Association between visual characterization of root caries and histological severity In vitro

Transcription

1 University of Iowa Iowa Research Online Theses and Dissertations Fall 2010 Association between visual characterization of root caries and histological severity In vitro Carolina Elizabeth Mendieta Facetti University of Iowa Copyright 2010 Carolina Elizabeth Mendieta This thesis is available at Iowa Research Online: Recommended Citation Mendieta Facetti, Carolina Elizabeth. "Association between visual characterization of root caries and histological severity In vitro." MS (Master of Science) thesis, University of Iowa, Follow this and additional works at: Part of the Other Dentistry Commons

2 ASSOCIATION BETWEEN VISUAL CHARACTERIZATION OF ROOT CARIES AND HISTOLOGICAL SEVERITY IN VITRO by Carolina Elizabeth Mendieta Facetti A thesis submitted in partial fulfillment of the requirements for the Master of Science degree in Operative Dentistry in the Graduate College of The University of Iowa December 2010 Thesis Supervisor: Associate Professor Justine L. Kolker

3 Graduate College The University of Iowa Iowa City, Iowa CERTIFICATE OF APPROVAL MASTER'S THESIS This is to certify that the Master's thesis of Carolina Elizabeth Mendieta Facetti has been approved by the Examining Committee for the thesis requirement for the Master of Science degree in Operative Dentistry at the December 2010 graduation. Thesis Committee: Justine L. Koker, Thesis Supervisor James S. Wefel Deborah V. Dawson John Warren Ronald L. Ettinger Deborah R. Cobb

4 To my mother, for her constant support and love. To my American host parents, for always being there for me. To all my family and friends, who are my well wishers. To my mentors, for their willingness to teach. ii

5 ACKNOWLEDGMENTS I would like to express my deeply gratitude to Dr. Justine L. Kolker, my thesis advisor and mentor, who has constantly supported me throughout this thesis project. I greatly admire her passion and excellence in Operative Dentistry. Her continued encouragement on this study gave me a wonderful learning experience academically and personally. I would like to deeply thank Dr. James S. Wefel for being a big part of my research committee and sharing his research experience in my thesis project. I would also like to thank him for all the extended collaboration on the Literature and Methodology of this study. I greatly appreciate all the help provided by Mary Margaret B. Hogan and Jeffrey D. Harless during the experimental work of this project. They were always willing to help me with the best humor and kindness. I feel very fortunate to have Dr. Deborah Cobb as my graduate program director and a valuable committee member. Her so approachable personality made my residency program easier and enjoyable. I would like to acknowledge Dr. Deborah Dawson and Colleen Kummet for their wonderful work analyzing the statistics of this thesis and their valuable suggestions. Additionally, I would also like to acknowledge Dr. Ronal L. Ettinger and Dr. John Warren for all their valuable insights. A special recognition to all the friends and faculty members of the Operative Dentistry department. iii

6 TABLE OF CONTENTS LIST OF TABLES...vii LIST OF FIGURES... ix CHAPTER I: INTRODUCTION... 1 Purpose of the study... 3 Clinical Relevance... 5 CHAPTER II: LITERATURE REVIEW... 6 Tooth hard tissues... 6 Enamel... 6 Dentin... 7 Cementum Root caries Systems to examine sections of root caries Diagnosis of root caries Cavitation or surface loss Texture Color Location Studies determining root caries activity Diagnostic tests The International Caries Assessment and Detection System (ICDAS) CHAPTER III: MATERIALS AND METHODS Overview Research questions Calibration phase Selection and preparation of teeth Visual/tactile assessment of root caries lesions Post-calibration phase Selection and preparation of the teeth Visual/tactile assessment of root caries lesions Preparation of the histological specimens Histological evaluation Intra-examiner reliability of histological measurements Visual/tactile depth of surface loss classified according to ICDAS Statistical analysis iv

7 Reliability testing Visual/tactile assessments for the post-calibration phase Histological assessment Correlation between the visual/tactile criteria and histological evaluation ICDAS with depth of of surface loss and depth of demineralized dentin tubule involvement CHAPTER IV: RESULTS Introdution Calibration phase Intra-inter examiner calibration for visual/tactile assessment Post-calibration phase Section Reliability of the visual/tactile assessment Intra-rater agreement Inter-rater agreement Reliability of the histological measurements Section Visual/tactile characteristics with histological findings Reationship between depth of surface loss and visual/tactile characteristics Reationship between depth of demineralized dentin tubules and visual/tactile characteristics Histological depth of surface loss Histological depth of surface loss and texture Relationship between depth of demineralized dentin tubule involvement and visual/tactile characteristics Section ICDAS related to depth of surface loss ICDAS related to depth of demineralization CHAPTER V: DISCUSSION CHAPTER VI: SUMMARY AND CONCLUSIONS REFERENCES v

8 LIST OF TABLES 1. Recompilation of clinical characteristics of root caries lesions provided by the first investigators Authors that described root caries activity according to loss of suface integrity Authors that described root caries activity according to texture Authors that described root caries activity according to color Authors that described location of root caries lesions Exact agreement between 2 assessments (n=45 teeth) Exact agreement between the 2 assessments (n=45 teeth) based upon (unweighted) kappa Exact agreement between the 2 assessments (n=45 teeth) based upon weighted kappa Intra-rater exact agreement between 2 visual/tactile assessments (n=63 teeth) Simple kappa agreement between 2 intra-rater visual/tactile assessments (n=63 teeth) Weighted kappa agreement between 2 intra-rater visual/tactile assessmentst (n=63 teeth) Inter-rater exact agreement between 2 visual/tactile assessments (n=63 teeth) Simple kappa agreement between 2 inter-rater visual/tactile assessments (n=63 teeth) Weighted kappa agreement between 2 inter-rater visual/tactile assessmentst (n=63 teeth) Descriptive statistics of mean depth of surface loss and mean depth of demineralized dentin tubule involvement Mean depth of surface loss by ICDAS vi

9 17. Mean depth of demineralized dentin tubule involvement by visual/tactile surface loss Studies on reliability of visual/tactile diagnosis of root caries lesions vii

10 LIST OF FIGURES 1. Dentinal tubules seen in a longitudinal section through dentin Polarized light microscopy picture of a stagnating root caries lesions ICDAS Root Caries Classification System Photographs taken of root caries lesions wet and dry Samples of color classification Periodontal probe used to measure the clinical depth of surface loss Dental instrument used to test the texture Teeth by depth of surface loss, texture and color (2 nd Assessment) Teeth mounted in mandrels to be sectioned in a hard-tissue microtome A hard-tissue microtome used to create sections od the lesion Root caries lesion sample after being imbibed in water being assessed under the Polarized Light Microscopy Histological assessment under the Polarized Light Microscopy Polarized Light Microscopy Olympus Sample of the three main specimens obtained after sectioning and their histological assessment Computer software Pro-Light Plus used to trace measurement points Computer software Pro-Light Plus after tracing measurement points Sample of the histological measurement of a root caries lesion Final sample size origin Comparison of intra and inter- rater simple kappa values Box Plots of log mean depth of surface loss by visual/tactile depth of surface loss Box plots of log mean depth of surface loss by texture Box plots of log mean depth of surface loss by visual/tactile depth of surface loss and texture group Box plots of log mean depth of demineralized dentin tubule involvement by texture viii

11 24. Box plots of log mean depth of surface loss by visual/tactile depth of depth of surface loss Box plots of log mean depth of demineralized dentin tubule involvement by visual/tactile depth of surface loss Root caries classification system proposed by this thesis study ix

12 1 CHAPTER I INTRODUCTION Root caries is a lesion located on the root surface of a tooth, usually close to or below the gingival margin. It is a dynamic process resulting in lesions of various clinical severities and treatment needs (59). The lack of a standard definition of root caries has hindered the search for a description of initial stages of root caries lesions (43). The clinical diagnosis of this type of lesion presents many challenges to dentists. Changes in color and appearance in root caries are much more subtle than in coronal caries (54). There are usually no reported symptoms of root caries. Due to this, the most commonly used clinical method to describe root caries utilizes visual and tactile specifications (11). The gingival margin is a plaque stagnation area; when the gingival margin recedes the cemento-enamel junction (CEJ) becomes exposed and mechanical cleaning is more difficult, resulting in continuous stagnation of plaque. As people age, their gums recede and root surfaces are exposed, making them more susceptible to root caries (23). Older individuals tend to be more vulnerable to root caries due to co-morbidities requiring multiple medications that reduce salivary flow and may result in a dry mouth (84); (78). The best available estimates suggest that from 40% to 86% of people over age of 30 are affected by root caries with the attack rate being highest for people over 50 years of age. Because of the methods of assessment, the true prevalence of root caries may be underestimated by as much as 30%. Severe root caries have been described as perhaps the most frustrating aspect of geriatric dental care (19).

13 2 Although root caries is an increasingly significant clinical problem, the development and validation of reliable indices for the determination of the clinical prevalence and severity of lesions has not been rigorously addressed (17). An evaluation of the literature shows how difficult it has been for researchers to completely agree on a defined common diagnostic criterion to recognize an active root lesion from an inactive one (30); (53); (54); (8); (66); (56); (19); (24). A recent review of 29 caries detection criteria systems concluded that the majority of the current caries detection systems, both for coronal and root caries, were ambiguous and did not measure the disease process at different stages (49). This review also found that while new caries detection criteria measured different stages of the caries process, there were inconsistencies in how the caries process was measured. Additionally, there was a gap between European and American systems for caries detection and inconsistencies among the research criteria for measuring dental caries (49). By and large, especially in the USA, the detection of dental caries has been synonymous with the presence of cavitation (49). Among the research community in Europe, the understanding of dental caries appears to be more detailed than the dichotomous approach used in the United States. In Europe the criteria for measuring caries in that the clinical stages of the disease process which precede cavitation are acknowledged and often recorded (49). A group of caries researchers integrated the best features of other systems and proposed a new system which was named the International Caries Detection and Assessment System (ICDAS) (49).

14 3 ICDAS classifies dental caries by the stage of the carious process. This criteria system varies from prior criteria in that it stages non-cavitated lesions. The ICDAS committee developed a wardrobe concept where users can decide at what stage (non-cavitated or cavitated) and severity they wish to measure dental caries. When using ICDAS, it is recommended that the ICDAS definitions are explicated for whatever stage(s) of dental caries is chosen for the respective study. The ICDAS criterion for assessing root caries has face validity, but it has not been tested for reliability and validity in any laboratory epidemiological or clinical studies. The only stipulation is the requirement that the ICDAS definitions are used for whatever stage of dental caries is chosen for a specific study. Hence, data on the validity and reliability of the ICDAS criteria for detection of root caries are not yet available. However, the corresponding ICDAS system for the detection of dental caries on coronal tooth surfaces has been shown to be reliable (49). Purpose of the Study The purpose of this study was to evaluate the correlation between root caries lesion clinical characteristics, such as color, depth and texture and the histological severity of cementum, dentin and pulpal involvement. Subsequently, the correlation between the ICDAS root caries classification system and the histological involvement was evaluated. This study assessed whether there was an association between visual characterization of root caries and histological severity in 100 extracted human teeth with root caries lesions. Photographs of the carious lesions, while both wet and dry, were obtained in order to simulate a clinical situation. A clinical assessment of the lesion

15 4 characteristics was recorded. Root caries lesions were sectioned based on the size of the lesion in a Hard Tissue Microtome* in order to obtain two to three samples of each lesion. The root caries lesion samples were evaluated under a Polarized Light Microscopy** to measure the level of lesion involvement. * Series 1000 Deluxe Silverstone-Taylor hard-tissue microtome - Scientific Fabrications, Colorado, U.S.A. ** Polarized Light Microscopy Olympus with a digital camera attached BX-50, Japan.

16 5 Statistical analyses were then completed to assess the correlation between the visual caries diagnosis and histological measurements. This thesis reviews different criteria used to classify root caries lesions by clinical observation. An overview of the concept of root caries and the challenges that dentists faced as they try to determine lesion severity was included. The thesis also describes and discusses the development and testing of the new root caries scoring system proposed by ICDAS. Clinical Relevance 1. Evaluating the validity of visual caries diagnosis methods could provide dentists with a better understanding of root caries. This gain in dentist s knowledge could potentially lead to more appropriate treatment selection for patients. 2. This study is one of the first research projects focusing on the assessment of root caries that includes ICDAS root caries criterion. 3. Concluding information may prolong oral health status by providing practitioners with the diagnostic tools to recommend and provide earlier root caries management.

17 6 CHAPTER II LITERATURE REVIEW The following topics are discussed in this chapter: hard tissues (dentin and cementum), root caries, the diagnostic criteria of root caries (lesion depth, texture, color, contour and location) and a relatively new caries classification system called ICDAS (International Caries Detection and Assessment System). Tooth hard tissues The hard tissues of the teeth are enamel, dentin and cementum. Enamel, the hardest of the three, covers the coronal part of the tooth, while cementum covers the cervical part of the tooth. Both the enamel and cementum cover the dentin underneath. Dentin forms the bulk of the tooth substance and gives the basic shape to each tooth; however, the thickness of enamel may vary considerably and modify the shape of the tooth, especially over the cusps (40). Cementum is the thin layer of calcified tissue covering the dentin of the root. It is pale yellow with a dull surface and is softer than dentin. In general, cementum is more permeable than dentin. The relative softness of cementum, combined with its thinness, means that it is readily removed by abrasion when the root surface is exposed to the oral environment (13). Enamel Mature enamel is a crystalline material that is the hardest calcified tissue in the human body. In its mature state, it is noted for its almost total absence of organic matrix. Enamel is not a static tissue because it can undergo mineralization changes. Mature enamel is, by weight, 96% mineralized or inorganic materials, 1% organic material, and 3% water.

18 7 This crystalline formation of mature enamel consists of mainly calcium hydroxyapatite with the chemical formula of Ca 10 (PO 4 ) 6 (OH) 2 (13). Enamel rods or prisms are the crystalline structural unit of enamel. Most enamel rods extend the width of enamel from the dentino-enamel junction (DEJ) to the outer enamel surface. In permanent teeth, the enamel rods near the CEJ tilt slightly toward the root of the tooth. Consequently, each enamel rod is oriented somewhat perpendicular to the DEJ and the outer enamel surface (from less than 90º to 60º). Thus, each enamel rod varies length and direction in different coronal locations (13). Dentin Dentin is the mineralized tissue that forms the bulk of the tooth. In the crown, dentin is covered by enamel, while the root it is covered by cementum. It is a rigid but elastic tissue, consisting of large numbers of small parallel tubules in a mineralized collagen matrix. The tubules contain the long processes of the cells responsible for forming the tissue, the odontoblasts, as well as a small volume of extracellular (dentinal) fluid. The cell bodies of the odontoblasts line the deep surface of the dentin, defining the outer border of the dental pulp (18). Fresh dentin is pale in color and contributes to the appearance of the tooth through the translucent enamel. Dentin is harder than bone and cementum, but softer than enamel. Its organic matrix and tubular architecture provide it with greater compressive, tensile and flexural strength than enamel. Dentin is permeable, depending on the size and patency of the tubules, which will decline with age. The gross composition of dentin approximates to 70% inorganic, 20% organic and 10% water by weight (18).

19 8 The inorganic, mineral component is in the form of calcium hydroxyapatite crystals. The crystallites are calcium poor and carbonate rich in comparison to pure hydroxyapatite and, although similar in shape, are very much smaller than those in enamel. The organic matrix of dentin, in which the crystallites are imbibed, has a composition similar to that of bone. The organic matrix consists of fibrils imbibed in an amorphous ground substance. The fibrils are collagen and comprise over 90% of the organic matrix (18). Dentin includes primary, secondary and tertiary dentin. Based on structure, primary dentin is composed of mantle and circumpulpal dentin. Primary dentin forms the body of the tooth; secondary dentin forms only after tooth eruption and is a narrow band that borders the pulp. Tertiary, or reparative dentin, is formed only in response to trauma to the pulp (6). Dentin is permeated by tubules which run from the pulpal surface towards the DEJ and CEJ. In vivo, the tubules may contain cellular processes, which are derived from cells lining the pulpo-dentinal junction (the odontoblasts). The tubules taper from about 4µm in diameter at their pulpal ends to 1µm or less at their periphery. The surface area of the dentin, near the pulp, is composed of tubules; while near the enamel-dentin junction, the tubules comprise only about 4% of the tissue by volume. The tubules follow a curved, sigmoid course the primary curvatures. The convexity of the primary curvatures nearest the pulp chamber faces rootward. In the root and beneath the cusps, the primary curvatures are less pronounced with the tubules running a straighter course (13).

20 9 E P D C Figure 1. Dentinal tubules seen in a longitudinal section through dentin. E. enamel; D. dentin; P. pulp and; CEJ. cementoenamel junction. (18)

21 10 Cementum Cementum, a hard tissue, is thickest at the apex of the tooth and in the inter-radicular areas of multirooted teeth (50 to 200 µm) and thinnest at the CEJ at the cervix of the tooth (10 to 50 µm). Cementum has no innervations and is avascular, receiving its nutrition by way of its own cells from the surrounding periodontal ligament. Like dentin and pulp, cementum can form throughout the life of the tooth (13). Mature cementum is by weight 65% mineralized or inorganic material, 23% organic material, and 12% water. This crystalline formation of mature cementum consists of mainly calcium hydroxyapatite. In certain situations when cementum is initially exposed, it is a dull, pale yellow color and lighter than dentin, but darker than enamel s whitish shade. When instruments are used, cementum feels grainy due to its higher mineral level compared to the harder, smoother enamel surfaces (13). Cementum is composed of a mineralized fibrous matrix and cells. The fibrous matrix consists of both Sharpey s fibers and intrinsic non-periodontal fibers. Sharpey s fibers are a portion of the collagen fibers from the periodontal ligament that are each partially inserted into the outer part of the cementum at 90º to the cemental surface (as well as the alveolar bone on the other end). These fibers are organized to function as a ligament between the tooth and alveolar bone. The intrinsic non-periodontal ligament fibers of the cementum are collagen fibers made by the cementoblasts and laid down in a non-organized pattern; yet, all these fibers run parallel to the dentino-cementum junction (DCJ) (13). Three patterns of cementum and enamel may be present at the CEJ (12). The classical view was that certain patterns dominated in certain oral cavities. New studies done

22 11 with the scanning electron microscope indicate that the CEJ may exhibit all of these patterns in an individual s oral cavity and that there is considerable variation when one tooth is traced circumferentially. In one pattern, the cementum overlaps the enamel at the CEJ, where the cementum exhibits a uniform placement and roughness with an explorer. In a second pattern, the cementum and enamel may meet end to end. A third pattern at the CEJ is that a gap may exist between the cementum and enamel, exposing dentin. (13). There are two basic types of cementum: acellular and cellular. Acellular cementum consists of the first layers of cementum deposited at the DCJ and thus is also called primary cementum. It is formed at a slow rate and contains no embebbed cementocytes (cementum cells) (13). At least one layer of acellular cementum covers the entire root, and many layers cover one third of the cervical area near the CEJ. The width of acellular cementum never changes. The other type of cementum is the cellular cementum, sometimes called secondary cementum. It consists of the last layers of cementum deposited over the acellular cementum, mainly in the apical third of the root. Cellular cementum is formed at a faster rate than acellular, and thus many embebbed cementocytes are found within it. This type of cementum is common in inter-radicular areas (13). Similar to bone, cementum undergoes histological removal within the tissue as a result of trauma. This removal involves resorption resulting in reversal lines. When stained histologically, reversal lines appear as scalloped lines. Cementum is less readily resorbed than bone because cementum, unlike bone, does not undergo continual remodeling or repair, being an important consideration during orthodontic tooth movement. Repair of traumatic resorption of cementum involves apposition of cementum by cementoblasts in the adjacent

23 12 periodontal ligament. This apposition of cementum is noted by its layers of growth, or arrest lines, which, when stained, look like smooth growth rings in a section of a tree (13). Root caries The root caries process may be defined as a dynamic equilibrium similar to enamel caries. The caries process produces demineralization due to acid on exposed root surfaces and results in a lesion body when viewed by the light microscope. The conversion of active to inactive root caries requires the formation of a mineralized layer, yet the loss of surface tissues and contour remain clinically (94). Recession of the gingival margin is an inevitable result of poor oral hygiene and gradual loss of periodontal attachment with age. Even in populations with regular oral hygiene, some recession occurs and its pattern of distribution within elderly populations is very common (30). In current populations, even adolescents experience some exposure of the cervical root surfaces in several teeth due to inappropriate plaque control procedures (30). Despite the fact that the presence of exposed root surfaces is a pre-requisite for root caries to occur, multiple other factors influence the level of caries risk such as oral hygiene, frequency of consumption of fermentable carbohydrates and salivary aspects such as flow, remineralization, buffer capacity, cleaning properties and defense factors (94); (30). It is occasionally claimed that root surface caries occurs within a deepened periodontal pocket. It is most likely, however, that the periodontal inflammation has been initiated by caries along the gingival margin (32). As a result of gingiva inflammation, the edema and swelling of gingiva have subsequently given rise to the impression that a carious lesion is hidden in the pocket (32).

24 13 The etiology of root caries formation appears to be similar to that of enamel caries (93). A susceptible tooth surface (exposed root), acidogenic microflora and fermentable carbohydrates are all needed to produce caries (93). Although different microflora were thought to be the causative agents of root caries, Mutans Streptococci still play a major role in root caries, as well as enamel caries (59); (15). A. viscosus has been identified as the single most predominant species in root caries plaque (41). In the late 1980 s, the root caries process was considered to be similar to that of the enamel where the surface layer was demineralized, but not cavitated, even though the carious lesion had reached dentin (31). The surface of the root caries lesion is infiltrated by microorganisms very early on in development. Even early on in root caries development, microorganisms may be found in the many exposed dentinal tubules (31). The dentin response is similar to that of coronal caries in that it corresponds to the involved area of the root. The pulpodentinal organ responds with increased mineralization deep within the tissue, resulting in a zone of higher mineral content in the involved areas. Likewise, tertiary, reactive dentin is frequently observed at the pulpal surface of the dentin corresponding to the involved tubules (31). During the root caries process, alternating episodes of demineralization and remineralization (demin-remin process) can occur on a daily basis (93). The demineralization process results from the interaction of host, microflora and diet, while remineralization occurs from salivary calcium and phosphate during periods of neutral ph (93). A much greater proportion of organic material degradation occurs on root than on

25 14 enamel. Therefore, it can be considered that during root caries process an important protein degradation occurs (93). In a study done by Arnold (4), the severity of root caries lesions developed by Gaengler (36) was described. In the Arnold study (4), the authors aimed to investigate the nature of the different dentin zones in root caries lesions and determine the threedimensional extension of these zones and their relation to sound dentin. The criteria given in this study about the depth of lesion, the degree of demineralization and the progression of the lesion of root caries are important information to relate to the histological assessment of the root caries lesions analyzed in this thesis study. According to Gaengler (36) three major categories of root caries lesion can be distinguished: initial lesions, stagnating lesions and progressing lesions. Clinically progressing lesions and stagnating lesions are distinguished by their coloration. Progressing lesions present a soft and yellowish surface, whereas stagnating lesions present a hard and brown to black surface (4); (7). Initial root caries lesions exhibit a translucent dentin directly underneath the surface in which the dentinal tubules are partly occluded by peritubular dentin formation (79); (68). Stagnating dentin lesions differ from progressing dentin lesions mainly by their higher mineral content in the surface layer and the absence of viable bacteria in dentin tubules (4). In a study done by Schupbach (81), three different zones in progressing root caries lesions where distinguished: the superficial layer of bacterial penetration (surface zone), the demineralized dentin and the translucent dentin. In more advanced lesions, the surface zone and the demineralized dentin were not demarcated. Schupbach (81) observed with polarized light microscopy that odontoblasts produced intratubular dentin as a biological reaction to the carious attack, which obliterated the dentin tubules and was the cause for the development of the

26 15 hypermineralized translucent dentin. On the other hand, the study done by Arnold (4) showed that the dentin tubules within the hypermineralized translucent dentin are not completely obliterated by intratubular dentin. It could also be demonstrated that dead tracts are intermittent between hypermineralized dentin (4) Fig. 2. These scatted dead tracts are important because they may be the reason for further progression of the caries lesion due to bacterial invasion (4). The histological features described in the latter study are highly important for the histological evaluation of this thesis study. When measuring the depth of the root caries lesion, the area to be considered will be the demineralized dentin. It is important to be able to identify the different zones of root caries lesions for better accuracy of the measurements.

27 16 Figure 2. Polarized light microscopy picture of a stagnating root caries lesion at the CEJ showing demineralized dentin on the surface, the hypermineralized translucent dentin and dead tracts. (4)

28 17 The aim of a study done by Wefel (92) was to obtain information about the detailed histopathology of naturally occurring root caries. Fifty extracted human teeth presenting some degree of root caries were longitudinally sectioned and examined with transmitted light, polarized light microscopy and microradiography. The polarized light microscope is a microscopy system designed to observe and photograph specimens that are visible primarily due to their optically anisotropic character. Optical anisotropy is the difference in the optical properties of a medium as a function of the direction of propagation of optical radiation (light) in the medium and of the state of polarization of the radiation. Optical anisotropy is seen especially in crystal optics, and is the phenomenon of double refraction. In order to accomplish this task, the microscope must be equipped with a polarizer positioned in the light path somewhere before the specimen, and an analyzer (a second polarizer) placed in the optical pathway between the objective rear aperture and the observation tubes or camera port. Image contrast arises from the interaction of plane-polarized light with a birefringent (or doubly-refracting) specimen to produce two individual wave components that are each polarized in mutually perpendicular planes. The velocities of these components are different and vary with the propagation direction through the specimen. After exiting the specimen, the light components become out of phase, but are recombined with constructive and destructive interference when they pass through the analyzer (64). Microradiography is the process of producing enlarged images of the interior by penetrating thin, usually small specimens with low-energy x-rays. The magnification can be obtained geometrically during the exposure by subsequent enlargement of the initial image through optical or electronic means, or by a combination of both processes. As with radiography on other size scales, microradiography shows the spatial distribution of mass and elemental composition of the

29 18 sample (88). The 50 teeth involved in this study had some teeth exhibiting a spectrum of carious lesions and others that had apparently no root caries. This was to see if the early, small root caries lesions could be identified histologically. Longitudinal sections of root caries, approximately 100µm in thickness were cut for evaluation. Observations were performed using a polarized light microscope for transmitted light and polarized light microscope. For transmitted light photomicrographs, the analyzer was removed from the polarized light microscope. The histological features evaluated were: lesion body, surface zone, dentinal tubular reaction, reparative dentin on pulpal margin and structureless areas (loss of normal histological appearance). The dentinal tubular reaction they observed included dead tracts and/or translucent zones (sclerotic dentin) and was used to describe an altered appearance of the dentinal tubules below or adjacent to the body of the lesion (93). Three imbibition media were used for these evaluations: air, water and quinoline. Subsequently, under microradiographic examination they observed the presence of a radiopaque surface, radiolucent body and the extent of the demineralization of the lesion. Comparing the three root caries histological evaluation techniques, they found that the demarcation between cementum and dentin was more easily seen in polarized light microscope because the cementum was negatively birefringence with respect to the CEJ as opposed to the positively birefringence dentin when imbibed in water (94). Birefringence, or double refraction, is the decomposition of a ray of light into two rays (the ordinary ray and the extraordinary ray) when it passes through certain types of specimens depending on the polarization of the light (64). Under polarized light microscopy, a normal root surface shows a clear demarcation between cementum and underlying dentin since the sign of birefringence are opposite (93). Neither transmitted light nor microradiography could easily

30 19 distinguish cementum from underlying dentin in normal root sections (93). Using microradiography, it was seen that initial lesions were determined by the presence of subsurface demineralization (body of the lesion) which was seen as a radiolucency. Many samples in this study showed a biological response of sclerotic dentin and/or reparative dentin at the pulpal surface. Wefel (93) considered it interesting to note that very small lesions at the outer surface showed large tubular reactions that followed an S shaped course from pulp to outer surface. The use of quinoline as an imbibition media for polarized light microscopy often showed the full extent of the lesion better than the use of water alone. This fluid more clearly defined the extent of the lesion and may show a phenolic reaction when using polarized light microscopy. This chemical phenomenon between quinoline and carious lesion is a reversible process, and has been termed as phenol reaction by Von Ebner (26). This chemical process occurs when the collagen fibers have lost some of the mineral face and the oriented absorption of phenol molecules reverse the sign of birefringence (93). They also identified a translucent area deep to the body of the lesion when using quinoline. This area demarcated by the quinoline often showed a reversal in the sign of birefringence, but was not completely devoid of mineral. This area has been termed the frontal zone, which may be similar to the translucent zone in enamel caries in that it seems to denote the advancing front of the lesion (93). Wefel (93) found that, for more advanced lesions, a loss of surface and cementum was often present. Microradiographically, only 40% of the lesions analyzed showed complete radiopaque surface layers. The presence of this radiopacity may be a sign of an inactive root surface lesion; however, it was not possible to clearly make that distinction in this study. This particular study is very useful because it describes how root sections are visualized with

31 20 different systems, specifically polarized light microscopy. Also, based on this study, it appears that polarized light microscopy has several advantages over transmitted light because in light microscopy the demarcation between cementum and dentin is quite obvious as the tissues have opposite signs of birefringence with respect to the CDJ. In natural root caries lesions, the unsupported collagen swells upon hydration and shrinks when allowed to dry (93). Mineral loss on a root surface is often reflected in the extensive softening when probed, and the root surface tends to be extremely vulnerable to mechanical damage (8). Within one to three months, where a root surface has been covered by plaque, a continuous subsurface loss of mineral occurs while there is a buildup of mineral in the surface layer. This is evidenced by a significantly larger size of the apatite crystals in this zone than in normal cementum (67), and most likely reflects a redeposition of minerals in the surface layer. The bacteria seem to split the collagen fibers of the cementum and often accumulate at the CEJ (67). The progression of root caries has been variously described as tending to spread laterally and sometimes without obvious cavitation, and as varying from no surface defect to pulpal involvement (42). In addition to the progression of microorganisms toward the pulp, a lateral spread of microorganisms has been observed, occurring independently from the side-branches of the dentinal tubules. This lateral spread can lead to intertubular dentin becoming heavily infected with microorganisms. This could explain the saucer shape of root caries (87). Since dentin and cementum contain more organic material than enamel, organic breakdown is likely to occur by the acid dissolution of these tissue minerals, leading to a root surface lesion with cavitation. Root caries lesions have been

32 21 frequently found without any signs of adjacent enamel demineralization. This suggests that the process may occur at ph levels above the enamel critical ph, which is (89). Clarkson (22) designed an in vitro study to investigate the effects of proteolitic enzymes and ph on caries lesion formation in vitro. Twelve caries-free human teeth were sectioned with a hard tissue microtome into nine sections, each approximately 100µm thick. Each section was examined microscopically to insure intact surfaces and sound enamel, cementum and dentin. Then, the sections were painted with a clear acid-resistant varnish leaving exposed areas on the enamel and root surface. The 108 sections of human teeth with exposed areas on enamel and root surface where placed into an enzyme/buffer system maintained at ph levels of 4.0, 5.5 and 7.0. One third of the vials contained sections and buffer only (controls), the other two thirds of the vials contained sections equally divided between collagenase and protease. After 21 days, the sections were imbibed in water and quinoline to be examined under polarized light microscopy. The results showed consistent surface erosion on the root surface only at a ph of 5.5 in the buffer containing enzymes. While at a ph of 4.0, histological observations showed caries-like lesion formation. Sections placed in ph 5.5 buffers only, which served as controls, showed obvious lesion formation on the root surface, but with histologically intact surfaces. This interaction occurring between enzymatic activity and acid demineralization suggested that enzymes may have a contributory effect on caries lesion formation in root surfaces. This study was helpful in understanding caries-like formation in root caries at specific ph s and types of enzymes involved in the process, as well as reading the sections imbibed in water and quinoline with a polarized light microscopy.

33 22 Systems to examine sections of root caries Various techniques have been used to study depth of root caries lesions and their resulting mineral loss. Polarized light microscopy, scanning electron microscopy and microradiography have been commonly used for this purpose. Another method involves projecting the slides photographed from the polarized light microscope on a sonic digitizer, which then measures the area and the depth of the lesion at specific intervals. These methods were productive in first measuring the mineral content in a given area by microradiography and then assessing the ultrastructure with scanning electron microscopy (40). An in vitro study completed by Phanksol (69) used an acidified gel system to produce artificial caries lesions in root surfaces and enamel. The teeth were exposed to the gel system for two weeks, after which the lesions were examined by polarized light microscopy and photographed by standardized techniques. The depths of penetration of the lesions were measured from these standardized photomicrographs. The photographic slides of each specimen were projected onto a screen with a projector at a 5m distance. For each specimen, the distance from the surface to the deepest portion of the lesion was measured with a millimeter ruler and calibrated to a standard scale. The carious lesions in the root surfaces were deeper than those in the enamel for all of the teeth. After two periods of exposure to the gel system (one after 2 weeks and the other one after 4 weeks), the lesions in the root surfaces of anterior teeth were deeper than those in pre-molars. This study shows how long it could take for the different degrees of destruction of the caries process to occur on root surfaces and how they were measured.

34 23 This fact is important to take into account for the depth of penetration; it is a variable that will be used in this research thesis project. Phanksol s study (69) was done prior to the advent of computer measurement systems, so the measurements may not be that accurate due to measuring the depths with the naked eye which then required the use of mathematical equations. Thus, current and future research should be more accurate by making measurements with computer software and noting all the points outlining the area of interest. An in vivo experiment was conducted by Arends (2) using combined methods to examine the ultrastructure of in vivo demineralized dentin in which the mineral content was determined by microradiography prior to scanning electron microscopy observation. An evaluation of the results showed that with in vivo demineralized dentin, the mineral loss occurred in substantial amounts both from the intertubular and peritubular regions of the dentin. The demineralized dentin tubules enlarged by about 30% when compared with sound dentin when viewed under scanning electron microscope. In a study done by Wefel it was (94) noted that one of the drawbacks of microradiography was that the specimens tended to shrink on preparation for SEM due to the desiccation of the specimens resulting in a less pronounced carious lesion. Similarly, studies found that dentin shrinks to an extent of 18% during sample preparation (37).

35 24 Diagnosis of root caries Clinical diagnosis is the process of recognizing diseases by their characteristic signs and symptoms. It is an imperfect process because there is considerable variation both in the signs and symptoms of disease in individual subjects and in the interpretation of those signs and symptoms by different clinicians (11). The dynamic nature of root caries lesions may make its diagnosis difficult (59). There are usually no reported clinical symptoms of root caries, although pain may be present in advanced lesions. The most commonly used clinical signs to describe root caries utilize visual (contour, surface, cavitation, color) and tactile (surface texture) specifications (11). The clinical investigators who first studied root caries provided clinical descriptions of the signs of root caries lesions because there were no reported clinical symptoms of root caries although pain may be present in advanced lesions (42); (87); (44); (9); (52); (89); (16); (65). Table 1. Cavitation or surface loss Some of the following definitions of root caries do not mention cavitation as a necessary factor for presence of root caries while other definitions describe cavitation in association with active and inactive carious lesion. Root caries lesions were defined in the 1970 s as cavitations below the CEJ (88). Later, the definition changed by the statement that this type of lesion can be cavitated (30); (41); (50); (66). Then, cavitation was considered as a sign of active lesions, whether soft or discolored; however, this definition was later modified to suggest that cavitated lesions without softening were inactive (53). Others stated that active root caries were cavitated or softened areas in the root surface (44). In a study of extracted teeth (56), the description of root caries varied from slight surface etching to a

36 25 cavity of 3mm ; only 3% of the lesions had progressed to a depth sufficient to involve the pulp and cavitation was defined as the loss of surface continuity. Table 2 lists author s description of root caries activity according to loss of surface integrity. Texture Primary root caries is currently identified by a diagnosis of change in texture of the root surface. Tactile diagnosis of caries has used probe tug back as a sign of the presence of caries. This has served the clinician well in coronal caries where the caries extends into the dentine (12). Non-carious dentine and cementum are soft calcified tissues and can produce some tug back on the probe in the absence of caries. Assuming that the probing pressure used is the same, the presence of tug back on the root surface is, therefore, more likely to result in a false positive diagnosis of dental caries (12). Nevertheless, texture, is used extensively used by clinicians to aid in the determination of root caries. Because of the fundamental differences in coronal and root caries, coronal caries is more likely to be confidently diagnosed at an earlier stage than root caries using visual/tactile methods (12). Setting aside the argument that probing can hasten the development of caries, the disadvantage of diagnosing root caries using visual/tactile methods is that a larger or more advanced surface defect needs to be present before a positive diagnosis can be made (12). This tactile criterion has been used to classify root lesions according to their activity. Unfortunately, color has not been found to be well correlated with root caries activity and probing pressure can be highly variable (12). It has been stated that inactive lesions may be even harder than the surrounding non-diseased root surface (66). Inactive root caries have been defined as hard (30); (53); (54), while several investigators defined active root caries

37 26 lesions as soft because there was an easy penetration into the dentin with an explorer (42); (88); (52); (90); (16). Similarly, it was stated that active root caries lesion was one in which an explorer point could easily be inserted with moderate finger pressure (44) and that an active lesion was where an explorer could easily enter and display some resistance to withdrawal (9). Furthermore, the term leathery has been used to describe passive and remissive lesions, which are softer than surrounding cementum, but not as soft as active lesions. In addition, texture may be indicative of slowly progressive lesions (8); (24); (30); (53); (54). Table 3 lists the authors and accompanying description of root caries lesion texture. Color Clinical researchers have used various criteria to assess the clinical severity and needed treatment of individual root caries lesions. Color has been used extensively as a diagnostic feature of root caries to determine its severity and carious activity. It was reported in a conference consensus statement that active root lesions are lightly discolored (yellow-brown), while inactive lesions are dark brown to black (29). Also, arrested lesions were defined as being almost black (24). A Root Caries Severity Index was developed by Billings (19) using color for classification of these lesions. According to this Index, the darker the color the more extensive the root caries lesion is. It was also stated in this study that early lesions were light tan to brown, while lesions involving the pulp were brown to dark brown and that inactive lesions could be light brown to black. It is important to note, however, that they did not specify the range of colors for active lesions. Other authors have described active lesions as: yellow to orange (62) yellow to light brown (30) and light to

38 27 dark brown (56). This study will also look at how the color of a root caries lesion can be compared to lesion severity using histological depth. Table 4 lists the authors and description of root caries lesions. An in vivo study done by Lynch (59) aimed to assess the microbiological content of 395 primary root caries lesions classified according to their color in order to better understand the clinical significance of color and its usefulness as an indicator of clinical severity. For each patient, the lesion color was recorded after visual comparison with a standard guide of four shades (yellow, light brown, dark brown and black) prepared from photographs of primary root caries lesions. Cavitation was also recorded as loss or no loss of surface contour (Specification on how contour loss was measured was not provided.) The texture of lesions were classified into three grades: hard where the texture of concern was touched by an Ash No. 6 probe and was as hard as the surrounding sound tooth tissue; leathery when penetrated with the probe using modest pressure it was not easily penetrated and had no resistance to withdrawal; soft easily penetrated by the probe under modest pressure with resistance to withdrawal of the probe. The relationship between the physical dimensions of the lesions and the distance from the gingival margin color, texture and microflora were also investigated. The lesions were sampled using standard, customary pressure by applying an excavator to transverse the whole lesion. Samples from individual lesion were then analyzed by determining the number of colony forming units (CFU) of Actinomyces species, Streptococcus mutans, Streptococcus sobrinus, Lactobacilli and yeast. They found that soft primary root caries lesions yielded significantly more bacteria than hard lesions, while leathery lesions yielded an intermediate number of bacteria. In this

39 28 study, they classified soft, leathery and hard lesions according to their color and there was no indication that lesion color was significantly associated with the total number of bacteria. This suggested that color was not a useful criterion for diagnosing the severity of primary root caries lesions. The purpose of this study was to compare root caries lesions classified according to color, but they ended up classifying lesions in several criteria like texture, cavitation, dimension and distance from the gingival margin. Then they evaluated the microflora of these lesions to determine if there was a relationship between the amount and different types of bacteria and the different types of lesions. The study is relevant, but at the same time the methodology was not well specified, resulting in the mixture of multiple variables. Relationships were poorly described and there was no evidence to make such conclusions. Location There are conflicting views about root lesions in the area of the CEJ. The question is: does caries start on the root or the crown? The location issue is whether or not caries in the area of the CEJ should be classified as root caries extending onto the crown, coronal caries extending onto the root, or both. It is a measurement issue more than a diagnostic issue. Root caries more often occurs supragingivally, at or close to (within 2 mm) the CEJ. This phenomenon has been attributed to the location of the gingival margin at the time conditions were favorable for caries to occur (8); (59). The location of root caries has been positively associated with patient age and gingival recession (12). This is consistent with the concept that root caries occurs in a location adjacent to the crest of the gingival where dental plaque

40 29 accumulates. Root caries occurs predominantly on the proximal (mesial and distal) surfaces, followed by the facial surfaces (10); (12); (82). The Root Caries Index proposed in 1980 (52) stated that all roots displaying gingival recession were at risk; as long as the CEJ was exposed. More recently, however, it has been demonstrated that from 10-20% of root caries can occur subgingivally (20); (58); (87). On the other hand, it was stated that a root caries lesions can occur anywhere on the root surface (42). Further studies (88) described root caries as lesions located on the root surface which may have involved enamel or not. Similarly, it was described that these lesions were located at the CEJ or wholly on the root surface (9). Later, root caries were defined as confined to the root surface with the possibility of undermining the enamel (53). Later, it was stated that more than half of a root caries lesion should be located on the cementum to be considered root caries (90). Table 5 lists the authors and description of location of root caries lesions. Studies determining root caries activity In a study by Ekstrand (28), the development and evaluation of two root caries control programs for home-based frail people older than 75 years were investigated. The purpose of this study was to develop and examine the validity of a system for determining lesion activity on root surfaces and to compare the effectiveness of two preventive programs in controlling root caries in elderly people using this system. This study included an in vivo and in vitro component. The authors developed a root caries activity scoring system based on the literature and the appearance of root caries lesions in 100 extracted teeth. The scoring system consisted of having four clinical characteristics to judge the activity of root caries lesions. The first clinical characteristic was

41 30 texture, divided into hard, leathery and soft categories with the respective scoring numbers of 0, 2 and 3. The second clinical characteristic was the contour of the surface, divided into well-defined and ill-defined contours with a designation of 1 or 2. The third clinical characteristic was distance of the lesion to the gingival margin divided in 1mm from the gingival margin and 1mm from the gingival margin with a respective scoring of 1 or 2. The fourth clinical characteristic was color of the lesion divided into dark brown/black and light brown/yellowish with scoring of 1 or 2 respectively. The teeth were selected in such a manner that the entire range of clinical appearances of root caries lesions was represented within the sample. The individual scores were summed to give a total score. A total score of 3 to 5 characterized lesions as arrested, while a score of 6 to 9 characterized lesions as active. The reproducibility of the devised scoring system was evaluated by training a clinical examiner to use the scoring system and then examine 28 patients according to these scoring criteria. The accuracy of the devised scoring system was completed by making impressions of the full arch in ten of the 28 patients using Lactic Acid Locator Clinpro Cario Diagnosis, 3M ESPE Seefeld, Germany. This material detects lactic acid associated with the demineralization of hard dental tissue and has the potential to be used as construct validity for lesion activity. The scoring system used in this study was unique, but the authors did not explain how they designed and developed the Index. Also, due to the lack of information provided regarding validation, the validity and reliability of the system used is questionable.

42 31 The clinical component of this trial included dividing the patients into three groups. The first group had their teeth brushed once a month by a dental hygienist with 1450 ppm fluoride toothpaste. The second group used 5000 ppm fluoride toothpaste at home twice a day. The third group used 1450 ppm fluoride toothpaste at home twice a day. At the baseline appointment patients completed forms about social background and medical history. They also had a clinical examination including a saliva test. The authors did not mention the specific kind of saliva test used. The presence of buccal plaque was scored on five selected teeth. The dental status of the teeth was classified according to the following system: 1) teeth present 2) retained roots 3) bridge extension. The caries status of the root surfaces of the teeth present were classified according to the following system: 0 - sound; 1 - primary caries; 2 restored with plastic material; 3 same as 2, but with caries; 4 crowned and no caries; 5 same as 4, but with caries; 6 could not be recorded (attrition, etc). Activity of the root caries lesions were characterized by means of the devised scoring system. After eight months, they again evaluated changes in the teeth using the Clinpro impression material. The results showed an Intra-examiner reproducibility of the root caries scoring system of 0.86 (Kappa). The sensitivity and specificity was 0.86 and An evaluation of the data from 88% of the total patients who completed the study disclosed that there were no inter-group differences at the baseline examination concerning relevant conditions. At the end of the study, the root caries status of participants in groups one and two had improved significantly when compared with group three. No significant differences were observed between groups one and two. The reliability of their scoring system to determine root caries activity was questioned because color and location were clinical characteristics that had not been proven to be in direct relationship with root caries activity.

43 32 For example: in the case of having a root caries lesion that was leathery (2 points), welldefined contour (1 point), 1mm from the gingival margin (1 point) and light brown/yellowish in color (2 points) would result in a total score of 6 points; meaning that the root caries lesion was active. However, this is not absolutely true since all the characteristics led to an arrested lesion except for color. Thus, this specific clinical characteristic has not been proven to be in correlation with root caries activity. In addition, the impression material to detect lactic acid and the activity of root caries had not been fully validated.

44 33 Table 1. Recompilation of clinical characteristics of root caries lesions provided by the first investigators.

45 34 CAVITATION Table 2. Authors that described root caries activity according to loss of surface integrity.

46 35 TEXTURE Table 3. Authors that described root caries activity according to texture.

47 36 COLOR Table 4. Authors that described root caries activity according to color.

48 37 LOCATION Table 5. Authors that described location of root caries lesions.

49 38 Diagnostic tests The use of specific tests can enhance the diagnosis and prediction of root caries, but the development of a useful test is hindered by the lack of an accurate clinical standard of diagnosis. A powerful predictive tool for root caries would likely result from the combination of risk assessment measures and a valid diagnostic test developed with the use of standardized and accurate methods of clinical diagnosis. Explorer probing of root caries lesions can influence the remineralization of the lesion. In an in vitro study performed by Warren (91), root surface lesions were created on ten extracted teeth by exposing them to a demineralizing solution for 21 days. One side of each lesion was then randomly selected and probed with an explorer, while the other side was not probed. The teeth were then suspended in a remineralized solution containing ten parts per million (ppm) fluorides. The solution was changed in three to four day intervals and the teeth remained in the solutions for 21 days to assure a high level of remineralization. Sites of remineralization were seen, but defects remained in the probed teeth. This pilot study suggests that probing of root surfaces may create defects that do not fully remineralize. This study is useful because it shows that too much pressure when probing can alter the tissue of the lesion and may also alter the measurement and characterization of the root surfaces. International Caries Detection and Assessment System (ICDAS) Over the last several decades, a number of measurement criteria have been developed to identify the presence of enamel and dentin caries. As the understanding of

50 39 dental caries progressed, the clinical criteria systems focused on the assessment of the disease process at only one stage: the decayed stage. Several investigators reviewed 29 caries detection systems and concluded that the majority of the current caries detection systems do not measure the disease process at its different stages (47). In 2002, a group of caries researchers, epidemiologists and restorative dentists met to determine the different definitions of caries status (72). There was a need to detect dental caries at the non-cavitated stages (72). This group selected a foundation for a new system, based on the work of Ekstrand (27), where their purpose was to integrate the best features of other systems, (71); (45); (34); (46); (75); (21) forming a new system which was named the International Detection and Assessment System (ICDAS). An ICDAS committee developed a standardized system based on the best evidence available. This led to better decisions about diagnosis, prognosis and clinical management of dental caries at the individual and public health levels (72). This system was designed to detect six stages of the carious process for coronal caries, ranging from the early clinically visible changes in enamel caused by carious demineralization to extensive cavitation; creating another system to classify two stages (code 1 and code 2) of root caries. There is also code 0, which means non-carious. The current version of ICDAS does not yet include an assessment of lesion activity. One important goal for ICDAS is to provide flexibility for clinicians and researchers to choose the stage of caries process and other features that fit the needs of their research or practice. Hence, the ICDAS developed the wardrobe concept where the users can decide at what stage (non-cavitated or cavitated) and severity they wish to measure dental caries. The ICDAS criteria for assessing root caries has face validity, but have not yet been tested in any epidemiological or clinical studies (47).

51 40 The system to detect and classify root caries lesions consists of assigning one score per root surface: the facial, mesial, distal and lingual surfaces. Code E - if the root surface cannot be visualized directly, and is therefore excluded. Surfaces covered entirely by calculus can be excluded or, preferably, the calculus can be removed prior to determining the status of the surface. Removal of calculus is recommended for clinical trials and longitudinal studies. Code 0 the root surface does not exhibit any unusual discoloration that distinguishes it from the surrounding or adjacent root areas, nor does it exhibit a surface defect either at the cement-enamel junction or wholly on the root surface. The root surface has a natural anatomical contour or, in the case of the root surface, may exhibit a definite loss of surface continuity or anatomical contour that is not consistent with the dental caries process. This loss of surface integrity usually is associated with dietary influences or habits such as abrasion or erosion. These conditions usually occur on the facial surface. These areas typically are smooth, shiny and hard. Abrasion is characterized by a clearly defined outline with a sharp border, whereas erosion has a more diffuse border. Neither condition shows discoloration. Code 1 there is a clearly demarcated area on the root surface or at the cement-enamel junction that is discolored (light/dark brown, black), but there is no cavitation (loss of anatomical contour 0.5 mm) present. Code 2 there is a clearly demarcated area on the root surface or at the cement-enamel junction that is discolored (light/dark brown, black), but there is cavitation (loss of anatomical contour 0.5 mm) present (46). Figure 2 shows a flow chart of this system. The results of this thesis project will be compared to the criteria set forth by ICDAS.

52 41 ICDAS ROOT CARIES CLASSIFICATION Can the root surface be visualized directly? NO / E YES After 5 sec of drying, is there a color change present? (discoloration=light/dark brown, black) NO No caries/0 YES Is cavitation present? (loss of anatomical contour 0.5 mm) NO / 1 Non cavitated root caries YES / 2 Cavitated root caries Figure 3. ICDAS Root Caries Classification System. (44)

53 42 CHAPTER III MATERIALS AND METHODS Overview of Methods An in vitro study was performed to evaluate the correlation between specific visual and tactile characteristics used for detecting root caries (surface loss, texture, and color) and the histological assessment of root caries lesion depth (based on cementum, dentin and pulpal involvement). This study was limited to root surfaces lesions located at the cementoenamel junction (CEJ). One hundred extracted teeth with root caries lesions were visually and tactilely evaluated and classified according to: color (light, medium, or dark), texture (hard, leathery, or soft), ; and surface loss ( <0.5mm or 0.5mm). After visual clinical assessment, the root caries lesions were sectioned with a hard-tissue microtome* obtaining two to three samples from each lesion (center of the lesion, right and/or left of the lesion). Samples were then photographed using a polarized light microscope**. The photographs were evaluated and measured for lesion surface loss and depth of demineralized dentin tubule involvement using computer software***. The resulting measurements from the two to three samples were averaged into a mean values for surface loss and depth of demineralized dentin tubule involvement. Both descriptive and analytical statistics were completed. * Series 1000 Deluxe Silverstone-Taylor hard-tissue microtome - Scientific Fabrications, Colorado, U.S.A. ** Polarized Light Microscopy Olympus with a digital camera attached BX-50, Japan *** Imagining Software Version Image Pro-Plus, Maryland, U.S.A.

54 43 Research questions This study addressed the following general questions: 1) Are the specific visual and tactile characteristics used for detecting root caries: surface loss, texture, and color correlated with histological depth of root caries lesions? 2) Are the specific visual and tactile characteristics used for detecting root caries (surface loss, texture, and color) and histological depth of root caries lesions related to ICDAS root caries classification? This study was carried out in 2 sections, the calibration phase and the postcalibration phase. The purpose of having a calibration phase was to obtain adequate intra and inter-examiner calibration for the visual/tactile assessment, to determine the most meaningful visual/tactile characteristics (among texture, color, surface loss, lesion location in relationship to the CEJ, and lesion contour), and to determine the overall sample size of the study. Calibration phase Selection and preparation of the teeth From anonymous donors fifty extracted teeth with carious root surface lesions were randomly selected. Most of the lesions were small to medium in size. Large lesions that most likely extended to the pulp and would be in need for root canal treatment were excluded. Root caries lesions were located on the facial, lingual, or proximal surfaces of the tooth. Both maxillary and mandibular teeth were included.

55 44 Prior to the visual/tactile evaluation bacterial stains and calculus were removed from the extracted teeth with an ultrasonic system. This was followed by washing the teeth with soap and water. With visualization the area of the root caries lesion that appeared to be the most severe was located and selected as the area of interest. This area outlined and marked vertically with a thin black permanent marker to delineate exactly the area that should be ideally obtained through sectioning with the hard-tissue microtome blade. Teeth were stored in double-de-ionized/distilled water containing thymol. Then, photographs of each lesion were taken wet and dry to replicate clinically when the lesion is covered with saliva and then viewed after air drying. All photographs were taken with a fixed magnification of 1:1 and an aperture of 57, this was achieved with a clinical photographic camera* with manual adjustment. For photographs, each tooth was placed on a professional grey background being stabilized by a piece of wax** (Fig. 4). *Nikon D80 digital camara 10.2-MP, Japan **Wax square ropes/white Heraceus Kulzer, Inc., New York, U.S.A.

56 45 Visual/tactile assessment of root caries lesions The visual/tactile assessment was completed jointly by 2 examiners twice. The second assessment was completed approximately one week after the first assessment. For these assessments, teeth were removed from their storage container, and then gently dried with a paper towel. If necessary, to avoid complete desiccation a moist microbrush* (with water) was used to rehydrate the lesion. Each root caries lesion was assessed in an area with white ceiling lights and an additional lamp with an incandescent bulb was used. The 50 teeth with root caries lesions were then classified according to the visual/tactile characteristics; color, surface loss, texture, location and contour. Color was divided into 3 groups: light (yellow, orange and tan), medium (light brown and brown) and dark (dark brown and black) (Fig.5). Surface loss was defined as the loss of surface integrity of the lesion and was measured with a periodontal probe** (Fig. 6) and classified as either: lesion with a depth less than 0.5mm and lesion with a depth equal to or greater than 0.5mm. *Flexible disposable applicator Kerr, Los Angeles, California, U.S.A. **Fox Probe, Starlite Mfg. Co., USA

57 46 Texture was classified using a thymozin PFI-3 burnisher* (Fig. 7) with a width of approximately 0.5 mm. The use of this burnisher is compatible to using a ball ended probe. Poking the dentin with a sharp probe was avoided for it could easily puncture the dentin causing irreversible damage that may lead the examiner to overestimate the depth and tactile feel of the lesion. Using a ball ended instrument allowed for increased tactile sensation as the instrument was dragged across the lesion. Texture was classified into one of three grades: hard (lesion was as hard as the surrounding tooth tissue), leathery (lesion was not as hard as the surrounding sound tissue and there was slight resistance as the burnisher was dragged across the lesion, but the tip of the burnisher did not fully penetrate the surface of the lesion) or soft (lesion was easily penetrated by the burnisher under modest pressure with no resistance to withdrawal of the instrument). Lesion location was identified as either at the CEJ or apical to the CEJ. Lesion contour was divided into those lesions that had a well defined and delineated contour and those with a rough, irregular, ill defined contour. After the 50 teeth were assessed and classified twice (the two visual/tactile assessments were done by two examiners together: J.K and C.M, with the purpose of calibration between the two examiners) into the 5 categories mentioned above the agreement of the two assessments were evaluated statistically. * Thymozin Instrument No. 3, restorative instrument Lustra, DENTSPLY. York, PA. USA

58 47 A B Figure 4. Photographs taken of root caries lesions a) Wet - b) Dry.

59 48 A B C Figure 5. Samples of color classification: A light; B medium; C dark

60 49 Figure 6. Periodontal probe used to measure the clinical depth of surface loss of the root caries lesions. Figure 7. Dental instrument used to test the texture of the root caries lesions. Ball tip Thymozin #3. Dentsply, USA.

61 50 Dual assessments were completed on 50 root surface lesions and the following characteristics were recorded: location, color, texture, contour and depth. Descriptive statistics were used to explain the agreement between the two assessments of the teeth. The percent of exact agreement was calculated for each characteristic and for all characteristics combined. Also the level of agreement beyond that due to chance was measured using the simple kappa coefficient measuring exact agreement. For characteristics with more than 2 ordinal levels, a weighted kappa coefficient using Cicchetti-Allison weights was calculated. For both the simple and weighted kappa coefficients, the value of zero corresponds to the case of no agreement beyond that expected by chance. Kappa values below 0.40 are considered to reflect poor agreement beyond that expected by chance alone, values of are interpreted as representing fair to good agreement beyond chance, and values above 0.75 represent excellent agreement (34). All analysis utilized SAS 9.2 statistical software and the Type I error was set at =0.05. Cross-tabulations of the first and second assessments for the visual/tactile characteristics color, texture, contour and depth of surface loss were done to determine discrepancies in the combination of the visual/tactile characteristics. Based on the results of the calibration phase (described in detail in the Results section) it was decided to remove both the location and contour characteristics. The characteristics: texture, depth of surface loss and color remained in the study. The target total sample size was set at 100 teeth; this decision was made based on the period of time available to perform this in vitro study. Additionally this sample size seemed to be reasonable since there were no other published studies with similar aims. Assuming

62 51 that approximately 30 teeth would be lost due to complications during sectioning a total of 130 teeth was selected. Since 50 teeth were selected in the calibration phase an additional 80 teeth were then selected in the post-calibration phase. The 50 teeth in the calibration phase where categorized based on the combination of surface lose, texture, and color as shown in Figure 8. To ensure having lesions in the full range of possible combinations of visual/tactile characteristics the 80 teeth selected in the post-calibration phase were systematically selected based on the combination of characteristics absent or limited in number in the calibration phase.

63 52 Dark n=0 Soft n=1 Medium n=1 Light n=0 Dark n=4 <0.5mm n=28 Leathery n=21 Medium n=9 Light n=8 Dark n=1 Hard n=6 Medium n=1 Light n=4 All Teeth n=45 Dark n=4 Soft n=12 Medium n=2 Light n=6 Dark n=3 0.5mm n=17 Leathery n=5 Medium n=2 Light n=0 Dark n=0 Hard n=0 Medium n=0 Light n=0 Figure 8. Teeth by depth of surface loss, texture and color (2 nd Assessment).

64 53 Post-calibration phase Selection and preparation of the teeth From anonymous donors eighty extracted teeth with carious root surface lesions at the CEJ were selected. Similar to the calibration phase, most of the lesions were small to medium in size. Large lesions that most likely extended to the pulp and would be in need for root canal treatment were excluded. Root caries lesions were located on the facial, lingual, or proximal surfaces of the tooth. Both maxillary and mandibular teeth were included. Similar to the calibration phase, the teeth were cleaned; the area of the lesion with the greatest carious involvement was marked; photographs were made and the teeth were stored in double distilled water with thymol. Visual/tactile assessment of root caries lesions Visual tactile/assessment was performed in the same way as it was done for the calibration phase; but this time a triple assessment was completed for the 80 teeth. The first assessment was independently completed by examiner C.M. Approximately one week later the second assessment was completed independently by examiner C.M. for intra-rater reliability testing. The third assessment was independently completed by examiner J.K. for inter-rater reliability testing when compared to the first assessment by C.M. Visual/tactile characteristics considered for these 80 teeth were texture, color and depth of surface loss. The classification that was used in the calibration phase was also used in the post-calibration phase: for texture (hard, leathery, soft), for color (light, medium, dark), and for depth of

65 54 surface loss (<0.5mm or 0.5mm). The results of each of the three assessments were saved on Excel Microsoft Office files for statistical analysis. Preparation of the histological specimens In order to facilitate sectioning lesions with a hard-tissue microtome the coronal part of the teeth were removed with a dental model trimmer*. The teeth were then mounted in a specially designed metal mandrel using dental sticky wax** (Fig. 9). The mandrel was attached to the microtome and each root caries lesion was then sectioned with a hard-tissue microtome (Fig. 10 and 11) obtaining three to five sections of approximately 100 µm thickness from each lesion. Tooth sectioning was done locating the microtome blade left to the most severe area of the root caries lesion, which was previously marked with a permanent marker, and from that location the microtome blade sectioned the lesion from left to right obtaining the 3 most representative sections of each root carious lesion. These 3 sections were labeled as: C - the section obtained from the center of the lesion; L - the section obtained from the left side of the center sample and; R - the section located on the right of the center sample. For most of the root caries lesions, it was possible to obtain 3 sections but in some cases the third specimen was lost, damaged or the lesion was too small to obtain 3 specimens, thus in those cases only 2 specimens from each lesion were obtained. * Dental model trimmer - Whip Mix Corporation, Kentucky, U.S.A. ** Regular Sticky wax - Whip Mix Corporation, Kentucky, U.S.A.

66 55 Specimens were carefully removed from teeth with a blade and placed in lab specimen containers imbibed in distilled water. The thickness of each section was measured using a digital micrometer* and if there was a need to decrease the thickness of the specimen it was done by rubbing the specimen against a sand paper** in a figure 8 shape movement until the digital micrometer read a number around 100 µm. * Digital micrometer Moore & Wright, England. ** Sandpaper grit M Imperial Wetordry, USA.

67 56 Figure 9. Teeth were mounted in special mandrels to be sectioned in a Hard-Tissue Microtome. Figure 10. A Silverstone-Taylor Hard Tissue Microtome, used to create sections of the lesion.

68 57 Figure 11. Root caries lesion after being sectioned in the hard-tissue microtome. The area demarcated in yellow shows the 3 samples taken for histological assessment.

69 58 Histological evaluation Eight teeth from the calibration phase and 17 from the post-calibration phase were excluded from histological evaluation due to suffering damage during sectioning or the histological photographs were unclear, making accurate measurements very difficult. One hundred teeth were histologically evaluated. Each section imbibed in water was evaluated (Fig. 12) with a polarized light microscopy and photographed using digital photography (Fig. 13). Microscope photographs were taken under a magnification of 2X and 4X, obtaining 2 photographs per section (Fig. 14). The 2 to 3 most representative microscopic photographs from each section were used for the measurements, either the 2X or 4X photograph. The measurements included the depth of surface loss and depth of demineralized dentin tubule involvement in the carious process. Measurements were made using computer soft-ware (Fig. 15). For the measurements first a straight line was created which represented the original shape and location of the root (cementum). This was done by drawing a line (electronically using the mouse) from the sound cementum coronally to the sound enamel or the estimation of the prior location of sound enamel. Then one dot was electronically placed at the deepest point of the cavitation. The computer software then calculated the perpendicular distance between the straight line and the dot obtaining an estimation of the depth of surface loss in millimeters. In a similar manner, the deepest point of demineralized dentin tubule involvement was electronically indicated with a dot. Once again the computer software calculated the perpendicular distance between the initial straight line and the dot, obtaining an estimated depth of demineralized dentin tubule involvement in millimeters.

70 59 Under polarized light microscopy this demineralized dentin tubule involvement (the body of the lesion) was determined by noticing differences in color and texture of the area below the cavitation, it appeared to be darker than the surrounding dental tissue. The course of the dentinal tubules appeared to be disrupted at the deepest aspect of the lesion and less prominent within the body of the lesion (Fig. 16). The demarcation between cementum and dentin was quite obvious as the tissues had opposite signs of birefringence with respect to the CDJ (cement-dentinal junction). Thus, the contrast between the 2 tissues allowed for detection and classification (92). All lesions with unclear histological appearance for measurements were excluded. When the demineralized dentin tubule involvement (body of the lesion) and the sclerotic dentin blended with each other making the measurement of the depth of demineralized dentin tubule involvement very difficult or impossible, the lesion was excluded from the study. Also, lesions were excluded when the microscopic pictures displayed cracks and pieces of loose dentin tissue all over the cavitation of the lesion making measurements difficult. The resulting measurements from a total of 100 lesions/teeth were used for statistical analysis.

71 60 Intra-examiner reliability of histological measurements The examiner C.M. had histological measurement training by looking at 80% of all microscopic pictures several times with a trained researcher. During training the depth of surface loss and the depth of demineralized dentin tubule involvement were repeatedly identified. This training helped in recognizing the distinction of the body of the lesion from the sclerotic dentin. After the histological evaluation of the 100 lesions approximately one week later examiner C.M. performed a second histological measurement for intra-examiner reliability testing. The second set of measurements was recorded on 50 teeth by selecting all of the lesions labeled with odd numbers (Fig. 17). All measurement results were recorded on a Microsoft Office Excel file to be analyzed statistically.

72 61 Figure 12. Root caries lesion sample imbibed in water being assessed under the Polarized Light Microscopy.

73 62 Figure 13. Polarized Light Microscopy Olympus with a digital camera attached BX-50, Japan.

74 63 L C R Figure 14. Sample of the 3 specimens obtained after sectioning and their histological assessment.

75 64 Figure 15. This image shows the computer software Pro-Light Plus before determining the points for measurements.

76 65 Figure 16. This image shows the computer software Pro-Light Plus after the software determined the depth of surface loss and dentin tubule involvement.

77 66 FIRST MEASUREMENT SECOND MEASUREMENT A LEFT 2X A 1 LEFT 2X Intra-rater testing B CENTER 2X B 1 CENTER 2X Intra-rater testing C RIGHT 2X C 1 RIGHT 2X Intra-rater testing Figure 17. Sample of the histological measurement of a root caries lesion. A, B, C and A1, B1, C1 show measurements done for the first and second measurement record.

78 67 Visual/tactile depth of surface loss classified according to ICDAS Root Caries Classification System Teeth were classified using the International Caries Detection and Assessment System (ICDAS) for root caries. An ICDAS Code = 1 is defined as a clearly demarcated area on the root surface or at the cemento-enamel junction (CEJ) that is discoloured (light/dark brown, black) but there is no cavitation (loss of anatomical contour < 0.5 mm) present. An ICDAS Code = 2 is defined as a clearly demarcated area on the root surface or at the cemento-enamel junction (CEJ) that is discoloured (light/dark brown, black) and there is cavitation (loss of anatomical contour 0.5 mm) present. The visual/tactile depth of surface loss recorded during the calibration and post-calibration phases were used to classify all 100 lesions according to the ICDAS criteria. Then, ICDAS codes were analyzed statistically to compare their relationship with histological depth of surface loss and depth of demineralized dentin tubule involvement.

79 68 Statistical analysis Statistical analysis All analyses utilized SAS Enterprise Guide 4.2 statistical software. The level of statistical significance specified was Reliability testing 1. Visual/tactile assessment for the post-calibration phase For the post-calibration visual/tactile assessment 63 teeth were analyzed statistically. Both intra- and inter- examiner reliability testing was performed using kappa statistics The level of exact agreement beyond that due to chance was measured using the simple kappa coefficient. For ordinal visual/tactile characteristics with more than two levels (texture and color), a weighted kappa coefficient was also calculated using Cicchetti-Allison weights (33). For both the simple and weighted kappa coefficients, the value of zero corresponds to the case of no agreement beyond that expected by chance. Kappa values below 0.40 are considered as reflecting poor agreement beyond that expected by chance alone, values of as reflecting moderate agreement beyond chance, and values above 0.75 are considered to represent excellent agreement beyond that expected by chance (33). 2. Histological assessment Reliability testing for the histological measurement utilized the intraclass correlation coefficient (85) for mean depth of surface loss and the nonparametric Rothery intraclass

80 69 correlation for mean depth of demineralized dentin tubule involvement (76), since the latter measure did not conform to distributional requirements of the parametric intraclass correlation procedure. Correlation between the visual/tactile criteria of the lesions and their histological evaluation. A linear regression model including three fixed effects was used to evaluate the relationship of mean of depth of surface loss and mean depth of demineralized dentin tubule involvement with factors defined by the visual/tactile characteristics color, texture and depth of surface loss. All first and second-order interactions among these 3 factors were assessed. The rarity of some combinations of lesion characteristics resulted in unequal cell sizes and empty cells; therefore a general linear modeling approach was used for this study rather than three-way ANOVA. Appropriate model diagnostic procedures were used to assess the validity of model assumptions. As a result, only main effects were included in the final ANOVA models. Therefore, the Tukey-Kramer method was used in conjunction with an overall 0.05 level of significance to adjust for multiple pairwise comparisons when comparing specific cell mean depth of surface loss and mean depth of demineralized dentin tubule involvement between different levels of the three factors of interest.

81 70 ICDAS Root Caries Classification System with depth of surface loss and depth of demineralized dentin tubule involvement. A Student s t-test was used to determine whether the two groups defined by the ICDAS Root Caries Classification System differed with respect to the means of natural log transformed mean depth of surface loss and of depth of demineralized dentin tubule involvement depth.

82 71 CHAPTER IV RESULTS Introduction The findings of these 2 phases (the calibration and the post-calibration phases) of the study are presented in this chapter. In the calibration phase the results of the statistical analysis from the intra and inter-examiner calibration for visual/tactile assessment are presented. For the post-calibration phase the results are presented in 3 sections: 1) the results of the reliability testing on the visual/tactile and histological assessments; 2) the results of the visual/tactile characteristics significance on the histological appearance of the root caries lesions and; 3) the results of the relationship found between ICDAS root caries classification system with the histological assessment. Calibration phase Intra/inter-examiner calibration for visual/tactile assessment The percentage of exact agreement varied by visual/tactile criterion and ranged from 62.2% for contour and 95.6% for texture. All 4 visual/tactile criteria agreed on 44.4% of teeth. Conversely stated, for 55.6% of teeth at least one criterion differed between the 2 assessments (Table 6). The kappa values ranged from 0.24 (slight agreement) for contour to 0.92 (excellent agreement) for texture. Simple kappa coefficients were significantly greater than zero for all visual/tactile characteristics except for contour (Table 7). For color and texture, which were scored on an ordinal scale, a weighted kappa coefficient was also calculated (Table 8). The weighted kappa coefficient for color and

83 72 texture were 0.74 (fair to good agreement) and 0.93 (excellent agreement) respectively. 95% confidence intervals were given for the weighted kappa values. Agreement was highest, with kappa tests indicating excellent agreement, for the criterion texture and lowest for the criterion contour, where only slight agreement was observed. Color and depth of surface loss showed moderate agreement between the first and second assessments. Location and contour characteristics were removed from the study. Location was removed because out of 50 teeth, there were only 4 in which the location was classified as apical to CEJ for the first assessment and 5 based on the second assessment. Due to finding a distinct minority of root caries lesion with such location this criterion was removed, and the remaining 45 teeth were considered in the study. The characteristic contour was also removed from consideration due to the poor inter-examiner agreement. The characteristics: texture, depth of surface loss and color remained in the study.

84 Table 6: Exact agreement between 2 assessments (n=45 teeth). 73

85 74 Table 7: Exact agreement between the 2 assessments (n=45 teeth) based upon (unweighted) kappa.

86 75 Table 8: Exact agreement between the 2 assessments (n=45 teeth) based upon weighted kappa.

87 76 Post-calibration phase Following the initial calibration phase and the selection of the visual/tactile criteria of color, texture and surface loss to be considered in this study based on the statistical analysis of the calibration phase. From those 45 teeth belonging to the calibration phase, 8 teeth had to be excluded from the study due to damage, loss or not so clear histological pictures. Therefore, there were 37 teeth from the calibration phase remaining in the study. Since the sample size of the study was agreed to be 100 teeth, an additional 80 teeth were selected, the amount necessary to complete a total of 100 teeth for the study and some other extras to replace the teeth that had to be dismissed for some reason. All these additional 80 teeth presented small to medium size lesions, were located at the CEJ and were carefully chosen to ensure coverage of the full range of possible combinations of clinical characteristics. The visual/tactile assessment of these 80 teeth was done three times. Two assessments were done by the examiner C.M with a period of one week between assessments for intra-examiner reliability testing; and another visual/tactile assessment was done by the examiner J.K within the same month for inter reliability testing. After teeth sectioning and histological assessment of these 80 teeth, 17 teeth had to be excluded from the study due to microtome damage, loss of samples or not so good histological pictures. Thus, only the best 63 root caries lesion samples from those initial 80 teeth were kept in the study to complete a sample size of 100 teeth. Final sample size consisted in 37 teeth from the calibration phase and 63 teeth from the post-calibration phase Figure 18.

88 77 Figure 18. Final sample size consisted in 37 teeth from the calibration phase and 63 teeth from the post-calibration phase.

89 78 Section 1 Reliability of the visual/tactile assessment Intra-rater agreement The percentage of exact agreement between the 2 visual/tactile assessments of 63 teeth done by the same rater varied by criterion and ranged from 79.4% for texture to 87.3% for depth of surfaces loss. All 3 clinical criteria (color, texture, and surface loss depth) agreed on 65.08% of teeth. Conversely stated, for 34.92% of teeth at least one criterion differed between the two assessments (Table 9). The simple kappa coefficients were significantly greater than zero for all attributes. Table 10 displays the simple kappa coefficient measuring agreement adjusted for chance for each clinical characteristic. The kappa values ranged from 0.67 for texture and 0.73 for depth (good agreement) to 0.78 (excellent agreement) for color. For color and texture, a weighted kappa coefficient was calculated (Table 11). The weighted kappa coefficient gives additional credit to close but not exact agreement. For color and texture, the weighted kappa values were 0.82 (excellent) and 0.72 (good) agreements respectively. 95% confidence intervals were given for the weighted kappa values.

90 79 Table 9. Intra-rater exact agreement between 2 visual/tactile assessments (n=63 teeth).

91 80 Table 10: Simple kappa agreement between 2 Intra-rater visual/tactile assessments (n=63 teeth).

92 81 Table 11: Weighted kappa agreement between 2 Intra-rater visual/tactile assessments (n=63 teeth).

93 82 Inter-rater agreement The percentage of exact agreement between the 2 separate visual/tactile assessments done by the examiners C.M and J.K varied by criterion and ranged from 82.5% for color to 90.5% for depth. All 3 criteria (color, texture and surface loss depth) agreed on 68.3% of teeth. On the other hand, for about one-third of teeth, at least one clinical criterion differed between the 2 visual/tactile assessments (Table 12). The simple kappa coefficients, measuring the agreement of the two separate raters beyond that expected by chance, ranged from 0.73 and 0.80 (excellent) for color and depth respectively to 0.83 (excellent) for texture (Table 13). The weighted kappa coefficients ranged from 0.78 (good) for color and 0.86 (excellent) for texture (Table 14). Intra and inter-rater reliability for the visual/tactile criteria is shown in Figure 19.

94 83 Table 12: Inter-rater exact agreement between 2 visual/tactile assessments (n=63 teeth).

95 84 Table 13: Simple Kappa agreement between 2 Inter-rater visual/tactile assessments (n=63 teeth).

96 85 Table 14. Weighted kappa agreement between 2 Inter-rater visual/tactile assessments (n=63 teeth).

97 86 Figure 19. Comparison of intra and inter rater simple kappa values.

98 87 Reliability of the histological measurements The intraclass correlation for the log transformed mean depth of surface loss measures was r= (excellent agreement) and highly significant (p<0.0001). Because a suitable normalizing transformation for the mean depth of demineralized dentin tubule involvement could not be identified, the Rothery nonparamentric intraclass correlation was used. The Rothery correlation for the mean depth of demineralized dentin tubule involvement was r= (p<0.0001) indicating excellent intra-rater reliability for the histological measures. The median difference (first minus second measure) in the mean surface loss depth was mm while the median difference in the mean demineralized dentin tubule involvement was mm. Neither measure was significantly different from zero using the Wilcoxon signed rank test, indicating no systematic difference between the first and second measures taken by the same examiner (C.M). Overall reliability results Agreement overall for visual/tactile assessment and histological measurement reliability test was good or excellent in this post-calibration phase. For the visual/tactile criteria (color, texture and depth of surface loss), the intra-rater agreement was highest on color while the inter-rater agreement was highest for texture. For the histological measurement of surface loss depth the intraclass correlation was also excellent, both above the 0.90 level. More variation existed in the measurement of depth of demineralized dentin tubule involvement than the depth of surface loss; however there was no evidence that the

99 88 median differences in either measures differed from zero, indicating that there was no evidence of systematic differences between the first and second measurements. In summary, all measures, visual/tactile and histological appear to have good to excellent agreement. Section 2 Visual/tactile characteristics in combination with histological findings Table 12 describes all the group combinations of visual/tactile criteria (depth of surface loss, texture and color) from those 100 teeth included in the study compared with their histological mean depth of surface loss and mean depth of demineralized dentin tubule involvement. Means histological depth of surface loss and depth of demineralized dentin tubule involvement were transformed to natural log to normalize data. It was observed that the largest mean depth of surface loss and mean depth of demineralized dentin tubule involvement were in teeth with a lesion depth of 0.5mm and soft texture. There were no teeth with a depth of <0.5mm and soft texture. In addition, three cells have only one observation: deep lesions with hard texture and light or medium color and deep lesions with leathery texture and light color. Out of 100 teeth, 60% were shallow lesions, less than 0.5mm, with the remaining 40% lesions being greater or equal to 0.5mm. The most common texture was leathery (48%), with soft lesions comprising 27% and hard lesions representing 25% of the sample. For color, 41% of the lesions were medium, 35% were light, and 24% were dark.

100 89 * One observation was determined to be an outlier and excluded from the mean dentin tubules analysis. Table 15. Descriptive statistics of mean depth of surface loss and mean depth of demineralized dentin tubule involvement.

101 90 Relationship between histological depth of surface loss and visual/tactile characteristics Histological depth of surface loss A linear regression model was fit to the log transformed mean depth of surface loss data with three main effects (color, texture, and depth of surface loss) and all first-order and the second-order interaction terms. No evidence of second-order interaction between the clinical attributes existed (p=0.3957). When a reduced model with main effects and firstorder interactions was fit, the results indicated that the first-order interactions were not significant (range of p-values: ). The model was reduced further to include only main effects. Model diagnostics based on residual analyses confirmed the validity of the model assumptions. Visual/tactile depth of surface loss was significantly associated with the histological mean depth of surface loss (p<0.0001), as was texture of the lesion (p=0.0038). Color was not a significantly related to histological surface loss mean. The adjusted mean of the log-transformed depth of surface loss was 2.09 for lesions with a depth of <0.5 mm and 1.17 for lesions 0.5 mm; for texture the adjusted means were 2.08, -1.63, and 1.18 for hard, leathery, and soft lesions respectively. There was evidence of significant differences between both leathery and hard texture, and between soft and hard textures but not between leathery and soft textures, after adjustment for multiple comparisons using the Tukey-Kramer method in conjunction with an overall Type I error level of 0.05.

102 91 Histological depth of surface loss and texture Visual/tactile depth of surface loss and texture were significant characteristics in the main effects model for histological mean depth of surface loss (Fig. 20 and 21). Teeth were then recoded into subgroups based on their combination of visual/tactile characteristics depth of surface loss and texture. Two levels of visual/tactile depth of surface loss and 3 levels of texture made a total of 6 subgroups possible; from these 6 subgroups, 1 subgroup had no observations (<0.5 mm and soft texture). A one-way ANOVA was used to assess differences in mean histological depth of surface loss among the five existing subgroups. The test for overall subgroup effect was significant in this model (p<0.0001) and further multiple comparisons testing showed that lesions with the visual/tactile characteristic combination of: 0.5 mm depth and soft texture presented the greatest mean histological depth of surface loss. The means reflecting histological surface loss for the five subgroups defined by visual/tactile characteristics decreased in the following order: 0.5 mm depth and leathery texture; 0.5 mm depth and hard texture; <0.5 mm depth and leathery texture with a p<0.0001; and <0.5 mm depth with a hard texture and p< (Fig. 22). Figure 19 shows how subgroups 1 and 2 are not significantly different from each other while they are statistically different from subgroups 3, 4 and 5. At the same time subgroups 2 and 3 are not significantly different from each other but they are significantly different from subgroups 1, 4 and 5. Although, subgroups 3, 4 and 5 are not s significantly different one from the other, they differ significantly from subgroups 1 and 2. The results of this subgroup analysis can be affected by the sample size of each subgroup.

103 92 Figure 20. Box plots of log mean depth of surface loss by visual/tactile depth of surface loss, with mean and (standard deviation) on log scale.

104 93 Figure 21. Box plots of log mean depth of surface loss by texture, with mean and (standard deviation) on log scale.

105 94 Figure 22. Box plots of log mean depth of surface loss by visual/tactile depth of surface loss and texture group, with mean and (standard deviation) on log scale; n= sample size of each group.

106 95 Relationship between depth of demineralized dentin tubule involvement and visual/tactile characteristics. The analysis of the mean depth of demineralized dentin tubule involvement revealed an outlier in the data. One tooth was determined to have an invalid measure so it had to be excluded from this analysis, resulting in a total sample of 99 teeth. A linear model was fit to the mean of the log transformed depth of demineralized dentin tubule involvement data with three main effects (color, texture, and depth) and all first-order and the second-order interaction terms. No evidence of second-order interaction between the visual/tactile characteristics existed (p=0.9183). Subsequently a reduced model with main effects and first-order interactions was fit, and the results indicated that none of the first-order interactions were significant (range of p-values: ). The model was therefore reduced further to include only main effects; model diagnostics (on model excluding the outlier) confirmed the validity of model assumptions. Texture of lesion was a significant tactile characteristic in the mean demineralized dentin tubule involvement depth (p=0.0455). The adjusted means on the log transformed scale for hard, leathery and soft textures were 0.35, -0.34, and 0.09 respectively. After adjustment for multiple comparisons evidence was found of significant differences between leathery and soft texture (Fig. 23). The visual/tactile characteristics depth of surface loss and color did not appear to be significantly related to the mean demineralized dentin tubule involvement depth.

107 96 Figure 23. Box plot of log mean depth of demineralized dentin tubules by texture, mean and (standard deviation) on log scale.

108 97 Section 3 ICDAS classification system related to depth of surface loss The International Caries Detection and Assessment System (ICDAS) classifies root caries lesions according to the following clinical criteria as: Code 1 lesions <0.5 mm depth and Code 2 lesions 0.5 mm depth. The second objective of this study was to compare the results of the relationship between root caries lesion clinical characteristics and their histological severity with what ICDAS root caries classification. All 100 teeth included in this study were classified according to the classification proposed by ICDAS and the descriptive statistics were calculated for teeth in each code (Table 13). The mean depth of surface loss (log-transformed) of ICDAS code 1 root caries lesions was 2.26 (standard deviation= 0.76) compared to a mean depth of surface loss of ICDAS code 2 mean of 0.90 (standard deviation= 0.66). This difference was significant (p<0.0001) using a two-sample test and indicating that ICDAS code 2 teeth had more surface loss than those of ICDAS code 1. (Fig. 24)

109 98 Table 16. Mean depth of surface loss (Log) by ICDAS Root Caries Classification.

110 99 Figure 24. Box plots of log mean depth of surface loss by visual/tactile depth of surface loss.

111 100 ICDAS classification system related to depth of demineralized dentin tubule involvement. The mean of depth of demineralized dentin tubule involvement of root caries lesions was calculated for each ICDAS code. The mean depth of demineralized dentin tubule involvement for code 1, lesions with a depth 0.5 mm was (standard deviation= 0.57 compared to code 2, lesions with a depth of 0.5mm showed a mean of 0.04 (standard deviation= 0.41). A two-sample t-test confirmed that this difference was significant, indicating that ICDAS code 2 lesions had a greater depth of demineralized dentin tubule involvement in the carious lesion (p<0.0001) (Fig.23).

112 101 Table 17. Log mean depth of demineralized dentin tubule by visual/tactile surfaces loss (standard deviation).

113 102 Figure 25. Box plot of log mean depth of demineralized dentin tubule involvement by visual/tactile depth of surface loss.

114 103 CHAPTER V DISCUSSION Clinical disagreement in root caries diagnosis can be attributed to several factors. Variation in an examiner's visual acuity can obviously affect the interpretation of the presence or absence of cavitation and/or a color change on the root surface. Even more critical, however, is that there is frequently disagreement between examiners concerning the relative softness or hardness of the area examined due to differences in interpreting tactile sensitivity (12). It was stated in the literature that there are strategies which can be implemented to maximize examiner reliability and accuracy of root caries diagnosis such as provision of a suitable diagnostic environment, establishment of a standard classification scheme which uses objective criteria based on clinical signs, intensive instruction and calibration of examiners, and a standardized reporting method (77). In this thesis study examiner reliability was performed by setting visual/tactile diagnostic criteria including 3 clinical characteristics, texture, color, and surface loss depth. The diagnostic criteria used in this study were created from a recompilation of clinical signs established in the past regarding root caries diagnosis by other researchers. In accordance with the strategies established by Sackett (77) to improve inter-examiner calibration, the clinical examiners of this thesis study went through calibration training together before visual/tactile assessment. This thesis study reliability testing on the visual/tactile (color, texture and surface loss depth) assessment was good to excellent. The intra-rater agreement was highest on color while the inter-rater agreement was highest for texture. Despite the subjectivity

115 104 inherent in interpreting the clinical signs of root caries diagnosis, good to excellent interexaminer reliability has been reported in clinical studies (12); (14); (31); (39); (73); (91); (63); (10); (55); (57); (35); (72); (38). These measures of inter-examiner reliability that have been reported in the past involving clinical diagnosis of root caries showed a percentage agreement between 85% and 99%, which is impressively good for human clinical studies. However, it was apparent that in these reliability studies the presence of filled root caries surfaces dramatically enhanced the agreement (12). When only untreated root caries were diagnosed, the examiner reliability was reduced considerably (11); (75) Table 15. This examiner reliability result difference between filled and non-filled surfaces can be attributed to the fact that is easier to identify restored root caries surfaces than primary root caries lesions, and to agree on their clinical characteristics respectively. Also, intra-examiner reliability has been shown to be slightly better than inter-examiner reliability. Table 18. Studies on reliability of visual/tactile diagnosis of root caries found in the literature. (11);(75)

116 105 According to Banting (11), methodological systems that require the observation of multiple signs (characteristics) in order to confirm a root caries diagnosis have an increased accuracy of the diagnosis; although, sometimes this can cause false negative cases. On the other hand, the use of just one of the signs to define the presence of root caries probably overestimates the true rate of the disease (11). One characteristic that is often used to confirm the diagnosis of root caries is lesion texture. There are particular aspects of the root dentin that need to be considered when evaluating the texture of a root lesion by probing in order to avoid over-diagnosing of root caries since tactile diagnosis of root caries has used probe tug-back as a sign of caries. For example, the fact that calcified non-carious root surface is not as hard as enamel, therefore, if a sound root surface is probed with excessive force it may feel as tugging or pulling back and the examiner could misclassify that tooth structure as carious. In 1987 The National Institute of Dental Research published diagnostic criteria for root caries which supported the use of evaluating root caries texture for diagnostic confirmation. They stipulated that visual criteria related to location, shape and discoloration of the suspected area do not define root caries. However, the criteria stated that the texture of the root dentin must be evaluated tactilely and the softness of the dentin to an explorer tip must be met for a definitive diagnosis of root caries (65). Tactile methods can be used to determine surface loss as well as the texture of carious lesions. The presence of surfaces loss is often difficult to determine visually and thus probes are used to detect surface defects. It is not easy to glide a probe tip freely over cementum or root dentin due to the low degree of mineralization of these dental tissues (12),

117 106 which can make difficult the perception of the amount of surface loss of a root lesion as well as to detect caries activity, since most inactive root caries lesions exhibit leathery to hard texture that can be similar or harder than sound root surface. Tactile and visual characteristics of root caries lesions have a tendency to change according to different levels of hydration. When a lesion is dehydrated the color tends to become lighter and the texture becomes harder. When the texture is harder the depth of surface loss measurement may not be accurate for when the area is probed there may be more resistance to withdrawal of the probe. In this thesis study it was intended to avoid extracted teeth dehydration by keeping them in water before visual examination, and also wetting the root caries lesions with a microbrush during examination. Although the effort to maintain the carious lesions moist to avoid clinical characteristics distortion, it could have happened in some degree. Another important factor to take into account when evaluating visual/tactile signs of root caries lesions from extracted teeth is that some lesion characteristics tend to be different between fresh extracted teeth and stored extracted teeth. The differentiation in texture from sound to leathery and leathery to soft was very difficult in the stored teeth used for this thesis study; while determining texture in freshly extracted teeth could be much more apparent and easier. Another limitation during a visual/tactile examination is that some natural root caries lesions present several clinical characteristics within the same lesion. For example, a root caries lesion can have parts where it is leathery and others parts where it is hard; regarding color, it can be medium (light brown-brown) and dark (dark brown-black) within the same lesion; additionally the lesion can have different surface loss depths. Since the demin-remin

118 107 process could have happened at different grades within one lesion, the visual/tactile assessment was done over a specific area of the lesion that was identified using a permanent marker. Lesions of every color, texture and surface loss were noted during the visual/tactile examination. However, there as an absence of root caries lesions with the following characteristics: 0.5mm/soft/any color. The lack of this clinical observation can be due to the fact that when continuous demineralization of cementum and dentin occur the disintegration of tissues occurs rapidly resulting in soft dentin with surface cavitation > 0.5mm. When the demin-remin process is more balanced and there is little surface loss, the dentin is most likely hard or leathery. The results of this in vitro study suggest that the criteria texture and depth of surface loss considered during visual/tactile assessment were important factors in determining the histological surface loss. On the other hand, the clinical characteristic color did not have much relevance in the severity grade of histological sections. There is no other in vitro study reported in the literature that compared several visual/tactile characteristics of root caries lesions with their histological assessment in the same study. However, there are histological studies of primary root caries lesions demonstrating that soft, leathery and hard lesions exhibit different histological appearances which reflect the level of bacterial infection (59). For example, soft lesions were characterized by the presence of bacteria within and spreading laterally between dentin tubules with an accompanying loss of mineral and organic matrix. Hard lesions were fully mineralized with bacterial ghosts observed within the amorphous mineral of the remineralized lesion; while leathery lesions presented a

119 108 variety of appearance between those observed in soft and hard lesions (68); (79); (80); (81). Despite the fact that this thesis study did not deal with bacterial infection it is interesting to mention that other researchers found a clear association between clinical texture of root caries lesions and their histological appearance reflecting bacterial invasion. Another in vivo study done by Lynch (59), who also confirmed that texture is a useful criterion to diagnose severity of root caries lesions. In order to better understand how useful texture and color are as clinical severity indicators, the microbiological content of 395 root caries lesions was measured and classified according to texture and color. He found that there was no indication that color was significantly associated with the total number of bacteria. Nevertheless, soft lesions yielded significantly more bacteria than hard lesions, while leathery lesions yielded an intermediate number of bacteria. Although no correlation has been demonstrated in the past between color of root lesions and caries activity (43); (83); (59), it has been agreed by various investigators that discoloration of the root surface can be indicative of the presence of caries (79); (80); (81). According to Dreizen (25) the discoloration of root surface lesions may indicate a biomechanical reaction of the collagen exposed by demineralization; and/or an effect caused by extrinsic stains, thus it can be probable that very early lesions still can be colorless and clinically not detectable (59). Although this thesis study did not measure bacterial invasion it did find that the largest mean depth of surface loss and mean demineralized dentin tubules involved in the carious process happened in teeth with a lesion depth of 0.5mm and soft texture. For the histological measurement of surface loss the intraclass correlation was excellent, above the 0.90 level. More variation existed in the measurement of depth of demineralized dentin tubule involvement than in the one of depth of surface loss; however

120 109 there was no statistically significant difference between the measurements of demineralized dentin tubule involvement. The amount of surface loss was quite visually apparent, while the depth of demineralized dentin tubule involvement was much harder to visually pinpoint. The difficulty in determining an exact point to measure may be attributed to different pulpal reactions which affect the appearance of dentin tubules. The histological pattern observed in this thesis study was similarly to that described in Wefel (92). The body of the lesion appeared to be yellowish and darker when compared with the surrounding dentin. The pattern of the dentinal tubules seemed to be disrupted at the deepest aspect of the lesion. The positive birefringence of dentin seen under polarized light microscopy is increased during demineralization, and, therefore, no stark contrast is produced between demineralized and sound dentin (92). In Wefel study when sections were imbibed in quinoline a clearer distinction between normal dentin and the demineralized area could be seen. In addition to the appearance of a structureless area at the deeper aspect of the body of the lesion it showed an actual reversal in the sign of birefringence of the tissue. This reversal in the sign of birefringence was seen as a bright blue area compared to a yellow of normal dentin. This is a phenol reaction and is a reversible process. It occurs when the collagen fibers have lost some of the mineral phase and the oriented absorption of phenol molecules reverses the sign of birefringence (+ to -) and the area of demineralized dentin gets stained by this chemical facilitating its visualization through polarized light microscopy. However, shrinkage of the main body of the lesion may occur, which affects the depth measurements (92). The main reason quinoline technique was not used in this thesis study was the lack of time to apply this imbibing technique within the period of time considered to finish this thesis. Although there was a trial where sections belonging to the

121 110 first 30 teeth were imbibed in quinoline, the technique was dropped because this chemical has a very strong odor that being around it for long hours can produce general unwell. Also, quinoline is a liquid solution which after being exposed at room temperature for certain number of hours becomes a gel entrapping the dental tissue section within the body of the gel. Another evaluative technique that could have been used to assess these root caries sections is microradiography, where the surface layer can be perceived as a radiopaque area and the body of the lesion as a radiolucent area, and sometimes the presence of dead tracts can be perceived also as slightly more radiopaque oblique lines running across the radiolucency representing the body of the lesion (92). In the study done by Wefel et al. (92) the natural root caries histological features observed under polarized light microscopy using water, quinoline and microgradiography technique were very similar, obtaining basically the clearest visualization of the true lesion extension using quinoline and microradiography. The nature of different dentin zones in root caries lesions and the relationship of the zones to sound dentin were investigated by WH Arnold et al. (5). Their study described the morphological characterization of initial, progressing and stagnating root caries lesions using histological visualualization. Many of their observed histological findings were similar to those found in this thesis study. The initiation of root caries starts with subsurface demineralization of root cementum and dentin, resulting in a hypermineralized superficial zone and demineralized dentin underneath the surface. The nature of the hypermineralized superficial layer was unclear. It may be due to reprecipitation of hydroxyapatite from salivary Ca and P ions or by reprecipitation of ions from the dissolved underlying hydroxyapatite (5). With polarized light microscopy they also observed that the hypermineralized surface layer was present in advanced root caries lesions and suggested

122 111 that this surface layer can be developed as a biological reaction to the carious attack where the odontoblasts produce intratubular dentin which obliterates the dentin tubules. Their investigation showed that the dentin tubules within the hypermineralized translucent dentin are not completely obliterated by intratubular dentin and that dead tracts are intermittent between hypermineralized dentin (5). This histological pattern described by WH Arnold et al. was observed in many of the histological photographs of this thesis study; these scattered dead tracts that made the depth of demineralized dentin tubule involvement measurement difficult, are only partly obliterated dentin tubules which may be the cause for further progression of the caries lesion due to bacterial invasion. According to WH Arnold et al. (3); (4), the intertubular dentin of the hypermineralized translucent dentin is also demineralized to some extend and the hypermineralization of this zone is due to intratubular dentin deposition of Ca and P from the demineralizing zone towards sound dentin. This thesis study intended to measure the depth of the demineralized dentin tubule involvement, but on several histological sections the distinction of the demineralized zone (body of the lesion) from the translucent zone (hypermineralized or sclerotic dentin) was not clear due to little or no distinguishable difference in the dentin tubule pattern between these zones. As mention above, the presence of dead tracts made measuring the depth of tubule involvement difficult. These features were observed on sections imbibed in water. There were additional limitations regarding the histological measurement that need to be acknowledged. For instance, during histological assessment the surface loss could have been exaggerated, either due to loss of soft tissue during microtome sectioning and

123 112 manipulation or due to tissue shrinkage after dehydration. However, great effort was made to keep the root caries samples hydrated at all times. Furthermore, to reduce the subjectivity of placing a line electronically to estimate the original location and shape of the cementum the researcher had set a criterion. Even though an established criterion was used some difficulties in obtaining measurements did arise. When discussing histological severity of root caries lesions, severity is often defined as the lesion proximity to the pulp. In this thesis study, the root caries lesions were measured from the outer surface to the deepest point of the demineralized dentin tubules in millimeters. Since the overall percentage of the dentin involved from the outer surface to the pulp was not measured and calculated the measurements in this study do not inherently represent proximity to pulp. The severity of a root caries lesion depends on the dental anatomy of the tooth, since the dentin thickness from cementum to pulp varies among different types of teeth (anterior, premolar and molar) and surfaces (buccal, lingual, mesial and distal). From the 100 teeth used in this study, 65 were maxillary teeth mainly molars and 35 were mandibular teeth, mostly premolars. Although it was intended to collect a majority of the lesions located on buccal/facial or lingual surfaces of the teeth, there were also some on mesial and distal surfaces. In the future, it may be interesting to measure both depth and percentage of demineralized dentin tubule involvement (severity). There are no in vivo studies reported in the literature that compare clinical diagnosis with histological assessment of root surface lesions. Although this is disappointing, it is not surprising because of the difficulty and complications involved in conducting studies where teeth are examined clinically, removed and histologically evaluated. (48). One of the

124 113 complications of an in vivo study is that many of the root lesions can be restored and are restored. Only the lesions that are quite severe would need to be extracted. While most of the extracted teeth with root caries have such severe caries that sectioning and histological evaluation are impossible. However if a patient is having a full mouth extraction due to periodontal reasons there is a chance that root lesions of all severity could be obtained, yet periodontal disease and caries do not always go hand-in- hand. The International Caries Detection and Assessment System (ICDAS) attemps to improve caries detection of coronal and root lesions by presenting a system that provides practitioners and researchers with a better understanding of the stages of the carious process. Even though this system was developed a few years ago it is still open to more scientific validation and new contributions that improve the detection criteria for root caries. For root caries, ICDAS presents a classification system dividing lesions into 2 codes based on clinical depth of surface loss. ICDAS tries to use visual characters to classify lesion. The resulting classifications are correlated to the severity of carious lesions and their histological depth. One of the purposes of this thesis study was to classify the carious lesions according to ICDAS root caries classification and compare the accordingly histological depths of demineralized dentin tubules. This gives an association between the clinical depth of surface loss and histological depth of lesion. The results of this part of the thesis showed that there were significant differences in both depth of surface loss and the depth of demineralized dentin tubules between the 2 codes, in other words, the deeper the clinical surface loss is, the deeper the involvement of the dentin affected by the carious process is as well.

125 114 ICDAS mentions all of the visual/tactile characteristics reported in the literature regarding root caries diagnosis criteria, such as color (light /dark brown, black), texture (soft, leathery, hard) appearance (shiny or glossy, matte or non-glossy) and cavitation (loss of anatomical contour: <0.5mm and 0.5mm). Yet the actual ICDAS criteria only use the depth of surfaces loss to classify lesions. Although this thesis study did not investigate clinical signs of caries activity it is worth to mention that ICDAS also recognizes the importance of differentiating visual/tactile characteristics of active versus inactive root caries and the need to validate a better definition of clinical criteria associated with root caries activity. The determination of root caries activity probably is more closely related to decisions regarding treatment or management than to the determination of the presence of caries on the tooth root (48). In this thesis study texture was a significant clinical criterion to determine depth of surface loss and depth of demineralized dentin tubules involvement. The current ICDAS Root Caries Classification System only considers discoloration of the root and depth of surface loss divided into 2 codes. It would be beneficial to incorporate the tactile characteristic texture also to their decision tree since this clinical sign has been shown to be very relevant for root caries diagnosis in this thesis study and also in the literature. Therefore, a new root caries classification system is proposed (Figure 26). This system to detect and classify root caries lesions will consist of keeping one score per root surface type: Code E - if the root surface cannot be visualized directly, and is therefore excluded. Code 0 the root surface does not exhibit any unusual discoloration that distinguishes it from the surrounding or adjacent root areas, nor does it exhibit a surface defect either at the cement-

126 115 enamel junction or wholly on the root surface. Code 1 there is a clearly demarcated area on the root surface or at the CEJ that is discolored, has a hard texture and there is a surface loss of 0.5 mm present. Code 2 there is a clearly demarcated area on the root surface or at the CEJ that is discolored, has a hard texture and there is a surface loss of 0.5 mm present. Code 3 there is a clearly demarcated area on the root surface or at the CEJ that is discolored, has a leathery texture and there is a surface loss of 0.5 mm present. Code 4 there is a clearly demarcated area on the root surface or at the CEJ that is discolored, has a leathery texture and there is a surface loss of 0.5 mm present. Code 5 there is a clearly demarcated area on the root surface or at the CEJ that is discolored, has a soft texture and there is a surface loss of 0.5 mm present.

127 116 Figure 26. Root caries classification system proposed based on this thesis study results and an extended review of the literature.