Dentin, a tissue of ectomesenchymal origin, forms most of the mass of the teeth. Its properties are similar to bone in many respects, but it has no trapped cells inside and is not involved in calcium phosphate metabolism.
Accordingly, its remodelling and regeneration is limited.
Dentin structure and formation. Cells produce dentin, cementum and bone first by creating an organic matrix that contains high levels of collagen. Inorganic calcium phosphate is then deposited in this matrix. Dentin contains 70% minerals, 20% organic material and 10% water. The main organic components are collagen and a number of non-collagenous proteins that have a role in the mineralisation process.
Figure 1.45. Figure 1. – Constituents of dentin
Types of dentin are classified based on their formation. Mantle dentin has an amorphous structure with no tubules. It is produced by newly differentiated odontoblasts with processes not yet developed, located at the enamel-dentin boundary. Primary dentin is formed subsequently. It has a characteristic tubular structure, and forms the main mass of the tooth. Secondary dentin is produced after root formation is complete, throughout the entire life. Tertiary or reparative dentin is produced in response to harmful external stimuli and is a barrier formed at the damaged surface.
Figure 1.46. Figure 2. – (A) Primary, (B) secondary and (C) tertiary or reparative
dentin
Tooth development starts as a result of ectodermal-mesenchymal interactions. The dentin is produced by dental papilla odontoblasts which form a single layer between the dentin they produced and the pulp. Unlike bone tissue, dentin does not contain cells, only odontoblast processes. Odontoblasts are of ectodermal origin; the differentiation of their blastoid progenitors starts in response to triggers that come from the inner enamel epithelium.
During initial mantle dentin formation, odontoblasts secrete (in addition to collagen) calcium phosphate via matrix vesicles. Simultaneously, odontoblasts become elongated, polarized, and are arranged in a tight columnar pattern. With the thickening of the dentin layer, the odontoblast cell body retreats, leaving behind thin cytoplasmic processes in the newly formed microscopic channels called dentinal tubules. In contrast to ameloblasts, which are of epithelial origin, an odontoblast never forms tight junctions around its entire circumference. The cell body is very rich in endoplasmatic reticulum and in Golgi apparatus. However, beyond the terminal bar, odontoblast processes are devoid of these organelles.
Figure 1.47. Figure 3. – Differentiation of odontoblasts
Figure 1.48. Figure 4. – Formation of mantle dentin during the early phase of
mineralization
Simultaneously with mantle dentin mineralisation, stable odontoblast processes and surrounding dentin tubules are formed by the cells. Predentin is located between odontoblast cell bodies and the mineralisation front, and is a partially mineralised ground substance.
Figure 1.49. Figure 5. – Dentin is produced by odontoblasts
Figure 1.50. Figure 6. – Mature secretory odontoblast
The secretory activity of odontoblasts shows a distinct spatial pattern. Cells produce collagen, proteoglycans and calcium ions at their neck, at the root of the processes. After demineralization, the organic structural framework of dentin, composed of collagen fibers, can be clearly observed. However, the exocytosis of phosphoproteins and proteases occurs from the processes at the mineralisation front. Proteases constantly degrade proteoglycans and phosphoproteins. Decreasing the amount of proteoglycans promotes mineralization, while phosphoproteins are instrumental by serving as phosphate donors.
Figure 1.51. Figure 7. – Involvement of hard tissue proteins in mineral formation
Dentin sialophosphoprotein (DSPP) or phosphoforin is a strongly acidic protein produced by odontoblasts. It is the most important non-collagenous component of dentin, and is also essential as a heterogenous mineralisation nodule and a phosphate donor. Its importance is shown by the fact that its genetic mutations cause dentinogenesis imperfecta which is associated with significant dentin damage.
Figure 1.52. Figure 8. – Dentinogenesis Imperfecta
Dentin permeability. The tubular structure of dentin makes it permeable if for any reason, such as tooth decay or abrasion, it opens to the surface. The formation of dentin tubules is defined by the odontoblast processes.
Figure 1.53. Figure 9. – Collagen fibers around tubules
Figure 1.54. Figure 10. – Components of dentin
The diameter of dentin tubules is 1 to 3 µm so they act as a bacterial filter by preventing the entry of microorganisms. Their density is greatest in the upper third of the pulp chamber, and is lower towards the root.
The processes of the odontoblasts contain dentin fluid and mineral deposits.
Figure 1.55. Figure 11. – The empty dentin tubules provide the basis for permeability
longitudinal section
Figure 1.56. Figure 12. – Permeability: the number and the diameter change depending on the dentin tubules
The opening of dentin tubules results in increased activity of sensory nerves in the pulp, thus causing pain, so it is a major problem in dental practice. Through the dentin tubules, fluid and mass transport can occur basically in two ways: convective transport, which facilitates mass transfer, and diffusive transport, whereby solutes are transported.
Figure 1.57. Figure 13. – Neuronal network of pulp/dentin
Convective transport has the main role and can be driven by the pressure difference between the outer and inner pulp space, for example in inflammation, or tooth decay caused by high-sugar solutions, or when the dentin is being dried by air or cotton wool, or when biting on a dislocated filling.
The extent of fluid flow is described by the Hagen-Poiseuille equation. The extent of convective transport is determined above all by the diameter of the dentin tubules. The dentin tubule is not an ideal pipe, because the odontoblast processes, collagen fibers, nerves and also mineral and protein precipitates narrow it. Thus blockage or at least narrowing of these tubules can be important in dental practice.
Figure 1.58. Figure 14. – Hagen–Poiseuille equation – fluid movement – basis of the hydrodynamic theory
Figure 1.59. Figure 15. – Increase of outward fluid movements from the pulp during inflammation
The role of diffusive transport is secondary because the penetration of substances by diffusion is much slower than the development of pressure differences. Diffusive transport results in harmful chemicals appearing in the pulp. Of these, toxins released from the cell wall of bacteria are especially important. Toxins may diffuse through the channels to the pulp. The rate of diffusive transport is described by Fick’s second law. The rate of diffusion is determined especially by the diffusion area, the length of the tubule and the concentration difference.
Figure 1.60. Figure 16. – Diffusion – Fick’s 2nd law
Figure 1.61. Figure 17. – In a caries lesion, cariogenic bacteria invade the dentinal
tubules, demineralizing sclerotic and peritubular dentin in the process
Figure 1.62. Figure 18. – Dentine sensitivity
Dentin permeability is extremely important in the so-called hypersensitivity, which is often caused by the very thin or partially missing overlapping area between enamel and cementum at the tooth neck. Treatment options include the closing of tubules with poorly soluble calcium salts or protein precipitates. In addition, a commonly method in dental practice is to hyperpolarize or depolarize nerve fibers by altering the ionic milieu, for example by using a high potassium toothpaste.
Figure 1.63. Figure 19. – Dentine hypersensivity – Treatment
4.1. Test – Dentinogenesis and disturbances; formation of
primary-, secondary- and tertiary dentin; dentin permeability
(answers)
1. Caries induced tooth pain is primarily induced by:
A. bacterial invasion B. opening of dentin tubules C. increased depolarisation D. increased hyperpolarisation 2. Usual diameter of dentin tubules:
A. 0,2-1 μm B. 20-100 μm C. 0,2-1 nm D. 2-10 μm
3. Dentin hypersensitivity can be diminished by ….. of dentin tubules A. drying
B. cleaning C. closure D. opening
5. 1.5. Amelogenesis – Gabor Varga
Tooth enamel is the hardest tissue of the body: the matured enamel consisting of 96% hydroxyapatite crystals and containing only a few percent of protein and water. It perhaps surprising that enamel is secreted by epithelia. However, it is very different from other epithelial products like the fluid-rich secretions of salivary glands, pancreas and liver.
Figure 1.64. Figure 1. – The arrangement of ameloblasts during enamel formation
The secretion of enamel by ameloblasts is a two-stage process. The first step is the building of a slightly mineralized matrix structure and the second step is the remodelling of this matrix to a highly mineral-rich structure.
Figure 1.65. Figure 2. – Amelogenesis
Ameloblasts originate from the inner enamel epithelium and have several forms according to their functional state during their life cycle. These are the morphogenic, inductive, early secretory, late secretory, maturation ruffled-ended, maturation smooth-ended and protective forms.
Figure 1.66. Figure 3. – Formal and structural changes of ameloblasts during enamel formation
These ameloblast forms change according to a strict time schedule and each has a specific functional role in particular phases of amelogenesis.
Figure 1.67. Figure 4.
In the secretory phase of amelogenesis ameloblasts are tall, columnar cells rich in mitochondria, endoplasmic reticulum and Golgi apparatus according to their active transport processes. Ameloblasts can be clearly distinguished from odontoblasts by their tight junctions. Ameloblasts have characteristic epithelial tight junctions throughout their lifetime which close the intracellular space and separate the apical and basolateral surfaces of the cell. These tight junctions allow the maintenance of extreme concentration gradients between the apical and basolateral extracellular spaces. The terminal bars prevent the entry of organelles into the Tomes process. The Tomes process of the ameloblast is a short, lance-shaped structure that provides the surface for the secretory transport processes. Calcium and phosphate ions (necessary for mineralization) are actively transported into the mineralization space in a basolateral to apical direction. The molecular mechanism of this mineral transport process is only partially understood at present.
Figure 1.68. Figure 5. – Secretory ameloblasts – formation of prismatic enamel (PE) and
interprismatic enamel (IPE)
The enamel matrix is not homogeneous. During enamel development central and lateral (also called prismatic and interprismatic) crystal rods are formed. Central rods are formed right below the Tomes process and have higher density. Interprismatic rods are not directly below the processes but are lateral to them, therefore the mineralization efficiency is lower there and less dense crystal is formed than in the prismatic area.
After the secretory phase the enamel is 30% mineralized. In this phase the mineral content is condensed into thin, parallel crystal ribbons while the space between the crystals is filled by matrix with high amelogenin content.
Figure 1.69. Figure 6. – Parallel running crystallites (Kr) in the early phase of enamel development
The enamel maturation. During the maturation phase ameloblasts change their morphology and cyclically transform between ruffle-ended and smooth-ended forms. The result of this cyclical modulation is that ameloblasts have a double function in this phase: they have to secrete calcium and phosphate and neutralize the protons liberated during hydroxyapatite crystal growth while they also have to reabsorb and degrade amelogenin cleaved by kallikrein-4 and MMP-20.
Figure 1.70. Figure 7. – Maturation ameloblast phenotypes
The essence of the process is that the protein matrix with high amelogenin content is degraded while in parallel the crystals expand in thickness until the whole matrix is eliminated and replaced by the tightly packed and practically impermeable crystal structure. The smooth-ended ameloblasts produce and secrete enzymes to
degrade and reabsorb the matrix that is no longer necessary. In the smooth-ended form, electrolyte transport is the primary function. But its mechanism and control is not known in detail. The papillary cells above the ameloblasts may have an important role in supporting the transport activity of the ameloblast cells and the removal of unnecessary materials. The ruffle- and smooth-ended forms interconvert cyclically.
Figure 1.71. Figure 8. – Hypothetic model for pH regulation by ruffle ended ameloblasts to neutralize liberated H
+The result of amelogenesis is an almost completely impermeable structure. There is some density difference between the crystal rods and the matrix but the whole enamel is 96% mineralized and it is the hardest tissue in the body.
Figure 1.72. Figure 9. – Cross sectional arrangement of enamel cristal rods (prisms)
Figure 1.73. Figure 10 . – Structure of the matured enamel
After the maturation phase, the ameloblasts de-differentiate, becoming short and cuboid rather than tall and columnar, and make a protective layer until eruption.
Secretory ameloblasts secrete numerous proteins whose functions are not completely clear but are essential for the construction and remodelling of enamel.
Figure 1.74. Figure 11. – Amelogenesis – list of enamel proteins
Amelogenin is coded by two genes, by AMELX and AMELY located on the X and Y chromosomes.
Significantly more protein is produced from AMELX. Among the enamel proteins amelogenin is present in the largest quantity. Its exact role is unknown. It is a small, globular protein when produced, which then forms nanospheres during the self-assembly of the proteins. The nanospheres interact with each other and create grid-like sheets to control the formation and orientation of enamel crystals. During the early phase of maturation enamelysin, and later kallikrein-4, disrupt the grid structure and break down the nanospheres. The building blocks become degraded and can be taken up by the cells while the space left behind is filled with the thickening crystals. By the end of the maturation phase amelogenin has almost completely disappeared from the enamel.
Figure 1.75. Figure 12. – Concept of the role of amelogenins in the mineralization of
enamel
Ameloblastin is the second most abundant protein in the enamel. Its functions are not completely established but one of them is to influence ameloblast function. Extracellular ameloblastin initiates cell differentiation via specific receptors. It also inhibits cell proliferation by the activation of tumor suppressor gene 21 and p27. In addition it supports amelogenin secretion by blocking the transcription factor MSX2. Overall it supports differentiation and matrix formation.
Figure 1.76. Figure 13. – Role of ameloblastin in the regulation of ameloblast function
Enamelin is secreted by the ameloblasts. Although its exact function is unknown, it is able to interact with amelogenin and other matrix proteins and regulates the growth of enamel crystals.
Enamelysin (MMP-20) is a protease produced in the early stage that is able to degrade the enamel matrix proteins. Kallikrein-4 (KLK-4), also called protease 2, is produced and secreted in the maturation phase. It eliminates proteins that were not degraded by enamelysin.
Mutations of the AMELX gene can cause amelogenesis imperfecta (AI). Mutations of AMELY are unknown.
Deletion of the AMELX gene in mice results in the formation of a very thin enamel. Mutations in ameloblastin can also cause AI. In addition the thickness and prismatic organisation of enamel are found to decrease in enamelysin knock-out mice. Three different mutations of enamelin have been described so far, and active proteases can also cause the disease.
Figure 1.77. Figure 14. – Amelogenesis imperfecta
Figure 1.78. Figure 15. – Structure of the X-chromosomal copy of the human amelogenin gene
5.1. Test – Amelogenesis (answers)
1. Cells forming enamel:
A. osteoblasts B. odontoblasts C. cementoblasts D. ameloblasts
2. Percentual representation of amologenin among proteins in non-maturated enamel:
A. 5-10%
B. 23-30%
C. 40-50%
D. 60-70%
E. 90%
3. Which protein gene mutations may lead to Amelogenesis imperfecta A. collagen
B. kallikrein-4 C. proteoglycan D. foszfoforin