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1 口腔胚胎及組織學 Oral embryology & histology Enamel 學習目標 能了解頭臉部及口腔之發育 結構與功能, 並發展能有效吸收與傳播相關資訊之辭彙, 將胚胎及組織學上之基本概念, 應用於臨床上之診斷與治療 臺北醫學大學牙醫學系鄧乃嘉醫師 參考資料 Summary 1.Illustrated Dental Embryology, Histology, and Anatomy: Mary Bath-Balogh, Margaret J. Fehrenbach, 2nd Edition, Elsevier Saunders, Esstential of Oral Histology and Embryology--A clinical approach: James K Avery, Denial J Chiego,Jr 3rd Edition, Elsevier Mosby, Ten Cate's Oral Histology: Development, Structure, and Function: Nanci Anatonio, 6th ed. Mosby, 2003 提供學生探討頭臉部及口腔之發育, 認識牙齒及口腔組織之正常顯微結構, 並了解其功能與結構之關係, 進而對生理 病理與臨床牙科學上之問題能做比較與思考, 為各牙科臨床科目之重要基礎課程 ENAMEL Physical characteristics Enamel's primary mineral is hydroxyapatite, which is a crystalline calcium phosphate. The large amount of minerals in enamel accounts not only for its strength but also for its brittleness. Dentin, which is less mineralized and less brittle, compensates for enamel and is necessary as a support. Unlike dentin and bone, enamel does not contain collagen. Instead, it has two unique classes of proteins called amelogenins and enamelins. While the role of these proteins is not fully understood, it is believed that they aid in the development of enamel by serving as a framework the hardest and most highly mineralized substance of the body, and with dentin, cementum, and dental pulp is one of the four major tissues which make up the tooth. It is the normally visible dental tissue of a tooth and must be supported by underlying dentin. 96% of enamel consists of mineral, with water and organic material composing the rest Enamel varies in thickness over the surface of the tooth and is often thickest at the cusp, up to 2.5 mm, and thinnest at its border, which is seen clinically as the cementoenamel junction (CEJ) 1

2 Structure of enamel The basic unit of enamel is called an enamel rod. Measuring 4 μm - 8 μm in diameter an enamel rod, formerly called an enamel prism, is a tightly packed mass of hydroxyapatite crystals in an organized pattern. In cross section, it is best compared to a keyhole, with the top, or head, oriented toward the crown of the tooth, and the bottom, or tail, oriented toward the root of the tooth. The area around the enamel rod is known as interrod enamel. Interrod enamel has the same composition as enamel rod, however a histologic distinction is made between the two because crystal orientation is different in each. The border where the crystals of enamel rods and crystals of interrod enamel meet is called the rod sheath. Scanning electron microscope views of (A) the enamel layer covering coronal dentin, (B) the complex distribution of enamel rods across the layer, (C and D) and perspectives of the rodinterrod relationship when rods are exposed (C) longitudinally or (D) in cross section. Interrod enamel surrounds each rod. R, Rod; IR, interrod; DEJ, dentinoenamel junction. Hydroxyapatite A and B, Highresolution scanning electron microscope images showing that crystals in rod and interrod enamel are similar in structure but diverge in orientation. P6 3 /m Ca 5 (PO 4 ) 3 OH D.R.Simpson, 1972) P Ca OH,F Transmission electron microscope images of a rod surrounded by interrod enamel from (A) young and (B) older forming enamel of a rodent. The crystals that make up the rod and interrod enamel are long, ribbonlike structures that become thicker as enamel matures. They are similar in structure and composition but appear in different planes of sections because they have different orientations. 2

3 Cross-sectional profiles of (A) recently formed, secretory stage enamel crystals and (B) older ones from the maturation stage. Initially the crystals are thin; as they grow in thickness and width, their hexagonal contour becomes apparent. B, The linear patterns seen in older crystals are a reflection of their crystalline lattice. Electron micrograph of mature enamel crystals. The outline is irregular as they press against each other. Rod and interrod Scanning electron microscope images showing various aspects of rat incisor enamel. A, The enamel rods (R) are arranged in rows with alternating orientations. B, The alternating row arrangement is also evident in the interrod (IR) cavities that accommodate the enamel rod. C, Rod and interrod enamel are made up of thin and long apatite crystals. The rod is shaped somewhat like a cylinder and is made up of crystals with long axes that run, for the most part, parallel l to the longitudinal l axis of the rod The interrod region surrounds each rod, and its crystals are oriented in a direction different from those making up the rod Fine structure of enamel. A, Crystal orientation along three faces of an enamel block. B to D, Transmission electron micrographs of the three faces. A and B, Decalcified preparation of cat secretory stage enamel. The organic matrix near the ameloblasts is younger and shows a uniform texture. t The distal portion of Tomes process (dptp) penetrates into the enamel. In deeper areas, near dentin, matrix is older and partly removed. Matrix accumulates at the interface between rod (R) and interrod (IR) to form the rod sheath (arrowheads). 3

4 Colloidal gold (black dots) immunocytochemical preparation showing the presence of amelogenin in the organic matrix that accumulates to form the rod sheath in maturing cat enamel. Rodents have no well-defined rod sheath; however, in decalcified preparations of maturing enamel a concentration of organic matrix (arrows) seems s to occur around most of the periphery of the rod (R), except at the zone of confluence (*) with interrod (IR). This matrix, like the one at other sites, is immunoreactive (black dots as enlarged in inset) for amelogenin. Structure of enamel Interrod partition associated with four rod profiles. At certain sites (arrows, zone of confluence) crystals from interrod enamel enter the rod. The arrangement of the crystals within each enamel rod is highly complex. Both ameloblasts (the cells which initiate enamel formation) and Tomes' processes affect the crystals' pattern. Enamel crystals in the head of the enamel rod are oriented parallel to the long axis of the rod. When found in the tail of the enamel rod, the crystals' orientation diverges slightly from the long axis. The arrangement of enamel rods is understood more clearly than their internal structure. Enamel rods are found in rows along the tooth, and within each row, the long axis of the enamel rod is generally perpendicular to the underlying dentin. In permanent teeth, the enamel rods near the cementoenamel junction (CEJ) tilt slightly toward the root of the tooth. Understanding enamel orientation is very important in restorative dentistry, because enamel unsupported by underlying dentin is prone to fracture. Amelogenesis 1. Produces a partially mineralized (approximately 30%) enamel. Once the full width of this enamel has been deposited.. 2. Significant influx of additional mineral coincident with the removal of organic material and water to attain greater than 96% mineral content. This mineral influx makes the crystal formed during the first step grow wider and thicker Amelogenesis 1. Presecretory stage: differentiating ameloblasts acquire their phenotype, change polarity, develop an extensive protein synthetic apparatus, and prepare to secrete the organic matrix ti of enamel. 2. Secretory stage: ameloblsts elaborate and organize the entire enamel thickness, resulting in the formation of a highly ordered tissue. 3. Maturation stage: ameloblasts modulate and transport specific ions required for the concurrent accretion of mineral. 4

5 Light microscopy of amelogenesis Enamel matrix formation as seen with the light microscope. The Tomes processes of ameloblasts jut into the matrix, creating a picket-fence appearance. initial secretory stage histodifferentiation stage morphogenetic stage Secretory stage secretory stage The various functional stages in the life cycle of the cells of the inner dental epithelium. 1, morphogenetic stage; 2, histodifferentiation stage; 3, initial secretory e stage (no Tomes process); 4, secretory stage (Tomes process); 5, ruffle-ended ameloblast of the maturative stage; 6, smoothended ameloblast of the maturative stage; 7, protective stage. secretory stage secretory stage secretory stage initial enamel inner enamel outer enamel Composite plate illustrating the morphologic changes that rat incisor ameloblasts undergo throughout amelogenesis. Am, Ameloblasts; BL, basal lamina; BV, blood vessel; D, dentin; E, enamel; IE, secretory stage inner enamel; InE, secretory stage initial enamel; Me, early maturation stage; Ml, late maturation stage; Mm, midmaturation stage; Od, odontoblasts; OE, secretory stage outer enamel; PD, predentin; PS, presecretory stage; TP, Tomes process; Tr, maturation stage transition. maturation early maturation late stage transition maturation 5

6 Representative micrographs of amelogenesis in the cat. A, Tooth formation shows an occlusal-to-cervical developmental gradient so that on some crowns finding most of the stages of the ameloblast life cycle is possible. The panels on the right (B corresponds with B1 and C with B2) are enlargements of the boxed areas: B, Secretory stage, initial iti enamel formation; C, secretory stage, inner enamel formation. D and E are from the incisal tip of the tooth (see Fig. 7-15). D, Midmaturation stage, smooth-ended ameloblasts; and E, late maturation stage, ruffle-ended ameloblasts. Am, Ameloblasts; D, dentin; E, enamel; N, nucleus; Od, odontoblasts; PL, papillary layer; RB, ruffled border; SB, smooth border; SI, stratum intermedium. ruffle-ended ameloblast of the maturative stage & smooth-ended ameloblast of the maturative stage Scanning electron microscope view of the enamel organ during the maturation stage. Cells from the stratum intermedium, stellate reticulum, and outer dental epithelium amalgamate into a single layer. Blood vessels invaginate deeply into this layer to form a convoluted structure referred to as the papillary layer. BV, Blood vessel; N, nucleus. Features of amelogenesis as seen through the light microscope. At A the inner dental epithelium consists of small, columnar undifferentiated cells. At B these cells elongate and differentiate into columnar ameloblasts that induce the differentiation of odontoblasts and then begin to secrete enamel (C). At D ameloblasts are actively depositing enamel matrix. Light microscopy of amelogenesis When enamel maturation is completed, the ameloblast layer and the adjacent papillary layer together constitute the reduced dental epithelium. Only an enamel space is visible in this histologic preparation because at this late developmental stage, enamel is heavily calcified and therefore is lost during decalcification. KEY 1. Inner dental epi. Became the ameloblast 2. Initial layer of enamel, which does not contain any rods 3. Tomes process 4. Postsecretory transition stage occurred just after the full thickness of enamel is completed 5. Papillary layer 6. Reduced dental epithelium 7. Protective phase 8. Junctional epithelium 6

7 Electron microscopy of amelogenesis Morphogenetic phase Presecretory stage 1. Morphogenetic phase 2. Differentiation phase mantle predentin ameloblasts Differentiating ameloblasts extend cytoplasmic projections (*) through the basement membrane (BM), separating them from the forming mantle predentin. The basement membrane is fragmented and removed before the active deposition of enamel matrix. mv, Matrix vesicle; sg, secretory granule. In Situ Hybridization In Situ Hybridization In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ), or, if the tissue is small enough (e.g. plant seeds, Drosophila embryos), in the entire tissue (whole mount ISH). This is distinct from immunohistochemistry, which localizes proteins in tissue sections. DNA ISH can be used to determine the structure of chromosomes. Fluorescent DNA ISH (FISH) can, for example, be used in medical diagnostics to assess chromosomal integrity. RNA ISH (hybridization histochemistry) is used to measure and localize mrnas and other transcripts within tissue sections or whole mounts. Colloidal gold immunocytochemical preparations illustrating the expression of amelogenin by differentiating ameloblasts. A, Amelogenin molecules are immunodetected (black dots) extracellularly early during the presecretory stage, before the removal of the basement membrane (BM) separating ameloblasts from the developing predentin matrix. Thereafter, enamel proteins (B) accumulate as patches (arrowheads) at the interface with dentin and then (C) as a uniform layer of initial enamel. djc, Distal junctional complex; im, infolded membrane; mv, matrix vesicle; Odp, odontoblast process; pptp, proximal portion of Tomes process; sg, secretory granule. A, Differentiated ameloblasts develop a junctional complex at their distal extremity. The cell extension above the complex is Tomes process and is divided into two parts. The proximal portion of Tomes process (pptp) extends from the junctional complex to the surface of the enamel layer, whereas the more distal portion (dptp) penetrates into enamel. B, Cross-sectional view of ameloblasts at the level of the distal junctional complex. This beltlike complex extends around the entire circumference of the ameloblast and tightly holds the cells together. Bundles of microfilaments (Cell web) run along the cytoplasmic surface of the complex. dcw, Distal cell web; DEJ, dentinoenamel junction; sg, secretory granule. Differentiation phase 7

8 Secretory stage Golgi complex is extensive and form a cylindrical organelle surrounded by rough endoplasmic reticulum Rod and interrod A, Cytochemical preparation for an enzyme resident in the Golgi complex showing the extent of this organelle throughout the supranuclear compartment of secretory stage ameloblasts. B, Scanning electron microscope image of a cross-fractured ameloblast. The Golgi complex has a cylindrical configuration and is surrounded by rough endoplasmic reticulum (rer). N, Nucleus; SI, stratum intermedium. SEE Fig 7-26 Comparative (A) scanning and (B) transmission electron microscope views of cross-cut secretory stage ameloblasts. The Golgi complex is located centrally and surrounded by cisternae of rough endoplasmic reticulum (rer). The preparation in B is immunolabeled (black dots) for amelogenin. Labeling is found not only in the Golgi complex and secretory granules (sg) but also in organelles involved in protein degradation such as multivesicular bodies (mvb). C, The organization of secretory stage ameloblasts as would be revealed in a section along their long axis. Immunocytochemical preparations for amelogenin. A, Immature (isg) and mature (msg) secretory granules are found on the mature face of the Golgi complex. B, Secretory granules are translocated into Tomes process and accumulate near secretory surfaces, recognized by the presence of membrane infoldings (im). dptp, Distal portion of Tomes process; IR, interrod; R, rod; rer, rough endoplasmic reticulum; RGS, rod growth site; sg, secretory granule; tf, tonofilaments. When initial enamel forms, the ameloblast only has a proximal portion of Tomes process (pptp). The distal portion develops as an extension of the proximal one slightly later when enamel rods begin forming. dcw, Distal cell web; sg, secretory granules. Double-labeled immunocytochemical preparations; the fine black dots indicate the presence of ameloblastin (AMBN), whereas the larger ones that of amelogenin (AMEL). A, Both proteins are processed simultaneously in the Golgi complex. B, Although the majority of secretory granules (sg) found near the Golgi complex or in Tomes process contain both proteins (4), some show the presence of only ameloblastin (2), only amelogenin (3), or neither protein (1). This indicates that both proteins are cosecreted. m, Mitochondria. 8

9 Transmission electron micrographs of initial enamel formation. A, This micrograph shows the close intermingling of dentin collagen with the ribbonlike crystals of enamel. B, A higher magnification shows the apparent continuity between calcified collagen and enamel crystallites. In three dimensions, interrod (IR) enamel surrounds the forming rod (R) and the distal portion of Tomes process (dptp); this portion is the continuation of the proximal portion (pptp) into the enamel layer. The interrod (IGS) and rod (RGS) growth sites are associated with membrane infoldings (im) on the proximal and distal portions of Tomes process, respectively. These infoldings represent the sites where secretory granules (sg) release enamel proteins extracellularly for growth in length of enamel crystals and, consequently, the thickening of interrod and rod enamel. Scanning electron micrograph of the surface of a developing human tooth from which ameloblasts have been removed. The surface consists of a series of pits previously filled by Tomes processes the walls of which are formed by interrod enamel. In cross section the distal portions of Tomes processes (dptp) appear as ovoid profiles surrounded by interrod enamel (IR). They decrease in size toward the dentinoenamel junction (dashed arrow) as the rod (R) grows in diameter. The crystals making up rod blend in with those of interrod enamel (small arrows, zone of confluence) at the point where the rod begins forming. RGS, Rod growth sites; sg, secretory granule. Light microscope radioautographic preparations following administration of 3Hmethionine to radiolabel secretory products (mainly amelogenins) of ameloblasts. The black silver grains over enamel indicate the presence of newly formed amelogenins. A and B, As expected, secretory stage ameloblasts actively secrete proteins during inner (IE) and outer (OE) enamel formation. C and D, The presence of grains over the surface enamel during the transition phase (Mt) and early maturation (M) indicates that ameloblasts still produce amelogenins during the early part of the maturation stage. BV, Blood vessel; PL, papillary layer; SI, stratum intermedium; TP, Tomes process. A and B, Scanning electron microscope illustrations showing the complex trajectory of rods in the inner two thirds of the enamel layer. B, The rods are organized in groups exhibiting different orientations; this illustration shows four adjacent groups. 9

10 The (A) first (initial) and (B) last (final) enamel layers are aprismatic, that is, they do not contain rods. Maturation stage Amelogenesis is a rather slow process, may take around 5 years, up to about 2/3 formation time can be occupied by the maturation ti stage Transitional phase, occurred just after the full thickness of enamel is completed Maturation proper Scanning electron microscope views of the (A) ruffle-ended and (B) smooth-ended apices of maturation stage ameloblasts. m, Mitochondria. Maturation proper Related to calcium transport and alternations in permeability of the enamel organ Ruffle-end: secret [bicarbonate ions] HCO 3 Ca polypeptide The functional morphology of ruffleended and smoothended maturation stage ameloblasts. Ca 10

11 Enamel protein Charge selective property At the start of the maturation stage, ameloblasts deposit a basal lamina like structure (BL) against the enamel surface to which it adheres firmly. Cytochemical detection (black dots) of sugar residues using lectins indicates that this structure is rich in glycoconjugates. Inset, The cell surface attaches to the basal lamina by means of hemidesmosomes (HD). Amelogenins: 90% regulate growth in thickness and width, nucleate crystals Nonamelogenin 1. Ameloblastin: promote mineral formation and crystal elongation 2. Enamelin: the molecule binds HA, crystal nucleation and growth 3. Sulfated glycoprotein 4. Tuftlin: cell signaling?? For DEJ 5. Enzymes Metalloproteinase: enamelysin MMP20 Serines proteinase: bulk degradation Phosphatase 6. Dentin phosphoprotein/ dentin sialoprotein Hypothetical scheme for amelogenin-mediated enamel biomineralization. A, Transmission electron microscope image illustrating the relationship of rod enamel crystals to a distal portion of Tomes process (dptp) and surrounding interrod enamel. The elongating extremity of the rod crystals abut the infolded membrane (im) at the secretory surface. B, In cross section, newly formed crystals appear as small, needlelike structures (arrows) surrounded by granular organic matrix. sg, Secretory granules. Four phases of enamel mineralization. Comparative immunocytochemical preparations illustrating the differential distribution of (A) amelogenin (AMEL) and (B) ameloblastin (AMBN), here in relation to a distal portion of Tomes process (dptp). Amelogenins are less concentrated in a narrow region near the secretory surface on the process (fewer black dots occur between the cell and the dashed line than beyond), whereas most of the ameloblastin is found in this region. sg, Secretory granule. 11

12 Striae of Retzius Longitudinal ground section showing disposition of the striae of Retzius (arrows).the outermost layer is the enamel, the two sections adjacent to the enamel represent the dentin, and the pulp chamber is in the center. 應為 enamel rod 每一段時間之 rod 結構上不同造成之年輪現象 參與細胞增加而產生? Cross striations In scanning electron microscopy, periodic varicosities and depressions are seen along enamel rods (R) in (A) rodent and (B) human teeth, producing the impression of crossstriations along their length. IR, Interrod enamel. Light microscope view of striae of Retzius in a ground section. In cross section, the striae appear as a series of concentric, dark lines (arrowheads). An enamel lamella can be seen running from the outer surface to the dentinoenamel junction. Human enamel is known to form a rate of approximately 4um/day Bands of Hunter and Schreger Longitudinal section of enamel viewed by incident light. The series of alternating light and dark bands of Hunter and Schreger are apparent. Higherpower view of a band of Hunter and Schreger as viewed by incident light. viewed under transmitted light. The differing orientation of enamel rods is clearly evident. The differing orientation of enamel rods 12

13 Enamel tufts & lamellae Enamel tufts: abrupt changes in the direction of groups of rods that arise from different regions of the scalloped DEJ Lamellae: longitudinal oriented defects filled with organic material (enamel organ or connective tissue) Enamel tufts & lamellae Transverse ground section of enamel. Enamel tufts are the branched structures extending from the dentinoenamel junction (DEJ) into the enamel (arrowheads). The junction is seen as a scalloped profile. Dentinoenamel junction. A, Ground section. B, Demineralized section after the enamel has been lost. The scalloped nature of the junction when seen in one plane is striking. C, A transmission electron micrograph shows the intermingling of dentin and enamel crystals. D, A low-power scanning electron micrograph of a premolar from which the enamel has been removed shows that the scalloping is accentuated where the junction is subjected to most functional stress. Geologic faults Enamel spindles Freeze-fracture preparation at the dentinoenamel junction (arrowheads). The distinctive appearance of the collagenous dentin and noncollagenous (initial) enamel layer is notable. Enamel spindles (arrows) in a ground section extend from the dentinoenamel junction into the enamel and most frequently are found at cusp tips. Odontoblast processes extend into the ameloblast layer 13

14 perikymata Scanning electron micrograph of the labial surface of a tooth, showing the perikymata. (Courtesy D. Weber.) Ground section of enamel showing the relationship between the striae of Retzius and surface perikymata. Gnarled enamel Bundles of enamel rods appear to intertwine in a highly irregular manner in the cusp region of teeth to form gnarled enamel. The phenomenon of gnarled enamel appears to be an optical illusion. The relationship between the striae of Retzius and surface perikymata (arrows). Gnarled Enamel Aging Discoloration Reduced permeability Modifications in the surface layer 14

15 Defects of amelogenesis Febrile diseases Tetracycline-induced disturbaneces Fluoride ion >5ppm Dentition of a patient who had two illnesses at separate times. The enamel defects, separated by normal enamel, are clearly visible. Acid etching END Scanning electron micrographs of etching patterns in enamel. A, Type I pattern: rod preferentially eroded. B, Type II pattern: rod boundary (interrod) preferentially eroded; C, Type III pattern: indiscriminate erosion. D, Junction between type I and type II etching zones. 15