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

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) 5. Root formation (formation of dentin and cementum of root) 6. Eruption Oral Histology and Embryology by Leslie P. Gartner, 1988

Bud stage 1. oral epithelium 2. dental lamina 3. tooth bud 5 1. oral epithelium 2. dental lamina 3. tooth bud 4. ectomesenchymal cells 5. vestibular lamina 1 2 3 4 http://www.iob.uio.no/studier/undervisning/histologi/index.php

Cap stage 3 1. Enamel organ (=dental organ) 2 2. Dental papilla 1 3. Dental sac (=dental follicle) 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 2 4 5 6 7 transient structure during cap stage http://www.iob.uio.no/studier/undervisning/histologi/index.php

Bell stage 1. outer enamel ep. 2. inner enamel ep. 3. stellate reticulum 4. stratum intermedium 4 1 2 3 http://www.iob.uio.no/studier/undervisning/histologi/index.php

Appositional stage 1 1. oral ep. 2. outer enamel ep. 2 3. stellate reticulum 4. inner enamel ep. 5. dental papilla 6. cervical loop 2 3 4 5 blood vessels 6 http://www.iob.uio.no/studier/undervisning/histologi/index.php

Appositional stage 8 1. dental papilla 2. preameloblasts 9 3. Preodontoblasts 4. odontoblasts 5. predentin 6. ameloblasts 7. dentin 8. stratum intermedium 9. enamel 9 6 7 5 Please remember the order of differentiation. Preameloblasts  preodontoblasts  odontoblasts  dentin  ameloblasts  enamel 2 4 3 1 http://www.iob.uio.no/studier/undervisning/histologi/index.php

Development of permanent dentition Embryonic age 1 2 3 4 5 6 7 8 months Primary incisor bud Cap bell mineralization of crown Permanent incisor bud Cap bell mineralization tooth germs of primary teeth Although permanent teeth begin to erupt at the age of 6, they begin to develop when the primary teeth develop. The tooth germs of permanent teeth are located at the lingual side of primary tooth germs. Some of the permanent teeth are formed next to the primary teeth (succedaneous teeth) but the tooth buds of molars are formed at the dental lamina that has grown distally without predecessors (accessional teeth). The tooth buds of succedaneous teeth roughly start to begin at the bell stage of the primary tooth germ. dental lamina tooth germs of permanent teeth

Formation of roots Hertwig epithelial root sheath (HERS) - Apical extension of cervical loop - Inner+outer enamel ep. - Not making enamel - Framework of root formation As the mineralization of crown continues, the epithelial cells at the cervical loop proliferate and migrate downward to form Hertwig epithelial rooth sheath (HERS). Although HERS is derived from enamel epithelium, it does not generate enamel. Instead, it ‘guides’ the root formation and serves a framework of rooth formation. As the HERS grows, it makes horizontal invagination (epithelial diaphragm) to define the size, shape and number of roots. root sheath epithelial diaphragm (size/shape/number of roots) Oral Histology and Embryology by Leslie P. Gartner, 1988

Formation of roots root dentin cementoblasts cementum fibroblasts odontoblasts cementoblasts cementum fibroblasts periodontal ligament osteoblasts alveolar bone The root formation is initiated by the disintegration of HERS. The cells in dental papilla are differentiated into odontoblasts and begin to make root dentin. Ectomesenchymal cells in dental follicle migrate onto dentin through disintegrating HERS and become differentiated into cementoblasts and fibroblasts. Cementoblasts synthesize cementum and fibroblasts synthesize collagen fibers for periodontal ligament. The dental follicle cells left outside the HERS are also differentiated into osteoblasts and fibroblasts to form alveolar bone and collagen fibers, respectively. The fibers synthesized by fibroblasts are embedded in cementum and alveolar bone at both ends. As the fibers mature, they are organized forming big bundles and become periodontal ligaments. disintegrated Oral Histology and Embryology by Leslie P. Gartner, 1988

Formation of roots odontoblasts cementoblasts cementum dentin dental sac HERS dental sac cells becoming cementoblasts 4 1 3 2 5 1. Formation of root dentin by odontoblasts 2. Disintegration of HERS 3. Migration of ectomesenchymal cells from dental sac to the root dentin through the disintegrated HERS 4. Differentiation of ectomesenchymal cells into cementoblasts and fibroblasts 5. Formation of cementum and collagen fibers - cementoblasts : secret cementoid (matrix) and mineralize cementum on root dentin - fibroblasts : synthesis of collagen fibers of periodontal ligament * Sharpey’s fiber : collagen fibers embedded in cementum

Nerves within tooth pulp Cap stage : axons enter dental follicle Sensory and sympathetic nerves Form subodontoblastic plexus Project into dentinal tubules No motor and parasympathetic nerves dental follicle axon Bell stage : axons enter dental papilla following the initiation of dentinogenesis dentin axon Sensory nerve terminals within mouse tooth pulp

Tooth eruption 1 2 3 4 5 6 1. oral ep. 2. connective tissue 1 mm 1. oral ep. 2. connective tissue 3. alveolar bone 4. Enamel 5. Dentin 6. HERS 1 2 3 4 5 6 http://www.anat.ucl.ac.uk/research/arnett_lab/

Tooth eruption Axial movement toward oral epithelium begin when the root formation begin. Source of erupting force : contraction of fibroblasts generating periodontal ligaments? 1 mm Tooth eruption means the axial movement of tooth toward the oral epithelium. Tooth eruption begins while the root formation occurs. The source of erupting force is not clear. One hypothesis suggests that fibroblasts within the periodontal ligament are able to contract and such contraction exert force to move the tooth axially. http://www.anat.ucl.ac.uk/research/arnett_lab/

Tooth eruption Alveolar bone and connective tissue are resorbed as teeth erupt. osteoclasts reduced enamel ep. As the tooth erupt, epithelial cells in the enamel organ become degenerated and form a reduced enamel epithelium. http://www.anat.ucl.ac.uk/research/arnett_lab/

Changes in epithelium during eruption enamel cuticle oral ep. The reduced enamel epithelium is eventually fused with oral epithelium and only small part of it is left as a junctional epithelium connecting tooth and oral epithelium. Enamel cuticle (Nasmyth’s membrane) is a thin layer of epithelial cells covering the surface of tooth emerging to oral cavity. It may be the remnants of epithelium covering the erupting tooth. junctional ep. reduced enamel ep.

Periodontal tissues oral ep Junctional ep Periodontal ligament In this histological section, junctional epithelium is detached from the tooth surface that is an artifact of the sample preparation. Oral epithelium does not directly attach to the tooth surface but is interposed by junctional epithelium. Periodontal ligament Alveolar bone http://www.iob.uio.no/studier/undervisning/histologi/index.php

Relationship of primary teeth and succedaneous permanent teeth D : deciduous tooth P or S : succedaneous tooth s When the deciduous teeth erupt, the tooth germ of succedaneous teeth are already formed. When the primary teeth are emerged to the oral cavity, the apex of the root is open. When the root formation is completed, the root apex has only a tiny opening that is called an apical foramen. As the succedaneous teeth erupt, the roots of primary teeth are resorbed. As the eruption continues, the roots of primary teeth are getting shorter and eventually the primary teeth fall off. erupting erupting resorption of root open apex

Summary of tooth development Oral epithelium oral epithelium Dental lamina Inner enamel ep ameloblasts enamel Outer enamel ep HERS guide root formation Stratum intermedium reduced enamel ep junctional ep. Stellate reticulum fibroblasts pulp Some cells in dental papilla remain to be undifferentiated within tooth pulp. These cells are pleuripotent stem cells that have a potential to differentiate into variety of cell types even following the completion of tooth formation. Dental papilla odontoblasts dentin Dental sac cementoblasts cementum Ectomesenchyme fibroblasts periodontal ligament osteoblasts alveolar bone

Most odontogenic epithelial cells degenerate following the completion of tooth formation Oral epithelium oral epithelium Dental lamina Inner enamel ep ameloblasts enamel Outer enamel ep HERS guide root formation Stratum intermedium reduced enamel ep. junctional ep. Stellate reticulum fibroblasts pulp Most odontogenic epithelial cells (dental lamina, ameloblasts, HERS, reduced enamel epithelium) are fated to degenerate following the completion of tooth formation. In contrast, the cells of ectomesenchymal origin remain and continued to function to generate the tooth and periodontal structures. Therefore, dentin and cementum are synthesized during the whole lifetime of the teeth. Dental papilla odontoblasts dentin Dental sac cementoblasts cementum Ectomesenchyme fibroblasts periodontal ligament osteoblasts alveolar bone

Remnants of odontogenic epithelial cells Dental lamina  epithelial pearl Epithelial pearl : remnants of dental lamina observed during the developmental stages

Remnants of odontogenic epithelial cells Dental lamina  epithelial pearl HERS  epithelial rests of Malassez in periodontal ligament Potential etiology of pathological conditions - Supernumenary teeth - Odontogenic cysts - Odontogenic tumors (ex: ameloblastoma) Epithelial rests of Malassez : Remnants of HERS Since odontogenic epithelial cells have high potential of proliferation, the uncontrolled growth and differentiation of remnants results in numerous pathological conditions.

Molecular mechanism of tooth development Many genes control tooth development but not completely understood shape, number of cusp (incisor vs molar) size number (2 vs 3 molars…..) location (mesio-distal, maxillo-mandibular….) timing of formation and eruption Future of dentistry? Control the number and location of teeth In vitro formation of tooth The genes that are involved in tooth development processes are actively studied. For further studies, refer to the references included in the next slide.

Further studies <Must > <Strongly recommended> 1. Oral Histology and Embryology by Leslie P. Gartner, 1988. Chapter 1-2. 2. University of Maryland BCDS learning center a. Odontogenesis I - Development of the Enamel Organ b. Odontogenesis II - Appositional Stage - Development of the Crown c. Odontogenesis III - Appositional Stage - Development of the Root d. Tooth Development Practical III <Strongly recommended> 1. Ten Cate’s Oral histology by Antonio Nanci, 7th Ed., Elsevier, 2008. 2. The cutting-edge of mammalian development; How the embryo makes teeth. Nature Reviews, 5:499-508, 2004 3. Biomineralization during early stages of the developing tooth in vitro with special reference to secretory stage of amelogenesis. Int J Dev Biol, 39:203-212, 1995. University of Maryland BCDS learning center http://www.dental.umaryland.edu/dentaloffices/student_tech_ctr/applications/dentalstudents.html

Acknowledgements The images in the lectures are derived from these websites with permission. University of Oslo, Oral and Craniofacial Histology web site Dr. Tim arnett at the University College of London Oral Histology and Embryology by Leslie P. Gartner, 1988. University of Maryland BCDS learning center

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

Question #1 The hardest tissue found in the oral cavity is: A. enamel B. dentin C. cementum D. bone E. enamel cuticle

Question #1 The hardest tissue found in the oral cavity is: A. enamel B. dentin C. cementum D. bone E. enamel cuticle

Question #2 The dental lamina: A. appears at 12 weeks of gestation B. is synonymous with the vestibular lamina C. is derived from neural crest material D. is derived from ectoderm E. will give rise to cementum

Question #2 The dental lamina: A. appears at 12 weeks of gestation B. is synonymous with the vestibular lamina C. is derived from neural crest material D. is derived from ectoderm E. will give rise to cementum

Question #3 Collagen in not the major component of matrix of: A. dentin B. enamel C. cementum D. periodontal ligament

Question #3 Collagen in not the major component of matrix of: A. dentin B. enamel C. cementum D. periodontal ligament

Question #4 One of the following is not present in the bell stage of tooth development: a. outer enamel epithelium B. inner enamel epithelium C. stellate reticulum D. stratum intermedium E. enamel knot

Question #4 One of the following is not present in the bell stage of tooth development: a. outer enamel epithelium B. inner enamel epithelium C. stellate reticulum D. stratum intermedium E. enamel knot

Question #5 Cementogenesis starts prior to : A. amelogenesis B. dentinogenesis C. disintegration of dental lamina D. formation of apical foramen E. disintegration of Hertwig’s epithelial root sheath

Question #5 Cementogenesis starts prior to : A. amelogenesis B. dentinogenesis C. disintegration of dental lamina D. formation of apical foramen E. disintegration of Hertwig’s epithelial root sheath

Question #6 Two of the following is not the product of dental sac: A. dentin B. cementum C. alveolar bone D. periodontal ligament E. enamel

Question #6 Two of the following is not the product of dental sac: A. dentin B. cementum C. alveolar bone D. periodontal ligament E. enamel

Question #7 One of the following is not the component of tooth germ: A. Dental papilla B. Enamel organ C. Dental follicle D. Vestibular lamina

Question #7 One of the following is not the component of tooth germ: A. Dental papilla B. Enamel organ C. Dental follicle D. Vestibular lamina

Question #8 One of the following remains after the completion of tooth development: A. amelogenin B. Hertwig’s epithelial rooth sheath C. ameloblasts D. odontoblasts

Question #8 One of the following remains after the completion of tooth development: A. amelogenin B. Hertwig’s epithelial rooth sheath C. ameloblasts D. odontoblasts

Question #9 Which one of the following is derived from ectomesenchyme? Ameloblasts Odontoblasts Stellate reticulum Stratum intermedium Reduced enamel epithelium

Question #9 Which one of the following is derived from ectomesenchyme? Ameloblats Odontoblasts Stellate reticulum Stratum intermedium Reduced enamel epithelium

Question #10 Odontoblasts are characterized by being located on external surfaces of roots being shed from the tooth at the time of eruption differentiating first at the cervical region of a forming tooth secreting a non-fibrous matrix composed of chains of amino acids possessing long cytoplasmic processes which lie within dentinal tubules

Question #10 Odontoblasts are characterized by being located on external surfaces of roots being shed from the tooth at the time of eruption differentiating first at the cervical region of a forming tooth secreting a non-fibrous matrix composed of chains of amino acids possessing long cytoplasmic processes which lie within dentinal tubules

Question #11 The pulp chamber of a mature tooth contains Cells that form periodontal ligament An enamel lining for thermal protection Blood vessels and nerves cementoblasts ameloblasts

Question #11 The pulp chamber of a mature tooth contains Cells that form periodontal ligament An enamel lining for thermal protection Blood vessels and nerves cementoblasts ameloblasts