Visualizing synthetic dental biofilm on teeth using micro computed tomography

N. Vyas1, A. D. Walmsley

2, E. Pecheva

2, H. Dehghani

3, L. Grover

4, R. L. Sammons

2

1Physical Sciences of Imaging for Biomedical Sciences (PSIBS) Doctoral Training Centre,

College of Engineering & Physical Sciences, University of Birmingham, Birmingham, B15 2TT, UK, nxv24@bham.ac.uk 2 School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, St

Chad's Queensway, Birmingham, B4 6NN, UK 3 School of Computer Science, University of Birmingham, Edgbaston, Birmingham, B15 2TT,

UK 4 School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham, B15

2TT, UK

Aims Dental plaque (biofilm) and calculus form naturally over time on tooth surfaces and dental implants. These need to be regularly removed in order to prevent periodontal diseases. Deep cleaning methods such as ultrasonic scaling are used to remove biofilm which cannot be removed by everyday dental hygiene. It is therefore important to investigate the effects of ultrasonic scaling instruments in order to understand the different processes involved in removing dental plaque and calculus, so more efficient instrumentation can be developed in the future. Bacterial biofilm is difficult to grow in a homogenous layer, and the unpredictable nature of its growth means that consistent results cannot be obtained each time. It would therefore be advantageous to use a material which has similar properties to biofilms, which can be used instead to image biofilm disruption from ultrasonic scalers. MicroCT would be able to non-invasively image the whole tooth with micrometre resolution, giving an excellent overview of biofilm localisation, such as where the most and least disruption occurs. The aim of this project is to investigate the possibility of using microCT to image an artificial biofilm.

Method Robinson et al. (2014)

1 have formulated a hydrogel for mimicking biofilm in the root canal,

created by dissolving 3 g of gelatine (Merck, Whitehouse Station, NJ, USA) and 0.06 g of hyaluronan (sodium hyaluronate 95%, Fisher, Waltham, MA, USA) in 45 ml of filtered water at

50C. In this work, two variations of hydrogel were produced; one as mentioned above, and another with 35 ml filtered water and 10 ml Lugols stain (1% iodine and 2% potassium iodide), to investigate whether this could enhance the contrast in microCT. The hydrogels were then centrifuged at 3000rpm for 10 minutes to remove air bubbles, and left to solidify at room temperature. In order to see whether the hydrogel can be imaged on teeth using microCT, a plastic model tooth (Frasaco, Germany) and a natural tooth crown were imaged in microCT before and after application of the hydrogel (stained and unstained). The pre- and post- scans were carried out with a SkyScan 1172 micro-CT scanner (SkyScan, Kontich, Belgium) using 5.4m resolution, 38kV voltage, 100A current, without any filter. The settings used for the tooth crown were: 7 m resolution, 66 kV/37kV voltage, 145/175 A current, with a 0.5mm aluminium filter in place (two scans were done for dual energy analysis). The samples were not moved between scans. The resulting slices were reconstructed with SkyScans NSRECON package using uniform attenuation coefficients for the model tooth and the tooth crown images. Image subtraction was performed in MATLAB, 3D visualisation and image merging was done using ImageJ and dual energy analysis was performed with DEhist.

Results In order to determine whether the hydrogel was actually being visualised in the images, the pre- and post- scan images were first subtracted in MATLAB (fig. 1). Only hydrogel remained, confirming that it was being visualised accurately.

Figure 1: (a) Reconstructed microCT slice of hydrogel on tooth model. (b) Reconstructed

microCT slice of tooth model before application of hydrogel. (c) Image b subtracted from image a, to confirm that the object seen on the tooth model in image a is the hydrogel.

The pre- and post- images were then merged on top of each other using ImageJ, in order to form a 3D visualisation showing the hydrogel clearly on the tooth (fig. 2). This confirms that microCT can be used to image hydrogel and its localisation. Further improvements will be made to the method of applying the hydrogel to the tooth to get homogenous converge and surface thickness on the tooth model. Other hydrogels will also be developed which mimic oral plaque or calculus more accurately. Methods to prevent air bubbles from forming in the hydrogel will also be devised. Disruption of the hydrogel from ultrasonic scalers will then be investigated by imaging before and after scaling via microCT.

Figure 2: Pre- and post- scans of tooth model superimposed to show where the unstained

hydrogel (green) has been applied. The DEhist application program was used to perform dual energy segmentation of the hydrogel on the tooth crown (fig. 3). Low energy images have more noise, but show the hydrogel with better contrast, whereas the high energy images show clearer borders, but the hydrogel cannot be imaged due to its lower x-ray absorption coefficient. Therefore merging the two images should give good contrast for both the enamel of the tooth crown and the hydrogel. A Gaussian blur with a radius of 1 was applied to the images (using ImageJ) to

Figure 3: Dual Energy analysis of hydrogel on a tooth crown using the DEhist program. (a) Low energy reconstruction slice of hydrogel applied to a tooth crown, taken at 37 kV (b) high energy image taken at 66 kV. (c, e) dual energy phase space plots with corresponding segmented images (d, f) without Gaussian blur applied to images. A lot of the noise in the image has also been included in the segmentation. (g, i) dual energy phase space plots and corresponding segmented images where (h) is with a Gaussian blur with radius 1, and (j) is with a Gaussian blur of radius 3. The red is the tooth crown and the green is the hydrogel. (k) 3D renderings of the combined data sets showing hydrogel (green) on the tooth crown (red).

remove noise before processing in DEhist. If this was not applied (fig 3 (d, f)), the noise was included in the segmentation. The 3D image shows the hydrogel coverage on the whole tooth (fig 3 (k)). This result will be validated by imaging the sample with other techniques such as digital imaging and light microscopy.

Conclusion This work shows an application of the Bruker DEhist dual energy program to image materials with low x-ray absorption coefficients on mineralised tissue. A similar methodology can be applied to other fields, such as imaging connective tissue interfaces

2. In this study, an artificial

dental plaque material could be imaged on tooth models and tooth crowns using microCT, and dual energy analysis could be performed to segment the artificial plaque from the tooth crown. This novel application of microCT will provide new insights into exactly how ultrasonic scalers remove plaque, which will eventually lead to improved ultrasonic scaler tip design.

References:

1. R.G. Macedo, J.P. Robinson, B. Verhaagen, A.D. Walmsley, M. Versluis, P.R. Cooper & L.W.M. van der Sluis, A novel methodology providing new insights into the ultrasonic removal of a biofilm-mimicking hydrogel from lateral morphological features of the root canal. International Endodontic Journal, 2014

2. A. Bannerman, J. Z. Paxton & L. M. Grover, Imaging the hard/soft tissue interface Biotechnology letters, 1-13, 2013