9 Enamel development

scientific diagram of signaling in amelogenesis
Figure 9.1: An illustration of the large number of and complicated interplay between morphogens involved in amelogenesis. Image credit: "The sequential and reciprocal regulatory signaling between epithelium (red) and mesenchyme (blue)" by Thesleff, I. and Tummers, M, Stembook.org, is licensed under CC BY 3.0

Overview

Sola dosis facit venenum (the dose makes the poison)Paracelcus

Even though dentin formation begins before enamel, we begin with enamel formation. This way, the chapter on dentin formation can remain close to pulp, which is important because they share the same from . Enamel is produced by cells derived from the . This leads to key molecular differences between the of enamel versus the ECM of dentin, cementum and bone tissue.

One major concept we focus on is that and undergo signaling, meaning you can’t get odontoblasts without ameloblast signals, and you can’t get ameloblasts without odontoblast signals. It takes a team effort. In this chapter, we mention three (ones we’ve covered before), and two  . Hopefully, Fig. 9.1 illustrates that the process is more complex. Don’t memorize the morphogens unless your instructor indicates they are on your exams. The important concept is that when we get to chapter 10 and discuss how dentin undergoes regeneration, we should question how it is similar to  embryonic , and how it is different as well.

photo of crystals
Figure 9.2: Fluorapatite crystals form in nature, shown here with quartz crystals. “Apatite-(CaF), Quartz” by Rob Lavinsky, irocks.com is licensed under CC BY-SA 3.0

One reason to study embryology is to learn about how tissues are made in the first place. When we do, we often learn how to repair tissues  better. For bone and , learning how into adult cell types has led to advances in , and . Could we one day re-grow enamel using stem cells? In this chapter, think about why creating  new ameloblasts might not be as useful to us as new osteoblasts or fibroblasts (hint: location, location, location). Then, focus on how enamel matrix is formed. Secrete some proteins and minerals and turns into teeth and bones, right? Fig. 9.2 shows fluorapatite crystals. A mouth full of those would look impressive, but wouldn’t be very functional. Calcium and phosphate react to form a powder, you can probably find a bottle of it in your school’s chemistry lab. To get these chemicals to react in a way that creates human teeth requires more than chemistry. So perhaps the biggest concept in this chapter is how ameloblasts create crystals in the shape and density of a tooth, rather than in the shape of Fig. 9.2. To understand tooth , you must understand how its is created.

In this chapter and the next, we discuss , and Tomes’ fibers (we use the name instead). If you wish, take a look at:

Amelogenesis

animation of amelogenesis and dentinogenesis
Figure 9.3: Animated representation of odontoblast and ameloblast formation.

Induction (or initiation)

This paragraph should be review. starts at the of tooth development. By this stage, cells have to produce two layers of called the inner enamel epithelium () and outer enamel epithelium (). Between the IEE and OEE, more epithelial cells are scattered, namely the and cells. Collectively, these cells are known as the .

Now let us add to what we learned in the previous chapter. The grows around a cluster of known as the . Initially, a separates the two. The first visible sign of occurs when the cuboidal cells next to the dental papilla elongate, becoming more columnar in shape. Their nuclei move to the side of the cell, while organelles needed for secretion accumulate on the side. We call these elongated cells . It is not certain what triggers this initial change in , evidence suggests a member of the family of activates signals. The ability to undergo halts, meaning no new pre-ameloblasts form after the bell stage. The disappearance of the basement membrane allows these cells to move more easily, due to the loss of attachments.

Pre-ameloblasts secrete a number of short-range , including a member of the family (Fig. 9.3). This neighboring of the to into and begin secretion of . This puts pre-ameloblasts in direct contact with pre-dentin. on pre-ameloblasts contact a form of found in pre-dentin (type I), as opposed to the type in the basement membrane (type IV). In addition, newly formed odontoblasts secrete several morphogens, including . The important concept is that half of the signal that induces the differentiation of comes from the pre-dentin side.

The second half of that signal comes from the opposite direction, the . One such signal produced by stellate reticulum cells includes a member of the family. This leads to the activation of in the pre-ameloblasts, which up-regulate the of involved in the secretion of enamel. Transcription factors involved in amelogenesis include MSX-2, a gene, and RUNX2, a transcription factor also involved in the differentiation of .  Visible under the microscope, pre-ameloblasts change shape again, becoming shorter, and develop a bump on the basal side known as . A summary of some molecules involved is in Table 9.1. The names are less important than the general order of events: from to to transcription factor to gene expression.

Table 9.1: A partial list of molecules involved in ameloblast differentiation.
Morphogens
Receptors Transcription factors Genes up-regulated

Collagen

(pre-dentin)

BMP

(Odontoblasts)

Integrins

BMP-R

MSX-2

DLX1

RUNX2

Enamelin

Amelogenin

Matrix Metalloproteinase

FGF (Stellate Reticulum) FGF-R

photo of a thin slice of tooth
Figure 9.4: A thin section highlights enamel’s colorless state. Image credit: “18036” is in the Public Domain, CC0 / cropped

Apposition

After changing , secrete enamel matrix from . This ECM is similar to the of , but with important differences. Like bone matrix, enamel matrix is first secreted in a softer form (), consisting mostly of proteins and water, and it mineralizes later. Enamel matrix contains Calcium-Phosphate crystals and organic components. Enamel, after it matures, has a higher mineral percentage, making it the hardest substance in the human body. Unlike bone tissue, however, enamel matrix has very little or no . Instead, ameloblasts secrete other proteins known as and . These proteins act as nucleation centers which help induce Calcium and Phosphate to form crystals. We can think of these proteins as for non-living chemicals rather than cells. The important fact to remember here is that collagen is a protein commonly made by (and other cells derived from ), which most often from the . Ameloblast , on the other hand, is from the . Presumably the connective tissue like collagen are and packed away around , and are not available for .

The shape of the in the determines the shape of the crown. No new ameloblasts form because they lose the ability to undergo once they into . Furthermore, no can contact both pre-dentin and once thick layers of enamel and (mature) dentin push the two apart. Therefore, no new ameloblasts form by differentiation, either. Because of how the pre-ameloblasts line up in the bell stage, mostly pointing towards the oral cavity, enamel is thicker in the masticatory surfaces and thinner in the cervical areas.

photo of a tooth in cross section
Figure 9.5: Enamel is deposited appositionally, as seen by the Lines of Retzius. Image credit: "Tooth of Paranthropus robustus SKX 21841 from Swartkrans" by Didier Descouens  is licensed under CC BY 3.0 / red box added

Enamel is secreted by from , laying down new layers of over older layers, pushing the ameloblasts further away from dentin. Ameloblasts, like many human cells, do not work at a uniform speed. About every 4-12 days there is a change in the enamel deposition rate. As a result of deposition of enamel speeding up and slowing down, visible bands of lighter and darker enamel (less dense and more dense) are visible when viewed in cross section. These visible bands are known as (or striae of Retzius, Fig. 9.5). One such band, the , is particularly dark because the ameloblasts rested during the day we were born. After birth, the edges of the Lines of Retzius may be visible on the surface of teeth, especially the lingual surface of anterior teeth, in which case they are called (in older texts, the imbrication lines of Pickerill). Over time, chemical and physical abrasion wear perikymata away. Counting the number of perikymata and multiplying by a number between 4 and 12 yields an estimate of how long it took the crown of that tooth to develop. This has no use in dental hygiene. It does represent a pattern unique to each individual, and thus may act like a fingerprint, with possible applications in forensics and biometric identification (a tooth print instead of a fingerprint).

animation of enamel rods
Figure 9.6: Enamel rods are each produced by one small group of ameloblasts.

Together in a line, all of the   produce horizontal layers of enamel, new on top of old. But roughly one ameloblast produces a single (or prism) in a perpendicular direction (it takes 4 ameloblasts to make one enamel rod, but each ameloblast works on 4 different rods). Because the rods are rounded (almost hexagonal), and not square, there are gaps between them. These gaps aren’t empty, they are filled with a slightly different form of enamel, called (while the enamel within rods is called ).

photo of a thin slice of tooth
Figure 9.7: Hunter-Schreger bands are the enamel produced by a group of ameloblasts, and are lines that run perpendicular to the DEJ. Image credit: “18036” is in the Public Domain, CC0 / cropped

If you look closely to Fig. 9.6, you might notice (HSB), which are lines running perpendicular to the and the . Each line represents the contributions of a small group of ameloblasts. If it’s a little hard to see, it is because each Hunter-Schreger band is only 1/10th of a millimeter thick. What you are looking for is a pattern similar to the fore edge of a textbook (all the pages as a group). Ameloblasts do not work in straight lines, the bands curve slightly– like the pages of that $375 textbook you dropped in water and can’t sell back to the bookstore. Furthermore, different regions have different curve angles and patterns. These curves aren’t accidental, they increase the tensile strength of enamel. If you have ever chopped wood, you know it is easy to do if you land the axe along the grain. Hit at an angle, and it can hurt you instead. The curving of Hunter-Schreger bands ensures there is no single grain along which a tooth can easily shear. Cracks develop in enamel, but cracks usually stop part-way through. The signals that guide rod curvature have been identified (antagonists of Wnt morphogens). The end result is that enamel, despite having the brittleness of glass, is the most resilient tissue in the human body. Dentin has similar curves (plural) which, unlike enamel, get specific names. As a fun side-note, or of relevance to those planning on pursuing a career as a veterinary dental hygienist at your local zoo, the Hunter-Schreger bands of hyenas occurs in a zig-zag pattern, increasing the tensile strength of their enamel even more, allowing them to chew through bones.

Maturation

Enamel is first secreted as a protein-rich gooey substance called . Two major organic components of enamel are the proteins and . also secrete a digestive enzyme. This enzyme, a , digests , making it easier for ameloblasts to migrate though the of overlying . This is necessary because as ameloblasts deposit pre-enamel from , it pushes the cells outwards. If you recall, matrix metalloproteinases are also involved in the proper migration of tissues during formation of the palate as well as in . Matrix metalloproteinases serve a second role by processing enamel proteins made by the ameloblasts after they are secreted into the . The enzymes modify (not digest) amelogenins and enamelins, improving their ability to catalyze the crystallization of calcium and phosphate. This creates a delay between the secretion of pre-enamel and its mineralization. Without a delay, ameloblasts could become trapped by their own ECM, halting .

Unlike dentin and cementum, enamel undergoes a significant amount of after mineralization. Once pre-enamel has been laid down and mineralizes, ameloblasts switch tasks and cause the hard enamel to mature into even harder enamel. Most of the proteins are removed, replaced by fluids which crystallize. and are removed by endocytosis and by secretion of protein-digesting enzymes. These enzymes include the whose first cuts to amelogenin and enamelin improved their function, but more cutting breaks them down. Removal of protein increases the relative mineral content of enamel, increasing its hardness compared to bone, dentin and cementum. We can therefore think of the protein secreted by ameloblasts as a that ensures enamel is made quickly, at about 4 μM per day. In contrast, fluoride-based re-mineralization of tooth enamel is not measured using a small ruler, suggesting the rate of re-mineralization is too slow to see under the microscope.

A number of different electrolytes and molecules are incorporated into enamel in small amounts during the maturation stage. One important electrolyte is fluoride (F). Fluoride ions are negatively charged, and incorporate into calcium hydroxyapatite crystals by replacing a small number of hydroxide (OH) ions. This has important clinical effects. First, a small amount of fluoride increases the strength of crystals. Secondly fluoride ions found in either saliva or in enamel neutralize acids released by bacteria, reducing enamel demineralization and preventing caries. Excessive fluoride levels, however, affect the activity of ameloblasts during the maturation stage of amelogenesis, reducing their removal of protein . This may create a visual change in the form of white lesions on the surface of teeth. These lesions are where the enamel has a higher protein and lower crystalline content, known as . As with any chemical, the dose makes the poison. Topical application, from toothpaste or gels, allows fluoride to incorporate into the surface layers of enamel, which has considerable benefits. The greatest potential benefit is when fluoride exposure occurs during the apposition and maturation stages of amelogenesis, early in embryonic development.

Clinical considerations

schematic of remineraliztion
Figure 9.8: Schematic of enamel re-mineralization using a bioactive compound. Legend: DK-6 = control (inactive) peptide. DE-11 = active peptide. CaP = calcium phosphate. HA = carbonated hydroxyapatite. AS = acetic acid solution (to mimic caries). Image credit: Schematic of the adsorption of DE-11 on the demineralized enamel surface for remineralization restoration by Wang, K. et al is licensed under CC BY-NC 4.0

Enamel re-mineralization

Certain chemicals help induce calcium and phosphate to react and add to the crystalline matrix of enamel. The most common is fluoride. In addition to its role in neutralizing acids, Ffuoride nucleates crystals (see also Fig. 9.2). Topical gels and toothpastes that promote re-mineralization frequently contain Fluroride plus calcium and phosphate in forms that dissolve in saliva. Other agents are added to help fluoride to stick to the surface of enamel. After all, promoting the formation of calcium-phosphate powder in saliva won’t help teeth. One such agent is  casein-phosphopeptide. Casein is a protein found in milk. Its stickiness makes it useful in cheese-making and glue (including the original formula of Elmer’s glue).

Increased understanding of protein has allowed researchers and companies to develop better tools for assisting in enamel re-mineralization.The goal is to mimic the ability of and proteins to nucleate crystals from a solution containing calcium and phosphate. These products do not need to contain amelogenins, enamelins, or even proteins. For instance, xylitol and certain glass compounds nucleate calcium hydroxyapatite crystals. Xylitol, a sugar alcohol extracted from birch trees, has the dual benefit of activating sweet taste buds. It can be used to replace simple sugars in foods, removing substrates which harmful oral bacteria use for energy when secreting enamel-dissolving acids (just don’t let your dog eat any).


electrn micrgraph of acid etched enamel
Figure 9.9: Acid etching demineralizes rod and interrod enamel at different rates, producing a rough surface suitable for bonding. Image credit: "This work" by Janice Carr, USCDCP is in the Public Domain, CC0

Surface etching

Because is slightly different from , applying an acid to the crown surface wears the two at different rates—a process called . This creates an uneven surface, providing more surface area to create a stronger bond between the tooth and a surface sealant (think about how and interdigit with one another for a stronger connection). Treatment with a weak acid also removes surface debris which interferes with bonding of an adhesive, known as a smear layer.


photo of a mouth with bulemia
Figure 9.10: Erosion of enamel caused by bulemia. Image credit: "Oral manifestation of bulimia by Jeffrey Dorfman is licensed under CC BY 4.0

Erosion (or corrosion)

Prolonged exposure to a weakly acidic environment, or a shorter exposure to a highly acidic environment, can cause of tooth enamel. Positively charged H+ ions of an acid react with negatively charges OH and OPO33- ions of crystals, dissolving them. Bulimia and acid reflux bring highly acidic hydrochloric acid of the stomach in contact with teeth, causing erosion. , caused by the abuse of drugs such as methamphetamines, the use of certain medications, chemotherapy, or associated with old age, reduces the protective buffering ability of saliva to protect tooth enamel. Acidic foods or sugary foods may also contribute to enamel erosion. The simple sugars in many foods are nutrients oral bacteria metabolize and use to produce energy, which they then expend to create and secrete enamel-eroding acids.

radiograph of caries
Figure 9.11: Dental caries (red arrows) caused by impacted 3rd molars (green arrows). Image credit: "Impacted wisdom teeth caries" by Coronation Dental Specialty Group is licensed under CC BY-SA 3.0

Early detection of erosion (a , or tooth decay) can be done during a regular examination. Early signs of enamel erosion includes the appearance of a white lesion on the surface of enamel, which may be softer to the touch, and appear more radiolucent on a dental radiograph. Treatment often involves removal of the carious tissue followed by replacement with a material.


illustration of abfraction
Figure 9.12: Three common patterns of tooth abfraction, all along the dentin-enamel junction. Image credit: "Own work” By Ebd2015 is licensed under CC BY 3.0

Abfraction

When non-carious tooth material is lost by force, it is called . Abfraction frequently occur along the . Chewing causes flexion forces at the cervical region of the tooth. Treatment of this type of lesion should involve reducing the stress that caused it in the first place, such as parafunctional habits or tooth occlusion. Because enamel is stronger than dentin, dentin is frequently lost in response to force transmitted through the enamel to the DEJ.

Developmental disturbances

photo of a mouth with enamel hypoplasia
Figure 9.13: An example of the pitting and white lesions caused by enamel hypoplasia. Image credit: "Symmetrical enamel hypoplasia of grade I on permanent incisors in a CD patient" by Maurizio Procaccini et al, Head & Face Medicine is licensed under CC BY 2.0

Enamel hypoplasia

Reduced enamel formation during embryonic development is called . This occurs to primary and/or succedaneous teeth. Hypoplasia in an embryo is often triggered by health changes in the mother, such as a gastrointestinal disorder or a syphilis infection during pregnancy. If ameloblasts cannot acquire enough minerals during the time they are active, upon tooth eruption the surface enamel may contain white lesions, or exhibit pits and grooves. The enamel is more brittle and susceptible to and . Furthermore, increased sensitivity to temperature and increased incidence of dental caries and periodontal disease are expected. Because is complete long before birth, there is currently no way to significantly assist in enamel production after tooth eruption occurs. Therefore, preventative treatment becomes much more important, and full-coverage crowns might be required.


photo of a mouth with amelogenesis imperfecta
Figure 9.14: An example of a patient with amelogenesis imperfecta. Image credit: "Amelogenesis imperfecta" by 신연아 is licensed under CC BY 3.0

Amelogenesis imperfecta

Another cause of enamel hypoplasia is the genetic condition . There are a number of different that, when mutated, cause this disease. For instance, mutations in genes for and leads to forms of amelogenesis imperfecta by reducing the ability of scaffolds to rapidly nucleate crystal formation, and as a result the enamel is hypo-mineralized. Another gene mutation that can be inherited is in a . Without removal of ameloginins and enamelins by the matrix metalloproteinase enzyme, ameloblasts cannot create the high mineral content of healthy, mature enamel. Because amelogenesis imperfecta is caused by different mutations to a number of different genes, the symptoms occur on a spectrum, from mild to severe.


photo of a tooth with an enamel pearl
Figure 9.15: An enamel pearl. Image credit: "Own work", By Wicketcity is licensed under CC BY-SA 4.0 / cropped

Enamel pearl

The within does not normally into because of the lack of from . However, a small region of the IEE might mistakenly differentiate into ameloblasts along the root and produce a small amount of enamel over cementum, known as an There is no harm in having pearls, other than they may be confused with a calculus during a scaling procedure. If the wrong type of dental scaler is used, the instrument tip may break off when it encounters the much harder enamel pearl. They can be identified by radiography, because enamel is more radiopaque than cementum. Enamel pearls generally occur on the furcations of the multi-rooted molars.


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