Heat-induced alterations of dental tissues: Implications for the identification of fire victims

Transcription

1 Heat-induced alterations of dental tissues: Implications for the identification of fire victims Michael A. Sandholzer 1, Anthony D. Walmsley 1, Philip J. Lumley 1, Gabriel Landini 1 1 School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, St Chad s Queensway, Birmingham B4 6NN, United Kingdom Aims Micro-CT scanning technology has been increasingly used in various fields of forensic science, however rarely in forensic dentistry. Forensic dentists are regularly confronted with incidents involving high temperatures (e.g. airplane crashes, natural disasters, house fires) and therefore information gained from experimental research can provide useful information for estimating temperature based on tissue changes to then facilitate victim identification. The most reliable and frequently applied method for identification of fire victims is comparative dental radiography. This involves comparing dental features (e.g. tooth morphology, missing teeth, pathologies and restorations) between ante-mortem and postmortem dental radiographs. Very often investigators are faced with fragmented and isolated remains, removed from their anatomical position. Whilst the heat-induce dimensional changes in bone have been previously studied, precise data for heat-induced shrinkage of human teeth is still lacking. The aim of this study was to evaluate the heat-induced three-dimensional dental tissue shrinkage and provide an improved understanding of those alterations by means of micro-ct imaging. Method A total of 56 freshly extracted human teeth (ethical approval NHS-REC 09.H / Consortium R&D No. 1465) were scanned before and after a 30 minute exposures to temperatures between 400 and 1000 C. Micro-CT scans were performed with a SkyScan 1172 scanner (SkyScan, Kontich, Belgium). The pre- and postscans of the teeth were done at 13.5µm resolution using 80kV voltage, 100µA current and a 0.5mm Aluminium filter. The resulting slices were reconstructed with SkyScan NSRECON using a consistent attenuation coefficient range ( ). Following the reconstruction the image stacks were converted in ImageJ 1.45r to NRRD file format and 3-D fast rigid registration of pre- and postscans was performed using 3D Slicer 3.6 ( After the manual determination of the region of interest (ROI) the registered image stacks were cropped, the resulting images converted into binary images using a manual threshold in ImageJ and the resulting grey values exported to SPSS version 19 (IBM SPSS Inc., Chicago, USA) for statistical analysis. Results Macroscopic results

2 Progressive, temperature-dependent shifts from a natural colour to black/dark brown (400 C), brown ( C), greyish-blue (700 C), light grey (800 C), chalky-white (900 C) and white/pink (1000 C) were observed (Figure 1). Lifting of the crown was observed alongside the dentine-enamel junction starting at 400 C, leading to a full detachment and fragmentation of the crown above 600 C. Micro-CT results In all teeth of the lower temperature groups ( 600 C) the micro-ct sections revealed multiple small cracks in the apical dentine and cementum as well as few (often single) large longitudinal cracks. At higher temperatures (700 C-1000 C) multiple larger longitudinal and numerous transversal cracks were visible in the dentine, mainly deriving from the canals and the pulp chamber (Figure 1). Significant differences (ANOVA post-hoc LSD, α <0.05) in the dentinal shrinkage were present in higher temperature groups ( 700 C), whereas no significant statistical difference was found between lower temperature groups. The average shrinkage ranged between 4.78% (at 400 C) and 32.53% (at 1000 C). Pearson s correlation coefficient demonstrated a positive correlation between the temperature and the dentinal shrinkage at the 0.01 level (adjusted R 2 = 0.859). Conclusion The progressive colour alterations correspond with previous observations of isolated human teeth exposed to high temperatures and can give the investigator an indication of the temperature that the dental remains have been exposed to. By combining the information from the colour changes and the values on average shrinkage, it might be possible to account for shrinkage and predict the ante-mortem size of the teeth more precisely by rescaling the post-mortem radiographs, supporting the odontological identification process in cases where only isolated teeth are present.

3 Figure 1: Heat-induced alterations of premolars of the 700 C (A) & 900 C (B) group. Visualisation of the colour alterations (left), the dentinal shrinkage using the registered pre- and postscans median sections (middle) and cross-section in the coronal dentine (right). Scale bars represent 1mm.

4 3D fractography on biocompatible dental materials Kamil Al-Katib, Kun V Tian, Peter Nagy, Eszter Farago, Csaba Dobo-Nagy Department of Oral Diagnostics, Semmelweis University, 47 Szentkiralyi Budapest, Hungary, H-1088 Aims Glass ionomer cements (GIC) are widely used in dentistry as well as in bone surgery (3). The purpose of this study was to assess the usability of X-ray micro tomography (μct) on fractography. Method Discs of biocompatible glass ionomer cements (GIC) were prepared for Hertzian indentation (4) and subsequent fracture analyses. Specifically mm samples for reproducing bottom-initiated radial fracture, complemented for 2µm cubic voxel size optimal resolution μct analysis (SkyScan 1172, Kontich, Belgium). Alignment was 70kV, 0.5mm Al filter and 0.5º rotation step. Three volume of interest (VOI, 0.2 mm thick mm diameter cylinders) of each sample were chosen for more detailed pore analyses. Nanocomputed tomography (SkyScan 2011, Kontich, Belgium) analyses with 0.39µm voxel size, 40kV, no filter was performed to support the reliability of the μct results. To assess the accuracy of the µct results, three VOIs 20µm thick X 200µm diameter cylinders were chosen on both nano CT and µct scans of the same sample for porosity parameter determination, with all results subsequently compared using a Student s t test. Complementary 2-dimensional fractographic investigation was carried out by an optical stereo microscope (Meiji, China) at up to 909 magnification, as well as with a scanning electron microscope (Hitachi S-2460N, Tokyo, Japan) at up to magnification, under vacuumidentifying fracture characteristics (2). Results The μct analysis (CTAn software, Skyscan) delivered Pore Volume (10-5 mm 3 ), Porosity (%), Pore Surface / Volume Ratio (10 3 mm -1 ), Pore Thickness (μm) measurements the difference between the top layer value and bottom layer value was statistically significant at P=0.05 Pore surface / Pore Pore volume Pore surface Structure / 10-5 mm 3 / 10-2 mm 2 volume ratio / thickness / 10 3 mm -1 SMI µm Top layer 3.5 (1.3) a 3.8 (1.7) a 1.1 (0.3) a 6.4 (1.9) a 3.4 (0.3) Bottom layer 8.5 (3.7) 6.8 (2.2) 0.8 (0.2) 9.0 (2.6) 3.9 (0.4) Whole volume 27.9 (14.1) 25.2 (14.1) 0.9 (0.2) 9.1 (2.5) 3.7 (0.3) Table 1. Determined mean porosity parameters (SD) a: the difference between the top layer value and bottom layer value was statistically significant (P<0.05). 3D analysis of GIC samples allowed fracture branching and incomplete bottom-initiated cracking to be accurately determined. Radial orientation of crack lines typical to brittle materials (1). Specifically, cracks grew to link pores while propagating along glass-matrix interfaces.

5 Fig. 1. 3D rendered image represents incomplete bottom-initiated cracking Fig 2. Nano CT image showing that microcracks propagating along the interface of the matrix and glass cores Fig 3. Transparent rendered image where the entrapped air bubbles and cracklines are segmented and having milky-white appearance Conclusion The methodological development herein is exploitable on related biomaterials and represents a new tool for the rational characterisation, optimisation and design of novel materials for clinical service. References: 1. Goldman M. Fracture properties of composite and glass ionomer dental restorative materials. J Biomed Mater Res. 1985;19: Hull D. Fractography: Observing, measuring and interpreting fracture surface topography. Cambridge: University Press; p Nicholson JW. Glass ionomer dental cements: update. Mater Tech Adv Perf Mater. 2010;25: Wang Y, Darvell BW. Interactive effect of indenter size and sample thickness in Hertzian indentation test. Dent Mater. 2010;26:

6 Human tooth root canal geometry assessment through micro-ct images A. Garay 1, J.H. Legarreta 1, I. Macía 1, R. C. Aza 2 1 Vicomtech-ik4, Mikeletegi Pasealekua, 57 Teknologi Parkea Donostia, Spain, jhlegarreta@vicomtech.org 2 Clínica Dental Roberto Carlos Aza, Sor Ángela de la Cruz, Madrid, Spain Aims Successful tooth root canal preparation constitutes an essential first step in endodontic therapy in order to ensure an appropriate posterior canal irrigation and obturation. Any infected tissue surrounding the tooth needs to be removed prior to sealing the canal with gutta-percha. The preparation consists of shaping, possibly enlarging and disinfecting the root canal system, thus removing the damaged tissue by means of a series of manually or mechanically powered files, a process called instrumentation. The choice of the technique depends on the canal anatomy, and has an impact on the shaping ability and cleaning effectiveness. Root canal reciprocating motion techniques have gained acceptance during recent years 3. Reciprocating motion, a fully automatic technique, is believed to produce less deformation, to cut better and to advance faster, as well as avoiding unexpected file fractures. In addition, instrumentation time decreases with reciprocating techniques. However, further studies on the effect of reciprocating motion on the canal geometry are still needed Error! Reference source not found.. The purpose of the clinical study is to assess the effect of reciprocating motion compared to continuous rotation in root canal instrumentation using micro-ct images. Although not suitable for in-vivo root canal anatomy assessment, micro-ct scanning allows for a detailed and accurate post-exodoncy assessment of the effect of a given instrumentation technique on the root canal geometry 1. 3D reconstruction from obtained data allows for a clear visualization of the root canal system, dentin and enamel. This paper presents the first step of the whole process, the acquisition and reconstruction parameter configuration for root canal geometry assessment in a SkyScan 1172 micro-ct. Method A number of samples were randomly selected out of a population of 40 human teeth for a preliminary micro-ct acquisition set-up study. All 40 teeth had been extracted due to periodontal causes or orthodontic treatment. All had completely formed apexes without visible apical re-absorption signs and without any previous endodontic treatment. Tissue remainders were removed after exodoncy, and they were stored in 10% buffered formaldehyde solution. A high-resolution SkyScan KV Hamamatsu C Mp (SkyScan, Kontich, Belgium) micro-ct scanner was used for the study. The micro-ct incorporates a Hamamatsu 100/ KV; µa; (10 W max); <5 µm spot

7 size source and a Hamamatsu C Mp 12-bit cooled CCD fiber-optically coupled to scintillator detector. The teeth were vertically positioned with the plastic tube sample holders provided by SkyScan and fixed with radio-transparent foam. The dental piece were scanned using a total number of four configurations: 180º and 360º rotation for each two filters, Al 0.5 mm and Al+Cu. Scanning with no filter is an unfeasible choice given that the beam contains too much energy for tooth scan purpose even at very low voltages. Other authors 2 have used an Al 1 mm filter, which is halfway between the Al 0.5 mm and Al+Cu filters. The oversize scan method was needed in order to fully scan the samples at reasonable resolution. The acquisition parameters for the SkyScan Version 1.5 (build 12) F Control Program shown in Table 1 and Table 2 were found to yield a good contrast and to achieve the transmission requirement of 40 to 60% at reasonable scanning speed (time), even at full 360º rotation (although this has other drawbacks discussed below). Acquisition parameter Value X-Ray source Voltage (kv) 100 Current (µa) 100 Object position and magnification Pixel size (µm) Rotation (º) 180/360 Stage/camera parameters Camera position (Binning) 4x4 Exposure time (ms) 90 Acquisition Rotation step (º) 0.7 Averaging (frames) 2 Random movement Off Scan duration (hh:mm:ss) 00:04:53/00:08:28 Generated dataset size (GB) 0.73/1.3 Table 1. Micro-CT acquisition parameters for the study of root canal geometry in human tooth using an Al 0.5 mm filter. Acquisition parameter Value X-Ray source Voltage (kv) 49 Current (µa) 167 Object position and magnification Pixel size (µm) Rotation (º) 180/360 Stage/camera parameters Camera position (Binning) 4x4 Exposure time (ms) 420 Acquisition Rotation step (º) 0.7 Averaging (frames) 2 Random movement Off Scan duration (hh:mm:ss) 00:08:27/00:14:53 Generated dataset size (GB) 0.73/1.3 Table 2. Micro-CT acquisition parameters for the study of root canal geometry in human tooth using an Al+Cu filter.

8 N.B.: The tables and figures in this paper refer to the values for one of the selected samples. Increasing the resolution (decreasing the pixel size) increases the dataset size accordingly and results in a rather unmanageable dataset for processing and visualization purposes with a medium-range workstation. NRecon version was used for reconstruction purposes. A Volume Of Interest (VOI) encompassing the whole volume was set in each case in order to reduce the generated reconstruction dataset size (and thus, the computational load). The reconstruction parameters used are shown in Table 3, Table 5, Table 5 and Table 6. Reconstruction parameter Value Smoothing 7 Misalignment compensation -2.0 Ring artifact reduction 20 Beam hardening (%) 30 CS rotation (º) 0.0 Generated dataset size(gb) 0.8 Table 3. Reconstruction parameters for micro-ct scanned human tooth using an Al 0.5 mm filter and 180 acquisition. Reconstruction parameter Value Smoothing 5 Misalignment compensation -2.5 Ring artifact reduction 20 Beam hardening (%) 54 CS rotation (º) 0.0 Generated dataset size(gb) 0.8 Table 4.Reconstruction parameters for micro-ct scanned human tooth using an Al 0.5 mm filter and 360 acquisition. Reconstruction parameter Value Smoothing 5 Misalignment compensation -2.5 Ring artifact reduction 4 Beam hardening (%) 30 CS rotation (º) 0.0 Generated dataset size(gb) 0.7 Table 5. Reconstruction parameters for micro-ct scanned human tooth using an Al+Cu filter and 180 acquisition. Reconstruction parameter Value Smoothing 5 Misalignment compensation -2.5 Ring artifact reduction 4 Beam hardening (%) 30 CS rotation (º) 0.0 Generated dataset size(gb) 0.7 Table 6. Reconstruction parameters for micro-ct scanned human tooth using an Al+Cu filter and 360 acquisition. N.B.: The generated reconstruction dataset size depends on the dimensions of the VOI selected for reconstruction.

9 The same parameters were appliedd to all sub-scans (part) belonging to the same scan (segment). The acquisition and visualization process were carried out in i a workstation with a Dell Precision T5500 workstation, Windows 7 Professional SP11 32-bit, Intel Xeon E GHz, 4 GB RAM (2. 93 GB usable), 2.72 TB hard disk and an NVIDIA Quadro FX580 graphic card. The reconstruction process was performed in a cluster of eight Intel 2xSixCore 2,93GHz, 24 GB RAM processors. Results The reconstruction process shows that, for our purposes, using the Al 0.5 mm filter implies heavier post-processing compared to that required by the Al+ +Cu filter. There are two main reasons for this: the increased beam hardening and ring artifacts. The choice of the filter has an impact on the beam hardening. Using the Al 0.5 mm filter results in a higher the beam hardening effect since the lower beam energies are still present and penetrate less than the high energy part of thee spectrum.. Thus, a higher figure is necessary to correct it. Although performing a 360 scan should minimize the beam hardening, we found f that the combination of thee Al 0.5 mmm filter and 360 results in an even poorer performanc ce. Finally, it was verified thatt increasing the smoothing parameter for the Al 0.5 mm filter at 360 in order to reducee the beam hardening figure did not n yield improved results. Furthermore, ncreasing the smoothing figure results in a loss of detail. Ring artifact increase for the t Al 0.5 mm filter may be due too the increase in the beam hardening. Seee Figure 42. Figure 42. Ring artifacts in a reconstructed slice for thee Al 0.5 mmm filter and 360 acquisition. Figure 43, Figure 44, Figure 45, Figure 46, Figure 47 and Figure 48 show the result of the reconstruction process. Figure 43. Al 0.5 mm filter acquisition preview. Figure 44. Axial reconstruction for Al 0.5 mm filter and 180 acquisition. Figure F 45. Axial reconstrucr ction for Al 0.5 mm filter and 360 acquisitiona n

10 Figure 46. Al+ +Cu filter acquisition preview. Figure 47. Axial reconstruction for Al+Cu filter and 180 acquisition. Figure F 48.Axial reconstrucr ction for Al+ +Cu filter f and 360 acquisitiona n. Once the reconstructionss done, CTAn version was used to analyze the datasets. Our interest was on segmenting the root canal volume and having its statistics computed. For canal geometry assessment purposes, the region comprised between the isthmus and the apexx was considered. A CTAn custom processing pipeline was built in order to do so. The Al 0.5 mmm dataset posed further segmentation problems than those posed by the Al+Cu filter. Since the contrast between the regions was worse for the Al05 filter and given the increased post-processing activity during reconstruction, the segmentation parameter compromise e was harder to achieve. In order to analyze the geometry off the root canal, the 3D 3 analysiss performed by CTAn were recorded. Table 7, Tablee 8, Table 9 and Table 10 show the differences across the acquisitions. Parameter Value Root canal volume (mm ) Tooth volume (mm ) Root canal volume/tooth volume (%) 1.64 Root canal surface(mm ) Root canal surface/volume (%) Table 7. Morphometry parameters for micro-ct scanned human tooth using an Al 0.5 mm filter and 180 acquisition. Parameter Value Root canal volume (mm ) Tooth volume (mm ) Root canal volume/tooth volume (%) 1.39 Root canal surface(mm ) Root canal surface/volume (%) Table 8. Morphometry parameters for micro-ct scanned human tooth using an Al 0.5 mm filter and 360 acquisition. Parameter Value Root canal volume (mm ) Tooth volume (mm ) Root canal volume/tooth volume (%) 1.38 Root canal surface(mm ) Root canal surface/volume (%) Table 9. Morphometry parameters for micro-ct scanned human tooth using an Al+ Cu filter and 180 acquisition.

11 Parameter Value Root canal volume (mm 3 ) Tooth volume (mm 3 ) Root canal volume/tooth volume (%) 1.45 Root canal surface(mm 2 ) Root canal surface/volume (%) Table 10. Morphometry parameters for micro-ct scanned human tooth using an Al+Cu filter and 360 acquisition. It can be drawn from the results that the segmentation process has an impact on the morphometry figures. CTVol version was then used to load and visualize the generated 3D models. The results are shown in Figure 49Figure 49 Figure 50, Figure 52 and Figure 51. Figure 49. 3D model view in CTVol for Al 0.5 mm filter 180 acquisition. Figure 50. 3D model view in CTVol for Al 0.5 mm filter and 360 acquisition. Figure 51. 3D model view in CTVol for Al+Cu filter and 180 acquisition. Figure 52. 3D model view in CTVol for Al+Cu filter and 360 acquisition. As the above images show, the canal volume is extracted with higher accuracy at the apical region from the Al+Cu filter datasets. The discontinuity in one of the root canals found in the Al+Cu 180 acquisition, as it was verified through a slice-wise examination, was probably a result of the compromise between the segmentation parameters. The 3D models confirm the variability seen in the morphometry analysis. Furthermore, since the exact location of the reference plane in the lowest point of the pulp chamber may vary across the different segmentations, another uncertainty element is introduced in the system. Conclusion

12 According to this initial set-up study for root canal geometry assessment through micro-ct images, the SkyScan 1172 micro-ct has proven to be a valid tool to know the root canal geometry. It has been shown that the Al 0.5 mm presents disadvantages compared to the Al+Cu filter. It requires a higher scanning voltage, up to the limits of the X-Ray source, resulting in a restricted parameter adjustment range. Although it offers a good contrast, its signal-to-noise ratio is worse due to the lower current compared to the Al+Cu filter. Furthermore, it also requires further post-processing to eliminate the generated ring-artifacts and beam hardening. Although the influence of the filter type is unclear when looking the discrepancies in morphometry, the segmentation process proves to be more difficult, and probably less accurate, for the Al 0.5 mm filter. The variability of the segmentation process itself may account for the variations in the morphometry analysis. On the other hand, although the 360º scan doubles both the acquisition time and the initial dataset size, the reconstructed dataset size is comparable to the one produced by a 180º rotation scan. Moreover, the scan time is still reasonable at 360º rotation. Because through the 360º rotation the whole revolution angles are scanned, more information is obtained on the structures contained in the volume. This produces a more accurate reconstruction, which, in turn, may enable an easier segmentation and better root canal geometry assessment. Although there may exist slight variations from one dental piece to another, the Al+Cu filter at 360º rotation seems a reasonable standard configuration for routine scan of dental pieces. It is now our aim to compare the effect of different instrumentation techniques on the root canal geometry. Thus, we expect the comparison to yield information on reparation errors, such as straightening, elbows, clipping or ledging. It should be noticed that for instrumentation technique effect comparison purposes, morphometry features (including volume and surface) will need to be computed separately for each root canal. This is due to the fact that each root canal s geometry is different, and since it is instrumented separately, its geometry varies in a specific way. Furthermore, parameters such as the SMI lack of any significance if not referred to a single canal. Also, a registration framework will need to be developed to compare the changes in a root s canal shape before and after instrumentation. References 1. Peter O.A. Current challenges and concepts in the preparation of root canal systems: a review. J Endod, 30(8): , Sousa-Neto, M. D. et al., Flat-oval root canal preparation with Self-Adjusting File instrument: a micro-ct study, Proceedings SkyScan User Meeting 2011, , Leuven, Belgium, Yared G. Canal preparation using one only Ni-Ti rotary instrument; preliminary observations. Int Endond J, 41: , You S.Y. et al., Shaping Ability of Reciprocation Motion in Curved Root Canals: A Comparative Study with Micro-Computed Tomography, J Endod, 37(9):

13 Page 17 Novel method to evaluate sealing ability in endodontic therapy using Micro-CT A.Parrilli 1, M.G.Gandolfi 2, D.Caretti 3, F.Salamanna 1,C. Prati 2, M.Fini 1 1 Laboratory of Preclinical and Surgical Studies, Rizzoli Orthopaedic Institute, Via di Barbiano 1/10, Bologna 2 Odontostomatological Sciences Department, University of Bologna, Via S.Vitale 59, Bologna, Italy 3 Industrial Chemistry and Materials Department, University of Bologna, Viale del Risorgimento 4, Bologna, Italy Aims The success of endodontic therapy is given by total obturation of the root canal in all dimensions. Thus is become necessary to create a fluid-tight seal able to prevent ingress of bacteria and their toxins [1]. Gutta-percha is the most common root filling material in use today and in this study it was utilized in association with Thermafil system (Maillefer, Switzerland) technique. Besides gutta-percha alone is not able to provide a fluid-tight seal of the canal space and a sealer is required along with the core obturating material. It was here used a gold standard sealer (AH Plus, Dentsply Germany) that is an epoxy resin. In this paper an innovative method using a micro-computed tomography (micro-ct) is proposed to measure the interface volume of voids/gaps of obturated root canals as indirect index of sealing ability. Method Eight teeth were prepared by using ProTaper rotary NiTi (Maillefer, Switzerland) instruments to model root canals for endodontic therapy. All roots were filled by Thermafil size 30 (Maillefer, Switzerland) and AH Plus (Dentsply, Germany) and were immersed in 5mL of HBSS (Hank s Balanced Salt Solution) at 37 C to allow the sealers to set. Afterwards the teeth were analyzed using a micro-ct system (SkyScan 1172, Belgium) after 96 hours and six months from the endodontic treatment. The 8 filled roots were scanned at 100kV of source voltage and 100!A of source current using an aluminum filter of 0.5mm. Each sample was rotated until 180 degrees with a rotation step of 0.4 degrees and the pixel size was 4!m. The acquisition was carried out on two vertical connected and consecutive field of view and the obtained images were used to reconstruct the internal structure of the specimens by the software NRecon version (Skyscan, Belgium) with the same post alignment for each vertical connected scan. No corrections were used except for the specific misalignment for each acquisition and an accurate ring artefact reduction because of the small rotation step and the small pixel size (Figure 1). The analysis were performed using the software CTAnalyser version (Skyscan, Belgium). For each sample the dataset is composed of 3000 images corresponding of a length of the root canal of 4 mm. These datasets were divided in three vertical portions (coronal third, middle third and apical third), i.e. in three groups of 1000 images each. The voids percentage was calculated on each root third also to evaluate the sealers behavior as regards the root anatomy.

14 Page 18 Figure 1: Projection and crossectional image of a tooth after an endodontic tratment The threshold value used to binarize the images was selected from the grey levels diagrams of cross sections to supply a repeatable and operator independent method. To define the area and the volume where the voids percentage will be calculated, a repeatable and operator independent novel method was used. Firstly, the materials filling the root-canal (sealer and gutta-percha) were detected from the images using the threshold level that corresponds to the peaks separation point of dentin and materials on the grey levels diagrams and binarizing the images from that point to 255. In the second step, the bi-dimensional Region of Interest (2D ROI) was shrunk along all the vertical axis of each third on the binarized images of materials to create a Volume Of Interest (VOI). In the third step each 2D ROI was dilated of 5 pixels (corresponding to 20µm) in every direction and the obtained perimeter was used to delimitate a new ROI and consequently a new dilated VOI. In this way a three dimensional profile of VOI, composed by sealer, guttapercha and the superficial 20 microns of the dentin walls, was defined. In the fourth step the images of this new VOI were binarized to detect the voids inside the VOI to evaluate the sealing of each sealer exactly in the same neighbourhood of the canal. The voids were detected by the threshold level corresponding to the peaks separation of air and dentin and binarizing the images from 0 to that point. A 3D analysis was carried out to calculate the percentage of voids in every third for each sealer. Moreover a 2D analysis was carried out to evaluate how the percent changes of voids and gaps along the volume of the root. The same procedure was repeated after 6 months on the same teeth. Results The data (means and standard deviations) of the percentage of voids and gaps in filled root canals analyzed after 1 week and 6 months were carried out both 2D and 3D. All the result are shown in table 1 and 2 and demonstrate the good sealing ability of the system used for the endodontic therapy. Moreover 3D models were created and they prove to be very useful for an immediate understanding of geometry of entire sample and of its details even if with a complicated microscopic structure. Examples of models created from this study data is showed in Figure 2.

15 Page 19 2D voids Volume after 1 w eek of storage (%) 3D voids Volume after 1 w eek of storage (%) sample apical third middle third coronal third apical third middle third coronal third means SD Table 1 2D and 3D analysis at one week from endodontic treatment 2D voids Volume after 6 months of storage (%) 3D voids Volume after 6 months of storage (%) sample api mid cor api mid cor means SD Table 2 2D and 3D analysis at six months from endodontic treatment Figure 2: The root canal from the inside with the gaps detected highlighted in black

16 Page 20 Conclusion In this study the use of micro-ct was introduced as innovative methods to detect porosities and voids in the filling materials and at the interface of filled root canals. The method proved to be a powerful tool for visualising the porous microstructure of materials. Moreover, the non destructive nature of this technique permitted the three dimensional analysis of the teeth and allowed to evaluate the possible variations of sealing ability over time directly on the same root sample. The novel method here proposed allows measuring the presence of porosities at the interface between the obturation materials and dentin. Alternative method consisting in sectioning the roots may unfortunately create several artefacts, such as gutta-percha shearing, production of debris and smear layer that may mask the porosities and the marginal defects. Finally, the procedures to obtain a dried dentine section increase the risk of dentine and cement fractures. References: 1. Siqueira JF Jr, Endodontic infections: concepts, paradigms, and perspectives, Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 94, , R.S. Gatewood, Endodontic materials, Dent Clin N Am, 51, , 2007

17 Page 76 Assessment of Trabecular Bone Microarchitecture by Two Differing Cone Beam CT Comparison with the Gold Standard Micro-CT BT. Szabo 1, C. Dobo/Nagy 1, R. Mikusi 1 1 Independent Section of Radiology, Faculty of Dentistry, Semmelweis University, Budapest Aims The goal of this study was to determine parameters of the microarchitecture of trabecular bone by two differing CBCT instruments in a comparison with MCT. Method The skulls of three female monkeys (Maccaca fascicularis), terminated for other research purpose, were fixed and stored in paraformaldehide solution. These were scanned using Planmeca ProMax 3D smart cone beam CT instrument at a resolution of 200 µm isometric cube voxel and using i-cat cone beam CT instrument at a resolution of 250 µm isometric cube voxel. We analyzed the trabecular bone both in the maxilla and mandible, on the left side between the roots of the third molars. Following preparation of dental jaw sections with relevant molars (M3 and M2), the samples were scanned using microct (SkyScan 1172) at a resolution of 17 µm isometric cube voxel. The MCT scanning was used as a basis of comparison, as the MCT is regarded as the best recognised radiation imaging technique. These scanned images were used as a gold standard for comparison. The trabecular structure and texture was determined by CTan v software (SkyScan), using manual tresholding. Results Correlation coefficient values between the Planmeca ProMax 3D smart and micro-ct were: structural model index: 0.79, and trabecular thickness: Weak correlation was found between any derived parameters of icat and micro-ct (0-0.16). Figure 1: Maxillary 2nd and 3rd molars with ROI (left: i-cat image, right: Planmeca 3D smart image)

18 Page 77 Figure 2: Maxillary 2nd and 3rd molars with ROI (reconstructed MCT image) Conclusion A strong correlation between Planmeca ProMax 3D smart and MCT suggests that highresolution cone beam computed tomographs might provide reliable derived parameters presenting the trabecular bone microarchitecture. MCT seems to be a useful tool for a comparison on dental CBCT systems ability in bone microarchitecture analysis. References: 1. Kovacs Miklós, Fejérdy Pál, Dobó Nagy Csaba: Metal artefact on head and neck conebeam CT images, Fogorvosi szemle, 5, , Carl W. Newton, Michael M. Hoen, Harold E. Goodis, Bradford R. Johnson, Scott B. McClanahan: Identify and Determine the Metrics, Hierarchy, and Predictive Value of All the Parameters and/or Methods Used During Endodontic Diagnosis, Journal of Endodontics, 12, , Michael V. Swain, Jing Xue: State of the Art of Micro-CT Applications in Dental Research, International Journal of Oral Science, 4, , Kıvanç Kamburo!lu, Hakan Kurt, Eray Kolsuz, Bengi Özta", #lkan Tatar, Hakan Hamdi Çelik: Occlusal Caries Depth Measurements Obtained by Five Different Imaging Modalities, Journal of Digital Imaging, 1-10, Gary Yip, Paul Schneider, Eugene W. Roberts: Micro-Computed Tomography: High Resolution Imaging of Bone and Implants in Three Dimensions, Seminars in Orthodontics, 2, , Mario Tanomaru-Filho, Regina K. P. Lima, Paula A. Nakazone, Juliane M. G. Tanomaru: Use of computerized tomography for diagnosis and follow-up after endodontic surgery: clinical case report with 8 years of follow-up, Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontology, 4, , Dan Brüllmann, Martin Seelge, Elmar Schömer, Ralf Schulze, Ulrich Schwanecke: Alignment of cone beam computed tomography data using intra-oral fiducial markers, Computerized Medical Imaging and Graphics, 7, , 2010

19 Page 78 Evidence of pulp disease in the inlayed teeth of the ancient Mayans: a micro-ct study M. A. Versiani 1, J. D. Pécora 1, M. D. Sousa-Neto 1 1 Department of Restorative Dentistry, Faculty of Dentistry, University of São Paulo, Avenida do Café s/n, Ribeirão Preto, São Paulo, Brazil, CEP Aims Root canals and their associated pulp chamber are the physical hollows within a tooth that are naturally inhabited by nerve tissue, blood vessels and other cellular entities, named pulp tissue. In a situation that a tooth is considered so threatened and infection of the root canal space is considered inevitable, the removal of the pulp tissue is advisable to prevent such infection that could lead to necrosis, calcification or internal resorption. Calcification is considered as the hardening of decaying of dead soft tissue represented by calcified nodules or amorphous deposits in the pulp, and the internal resorption as a condition when the dentine and pulpal walls begin to resorb centrally within the root canal 1. An old and fascinating human practice, body ornamentation can be achieved through a variety of means including clothing, piercings, tattooing, and scarification, among others. Another such method, artificial dental modification, is found in many areas of the world but is perhaps best known in Mesoamerica. The Mayas were a peaceful people with a highly developed culture who inhabited the Yucatan Peninsula. The nation's history began about 2500 B.C., but the culture flourished from about 300 A.D. to about 900 A.D. In their dental practiced, teeth were filed into points, ground into rectangles and drilled with small holes to permit the insertion of small round pieces of stone in over a hundred different patterns 2. The filing procedure was employed using a hard tube that was spun between the hands or in a rope drill, with a slurry of powdered quartz in water as an abrasive, to cut a hole through the tooth enamel. Then, the inlay was cemented into place. The stone inlay was ground to fit the cavity so precisely and the plant adhesive was so powerful that many have remained in the teeth for thousands of years 2,3. Although many studies involve description and classification of artificially modified teeth, few examine the consequences of this modification. Previous X- ray and scanning electron microscopy analysis have shown that most of the time the holes reached the dentine and even the pulp cavity 2,4-6. However, to date no study demonstrated the three-dimensional relationship between the tooth inlay and the pulp cavity in Mayan's teeth. The aims of the present study were to evaluate qualitatively the relationship between the inlay and the pulp cavity, and its influence within the pulp canal space, in ancient Mayan's teeth, using microcomputed tomography technique.

20 Page 79 Method Six well-preserved Mayan's teeth from Guatemala with approximately 1600 years old, donated by a private collector, presenting alteration of the labial surface of the crown with inlays of jade or hematite, classified as types E1 (one stone at the labial surface) and E2 (two stones at the labial surface) 7 were selected for this study. SkyScan 1074 (SkyScan, Kontich, Belgium) high-resolution micro-ct scanner was used to scan the teeth. The system consisted of a sealed air-cooled X-ray tube, kv / 40W / 800 µa, with a precision object manipulator with two translations and one rotation direction. The system also included a 14- bit CCD-camera based on a 1.3 Megapixel (1304 x 1024 pixels) CCD-sensor. Each tooth was vertically positioned on a metal holder in the center of the specimen stage and scanned (50 kv, 800 µa) with a pixel size of 22.5 µm, rotational step of 0.70 degree, rotational angle of 180 degrees, and a 3.1-second exposure time, using a 1-mm-thick aluminum filter. With the NRecon version (Skyscan) software, images obtained from the scan were reconstructed to show 2-dimensional (2D) slices of the inner structure of the teeth. Finally, the CTan version 1.11 and CTVol version 2.1 (Skyscan) softwares were used for the 3- dimensional (3D) volumetric visualization and qualitative analysis of the canal space assessed by an expert observer. Results The filing of the all analyzed teeth was of type E1 (Figures 1 to 5) except the lateral upper incisor that was type E2 (Figure 6). It is interesting to note that teeth with a thicker enamel layer, as canines and premolar (Figures 1 to 3), did not present significant alteration in the canal space, as pulp tissue was not injured. The interior anatomy of the root canal space and dentine are clearly visible in these teeth. On the other hand, the central and lateral incisors (Figures 4 to 6) showed that the cavities created to insert the inlay stones perforated the pulp chamber, resulting in an irreversible inflammatory reaction of pulp tissue leading to a massive internal resorption (Figures 4 and 5) and calcification of the pulp tissue (Figure 6). Figure 1: Right upper canine. 3D reconstruction in a frontal and lateral views of the external (a, b) and internal (c, d) anatomy of the teeth and the pulp canal space (e, f). Transversal (g, h) and axial (i, j, k) cut showing pulp canal space and dentine without evidence of disease [green arrow: dental inlay (hematite)].

21 Page 80 Figure 2: Right upper first premolar. 3D reconstruction in a frontal and lateral views of the external (a, b) and internal (c, d) anatomy of the teeth and the pulp canal space (e, f). Transversal (g, h) and axial (i, j, k) cut showing pulp canal space and dentine without evidence of disease [green arrow: dental inlay (jade)]. Figure 3: Right upper canine. 3D reconstruction in a frontal and lateral views of the external (a, b) and internal (c, d) anatomy of the teeth and the pulp canal space (e, f). Transversal (g, h) and axial (i, j, k) cut showing pulp canal space and dentine without evidence of disease [green arrow: dental inlay (jade)].

22 Page 81 Figure 4: Left upper central incisor. 3D reconstruction in a frontal and lateral views of the external (a, b) and internal (c, d) anatomy of the teeth and the pulp canal space (e, f). Transversal (g, h) and axial (i, j, k) cut showing pulp canal space and dentine with evidence of disease [red arrow: internal resorption; green arrow: dental inlay (hematite)]. Figure 5: Left upper central incisor. 3D reconstruction in a frontal and lateral views of the external (a, b) and internal (c, d) anatomy of the teeth and the pulp canal space (e, f). Transversal (g, h) and axial (i, j, k) cut showing pulp canal space and dentine with evidence of disease [red arrow: internal resorption; green arrow: dental inlay (hematite)].

23 Page 82 Conclusion Figure 6: Right upper lateral incisor. 3D reconstruction in a frontal and lateral views of the external (a, b) and internal (c, d) anatomy of the teeth and the pulp canal space (e, f). Transversal (g, h) and axial (i, j, k) cut showing pulp canal space and dentine with evidence of disease [red arrow: calcification; green arrow: dental inlay (jade)]. Considering the limitations of the present study, micro-ct analysis of ancient Mayan's teeth allowed to observe tridimensionally the anatomical relationship between the bottom of the artificial cavity prepared to hold an inlay, and the pulp chamber, evidencing a perforation of a depth of approximately 1.5 mm deep, with parallel walls and a convexly shaped basis only in the incisors. In these teeth, the pulpal response was readily detectable with the presence of internal resorption and calcification. References: 1. Hargreaves KM, Cohen S, "Cohen's Pathways of the Pulp", Mosby Elsevier, Whittington SL, Reed DM, "Bones of the Maya: Studies of Ancient Skeletons", University of Alabama Press, Romero-Vargas S, Ruiz-Sandoval JL, Sotomayor-González A, Revuelta-Gutiérrez R, Celis-López MA, Gómez-Amador JL, García-González U, López-Serna R, García- Navarro V, Mendez-Rosito D, Correa-Correa V, Gómez-Llata S, "A look at Mayan artificial cranial deformation practices: morphological and cultural aspects", Neurosurg Focus, 6, E2, Gwinnett AJ, Gorelick L, "Inlayed teeth of ancient Mayans: a tribological study using the SEM", Scan Electron Microsc, 2, , McKillop HI, "The ancient Maya: new perspectives", ABC-CLIO, González EL, Pérez BP, Sánchez JA, Acinas MM, "Dental aesthetics as an expression of culture and ritual", Brit Dent J, 2, 77-80, Romero J, "Dental Mutilation, Trephination, and Cranial Deformation", University of Texas Press, 1970.

24 Page 140 Flat-oval root canal preparation with Self-Adjusting File instrument: a micro-ct study M. D. Sousa-Neto 1, M. A. Versiani 1, J. D. Pécora 1 1 Department of Restorative Dentistry, Faculty of Dentistry, University of São Paulo, Avenida do Café s/n, Ribeirão Preto, São Paulo, Brazil, CEP Aims Root canal treatment is a dental procedure which is undertaken to treat the infected pulp of a tooth. The pulp is the technical name for the soft tissue inside the tooth 1. The ultimate goal of root canal preparation is to remove the inner layer of the dentin while allowing an irrigant solution to reach the entire length of the root canal. To deal with this complex problem, several techniques and modified instrument designs have been proposed. In modern endodontic practice, a move has been seen towards the use of engine-driven rotary preparation with nickel-titanium (NiTi) instruments, since they provide more centered and tapered preparations 2,3. Although many technical advances have been made in endodontics, canal preparation is still adversely influenced by the highly variable anatomy, especially in oval, flat, or curved root canals. In flattened canals, rotary files have failed to perform adequate cleaning and shaping leaving untouched fins or recesses on the buccal and/or lingual aspects of the bore created by the instrument. Therefore, efforts should be directed toward the development of strategies that maximize root canal preparation before filling 4. The Self-Adjusting File (SAF) (ReDent-Nova, Ra anana, Israel) has been devised with the purpose of sidestepping some of the limitations of rotary NiTi instruments 5. The SAF is a hollow file designed as a compressible, thin-walled, pointed cylinder, of either 1.5- or 2.0-mm diameter, composed of a thin nickel-titanium lattice. During its operation, the file is designed to adapt itself three-dimensionally to the shape of the root canal. Then, the file attempts to regain its original dimensions, thus applying a constant delicate pressure on the canal walls 5,6. Important technological advances for imaging dental structures have been introduced in recent. The development of X-ray micro-computed tomography (µct) has gained increasing significance in the study of hard tissues. µct offers a noninvasive reproducible technique for three-dimensional assessment of root canal systems and can be applied quantitatively as well as qualitatively 6. To date, root canal preparation with SAF has been quantitatively and qualitatively describe in different teeth, but not in mandibular incisors. Thus, the purpose of this study was to evaluate the root canal preparation in flattened root canals of mandibular incisors treated with either rotary or SAF, using three-dimensional µct analysis.

25 Page 141 Method Forty single-rooted freshly extracted human mandibular incisor teeth with fully formed apices were selected. Before use, teeth were washed in running water for 24 h, slightly dried, mounted on a custom attachment, and scanned in a desktop X-ray microfocus CT scanner (SkyScan 1174v2; SkyScan N.V., Kontich, Belgium) at an isotropic resolution of 19.7 µm. The system consisted of a sealed air-cooled X-ray tube, kv / 40W / 800 µa, with a precision object manipulator with two translations and one rotation direction. The system also included a 14-bit CCD-camera based on a 1.3 Megapixel (1304 x 1024 pixels) CCD-sensor. Root canals were accessed by using high-speed diamond burs, and patency of the coronal canal was confirmed. Coronal flaring was accomplished with proper burs. Specimens were then randomly assigned to two experimental groups (n = 20), according to the instrumentation technique: SAF (group A) and rotary (group B). In all groups, after root canal preparation, a final rinse with 5 ml of normal saline solution was performed, the root canals were dried with paper points, and teeth were resubmitted to a postoperative µct scan applying the initial parameter settings. Images were reconstructed from the apex to the level of the cementoenamel junction (NRecon v ; SkyScan) providing axial cross sections of the inner structure of the samples. For each tooth, evaluation was done for the full canal length in approximately 400 slices per specimen. CTAn v software (Skyscan) was used for three-dimensional analysis of volume and surface area. Increases of all analyzed parameters were calculated by subtracting the scores for the treated canals from those recorded for the untreated counterparts. CTVol software (Skyscan) was used for three-dimensional visualization and qualitative evaluation of the pre- and post-instrumented canals. Color-coded root canal models (green indicates preoperative, red postoperative canal surfaces) enabled qualitative comparison of the matched root canals before and after shaping. OnDemand 3D software (Cybermed Inc., Irvine, CA, USA) was used for the analysis of the fifteen superimposed cross-sections images of each specimen (n=300 per group) regarding the percentage of instrumented and non-instrumented walls. The root canal preparation was classified into two categories: (a) cross-section in which the whole perimeter or almost all perimeter was treated (80% or more of the perimeter treated) and (b) cross-section in which most of the perimeter was untreated (20% or less of the perimeter treated). The results were statistically analyzed with independent sample t test and chi-square test (with Yates' correction) between groups and paired sample t test within the group, with the null hypothesis set as 5%, using SPSS 17.0 for Windows (SPSS Inc., Chicago, IL, USA). Results The results of three-dimensional analysis are detailed in Table 1. Despite the mean increase of the canal volume was significantly higher with SAF (1.47 ± 0.67 mm 3 ) than rotary instrumentation (2.32 ± 1.0 mm 3 ) (P =.04), the same was not observed with the surface area (P >.05). Within group, volume and surface area showed significant statistical difference between pre- and postoperative results (P <.05). Preoperatively, root canal cross sections presented significantly flatter in the mesiodistal view than buccolingual aspect. Its geometry was changed after root canal preparation with both instruments. Superimposed µct reconstructions in all thirds demonstrated that the use of SAF resulted in a more uniform dentin removal along the perimeter of the canals than rotary instrumentation. The latter showed substantial untouched areas mainly on the lingual side of the canal. The percentage of mechanically untreated canal walls at coronal, middle and apical third, calculated by using superimposed µct data sets, were 8%, 35%, and 15% for SAF group and 38%, 56%, and 25% for rotary group. There was statistically significant difference between the instrumented and the noninstrumented walls between groups at coronal and middle thirds (Table 2). Cross sections and tridimensional analysis showed that the use of SAF resulted in a more homogenous preparation of the root canal walls compared to rotary instruments (Figures 1 and 2).

26 Page 142 Table 1: Morphometric 3D data (mean ± standard deviation, n=20 each) and their changes for root canal in lower incisors before and after preparation with SAF or rotary systems. Rotary (n=20) SAF (n=20) P Before Prep. 4.13± ± Volume (mm 3 ) Surface (mm 2 ) After Prep. 5.59± ± ! 1.47± ±1, % 41.63± ± P Before Prep ± ± After Prep ± ± ! 4.06± ± % 13.29± ± P Statistical significant difference between groups is marked with red font in the same line (Independent sample t test, P < 0.05). Statistical significant difference within group is marked with bold font in the same column (Paired sample t test, P < 0.05). Table 2: Statistical comparison of the percentage of root canal walls preparation using SAF system or rotary instruments at different thirds. Coronal Third Middle Third Apical Third Category Rotary SAF P Rotary SAF P Rotary SAF P 80% or more treated % or less treated Total (%) Statistical significant difference between groups is marked with red font in the same line (Chi-Square test with a Yates' correction,! =.05)

27 Page 143 Figure 1: Representative example of µct data of flat-oval-shaped root canal of a mandibular incisor prepared with the SAF system at the middle level of the root (yellow line in A). 2D analysis show the preoperative (B), postoperative (C), and superimposed reconstructions (D) of the root canal (green and red areas are preoperative and postoperative superimposed cross sections, respectively). Note that SAF instrumentation removed a uniform layer of dentin from root canal walls (D). In the qualitative evaluation, three-dimensionally reconstructed µct images show the root canal before preparation in a buccal (E) and distal views (G). Superimposed µct reconstructions in a buccal (F) and distal (H) views demonstrate the homogenous preparation of the canal surface after root canal preparation with SAF even in a root canal with very irregular walls. Figure 2: Representative example of µct data of a flat-oval-shaped root canal of a mandibular incisor prepared with K3 rotary system at the middle level of the root (yellow line in A). 2D analysis show the preoperative (B), postoperative (C), and superimposed reconstructions (D) of the root canal (green and red areas are preoperative and postoperative superimposed cross sections, respectively). Note that rotary instrumentation increased the diameter of the canal with a round cross section only in its buccal aspect (D). In the qualitative evaluation, three-dimensionally reconstructed µct images show the root canal before preparation in a buccal (E) and distal views (G). Superimposed µct reconstructions in a buccal (F) and distal (H) views demonstrate substantial untouched areas with rotary instruments even when used in a root canal with regular walls.

28 Page 144 Conclusion Within the limitations of this ex vivo study, it can be concluded that in the coronal third of the canal, mean increases of area and volume of the root canal, as well as the percentage of prepared walls, were significantly higher with SAF than rotary instrumentation. By using SAF instrument, flat-oval-shaped canals of mandibular incisors were homogenously and circumferentially prepared. The size and flare of the SAF preparation in the apical third of the canal were equivalent to those prepared with #40 rotary file with a.02 taper. References: 1. Hargreaves KM, Cohen S, "Cohen's Pathways of the Pulp", Mosby Elsevier, Fornari VJ, Silva-Sousa YT, Vanni JR, Pécora JD, Versiani MA, Sousa-Neto MD, "Histological evaluation of the effectiveness of increased apical enlargement for cleaning the apical third of curved canals", Int Endod J, 11, , Versiani MA, Pascon EA, de Sousa CJ, Borges MA, Sousa-Neto MD, "Influence of shaft design on the shaping ability of 3 nickel-titanium rotary systems by means of spiral computerized tomography", Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 6, , Sasaki EW, Versiani MA, Perez DE, Sousa-Neto MD, Silva-Sousa YT, Silva RG, "Ex vivo analysis of the debris remaining in flattened root canals of vital and nonvital teeth after biomechanical preparation with Ni-Ti rotary instruments, Braz Dent J, 3:233-6, Metzger Z, Teperovich E, Zary R, Cohen R, Hof R, "The self-adjusting file (SAF). Part 1: respecting the root canal anatomy - a new concept of endodontic files and its implementation", J Endod,4, , Peters OA, Paqué F, "Root canal preparation of maxillary molars with the selfadjusting file: a micro-computed tomography study", J Endod, 1, 53-7, 2011.

29 Page 176 A Preliminary Approach for Analysing Debris in the Complex Canal Network of Teeth Jonathan Robinson 1, Paul Cooper 2, Richard Williams 1, Hamid Deghani 1, Philip Lumley 2, Damien Walmsley 2 1 Physical Science of Imaging in the Biomedical Sciences (PSIBS), University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK. 2 School of Dentistry, University of Birmingham. Aims In dentistry, root canal therapy is the internal cleaning of a complex channel network contained within the tooth. The aim is to disinfect the canal and remove any remaining bacteria to prevent further infection prior to root filling. Irrigant solutions play an important role in this procedure as they allow canal debridement. Sodium hypochlorite (NaOCL) is an irrigant that is clinically recommended due to its antimicrobial and demineralising actions 1,2. Ethylene-Diamine-Tetra-Acetic acid (EDTA) is another commonly used irrigant that is also capable of demineralising tissue 1,2. Debridement of root canals and the anatomy of the isthmus (microcanals which link adjacent canals) have commonly been analysed using optical techniques such as stereomicroscopy) and scanning electron microscopy (SEM) 3-5. These approaches require mechanical crosssectioning of the tooth to enable imaging. Analysing debris removal in hard to reach areas such as the isthmus therefore would be better performed using an internal non-invasive imaging approach. MicroCT provides such an alternative imaging however investigators are currently unable to distinguish between structural and debris dentine as both exhibit the same opacity to x-rays 6. The aim of this study was to create a computational method to quantify debris, distinguishing structural dentine and debris and subsequently use this approach to analyse differences between canal preparations performed using two different irrigants (NaOCL and EDTA). Method Twelve lower first permanent molar teeth were pre-scanned and divided into 2 groups based on size of isthmus. Teeth were further subdivided into 3 groups comprising treatment with: i) no irrigation (positive control), ii) 17% aqueous EDTA (Vista Dental, Racine, US) and iii) 6% aqueous NaOCL (Vista Dental, Racine, US). Each group contained an equal number of teeth from groups 1 and 2. The canals were prepared by an A B Endodontic clinical expert using standard instruments (Dentsply Maillefer, Addlestone, UK). Fig. 1.MicroCT images of A) canal space and B) debris in the same canal. Analysis of the entire lengths of all teeth was performed prior to and after preparation using a Skyscan 1172MicroCT system (E2V). Using segmentation, erosion and dilation processes, two image sets per tooth was obtained for canal space and debris in the canal space (Fig. 1). Debris per unit pixel was calculated through summation of pixels in each image set. By calculation on pre- and post-treatment scans, percentage increase/reduction in debris was determined.

30 Page 177 Results A preliminary study initially quantified the debris in three teeth with large isthmuses. The use of no irrigant produced a 45% increase in debris whilst EDTA and NaOCL resulted in a 41 and 28% reduction in debris present. These results were in agreement with a qualitative analysis obtained by visual inspection of images. Qualitatively NaOCL appeared to be the most effective irrigant however this was dependent upon canal morphology. Both protrusions and micro-canals create unreachable areas, prohibiting the complete removal of debris (fig. 2). Figure 2.Images of root canal before (Top) and after (Bottom) preparation. Arrows indicate areas with accumulated debris. Conclusion The method presented to quantify debris in teeth provides promising results and is able to distinguish between structural and debris dentine whilst enabling quantification of the whole canal volume. Further work will apply this methodology to all samples and computational error will be determined using ball bearings with known volume. Data indicates that no irrigation resulted in effective root cleaning however further studies are planned to investigate whether the use of additional ultrasonic cleaning techniques improve debridement. References 1. Walmsley DA, Walsh TF, Burke FJ, Shortall AC, Lumley PJ, Hayes-Hal R, Restorative Dentistry, (Elsevier, Edinburgh), , Zehnder M, Root Canal Irrigants, J Endod, 32, , Peter OA, Barbakow F (2000) Effects of Irrigation on Debris and Smear Layer on Canal Walls Prepared by Two Rotary Techniques: A Scanning Electron Microscopic Study. J Endod 26: Gambarini G (1999) Shaping and cleaning the root canal system: A scanning electron Microscopic evaluation of a new instrumentation and irrigation technique. J Endod 12: Teixeira FB, Sano CL, Gomes BP, Zaia AA, Ferraz CC, Souza-Filho FJ (2003) A preliminary in vitro study of the incidence and position of the root 6. Paqué F, Laib A, Nat S, Gautschi H, Zehnder M (2009) Hard-Tissue Debris Accumulation Analysis by High-Resolution Computed Tomography Scans. J Endod 35:

31 Page 207 Comparative Accuracy of dental CBCT Equipment with MCT in the Characterisation of Root Canal System Csaba Dobo Nagy, Levente Pataky, Regina Mikusi, Independent Section of Radiology, Dental Faculty, Semmelweis University, Budapest, Hungary Objectives: The aim of this study was to compare three differing CBCT instruments with gold standard to quantitatively determine their respective proficiencies with respect to imaging root canal systems. Methods: The skulls of three female monkeys (Maccaca fascicularis), terminated for other research purpose, were skanned at their highest respective resolutions for each of the following instruments: Planmeca ProMax 3D smart, i-cat and NewTom VG. The root canal system of the left upper and lower molars were dynamically analysed, coronal to apical, in three plains from reconstructed images by two independent observers, each with more than ten years experience in oral radiology and endodontology. The observers were not provided with results from related microct analyses. The most apical level where the root canal lumen was visible, was used as the reference level (RL). Following preparation of dental jaw sections with relevant molars (M3 and M2), the samples were scanned using microct (SkyScan 1172) at a resolution of 17µm isometric cube voxel. These scanned images were used as a gold standard for comparison. Root canal lumen were analysed at the RL on the axial planes of the microct, with the help of CTAn software (SkyScan). Following the manual tresholding of images the area of the lumen, the major and minor diameters were subsequently determined. The aspect ratio as well as the mean thickness values were also calculated using SkyScan. Results: Using the Planmeca instrument, only one root canal end was invisible from 1.80 mm coronal to the apex. NewTom showed a total of 11 apical root canal lumen as being invisible, with a mean RL-apex distance of 2.79±1.34 mm. Finally, i-cat images showed two thirds of apical root canals (16 of 25) as being invisible, with a 3.62±1.45 mm mean length for invisible parts. In nine cases where the apical lumen was invisible on both CBCTs, the invisible part of the root canal was 2.31 mm longer on i-cat images. The cross section parameters of the root canal lumen were as follows: the mean minor diameter was ±86.85!m and 69.46±43.56!m while the major diameter was ±210.69!m and ±82.08!m on i-cat and NewTom images, respectively. The aspect ratios representing the cross sectional shape of the lumen were 3.11±1.39 and 3.00±0.98, respectively. Mean thickness of the root canal lumen at RL was 95.05±44.34!m and 55.06±18.52!m for i-cat and NewTom, respectively. Conclusion: Results indicate that MCT seems to be a useful tool for determining accuracy of clinicaly used CBCT equipments in a compararison.!

32 Page 219 Timelines in teeth: using micro-ct scanning to investigate mineralization in developing human enamel Janet Montgomery 1, Julia Beaumont 2, Kevin Mackenzie 3 1 Department of Archaeology,Durham University,Durham, DH1 3LE, England 2 AGES, University of Bradford, Bradford, BD7 1DP, England 3 Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, Scotland Aims The enamel of a human tooth crown holds a mini-archive of isotopic information recording the diet, climate and residence of its owner(1,2,3). This information allows us to reconstruct what an individual was eating, and what the environmental conditions were in the past. In particular, if we can construct a temporal sequence for an individual we can also determine how they moved around and utilized this environment, and what seasonal changes in temperature were experienced. If the pattern of mineralization can be established for each tooth type, then it would be possible to sample the enamel using this as a template. Archaeological teeth from individuals who have died while the teeth were forming offer a unique chance to investigate the different stages of mineralization. A pilot study suggested that Micro-CT scanning offered a non-destructive way of mapping the changes in density as teeth develop and mineralize(4). Method The samples illustrated are from 10th-11th century AD cemetery at Raunds, Northamptonshire, UK. Scanning was carried out at the Microscopy and Cellular Imaging Facility, University of Aberdeen using a SkyScan-1072 high-resolution desk-top micro-ct system. Teeth were scanned at 100kV/98.4uA using a 1mm Aluminum filter at a magnification of x23, giving a pixel resolution of 13.31!m. Back projection images were reconstructed using NRecon software, viewed in Dataviewer and false coloured in relation to greyscale values of the tooth. The reconstructed datasets were imported into CTAn and a region of interest (ROI) was selected of just the tooth, 3D models were then created and viewed in CTVol software. In order to obtain absoluterather than relative density, calibration of the scanner was carried out using three different methods: Bone mineral density phantoms (0.25g/cm 3 and 0.75g/cm 3 phantoms supplied by Skyscan), Two longitudinal sections from fully-mineralized teeth previously measured using micro-radiography; Calibration (using air and water) to Hounsfield units. Results The partially mineralized archaeological teeth showed surface bands of staining from the burial environment inferred as indicating different density and thus resistance to post-mortem change, which matched the differential mineralization seen in the scans. The density of the incisor teeth appears to decrease from the tip downwards, with a front of mineralization both at the junction between enamel and dentine, and on the surface (Figure 1). The molar tooth has a more complex pattern, with high density achieved at the cusp tips and the

33 P a g e 220 cervical margin. Areas which have a more complex shape within the enamel such as the occlusal fissures appear less dense. (Figure 2). Figure 1: Upper left central incisor, crown ½ formed : image from scan colouredusing Dataviewer to enhance the density range. Figure 2: upper right first permanent molar, crown complete. Figure 3: Upper left first permanent molar showing high density on the buccal wall surface, interpreted as beam hardening. Calibration has been difficult, with differing results obtained: Hounsfield units produces unlikely figures for the density of the enamel; and the teeth sections calibrated by micro-

34 Page 221 radiography show marked density differences when compared to readings obtained from the Micro CT using the phantoms for BMD. Problems have also arisen when analyzing fully developed teeth, with beam-hardening causing artefacts in the density of the surface enamel (Figure 3). (We are now using a much higher BH reduction of 50% to help minimize this artifact) Conclusion Micro-CT scans certainly allow the production of exciting images which suggest that we can indeed map the direction of mineralization in different stages of tooth development.technical issues are currently preventing true measurement of the density of different areas within the teeth, and beam-hardening is masking the variations within fully-mineralized teeth. References: 8. Fricke HC, O'Neil JR, and Lynnerup N Oxygen Isotope Composition of Human Tooth Enamel from Medieval Greenland: Linking Climate and Society. Geology 23: Montgomery J, Budd P, and Evans JA Reconstructing the Lifetime Movements of Ancient People: a Neolithic Case Study from Southern England. European Journal of Archaeology 3: Montgomery J, Evans JA, Powlesland D, and Roberts CA Continuity or Colonisation in Anglo-Saxon England? Isotope Evidence for Mobility, Subsistence Practice and Status at West Heslerton. American Journal of Physical Anthropology 126: Beaumont J Timelines in teeth: an evaluation of scientific methods used to assess the pattern of human enamel mineralization.masters thesis : University of Bradford.

35 Page 224 Micro-CT Analysis of Apical Anatomy of Vital and Necrotic Teeth M.R. R. S. Bardauil 1 1 University of São Paulo, SP, Brazil, Rua Lineu Prestes 2227 CEP Aims Root canal treatment is a procedure that requires, beyond the realm of technique, knowledge of the internal anatomy of the root canal, specifically the anatomical apex. Almost all methodologies studied thus far are destructive, laborious, and time-consuming and allow for only one-dimensional measurements. In the present study, we used micro-ct technology to evaluate the anatomical structures of the apical region in vital and necrotic pulp of human permanent teeth. Method This ex vivo study investigated the use of high-resolution micro-ct (SkyScan 1172, SkyScan, Kontich, Belgium), with 6.7-!m pixel resolution, to evaluate 3D slices of apical anatomy of vital (n = 21) (VP) and necrotic (n = 20) (NP) pulp teeth. The x-ray tube was operated at 100 kv, 10 W, and 100!A, with a 0.5-mm aluminum filter and a focal spot size of 5 µm. Scanning of the specimen was done with a 180 vertical axis rotation, and a single rotation step of 0.9. The teeth were fit with the crown positioned downward and the long axis perpendicular to the floor of the specimen-holder and the x-ray source. The scanning time for each sample was approximately 45 min. With NRecon volumetric reconstruction software (SkyScan, 1.4.0), acquired angular projections produced two-dimensional cross-sectional slices through the root apical third volume of interest. An automatic filter for beam-hardening compensation during reconstruction was used at a level of 40% and ring correction of 18%, lasting 20 min. The reconstructed set of slices was viewed with the DataViewer software program (SkyScan 1.3.2). Images were displayed as 3 orthogonal sections, centered at the root canal inside the reconstructed axial slice. Then, using the click and drag feature to control the 3 intersecting orthogonal sections, we set the images so that the emergence of the open foramen could be seen independently or jointly in both sagittal and coronal sections. The following data regarding the root apex were measured in sagittal and coronal sections: the cementum extension into both sides of the root canal, and diameters of the apical foramen and root canal at the cemento-dentino canal junction (CDJ) (Figure 1). Two calibrated examiners performed the measurements and additional features could be used, including magnification and gray scale. The inter-examiners agreement was confirmed by intraclass coefficient (ICC) and measurements were compared by Mann-Whitney test (Wilcoxon rank sum test - p"0.05). Results The results indicated that there was no statistically significant difference in relation to the extension of cementum into the root canal in both groups VP or NP with average distances of 0.32±0.1mm and 0.36±0.16mm respectively. The widest diameter of the apical foramen corresponded to the NP group, of ±131.78µm on average. The average diameter of the root at the CDJ was ±126,62µm for VP and 357,62 ±96,14µm for NP groups presenting no statistically significant difference.

36 Page 225 Figure 1: Diameters of the apical foramen (A) and root canal at the cemento-dentino canal junction (B) Figure 2: The extension of cementum into the root canal (A) and (B) Conclusion With Micro-CT, we were able to map the structures and architecture of the root apex. The high-resolution images could differentiate dentinal and cemental tissues. Radiographically, such differentiation could be assessed from the distinct radiopacities and morphological deposition characteristics of the tissues. The results of this study suggested that CDJ position was not influenced by pulp vitality. References: 1. Bergenholtz G, Spångberg L. Controversies in endodontics. Crit Rev Oral Biol Med 15:99-114, Ponce EH, Fernandez JAV. The cemento-dentino-canal junction, the apical foramen, and the apical constriction: evaluation by optical microscopy. J Endod. 29:214-9, Olson DG, Roberts S, Joyce AP, Collins DE, McPherson JC. Unevenness of the apical constriction in human maxillary central incisors J Endod.34:157-9, 2008.

37 Page Guillaume B, Lacoste JP, Gaborit N, Brossard G, Cruard A, Baslé MF, et al. Microcomputed tomography used in the analysis of the morphology of root canals in extracted wisdom teeth. Br J Oral Maxillofac Surg.44: , Peters OA, Laib A, Rüegsegger P, Barbakow F. Three-dimensional analysis of root canal geometry by high-resolution computed tomography. J Dent Res. 79: , Paqué F, Zehnder M, Marending M. Apical fit of initial K-files in maxillary molars assessed by micro-computed tomography. Int Endod J. 43:328-35, 2010.

38 Page 86 Characterization of root canal curvatures by description of the axis based on microct imaging C. Dobo-Nagy 1, A. Kovacs 2, L. Szilagyi 3, B. Benyo 3 1 Independent Section of Radiology, Semmelweis University, Budapest, 2 Dept. of Architecture of Representation University of Technology and Economics, Budapest, 3 Dept. of Control Engineering and Information Technology, Budapest University of Technology and Economics, Budapest, Hungary Introduction: Every root canal of the teeth has its own individual form; therefore, guidelines are required in both endodontic practice (root canal treatment) and research for the purpose of simplification. Knowledge of the root canal shape helps in making the selection of the best technique or choosing the most appropriate instrumentation of a given root canal treatment a priori. Cone-beam computer tomography (CBCT) was recently introduced in clinical dental radiology. This modality is essentially the same in the way of image records to that of the microct. Experiments carried out on extracted human teeth using microct serve as a basis of clinical use of CBCT. Purpose: The purpose of this study was to give a mathematical description of 3D centreline of root canals of extracted human teeth using microfocal x-ray source records. Materials and methods: Fifty one-rooted teeth were scanned with a microct (SkyScan 1172) where the voxel size was 10x10x10 micrometer. Approximately 1000 reconstructed cross sections of each root were used for further analysis. In this study we introduced a complete image processing procedure, which started with the enhancement of input microct slices, continued with 2D image segmentation based on an enhanced fuzzy c- means clustering [Szilagyi et al. 2003] identification of the centre points via a region growing method. During the automatic segmentation, the centre point was determined by fourclustered image first and sequential iteration of clustered images resulted in the binary image having a detected centre point (Fig. 1.). This centre of gravity was solved as a point of the root canal axis at the given level. Finally centres of gravity were determined at each reconstructed slices and the set of these centres of gravity were taken as the 3D root canal axis. Approximating a fourth degree polynomial function to the set of the centre of gravity provided a simple space curve. The Gaussian least square method was used to fit a polynom to the centre of gravity points. The root canal axis was determined and visualized by the MathCAD 14.0 software. Fitting of root canal axes and functions were determined with correlation coefficients. The basis of the computer graphycs alignment were (1) the determination of the set of curvature values, the mean curvature value and the maximum curvature value as well as the torsion value; and (2) view projections of the spatial polynomials being as plain curvatures. For this later analysis our previously described method was used [Dobo-Nagy et al.1995]. For comparison, third degree and fifth degree polynomial function approximation were also carried out. Results: The correlation coefficient value of the fourth degree polynomial function was found to be between for all of the cases. Approximation of curvatures by any other degree polynomial function resulted in lower correlation coefficient values. The 3D appearance of a root canal may highly different form its 2D views. The projections of the each 3D centreline from different views appeared as different plain curvatures (Fig. 2.).

39 Page 87 Conclusion: The centreline of the root canal was interpolated as a spatial fourth degree polynomial function. The repetition of the fourth degree polynomial approximation appeared to be the most reliable to date. We could simplify the 3D individual form of real root canals. This kind of mathematically based simplification might improve the endodontic practice if it were successfully embedded in a novel dental imaging modality (e.g. CBCT). References: C. Dobo-Nagy, J. Szabo, J Szabo (1995): A mathematically based classification of root canal curvatures on natural human teeth. J. Endodontol. 21(11): L. Szilagyi, Z. Benyo, SM Szilagyi, HS Adam (2003): MR brain image segmentation using an enhanced fuzzy c-means algorithm. Proc 25 th Fig. 1. Detailed presentation of image segmentation of recontructed microct slices First column shows the original recorded images; second column presents clustered images (four coloured image) with a preliminary centre of gravity; third column indicates segmented binary images with detected centre of gravity points at the end if iteration.

40 Page 88 Fig. 2. Different appearances of the same 3D centreline Two different projections of the same root canal are shown in this figure. Notice the big difference in their appearance. The left looks as a gradual simple curve, while the right looks as a severe double curvature. This example represent that the 3D visualisation is superior to the 2D view.

41 Page 108 Resin-composite restoration in a tooth cavity. N. Silikas 1, P. Tsakiridis, G. Eliades 2 1 School of Dentistry, The University of Manchester, nick.silikas@manchester.ac.uk 2 Department of Biomaterials, School of Dentistry, University of Athens, Greece Aims. To investigate in 3-D the adaptation of a resin-composite restorative material placed in a tooth cavity. To generate a video clip file from the reconstructed 2-D sections. Method. One tooth was scanned using the SkyScan 1072 micro-ct (SkyScan, Kontich, Belgium). The scanning conditions were 100 Kv, 98 A, 9.44 pixel size, rotational step of 0.90º and exposure time of 5 s. The specimen was reconstructed with 20 % Beam hardening correction. Results. The three areas of interest were identified. Dentine, enamel and the resincomposite material could clearly be differentiated and shown independently. A video clip (.wmp format) showing the adaptation in 3-D has been composed and each of the three main areas could be shown independently. Dentine Material Enamel Figure 1: Tooth cavity with resin-composite material in the middle (light grey), surrounded by dentine tissue (dark grey) and enamel (white). Conclusions. This will help clinicians to identify possible gaps between material and human tissue. Also, it will elucidate the 3-D adaptation of the material and indicate areas prone to failure. Furthermore, it can show the interfaces between material and enamel and material and dentine. The specimen can be re-examined after it is subjected to various procedures and then identify the areas of failure.