Morphology and Structural Characterization of Human Enamel and Dentin by Optical and Scanning Electron Microscopy

https://doi.org/10.5272/jimab.2019254.2744 Journal of IMAB Journal of IMAB - Annual Proceeding (Scientific Papers). 2019 Oct-Dec;25(4) ISSN: 1312-773X https://www.journal-imab-bg.org Original article

MORPHOLOGY AND STRUCTURAL CHARACTERIZATION OF HUMAN ENAMEL AND DENTIN BY OPTICAL AND SCANNING ELECTRON MICROSCOPY

Ekaterina Karteva1, Neshka Manchorova-Veleva1, Zhelyazko Damyanov2, Teodora Karteva1 1) Department of Operative Dentistry and Endodontics, Faculty of Dental Medicine, Medical University -Plovdiv, Bulgaria 2) Institute of Mineralogy and Crystallography “Acad. Ivan Kostov”, Bulgarian Academy of Sciences, Sofia, Bulgaria.

ABSTRACT rations. The transparency of enamel is also influenced by Purpose: The aim of this paper is to visualize and the stage of mineralization and its homogeneity. examine the structural features of enamel and dentin by ob- The enamel consists of 95-98 wt.% mineral content, servations in optical and scanning electron microscopes. mostly hydroxyapatite, 1-2 wt.% organic matter, and about Material/Methods: Optical microscopic investiga- 4 wt.% water. It is composed of enamel prisms – about 5 mil- tions were carried out on crown and root dentin of intact lion in incisors and up to 12 million in molars [2]. Their human teeth, extracted for orthodontic or periodontal rea- path is uninterrupted, starting from the enamel-cemental sons. The prepared samples were examined in transmitted junction (ECJ) and ending at the enamel surface or in the and reflected light by Leitz Orthoplan-Pol polarizing micro- aprismatic layer. Their direction is parallel to the long axis scope with digital image attachment using observations in of the tooth in the incisal edge and the molars cusps. Along plane polarized light, crossed nicols (polars) and through λ- the lateral regions of the crown, their direction is oblique to plate phase-advancing compensator. Gold-coated freshly the long axis, whereas in the cervical region they become broken pieces from the same samples were examined by perpendicular. In the internal enamel regions, they have a Philips 515 scanning electron microscope (SEM) operated distinct S-shaped curve. Enamel is brittle, with a high elas- at 30 kilo electron volts (keV). ticity modulus, but little tensile strength. It is supported by Results: Transmitted light photomicrographs of the dentin, which has a lower elasticity modulus and can enamel were obtained, showing detailed images of structural withstand higher values of masticatory pressure due to its characteristics like enamel spindles, tufts, lamellae and unique structure. Hunter-Shreger bands. The images from dentin visualized the Dentin structure is responsible for the mechanical lateral canals, mantle dentin, ortodentin, globular dentin and properties of the tooth. Dentin consists of 75% inorganic granular dentin. The SEM images showed cross-sectional and material, 20% organic matter and about 5% water and other longitudinal images of the dentinal tubules, peritubular den- substances. Its hardness is only about 1/5 of that of enamel, tin and the collagen network with hydroxyapatite crystals. with its highest values near the enamel cemental junction. Conclusions: The results of this study showed that the Its modulus of elasticity is about 1,67x106 PSI, which helps morphostructural characteristics of the hard dental tissues in support of the brittle enamel [3, 4]. Dentin is secreted and could be successfully observed and studied by the main tech- formed by odontoblasts, with its main morphological struc- niques of polarizing optical microscopy and SEM. The ob- ture – the dentinal tubule [4]. servations provided highly-contrast, detailed and informa- Due to their crystalline structure, relative transparency tive images of enamel and dentin. and anisotropy, enamel and dentin can be observed and studied by the techniques of polarizing optical microscopy re- Keywords: SEM, dentin, enamel, morphology, struc- vealing various features of their building ingredients, such ture, optical microscope as size, form of aggregates, inner structure, mutual relationships, optical characteristics, relative micro hardness, etc. [5]. INTRODUCTION In addition to the optical microscopy, the scanning electron Enamel is the most mineralized substance in the hu- microscopy can provide high-quality, detailed images giv- man body. Its width varies in different tooth regions, from 2 ing information about the morphology, topography and mm in the incisal edge of frontal teeth, up to 2.4 – 3 mm in structure of the objects studied [6]. The aim of this paper is the molar cusps [1]. Since enamel is translucent, its colour- to reveal and describe the features of enamel and dentin ing depends on the colour of the underlying dentin, the structures by observations in optical and scanning electron width of the enamel layer and the presence of discolou- microscopes.

2744 https://www.journal-imab-bg.org J of IMAB. 2019 Oct-Dec;25(4) MATERIALS AND METHODS Optical microscopic investigations were carried out to reveal inner structure, size and phase relationships of crown and root dentin of intact human teeth, extracted for orthodontic or periodontal reasons. Two thin and two polished sections were prepared by standard preparation techniques from representative samples of crown and root dentin. The thin sections were about 0.03 mm in thickness. They were cemented to glass slides with Canada balsam. Before cutting the samples were visually oriented in a parallel and cross sectional direction. The same orientation was also used for the polished sections – the samples were cemented in the epoxy resin matrix and finely polished. The prepared sections were examined in transmitted and reflected light by Leitz Orthoplan-Pol polarizing microscope with digital image attachment using the main techniques of mineral optics study – observations in plane po- Fig. 2. Transmitted light photomicrographs of root λ larized light, crossed nicols (polars) and through -plate dentin structure in plane polarized light under parallel phase-advancing compensator [8]. Observations of thin sec- nicols (A), crossed nicols (B), and λ-plate compensator (C). tions from transparent minerals (phases) and their aggre- The lumen of the root canal is visible. gates in plane polarized light provide information about their shape, form, grain size, quantity, color and its inten- sity, cleavage, inclusions, intergrowths, structures. Under crossed nicols, the anisotropic minerals (phases) show interference colors allowing to determine different optical characteristics and some structural details. The λ-plate compensator produced interference colors from higher order, thus providing advanced phase and structural recognition. Gold-coated freshly broken pieces from the same samples were examined by Philips 515 scanning electron microscope (SEM) operated at 30 kilo electron volts (keV) to reveal micromorphology, size and phase intergrowths of crown and root dentin’s structure.

RESULTS Fig. 1. Transmitted light photomicrographs of enamel and dentin structure in plane polarized light under parallel (A) and crossed (B) nicols.

J of IMAB. 2019 Oct-Dec;25(4) https://www.journal-imab-bg.org 2745 Fig. 3. Transmitted light photomicrographs of crown Fig. 4. Transmitted light photomicrographs of granu- dentin structure in plane polarized light under parallel lar dentin structure in plane polarized light under parallel nicols (A), crossed nicols (B), and λ-plate compensator (C). nicols (A), crossed nicols (B), and λ-plate compensator (C).

2746 https://www.journal-imab-bg.org J of IMAB. 2019 Oct-Dec;25(4) Fig. 5. Transmitted light photomicrographs of root Fig. 6. Secondary electron (SE) photomicrographs of dentin structure in plane polarized light under parallel coronary dentin (A) and root dentin (B) at different magni- nicols (A), crossed nicols (B), and λ-plate compensator (C). fications. A distinct atubular zone is visible.

J of IMAB. 2019 Oct-Dec;25(4) https://www.journal-imab-bg.org 2747 There are several characteristics of the enamel struc- tissues meet and acts to prevent the propagation of cracks ture that became visible during this study: from enamel into the dentin. It consists of scallops with 1. Enamel tufts (Fig. 1A). They are located in the in- convexities directed toward the dentin and concavities to- nermost layer of enamel, extending from the dentinoenamel ward the enamel. According to Marshall et al. [9], the DEJ junction into the enamel. The junction itself has a wave- consists of three levels, with scallops of 25-–100 µm, like appearance, and the tufts start from the tips of the thus microscallops of 2–5 µm and a smaller scale structure. formed folds. The morphological structure of dentin is defined by 2. Enamel spindles (Fig. 1A). They extend from the the dentinal tubule. The tubules are visible on our scan- dentinoenamel junction towards the enamel surface. ning electron microscopy images in two sections – longi- 3. Enamel lamellae (Fig. 1A). The lamellae are tudinal and cross-sectional (Fig. 6). The tubules are formed enamel cracks, located at different levels. during dentinogenesis around the odontoblasts’ processes 4. Hunter-Shreger bands (Fig. 1B). These lines be- (Tomes processes). They originate in the zone above the came visible under observations with crossed nicols. They pulp chamber, pass through the whole width of the dentin are not elements of “real” enamel structure, but a result of and terminate at the DEJ. The tubules are more densely light refraction and the wave-like direction of the prisms. packed near the pulp, whereas near the DEJ their lumen de- The bands appear as alternating light and dark lines with creases and the intertubular spaces increase. Each tubule different thickness. is enveloped in a highly mineralized layer of peritubular dentin. In between the tubules, the less mineralized inter- The following layers of dentin could also be ob- tubular dentin is located. The odontoblasts’ processes served: branch off in numerous lateral processes that create lateral 1. Mantle dentin. It is the first secreted layer of den- canals (canaliculi) (Fig. 1A) [10, 11]. The cross sectional tin, located directly below the DEJ (Fig. 1B). images with the optical microscope show the S-shaped di- 2. Orthodentin. This is the so-called “dentin proper”. rection of the dentinal tubules (Fig. 2). It resembles the path 3. Globular dentin (Fig. 1B). It is located between of the enamel prisms and increases the tooth’s resilience the mantle dentin and orthodentin. to masticatory pressure. 4. Predentin. This is the newly-secreted, not yet min- Mantle dentin is highly mineralized, with traces of eralized dentin matrix, located just above the pulp. organic material only, as there are no disturbances or de- 5. Granular dentin (Fig. 4). It is located on the sur- fects during its formation [12] (Fig. 1B). Korff fibers are face of the root dentin, under the cement layer. It is also abundant, which aids in the close alignment of hydroxya- called Tomes granular layer and is visualized as lines of patite crystals. Its thickness is about 15-30 µm. Compared dark granules that lie parallel to the outer surface of the to the other zones of dentin, it has fewer tubules. Ortho- dentin. dentin is less mineralized compared to mantle dentin. Korff fibers are fewer, with more ß-fibers, secreted from DISCUSSION the odontoblasts [13]. They are scattered, which reflects The enamel tufts, spindles and lamellae, as well as on the proper mineralization of this layer. Globular den- the dentinoenamel junction (DEJ) with its numerous lat- tin is the least mineralized layer of the three, appearing eral canals, are clearly visible on Fig. 1A. Enamel tufts are as lighter rounded areas (Fig.1B). The archlike darker ar- low-mineralized, with high organic content. They are eas, visible in between the globular dentin, are consid- thought to be a result of defects in ameloblasts’ function ered a result of incomplete mineralization, where the glob- and can act as “pathways” for caries progression. Spindles ules of dentin did not fuse completely. This is the inter- are formed by the entrapment of the processes of odonto- globular dentin and is most common in the coronal den- blasts between ameloblasts during the tooth’s develop- tin, near the DEJ and in dentinogenesis imperfecta. These ment. When the ameloblasts start secreting enamel, those areas are also known as Chermak’s zones [14]. In preden- processes are left “enclosed” into the enamel structure. It tin, separate globules can be visible, which is a sign of is thought that they can act like “pain receptors”, which commencing mineralization [13]. Granular dentin might can explain some cases of pain sensitivity during prepara- be a result of the disorientation of odontoblasts during tion. Enamel lamellae contain mainly organic matter, which root dentinogenesis, or an increased globular layer with can ease the penetration of acids and microorganisms in larger interglobular spaces (Fig. 4). caries development. There are 2 paths for lamellae forma- On Fig. 5 an atubular zone can be traced. This zone tion: a defect in ameloblasts’ development or mechanical is located in the root dentin in an intact tooth sample. Usu- pressure after enamel maturation. Hunter-Shreger bands are ally, such zones are a result of the occlusion of dentinal located in different regions of the enamel in different teeth, tubules due to carious decay or abrasion and are callled due to the various direction of the prisms. In incisors, they “tertiary” or “reactionary” dentin. In our samples, however, are located near the incisal edge, whereas in molars they such conditions are not present. A possible explanation of can be found anywhere from the cervical region till the the observed microscopic images can be found in the gen- cusps’ tips [1, 2]. esis and morphology of transparent dentin. It is character- The DEJ is a complex structure where two different ized by smaller crystallite size, which is a result of the spe-

2748 https://www.journal-imab-bg.org J of IMAB. 2019 Oct-Dec;25(4) cific conditions during its formation. According to the lit- The SEM images show areas in coronary and root erature, it is progressively accumulated with ageing in vi- dentin at different magnifications (Fig. 6). The coronary tal, as well as non-vital teeth. dentin photomicrographs show cross-sectional images of Transparent dentin is physiologically formed throug- the dentinal tubules with their lumen (Fig. 6A). The hout the lifespan of the tooth [15]. During its formation, peritubular dentin is visible, as well as a complex network accumulation of mineral substances in the lumen of of collagen fibers with scattered hydroxyapatite crystals, dentinal tubules occur, which resembles the formation of forming the intertubular dentin. The root dentin photo- sclerotic dentin. Its deposition begins 3-4 years after the micrographs show the dentinal tubules in a longitudinal eruption of the tooth, first in the apical part of the root and section, with remnants of collagen fibers forming the then more coronally, which can explain its location in our peritubular dentin in some of the sectioned zones (Fig. 6B). root sample. The amount of transparent dentin increases with age [16]. Its name is derived from its optical qualities CONCLUSIONS [17]. The most popular statement for the origin of trans- The results of this study showed that the morpho- parent dentin discusses the transportation of minerals from structural characteristics of the hard dental tissues could the intertubular matrix towards the dentinal tubules. This be successfully observed and studied by the main tech- process is called “dissolution - reprecipitation” of the min- niques of polarizing optical microscopy and SEM. The eral content [18, 19]. According to that view, peri- and in- observations provided highly-contrast, detailed and intertubular dentin are the sources of calcium ions released formative images of enamel and dentin, proving useful for under the influence of a certain event (pulp hypoxia, the future study of dental tissues in both norm and pa- apoptosis) and then reprecipitated into mineral content that thology. occludes the tubules.

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J of IMAB. 2019 Oct-Dec;25(4) https://www.journal-imab-bg.org 2749 Please cite this article as: Karteva E, Manchorova-Veleva N, Damyanov Z, Karteva T. Morphology and structural characterization of human enamel and dentin by optical and scanning electron microscopy. J of IMAB. 2019 Oct- Dec;25(4):2744-2750. DOI: https://doi.org/10.5272/jimab.2019254.2744

Received: 17/05/2019; Published online: 29/10/2019

Address for correspondence: Ekaterina Karteva, Department of Operative Dentistry and Endodontics, Faculty of Dental Medi- cine, Medical University –Plovdiv, 7, Dimcho Debelianov str., Plovdiv 4000, Bulgaria. E-mail: [email protected] 2750 https://www.journal-imab-bg.org J of IMAB. 2019 Oct-Dec;25(4)