Cone-beam computed tomography images: applications in endodontics

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Content i CONE-BEAM COMPUTED TOMOGRAPHY IMAGES: APPLICATIONS IN ENDODONTICS by Jing Guo A Dissertation Presented to the FACULTY OF THE USC HERMAN OSTROW SCHOOL OF DENTISTRY UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (CRANIO-FACIAL BIOLOGY) May 2015 Copyright 2015 Jing Guo ii Dedication To Kevin To my parents and parents-in-law To my whole family To my mentor: Dr. Reyes Enciso iii Acknowledgements A special thank to all my committee members: Dr. Reyes Enciso Dr. Michael L. Paine Dr. Stanley P. Azen Dr. Glenn T. Sameshima Dr. Parish Sedghizadeh In memory of Dr. James H.S. Simon iv Table of Contents Dedication ii Acknowledgments iii List of Tables vi List of Figures vii Abstract viii Chapter One: Introduction 1 1.1 Cone-Beam Computed Tomography 1 1.1.1 Physical Principles of CBCT 3 1.1.2 CBCT imaging principles 8 1.1.3 Quality of CBCT imaging 10 1.1.4 Data assessment 20 1.2 CBCT Applications in Endodontics 21 1.2.1 Detection of Vertical Root Fracture and Perforation using CBCT 21 1.2.2 Internal and External Root Canal Resorption 23 1.2.3 Root Canal Treatment Assessment using CBCT 25 1.2.4 Differential Diagnosis of Periapical lesions using CBCT 28 1.2.5 Evaluation of root and canal morphology using CBCT 31 1.3 CBCT The Accuracy and Reliability of Diagnostic Test 37 1.3.1 Accuracy 37 1.3.2 Reliability 40 Chapter Two: Evaluation of the reliability and accuracy of using Cone-beam CT for diagnosing periapical cysts from granulomas 43 2.1 Abstract 43 2.2 Introduction 45 2.3 Materials and Methods 48 2.3.1 Radiology 48 2.3.2 Histopathology 48 2.3.3 Diagnostic criteria 50 2.3.4 Evaluation of diagnostic criteria 50 2.3.5 Intra-and inter-rater reliability 52 2.3.6 CBCT comparison to Histopathological reports 54 2.4 Result 56 2.5 Discussion 60 Chapter Three: Evaluation of Root and Canal Morphology of Maxillary Permanent First Molars in a North American Population by Cone-beam Computed Tomography 65 3.1 Abstract 65 3.2 Introduction 67 v 3.3 Material and Methods 71 3.3.1 Subjects 71 3.3.2 Radiological evaluation 71 3.3.3 Statistics 72 3.4 Results 74 3.4.1 Prevalence of three-rooted maxillary first molars on left and right sides 76 3.4.2 Prevalence of three-rooted maxillary first molars according to gender 76 3.4.3 Percentage of MB2 canal in three-rooted maxillary first molars according to age, left and right side, and gender 76 3.4.4 Percentage of Vertucci’s classification of canal type patterns in three-rooted maxillary first molars 77 3.4.5 The relationship among ethnicities and gender on Vertucci’s classification of canal types 80 3.5 Discussion 83 3.6 Conclusion 88 Chapter Four: Evaluation of Root and Canal Morphology of Mandibular Permanent First Molars in a North American Population by Cone-Beam Computed Tomography 89 4.1 Abstract 89 4.2 Introduction 91 4.3 Materials and Methods 94 4.3.1 Subjects 94 4.3.2 Inclusion and exclusion criteria 94 4.3.3 Radiological evaluation 94 4.3.4 Statistics 95 4.4 Results 97 4.4.1 Number of Roots in Mandibular First Molars 99 4.4.2 Root Canal Configuration of Mandibular First Molars 101 4.5 Discussion 106 Chapter Five: Conclusion 110 Reference 113 vi List of Tables Table 1. Comparison of diagnostic results with the gold standard 38 Table 2. Comparison of diagnostic results of one rater for the calculation of intra-rater reliability 41 Table 3. CBCT diagnostic criteria for differential diagnosis of a periapical cyst 52 Table 4. Sensitivity, specificity, false positive rate, false negative rate, overall agreement, and AUC for three evaluators compared with histopathological diagnoses 58 Table 5. Frequency of MB2 occurrence in maxillary first molars in six age groups 78 Table 6. Frequency distribution and percentage of Vertucci's classification of canals for three roots according to gender in 628 three-rooted maxillary permanent first molars 79 Table 7. The frequency of MB2 occurrence in maxillary first molars in five ethnic groups 81 Table 8. Studies of prevalence of disto-lingual roots and canal morphology of mesial roots in different populations 93 Table 9. Vertucci's classification of root canals in 496 mandibular first molars 103 vii List of Figures Figure 1. Principle of CBCT 2 Figure 2. Focal spot size and penumbra effect (shadowing effect) 4 Figure 3. Image intensifier of a CCD based CBCT device 7 Figure 4. The collimator of a CT device 12 Figure 5. FOV, Matrix size, Pixel, and Voxel : 13 Figure 6. Streak artifact due to metallic restorations 18 Figure 7. Aliasing 18 Figure 8. Vertucci's classification of root canal patterns 32 Figure 9. An example of ROC curve 40 Figure 10. Study protocol flowchart 53 Figure 11. ROC curves and AUCs 55 Figure 12. CBCT images and histological reports of four cases 57 Figure 13. Axial sections of maxillary permanent first molars with different root and canal configurations 75 Figure 14. Frequency distribution of Vertucci's classification of mesiobuccal root canals in 458 maxillary first molars by ethnicity 82 Figure 15. Transverse sections of mandibular permanent first molars with different root and canal configurations: 98 Figure 16. Prevalence of number of roots by ethnic groups in mandibular first molars 100 Figure 17. Prevalence of canal configuration according to Vertucci's classification of mesial and distal roots by ethnicity 105 viii Abstract Cone-beam Computed Tomography (CBCT) is an revolutionary invention, which meets the demand for in vivo three-dimensional information for diagnostic tasks in dentistry. It has already been used as an auxiliary method in the research area of periapical lesion differential diagnosis, and has been shown to be an optimal method for pre-treatment identification of root and canal morphology. Periapical cysts and granulomas are common diseases occurring in periapical regions. The treatment of periapical cysts may require surgery, while periapical granulomas may heal under proper root canal therapy. Definitive diagnosis of a periapical cyst versus periapical granuloma is difficult using conventional dental radiographs alone. Histopathologic evaluation subsequent to biopsy is considered the 'gold standard' for definitive diagnosis. The high incidence of periapical granulomas results in a high rate of excessive surgeries for patients whose lesions may heal non-surgically. To treat a periapical granuloma non-surgically, the first step is to diagnose it without surgery. A non- surgical alternative to biopsies for differentiating periapical cysts from granuloma is the issue. Periapical lesions develop as a result of a pathological immune response to continuous stimuli from infected root canals. Pulpectomy should be conducted to remove inflammatory or necrotic pulp. Complete knowledge of internal root morphology has been an important issue in planning and executing root canal therapy. Successful treatment requires clinicians to have a basic ix knowledge of root canal morphology and possible anatomic variations. Maxillary and mandibular permanent first molars have multiple roots and canals, and have high rates of variation in anatomy. The accurate knowledge of the number of roots and canal morphology of these teeth prior to root canal therapy is a concern for endodontists. In Chapter Two, a set of six diagnostic criteria was established to differentiate periapical cysts from granulomas. The six criteria were based on the radiological characteristics of a periapical cyst (location, periphery, shape, internal structure, and effects on surrounding structure). If successful, these criteria will provide greater accuracy, and may improve agreement among raters as well as increase intra-rater reliability while diagnosing between cysts and granulomas. CBCT may exhibit potential for clinical applications in endodontics for the differential diagnosis of cysts versus granulomas. The goal of this study was to evaluate this set of six diagnostic criteria to find out the optimal number of criteria findings necessary for the differential diagnosis of periapical cysts (cavitated lesions) versus periapical granulomas. Moreover, to evaluate the intra-rater and inter-rater reliability of three independent evaluators using the criteria, and the accuracy, specificity, sensitivity and overall agreement of CBCT diagnoses and histopathological reports for three blinded evaluators. In Chapter Three and Four, CBCT scans of 1,484 patients were evaluated, of which 317 cases with bilateral maxillary first molars and 248 cases x with bilateral mandibular first molars were identified. Frequency of number of roots and Vertucci’s canal type for each root were evaluated for all maxillary and mandibular first molars. The presence of an additional mesiobuccal canal (MB2) and the occurrence of disto-lingual roots were recorded for maxillary and mandibular first molars, respectively. Differences by gender and ethnicity were calculated using Chi-square test and Fisher’s exact test. The intra-rater reliability was assessed using Cohen’s Kappa statistic. These two studies were aimed to evaluate the differences in number of roots and canal morphology of maxillary and mandibular permanent first molars according to gender, age and ethnicity in a North American population by means of CBCT images. 1 Chapter One: Introduction 1.1 Cone-Beam Computed Tomography Cone-beam technology is rapidly become a novel technique to assist dentists and specialists to improve patient care [1]. Cone-beam computed tomography (CBCT) (Figure 1) is mostly referred as cone-beam technology. A conically shaped beam is the most significant characteristic of CBCT compared with the traditional “fan-shaped beam” of conventional Computed Tomography (CT), which requires multiple complex scan movements to completely scan the area of interest. The conically shaped beam scans the patient in a circular path around the vertical axis of the head in 180° or greater, reducing the acquisition time of data, the exposure time for the patient, and the artificial artifacts due to patient movements. The rapid development of software has made it possible for clinicians to better interpret the information acquired from CBCT images, increasing the number of clinical applications of CBCT in dentistry. 2 Figure 1. Principle of CBCT 3 1.1.1 Physical Principles of CBCT The fundamental physical principles for CT and CBCT are the same. An X-ray tube is used for the generation of X-ray beams. Focal spot (Figure 2) is the area in the X-ray tube from which the resulting x-rays are emitted. The size and shape of the focal spot affect the ray width and the resolution of the images [2]. A small focal spot creates rays with narrow widths, which produce better image details. However, a small focal spot will concentrate heat and could damage the tube; a balance between resolution and heat concentration needs to be decided by operators when the device allows a choice of focal size. 4 Figure 2. Focal spot size and penumbra effect (shadowing effect) 5 After the beam is generated, it passes through the body and then through an image intensifier (II) (Figure 3). The role of the image intensifier is to interact with the incoming radiation received, and to release light photons. The light photons expose the lens of digital charge-coupled device (CCD) camera and transferred to the CCDs [3]. The CCDs then convert the light photons to digital values in order to be processed by computers. The initial and outgoing intensity of the beam of photons are recorded and the linear attenuation coefficients of each voxel are obtained as follows; Lambert-Beer Law is used to describe the attenuation of monochromatic x-ray beam through a homogeneous object [4]. The formula is: I = I o e –µx [2] where I 0 is the initial intensity measured without the object in the field of view, I is the outgoing intensity after transmitted through the object, x is the length of the X-ray path through the object, and µ is the linear attenuation coefficient of the material at the X-ray energy employed [2]. However, in the real world, the subject (human body) is not homogeneous, the attenuation of X-rays consequently can be described by: I = I o e -∫µx dx [2] Linear attenuation coefficient, µ, can be calculated. These coefficients constitute so-called ‘raw data’ that are then fed into an image reconstruction method that generates cross-sectional images via digital/analog converter, whose pixel values correspond to linear attenuation coefficients [5]. In CT 6 images, the resulting attenuation coefficients are usually expressed with reference to water (HU=0), and are given in Hounsfield units (HU): HU patient =1000 x (µ patient -µ water )/µ water The image intensifier/Charge-coupled device (II/CCD) detector is still widely used in conventional CT devices and in early CBCT units. Nowadays, a large area detector — Flat panel detector (FPD) is equipped in most CBCT units. FPD uses a layer of needle structured scintillator coupled to an array of photodiodes on the a- silicon plate [6], and is less complicated in structure and less bulky compared to II/CCD, however, PFD has the limitations of: a) bad pixels (non-functional pixels), b) linearity of response to the radiation spectrum (the output signal of each pixel should be linear with the input radiation density), and c) uniformity of response throughout the area of the detector (image uniformity of each pixel of the detector). These limitations cause FPD-based artifacts and affect the image quality. Thus, images from CBCT with flat panel detectors lack adequate grey scale sensitivity and do not directly represent Hounsfield Units [2]. 7 Figure 3. Image intensifier of a CCD based CBCT device 8 1.1.2 CBCT imaging principles CBCT imaging process includes data acquisition and imaging reconstruction. 1.1.2.1 Data Acquisition. The X-ray source and the detector of a CBCT scanner are fixed to a rotating gantry [7]. A subject is positioned within the gantry, and the conically shaped beam is then transmitted through the region of interest (ROI), and the detector measures the attenuated radiation. Multiple sequential planar projection images of the FOV are acquired in an arc of 180 o or greater [5]. These images, referred as the raw data, are not easily readable by clinicians. They appear similar to cephalometric radiographic images taken from a slightly different point of view. A process called image reconstruction is needed [5]. The total number of acquired projections depends on the exposure time, frame rate (number of projections acquired per second), and on the trajectory arc. A high number of projections provide more information to reconstruct the image, allowing for greater spatial and contrast resolution. However, this is usually accomplished with a longer scan time, a proportionally higher patient’s radiation dosage, and longer primary reconstruction time. A trade-off between resolution and patient’s radiation dosage needs to be made. Different from a one-dimensional detector of conventional CT, CBCT requires a two-dimensional receptor, aka the area detector (FPD) [5]. FPD lacks uniformity and linearity of response throughout the area of the detector, which effects the image quality [8]. Thus, detectors should be re-calibrated periodically. 9 1.1.2.2 Image Reconstruction. Hundreds of raw data images are then obtained at the data acquisition process. These data is then processed to create a volumetric dataset (voxels) using software algorithms [5]. This process is called the image reconstruction. Some parameters should be corrected before reconstruction of CBCT data, such as the adjustment of Frankfort horizontal and mid-sagittal plane, grayscale brightness levels, contrast range, and specific filters [5]. After correction, images are converted into a special representation called a sinogram, then processed by a reconstruction algorithm named filtered back-projection [5]. Filtered means the use of digital image processing algorithms, which are used to improve image quality or change certain image quality characteristics, for example, detail and noise [2]. The actual process used to reconstruct the image is the "back projection" [2]. As mentioned, the intensity data is a profile of the X-ray attenuation by the objects. It will be possible to reconstruct an image if a number of the individual profiles are projected back onto an image area [2]. When individual views are superimposed, the image will be reconstructed. Nowadays, the most widely used filtered back projection algorithm is Feldkamp (FDK) algorithm [5]. Recently, a modified algorithm, the combination-weighted Feldkamp algorithm (CW-FDK) was developed, and Mori et al. [9] tested it in a phantom and concluded that this algorithm could reduce cone-beam artifacts and enhance reconstruction coverage to improve the image quality. 10 1.1.3 Quality of CBCT Imaging Quality, refers to the quality of the image itself, determined by the imaging method (CT, CBCT, etc), the characteristics of the equipment, and the imaging variables selected by the operator [2]. There are three basic CBCT imaging and diagnostic quality factors: image quality, viewing conditions, and observer performance characteristics [2]. a. Image Quality. Resolution; visibility of detail and blur; noise; and artifacts [2] are related to image quality. (1) Resolution There are two types of resolution: spatial resolution, which determines the proximity of details to be detected, and contrast resolution, which enables distinction between tissues of different radio-density [2]. Spatial resolution is determined by the size of the imaging voxels. As mentioned in the physical principle section, linear attenuation coefficient of each voxel is obtained (raw data images) and then fed to the image reconstruction algorithm [5]. Voxels are the individual volume elements produced in formation of the volumetric dataset, and determine the resolution of the images [8]. Voxels are determined by different factors in CT and CBCT. In conventional CT, voxel size is defined by the size of each voxel in three dimensions, i.e., slice thickness, matrix size, and FOV [8]. The slice thickness 11 in CT is determined by the collimator (Figure 4), which is assembled in front of the X-ray tube to control the physical size and shape of the X-ray beam [2]. Slice thickness (Figure 5) corresponds to the depth of a voxel. Slice thickness, together with matrix size, and FOV affect voxel dimensions, then image quality of CT [2]. Matrix size is the number of rows and columns for a particular image (number of voxels in each direction) and the principal factor to determine the size of the individual pixels [2]. Given that each pixel has only one numerical value or shade of grey, a certain area with more and smaller pixels (large matrix size) is of higher quality than another image with less and larger pixels (Figure 5). Field of view (FOV) (Figure 5) also affects image details. FOV is the diameter of the body region area being imaged. Pixel size = FOV/matrix size, so that a large FOV needs a large matrix to produce the same details as a small matrix in a smaller FOV [2]. In CBCT, however, all the information in the FOV is acquired in one single gantry rotation regardless of the slice thickness [10]. The slice thickness only affects how the information is partitioned from the FPD during the reconstruction process [10]. CBCT provides voxel resolutions that are isotropic, allowing the reconstruction with equally fidelity in sagittal, coronal, and transversal plate [4]. The voxels’ dimensions (as well as spatial resolution) are primarily dependent on pixel size of the area detector [8]. A smaller pixel detector is desired to acquire higher spatial resolution, however, the smaller pixels would capture less X-ray photons, and thus more image noise occurs [8]. 12 Figure 4. The collimator of a CT device 13 Figure 5. FOV, Matrix size, Pixel, and Voxel : A large matrix size results in a smaller pixel size with the size of the detector fixed. 14 The most fundamental characteristic of an image is the contrast [2]. Whether an object within the body will be visible in an image depends on having sufficient physical contrast relative to surrounding tissue (to be determined or distinguished from its background) [2]. Contrast resolution, is another primary characteristic of an imaging system that establishes the relationship between image contrast and object contrast. Object contrast varies in different tissues of human bodies, for example, bone is of high object contrast while soft tissue is low. When the imaging system has a lower contrast resolution, only objects with a high object contrast will be visible in the image. The contrast resolution is given by the possible number of pixel values (number of bits per pixel). If the imaging system has a high contrast resolution, the lower-contrast objects will also be visible, and thus increasing the image contrast. To adjust and increase the contrast in the displayed image, a method called windowing may be used. Windowing is the selection of the range of pixel values that will be converted into the full gray scale or brightness range (to control the image contrast). When a window level is selected, any segment of the CT scale can be expanded to cover the entire gray scale range. Thus, a smaller window level will lead to a higher image contrast [2]. (2) Visibility of detail and blur Resolution describes the ability of an imaging system to distinguish or separate (i.e. resolve) objects that are close together [2]. The resolving capability of an imaging system is determined by the amount of blur. Motion 15 and a large X-ray tube focal size (Figure 2) may cause blur. Blur limits the visibility of details such as small objects. The higher is the blur, the less visibility of small objects or details there is [2]. (3) Noise Noise is the random variation (non-uniform) in image density or brightness. It affects objects with small size and low contrast in the images. X- ray photon density has an effect on image noise, i.e., quantum noise. Quantum noise is the result of the random manner in which the X-ray photons (X-ray quanta) are distributed on the detector [2]. The uneven distribution of X- ray quanta shows up in the image as noise. Quantum noise could be reduced by increasing the radiation exposure which is used to generate the image, thus a balanced relationship between quantum noise and radiation exposure to patients is an important issue [11]. Another problem of the X-ray computed tomography technique is the scatter radiation. As an X-ray beam passes through the object, the photons will either penetrate, be absorbed , or produce scatter radiation due to the Compton effect. The Compton effect causes the change in photon direction. Thus, a portion of the outgoing photons bounce off or “scatter” after interacting with the object. Scatter causes distortions and contrast loss in the reconstructed images [12], especially when the X-ray system has a large area detector (i.e. FPD in CBCT devices). The contrast reduction produced by the scatter can be estimated using scatter-to-primary ratio (SPR). SPR is the ratio of scattered radiation intensities to the primary 16 radiation intensities, which may be high in certain areas of CBCT images [12]. Scatter correction methods may be used to suppress scatter radiation and improve the contrast-to-noise ratio (CNR) of the reconstructed images, and thus improve the quality of CBCT images [12]. Three parameters related to X-ray tube that can be adjusted by operators in some units to improve image quality are: Milliamperes (MA), exposure time, and Peak Kilovoltage (KVp). Different combinations of these parameters will have different effects on patient exposure, heat production, and image contrast and blurring. MA, the electrical current that flows through X-ray tube, is usually coupled with the selection of focal spot size [13]. A higher MA results in a higher focal spot size, which also enlarges the penumbra effects blurring the images [2]. Penumbra effects (Figure 2) are blurred edges in the images (halo effect). Exposure time is proportional to the quantity of radiation (exposure) produced by the X-ray tube. Short exposure time could minimize motion blurring and reduce patient motion artifacts. However, if the exposure time is too short, image noise will increase due to the reduction of photon intensity. KV, the voltage or potential, affects several factors of the X-ray generation. With an increase of KV, the exposure from the tube and penetration increase. A reduced tube voltage will lead to a higher image-noise-ratio since less KV means lower concentration of photons and an increase in quantum noise [2]. 17 (4) Artifacts Artifacts are details that do not represent physical structures or are present in obscure parts of the image causing misinterpretation. When penetration through the object occurs, lower energy photons are absorbed in preference to higher energy photons increasing the mean energy of the beam [8]. This effect is called the beam hardening effect. Two artifacts resulting from the beam hardening effects are streaks and cupping. Streaks are produced between two dense objects in the image (i.e. braces, implants, or metallic restorations) (Figure 6). Cupping artifacts occur because the center beams become harder in the center of the object than those passing through the edge of the object, which results in the appearance of a “cup” of the object in the image [14]. Cupping artifacts are demonstrated when a uniform cylindrical object is imaged [14]. Another artifact is called aliasing. Aliasing shows as linear radiolucent lines throughout the image given that too few projections are provided for the reconstruction (Figure 7) [8]. Scatter radiation also cause dark streaks between two high attenuation objects with surrounding bright streaks [15] (Figure 6). As mentioned in the noise section, scatter causes X-ray photons to change direction, and the scattered photons end up in different areas on the flat panel detector. When high attenuation objects present (i.e. dental implants), the metal blocks all photons and the detector will only detect scattered photons. The streaks then will be presented [15]. 18 Figure 6. Streak artifact due to metallic restorations Figure 7. Aliasing 19 b. Viewing Conditions. The ability of an observer to detect the object depends on a combination of factors including object contrast, background brightness and texture, glare, and distance between the image and the observer [2]. Objects with higher contrast comparing to surrounding and background are easier to see or detect. Low brightness of background causes the objects with small or relatively low level of contrast harder to be detected. Visual ability will increase when the distance between the image and the observer decreases, however, after the peak value (about 2 ft); the ability will decrease due to focus difficulty. Also, glare scatters over some areas within the visual field, reducing the contrast sensitivity [2]. c. Observer performance characteristics. CBCT imaging and diagnostic quality as mentioned above depends on image quality, viewing conditions, and observer performance characteristics . Individual observer often influences whether a specific observation is successful, as each observer has his/her own criteria. The quality of an image relates to how well it could present the actual diagnostic information to the observer. The accuracy of the diagnosis can be tested using the concept of the receiver operating characteristic (ROC) curve [2]. 20 1.1.4 Data assessment CBCT images allow observers to have a three-dimensional experience of the region of interest; for instance by allowing for the visualization of volumetric data by selective display of voxels within a dataset. For 3d visualization of a specific structure, e.g. bone or the upper airway, a process called segmentation is required [16]. Segmentation is defined as the construction of 3D virtual surface models to match the volumetric data. Image thresholding is the basis for segmentation, converting CBCT images to a binary format [16]. Thresholding techniques vary among different software programs. The operator by choosing the wrong threshold for a certain structure may lead to misinterpretation. 21 1.2 CBCT Applications in Endodontics CBCT is a novel radiological technique in the field of Endodontics compared to other visual techniques, for example periapical radiographs (PR) and dental operating microscope. The advantages of faster scanning time compared to CT, high resolution, and the ability to visualize structures three- dimensionally allow CBCT to provide more patient-specific information. Hundreds of studies were conducted by means of CBCT; however, most of them were using CBCT as an auxiliary technique. As the increasing use of CBCT in Endodontics, some in vivo and ex vivo studies were conducted and aimed to evaluate the use of CBCT in Endodontics, and compared CBCT with other routinely used dental techniques, most frequently, PR. For ex vivo studies, the accuracy of CBCT was compared with Micro-CT and other visual technique such as light microscope. In this section, studies related to the applications of CBCT in Endodontics were further discussed. 1.2.1 Detection of Vertical Root Fracture and Perforation using CBCT 1.2.1.1 Detection of Vertical Root Fracture. Vertical root fracture (VRF) is a complication for root canal therapy, which often leads to root or tooth extraction [17]. The correct diagnosis of vertical root fracture (VRF) is 22 challenging for endodontists, and may effect the treatment planning. The most widely used complementary method for diagnosing VRF is PR. However, in many cases, VRF is not visible in the periapical image due to the two- dimensional aspect, the superimposition among tissues, or the location of the fracture line hidden by dentin structure or by dense filling materials [18]. CBCT is considered to be an effective method in the diagnosis of VRF due to its three-dimensional representation of tooth structure. Brady et al. [19] conducted an ex vivo and cross-sectional study to evaluate the diagnostic accuracy of PR and CBCT for the detection of artificially induced incomplete and complete VRFs, and to determine whether the width of the VRFs had an impact on the diagnostic accuracy of the imaging systems. The authors induced 30 incomplete VRFs using non-endodontically treated human mandibular premolars and molars. After using optical coherence tomography to measure the widths of these incomplete VRFs, 15 of them were induced to complete VRFs. CBCT scans and PR were taken prior to and after fracture induction, and the results showed that CBCT scans were significantly more accurate than PR for the detection of incomplete and complete VRFs [19]. 1.2.1.2 Detection of Root Perforation. Root perforation makes possible a communication between root canal system and the external root regions [20]. This undesirable incident might occur at any step in root canal therapy, and results in endodontic treatment failures. The diagnosis of root perforation may 23 be a challenge using PRs due to the lack of clinical symptoms and the fact that PRs provides limited abilities to detect image root perforation. The possibility of CBCT to present sagittal, coronal, and transversal plate images enable it to be an effective method for root perforation detection. Shemesh et al. [21] compared the sensitivity and specificity of CBCT and PRs in detecting root perforations after root canal therapy, and the results indicated high sensitivity and specificity of CBCT scans (0.86 and 0.70, respectively). Haghanifar et al. [22] determined the sensitivity and specificity of cone-beam computed tomography (CBCT) and digital periapical radiography in the detection of mesial root perforations of mandibular molars. Root perforations were induced at the furcation region in 24 mandibular molars. CBCT and periapical radiographs in three angles were used to detect the presence of root perforation in root-filled canals and non-obturated canals. The results indicated that CBCT showed high sensitivity and specificity (92% and 100%, respectively) in the detetion of root perforation in non-obturated canals. 1.2.2 Internal and External Root Canal Resorption Resorption is the condition that a physiological or pathological process resulting in loss of dentin, cementum, or bone [23]. It may occur within the root in all directions. Their radiolucency of sizes and positions may not be detected 24 in periapical radiographs due to the superimposition of hard tissues. Root Resorption has been classified into internal and external resorption. Internal root resorption (IRR) is the progressive destruction of intraradicular dentin and dentin tubules associated with the inflammatory process of the pulp [24]. The diagnosis of IRR is based on clinical and radiological evidence. Clinically, visual examination based on color change in the crown (reddish area) may be used as the evidence of IRR. The color change in tooth crown is due to the presence of granulation tissue showing through the resorbed area [25]. Round to oval radiolucent enlargement of the pulp space is shown in radiographs of IRR. The margin of the radiolucency is smooth and defined with distortion of the original root canal outline [25]. Root perforation may accompany with IRR. Early diagnosis of IRR is important for a good treatment outcome [26]. External root resorption (ERR) is characterized by the reduction in root length and the presence of defects on the root surface [27]. Orthodontic movements, dental trauma, pressure from the adjacent teeth or pathologic conditions, and reimplantation may be the causative factors [27]. ERR diagnosis is based on radiological evidences. CBCT is believed to be effective in the detection of IRR and ERR. Kamburoglu et al. [28] compared CBCT with intraoral radiographs in 90 single- 25 rooted mandibular anterior teeth with ex vivo simulated IRR and ERR, and the comparison was conducted by three observers twice. The results concluded that both intra- and inter-observer agreements were statistically significantly higher for CBCT. Also, the authors indicated that CBCT images performed better than intraoral radiographs in both ex vivo simulated IRR and ERR [28]. In vivo studies were also performed for the accuracy of IRR and ERR detection using CBCT. Patel et al. [29] compared intraoral radiographs and CBCT images taken from patients with IRR, ERR, and no resorption, and the results were similar with the ex vivo study [28], showing that CBCT was effective and reliable in detecting the presence of resorption lesions. 1.2.3 Root Canal Treatment Assessment using CBCT Root canal therapy is the most commonly used treatment method in endodontic diseases. Root canal shaping and root canal obturation are key stages for endodontic treatment. Predictive successes could be made if treatments were properly performed. CBCT scanning is considered as an efficient method for the assessment of root canal instrumentation techniques for root canal shaping and obturation [30] [31] [32] [33]. 1.2.3.1 Root Canal Transportation. The main objective of root canal shaping is to effectively shape and clean the root canal system while keeping its original configuration [30]. The term root canal transportation is defined as 26 “The removal of canal wall structure on the outside curve in the apical half of the canal due to the tendency of files to restore themselves to their original linear shape during canal preparation” by the American Association of Endodontics (AAE) [20]. With the prevalence of automatic nickel-titanium (Ni- Ti) instruments, optimal root canal preparation with shorter working time, safer and more accurate orientation, and less frequency of procedural errors are achievable [30]. Root canal transportation, however, still remains as a complication when the dentist encounters curved canals. The ability of CBCT in providing three-dimensional information increases its use in the assessment of root canal transportation and can help the dentist to assess root canal centering ability of rotary instrumentation. Tambe et al. [31] compared three rotary systems using CBCT to test their ability for maintaining curved root canal geometry. The result indicated that there were significant differences among different files [31]. Using rotary systems for root canal preparation with and without glide path has also been evaluated using CBCT. A smooth and sufficiently wide glide path created by manual hand files or rotary Ni-Ti path gliding files is recommended prior to a subsequent canal preparation [32]. Elnaghy et al. [33] compared the volume of removed dentin, transportation, and centering ability of one commonly used rotary system (ProTaper Next, Dentsply Maillefer, Ballaigues, Switzerland) with and without glide path preparation by using 27 CBCT. Better performances with lesser canal aberrations were observed when using ProTaper Next with ProGlider (PG) (Dentsply Maillefer) Ni-Ti rotary instruments [33]. 1.2.3.2 Root Canal Obturation Assessment. The quality of root canal obturation was assessed in PR. However, details are lost when compressing three-dimensional information into two-dimensional periapical radiographs. Periapical radiographs alone do not provide important features of bucco- lingual/bucco-palatal portions as well as the voids inside the gutta-percha. Møller et al. [34] compared six intraoral digital receptors and a CBCT scanning system for detection of voids in root fillings using Micro-CT as gold standard. The result indicated that all intraoral digital receptors underestimated the extension of voids in gutta-percha, and CBCT resulted in a higher proportion of correctly observed voids, though with several false-positive recordings [34]. 1.2.3.3 Root Canal Re-treatment. Teeth may not respond to root canal therapy if the prevention and control of the intra-canal infection fails [35]. The first choice to reduce or eliminate the persistent infection is root canal re- treatment [36]. Complete removal of gutta-percha is the key step to infection control, followed by steps of effective cleaning, shaping and root canal re-filling. Rotary Ni-Ti systems are considered to be efficient, effective, and safe when 28 used to remove root canal fillings [37]. Rotary systems with specific ability to conduct root canal re-treatment have bloomed in the market. Studies that compared the effectiveness of different systems have been reported [38-40]. CBCT, with its ability to calculate the area of gutta-percha, is now used as an auxiliary method in studies of effectiveness of root canal re-treatment. Marfisi et al. [36] used CBCT to evaluate the gutta-percha residue after the re-treatment by three rotary Ni-Ti systems of straight canals filled with gutta- percha or Reslion. The result showed that no system could entirely remove the intra-canal fillings, and the study also indicated that CBCT enables three- dimensional appraisal when performing root canal re-treatment. 1.2.4 Differential Diagnosis of Periapical lesions using CBCT Periapical lesions develop as a result of a pathological immune response to continuous stimuli from infected root canals [41]. Numerous sequelae may follow untreated pulp necrosis and are dependent on the virulence of the microorganisms involved and the integrity of the patient's overall defense mechanisms [42]. Bacterial infection of dental pulp ultimately results in the formation of dental periapical lesions mainly consisting of periapical granulomas and periapical cysts [41]. Periapical inflammatory processes are stimulated and sustained by necrotic pulpal tissue debris, inflammatory cells, and anaerobic bacteria [42]. The longer the inflammation persists, the less 29 effective the host reaction becomes, and then microbes invade and toxins spread into the apical area [42]. Periapical granulomas may arise from a periapical abscess or develop as the initial periapical pathosis. Periapical granulomas are composed by granulation tissue and fibrous tissue infiltrated by variable numbers of neutrophils, lymphocytes, plasma cells, and macrophages [42]. Epithelial rests of Malassez may be identified within the granulation tissue. Periapical cysts develop from pre-existing periapical granulomas [42]. Periapical cysts are the result of the stimulation of the epithelial rests of Malassez in response to the inflammation and the body’s response to separate the inflammation from the surrounding bones [42]. Histologically, periapical cysts are lined by non-keratinized stratified squamous epithelium of variable thickness with a chronic inflammatory cell infiltration [42] . The traditional diagnosis of apical cyst is based on histopathological examination of biopsy tissue, which means that the only way to confirm the diagnosis is surgical [43, 44]. Water-soluble contrast media [45], Papanicolaou smears [46] and albumin tests [47] were used to make differential diagnoses of periapical cysts from periapical granulomas in non-surgical ways. Efforts were made in using radiological techniques for the differential diagnosis. Ricucci et al. [48] used paralleling PRs to compare the presence or absence of 30 radiopaque lamina of 57 human periapical lesions. All the PRs were scanned and standardized, images were evaluated on a computer screen by two observers. Periapical surgeries were conducted and serially sectioned histological specimens were obtained. The results indicated that only 3% (3 out of 10) lesions with radiopaque lamina were histologically periapical cysts, while as high as 15% (7 out of 47) lesions were periapical cysts though there was no radiopaque lamina shown. Thus, the authors concluded that the diagnosis of periapical lesions could not be made on the basis of the presence or absence of a radiopaque lamina [48]. CBCT scanner’s reliability for differential diagnosis of periapical cysts from periapical granulomas has been shown. The accuracy of differential diagnosis of periapical lesions using CBCT is controversial. Simon et al. [49] compared the differential diagnosis of large periapical lesions (granuloma versus cyst) to traditional biopsy using CBCT. In this study, 17 large lesions were scanned and gray value measurements of the imaged lesion were evaluated. Preoperative diagnoses of all lesions were made based on the gray value measurements. Histological reports were made and compared with the CBCT diagnoses. The results showed that 13 out of 17 cases were of consistent diagnoses between biopsy reports and the CBCT diagnoses. In the four inconsistent diagnosed cases, the CBCT read periapical cysts while the oral pathologist’s diagnoses being periapical granuloma. A possibility that the sections of lesions may not represent the full lesion and may have missed the epithelial area, the authors concluded that CBCT might provide a more 31 accurate diagnosis than biopsy and histology providing a diagnosis without invasive surgery and/or waiting a year to see if nonsurgical therapy is effective [49]. Rosenberg et al. [50] used CBCT images to differentiate periapical cysts from granulomas. In that study, two radiologists independently analyzed CBCT images from 45 patients and provided a diagnosis using a set of possible results (cysts, likely cysts, granulomas, likely granulomas, and others). The results indicated a weak (poor) agreement between two radiologists (κ=0.14), and an overall accuracy (AUC, Area Under the Curve) of 0.65 for the first radiologist and 0.51 for the second compared to the biopsy [50]. A large set of possible diagnostic options might have resultee in the moderate to weak agreement between radiologists. To further explore the ability of CBCT for the differential diagnosis of periapical cysts from periapical granulomas, a quantitative measurement based on a set of six diagnostic criteria was established [51]. Details are shown in Chapter Two. 1.2.5 Evaluation of root and canal morphology using CBCT Knowledge of the number of roots and the internal root canal morphology is fundamental for thorough cleaning of the canal system and the success of root canal treatment [52]. Clinicians are required to have basic knowledge of root and canal morphology and possible anatomic variation [53]. 32 Figure 8. Vertucci's classification of root canal patterns The canal morphology was thoroughly explored and classified into eight types by Vertucci et al. [54]. This classification was taken as a reference in many studies. In this Vertucci’s classification, the root canal configurations present within the roots of human permanent teeth can be classified into eight types [54] (Figure 2): 33 Type I. A single canal extends from the pulp chamber to the apex. Type II. Two separate canals leave the pulp chamber and join short of the apex to form one canal. Type III. One canal leaves the pulp chamber, divides into two within the root, and then merges to exit as one canal. Type IV. Two separate and distinct canals extend from the pulp chamber to the apex. Type V. One canal leaves the pulp chamber and divides short of the apex into two separate and distinct canals with separate apical foramina. Type VI. Two separate canals leave the pulp chamber, merge in the body of the root, and re-divide short of the apex to exit as two distinct canals. Type VII. One canal leaves the pulp chamber, divides and then rejoins within the body of the root, and finally re-divides into two distinct canals short of the apex. Type VIII. Three separate and distinct canals extend from the pulp chamber to the apex. Various findings of the canal morphology have been reported [55-58]. Benjamin et al. [55] examined 364 extracted human mandibular incisors radiographically using metallic endodontic probes inserted into the canals to explore the presence of two canals. The authors found that 41.4% of those mandibular incisors had two separate canals. A similar result was concluded by 34 Kartal et al. [56]. The authors used clearing technique and examined a number of 100 central and lateral mandibular incisors and concluded that the chance of having two canals in mandibular incisors was 50%. In this study, two more canal types were explored which were not defined in the Vertucci’s classification (2-3-1, and 1-2-1-3). Studies of human permanent premolars were also conducted. Kartal et al. [57] investigated 600 extracted maxillary first (300 teeth) and second premolars (300 teeth) using the clearing technique to explore their canal type of Vertucci’s classification. The results indicated that 89.64% of maxillary first molars demonstrated two canals (Type II to Type VII), while 1.66% of them had three canals. For the maxillary second premolars, 48.66% of which was of Type I, while 50.64% was Type II to Type VII (two canals). Singh et al. [58] studied the canal morphology of mandibular premolars in a South Asian Indian population using the clearing technique. In that study, 100 mandibular first premolars and 100 mandibular second premolars were collected and evaluated. The results showed that 80% of mandibular first molars had type I, 6% had type II, 10% had type IV, 2% had type V, and 2% teeth had type IX root canal anatomy. For the mandibular second premolars, 58% of teeth had a single canal, and 42% had two canals. An additional canal in the mesiobuccal root (MB2) of maxillary first molar has generated more researches than any root as for the inability of finding the MB2 is a reason for the root canal therapy failure [59]. An in vivo study of evaluating the root and canal morphology of maxillary first molar in a US population [60] is discussed in details in Chapter Three. 35 Like the variation of canal morphology, the number of roots may also vary. In the mandibular premolar study [58], 94% of mandibular first premolars had one root, whereas 6% were two-rooted. For the mandibular second premolars, 8% of which were two-rooted and fused. One important variation for the mandibular first molar is the presence of an additional root located lingually (the radix entomolaris) or buccally (the radix paramolaris) [61]. Details about the root and canal morphology of mandibular first molar are presented in Chapter Four. In vivo and ex vivo studies of evaluating the root and canal morphology using different techniques had been conducted to enrich the document of Endodontics. In in vivo studies, CBCT scans are superior in detecting the number of roots and the canal morphology compared to PRs [62]. Variations such as dilacerations and unusual number of roots and canals could be easily detected when images are visualized in a three-dimensional way. The accuracy of CBCT in determining the number of roots and canal morphology has been compared with Micro-CT, which is considered as the gold standard for laboratory studies for root and canal anatomy analysis [63]. Domark et al. [64] used CBCT images to determine the number of canals in meisobuccal root of maxillary molars and compared the counts with Micro-CT (gold standard), and there was no statistically significant difference between the counts of CBCT and Micro-CT [64]. Paes da Silva Ramos Fernandes et al. [65] also compared the 36 accuracy of CBCT in the identification of various internal anatomic patterns in mandibular incisors. The results concluded that all CBCT devices (Kodak 9000 3D [Carestream Health, Rochester, NY], Veraviewepocs 3De [J Morita MFG Corp, Kyoto, Japan], NewTom 5G [QR Srl, Verona, Italy]) are accurate in identifying the canal pattern mandibular incisors compared with Micro-CT. 37 1.3 The Accuracy and Reliability of Diagnostic Test 1.3.1 Accuracy Accuracy refers to the closeness of the measured value to the reference or gold standard. In diagnostic tests, accuracy is how the diagnostic method could reflect the disease status. The gold standard is the method that is widely recognized as the best available method that could reflect the true disease status [66]. In diagnostic tests, accuracy is used to measure the usefulness of a test method, evaluating how accurate the test is. Sensitivity (the probability the diagnostic test is positive for disease for a patient who truly has the disease) and specificity (the probability the diagnostic test is negative for disease for a patient who truly does not have the disease) are used to assess the diagnostic ability of the testing method, for example, the diagnostic ability of CBCT to differentiate periapical cysts and granulomas. The test results of the new diagnostic method being tested are compared with the gold standard, which is shown in Table 1. 38 Table 1. Comparison of diagnostic results with the gold standard The gold standard Positive Negative Total Diagnostic test results Positive TP FP TP+FP Negative FN TN FN+TN Total TP+FN FP+TN N TP – true positive, FN – false negative, FP – false positive, TN – true negative, N – sample size Based on the true positive (TP), false negative (FN), false positive (FP), and true negative (TN), an estimated overall accuracy and predictive values for the diagnostic test could be calculated: 1. Overall accuracy = !" ! !" ! 2. Sensitivity = !" !" ! !" 3. Specificity = !" !" ! !" 4. Positive predictive value = !" !"!!" 5. Negative predictive value = !" !"!!" An ideal test method should have both high sensitivity and specificity. 39 Receiver operating characteristic (ROC) test [67], a plot of the true positive rate against the false positive rate for the different possible cut points of a diagnostic test, will show the largest Area Under the Curve (AUC) at the highest sensitivity and specificity trade-off. An ROC curve is constructed by plotting the FP rate (1-specificity) along the X-axis against the sensitivity on the Y-axis. The curve begins at the (0, 0) coordinate, which means all test results are negative [67], and ends at (1, 1) coordinate, meaning that all test results are positive [67]. The reference line is the connection of (0, 0) and (1, 1) coordinates, which represents a chance accuracy [67]. The closer the curve follows the left-hand border (with higher specificity) and then the top border (with higher sensitivity) of the ROC space, the more accurate the test (Figure 9). Accuracy was noted as good if AUC>0.80, moderate if 0.60≤AUC≤0.80, poor if AUC< 0.60 [68]. 40 Figure 9. An example of ROC curve 1.3.2 Reliability Reliability is the degree to which the measurement method is consistent, and it refers to the closeness of two measurements, no matter the result is correct or not [66]. Gold standard is not needed to calculate the reliability of measurements. Reliability could be analyzed in three ways: internal consistency, test-retest consistency, and inter-/intra-rater reliability. Internal consistency is based on the correlation of different items on the test, which measure the same construct or idea (this applies mostly to questionnaires). Test-retest consistency evaluates whether the results will change over time 41 (repeated measurement). Pearson’s correlation coefficient could be used to test-retest reliability. Inter-rater reliability is the agreement of two or more evaluators, and intra-rater agreement is the agreement of one evaluator against himself/herself using the same methods on two separate occasions [69]. Intra- class correlation coefficient (ICC) and Pearson’ correlation coefficient could be used for continuous data, while kappa coefficient and overall percent agreement could be used for binary data. Cohen’s Kappa (Kappa coefficient) was used in our study to calculate intra-rater reliability. Kappa has been referred to as the ‘chance-corrected agreement rate’ [70]. When comparing the results of the same rater at two separate times, a 2x2 table (Table 2) could be made: Table 2. Comparison of diagnostic results of one rater for the calculation of intra- rater reliability Rater One Time 2 Yes No Total Rater One Time 1 Yes a b a+b No c d c+d Total a+c b+d N 42 The Kappa coefficient could be calculated using the formula: !∗(!"!!") !!! ∗!!!∗(!"!!") . Agreement was noted as excellent if κ≥0.75, good if 0.60≤κ<0.75, intermediate if 0.4≤κ<0.60, and poor if κ<0.4 [69]. Cronbach’s coefficient alpha was used in our study to calculate inter- rater reliability among three evaluators with binary data [70]. Agreement was noted as excellent if α≥0.80, good if 0.60<α<0.79, intermediate if 0.40<α≤0.60, and poor ifα≤0.40 [71]. 43 Chapter Two: Evaluation of the reliability and accuracy of using Cone- beam CT for diagnosing periapical cysts from granulomas 2.1 Abstract Introduction: The purpose of this study was to evaluate the reliability and accuracy of Cone-Beam Computed Tomography (CBCT) against the histopathologic diagnosis, for the differential diagnosis of periapical cysts (cavitated lesions) from (solid) granulomas. Methods: Thirty-six periapical lesions were imaged by CBCT scan. Apicoectomy surgeries were conducted for histopathological examination. Evaluator 1 examined each CBCT scan for the presence of six radiological characteristics of a cyst (location, periphery, shape, internal structure, effects on surrounding structure, and perforation of the cortical plate). Not every cyst demonstrated all radiological features (e.g. not all cysts perforate the cortical plate). For the purpose of finding the minimum number of diagnostic criteria present in a scan to diagnose a lesion as a cyst, we conducted six ROC (Receiver Operating Characteristic) Curve analyses comparing CBCT diagnoses with the histopathologic diagnosis. Two other independent evaluators examined the CBCT lesions. Statistical tests were conducted to examine the accuracy, inter-rater reliability, and intra-rater reliability of CBCT images. Results: Findings showed that a score of ≥4 positive findings was the optimal scoring system. The accuracies of differential diagnoses of three evaluators were moderate (AUC=0.76, 0.70, and 0.69 for 44 evaluators 1, 2, and 3, respectively). The inter-rater agreement of the three evaluators was excellent (α=0.87). Intra-rater agreement was good to excellent (κ=0.71, 0.76, and 0.77). Conclusion: CBCT images can provide a moderately accurate diagnosis between cyst and granuloma. Key words: Cone-beam Computed Tomography, Differential diagnosis, Periapical cyst, Granuloma, Biopsy 45 2.2 Introduction The traditional diagnosis of apical cyst is based on histopathological examination of biopsy tissue, which means that the only way to confirm the diagnosis is surgical [43, 44]. Previously, researchers attempted to make an accurate diagnosis of cyst as compared to granuloma, using: periapical radiographs [48], water-soluble contrast media [45], Papanicolaou smears [46] and albumin tests [47]. However, none of these were accurate. Recently, studies on pre-operative differential diagnosis by using advanced imaging technology, such as CT [72] and cone-beam CT [73, 74], have become available in the literature. Aggarwal et al. [72] used CT scans to differentiate periapical lesions; of 17 lesions evaluated, 12 were accurately diagnosed wherein the imaging results concurred with the histopathological diagnosis, indicating that CT scans could provide relatively accurate diagnosis without invasive surgery. Cotti et al. [74] also reported positive results for CT scans. Shrout et al. [73] indicated that granulomas had a narrower range and lower grey values than did cysts using CBCT, which could be used for the differential diagnosis of periapical lesions according to the authors. Researchers have evaluated the accuracy of CBCT imaging to detect apical periodontitis (AP) as compared with periapical (PA) radiographs. Estrela et al. [75] evaluated a new periapical index based on CBCT for the identification of AP [75]. More AP was identified by CBCT (60.9%) than by periapical 46 radiographs (39.5%) after examining 1014 images. The authors concluded that a periapical index based on CBCT might offer an accurate diagnosis of AP. Their study provided convincing evidence of accurate diagnosis of AP by CBCT images. Tsai et al. [76] used simulated periapical lesions in human cadavers to assess the diagnostic accuracy of CBCT and PA radiographs. The authors indicated that CBCT demonstrated excellent accuracy when simulated lesions were larger than 1.4mm, fair to good when between 0.8-1.4mm, and poor when less than 0.8mm. PA radiographs demonstrated poor accuracy for all lesion sizes. Rodrigues et al. [77] reported a rare case of lymphangioma mimicking AP, indicating that CBCT could be useful for the diagnosis of well- circumscribed lesions. Rosenberg et al. [50] used CBCT images to differentiate periapical cysts from granulomas with inconclusive findings, indicating a weak agreement between two radiologists (κ=0.14), and an overall accuracy of 0.65 for the first radiologist and 0.51 for the second as compared to biopsy results. Simon et al. [49] differentiated periapical cysts (cavitated lesions) from granulomas using CBCT imaging (NewTom 3G). Seventeen periapical lesions were scanned, and the grey values of each lesion were measured. The lesions were then surgically removed for biopsy examination. In 13 out of 17 lesions, the diagnoses of CBCT images were consistent with pathological reports. The authors indicated that CBCT images might provide more accurate diagnosis than pathological reports. 47 The use of CBCT scans for endodontic diagnosis is controversial due to concerns relating to higher exposure of radiation to patients than necessary, long duration of scanning, and expensive cost as compared to conventional dental radiographs [78]. Though CBCT is widely used in orthodontics [79], dental implant treatment planning [80], and the detection of obstructive sleep apnea [81], the use of grey values in the diagnosis of periapical lesions exhibits some disadvantages according to a recent publication [79]. Authors of this publication concluded that grey values could be easily affected by the field of view (FOV) and spatial resolution selections [79], so in our current study we used a set of diagnostic criteria based on the radiological characteristics of apical cysts instead of using grey values. The main purpose of the present study was to evaluate a set of diagnostic criteria for differentiating periapical cysts from granulomas according to their CBCT imaging characteristics. The criteria were established based on the radiological characteristics of periapical cysts [82, 83]. The goals of this study were (I) to evaluate this set of six diagnostic criteria and to find out the optimal number of criteria findings necessary for the differential diagnosis of periapical cysts (cavitated lesions) versus granulomas; (II) to evaluate the intra- rater and inter-rater reliability of three independent evaluators using six criteria for the differential diagnosis of cysts versus granulomas; (III) to evaluate the accuracy, specificity, sensitivity and overall agreement of CBCT diagnosis and histopathological reports for three blinded evaluators. 48 2.3 Materials and Methods Thirty-six periapical lesions diagnosed by clinical symptoms and radiographic findings were selected for the study. The inclusion criteria of lesions were a minimum average diameter of 5mm from sagittal views of CBCT images. The protocol was approved by the Institutional Review Board of the University of Southern California (#UP-12-00506). 2.3.1 Radiology Periapical radiolucency was imaged by Kodak 9000 3D (Carestream Health Inc., NY) at the Redmond Imaging Center, University of Southern California. The technician acquired a fixed field of view volume of 3.75cm x 5cm. The pixel size was 76µm and 15-bit pixels (32,768 grey values). The scanner was operated at 70-90 kV and 8-10 mA. DICOM (The Digital Imaging and Communications in Medicine) format images were exported from Kodak 9000, and imported into InVivo Dental Application 5.1.6 software (Anatomage Inc, CA). 2.3.2 Histopathology Root-end resections were performed as per routine clinical protocols. Histopathological specimens were obtained for microscopic examination, stained by Hematoxylin and Eosin (H&E), and analyzed. All histopathological specimens were examined by a board certified oral pathologist (PS) without 49 prior knowledge of clinical symptoms of patients, CBCT diagnosis, and surgical observation of lesions. Pathology microscopic criteria for diagnosis of cyst versus granuloma: For a granuloma diagnosis, there had to be no histopathologic evidence of cystic organization, no evidence of squamous lining epithelium as specifically described for cyst diagnosis to follow, the presence of soft connective tissue morphologically consistent with granulation tissue, and the presence of inflammatory cell infiltrates, foamy histiocytes or multi- nucleated giant cells. For a cyst diagnosis, there had to be histopathologic evidence of fibrous connective tissue with cystic organization and more than one definitive foci of stratified squamous lining epithelium comprising ≥50 cohesive cells attached by visible cell-cell junctions on high power magnification (60x original), and the presence or absence of inflammatory cell infiltrates within the wall or the lining of the cyst. Epithelial islands or odontogenic rests located or embedded within fibrous connective tissue were not considered evidence of lining cystic epithelium. Overt evidence of keratinaceous debris within an epithelial-lined lumen, and the presence of cholesterol clefts and Rushton bodies, erythrocyte extravasation and hemosiderin deposition and occasional multi-nucleated giant cells was considered supportive of a cyst diagnosis along with the aforementioned criteria and the acknowledgment that histopathologic findings can overlap between cysts and granulomas and there is no single pathognomonic feature to discriminate the two lesions histopathologically. A second evaluation of all 50 biopsy slides was taken by the same oral pathologist after two weeks to calculate the intra-rater reliability. 2.3.3 Diagnostic criteria A set of diagnostic criteria (Table 3) for periapical cyst was established based on radiological characteristics (location, periphery, shape, internal structure, effects on surrounding structure, and perforation of the cortical plate) [82, 83]. For each lesion, evaluator 1 (JG), without prior knowledge of the patient or the histopathological report, examined the DICOM images, and tabulated the data. For each criterion, if radiological evidence was found, the lesion would be scored one point. A cumulative score for each lesion was documented. The final score for each lesion was a number between 0 and 6 according to the number of positive findings. 2.3.4 Evaluation of diagnostic criteria For each lesion, we computed a cumulative score 0 to 6 as explained in the prior section. A score of 1 meant that only 1 of the criteria in Table 3 was determined to be present in the images. Due to the limitations of CBCT scans, and the fact that most cysts do not show all six characteristics (e.g. not all cysts may perforate the cortical plate), all the lesions did not demonstrate the six radiological findings. For the purpose of finding the optimal number of diagnostic criteria (scoring system) to determine if a lesion was a cyst, we conducted six ROC (Receiver Operating Characteristic) Curve analyses [67]. 51 ROC provides measures of accuracy, which is constructed from the sensitivity and specificity of a diagnostic test. The measure of accuracy was the Area Under the Curve (AUC), representing the average specificity over all sensitivities [67]. For the first ROC Curve, a diagnosis of cyst corresponded to a score≥1. The diagnosis of a cyst in the sixth analysis corresponded to a score=6. Therefore, six ROC analyses were conducted based on the histopathological diagnoses from the oral pathologist (cyst=yes/no) and the six CBCT diagnoses according to the 6 scoring systems (≥1, ≥2, …, =6). The AUC was calculated for each scoring system, ranging in value from 0.5 (chance) to 1.0 (perfect discrimination or accuracy) [67]. The largest of the six AUC was selected as the best scoring system, and was chosen for the diagnosis of cyst. Three independent endodontist evaluators (JG, OS and TC), blinded to the histopathological reports, examined the images of 36 lesions using the 6 criteria in Table 3. A lesion was diagnosed as a cyst if the score was equal or above 4 (the optimal scoring system). 52 Table 3. CBCT diagnostic criteria for differential diagnosis of a periapical cyst Located at the apex of the involved tooth Well-‐defined corticated border Shape is curved or circular Internal structure of lesion is radiolucent Displacement and resorption of the roots of adjacent teeth with a curved outline Perforation of cortical plate 2.3.5 Intra-and inter-rater reliability To assess the intra-rater reliability of the three evaluators, all 36 cases were examined for a second time six months later. Intra-rater reliability of each evaluator was conducted with Cohen’s Kappa. Cohen’s Kappa assesses the extent to which a rater agrees in two evaluations of the same set of cases [69]. Agreement was noted as excellent if κ≥0.75, good if 0.60≤κ<0.75, intermediate if 0.4≤κ<0.60, and poor if κ<0.4 [69]. Inter-evaluator agreement between the three evaluators was conducted using Cronbach’s Alpha (α) tests. Cronbach’s Alpha is to assess the internal consistency among multiple raters [71]. Agreement was noted as excellent if α≥0.80, good if 0.60<α<0.79, intermediate if 0.40<α≤0.60, and poor ifα≤0.40 [71]. Figure 10 describes the overall protocol for the study. 53 Figure 10. Study protocol flowchart ROC, Receiver Operating Characteristic. AUC, Area Under the Curve. 54 2.3.6 CBCT comparison to Histopathological reports Using the best scoring system (≥4 criteria), CBCT imaging was compared to histopathological diagnoses. Sensitivity, specificity, false positive rate, false negative rate, overall agreement and AUC’s were calculated for each of the three independent evaluators. Accuracy was noted as good if AUC>0.80, moderate if 0.60≤AUC≤0.80, poor if AUC< 0.60 [68]. SPSS software version 17.0 (SPSS Inc, Chicago, IL) was used in this study with significance accepted at a value of p<0.05. 55 Figure 11. (A-‐B) ROC curves and AUCs: A). ROC curves and AUCs for six scoring systems (evaluator 1) compared with the histopathological diagnoses. B) ROC curves and AUCs using the optimal scoring system (evaluators 1, 2 & 3) compared with the histopathological diagnoses. The reference line (solid line) represents chance (50%) accuracy. 56 2.4 Result The intra-rater agreement for the oral pathologist was excellent (κ=0.93). Among the six AUCs (Figure 11A), one for each scoring system, a score≥4 positive finding exhibited the highest AUC (0.76), therefore it was used in this study for the differential diagnosis of a cyst. A lesion was diagnosed as a cyst if four or more of the six criteria evaluated were considered as ‘yes’ by the evaluator. Intra-rater agreement was good for evaluator 1 (κ=0.71), and excellent for evaluator 2 (κ=0.76) and evaluator 3 (κ=0.77). Results of Cronbach’s Alpha indicated that the inter-agreement between the three evaluators was excellent, and it was not due to chance agreement (α=0.87). When comparing each evaluator’s ratings to the histopathologic diagnosis (PS), we found a high mean overall agreement (mean overall rate=0.77). For evaluator 1, 30 out of 36 lesions had the same diagnosis as the histopathological reports (overall agreement= 0.83; AUC= 0.76, Figure 5B). For evaluator 2, 27 out of 36 lesions were in agreement (overall agreement= 0.75; AUC=0.70, Figure 2B), while 26 out of 36 for evaluator 3 (overall agreement= 0.72; AUC= 0.69, Figure 2B). Table 2 presents the sensitivity, specificity, overall agreement, false positive rate, false negative rate, and AUC for all three evaluators compared with the histopathological results from the board certified oral pathologist (Table 4). 57 Figure 12. CBCT images and histological reports of four cases: (A-‐F) A lesion with consistent diagnoses between CBCT evaluation and histopathological diagnosis as a periapical cyst. A) Three-‐dimensional reconstruction image; B) CBCT sagittal view; C) CBCT coronal view; D) CBCT axial view; E) ×40 histopathological photomicrograph; F) ×100 histopathological photomicrograph. (G-‐L) A lesion with inconsistent diagnoses between CBCT evaluation (inter-‐ evaluator agreement of a periapical granuloma) and histopathological diagnosis (periapical cyst). G) Three-‐dimensional reconstruction image; H) CBCT sagittal view; I), CBCT coronal view; J) CBCT axial view; K) ×40 histopathological photomicrograph; L) ×100 histopathological photomicrograph. (M-‐R) A lesion with consistent diagnoses between CBCT evaluation and histopathological diagnosis as a periapical granuloma. M) Three-‐dimensional reconstruction image; N) CBCT sagittal view; O) CBCT coronal view; P) CBCT axial view; Q) ×40 histopathological photomicrograph; R) ×100 histopathological photomicrograph. (S-‐X) A lesion with inconsistent diagnoses between CBCT evaluation (two out of three evaluators diagnosed it as a periapical cyst) and histopathological biopsy (periapical granuloma). S) Three-‐dimensional reconstruction image; T) CBCT sagittal view; U) CBCT coronal view; V) CBCT axial view; W) ×40 histopathological photomicrograph; X) ×100 histopathological photomicrograph. 58 Table 4. Sensitivity, specificity, false positive rate, false negative rate, overall agreement, and AUC for three evaluators compared with histopathological diagnoses Evalua tor Sensitivi ty Specific ity Overall rate False positive rate False negative rate AUC 1 0.60 0.92 0.83 0.08 (2/26) 0.40 (4/10) 0.76 2 0.60 0.81 0.75 0.19 (5/26) 0.40 (4/10) 0.70 3 0.60 0.77 0.72 0.23 (6/26) 0.40 (4/10) 0.69 Sensitivity= true positive/(true positive + false negative); Specificity= true negative/(true negative + false positive); Overall rate= (true positive + true negative)/total number; False positive rate= false positive/(false positive + true negative); False negative rate= false negative/(false negative + true positive); AUC, Area Under the Curve. 59 Figure 12 (A-F) shows an example of a lesion with consistent diagnoses of periapical cyst by three evaluators and the histopathological report. Figure 12 (G-L) shows an example of an inconsistent differential diagnosis between three evaluators (a periapical granuloma) and the histopathological report (a periapical cyst). Figure 12 (M-R) shows an example of a lesion with consistent diagnoses of a periapical granuloma by three evaluators and the histopathological report. Figure 12 (S-X) shows an example of a lesion with inconsistent diagnoses between CBCT evaluation (two out of three evaluators diagnosed it as a periapical cyst) and the histopathological report (a periapical granuloma). 60 2.5 Discussion Surgical biopsy and pathological examination is standard procedure when a distinction between periapical cyst and granuloma is necessary. Nair [44] concluded that 85% of all periapical lesions were periapical granulomas. Lin et al. indicated that most reactive periapical lesions (granulomas and radicular cysts) may heal by nonsurgical root canal therapy except apical true cysts [84, 85]. Apical true cysts (periapical cavities completely enclosed in epithelial lining), particularly larger lesions, may require surgical intervention because they are self-sustaining and not dependent on presence or absence of intracanal infection or inflammation [44, 84]. However, there is no direct evidence to show that large cysts or granulomas can or cannot heal after nonsurgical root canal therapy; indirect clinical evidence appears to indicate that radicular cysts may heal or regress after nonsurgical root canal therapy, while apical true cysts require surgery [84]. The key factor in establishing that apical true cysts can or cannot heal after nonsurgical root canal therapy would be the pretreatment identification of an apical lesion as an apical true cyst [84]. Currently, there is no predictable method of identifying pretreatment apical lesions as an apical true cyst. Post-treatment biopsy alone cannot be used to imply that apical true cysts can or cannot heal after nonsurgical root canal therapy; this determination requires definitive pretreatment identification, which is impossible at this time [84]. If we were able to predict the presence of apical true cysts before nonsurgical root canal therapy, we could conduct outcome 61 studies in animals and humans to test the hypothesis of whether inflammatory cysts can or cannot regress after nonsurgical root canal therapy [84]. Thus, as one potential step towards this end, we evaluated in the current preliminary study whether CBCT could represent a feasible biopsy- independent alternative for differentiating apical cysts from granulomas. CBCT has been considered to be one nonsurgical alternative used in the differentiation between apical cysts and granulomas [49]. Tsai et al. [76] compared CBCT and PA radiographs and the results showed that CBCT could demonstrate excellent accuracy when simulated lesions were larger than 1.4mm while AP radiographs demonstrated poor accuracy for all lesion sizes. Rodrigues et al. [77] used PA radiographs, CBCT, and MRI as methods to evaluate a lymphangioma mimicking AP, concluding that CBCT could diagnose well-circumscribed lesions. However, its use is still controversial. CBCT provides grey values rather than Hounsfield Units (HUs), which are easily affected by field of view, spatial resolution selection, hard beaming, scattering, and the number of basis projections [80]. On the positive side, CBCT has advantages in the field of endodontics due to better representation of soft tissue, lower exposure to radiation, and less cost compared with conventional CT [86]. In this study, a set of six diagnostic criteria was evaluated to differentiate periapical cysts from granulomas. The six criteria were based on the radiological characteristics of a periapical cyst (location, periphery, shape, 62 internal structure, and effects on surrounding structure) [83]. In some cases, it is difficult to differentially diagnose a periapical cyst from a granuloma [83]. The reason is that a granuloma might present with the same radiological characteristics as a cyst. Also, not every cyst will show all radiological features (i.e, not all cysts may perforate the cortical plate) [83]. Thus, an optimal number of criteria were assessed (highest AUC of six diagnostic criteria), in order to differentiate a cyst from a granuloma with the most accuracy. Using ROC analyses to assess the accuracy of each scoring system, we found that 4 or more positive findings (criteria on Table 3) were the optimal choice for a lesion to be diagnosed as a cyst (AUC=0.76). All three evaluators in our study showed moderate accuracy when compared to histopathological diagnoses from the board certified oral pathologist (AUC=0.76, 70, and 0.69). In the present study, the results indicate that CBCT images could provide good to excellent accuracy for differential diagnosis between cyst and granuloma, with excellent inter-rater (α=0.87) and good to excellent intra-rater reliabilities. Mean overall agreement of 3 evaluators was 0.77, which was the same as that in a previous study by Simon et al. [49] using CBCT grey values. In that study, 13 out of 17 CBCT diagnoses coincided with the biopsy reports (overall agreement=0.77). According to the authors, in the four cases of split diagnosis, the pathological reports were inaccurate to diagnose AP because the biopsy examination might not represent the entire lesion and epithelium 63 may be present but not identified in viewed sections. The authors concluded that CBCT imaging might be more accurate than biopsy examination. In a recent study by Rosenberg et al. [50] two radiologists independently analyzed CBCT images from 45 patients using a set of diagnostic criteria. The results indicated a weak (poor) agreement between two radiologists (κ=0.14), and an overall accuracy (AUC) of 0.65 for the first radiologist and 0.51 for the second compared to the biopsy. The moderate to weak agreement between radiologists might be due to the large number of possible diagnoses (cysts, likely cysts, granulomas, likely granulomas, and others), while only 2 possible differential diagnoses were taken into account in this paper (cyst/granuloma). In this study we also attempted to reduce uncertainty by reducing the number of criteria to 6, and making the diagnosis of a cyst a quantitative measure (presence of 4 or more radiological criteria). In the previous study, radiologists chose the best diagnosis for each lesion based on the diagnostic criteria without a clear scoring system; however, in the present study, evaluators only examined the lesion based on the six pre-defined criteria, and reported a cyst diagnosis if 4 or more positive findings were present. The authors believe that this quantitative scoring system reduced uncertainty and increased inter-rater and intra-rater reliability, as well as accuracy compared to prior studies. One of the limitations in our study was small sample size. Further studies on a larger number of cases will be required to confirm the findings of 64 the present study. Moreover, the small amount of periapical cyst lesions (10/36) may also have contributed to the high rates of false negatives (4/10, 4/10, and 4/10 for the three evaluators). Another limitation is the inclusion of only two types of periapical lesions. On the positive side, the accuracy of all evaluators was moderate, and the inter- and intra-rater reliability was good to excellent. Finally, the cross-sectional nature of this study and the level of evidence it represents are insufficient to make any recommendations or claims regarding the clinical utility of CBCT for the diagnosis of reactive endodontic lesions. Well- controlled human studies that are longitudinal in nature would be required to demonstrate clinical relevance and efficacy of CBCT in the current context, particularly given the radiologic standard of care ALARA (as low as reasonably achievable) for radiation exposure and safety to patients. Accordingly, the current study is not meant to provide clinical evidence or rationale for subjecting patients to the additional radiation of CBCT. In summary, CBCT images provided a moderately accurate differential diagnosis between cysts and granulomas when the apical lesion presented with a minimum average diameter of 5 mm. CBCT exhibits potential for further research in Endodontics for the differential diagnosis of cysts versus granulomas. 65 Chapter Three: Evaluation of Root and Canal Morphology of Maxillary Permanent First Molars in a North American Population by Cone-beam Computed Tomography 3.1 Abstract Introduction: The purpose of this study was to evaluate the number of roots and canal morphology of maxillary permanent first molars in North America. Methods: 317 cases with bilateral maxillary first molars were included. All images from Cone-beam Computed Tomography (CBCT) were carefully reviewed by two endodontists. Frequency of number of roots, presence of an additional mesiobuccal canal (MB2), and Vertucci’s canal type for each root was tabulated. Age, gender and ethnicity differences were calculated with the Chi-square test. The intra-rater reliability was assessed using Cohen’s Kappa statistic. Results: The fused root rate was 0.9%. The occurrence of three-rooted maxillary first molars differed between left and right sides (p=0.03). MB2 occurrence only showed statistically significant differences among age groups (p=0.005). In the mesiobuccal roots, the most common Vertucci’s classification of canal types were Type IV (2-2, 41.9%), Type I (1, 28.3%), and Type II (2-1, 26.3%). There was a statistically significant difference in Vertucci’s classification of canal type among five ethnic groups (African- American, Asian, Hispanic, Other, and Non-Hispanic White, p<0.001). Conclusion: CBCT facilitates the identification of root and canal configuration. 66 The information gained about the tooth anatomy and canal morphology before treatment could potentially facilitate root canal therapy. Key Words: Cone-Beam Computed Tomography, tooth anatomy, root canal morphology 67 3.2 Introduction Complete knowledge of internal root morphology has been an important issue in planning and executing root canal therapy [52]. Successful treatment requires clinicians to have a basic knowledge of root canal morphology and possible anatomic variations [53]. Many efforts have been made in the in vitro examination of root canal morphology, using the tooth-clearing technique with the naked eye [87-89] or with the aid of a microscope [90]. Micro-computed tomography (Micro-CT) is an advanced radiological technique that provides non-destructive evaluation of root canal morphology and root anatomy [91]. Micro-CT has been generally considered as a reliable approach to analyze root canal anatomy [64]. However, all the in vitro techniques (tooth-clearing technique or micro-CT) can only be applied to extracted teeth, limiting the applicability of these techniques in clinical practice and being incompatible with root canal therapies where the goal is to save the tooth. In vivo methods include periapical radiographs and CBCT. Periapical radiographs provide only two-dimensional information of three-dimensional structures, which might lead to misunderstanding of root anatomy due to superimposition of roots [20]. CBCT has been shown to be a good method for initial identification of maxillary first molar internal morphology [92]. Filho et al. compared ex vivo (140 extracted teeth), clinical (291 patients), and CBCT approaches (54 randomly selected teeth) for the number of additional root canals and their locations, the number of foramina, and the frequency of canals 68 [20]. 54 maxillary first molars examined with CBCT had 4 canals (37.05%), and 90.9% of the teeth that had additional canals comprised one foramen, whereas 9.1% comprised 2 foramina. The authors indicated that operating microscope and CBCT have been important for locating and identifying root canals, and CBCT can be used as a good method for initial identification of maxillary first molar internal morphology[20]. Plotino et al. [93] 161 maxillary first molars, 157 maxillary second molars, 117 mandibular first molars, and 161 mandibular second molars) to establish the symmetry in root and canal anatomy between left and right sides using CBCT scanning. A percentage of symmetry that varied from 70%-81% was reported. The authors suggest taking into high consideration the asymmetry of canal configuration when treating two opposite molars in the same patient. CBCT scanning could also facilitate the identification of rare canal configurations according to the authors. Martins et al. [94] reported two rare cases of C-shaped configuration in upper first molars using a dental operating microscope. CBCT scanning was used and the same canal configuration on the opposite tooth was identified. Number of roots and canal morphology may vary by gender, age or ethnicity [52, 95-97]. Sert et al. [52] used the tooth-clearing technique and examined 200 maxillary first molars (100 from males and 100 from females). The prevalence of MB2 was 35% for males and 30.2% for females. Kim et al. [95] used CBCT to evaluate the morphology of maxillary first and second molars in a Korean population. The authors found that having additional canals 69 in the mesiobuccal root is the most common variation in maxillary molars (63.6% in 3-rooted maxillary first molars and 34.4% in 3- or 4-rooted maxillary second molars), and the root and canal configuration of this Korean population was different from other studies [95]. Pattanshetti et al. [96] treated 110 three- rooted maxillary first molars in Kuwait. Of those, 58% had one canal in the mesiobuccal root and 42% had two canals, with a high occurrence of Weine type II canal configuration (2-1) in the Kuwaiti population. The authors also indicated that all distobuccal and palatal roots had one single canal [96]. Zheng et al. [97] examined 775 maxillary first molars in a Chinese population and indicated that the occurrence of MB2 decreased with age. Reis et al. [98] demonstrated the effectiveness of CBCT scanning in mapping MB2 canals present in different thirds of the root. A total of 343 teeth (79 right and 79 left maxillary first molars, and 94 right and 91 left maxillary second molars) from100 male and 100 female patients were analyzed in vivo using CBCT scanning to identify root number and the presence of MB2 canals in different thirds. The authors indicated that the prevalence of MB2 canals decreased as the root canal approaches the apical third and as age increases. The prevalence of MB2 in the US population has been evaluated using in vivo observations with the aid of microscopes [90, 99] or with the ex vivo tooth-clearing technique [89]. Stropko [90] examined 1096 conventionally treated maxillary first molar records and found the prevalence of MB2 was 93.0% with the aid of a dental surgical microscope. Sempira et al. [99] treated 70 130 maxillary first molars using a microscope to record the occurrence of MB2, and the prevalence of MB2 was 33.1%. The authors indicated that a surgical microscope did not increase the number of second mesiobuccal canals located when compared with those reports without microscope use. Ibarrola et al. [89] evaluated 87 extracted maxillary molars and decalcified the mesiobuccal roots to observe the occurrence of MB2, and the prevalence was 77%. However, to our knowledge, little effort has been dedicated to the examination of number of roots and canal configuration of maxillary permanent first molars in North America by using CBCT. The aim of this study was to evaluate the differences in number of roots and canal morphology of maxillary permanent first molars according to gender, age and ethnicity in a US population by means of CBCT images. 71 3.3 Materials and Methods 3.3.1 Subjects CBCT images of maxillary first molars were collected from 1,484 patients who had undergone CBCT scanning for orthodontics or implant treatment planning at the Redmond Imaging Center, Ostrow School of Dentistry at the University of Southern California between July 2007 and July 2012. The research protocol was approved by the Institutional Review Board of the University of Southern California (#UP-13-00024). Inclusion criteria The following cases were included in the study: 1. Cases with bilateral maxillary permanent first molars. 2. Fully matured and erupted bilateral maxillary permanent first molars. 3. Molars without root canal fillings, posts, crown restorations, apical periodontitis or any other odontogenic or non-odontogenic pathology. Exclusion criteria Molars with fused roots were excluded due to the difficulty of identifying mesiobuccal, distobuccal, and palatal roots. 3.3.2 Radiological evaluation All the CBCT images were acquired with a Sirona Galileos device (Sirona Dental Systems, Inc., Long Island City, NY). The technician acquired a 72 fixed field of view volume of 15 cm x 15cm x 15 cm. The 3D Resolution (isotropic voxel size) was 0.3/0.15 mm. The scanner was operated at 85 kV and 5-7 mA. The Digital Imaging and Communications in Medicine (DICOM) format images were exported from Galileos and imported into InVivo Dental Application 5.1.6 software (Anatomage Inc, San Jose, CA). Serial sagittal, coronal, and axial views of CBCT images were examined carefully by two endodontists independently until an agreed diagnosis was reached for each case. The DICOM images were examined and the number of roots, the number of root canals, and the canal morphology (defined by Vertucci’s classification [54]) were tabulated for each maxillary first molar. Gender, ethnicity, and age were also collected. 20 randomly chosen cases (40 teeth) were examined and diagnosed a second time by the same two endodontists, 2 weeks apart, and the intra-rater reliability was calculated using Cohen’s Kappa. Agreement was noted as excellent if κ ≥0.75, good if 0.60≤ κ<0.75, intermediate if 0.4≤ κ<0.60, and poor if κ<0.4 [100]. 3.3.3 Statistics The prevalence of three-rooted maxillary first molars was calculated. Descriptive statistics (age, ethnicity, gender) were calculated. Pearson’s Chi- square test was used to analyze the differences in prevalence of three-rooted maxillary first molars between left and right sides and by gender. Differences by age in prevalence of MB2 were also analyzed with the Chi-square test. 73 Differences by ethnicity in canal morphology were examined using Fisher’s exact test. SAS Software 9.3 (SAS Institute Inc., Cary, NC, USA) was used in this study with a significance value of p=0.05. 74 3.4 Results The intra-rater reliability was 0.81 (excellent). 1,484 CBCT scans were examined for inclusion criteria. 317 cases with bilateral maxillary permanent first molars met all the criteria and were included in the study. The average age of the patients was 40 years old, among whom 161 were female patients (average age of 38 years old) and 156 were males (average age of 42 years old). Among these 634 maxillary first molars, 6 (0.9%, 6 out of 634) were two-rooted, and 628 (99.1%, 628 out of 634) were three- rooted. No single-rooted maxillary first molar was detected. Fused root molars were excluded due to the difficulty of identifying mesiobuccal, distobuccal, and palatal roots. Examples of different canal morphologies are shown in Figure 13 (A-D). 75 Figure 13. (A-‐D) Axial sections of maxillary permanent first molars with different root and canal configurations: (A) Fused distobuccal and palatal roots; red arrow denotes mesial root and green arrow denotes fused root; (B) A maxillary first molar with three separate roots, red arrow denotes mesiobuccal root, white arrow denotes distobuccal root, and green arrow denotes palatal root; (C) One Fused mesiobuccal and distobuccal root; red arrow denotes the buccal fused root and green arrow denotes the palatal root; (D) Fused mesiobuccal and distobuccal roots; red arrow denotes the fused root, and green arrow denotes the palatal root. 76 3.4.1 Prevalence of three-rooted maxillary first molars on left and right sides The prevalence of three-rooted maxillary first molars was higher on the right side (100%, 317 out of 317) compared to the left side (98.1%, 311 out of 317) (p=0.03). 3.4.2 Prevalence of three-rooted maxillary first molars according to gender Regardless of side, the prevalence of three-rooted maxillary first molars in females was 50.8% (319 out of 628) and in males was 49.2% (309 out of 628). The overall occurrence of three-rooted maxillary first molars in females and in males showed no statistically significant difference. 3.4.3 Percentage of MB2 canal in three-rooted maxillary first molars according to age, left and right side, and gender The prevalence of bilateral MB2 occurrence was 65.6% (208 out of 317 cases). The overall occurrence of MB2 in three-rooted maxillary first molars was 68.2% (428 out of 628 teeth). The frequency distribution of MB2 by six age groups (10-20, 20-30, 30-40, 40-50, 50-60, and 60+) is shown in Table 5. The highest occurrence of MB2 among all age groups was age group 60+ (80.0%, 67 out of 86), while the lowest was age group 30-40 (60.0%, 61 out of 109). There was a statistically significant difference of MB2 occurrence among six age groups (p=0.005). The occurrence of MB2 in the 60+ years old was 77 statistically significantly higher than in the younger than 60 (p=0.023), while the 30-40 years old had a statistically significantly lower occurrence of MB2 than the other age groups combined (p=0.002). The occurrence of MB2 in males was 69.4% (215 out of 310), and in females was 67.0% (213 out of 318). The occurrence of MB2 on the left side was 67.5% (210 out of 311), and 68.5% on the right side (217 out of 317). No statistically significant differences were found by gender or by sides (p=0.523 and p=0.864, respectively). 3.4.4 Percentage of Vertucci’s classification of canal type patterns in three-rooted maxillary first molars The canal configurations of maxillary first molars were analyzed, and the results for three-rooted maxillary first molars are shown in Table 6. The bilateral symmetry of mesiobuccal roots was 97.5% (309 out of 317). Of the 628 maxillary first molars, Type IV pattern (2-2, 41.9%, 263 out of 628) was the most common root canal type, followed by Type I pattern (1, 28.3%, 178 out of 628) and Type II pattern (2-1, 26.3, 165 out of 628). The frequencies of mesiobuccal root canal types in type I pattern (p=0.076), type II pattern (p=0.086), and type IV pattern (p=0.35) did not differ between females and males. 78 Table 5. Frequency of MB2 occurrence in maxillary first molars in six age groups Age groups (years) 10-‐20 20-‐30 30-‐40 40-‐50 50-‐60 60+ total Teeth w/MB2 n (%) 71 (67.6) 76 (72.4) 61 (60.0) 94 (74.6) 59 (60.8) 67 (80.0) 428 (68.2) Teeth w/o MB2 n (%) 34 (32.4) 29 (27.6) 48 (40.0) 32 (25.4) 38 (39.2) 19 (20.0) 200 (31.2) Total 105 105 109 126 97 86 628 p=0.005 79 Table 6. Frequency distribution and percentage of Vertucci's classification of canals for three roots according to gender in 628 three-rooted maxillary permanent first molars Mesiobuccal root Distobuccal root Palatal root Female n (%) Male n (%) Total No. of teeth (%) Female n (%) Male n (%) Total No. of teeth (%) Female n (%) Male n (%) Total No. of teeth (%) Type I (1) 99 (15.8) 79 (12.5) 178 (28.3) 317 (50.4) 309 (49.2) 596 (100) 318 (50.6) 310 (49.4) 628 (100) Type II (2-‐1) 74 (11.8) 91 (14.5) 165 (26.3) -‐ -‐ -‐ -‐ -‐ -‐ Type III (1-‐2-‐1) 2 (0.3) 5 (0.8) 7 (1.1) 1 (0.2) -‐ -‐ -‐ -‐ -‐ Type IV (2-‐2) 139 (22.1) 124 (19.8) 263 (41.9) -‐ -‐ -‐ -‐ -‐ -‐ Type V (1-‐2) 4 (0.6) 11 (1.8) 15 (2.4) -‐ 1 (0.2) -‐ -‐ -‐ -‐ total 318 (50.6) 310 (49.4) 628 (100) 318 (50.6) 310 (49.4) 628 (100) 318 (50.6) 310 (49.4) 628 (100) 80 3.4.5 The relationship among ethnicities and gender on Vertucci’s classification of canal types Data on five ethnic groups (African-American, Asian, Hispanic, Other, and White non-Hispanic) was collected and 458 maxillary first molars with known ethnicities were analyzed according to the presence of MB2 by ethnicity (Table 7). Maxillary first molars were also analyzed by Vertucci’s classification of canals of mesiobuccal roots by ethnicity (Figure 14). Among five ethnicities, cases from the group Hispanic made up 36.2% of all cases (166 out of 458), followed by the group of other ethnicities (104 out of 458), Non-Hispanic White (102 out of 458), Asian (60 out of 458), and African-American (26 out of 458). Type IV pattern (2-2) was the most common canal type among all ethnic groups followed by Type I pattern (1) and Type II pattern (2-1). There was no statistically significant difference in the occurrence of MB2 among five ethnic groups (p=0.123) (Table 3), however, there was a statistically significant difference in canal morphology between the ethnicities (p<0.001). 81 Table 7. The frequency of MB2 occurrence in maxillary first molars in five ethnic groups Ethnic groups African-‐ American Asian Hispanic Other Non-‐ Hispanic White Total Cases w/ MB2 n (%) 17 (65.4) 36 (60.0) 121 (72.5) 63 (60.6) 74 (72.5) 311 Cases w/o MB2 n (%) 9 (34.6) 24 (40.0) 46 (27.5) 41 (39.4) 28 (27.5) 147 Total 26 60 167 104 102 458 82 Figure 14. Frequency distribution of Vertucci's classification of mesiobuccal root canals in 458 maxillary first molars by ethnicity 83 3.5 Discussion The correct understanding of the number of roots and canal morphology is essential in root canal therapy. With high rates of variation in maxillary first molars [52, 54, 90, 95, 96], the accurate knowledge of the number of roots and canal morphology prior to root canal therapy is a concern for endodontists. Wolcott et al. [101] examined the treatment records of 3,578 first molars consecutively over a 5-yr period , and found that the overall prevalence of a MB2 canal was 60% (2,133 teeth). The authors indicated that a significant difference in the prevalence of a MB2 canal between initial treatment records (58%) and retreatment records (66%) suggested that failure to find and treat existing MB2 canals will decrease the long-term prognosis. CBCT is considered a useful approach to make pre-intervention diagnosis [62, 64, 102]. Matherne et al. [62]used CBCT as a reference standard to identify root canal systems in vitro compared with charged coupled device (CCD) and photostimulable phosphor plate (PSP) digital radiography [62]. Seventy-two extracted teeth were exposed with CCD, PSP, and CBCT radiography. The endodontist evaluators failed to identify 1 or more root canal configurations with CCD in 41% of teeth and 40% for PSP, and the authors indicated that evaluation of CBCT images resulted better in identifying root canal system. Blettner et al.[102] tested twenty completely intact maxillary first and second molars for the existence of the MB2 canal. The accuracy of CBCT 84 scanning and clinical sectioning (gold standard) in the identification of the MB2 canal was compared. The authors indicate that CBCT scanning is a reliable method to detect the MB2 canal. Domark et al. compared digital radiography, CBCT, and Micro-CT (the reference standard) in the determination of the number of canals in mesiobuccal roots of maxillary first molars. The author stated that for cadaver maxillary molars, there was no difference in canal counts between CBCT and Micro-CT [64]. In this retrospective study, 1,484 previously acquired CBCT scans were screened for bilateral first molars. 628 three-rooted maxillary first molars were included in the study with type IV pattern (41.9%), type I pattern (28.3%), and type II pattern (26.3%) were the most common canal types in mesiobuccal roots regardless of gender and ethnicity. For distobuccal and palatal roots, 99.6% and 100% cases, respectively, was of type I pattern. These findings were consistent with Vertucci’s study [54]. Vertucci [54] used the tooth-clearing technique to study 2,400 permanent teeth, and summarized the classification of root canals (type I to type VII). Among 100 maxillary first molars, type I, type II and type IV were the most common types for mesiobuccal roots, while for both distobuccal and palatal roots it was type I. The prevalence of bilateral MB2 occurrence was 65.6%, and the overall MB2 occurrence in three-rooted maxillary first molars was 68.2%, which is consistent with the Korean study [95]. These high prevalence rates suggest that 85 endodontists need to be aware of the occurrence of MB2, in particular for patients who had a known MB2 on one side. The presence of MB2 did not differ between gender and position, which was consistent with previous studies [95, 97]. In the Korean population study, the prevalence of MB2 was 63.59%, with 88.10% symmetry MB roots bilaterally [95]. Zheng et al. [97] examined 775 CBCT images of maxillary first molars in a Chinese population, and found the prevalence of MB2 was 52.24%. 71.11% of the additional mesiobuccal root canals in subjects with bilateral qualifying molars were symmetric [97]. The distribution of MB2 occurrence in the present study by age was different from some previous studies [97, 103]. In the Chinese population study, the highest prevalence of MB2 was in the age group of 20-30 years old [97]. Neaverth et al. [103] studied 228 maxillary first molars during endodontic therapy, and found that the prevalence of MB2 was 77.2% (176 teeth), of which 61.8% (141 teeth) had two foramina. The authors also concluded that the age group of 20-40 year-olds possessed the highest rate of MB2 occurrence. The differences among the present study with others may be explained by differences in ethnicity, method of evaluation and sample size. Although in the present study, the occurrence of MB2 in the age group of 30-40 was statistically significantly lower than other groups, the rate was still as high as 60.0%, which suggests that endodontists should pay attention to the presence of MB2 in all age groups. 86 Root canal morphology in populations with different ethnic origins has been explored in prior studies, such as Turkish [52], US [89, 90, 103], Korean [95, 104], Japanese [105], Burmese [106], Thai [107], Indian[108], and Chinese populations [97]. Variances in the presence of MB2 may because of ethnic background. Neelakantan et al. [108] investigated the root and canal morphology of maxillary first and second molars in an Indian population using CBCT. Two hundred and twenty extracted maxillary first molars were examined and among buccal roots of three-rooted first molars, Type I (51.8%) and Type IV (38.6%) were shown. The authors indicated that the root number and canal morphology of Indian maxillary molars showed features that were different from both Caucasian and Mongoloid traits. However, little research has been done to explore the differences of MB2 occurrence and canal type among different ethnicities by CBCT. Only descriptive data were shown in these studies, indicating that in all ethnic groups, type I pattern, type II pattern, and type IV pattern were most common in mesiobuccal roots of three-rooted maxillary first molars [52, 89, 90, 95, 97, 103, 105-108]. In the present study, we compared five common ethnic groups living in the US. The results indicated that there was no difference in the occurrence of MB2 among ethnic groups; however, a statistically significant difference was found by canal morphology, indicating that careful exploration of canal types should be taken when treating patients from different ethnic origins. 87 The main limitation of the present study was the relatively small sample size of some ethnic groups (Asian, African-American). Further research is warranted to investigate the differences in canal morphology among different ethnic groups with larger sample sizes. 88 3.5 Conclusion CBCT facilitates the identification of root and canal configuration. In this retrospective study, the bilateral MB2 occurrence was 65.6% with significant differences among age groups. The most common Vertucci’s classification of canal types were Type IV (2-2), Type I (1), and Type II (2-1), and there was a statistically significant difference among ethnic groups. The information gained about tooth anatomy and canal morphology before treatment could potentially facilitate root canal therapy. 89 Chapter Four: Evaluation of Root and Canal Morphology of Mandibular Permanent First Molars in a North American Population by Cone-Beam Computed Tomography 4.1 Abstract Introduction: The purpose of this study was to evaluate root and canal morphology of mandibular permanent first molars in a North American population. Methods: A total of 248 cases with bilateral mandibular first molars were evaluated after meeting inclusion and exclusion criteria. All scans from Cone-Beam Computed Tomography (CBCT) were carefully reviewed by two endodontists. The number of roots, occurrence of disto-lingual root, and Vertucci’s classification of canal types for each molar were recorded. Differences by gender and ethnicity were calculated using Chi-square test and Fisher’s exact test. The intra-rater reliability was assessed using Cohen’s Kappa statistic. Results: The symmetry rate was 85.5%. The occurrence of three-rooted mandibular first molars in Asians was statistically significantly different as compared to other ethnic groups (p<0.0001). The most common type of root and canal morphology for mandibular first molars was two separate canals in the mesial root and one canal in the distal root. There was a statistically significant difference in Vertucci’s classification of canal types among five ethnic groups (African-American, Asian, Caucasian non-Hispanic, Hispanic, and Others, p<0.0001). Conclusion: CBCT can assist in the 90 identification of root and canal morphology of mandibular permanent first molars. The information gained about the root and canal morphology from CBCT before treatment could facilitate root canal therapy. Key Words: Cone-beam computed tomography, tooth anatomy, root canal morphology, mandibular first molar, Endodontics 91 4.2 Introduction Prior and complete knowledge of internal root and canal morphology is essential in planning and executing root canal therapy [52]. Mandibular permanent first molars, usually the earliest permanent teeth to erupt, are the most common extracted teeth [109] and the most frequent reason is caries [110]. Mandibular first molars typically have two roots (mesial and distal) and three root canals (two in the mesial root and one in the distal root) [52, 111-114]. In vitro and in vivo studies have been conducted for detecting the number of roots and canal morphology. Noninvasive in vitro techniques such as tooth-clearing technique and Computed Tomography (Micro-CT) offer complete detection of root and canal morphology of samples in two- or three-dimensions [115]. However, only extracted teeth are eligible for non-destructive in vitro techniques, limiting the applicability of these techniques in clinical practice for pre-treatment diagnosis of root and canal morphology. Cone-Beam Computed Tomography (CBCT) is an advanced and non-invasive in vivo method which can provide three-dimensional information, and has been shown to be an optimal method for pre-treatment identification of root and canal morphology [116]. The presence of variation in number of roots and canals has been reported frequently using extracted teeth, transparent tooth technique and 92 dissecting microscope, and CBCT [52, 111-114, 117-127]. A summary of studies of prevalence of disto-lingual roots and canal morphology of mesial roots are presented in Table 8. There are also two literature reviews on gender and ethnicity differences in root and canal morphology. In a review by de Pablo et al. [128], 41 studies were identified including a total of 18,781 teeth. The incidence of a third root in their review was 13%, which was strongly correlated with the ethnicity of the studied population. Abella et al. [129] concluded that the number of roots in the mandibular first molar is directly related to ethnicity; the prevalence of disto- lingual roots of mandibular first molars was 14.4% and was correlated with ethnicity. However, to our knowledge, little effort has been dedicated to detecting the root and canal configuration of mandibular permanent first molars from various ethnic groups in North America using CBCT. The aim of this study was to evaluate the differences in root and canal morphology of mandibular first molars by gender and ethnicity in a North American population by means of CBCT images. 93 Table 8. Studies of prevalence of disto-‐lingual roots and canal morphology of mesial roots in different populations Population Technique Sample size %DL Canal morphology of mesial roots Year Authors Type II (n,%) Type IV (n,%) Type V (n,%) Chinese In vivo (Micro-‐ CT) 122 31.97% -‐ -‐ -‐ 2010 Gu et al. [117] Chinese In vivo (CBCT) 232 29.00% -‐ 178 (81%) 33 (15%) 2011 Zhang et al. [111] Chinese In vivo (CBCT) 558 25.80% -‐ 385 (93.90) -‐ 2010 Wang el al. [112] Korean In vitro (Serial cross-‐sectional CT) 3,088 24.50% -‐ -‐ -‐ 2010 Song et al. [118] Korean In vivo (CBCT) 780 24.50% -‐ -‐ -‐ 2013 Jang et al. [119] Taiwanese In vitro (CBCT) 166 21.09% -‐ -‐ -‐ 2007 Tu et al. [114] Taiwanese In vitro (transparent teeth) 183 20.00% 54 (30%) 101 (55%) 3 (2%) 2009 Chen et al. [113] South Indian In vivo (periapical radiograph) 1,000 18.60% -‐ -‐ -‐ 2011 Chandra et al. [121] Thai In vitro (transparent teeth) 118 13.00% -‐ -‐ -‐ 2002 Gulabivala et al. [120] Brazilian In vivo (CBCT) 234 11.00% -‐ -‐ -‐ 2013 Silvia et al. [122] Burmese In vitro (transparent teeth) 139 10.00% -‐ -‐ -‐ 2001 Gulabivala et al. [123] Turkish In vivo (CBCT) 823 2.06% -‐ -‐ -‐ 2013 Demirbuga et al.[124] German In vivo (periapical radiograph) 1,024 1.35% -‐ -‐ -‐ 2008 Schäfer et al. [125] South-‐ Eastern Turkish In vivo (CBCT) 966 0.50% 5 (1%) (F) 5 (1%) (M) 428 (89%) (F) 455 (93%) (M) 14 (3%) (F) 3 (0.6%) (M) Nur et al. [126] White In vivo (CBCT) 117 0 48 (41.00) 73 (62.40) -‐ 2013 Plotino et al. [127] Turkish In vitro (transparent teeth) 200 -‐ 41 (41%) (F) 47 (47%) (M) 45 (45%) (F) 41 (41%) (M) 2 (2.00) (F) 0 (M) 2004 Sert et al. [52] F, Female; M, Male. 94 4.3 Materials and Methods 4.3.1 Subjects CBCT scans of 1,484 subjects were retrospectively assessed for eligibility. All the subjects had undergone CBCT scanning for the purpose of orthodontics or implant treatment planning at the Redmond Imaging Center, Ostrow School of Dentistry at University of Southern California in the time spanning from July 2007 to July 2012. The research protocol was approved by the Institutional Review Board of the University of Southern California (#UP-13- 00024). 4.3.2 Inclusion and exclusion criteria Fully matured and erupted bilateral mandibular permanent first molars from any ethnic group were included in this retrospective study. Cases were excluded if they had root canal fillings, posts, crown restorations, apical periodontitis or any other odontogenic or non-odontogenic pathology associated with the mandibular permanent first molars. 4.3.3 Radiological evaluation The CBCT images were taken with Sirona Galileos (Sirona Dental Systems, Inc., Long Island City, NY). The technician acquired a fixed field of view volume of 15 x 15 x 15 cm 3 . The resolution in the volume (voxel size) equals 0.3 x 0.3 x 0.3 mm 3 . The scanner was operated at 85 kV and 5-7 mA. 95 The Digital Imaging and Communications in Medicine (DICOM) format images were exported from Sirona Galileos and imported into InVivo Dental Application 5.1.6 software (Anatomage Inc, San Jose, CA). Each case was independently examined by two endodontists. Whenever disagreement in the diagnosis occurred, a consensus was reached by discussion. Sagittal, coronal, and axial views for each case were evaluated. The number of roots and canal morphologies using Vertucci’s classification [54] were tabulated for each mandibular first molar. Age, gender, and ethnicity for each case were also collected. To calculate intra-rater reliability, 20 cases (total 40 teeth) were randomly chosen, and evaluated for a second time by the same two endodontists, one month apart. The intra-rater reliability was calculated using Cohen’s Kappa statistic. The agreement was regarded as excellent if κ ≥0.75, good if 0.60≤ κ<0.75, intermediate if 0.4≤ κ<0.60, and poor if κ<0.4 [100]. 4.3.4 Statistics Descriptive statistics (age, ethnicity, and gender) were calculated and illustrated. The prevalence of number of roots was calculated for the left- and the right-side as well as by gender. Pearson’s Chi-square test was used to analyze the differences in prevalence of number of roots in mandibular first molars by side and gender. Percentages of Vertucci’s classification of canal 96 type patterns in roots were also analyzed by side and gender using the Chi- square test. Differences in the canal morphology in different ethnic groups were examined using Fisher’s exact test. SAS Software 9.3 (SAS Institute Inc., Cary, NC, USA) was used in this study with a significance value of α=0.05. 97 4.4 Results CBCT scans of 1,484 subjects were examined for inclusion criteria. Among all these scans, 248 cases presented with bilateral mandibular permanent first molars and met all inclusion criteria. Each case was independently examined by two endodontists with excellent intra-rater reliability (κ=0.82). Final evaluation of root and canal morphology was reached by consensus. The average age of the recruited patients was 40.0 years old, among whom 121 were female patients (average age of 38.2 years old) and 117 were males (average age of 41.4 years old). Among all 248 cases, 212 cases had symmetric number of roots and canal types, with a symmetry rate of 85.5% (212 out of 248). In summary, 225 cases had bilateral first molars with two roots (90.7%, 225 out of 248) and 8 cases had three roots (3.3%, 8 out of 248). Another 15 cases had unilateral three roots (6.0%, 15 out of 248). The total number of two- rooted first molars was 465 (93.8%, 465 out of 496), while 31 (6.2%, 31 out of 496) were three-rooted. No single-root mandibular first molar was detected. Examples of different canal morphologies are shown in Figure 15 (A-D). 98 Figure 15. (A-‐D) Transverse sections of mandibular permanent first molars with different root and canal configurations: (A) Two-‐rooted mandibular first molar with two canals; red arrow denotes mesial root and blue arrow denotes distal root; (B) Two-‐rooted mandibular first molar with three canals; red arrow denotes mesial root and blue arrow denotes distal root; (C) Two-‐rooted mandibular first molar with four canals; red arrow denotes mesial root and blue arrow denotes distal root; (D) Bilateral mandibular first molars with three roots and four canals; red arrow denotes mesial root, blue arrow denotes disto-‐buccal root, and green arrow denotes disto-‐lingual root. 99 4.4.1 Number of Roots in Mandibular First Molars 4.4.1.1 Prevalence of Number of Roots by Ethnicity Among all mandibular first molars, ethnicity was known for 472 teeth (20 African-American, 66 Asian, 148 Hispanic, 146 Caucasian non-Hispanic, and 92 Others). The descriptive statistics of the number of roots by ethnicity is shown in Figure 16. Among all 31 three-rooted mandibular first molars, 15 molars (22.7%, 15 out of 66) were from the ethnic group of Asians (3.4%, 16 out of 407). The prevalence of three-rooted mandibular first molars in Asians was statistically significantly higher than all other non-Asian cases (p<.0001) as well as the prevalence of two-rooted mandibular first molars (p<.0001) (Figure 16). 4.4.1.2 Prevalence of Number of Roots by Side The prevalence of two-rooted mandibular first molars was 94.8% on the right side (235 out of 248) and 92.7% on the left side (230 out of 248) (p=0.42). For three-rooted mandibular first molars, the prevalence was 5.2% on the right side (13 out of 248) and 7.3% on the left side (18 out of 248) (p=0.42). 100 Figure 16. Prevalence of number of roots by ethnic groups in mandibular first molars 101 4.4.1.3 Prevalence of Number of Roots by Gender Regardless of side, the prevalence of two-rooted mandibular first molars in females was 94.7% (248 out of 262) and in males was 92.7% (217 out of 234). The overall occurrence of two-rooted mandibular first molars in females and in males showed no statistically significant difference (p=0.38). 4.4.2 Root Canal Configuration of Mandibular First Molars 4.4.2.1 Percentage of Vertucci’s Classification of Canal Type Patterns in Two-rooted Mandibular First Molars The canal configurations of mandibular first molars were analyzed and the results are shown in Table 9. Of the 465 two-rooted mandibular first molars, 312 mesial roots exhibited the Type IV configuration canals (2-2, 67.1%, 312 out of 465), which was the most common type. For type IV, there were no statistically significant differences between sides nor by gender (p=0.62 and p=0.07, respectively). The most common type of distal root was type I (1, 90.3%, 420 out of 465), and there were no statistically significant differences in the occurrence of type I in distal roots between sides or by gender (p=0.93 and p=0.08, respectively). 4.4.2.2 Percentage of Vertucci’s Classification of Canal Type Patterns in Three-rooted mandibular first Molars 102 Of the 31 three-rooted mandibular first molars, the highest percentage of canal configuration in mesial roots was Type IV (2-2, 61.3%, 19 out of 31), and the most common canal configuration in disto-buccal and disto-lingual roots was Type I, with a frequency of 93.5% (29 out of 31) and 100%, respectively (Table 9). 4.4.2.3 Differences in Canal Configuration of Two-rooted Mandibular First Molars by Ethnicity Five race groups (African-American, Asian, Caucasian non-Hispanic, Hispanic, and Others) were present in our study. 442 two-rooted mandibular first molars with known races were analyzed according to Vertucci’s classification of both mesial and distal canals (Figure 17A and 17B). There was a statistically significant difference in canal morphology among all ethnicities in mesial roots (p<0.0001), however there was no statistically significant difference between Type I and other types of canal configuration in distal roots (p=0.21). 103 Table 9. Vertucci's classification of root canals in 496 mandibular first molars Two-‐rooted Three-‐rooted Mesial (n, %) Distal (n, %) Mesial (n, %) Disto-‐buccal (n, %) Disto-‐lingual (n, %) Type I (1) 69 (14.9) 420 (90.3) 3 (9.7) 29 (93.6) 31 (100) Type II (2-‐1) 59 (12.7) 5 (1.1) 5 (16.1) 1 (3.2) -‐ Type III (1-‐2-‐1) 1 (0.2) 7 (1.5) -‐ -‐ -‐ Type IV (2-‐2) 312 (67.2) 9 (1.9) 21 (67.7) -‐ -‐ Type V (1-‐2) 23 (5.0) 24 (5.2) 2 (6.5) 1 (3.2) -‐ Total 465 (100) 465 (100) 31 (100) 31 (100) 31 (100) 4.4.2.4 Differences in Canal Configuration of Three-rooted Mandibular First Molars by Ethnicity The mesial roots of 30 three-rooted mandibular first molars with known ethnicity were analyzed. Type IV (2-2) canal configuration was the most 104 common type in the mesial roots in this kind of molar (66.7%, 20 out of 30). All samples from the Asian group showed Type IV (2-2) canal configuration in mesial roots (100%, 13 out of 13). In the Caucasian non-Hispanic group, 71.4% (5 out of 7) was of Type IV canal configuration. 105 Figure 17. Prevalence of canal configuration according to Vertucci's classification of mesial and distal roots by ethnicity 106 4.5 Discussion High prevalence of caries in adult mandibular first molars [110] makes it the most common tooth to receive root canal therapy and/or oral surgery. Precise pre-diagnosis of the number of roots and canal morphology is essential in root canal therapy and a main concern for endodontists. Noninvasive approaches such as CBCT could provide accurate pre-intervention diagnosis [116]. Neelakantan et al. [116] investigated the accuracy of CBCT in evaluating root canal morphology using the canal staining and clearing technique as the gold standard method. The authors concluded that CBCT was as accurate as the canal staining and tooth clearing technique in identifying root canal systems. In this retrospective study, CBCT was used as a non-invasive approach to gain thorough information of root and canal morphology of mandibular first molars. Ethnic variations of root and canal morphology are a challenge for clinicians. One significant variation among ethnicities is the presence of a disto- lingual root in the mandibular first molars. Several studies have reported a higher occurrence of three-rooted mandibular first molars in Asians than in other ethnic groups (15-21). Gu et al. [117] examined 122 extracted mandibular first molars using Micro-CT, and the frequency of three-rooted mandibular first molars was 31.97% (39/122). Zhang et al. [111] evaluated 232 mandibular first molars selected from a Chinese population using CBCT, and found out the 107 occurrence of three-rooted molars was 29%. Wang et al. [112] examined 558 samples using CBCT to evaluate the root and canal morphology of mandibular first molars in a Western Chinese population. The authors concluded that 25.8% had a separate disto-lingual root. Song et al. [118] examined 3,088 first molars in a Korean population. Serial cross-sectional CT images were used, and the authors indicated the prevalence of disto-lingual roots in mandibular first molars was 24.5% (Table 6). Compared to the occurrence of disto-lingual roots in Asian sample populations, studies from other ethnic origins all reported lower frequency of three-rooted mandibular first molars. Silvia et al. [122] reported a frequency of 11% in 234 cases from Brazil. Demirbuga et al. [124] studied 823 CBCT images of Turkish individuals and indicated that three roots were identified in 2.06% of the sample. Schäfer et al.[125] used full-mouth periapical radiographs to examine a total of 1,024 mandibular first molars. The authors indicated an overall prevalence of 1.35% in this German sample. No three-rooted mandibular first molars were found in the study conducted in a Caucasian population from Italy by Plotino et al. [127] (Table 8). In this retrospective study, 1,484 CBCT scans were evaluated and 248 cases were included with bilateral mandibular first molars. The Asian group had a statistically significant higher prevalence of three-rooted molars (22.7%, 15 out of 66, Figure 16) compared to all other ethnic groups (p<.0001). Our results 108 are consistent with the studies above (15-21). This suggests the possibility of an additional root in patients originally from Asia, and an access extension in a disto-lingual direction may be necessary for these patients. As for the Vertucci’s classification of canal types, Type IV configuration (2-2) was the most common canal configuration type in mesial roots for two- rooted mandibular first molars (2-2, 65.9%, 312 out of 465), followed by Type I (1, 14.9%, 35 out of 465). The most common type of distal roots was Type I (1, 90.3%, 420 out of 465). These results are consistent with previous studies. Zhang et al. [111] examined 232 mandibular first molars and found out 220 (95%) contained two canals in mesial, amongst which type IV (81%) was the most prevalent. Also, the authors reported that all of the disto-lingual roots had the Type I configuration. Demirbuga et al. [124] recorded five variants in the root canal morphology of 823 mandibular first molars. The most common in mesial roots was Type IV (63.7% in female, 72.3% in male). Of the distal roots, 83% had one canal. The most commonly observed root and canal morphology in the present study for mandibular first molars was two separate canals in mesial root and one canal in distal root. This result is consistent with studies from Brazil [122], Jordan [130], Korea [131], and Italy [127]. One notable trend is that among the five ethnic groups in our study (African-American, Asian, Caucasian non- Hispanic, Hispanic, and Others), there was a statistically significant difference 109 in canal morphology among all ethnic groups in mesial roots (p<0.0001), suggesting that clinicians should carefully explore for variations in canal morphology when treating patients from different ethnic origins. This retrospective study has some limitations including the relatively small sample size of some ethnic groups such as African-American and Asian groups. Further research is warranted in the investigation of root and morphology of mandibular first molars among different ethnic groups with larger sample sizes. Also, due to the resolution of the scanner used in this paper (Sirona’s Galileos with pixel size at 250µm), some intricate anatomic details may not be detected by CBCT. Further development of the technology with higher resolution and lower X-ray exposure may bring more opportunities for in vivo research. In this retrospective study, CBCT was used to identify the root and canal morphology of mandibular first molars from a North American population. The prevalence of disto-lingual roots in Asians was statistically significantly higher as compared with other ethnic groups. The most common type of root and canal morphology for mandibular first molars was two separate canals in the mesial root and one canal in the distal root, and there was a statistically significant difference of canal morphology among five ethnic groups. Information gained before treatment using CBCT about the root and canal morphology could facilitate root canal therapy. 110 Chapter Five: Conclusion Endodontic therapy is required when pulpitis and periapical lesions occur. Maxillary and mandibular first molars are believed to have complicated anatomies due to the high rates of variation in the number of roots and canal morphology. Caries is the most common reason to injury the pulp and cause pulpitis. The high rates of caries in maxillary and mandibular first molars due to their early eruption pattern may increase the need for endodontic therapy. An additional canal (MB2) in the mesiobuccal root is the most prevalent variation for maxillary first molars. The occurrence of MB2 varies by age and ethnicity. The radix entomolaris and the radix paramolaris are common variations for mandibular first molars. The additional root presents buccally or lingually, whose occurrence rate varies significantly among different ethnic groups. Inability of locating, instrumenting, and obturating the MB2 in maxillary first molars or the additional root in mandibular first molars would lead to treatment failures of these teeth. Thus, the information gained about tooth anatomy and canal morphology before treatment could potentially facilitate root canal therapy. Periapical cysts are by far the most common cysts of the jaws. The proliferation of the rests of Malassez within the periodontal ligament causes these inflammatory cysts. A histological report with the evidence of stratified squamous lining epithelium is considered as the reference for diagnosis of periapical cysts. Radiologically, it is a difficult task to make differential 111 diagnosis using periapical radiographs only. With a high incidence of periapical granulomas, a high rate of biopsies would be conducted; However, according to some authors those lesions may heal non-surgically. In this dissertation, three studies were conducted using CBCT as an aid to solve endodontic issues. CBCT, with its ability to acquire in vivo three- dimensional information for diagnostic tasks, harbors the potential to become a routine diagnostic and pre-treatment evaluation instrument in Endodontics. In Chapter Two, a set of six diagnostic criteria based on the radiological characteristics of a periapical cyst (location, periphery, shape, internal structure, and effects on surrounding structure) was established to differentiate periapical cysts from granulomas. An optimal scoring system (with four or more criteria would make a diagnosis of periapical cyst) was confirmed with a moderate accuracy and good to excellent reliability. Limitations of the study include small total sample size, small number of cysts in the sample and the inclusion of only two types of lesions. Further studies on a larger sample size with more periapical conditions will be required to confirm the findings of the present study. In Chapter Three and Four, root and canal morphology was evaluated for maxillary and mandibular first molars from a US population. A high incident rate of bilateral MB2 in maxillary first molars was noticed, and the most common Vertucci’s classification of canal types of mesiobuccal roots in these molars 112 was Type IV (2-2) with significant differences among five ethnic groups. Also, the prevalence of disto-lingual roots of mandibular first molars in Asians was statistically significantly higher as compared with other ethnic groups. The most common canal pattern for mandibular first molars was two separate canals in the mesial root and one canal in the distal root, and there was a statistically significant difference of canal morphology among five ethnic groups. 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Who is reading the digital radiography and the cone beam computed tomography?

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Three-dimensional assessment of tooth root shape and root movement after orthodontic treatment: a retrospective cone-beam computed tomography study

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Creator Guo, Jing (author)

Core Title Cone-beam computed tomography images: applications in endodontics

School School of Dentistry

Degree Doctor of Philosophy

Degree Program Craniofacial Biology

Publication Date 09/10/2015

Defense Date 02/23/2015

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Biopsy,cbct,cone-beam computed tomography,differential diagnosis,disto-lingual root,endodontics,mandibular first molar,maxillary first molar,MB-2,OAI-PMH Harvest,periapical cyst,periapical granuloma,root canal morphology,root canal therapy,tooth anatomy

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Advisor Enciso, Reyes (committee chair), Azen, Stanley P. (committee member), Paine, Michael L. (committee member), Sameshima, Glenn T. (committee member), Sedghizadeh, P. Parish (committee member)

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