms were grown for 72 h on 10 mm-length glass slides pre-conditioned with porcine gastric mucin. Biofilm stiffness was measured by performing uniaxial-compression tests. We developed an in-vitro interproximal model which allowed the parallel insertion of two biofilm-colonized slides separated by a distance of 1 mm and enabled high-speed imaging of the removal process at the surface. S. mutans biofilms were exposed to either a water microspray or an air-only microburst. High-speed videos provided further insight into the mechanical behaviour of biofilms as complex liquids and into high-shear fluid–biofilm interaction. We documented biofilms extremely transient fluid behaviour when exposed to the high-velocity microsprays. The presence of time-dependent recoil and residual deformation confirmed the pivotal role of viscoelasticity in biofilm removal. The air-only microburst was effective enough to remove some of the biofilm but created a smaller clearance zone underlying the importance of water and the air–water interface of drops moving over the solid surface in the removal process. Confocal and COMSTAT analysis showed the high-velocity water microspray caused up to a 99.9% reduction in biofilm thickness, biomass and area coverage, within the impact area

Removal of interproximal dental biofilms by high-velocity water microdrops

The influence of the impact of a high-velocity water microdrop on the detachment of Streptococcus mutans UA159 biofilms from the interproximal (IP) space of teeth in a training typodont was studied experimentally and computationally. Twelve-day-old S. mutans biofilms in the IP space were exposed to a prototype AirFloss delivering 115 µL water 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 biofilms from different locations in the IP space. The 3D geometry of the 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 (?w) distribution caused by the drops on the tooth surface. A qualitative agreement and a quantitative relationship between experiments and simulations were achieved. The wall shear stress (?w) generated 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

System and method for detecting halitosis

A system for detecting halitosis is disclosed that comprises a gas sensor (116) for generating a sensor signal signalling the detection of compounds indicative of halitosis exhaled through an oral cavity; an image sensor (114) for capturing an image (500, 510) of a dental condition and/or tongue condition in said oral cavity; and a processor (110) communicatively coupled to the gas sensor and the image sensor and adapted to process the sensor signal and the image in order to determine if in case of the sensor signal signalling the presence of a compound indicative of halitosis, said halitosis originates from said oral cavity by determining the dental condition and/or tongue condition in said image. A method for detecting halitosis and a computer program product implementing the method are also disclosed

Fluid-driven Interfacial instabilities and turbulence in bacterial biofilms

Biofilms are thin layers of bacteria embedded within a slime matrix that live on surfaces. They are ubiquitous in nature and responsible for many medical and dental infections, industrial fouling and are also evident in ancient fossils. A biofilm structure is shaped by growth, detachment and response to mechanical forces acting on them. The main contribution to biofilm versatility in response to physical forces is the matrix that provides a platform for the bacteria to grow. The interaction between biofilm structure and hydrodynamics remains a fundamental question concerning biofilm dynamics. Here we document the appearance of ripples and wrinkles in biofilms grown from three species of bacteria when subjected to rapid high-velocity fluid flows. Theoretical treatment of the process as a Kelvin-Helmholtz instability indicates that the rippling process was primarily due to physics rather than chemistry or biology. The analysis also predicted a strong dependence of the instability formation on biofilm viscosity explaining the different surface corrugations observed. Turbulence through Kelvin-Helmholtz instabilities occurring at the interface demonstrated that the biofilm flows like a viscous liquid under high flow velocities applied within milliseconds. Biofilm fluid-like behavior may have important implications for our understanding of how fluid flow influences biofilm biology since turbulence will likely disrupt metabolite and signal gradients as well as community stratification