Dentinogenesis

Pulpodentin complex: Why named so? o Dentine and dental pulp are always discussed and studied together since they both originate from the same embryological tissue (which is the dental papilla) and so they have common features o Enamel is a dead tissue (e.g. NOT replaceable) WHILE dentine & dental pulp are vital tissues (e.g. replaceable) As long as pulp is there dentine is vital, BUT once pulp is removed (e.g. root canal treatment) dentine becomes non-vital Cells that form dentine and provide vitality to dentine (e.g. odontoblasts ) exist in the peripheral part of dental pulp and so if dental pulp is removed, then odontoblasts are lost, then dentine becomes non-vital And so dentine & dental pulp are always connected together embryologically & physiologically

** Dentinogenesis is a continuous process that ONLY stops if pulp is removed or tooth is lost ** Odontoblasts are programmed to deposit dentine upon external stimulus (e.g. injury) and also continuously throughout life Why important to Endodontics? o Endodontics is the science dealing with treatment of dental pulp problems (e.g. pulpitis) Why learn development of pulpodentin complex? o Because after learning the way dentine and dental pulp develop, we will have excellent understanding about the response of these tissues to injuries and then treatment of these injuries ** This picture reviews the different developmental stages the tooth germ passes through ** Bud stage cap stage bell stage ** The area enclosed inside the concavity of enamel organ is called dental papilla (which forms dentine & dental pulp) ** The area surrounds the enamel organ is called dental follicle (which forms cementum, alveolar bone, periodontal ligament and gingiva) ** This picture reviews the way hard tissue formation takes place in the tooth ** Dentinogenesis precedes amelogenesis ** Hard tissue formation begins at cusps tips & incisal edges and then proceeds cervically
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** The full cusp is formed, while we dont have enamel at the cervical part of the tooth yet (e.g. the full thickness of enamel is achieved occlusally, while we dont have enamel at the cervical margin yet) ** The last layer of enamel forms cervically (and after this layer, enamel formation stops forever) ** Dentin formation continues and with time dental pulp becomes smaller and smaller Dentinogenesis: Dentinogenesis = the process of dentine formation Starts after tooth germ has reached the bell stage (e.g. late bell stage) Early bell stage = morphodifferentiation Late bell stage = histodifferentiation (hard tissue formation) Starts at the cusp tips and incisal edges and proceeds downward toward cervical area Dentinogenesis precedes amelogenesis all the time (we have dentine formation just short time before we have enamel formation) Enamel organ is now fully formed (we should establish the 3D shape of the crown (e.g. morphogenesis ) before dentinogenesis begins PLUS we should have differentiated IEE) Internal Enamel Epithelium pre-ameloblasts (differentiated IEE) dental papilla cells differentiate into odontoblasts first layer of dentine basal lamina breaks down ameloblasts first layer of enamel This is called the reciprocal interaction (e.g. epithelial-mesenchymal intercation) between ectoderm (IEE of enamel organ) and ectomesenchymal tissue (dental papilla) Dental papilla divides into 2 populations of cells (one group remains attached to basement membrane and differentiates into odontoblasts to lay down dentine, and the other group migrates toward the center of dental papilla and remains undifferentiated to be ready to produce dentine on call) Once dentinogenesis begins, we start calling the dental papilla the dental pulp Dentinogenesis differs from amelogenesis in: Continues throughout life Dentinogenesis continues as long as teeth are vital (e.g. the pulp is there) BUT amelogenesis stops once the whole thickness of enamel is established Doesnt involve maturation In amelogenesis we have initial mineralization (30%, during the secretory stage ) and then full mineralization (96%, during the maturation stage ), so maturation is needed to supply enamel with the high percentage of minerals it needs Dentinogenesis doesnt need maturation because dentine is only 70% mineralized (e.g. less mineralized than enamel and so, doesnt need extra minerals) Mineralization isnt immediate In dentinogenesis organic matrix of dentine is deposited first then after a gap of time (maximally 24 hours) mineralization begins BUT in amelogenesis mineralization of the organic matrix is immediate with NO time gaps
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 The unmineralized portion of dentine that will be mineralized later is called Predentine Odontoblasts differentiation: Neural crest cells are: They are ectodermal in origin BUT NOW present inside the mesoderm They are then called ectomesenchymal tissue Some of these neural crest cells migrate to the oral region and become involved in the structure of dental papilla and then they differentiate into odontoblasts which are the dentine forming cells Epithelial-mesenchymal interaction It is the interaction between the IEE of enamel organ & the cells of dental papilla Contact between oral epithelium (enamel organ) and ectomesenchyme (dental papilla) is essential for odontoblastic differentiation Basement membrane of IEE seems to be important for odontoblastic differentiation as it undergoes time-limited changes The disappearance of the basement membrane is important for the interaction and for the signaling molecules to be distributed between the two tissues (ectoderm and ectomesenchyme) to induce hard tissue formation We have certain times where the basement membrane disappears & other times where it reappears again and these time dependant changes are important in tissue differentiation Once dentin is formed by means of reciprocal interaction, the contact between dentine itself as a hard tissue and the overlying IEE cells is also important for enamel deposition

Interaction with dentine is also essential for enamel matrix deposition

Growth factors (TGF, IGF and BMP) have been found in IEE and found to be important in hard tissue formation

Signaling interaction: Influencing odontoblast differentiation & dental pulp formation Transcription factors Protein binding to DNA near the start of gene transcription Regulating gene expression Proteins binding to specific receptors on cell surface Leading to membranous changes intracellular events altered gene expression activating cell growth and differentiation

Growth factors

Signaling molecules: Bone morphogenetic proteins BMPs Fibroblast growth factors FGFs WNT factor Sonic hedgehog SHH Transcription molecules
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Msx1, Msx2 homeobox genes Lymphoid enhancer-binding factor 1 Pax9

Morphogenetic gradient: Most differentiated odontoblasts align at epithelial-mesenchymal interface Least differentiated toward the core of dental papilla

Cells at the upper part of the picture are ameloblasts at different stages of differentiation Cells at the lower part of the picture are odontoblasts at different stages of differentiation At first we only have IEE cells separated by basement membrane from ectomesenchymal cells of dental papilla Ectomesenchymal cells will divide to give us 2 populations of cells: One line represent cells that stay in contact with the basement membrane Second line represent cells that migrate to the core of dental papilla Cells which stay in contact with the basement membrane differentiate into pre-odontoblasts by the activity of the pre-ameloblasts because they send signals crossing the basement membrane Pre-odontoblasts differentiate into odontoblasts and then they start to deposit dentine (in the form of Predentine) Once dentine is formed, the basement membrane disappears

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 The disappearance of basement membrane allows pre-ameloblasts to come in direct contact with odontoblasts and the first layer of dentine which stimulates them to differentiate into ameloblasts then they start to deposit enamel Ameloblasts & odontoblasts deposit hard tissue and migrate away from each other ** Ameloblasts deposit enamel and migrate upward (toward the core of enamel organ) BUT odontoblasts deposit dentine and migrate downward (toward the core of dental papilla) Each ameloblast has a process called Tomes process which is important for the prismatic structure of the enamel. This process is short (doesn't extend through the full thickness of enamel) and doesnt remain in the body of enamel forever Each odontoblast has a process called odontoblastic process that is long (extending through the full thickness of dentine) and remains in the body of dentine forever At the final stages of dentinogenesis: o o o o Odontoblasts remain at the peripheral aspect of the dental pulp Odontoblasts appear as columnar cells with very long odontoblastic processes Odontoblastic processes must exist within dentine in tubules dentinal tubules The tips of these odontoblastic processes that cross the EDJ are called enamel spindles

Odontoblast differentiation: Odontoblastic differentiation passes into 3 different stages: o Induction signaling molecules sequestered in basal lamina to which peripheral papillary cells align o Competence After a predetermined cell divisions are completed, cells express specific growth factors receptors o Terminal differentiation Cyto-differentiation of odontoblasts: It is when differentiated cells are found at the periphery of dental papilla After division, daughter cells in contact with the basement membrane become pre-odontoblasts then odontoblasts Differentiation starts at cusp tips & incisal edges and then proceeds cervically along the external contour of the tooth until the whole crown is formed When cells begin to differentiate they undergo hypertrophy (increase in size) and the nuclei become located basally (towards the pulp polarized cell) to provide good space inside the cytoplasm for the synthesis and storage of the materials that are going to be secreted Golgi complex becomes pronounced above the nucleus and this means that the cell is now becoming active and synthesizing proteins Rough endoplasmic reticulum increases in size and flattens parallel to the long axis of the cell and this again means that the cell is active and synthesizing proteins Mitochondria, which is the organelle of energy in the cell that produces ATP Redistribution of the intracellular skeleton and this occurs due to the hypertrophy of the cell (elongation & different shape of the cell)

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Small cell processes extend through the full thickness of dentine toward the basement membrane of IEE and these are the odontoblastic processes which remain in contact with the basement membrane and keep the dentine vital ** The only cell in the body with processes living in hard tissue is odontoblast Each odontoblast has many odontoblastic processes but only one becomes dominant while the rest disappear Tight gap junctions & desmosomes occur between odontoblasts also linking with subodontoblastic cells Isolation between the pulpal area & dentin is very important to preserve the structure of dentine

Deposition of dentine matrix: Deposition of dentine organic matrix starts after the full differentiation of odontoblasts is completed Organic matrix is important to provide a framework (e.g. structure of dentine) that can be mineralized later Components of the organic matrix: Collagen (I (90%), V, VI, III?) Phosphoproteins Proteoglycans (ground substance of any connective tissue) Glycoproteins/sialoproteins Lipids, Serum-derived proteins and Growth Factors (GFs)

Major components: Collagen I Dentine Phosphoproteins (DPP) Has a role in mineralization

Dentine collagen: Dentine collagen is similar to that of bones and tendons Remember than enamel doesnt have collagen Dentine collagen is mainly composed of the 3 amino acids: Glycine 33% Alanine 15% Proline 13% Dentine collagen fibers are smooth and banded and each band is between 640-700 angstroms (the Angstrom is 0.1 nanometer) Protein fibrils are of varying lengths up to 700 angstrom and indeterminate thickness So we can find thin collagen fibrils or thick collagen fibrils, then these fibrils are welded together into bundles (fibers) then these fibers act together as a matrix (framework) to get calcified

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 Dentin sialo(phospho)protein: DSPP-null mice have: o Hypomineralized dentine o Widened Predentine ** Null mice = mice not having the DSPP ** This experiment proves that DSPP is important in dentine mineralization Gives rise to 2 proteins : o Dentin sialoprotein (DSP) doesn't have well-known functions o Dentin phosphoprotein (DPP) Produced by odontoblasts Initiator and modulator of formation & growth of apatite crystals Transported to the mineralization front and bind to collagen to promote formation of initial apatite

Dentine matrix protein 1: DMP1-null mice have: o Hypomineralized dentine o Widened Predentine ** This experiment proves that DMP1 is also important in dentine mineralization Regulating matrix mineralization Present in dentine and Predentine Localized in peritubular dentine Its fragments are nucleators of apatite formation

Bone sialoproteins: Unknown specific functions Believed to act as: o Nucleators for the formation of initial apatite crystals o Then as an inhibitor in directing the growth of the crystal (to direct mineralization in one direction only) Mainly in Predentine (dentine contains little or no BSP) Elevated in tertiary dentine (dentine forming upon external stimulus)

Osteopontin: Only minor amounts exist in dentine Elevated levels in tertiary dentine

Deposition of dentine matrix: Dentine is divided into two areas: o Mantle dentine: It is the superficial layer of dentine being formed FIRST just beneath the EDJ (e.g. beneath enamel)
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This layer of dentine is ONLY seen in the CROWN portion of the tooth It is a very thin layer (only up to 150 mm in thickness) It has different structure and composition from the reminder of dentine (which is called circumpulpal dentine) ** The process by which mantle dentine mineralizes is different from that of circumpulpal dentine Mantle dentine mineralizes by matrix vesicle mineralization WHILE circumpulpal dentine mineralizes by DPP-regulated mineralization ** Collagen bundles (organic matrix) lie at a right angle to the future EDJ

o Circumpulpal dentine: It is the reminder of dentine being formed LATER It is the dentine that surrounds the pulp This layer of dentine is seen in the crown and root portions of the tooth Deposition is in a regular incremental pattern Collagen I released from odontoblastic body Dentine Phosphoprotien is released from odontoblastic process a short distance from the cell body Odontoblasts are made of two parts: body & process Collagen is released from the odontoblastic body while the protein responsible for mineralization of the organic matrix (e.g. Dentine Phosphoprotien) is released from odontoblastic process Dentine Phosphoprotien has a role in mineralization of Circumpulpal dentine Dentine Phosphoprotien molecules that are deposited to mineralize dentine have to bypass Predentine to reach dentine Predentine exists because there's a gap of time between organic matrix deposition and mineralization (e.g. mineralization isn't immediate ) It also exists because the deposition of the organic matrix is from the odontoblastic body while the factors responsible for mineralization are from the odontoblastic process Predentine = the most recent dentine formed = unmineralized dentine close to pulpal side Korffs fibers: These are immature unmineralized collagen fibers which are present throughout dentine but its amount is limited If collagen fibers aren't mature , they won't be mineralized

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o Predentine: It is the non-mineralized form of dentine Organic matrix deposition precedes mineralization and there's a gap of time (maximally 24 hours) between the two processes ** In enamel we don't have pre-enamel because its mineralization is immediate The deposition of dentine organic matrix is rhythmatic, this means we have alternative periods of activity and quiescence (being inactive) and this rhythm is important in producing the incremental lines of dentine Short term rhythm: Diurnal (4 m/day) the distance between two incremental lines is 4 m and this means the odontoblast is capable of producing 4 m of dentine each day Long term rhythm: Odontoblasts take a long break The rhythmatic deposition of dentine can be either: Reduced by systemic disturbances like: Birth At birth theres sudden change in the environment and this shocks odontoblasts as well as ameloblasts (which are very sensitive cells) Illness Increased by: Injuries to pulpal nerves (like a trauma) Mineralization in the Circumpulpal dentine: Serum Ca+2 is taken up by odontoblasts and accumulates in the distal body and process of the odontoblast Under the control of DPP (Dentine Phosphoproteins ), Ca+2 is deposited onto a template formed by collagen I fibrils to build up as crystalline material So we will have a framework of collagen and on this framework mineralization will happen by the very organized deposition of calcium that is controlled by DPP DPP is absent in Dentinogenesis Imperfecta Dentinogenesis imperfecta = a condition in which the formation of dentine is not perfect because of the absence of a very important protein for the process of mineralization which is DPP Role of matrix proteins in mineralization: o DPP: Transport of ions to mineralization front
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 Determines the location of nucleation (the place where mineralization starts) to specific regions of collagen fibril surface Aids in the stabilization of the formed crystal DPP is a very important molecule that should exist in the mineralization of perfect dentine o Other proteins that exist in dentine and may have a role in mineralization: Osteonectin Osteopontin Bone sialoprotein Proteoglycan chondroitin sulphates 4 and 6

Matrix Vesicle Mineralization: The first process of dentine mineralization is DPP regulated mineralization and then comes the second process which is the Matrix Vesicle Mineralization (where DPP isn't important at all) This process of mineralization is limited to mantle dentine Occurs in addition to or in place of DPP-mediated nucleation on collagen fibrils Mantle dentine mineralization takes place either by DPP-regulated mineralization and/or by matrix vesicle mineralization Circumpulpal dentine mineralization takes place by DPP-regulated mineralization ONLY (Matrix vesicle mineralization only takes place in mantle dentine) Cell budding fragmentation to form matrix vesicles occurs in what will become mantle dentine Formation of membrane-bound organelles (30-200 nm) leads to concentration of phosphate ions and thus development of mineral crystals within the vesicles Crystals initiate mineralization Similar mineralization occurs after the development of a nidus (nucleator) of cell debris Mineralization front (junction between Predentine & dentine) can be irregular (spheres/calcospherites) Calcospherites and/or linear Each nucleation area enlarges to become a structure called Calcospherites which is a spherical part of dentine , when these structures get close to each other, they fuse to become mineralized Dentine As mineralization of dentine is more active, calcospherites join together irregularly and lead to irregular mineralization front As mineralization of dentine is inactive , calcospherites join together regularly and lead to linear smooth mineralization front Irregular mineralization fronts are common in primary dentine (dentine formed before tooth eruption) while linear mineralization fronts are common in dentine forming throughout life after tooth eruption Failure of calcospherites to fuse produces interglobular dentine often in peripheral dentine
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CROWN PORTION OF THE TOOTH

ENAMEL MANTLE DENTINE INTERGLOBULAR DENTINE DENTINE CIRCUMPULPAL DENTINE PREDENTINE SUPRA-ODONTOBLASTIC LAYER ODONTOBLASTS DENTAL PULP CELL FREE ZONE OF WEILL CELL RICH ZONE PULP CORE
Formation of root dentine: Root dentinogenesis is similar to coronal dentinogenesis EXCEPT peripherally First collagen is NOT deposited immediately against the basal lamina at the CDJ so there is a space between cementum & dentine which is going to be filled by the Epithelial Root Sheath with an amorphous ground substance and a fine-fibrillar non-collagenous matrix this layer is called the hyaline layer which is about 10 m thick ** Epithelial root sheath originates from an area in the crown called "cervical loop" and its function is to shape the root similar to the IEE which shape the crown ** The first layer of dentine in the crown is collagenous , while the first layer of dentine in the root is non-collagenous ** Hyaline layer is as structurless layer that is in direct contact with cementum on the external surface of the root The initial collagen fibrils in roots are deposited parallel to the CDJ (cemento-dentine junction) compared to the initial collagen fibrils in crowns that are deposited perpendicular to the DEJ Underneath the hyaline layer theres a layer called Granular Layer of Tomes and there are 2 hypotheses that explain its formation: Radicular odontoblasts develop several fine branches in an umbrella fashion Unmineralized interglobular areas Calcospherites are smaller and Interglobular areas are more numerous than those of coronal dentine Rate of root dentine formation is slower than that in the coronal dentine

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ROOT PORTION OF THE TOOTH

PULPAL CORE

CIRCUMPULPAL DENTINE

GRANULAR LAYER OF TOMES

HYALINE LAYER OF DENTINE

CEMENTUM

PDL

ALVEOLAR BONE

DENTINE
Formation of peritubular dentine: Peritubular dentine (forms immediately and makes the walls of the tubules that surround the odontoblastic processes) A Intertubular dentine (forms between the tubules ) B Small crystal in an amorphous matrix Peritubular dentine is a product of odontoblasts & plasma proteins that have diffused along the cell membrane Tubular occlusion: Age related (especially near root apices ) the diameter becomes smaller with age, this is useful in forensic dentistry as it is used to identify the approximate age of a dead body Caries this causes re-precipitation of minerals in an attempt to resist caries distribution Types of dentine: Primary dentine = all dentine that forms BEFORE root completion Secondary dentine = any dentine that forms AFTER root completion and throughout life without a stimulus (e.g. NO caries, NO injuries ) Tertiary dentine = any dentine that forms AFTER root completion in response to a stimulus (e.g. injuries, caries )

Formation of secondary dentine: Physiological slower-paced & down-regulated deposition after root completion ** Secondary dentine has slower deposition & lesser regulation compared to primary dentine formed before root completion Deposition of secondary dentine is asymmetric roof & floor of pulp chamber have faster rate of deposition compared to sides ** This is a real problem in the field of endodontics especially in old people where too much secondary dentine is deposited constricting the dental pulp too much and making the floor & roof close to one another (even if sides aren't that much affected)
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Secondary dentine deposition leads to decrease of pulp volume which leads to decrease in odontoblast population ** When pulp volume decreases, odontoblasts become crowded, and this decreases their productivity and decreases the quality and regularity of dentine they produce

Change in the direction of tubules but continuous with those of primary dentine because of the crowding and the less number of odontoblasts ** Same cell is responsible for both dentines Challenges Root Canal Treatment in old people

Formation of tertiary dentine: By definition, tertiary dentine is dentine forming in response to stimulus Severe stimuli leads to pulp necrosis (death of dental pulp) Less severe stimuli induce the production of tertiary dentine Tertiary dentine is formed by odontoblast newly differentiated from the pulpal mesenchyme after original cells have died ** Dental papilla divides into 2 populations of cells: one population remains attached to basement membrane and another population migrates to core of dental pulp and can be called when needed to produce tertiary dentine The new odontoblasts dont form DPP tertiary dentine mineralization is much affected Tertiary dentine resembles osteoid rather than tubular dentine ** Osteoid = bone forming upon injury ** Sometimes tertiary dentine isn't tubular at all and it doesn't resemble tubular dentine because its process of formation isn't regular and isn't organized A range of responses ranging from secretion of: o Regular tubular matrix (bearing a little different from primary dentine) o Very dysplastic matrix Classified into: o Reactionary o Reparative Reactionary tertiary dentine: o Secreted by surviving original odontoblasts subjected to milder stimulus Irritation from caries bacterial product Irritation from cavity preparation Leaching from restorative materials ** All of these stimuli injure original odontoblasts but dont kill them and so they are still able to restore dentine if injured Reparative tertiary dentine: o Secreted by a new generation of odontoblast-like cells after the death of the original odontoblasts forming primary and secondary dentine o Response to stronger stimuli
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o o o o

Can either be a sequel to reactionary dentinogenesis or independent Recruitment of pulpal progenitor cells Less permeable due to different (or even lost) tubularity Preceded by secretion of atubular fibrodentin matrix which may play a role in signaling for the true reparative dentinogenesis

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