Susceptibility of Mice Teeth Enamel Affected by Amelogenesis Imperfacta to Caries - Literature review Example

?Susceptibility of mice teeth e l affected by amelogensis imperfacta to caries, a study using x-ray microtomography Professor: Date: Table of Contents Table of Contents 2 1.Enamel 3 1.1.Dental Hard Tissues 3 - Enamel 3 - Enamel Mineral Composition 6 - Induced Caries 9 1.2. Human Enamel Structure 9 1.3. Enamel Morphology of Rodent Teeth 10 2.Amelogenesis Imperfacta (AI) 11 2.1. Genetics of AI 11 2.2. Phenotypes of AI 12 2.3.Induced AI 13 2.4.Clinical Importance of this Genetic Condition. 14 3. X-ray microtomography XMT 14 3.1. History 15 3.2. Basic Theory 16 3.3. Application 18 References 19 1. Enamel 1.1. Dental Hard Tissues Topographically, there are various tissues involved in the composition of teeth due to which physiological functions of the teeth have been possible. Since, the teeth have to undergo through various chemical and physical processes, therefore, they are susceptible to several compressive forces, wear and tear, and chemical acidic attacks from foods and bacteria. Hence, the tooth is protected by an external layer of dental hard tissue called enamel that shields the crown by a sheet of 2 millimeter occlusally and more in analogy to the cuspids and reduces cervically at the collar to just a few micrometers (Tencate 1998). There are three different dental hard tissues which are enamel, dentin and cementum. Ameloblasts produce enamel, odontoblasts produce dentin and cementoblasts produce cementum. - Enamel The most mineralized tissue within the entire body is enamel. It creates a layer of calcified tissue that is very thin, hard and translucent layer covering the anatomic crown of the tooth completely. Enamel is extremely hard since it primarily comprises of inorganic materials. Almost 95 percent of the enamel includes calcium and phosphate ions combined together to form strong hydroxyapatite crystals (Fincham and Belcourt 1981). Trace minerals are readily contained in hydroxyapatite forming the crystal lattice. These ions when charged negatively form carbonate or fluoride and also, they can form zinc, sodium, potassium or strontium when charged positively. The solubility of the enamel gets affected by the concentrations of these trace minerals. As for instance, the crystal structure becomes stronger with the presence of fluoride but its solubility decreases whereas its solubility can be increased by incorporating carbonate. Hence, the hydroxyapatite crystals contain less carbonate and more fluoride in the outer layer as compared to the crystals in the inner layer due to which the outer surface becomes less soluble in contrast to the deeper layers of the enamel. Almost 1 to 2 percent of the enamel comprises of organic materials, in particular the enamel-specific proteins that are known as the enamelins that have a high potential of binding hydroxyapatite crystals (Lussi and Hellwig 2001). Approximately 4 percent of the enamel composition comprises of water. All the components of enamel (water, organic and inorganic) are extremely organized. There are millions of carbonated hydroxyapatite crystals aligned in thin and long structures, with diameter of about 4 to 8 ?m (Boyed 1997). These structures are called rods that look like key-hole shaped structures when viewed in cross section. It has been found that the number of rods in a tooth varies from five million to 12 million in the lower lateral incisor and upper first molar, respectively. Usually, the rods stretch from the dento-enamel junction to the surface of the tooth at right angles. A rod sheath comprising of a protein matrix of enamelins is around each rod. Interrod cement or Interrod enamel is the space between these rods. Tiny spaces are present between rods where crystals are not formed, which are typically called pores. These pores enable the occurrence of fluid movement and diffusion making the enamel permeable. However, they also contribute to the hardness in the tooth and variations in the density due to which spots are formed that are more vulnerable to demineralization when the pH level of the mouth becomes too acidic (Robinson and Briggs 1981). Enamel is regarded as an exclusive mineralized tissue since it is generated through ectodermally derived ameloblasts that go through a chain of discrete differentiation phases that are associated with the different stages of enamel development. Ameloblasts are detached in the pre-secretory phase from the odontoblasts that has been derived from the adjacent neural crest cell with the help of a basement membrane that is removed later when the ameloblasts go in to their secretory stage. At this stage, an eosinophilic is secreted by the ameloblasts, which is an enamel extracellular matrix comprising of above 90 percent of AMELX whereas the remaining below 10 percent of its constituents includes non-amelogenin proteins like ENAM, ameloblastin, and tuftelin along with the enzymes such as the kallikrein-4 (KLK4) and proteases enamelysin. The enamel matrix commence to bio-mineralize when it is being secreted in the absence of the non-mineralized ‘pre-enamel’, like the one in other mineralized tissues of the tooth. The proteins in the extracellular matrix are processed enzymically in the secretory phase in a way that nascent molecules are transformed in to minute fragments that provide more thickness to the matrix. When the ameloblasts go through their maturation phase then the organic matrix is fully degraded so as to enable the secondary growth of the HAP- crystals which will eventually obstruct the spaces that have been occupied earlier by the proteins of the enamel extracellular matrix. Subsequently, the mature erupted tissue completely loses the ameloblasts atrophy as well as the cellular layer. - Mineralization Enamel is produced by the epithelial cells known as ameloblasts. The ameloblasts break down just prior to the eruption of the tooth from the gums due to which the enamel loses its ability to repair itself. This signifies that the enamel cannot restore itself when it gets damaged through decay or injury. The tooth is not completely mineralized at the time of eruption. Thus, phosphorous, calcium and fluoride ions are extracted from the saliva over a period of time to create a covering of enamel on the tooth which is around 10 to 100 ?m in thickness in order to fully mineralize the tooth (Elliott, Wong and Anderson 1998). The enamel comprises of about 96 percent of mineral and just 0.5 to 2 percent of organic material. HA is the inorganic component of the enamel that is replaced with the mineral components like carbonate and fluoride ions (Marthaler 2003). Enamel rods are created by the HA crystallites, which were previously known as the enamel prisms. - Enamel Mineral Composition Enamel is found to be the most mineralized dental hard tissue with its mineral content usually being above 97 percent in weight. Enamel basically comprises of many different types of calcium phosphates. In general, the calcium phosphate is reported to vary from 1.64 to 1.8 in normal enamel in correspondence to those of apatites (Dowker and Andreson 1999). Enamel hardly contains the stoichiometric form of hydroxyapatite that is carbonated at different levels varying from 4 percent to 6 percent. In normal teeth, the distribution of carbonate is usually homogenized whereas the hypo-mineralized enamel indicates a higher consistency of the carbonate. The enamel loses it stiffness in carbonated areas. The enamel layer includes fluorine that protects the teeth from acid attacks. In general, the fluorine spreads along a gradient increasing from the core towards the outer layers. Enamel structures have been found to include some elements with the help of relevant techniques like X-ray Microanalysis and Secondary Ion Mass Spectrometry that are used for detecting the elements at low concentration (Anderson and Elliott 1996). Elements like Fluorine, Chlorine, Sodium, Potassium, Magnesium and Strontium spread in a gradient from the dentine to the surface across the enamel layer. It has been found that Magnesium and Potassium are a bit higher in concentration in areas that are hypo-mineralized, particularly towards the surface whereas the concentration of Sodium has been reported to be higher near the areas where the enamel is defected (Downer, Drugan and Blinkhorn 2005). Nevertheless, the concentrations of Chlorine and Strontium are not directly related to the level of mineralization. Other techniques have reported traces of elements like Fe, Zn, Pb and Ba to be accumulated in enamel layer in teeth of children living in polluted areas or as a result of specific nutrition regimes among children. Teeth are composed of hardest tissues that are absolutely accustomed to their functionality. The mechanical and material attributes of the enamel relates to: the internal architecture, the morphology and the nano-scale properties of the mineralized dental tissue layers. Researchers have been yet unable to completely explain the mechanism of the formation of enamel. Like all the mineralized tissues in living beings, enamel is a composite of natural polymers that serve as a template for all the inorganic compounds. The existence of natural polymers is necessary in order to support the process of crystallization. Ameloblasts form the enamel matrix, which are formulated from the ectodermal epithelium when the dental lamina is formed and proliferation takes place as a result of invasion of the underlying mesenchyme by the neural crest cells. The ameloblasts are accumulated in a sequence at the base of dental papilla around the specialized mesenchymal cells that are the odontoblasts secreting the dentinal matrix. The dentinal matrix includes various molecular signals that comprises of structural polymers like the collagen type I, minerals, cytochines, dentine sialoprotein and signal molecules like the Wnt, fibroblast growth factor, Hedgehog, bone morphogenetic protein transforming growth factor (Ohshima, Wartiovaara and Thesleff 1999). Dentin sialophosphoprotein is found to be particularly essential in the formation of amelogenesis, dentino-enamel junction and aprismatic enamel. It has been reported that the over expression of dentin sialoprotein among trans-genic animals increases the rate of enamel mineralization. The mechanical properties and the hardness of the mineralized tissues in wild animals can be increased by including the dentin sialoprotein in the formation of aprismatic enamel. On the other hand, the over-expression of dentin phosphorprotein results in the hypo-mineralized enamels. - Degradation of hard tissue The ideal range of pH of saliva is from 5.5 to 6.5 (Robinson and Shore 2000). The threshold value of the saliva pH is 5.5 for the formation of dental caries where as the oral cavity may resume from the effect of dental degradation when the pH value of the oral cavity falls below the threshold value (Marsh and Nyvad 2008). However, demineralization of the enamel occurs more quickly due to the prolonged exposure to this threshold pH or when frequent cycling of the pH takes place from the optimal value to the value that is less than the threshold. Usually, the lowered salivary pH is due to the bacterial digestion of carbohydrates like sucrose and fructose that creates acidic by-products in dental plaque (Zero 1996). Moreover, dental erosion could also be the cause behind tooth demineralization. Dental erosion is defined as the irreversible and generally painless process of losing dental hard tissue as a consequence of a chemical reaction like dissolution or chelation that does not involve micro-organisms (Marsh 1999). Despite the fact that vulnerability to dental erosion among people varies because of the factors like oral pH value, buffering capacity, flow of the saliva, and formation of the pellicle, however, it has been noted that the consumption of soft drinks and citrus fruits that form acetic acid may be a major reason behind the development of the disease (Littman 2005). The pH value of the carbonated soft drinks is quiet low and they also include sugar and various other additives that contribute to the acid dissolution and/or erosion of the dental enamel. Other two important factors are attrition and abfraction that result in enamel erosion. Abfraction is considered to cause the erosive process by activating the enamel to erosion. Acid eroding dental enamel creates lesions that have a base area comprising of softened enamel having a depth of few microns and a high vulnerability to physical wear and tear. Erosion and attrition of enamel takes place at low oral pH levels that are below 6.0 (Shellis and Wahab 1993). The rise in the pH increases attrition, however, the level of the threat is based upon the applied load, the pH value of the medium, and the time period of the contact between the affected materials (Mandel 1983). At a pH value greater than or equal to 7, the erosion is nearly non-existent. When the corrosion takes place at low pH between the enamel surfaces then the stress cracks are created within the enamel which propagate further and also, release particles. The debris of these particulates get hooked and/or accumulated between the surfaces in-contact that result in to the transformation of two body abrading system in to a highly damaged three body abrading system. This process does not seem to occur in low pH media since the appearance of the opposing surfaces look smooth. Indeed, it looks as if the erosion regulates attrition such that the wear disappears due to an apparent effect of polishing on the surfaces in contact. Although enamel degradation of the tooth has been reported to be an intricate phenomenon, however, erosion seems to take over at pH levels. Undoubtedly erosion is responsible for damaging the enamel to a large extent. Nevertheless, erosion can be avoided to a significant degree by adjusting drinking habits, more elaborately, by decreasing the intake of beverages and acidic foods. Moreover, the beverages can be modified by the inclusion of citrate ions that effectively decreases erosion by altering the acidogenic potential (Margolis and Moreno 1992). - Induced Caries Induced caries such as lesions can be generated in dental enamel under controlled environment so as to demonstrate all the prime histological aspects of natural caries whereas the re-mineralization of such induced carious lesions can be exhibited with the help of the scanning electron microscopy. Lesions of enamel and other such induced caries are more homogeneously reproducible in contrast to natural caries (Kidd 2005). 1.2. Human Enamel Structure There are two functions of ameloblasts during amelogenesis. First, they produce the four important proteins and proteases of the enamel matrix, which are namely: amelogenin, enamelin, ameloblastin, and enamelysin (Fincham and Moradian 1999). Second, they cause the enamel maturation that leads to an increase in mineralization and the loss of organic matrix. Enamel is formed of extremely determined, organized, and tightly packed crystallites that are very long in relation to their thickness. In general, these crystallites stretch from the dentin in the base to the tooth surface and are arranged into bundles that are called prisms or rods (Davis and Wong 1996). As a result of its unusual attributes, the enamel copies and records the changes in the metabolic status of a person during its development. Thus, the enamel anomalies may indicate not only environmental but also systemic disturbances. 1.3. Enamel Morphology of Rodent Teeth The structure of the rodent incisor enamel consists of a uniserial lamellar pattern of rods present inside the inner enamel and parallel rods incisally directed in the outer enamel. When a mice of around 5 weeks of age was incised maxillary and mandibularly then its enamel was found to have the centrolabial thickness around 95 ?m in the mandibular incisor and almost 60 ?m in the maxillary (Wong and Elliott 2000). The angle between the rows of the rods and junction of enamel-dentine was found to be around 45 degrees in the mandibular incisor and around 70 degrees in the maxillary whereas the angle of decussation was between 30 to 80 degrees in the mandibular incisor and 50 to 95 degrees in the maxillary, which increased from the junction between the enamel and dentine to the outer enamel. The angle was found to be around 5 to 15 degrees in the mandibular incisor and 12 degrees in the maxillary between the outer enamel rods and the surface of the enamel (Moinichen and Lyngstadaas 1996). The exterior 1/2 to 1/3 of the outer enamel included iron and found to be more resistant towards acid when compared to the remainder of the enamel (Hebal and Stromberg 1986). The concentration of iron in the mandibular incisor was inadequate to provide the enamel with visible pigmentation (Lyngstadaas and Moinichen 1998). The layer of the enamel was found to be thick in the mouse mandibular incisor as the ameloblasts would have been in the zone of enamel secretion for longer duration as a result of a fairly slow rate of eruption. 2. Amelogenesis Imperfacta (AI) The amelogenesis imperfecta (AI) has been defined as a heterogeneous group of hereditary disorders in genes defined by the abnormalities in the dental development such that either the quality and/or quantity of the tooth enamel is affected while the presence of generalized or systemic disease have been discarded. AI has been found to have the following three types: hypoplastic-type which can be classified as the secretory defect, hypocalcified-type which can be classified as the crystallite nucleation defect, and hypomaturation type which can be classified as the maturational stage defect (Wright and Duggal 1993). According to the clinical manifestation and the inheritance mode, it has been presently categorized into 14 different sub-types (Backman and Ammeroth 1989). As for instance, the autosomal form and the C-linked form. These defects have been reported to occur as a consequence of mutation taking place in the gene encoding enamelin and/or gene encoding amelogenin (Seow 1993). The amelogenesis imperfecta has also been recognized as part abnormalities in multiple organs, as for instance, the hypothalamo-hypophyseal insufficiency, the cone-rod dystrophy and the renal failure (Aldred and Crawford 2003). Nephrocalcinosis and hypoplastic-type of AI have been usually found to exist along with the increased value of plasmatic creatine, delayed eruption of tooth, and low excretion of calcium (Witkop 1988). 2.1. Genetics of AI The findings of enamel in AI vary extremely from deficient formation of enamel to disorders in the content of minerals and proteins (Stephanopoulos, Garefalaki, and Lyroudia 2005). The expression of multiple genes is required for the production of enamel, which transfer the required matrix proteins and proteineases for controlling the intricate process of mineralization and growth of the crystals. The phenotypes of the AI are based upon the involvement of a particular gene, the mutation type and its location, and the respective change at the protein level. Other different inheritance patterns of amelogenesis imperfecta include autosomal dominant and autosomal recessive. 2.2. Phenotypes of AI Each of the basic classification of AI types is further divided in to various sub-types that are distinguished by the mode of their inheritance. The variation in the physical appearance of these various sub-types of AI makes it difficult to differentiate one type from the other. Some teeth will look like normal dentitions to the untrained eye whereas the other types of AI can be easily identified due to visible disfigurement (Gibson et al. 2001). The pathological patterns related to AI reflect impacted permanent teeth, enlarged follicles and ectopic eruption. One major problem in fully explaining the underlying molecular processes related to AI in people is the near failure or difficulty in the acquisition of the viable pre-eruptive teeth. On the other hand, the incisor teeth in adult rodents develop and evolve continuously across the life time and provide access to all phases of enamel formation in a single tooth. Thus, the rodent teeth serve as an excellent model for studying the fundamental events subject to dental development as well as for investigating the molecular pathogenesis involved in AI. In association to this, the experiments of gene targeting have affirmed a vital contribution of amelogenin, enamelysin and ameloblastin in the development of tooth enamel. Despite the fact that ameloblastin may serve as a molecule used for the purpose of cell adhesion that means necessary for maintaining the ameloblasts state of differentiation, however, amelogenin and enamelysin both are crucially essential for the development of enamel with full thickness comprising of crystal structure. Likewise, the study of dominant induction of N-ethyl-N-nitrosourea (ENU) inherited mouse mutations have revealed that enamelin is the prime element that initiate the generation of mineral crystal during the initial phases of enamel formation and also, the enamelin is necessary for forming full thickness enamel (Masuya et al. 2005). Such studies have also demonstrated that the rodents with a phenotype of X-linked enamel immediately suggests mutations in the Amelx gene since it is the only protein of the enamel matrix that has been recognized on the X-chromosome, at present (Masuya et al. 2005). Human mutations of AMELX that have been reported earlier comprising of those causing a complete loss of secreted protein. Mutations that result in a loss of the AMELX C-terminal, and mutations that impact the AMELX N-terminal region, such as a lectin-like, have found to bind tri-tyrosyl domain to N-acetyl-D-glucosamine (Wright wt al. 2003). 2.3. Induced AI Also, the AI demonstrates genetic variation since families have been found to show patterns of X-linked, autosomal dominant, and autosomal recessive inheritances. The genetic investigations of the impacted human families have disclosed the genes that are held responsible for AI. The recessive forms and the dominant forms of the X-linked hypoplastic and hypomaturation AI that are represented by AIH1 are found to be associated with mutations that influence amelogenin. On the basis of the location and type of the mutation having occurred in the amelogenin gene, the AIH1 can develop a wide range of phenotypes that start from the smooth hypoplastic to the hypomaturation or hypomineralized (Caterina et al. 2002). AHI2 is another dominant form of AI that is developed through mutations in the gene of enamelin mapped to the chromosome. It has been found recently that a novel mutation in the enamelin cause recessive form of AI in human that is denoted by ARAI (Masuya et al. 2005). Different mutations of the ENAM can develop a wide range of AI phenotypes that range from the hypoplastic to the pitted hypoplastic AI. Mouse mutants are taken as study models to learn about human AI, since gene functions can be studied through forward and reverse approaches to genetic investigations. The genes called Amelogenin (Amelx) and enamelysin that duplicates active protease during the secretory stage of the development of the enamel, were generated in mouse models. It was found through the amelogenin-null mice that Amelx is not necessary for creating the formation of the mineral crystals however it is needed to arrange the crystal patterns and regulate the thickness of the enamel (Aldred and Crawford 1997). 2.4. Clinical Importance of this Genetic Condition. The dental development can be genetically governed by means of an intricate series of events that can be classified in to two pathways, very schematically: type, size and position specification of each organ related to the oral cavity, and certain processes responsible for the creation and development of the tooth enamel and dentin. Many genes associated with initial positioning and development of the teeth, are related to signalling pathways and possess the functionality of morphogenesis regulation in the organ-morphogenesis at which they are linked to the signalling pathways. The mutations of these genes usually exhibit pleiotropic impact exceeding the study of dental morphogenesis and causing the disorders of syndromic development. MSX1, AXIN2 are two of the few genes that influence the initial stage of tooth development, which are linked to the tooth agenesis and its systemic aspects such like colorectal cancer and cleft palate (Aldred 2003). On the other hand, the structures of the genes associated with the enamel and dentin, for example, ENAM, AMELX, KKL4, MMP20, and DSPP, are extremely specific for tooth. Amelogenesis Imperfecta and other such genetic disorder are caused by the mutations in the following genes: ENAM, AMELX, KKL4, MMP20, and DSPP (Barron et al. 2010). 3. X-ray microtomography XMT 3.1. History Dental caries have been found to be more of a “cavity” in contrast to being a process of disease. The mechanism of caries among dental hard tissues is very intricate and dynamic from a microscopic point of view as it disturbs the balance between demineralization and remineralization and thereby, affects the nature of the layers of the bio-film environment of the surfaces of the dental hard tissues. Hence, the changes in the specimens that are obtained through the techniques employed to analyze them confines our understanding of these processes by far. These techniques usually involve intricate and sometimes even destructive methods like polarized light microscopy, chemical analyses, microprobe analysis, nano-indentation or cross-sectional micro-hardness determinations, electrical conductivity studies, iodine absorptiometry, and X-ray microtomography (XMT) (Wilmott 2003). XMT is actually a miniaturized version of Computed tomography (CT), a famous medical procedure for the non-destructive investigation of internal structures, having a resolution measured in microns instead of millimeters. Despite the fact that Elliott and Dover have been credited for first inventing the XMT in the year 1982, however, some researchers have revealed that in 1970’s, the simplest form of the microtomographic scanner was invented that consisted of a pinhole collimator along with a single detector (Davis and Wong 1996). Energy discriminating detection was conveniently employed in this first generation of microtomographic scanner. Also, the first generation of microtomographic scanner was able to measure attenuation coefficient through the monochromatic radiation, therefore, the mass density of the material could be known by determining the X-rays that have been penetrated in the material (Mosleh-Shirazi and Evans 1998). Nevertheless, its usage was confined due to the prolong time duration required for the acquisition of data. The second generation of microtomographic scanners consists of linear-array system that includes a linear detector array for acquiring one entire projection at a time. Therefore, the data collection in the second generation was much faster in comparison to the first generation of microtomographic scanners. Two-dimensional detector array was incorporated in the third generation of microtomographic scanners, which have a 2-dimensional detector array (Elliott, Davis, and Dover 2008). The cone creates the projections through the X-rays that were further employed for the reconstruction of a 3D picture of the specimen. Therefore, the third generation of microtomographic scanners were also called cone-beam microtomographic scanner. These scanners have the shortest time for data collection among the previous generations, however, the three dimensional reconstruction of the image is more intricate in contrast to the previous fan-beam system (Hounsfield 1973). Moreover, the images of internal features were possible to be constructed through micro computed tomography with the help of using their X-ray attenuation coefficients that make it possible to analyze even the small objects at a resolution equal to just a few micrometers. A present, the XMT has been identified as one of the non-destructive three dimensional techniques for material analysis in the area of dental hard tissues and therefore, it has been employed in different areas of dentistry such as the caries research (Elliot and Dover 1982). 3.2. Basic Theory The function of X-ray Microtomography (XMT) is based on the same primary principles as those used in the technology of X-ray CT scanners employed in hospitals. However, the spatial resolution of the XMT is conventionally hundred times higher or even more than that of the X-ray CT scanners (Davis and Elliott 1997). The reconstructed slices obtained by a scanned object are called X-ray tomograms that are used to record the relative distribution of density. Hence, the tomograms are useful in determining the location of distinct materials in the structure provided that the materials are known in the scanned object. A 3D structure can also be provided by stacking the tomograms (Wong and Elliott 2000). XMT provides a perfect non-destructive mechanism to visually see and record the micro-structural characteristics of the materials with a maximum of 4 microns pixel resolution (Wong et al. 2004). Various kinds of XMT systems are employed to study dental caries. The use of different X-ray sources is the distinguishing feature among these types of XMT systems. As determined by the Beer’s law, the artifacts of beam-hardening emerge from polychromatic sources since the incident X-ray beam attenuation is not exponentially associated with the thickness of the material (Anderson et al. 1996). Therefore, the energies of the lower X-ray from the polychromatic spectrum are easy to penetrate whereas the higher X-ray energies of the polychromatic spectrum are less attenuated. In CT images, another vital source of artifacts is scattering, particularly in the case of cone-beam geometry application, just like in XMT (Davis et al. 2010). In generally, the most commonly used parameter that should importantly be acquired in the investigation of caries is the density of the minerals of the tooth or its variation from a certain site. The changes in X-ray attenuation are used to determine the mineral density profiles. Researchers have found that the X-ray attenuation coefficient directly relates to the mineral density. In the reconstructed XMT slices, the grey levels directly relate to the attenuation coefficients within a given object. The XMT scan can provide linear attenuation coefficient (LAC) that can be used as the parameter denoting the mineral density of the dental hard tissues due to which the process of measurement and the data handling becomes easier (Davis and Elliott 2003). The LAC can be defined as the degree to which the energy beam’s intensity falls as it goes through a certain material. If the value of LAC is small then it suggests that the material to be analyzed is comparatively transparent whereas the larger LAC values exhibit the greater level of opacity. In general, the LAC will be lower when the material density is lower and the radiation energy is higher (Davis & Wong 1996). Since, the caries studies reflect that most of the X-ray attenuation is caused by the minerals present in the dental tissues, particularly the hydroxyapatite, and the LAC is not much affected by the normal changes in the chemical composition of the minerals in the enamel therefore any change in the LAC for enamel directly relates to the density of its mineral. Many researchers have studied the issue of developing calibration standards for application, which are also called “phantoms”, when investigating dental hard tissues (Potter et al. 2006). Phantoms can be commercially produced but they would be comparatively costly. Phantoms may also be developed from different materials like wire or post of pure aluminum having diverse dimensions. However, phantoms should be ideally made up of materials that are associated with the examined tissues. Since HAP-hydroxyapatite is that main mineral component found in the dental enamel, thus, pure crystalline hydroxyapatite can be used to represent enamel as it approximates the enamel composition. 3.3. Application In general, visual inspection and simple statistics obtained from the XMT tomograms are adequate for the engineers for taking effective decisions whereas on the other hand, the first step for investigating the structure related properties of interest is to acquire the structure. It is now possible to scan and reconstruct the real structures bearing the porous media with the help of using the advance three dimensional techniques of structure characterization. Computer models are also available for particles, which support towards the development of realistic structures. XMT provides packing structures in the format of digital volume. Granulation is a frequently used technique in the pharmaceutical industry to produce granular products. Granules manufactured in this fashion are usually aggregates of finer particles. The known properties such as the mechanical strength and dissolution of the aggregate particles are based upon their internal structures and dissemination of various components. XMT provides a perfect technique to acquire the required structural information. XMT is a very effective tool for the purpose of acquiring microstructures in a non-destructive manner. XMT has already proved its great capability in numerous applications for helping both academics and field engineers alike. 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