Tooth Development - I Man-Kyo Chung, DMD, PhD (mchung@umaryland.edu) Assistant Professor Department of Neural and Pain Sciences University of Maryland Dental School

Developmental histology of tooth zygote (a cell from fertilization) development Whole organism > organ > tissue > cell > cellular organelle Ex) human heart muscle myocyte nucleus Gross anatomy microscopic anatomy (histology) This lecture is about the microscopic anatomy of the developmental process of teeth. Methods) naked eye light microscopy electron microscopy

Two types of cells give rise to teeth zygote Enamel epithelial cells Dentin Pulp Proliferation (increase in number) ectomesenchymal cells Cementum Migration (change in location) Two types of embryonic cells undergo proliferation, migration, and differentiation to form teeth. Differentiation (specialization) Ameloblasts forming enamel Odontoblasts forming dentin Cementoblasts forming cementum

Early embryonic development <1 wk ~2 wks Oral Histology and Embryology by Leslie P. Gartner, 1988 bilaminar germ disc <trilaminar germ disc> Ectoderm (tissues covering outside the body / epidermis, hair etc) Three layers of embryonic cells (ectoderm, mesoderm and endoderm) will be developed to specified tissues in the future. Mesoderm (connective tissues / heart, muscle, blood cells, bone, dermis etc) Endoderm (tissues lining inside the body / lung, stomach, intestine, liver etc) ~3 wks

Tissues originated from ectoderm besides epidermis epithelium 3. connective tissues of head and neck (bone, cartilage, muscles, tooth etc) (Ectomesenchymal origin) ectodermal origin embryonic connective tissue Ectoderm undergoes invagination to mesodermal side and form two more differentiated structures; neural tube and neural crest. Neural crest gives rise to sensory ganglia and connective tissues of head and neck, including a part of tooth. Therefore, teeth are originated from two types of cells. Ectodermal origin  generate enamel Ectomesenchymal origin  generate dentin, cementum, and pulp neural crest 2. sensory ganglia neural tube 1. brain (neuroectodermal origin)

Location of tooth formation 6 weeks old embryo Coronal section nasal septum Maxillary process eye auricle tongue Mandibular process Tooth formation occurs at the oral epithelium of maxillary and mandibular processes of embryo. Oral epithelium of maxillary and mandibular processes Oral Histology and Embryology by Leslie P. Gartner, 1988

Stages of tooth development 1. Bud stage (epithelial ingrowth into ectomesenchyme) 2. Cap stage (further epithelial growth) 3. Bell stage (histo- and morpho-differentiation) 4. Appositional stage (mineralization) (formation of enamel and dentin of crown) Tooth develops by complicated continuous processes of proliferation and histomorphological differentiation. For convenience of explanation, the stages of tooth development are separated as presented. However, there is no clear cut between stages. * The figure only displays changes in the epithelium. * Note the disintegration of dental lamina in panel d. 5. Root formation (formation of dentin and cementum of root) 6. Eruption Oral Histology and Embryology by Leslie P. Gartner, 1988

Bud stage Dental lamina - outgrowth of horse shoe-shaped epithelial band from oral epithelium - one dental lamina per arch developing maxillalry bone - 10 tooth buds per lamina palate tongue Mx dental lamina This slide shows the frontal section of the head of ~6 week-old embryo. Thin (within several micrometers) sections were stained by hematoxilin and eosin to reveal the structure under light microscopy. A tooth bud of a mandibular molar is shown. Bud stage is represented by the epithelial outgrowth. outgrowth of dental lamina from oral epithelium. outgrowth of vestibular lamina from oral epithelium. outgrowth of tooth buds from dental lamina. tooth bud Mn developing mandibular bone http://www.iob.uio.no/studier/undervisning/histologi/index.php

Bud stage Mx vestibular lamina Mn vestibule *vestibule a furrow between gingival and buccal cheek a junction of freely movable cheek and gingiva attached to alveolar bone. Mn http://www.iob.uio.no/studier/undervisning/histologi/index.php

Bud stage ectomesenchymal cells http://www.iob.uio.no/studier/undervisning/histologi/index.php

Bud stage condensation of ectomesenchymal cells around tooth bud Purple dots represent nucleus of cells. Higher density of purple dots represent more dense distribution of cells. Note the higher density of ectomesenchymal cells around the tooth bud. condensation of ectomesenchymal cells around tooth bud http://www.iob.uio.no/studier/undervisning/histologi/index.php

Cap stage 3 1. Enamel organ (=dental organ) 2 2. Dental papilla 1 (proliferation of epithelial cells) 2 2. Dental papilla (condensation of ectomesenchymal cells) 1 3. Dental sac (=dental follicle) (capsule like condensation of ectomesenchymal cells) This is a tooth germ of maxillary tooth. At this stage, epithelial cells proliferate to form enamel organ. Ectomesenchymal cells form two different structures, dental papilla and dental sac. http://www.iob.uio.no/studier/undervisning/histologi/index.php

Future tooth products 3 1. Enamel organ 2 2. Dental papilla 1 dentin pulp 1 3. Dental sac Each structure will form tooth and periodontal structures in the future. cementum periodontal ligament alveolar bone connective tissues of gingiva http://www.iob.uio.no/studier/undervisning/histologi/index.php

Tooth germ means + + 3 1. Enamel organ 2 2. Dental papilla 1 3. Dental follicle Tooth germ = enamel organ + dental papilla + dental follicle. http://www.iob.uio.no/studier/undervisning/histologi/index.php

Differentiation of enamel organ Transformation of a cell mass into morphologically and functionally distinct components Enamel organ Secreting proteoglycan (absorbing water  increase extracellular space) desmosome Flattened (squamous or cuboidal) Star shaped Elongated and polarized (columnar) Differentiation represent the changes in the properties of the cells. In contrast, proliferation represent the increase in number. At this stage, proliferation and differentiation occur at the same time. Differentiation is obvious in a mass of epithelial cells (enamel organ). Differentiation may start by the secretion of proteoglycan from the epithelial cells to the extracellular space. The absorption of water by the proteoglycan increases the extracellular space. The cells in the epithelial layers are not separated but still maintain contacts between cells via the tight intercellular junction by desmosome. Such changes eventually make the cells look star-shaped. Inner (a side facing dental papilla) enamel epithelium undergoes independent differentiation processes to make them taller and polarized. The height of the cells : Squamous < cuboidal < columnar Polarized : the nucleus is located at one side of the cell. 1) Outer enamel epithelium 2) Stellate reticulum 3) Inner enamel epithelium

Enamel organ of cap stage 3 1) Inner enamel ep 2) Outer enamel ep 3) Cervical loop 4) Stellate reticulum 5) Enamel knot 6) Enamel cord 7) Enamel navel 1 2 4 (inner+outer enamel ep.) 5 6 Inner enamel ep : a layer facing dental papilla. The cells are tall and polarized. Outer enamel ep : a layer facing mesenchyme. The cells are cuboidal or squamous. Cervical loop : junction of inner and outer enamel ep. Stellate reticulum : a mesh like structure composed of star-shaped cells. Enamel knot : a condensation of stellate reticulum at the deepest part of enamel organ. Enamel cord : an extension of enamel knot toward outer enamel ep. Enamel navel : a depression on the outer enamel ep to which enamel cord is directed. 5-7 are transiently observed during cap stage. 7 transient structure during cap stage http://www.iob.uio.no/studier/undervisning/histologi/index.php

Enamel organ of cap stage 3 1) Inner enamel ep 2) Outer enamel ep 3) Cervical loop 4) Stellate reticulum 5) Enamel knot 6) Enamel cord 7) Enamel navel 1 generate enamel 2 4 root formation 5 (protect inner enamel ep.) 6 Inner enamel ep is the most important structure since it will generate enamel in the future. Cervical loop will proliferate further to form the shape of crown and eventually guide the formation of root. Stellate reticulum and outer enamel ep might play a role to protect inner enamel ep layer. Enamel knot may be a site where the calcification is initiated and therefore the location of future cusp tip. location of cusp? 7 transient structure during cap stage http://www.iob.uio.no/studier/undervisning/histologi/index.php

Tooth germ of late cap stage (tooth bud of permanent tooth) * Basement membrane : a structure composed of collagen fibers and attachement proteins that connects connective tissue and epithelium. It is a universal structure between connective tissue and epithelium, not a unique structure of the tooth germ. Oral Histology and Embryology by Leslie P. Gartner, 1988

Bell stage 1. Tooth shape is identifiable. 2. Further differentiation of enamel organ 1) outer enamel ep. 2) inner enamel ep. 3) stellate reticulum 4) stratum intermedium 4 1 2 3 Proliferation and differentiation of epithelial and ectomesenchymal cells continue and form a larger tooth germ with more complicated structures. http://www.iob.uio.no/studier/undervisning/histologi/index.php

Bell stage 1. Tooth shape is identifiable. 2. Further differentiation of enamel organ 1) outer enamel ep. 2) inner enamel ep. 3) stellate reticulum 4) stratum intermedium 4 1 2 3 Enamel organ is further differentiated into four distinct layers. http://www.iob.uio.no/studier/undervisning/histologi/index.php

stratum intermedium inner enamel ep basement membrane dental papilla - several layers of cells between the inner enamel epithelium and stellate reticulum may be derived from the retraction of stellate reticulum. show exceptionally high activity of alkaline phosphatase, an enzyme essential for mineralization http://www.iob.uio.no/studier/undervisning/histologi/index.php

Bell stage Oral Histology and Embryology by Leslie P. Gartner, 1988

Differentiation of inner enamel ep. differentiated to ameloblast without proliferation Cusp tip older taller polarized The cells in inner enamel epithelium at this stage are composed of a spectrum of cells with different degree of proliferation and differentiation. 1. The cells at the cervical loop – mainly proliferating The ‘proliferation’ of inner enamel epithelium mainly occur at the cervical loop. These cells are younger (recently proliferated and undifferentiated) and the shape is round and short. As the cells proliferate, the cells migrate to the direction of the future root. 2. The cells at the deep part of enamel organ – mainly differentiating The cells at the deepest part of the enamel organ stop proliferating and migrating. Instead, the cells mainly undergo ‘differentiation’ to prepare forming enamel. The cells become taller and polarized. younger shorter unpolarized root Keep proliferating and migrate downward http://www.iob.uio.no/studier/undervisning/histologi/index.php

cap stage bell stage appositional stage * Note the relative distance from inner enamel epithelium to outer enamel epithelium become shorter as the tooth germ develops. * The absolute size of the tooth germ become bigger as the development continues. The presented examples are scaled to the similar size for better contrast of the morphological changes.

cap stage bell stage appositional stage * The absolute size of the tooth germ become bigger as the development continues. The presented examples are scaled to the similar size for better contrast of the morphological changes.

Appositional stage Stage of mineralization 1 1. oral ep. 2. outer enamel ep. 3. stellate reticulum 4. inner enamel ep. 5. dental papilla 6. cervical loop 2 3 4 At the appositional stage, the mineralization starts at the deepest part of the enamel organ. Note that blood vessel does not exist within the enamel organ but outside outer enamel epithelium. Blood vessels supply oxygen and nutrients required for the mineralization. The oxygen and nutrients diffuse into outer enamel epithelium and stellate reticulum to reach inner enamel epithelium. Therefore, the distance from the blood vessels to inner enamel epithelium has to be short. Vascularization close to the tooth germ can be seen in bell stage as well. It increases greatly during appositional stage. 5 blood vessels 6 http://www.iob.uio.no/studier/undervisning/histologi/index.php

Epithelial and mesenchymal cells interact throughout tooth formation 1 bud Cap mineralization bell mesenchymal cells oral epithelium dental lamina 1 signal epithelium mesenchyme proliferation Epithelial and mesenchymal cells interact each other throughout the tooth formation. 1. Mesenchymal cells underlying oral epithelium induce the proliferation of oral epithelium to form dental lamina.

Epithelial mesenchyaml interaction 1 bud Cap mineralization bell mesenchymal cells oral epithelium dental lamina inner enamel epithelium 2 preameloblast 2 preodontoblast Preodontoblasts Preameloblasts a 1 a 1 b 2 b 2 c 3 c 3 d 4 d 4 2. Pre-ameloblasts (differentiated inner enamel epithelial cells to form enamel in the future) induce the differentiation of ectomesenchymal cells to preodontoblasts that will synthesize the matrix of dentin. e 5 e 5 f 6 f 6 g 7 g 7 h 8 h 8 i 9 i 9 j 10 mesenchyme epithelium k 11 l 12 m 13

inner enamel epithelium 3 1 Predentin (dentin matrix) bud Cap mineralization bell odontoblasts preameloblasts mesenchymal cells oral epithelium dental lamina inner enamel epithelium 2 preameloblast preodontoblast odontoblast 3 enamel matrix dentin Predentin ameloblasts 3. Dentin matrix (uncalcified predentin) induce the differentiation of preameloblast to ameloblast which synthesize the matrix of enamel. ameloblast dentin enamel matrix enamel

Appositional stage 6 a. predentin b. dentin c. enamel c 1. ameloblasts 2. preameloblasts 3. odontoblasts 4. preodontoblasts 5. dental papilla 6. stratum intermedium c 1 b a Please remember the order of differentiation. Preameloblasts  preodontoblasts  odontoblasts  dentin  ameloblasts  enamel 2 3 5 4 http://www.iob.uio.no/studier/undervisning/histologi/index.php

stratum intermedium ameloblasts enamel dentin predentin odontoblasts http://www.iob.uio.no/studier/undervisning/histologi/index.php

Developmental lobes tooth germ of mandibular molar groove (junction of 2 lobes) tooth germ of mandibular molar cusp Pit (junction of >3 lobes) Initiation of mineralization at 5 points continued mineralization toward cervical direction Coalescence of 5 segments Each tooth has multiple starting points of mineralization. The unit mineralizing segment is called a developmental lobe. Coalescence of lobes results in grooves and pits. Developmental lobes

Biomineralization Dentin = collagen + hydroxyapatite crystals Concrete = metal framework + cement Dentin = collagen + hydroxyapatite crystals Enamel = amelogenin + hydroxyapatite crystals resorbable The formation of tooth structure is initiated by the synthesis of matrix protein (collagen in dentin and amelogenin in enamel). The inorganic crystals are accumulated in the matrix protein to form hard tissues. Note that the matrix of enamel (amelogenin) disappears during the mineralization process to allow dense packing of inorganic crystals. In constrast, collagenous matrix of dentin is not resorbed. allows further packing of crystals inorganic component, hardness enamel >>> dentin

Essential components of calcification 1 2 Ca2+ + PO43- 3 Alkaline phosphatase (supply phosphate) Pyrophosphatase (hydrolyze pyrophosphate) ATP pyrophosphate AMP Ca10(PO4)6(OH)2 Calcium and phosphate form hydroxyapatite crystals. Alkaline phosphatase increases the local concentration of phosphate by the cleavage of organic phosphate at the site of mineralization. Although calcium, phosphate and alkaline phosphatase present abundantly in many tissues, calcification does not occur in every tissue since ‘pyrophosphate’ inhibits calcification even in the presence of calcium and phosphate. Therefore, inhibition of the role of pyrophosphate by pyrophosphatase is also necessary for the mineralization process. The odontoblasts and osteoblasts release matrix vesicles for mineralization. Matrix vesicles contain not only the essential components of calcification but also seeds of inorganic crystals where the growth of crystals can occur easily within organic matrix. Matrix vesicles Contain essential components Contain seed of crystal Released from odontoblasts to collagen matrix

Acid can reverse calcification Ca2+ + PO43- acid acidogenic bacteria Ca10(PO4)6(OH)2 Any type of acid can reverse the mineralization process easily. The remineralization of the demineralized tooth substances is very limited. Bone maintains a balance between mineralization by bone-forming cells (osteoblasts) and demineralization by bone-resorbing cells (osteoclasts) in order to maintain physiological concentrations of calcium and phosphate in the body. There is no such mechanism in tooth. Dental caries decalcification of inorganic tooth substances by acid produced by bacteria Only very limited remineralization can occur in tooth. (cf: bone maintains a balance between mineralization and demineralization )

Appositional stage Reduced stellate reticulum Aprismatic dentino- (alkaline phosphatase ) (predentin) Aprismatic dentino- enamel junction (collagen fibers of mantle dentin) Mantle This is a sketch of electronmicroscopic structures. Ameloblasts and odontoblasts are actively synthesizing the matrix to form enamel and dentin. Large part of cytoplasm is filled with cellular organelles, such as endoplasmic reticulum and golgi complex, for synthesizing protein matrix. Ameloblasts and odontoblasts keep moving away from the dentino-enamel junction as they synthesize matrix. Ameloblasts die away once they synthesize a full thickness of enamel. But odontoblasts remains and continuously synthesize dentin in the pulp. Stratum intermedium contains high concentration of alkaline phosphatase. Aprismatic enamel : a layer of first synthesized enamel Mantle dentin : a layer of first synthesized dentin Korff’s fiber : collagen fibers of mantle dentin odontoblasts Oral Histology and Embryology by Leslie P. Gartner, 1988