CHAPTER I INTRODUCTION Though there is a general agreement that a marked reduction in caries prevalence in recent decades, caries is still one of the most common diseases and increasing in many of the developing countries. The decreasing trend of dental caries may be substantially related to the oral health habits and the incorporation of fluoride in all possible ways, let alone water. Other factors such as the oral health education and delivery systems, the availability and utilization of the oral health services and the upward shift of socioeconomic status may all have contributed to this declining trend. However, the whole picture of the dental caries is not that straight forward. Until recently, dental caries remained one of the most prevalent diseases of the world population. According to the U.S Health and Human Services report (2000), 94% of the adults have experiences of tooth decay in their lifetime. Moreover, the prevalence of caries in less affluent societies is not declining. The competitive and busy life style of the developing countries, sugary diet and soft drinks may deleteriously concoct the upsurge of dental caries. Quite a few studies have been done to understand dental caries. Various instruments and techniques have been applied to characterize the mechanism of the carious process. Generally, the pathogenesis of caries formation can be simplified into two processes (Higuchi et al., 1969; Moreno and Zahradnik, 1974; Vogel et al., 1987). (1) Dissolution of the enamel inorganic constituents (2) Diffusion of ions in and out of the dental enamel Therefore, the prevention of dental caries is emphasized strategically (1) on the strengthening of the enamel structures to be able to resist acidic challenge and thereby to reduce dissolution and (2) to block out or lessen the diffusion process inside the enamel by sealing diffusion channels or pathways. One of the most promising technologies in caries prevention comes with the introduction of laser into dentistry. Excitingly, the application of lasers on the dental enamel has been related to both the preventive pathways: strengthening the enamel crystalline structure (Holocomb and Young, 1980; Hsu et al., 2000) and/or decreasing the diffusion process (Hsu et al., 2000). Although the laser was introduced into dentistry with high expectation a few years ago, it has not come out yet as a clinical preventive tool for dental caries. The diffusion phenomenon in enamel is a subtle and complicated process. Yet, it is one of the key principles and is related to many physiological, pathological and clinical processes in dentistry. The carious process, the topical delivery of dental therapeutics and the fluid dynamics of the pulpo-enamel continuum are a few examples, which are intrinsically related to the enamel diffusion. A few studies have been done to explain enamel diffusion. But due to the technical limitations and difficulties in designing research, most of the documented studies were qualitative or semi-quantitative. The reliable quantitative data on the enamel diffusion were scarce. Though the laser-induced enamel porosity changes have been demonstrated (Ying et al., 2004), the effect of laser on diffusion has not been quantified yet. At least four research methods have been applied in determining enamel diffusion, namely, conductometry measurement, diffusion cell method, penetration profile study and electromotive force measurement. Unfortunately, those methodologies applied are time consuming, difficult to conduct and reproduce, prone to error, and most importantly, not flexible to tailor for specific needs and characterization. Therefore, the enamel diffusion remains a mystery of the modern world. With the advent of fluorescence technology, there arose two classical methods to quantify diffusion process in biological tissues: Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery After Photobleaching (FRAP). Coupled with versatile Confocal Laser Scanning Microscopy (CLSM), it becomes easier to perform FRAP in biological specimens and consequently it becomes one of the most frequently and widely used techniques in biophysical and pharmaceutical research. However, no publication is available for dental hard tissues. With the consideration of all these factors together, the following diffusionrelated knowledge-gaps become important research topics in dentistry. (1) The enamel diffusion, being centrally related to most dental procedures, has not been characterized quantitatively and site-specifically. (2) There is a lack of technique that can be applied to measure diffusion in dental hard tissue directly and quantitatively. Ideally, the technique should be readily applicable, versatile, fast, accurate and reproducible. (3) Related to enamel diffusion, the effectiveness and the mode of action of dental laser need to be quantified. In this study we explored the applicability of FRAP coupled with CLSM in quantifying enamel diffusion site-specifically to reveal the effectiveness of Er: YAG laser treatment. Furthermore, the role and magnitude of contribution of organic matrix in the laser-induced retardation of enamel diffusion was quantified. Finally, another fluorescence technique under CLSM, called time-series analysis or fluorophore transport study (FTS), was used to substantiate the FRAP measurements and to measure the diffusion process that occurred through the intact natural enamel surface. Therefore, the objectives of this study can be tabulated as followed: (1) To evaluate the feasibility and accuracy of FRAP coupled with CLSM as a research tool for measuring diffusion in dental enamel. (2) To quantify micro-diffusion in lased and normal enamel sections site- specifically. (3) To verify “organic blocking theory” by quantifying the effect of laser on the normal enamel (OM+) and OM extracted enamel (OM-). (4) To explore the behavior of diffusion through the natural surface over time. CHAPTER II LITERATURE REVIEW 2.1. Human Dental Enamel Dental enamel is the hardest tissue in the human body and is the outermost structure that covers the crown of the tooth in the oral cavity. Moreover, mature enamel is a totally acellular tissue. Unlike dentine, it originates developmentally from ectoderm. 2.1.1. Structures of Enamel 2.1.1.1. Enamel Rod or Enamel Prism Enamel rods or enamel prisms are thin and long structures that extend from the dentino-enamel junction (DEJ) to the enamel surface. The width of the enamel rods are highly variable, but roughly they are around μm (Meckel et al., 1965). Obviously, the length of enamel prism is limited by the thickness of enamel, which is widely variable individually. Strictly speaking, the length of the enamel prism may be longer than enamel thickness since it takes some wavy course inside the enamel. The size of the prism in cross-section appears smaller near the DEJ (~3 µm) and larger near the outer surface (~ 6µm). It may be due to fan-like arrangement and larger available space toward the enamel surface. In cross section, the enamel rods are described classically as a keyhole shape and typically the head (keyhole) is directed occlusally or incisally and the tail part is oriented cervically (Fig 2.1). The cross-sectional appearance may vary from circular to some irregular outlines in some areas especially near the enamel surface or DEJ. The orientation and direction of the enamel prism is clinically important since fracture of the enamel usually occurs along the enamel prisms especially if they are left unsupported during any clinical procedure. Moreover, the diffusion process, one of the mechanisms involved in dental caries, is largely controlled by the arrangement of prism and interprismatic spaces. Head of the Enamel Prism Tail of the Enamel Prism Enamel Crystals Enamel Prism Fig 2.1 Enamel crystals arrangement and orientation in the enamel prism (Meckel et al., 1965). As the direction and arrangement of enamel prisms are important clinically, it is worth understanding the wavy course of enamel prism. It starts perpendicular to the DEJ, deviates in the horizontal plane, and takes a wavy course specific for each row of prisms. As it approaches the outer third of enamel, the prisms become parallel and meet the outer enamel surface at right angles. The number of enamel rods on the enamel surface is variable, averaging around 20,000 to 30,000 per square millimeter (Fosse, 1964). At the dentinal end, the number of rods may be higher about 10% (Schroeder, 1991) due to the smaller cross-sectional size. The structure and composition of the interprismatic zone is a highly contentious area. It is generally accepted that there is no fundamental difference between prism and interprismatic zones in terms of chemical composition since both contain 86% to 88% by volume of crystallites (Angmar-Mansson, 1970). The angle between crystals of two adjacent prisms can be as large as 60° and therefore, it was postulated that the microscopic appearance of distinct interprismatic areas, which is totally different from prismatic area, may be an optical phenomenon that arises from the different orientations of the crystals. However, using electron microscopy the presence of prism sheath has been clearly documented (Travis and Glimcher, 1964; Nakata et al., 1982). These prism junction areas are believed to be important in providing the principal diffusion pathways through the enamel because of extra spaces and condensation of the organic matrix (Frank, 1966). Enamel is a structurally highly variable tissue and a good example is the prismless enamel which is commonly seen at the surface of the deciduous teeth. The prismless enamel could be as thick as 15 – 40µm (Gwinnett, 1966a, Simmelink, 1994,). The prismless enamel is believed to arise during the final phase of amelogenesis. In this area, the inorganic crystals are arranged perpendicular to the enamel surface (Gwinnett, 1966b; Speirs, 1971). The prismless enamel is less common in the permanent and erupted teeth, and most commonly seen at the gingival third (Ripa et al., 1966, Gwinnet, 1967). These structural variations of enamel within a tooth may need site-specific tools to determine diffusion accurately. 2.1.1.2. Enamel Crystals Enamel crystallites can be viewed as the structural unit of enamel. Generally, the crystals are believed to be oriented parallel to the long axis of the enamel rods in the head region of the keyhole-shaped prism. However, X-ray diffraction and polarized light microscopy studies suggest that the enamel crystallites are not arranged parallel to the long axis of the prism (Poole and Brook, 1961; Carlstrom, 1964) but may possibly be orientated as much 30° away from the prism axis. The crystallites from head and tail regions within a single prism are oriented differently. Specifically, the orientation of crystallites changes gradually from parallel to the long axes of the prism in the upper part of the head region to 60-70° angles in the tail region (Meckel et al., 1965) (fig 2.1). In cross section, the crystallites are hexagonal in shape, slightly flattened, sized about 20-60 nm thick, 30-90 nm wide and variable length (Orams et al., 1976). Although the shape of the crystal is classically described as hexagonal, it may vary and become irregular sometimes. All these hydroxyapatite crystals in the keyhole-shaped enamel prism are embedded in the submicroscopic amount of organic matrix which amounts only 1-2 % by volume of mature enamel (Angmar-Mansson,1970) resulting in a tightly packed structural arrangement with less than nm spaces between crystals (Simmelink, 1994). The intercrystal spaces together with the filled-up organic matrix, though small compared to interprismatic spaces, may theoretically contribute to a certain fraction of overall diffusion because the size and organic content of the intercrystal space may have a sieving effect on certain molecules. 2.1.1.3. Enamel Tufts, Lamellae, Spindles and Cracks These structural variations can be viewed as localized enamel defects. Lamellae are improperly mineralized areas during maturation while tufts are areas with defective maturation of young enamel proteins. Lamellae extend from the enamel surface toward the DEJ, but tufts extend form the DEJ, typically from the crest of the scallop, to about one third of the enamel thickness. Enamel lamellae and tufts are areas filled up with organic materials. But lamellae can be developed after maturation due to strain and, in this case, the areas may be filled with exogenous material from the oral cavity. Enamel spindles are developmentally related to dentine and spindle-shape areas with higher organic content, extending from the DEJ into the enamel. Enamel spindles may trap odontoblastic processes inside the enamel and can be found elsewhere along the DEJ (ten Cate, 1998). The accumulation of higher organic content may make these areas physicochemically different from other parts of enamel. Hence, these areas may have different behavior of diffusion, hypothetically. 2.1.1.4. Striae of Retzius Striae of Retzius are incremental lines of daily growth in enamel, around µm apart and named after the anatomist Anders Retzius (1796-1860). Striae or incremental lines of Retzius are concentric growth rings run from the DEJ obliquely toward the occlusal surface and appear as brownish under transillumination in the ground coronal section. Lines of Retzius are believed to correspond to the resting phase of active secretory process during amelogenesis. Under scanning electron microscopy, hydroxyapatite crystals in Striae of Retzius are irregularly arranged and fewer than cross striations. Normally, one special line of Retzius formation appears shortly after birth and, therefore, it may denote nutritional changes and disturbances in the growth pattern. This accentuated line of Retzius formed shortly after birth is termed neonatal line (ten Cate, 1998). Lines of Retzius that reach the enamel surface and form horizontal ridges are called Perikymata or imbrication lines that are more prominent on the facial surface of the crown. There is no literature available documenting whether this developmental pattern is related to diffusion channels or involves in any transportation mechanisms. 2.1.1.5. Cross Striations of the Prisms Cross striations are believed to be formed by diurnal variation in the secretion rate of ameloblasts during enamel formation. They appear at regular intervals of approximately 4µm distance in longitudinal ground sections if etched slightly and appear as dark parallel bands passing through the prisms at right angles. Cross striations can be seen as hypomineralised zones in microradiographs (Gustafson and Gustafson, 1967). 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Archs Oral Biol. 20: 317-325. 162 [...]... exchange of labeled materials between immersion medium and enamel Therefore, the authors commented that the very high value of DCs which did not differed much from DC of water (10-5 cm2/s) can be due to the presence of cracks In another extreme, very low values can be suspected of the chemical reaction of the diffusion ions with the surface of the enamel crystals The authors concluded that the DC of sound... temperature more than the diffusion in bulk water, which they suggested as an indication of high activation energy of diffusion (Zahradnik and Moreno, 1975) Moreover, the complex diffusion process shows by the enamel membrane has been related to thermodynamic properties of constituents (Burke and Moreno, 1975) 2.2.4 Enamel Diffusion Measurements A few techniques have been used to characterize the diffusion. .. strengthens the bond between enamel and dentine The DEJ, the area where caries may rapidly spread laterally, has a higher organic content 11 2.1.1.9 Water Structure of Enamel The water structure of enamel is one of the least understood areas in enamel studies The water content of enamel may vary with age and tooth type (Moreno and Zahradnik, 1973) and with relative location of enamel in the tooth (Brudevold... radioactive tracer, i.e a radio-labeled ion, is added to the solution After a certain period, the sample is removed from the labeled solution, washed and dried The enamel blocked is cut out, the surface area of block is determined and the surface is ground into parallel layers of a 27 few micron thicknesses by a series of abrasive paper The weight of the layer removed is determined by a microbalance... quantitative measurements on enamel diffusion are scant It may be related to the difficulties in designing the experimental techniques, time-consuming nature of enamel section preparation and, worst of all, restrictive diffusion behavior of enamel membrane To sum up, the enamel diffusion still remains a modern mystery which deserves more research for a better understanding of the molecular sieve behavior,... water structures in relation to the transport mechanism and, last but not least, the role played by organic matrix 2.3 Laser in Dentistry 2.3.1 Introduction The first laser was a pulsed ruby laser of 0.694 µm wavelength developed by Theodore H Maiman of Hughes Aircraft Corporation in 1960 In dentistry, laser research, according to documents available, started since 1963 by Stern and Sognnaes Earlier... concentrations of ions in the removed materials in the successive layers are measured and plotted against the distance from the original enamel surface (fig 2.4) Grinding by layers Enamel block Tracer solution Covering material Concentration Tracer concentration in each layers is translated into profile plot Depth Fig 2.4 The illustration of the procedures involved in the tracer transport study The experimental... 28 where ‘erfc’ is the complementary to error function (erf) and expressed as ‘erfc y =1-erf y’, ‘c0’ is tracer concentration at the surface, and ‘t’ is diffusion time Driessens (1982) suggested that in applying this technique for enamel, the time necessary for the formation of the penetration profile must be long compared to the sampling time of enamel for determination of the penetration profile... by using nitroxide spin-labeled solution, the concentration profile inside the enamel is detected by EPR spectrometer at different time intervals From the time-dependent distribution function of EPR spectra, the corresponding transport parameters can be calculated by deconvolution of the field gradient (Skaleric et al., 1987) Another interesting method is the clearance study in which the slabs of enamel. .. hypothesized, as the phosphate diffusion profile was significantly different from that of calcium and fluoride, that the difference in surface potential of enamel or difference in charges between ions may affect the diffusion 2.2.3.4 Water structure Segmental studies conducted by 24 Na and 18 F demonstrate that the uptake of these ions by enamel is fastest at the deepest part of enamel, that is near the . effectiveness of Er: YAG laser treatment. Furthermore, the role and magnitude of contribution of organic matrix in the 3 laser- induced retardation of enamel diffusion was quantified. Finally, another. difference in orientation of groups of enamel rods and known as Hunter-Schreger bands named after J. Hunter (1729-1793) and D. Schreger 1766-1825). 2.1.1.7. Gnarled Enamel Gnarled enamel. quantitative data on the enamel diffusion were scarce. Though the laser- induced enamel porosity changes have been demonstrated (Ying et al., 2004), the effect of laser on diffusion has not been