25/26: crown development ii and enamel i-

Transcribed by Chris Bedoya April 21, 2014

Craniofacial Biology Lectures 25 – Crown Development II by Dr. Wishe

Slide 1 – Dog sippin on a mimosa Good morning. This is where we left off last time, except we’ll just assume our friend is drinking some OJ, and not a mimosa. Everybody alive? Eyes open? Good.

Slide 2 – Fig 5-8 Dentin FormationIn terms of discussion, the last thing we actually spoke about was the dental

lamina in terms of the formation of the molars, and I forget if I did or didn’t mention toothogenesis at that point in time, but this is a fairly common developmental anomaly. And it could happen on one side, or on both sides, but it turns out that the 3rd molar happens to be the tooth that is frequently missing in development of your dentition. And that’s no big deal, because there really isn’t enough room for the most part, to have your 3rd molars. A lot of people have problems because of the lack of space, and they become impacted, and it doesn’t erupt into the oral cavity in a nice straight pattern. It tends to incline to one side, it tends to incline towards the 2nd molar, and this could produce problems later on.

So this particular picture, we’re moving along in terms of development. And I like to discuss enamel development before dentin formation, everybody is different. But before I can say any words about enamel development, I have to start off briefly with some dentin formation. You should recall that I did mention somewhere along the line in the last lecture, that in order to have enamel formation, dentin has to form first. And that’s what this picture is essentially showing us. Let’s get my highlighter on.

So just as a review, this outer blue layer is your outer enamel epithelium. This region where the cells seem to be spread out, they kind of look like mesenchyme cells, but they’re not. That’s your stellate reticulum. Over here you have your inner enamel epithelium, which becomes your ameloblasts. And sitting right on top of it is this layer of cells known as your stratum intermedium, and these cells are more or less perpendicular to the underlying inner enamel epithelium. Going a little further down, your dental papilla. And all these cells represent your odontoblasts. Now the inner enamel epithelium releases born morphogenic protein, which stimulate a certain number of the mesenchyme cells of the dental papilla, and specifically it stimulates your msx1 genes. This then converts some of these mesenchyme cells to become odontoblasts. And once they become odontoblasts, they start moving around and begin to lineup just like you see them over here. But as you look at the picture there seems to be a fairly regular pattern of organization, in reality it’s not as perfectly lined up as the picture shows you. But the inner enamel epithelium, which become the ameloblasts, are like little soldiers lined up shoulder to shoulder. They maintain a perfect linear order, if you want to put it that way. Bone morphogenic proteins continued to be released from the inner enamel epithelium, and they now stimulate the newly formed odontoblasts to start producing dentin. So what you see in here in yellow is your early formation of dentin.

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Just to refresh your memory, the structure that looks like a loop is known as your cervical loop, and is formed by the junction of the inner and outer enamel epithelium, and it plays a role in root formation.

Slide 3 – Fig 5-13 AmelogenesisHere we have a little bit later picture. And technically what I’m showing you

now can fit into the what is known as your apposition phase, in terms of laying down your various layers…and so the amount of the yellow, the amount of dentin, is increasing in amount. And then you’ll see, I don’t know lets call this brownish area, brownish kind of tissue, once you have dentin formation the presence of the dentin induces the inner enamel epithelium to really completely mature and produce enamel as a tissue.

Slide 4 – Fig 5-9 Apositional StageThis is a picture, which isn’t so great, but basically is showing you what we

saw in the precious diagram. And this rectangular box is what’s being blown up in this bottom picture. So this is your pulp, and here are your odontoblasts. This mustard-type color represents the dentin that has formed. And if we switch to the other side, these are your ameloblasts, and this region represents the enamel. And as you look at the enamel you see almost like, something that looks like this (draws a horseshoe-shape upside down) those could be called the heads of the rods.

Slide 5 – Fig 5-19 Incremental GrowthWhat we’re actually witnessing in this picture is your actual incremental

growth. Whether we talk about enamel or dentin, it’s all by increments. And both the dentin and enamel will state to form at the most occlusal, cuspal surface of the actual tooth. So diagram one is showing you a little dentin that’s formed, diagram 2 you can see like there are 2 layers, 2 increments of dentin. And if we go to the bottom diagram, we now have 4 layers. So that’s your increments. But once you get dentin forming, you’ll notice in this picture, enamel now begins to form. And enamel keeps adding on once again, in terms of increments. So both tissues will form in terms of your organic matrix at the most cuspal/coronal part of the tooth, then it proceeds in a cervical direction. This picture is just showing you more enamel. And finally this last picture shows quite a large number of dentin increments, and enamel increments. Let me also point out to you right over here you sort of see a dark line, just over here you see the dark line again, there it is, and over here. That represents the DEJ, which separates the enamel and the dentin. And in terms of mineralization we have the same type of pattern, it will start off at the most occlusal, cuspal, coronal part of the tooth and proceed cervically.

Slide 6 – Fig 5-20 Enamel MineralizationAnd this is supposed to illustrate your pattern of enamel mineralization. This

sort of pinky colored tissue is your dentin, and this picture is just meant to show you the mineralization of enamel. “A” shows you two layers of enamel, “B” there’s three, and “C” there’s also three, but the difference between B and C, it’s showing you the mineralization pattern. And the enamel that becomes mineralized first will be

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closest to the DEJ. If you try to mineralize the enamel over here, like by the ameloblasts, you would not be able to mineralize all of the tissue. It’s like the example I used in class last week. You’re out there, I’m up here, I put a cement wall between us, you can’t get passed the cement wall.

As we go from “D” to “F” it’s showing you more and more of the tissue becoming mineralized. This one is less mineralized, and that’s even less. So this picture isn’t depicting anything about dentin, but it’s just showing you that enamel mineralized also in increments.

Slide 7 – Fig 4 Tooth Development AppositionHere’s a nice picture that shows us apposition of the various tissues. Some

place in the head area is all mesenchyme, here’s your developing tooth, and although dental papilla is shown in the picture, at this particular point in time, the dental papilla should have become much more vascular, and we should be able to actually call this your pulp. These are the odontoblasts, which were formed form the mesenchyme of your dental papilla. There’s your dentin. And I don’t know how well you can see it on the screen, but you can probably see it on your computer, but there are little lines between the odontoblasts and the actual dentin. (Starts drawing on screen) If we look at an odontoblast, actually let’s call this the DEJ. We continue looking at it. The odontoblast gives rise to a process, and it’s called your odontoblastic process. And what happens is that the odontoblast gives rise to the process and then from the process, dentin is released outside the process to surround it. And as more and more dentin is being produced, the odontoblast doesn’t have much of a choice, it’s going to recede back into the pulp. And where the process used to be is where you’re going to find your dentin that has been laid down. And in next stage, would look like this. Here’s our odontoblast, and here’s your dentin that’s being laid down. We’ll come back to that when we do dentin formation. So the little thing that you’re seeing here happens to be the odontoblastic process. What about the enamel. Keep in mind that the enamel organ (starts drawing) originally looked like this, and there are a lot of loosely dispersed cells in this area, and they’re called the stellate reticulum. They look like mesenchyme cells but they weren’t, they’re all ectodermally-derived. And they seem to be spread out, but they are attached to each other by desmosomes. And it contains fluid which cushions, protects, their underlying enamel epithelium. The same thing with enamel, once you produce enamel it has to go some place, so the ameloblasts move back into the stellate reticulum, narrowing the space itself. And where the ameloblasts used to be, is where the enamel now gets deposited. So by the time you’re all through, your stellate reticulum should be completely gone. And then you’ll have the remnants of your ameloblastic layer, your stratum intermedium, if there’s any stellate reticulum left, and the outer enamel epithelium, and you put all these layers together we get a structure known as the reduced enamel epithelium, which we will come back to in a while.

So here are out ameloblasts nicely lined up. You don’t see a stellate reticulum anymore. And this primary tooth has detached from the surface epithelium, oral epithelium, but we see a structure over on the left. Coming off your dental lamina is a structure known as your successional lamina, that’s really where the permanent

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tooth bud arises from. We’re not interested in that in this particular picture, but that’s what gives rise to your secondary dentition.

Slide 8 – Fig 5-25 Crown Almost Completely Formed Here we have a multi cusped tooth forming. There’s your pulp, three’s your

dentin, and there’s a big space called the enamel space. Doesn’t seem to be any enamel located within it, except down in this neck of the woods. When we decalcify a tooth, we remove the calcium. And enamel is the most highly calcified tissue that exists. Like 96% hydroxyapatite. If you’re gonna de-calcify the tooth, the enamel is gonna go bye bye. That’s why we have this enamel space. This is just a little remnant of your enamel that wasn’t quite removed. Then we have this layer , which seems to go all the way around this developing enamel organ, that’s the reduced enamel epithelium. The remnants of all those structures I mentioned before: your ameloblastic layer, stratum intermedium, stellate reticulum, and outer enamel epithelium.

Slide 9 – Fig 6-24 REEThis is just another picture of the same thing from a different book. And as

you look at this you can see the reduced enamel has a certain thickness because it is a combination of various layers joining together.

Slide 10 – Fig 5-27 Root FormationThis is just to briefly introduce you to root formation. Dr. Craig will be talking

about that in the upcoming future. But remember we had a cervical loop, here’s your loop, outer and inner enamel epithelium joined up to form the loop, and from this loop you get forming what is an epithelial root sheath, and that’s what you’re seeing down over here. Same thing in this picture, as well as in diagram C. So with root formation, eventually you wind up with this sheath, sort of growing in a more horizontal direction, and the two ends don’t meet and join. There’s a space that’s created in here. Anyone know what that space might be? Apical foramen. That’s my discussion of the root. And what you’re also seeing as represented by this pink color happens to be your dentin. Here are your odontoblasts. And pre-dentin we really haven’t mentioned up to this point in time, but pre-dentin is uncalcified dentin. Remember in bone we had osteoid, that was uncalcified bone. You’ll find the same thing with cementum, cementeoid, uncalcified cementum. Enamel, the organic matrix forms and almost immediately mineralizes. So you don’t really have a pre-enamel per se, because it’s so short lived.

Slide 11 – Fig 5-29 Epithelial Rests of MalassezIn the periodontal ligament, which this is representative of, here’s your

dentin, and the cementum, and here’s your alveolar bone, you will find these little groups or clusters of cells, they’re called cell Rests of Malassez. And they’re just there from the break up of the epithelial root sheath as you’re forming the root. They can stay there, they can form something called enamel pearls. They could possibly turn to cysts, etc. But that will be covered under your PDL.

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Slide 12 – Fig 7-2 Keyhole HypothesisNow the theories of how enamel rods are formed. And it’s known as the

Keyhole Hypothesis. There are two theories, and to me it’s just a matter of changing of terminology. One theory doesn’t help us understand how enamel is formed more than the other. As you look at the structure in purple, that’s where the term keyhole came from. At one time most of the locks used to have a shape like this, and you had this big skeletal key. And if you look at these cold castles in various countries, you’ll find that type of locking mechanism still in existence. Maintains the old pattern. As we look at this picture, we see these 6-sided figures, these hexagons, and that’s supposed to represent a cross-section through your ameloblasts. According to the original theory of terminology, 1 ameloblast, this forms the head or body of your enamel rod. Then one, two, and you don’t really see it but it’s down over here, 3 ameloblasts form this part of the rod, known as the tail. And if you play with this diagram you will figure out that each ameloblast will help in the formation of 4 enamel rods. The lines that we see off to the side here represent the deposition of the hydroxyappetite crystals, and we have a better picture of that.

The second theory states that this structure is actually the enamel rod. When you use the word rod, you have an impression of something circular that looks like this. And that certainly fits the bill. What we were calling tail now becomes known as the inter-rod substance. And we’re going to use the same mathematics that we did before. 1 ameloblast forms the enamel rod. 3 ameloblasts will form the inter-rod substance. Does that actually helps us with understanding how enamel forms? I don’t really think it does. It’s just a different way, a different philosophy of looking at it.

Slide 13 – Fig 3-49 Keyhole HypothesisAnd here’s a nice picture again showing you the keyhole hypothesis. And

what’s nice about this particular picture you can see that this ameloblast, plus that, and a little piece up here, that’s 3 ameloblasts are going to form the tail, or inter-rod substance.

Slide 14 – Fig 7-3 Keyhole HypothesisHere we have an enamel rod that’s actually been pulled out. So the book that

we are using for the course by Tencotti (sp?) goes with the idea of rode and inter-rod substance. As we look at this picture, we have a head, or body, and a tail.

Slide 15 – Fig 7-5 Keyhole HypothesisAll the dashes that you see here actually represent the HAP crystals, which

are depositing. We start off with calcium, it gets organized into a crystalline structure. If you look at the head or body, most of these crystals are oriented parallel to the long, or axial direction of the rod. As you get down towards the tail, you can use the word oblique. So they’re extending in the tail, or inter-rod substance if you like, up to an angle of 60-65 degrees.

Slide 16 – Fig 3-5 Keyhole Hypothesis

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Here’s a nice EM picture showing us the keyhole hypothesis. And the reason you can see this part versus that part is because the difference in orientation of the crystals, and as the lights hit the crystals, if the crystals are in different directions, that’s gonna give you two different images. So technically this is the body or head of the rod, that’s the tail. I’m essentially drawing a circle now around this, to match the new theory of terminology, and that’s your enamel rod. It looks like this. And that’s your tail, or inter-rod substance. Since enamel is 96% HAP, what else makes up enamel? You’re gonna have 2% water, 2% organic matrix. And the matrix is going to go around the rod, in essence what I drew in red, that’s the organic material, and it’s called a rod sheath. So 2% isn’t a lot of protein, but it’s sort of encapsulating, de-limiting one rod from another.

Slide 17 – Fig 6-24 REEThis we already saw serves no purpose at this particular time.

Slide 18 – DVD – Dentin FormationThis is showing you a type of slide we used to use in the conference when we

did study formation of your dentition. This again is your pulp. You can see little triangular processes here, these re the lighter areas, and that’s what I drew in one of these pictures before, they’re extensions of your odontoblast cell body. Then you see sort of a lighter pink tissue surrounding the process, that’s your pre-dentin, or uncalcified dentin. When we look at this region, and this double arrow is covering it, that’s the more mature tissue of which calcium has been deposited. This arrow head is pointing to this particular area here (highlights border between black and dark pink sections) there’s artificial separation which there shouldn’t be, but that’s your DEJ. Switching to the left side of the picture, these are your ameloblasts. And you can see some sort of fuzziness right over here, that’s where you’ll have your ameloblastic processes, or Tomes’ processes. And this region represents enamel. The darker part of the enamel is more mineralized than the lighter part.

Slide 19 – DVD – Globular CalcificationJust another view. Again, your pulp would be up here on top. Odontoblastic

processes, you can see how they’re actually lined up compared to the cells right over here. These are the ameloblasts (below, purple/white area leaning to right slightly), and those are the odontoblasts (top row). You can see the processes a little clearer in this picture. This arrow is pointing to it (top left arrow), this is your dentin that has formed. This picture also has something that looks like this, sort of like a scalloped edge (boundary between middle pink layer, and light white/yellow layer above it). In actuality this is also part of it, same thing here, these are known as calcoglobules, which we will return in our discussion in our discussion of dentin. There are 2 patterns of mineralization: linear and globular. We’ll get into that at a later time.

Slide 20 – Fig 8-16 Mineralization: Linear, GlobularAgain from your textbook, as you look at picture A, it seems to be more

uniform in coloration throughout. But look at B, you see certain areas, darkened

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areas. Circular in fashion. This is illustrating the globular calcification, whereas this is a linear calcification.

Slide 21 – Dog PicHow about a little peep?

(Opens up PP: Mature Enamel 2014)

Slide 22 – Mature Enamel 2014

Slide 23 – Blank Slide, Wishe ended up drawing a bunch of different pictures hereSo now we come to the life cycle of the ameloblast . And there are various stages that we can identify. Pre-secretory stage, secretory stage, maturation stage, protective stage, and desmolytic stage. So pre-secretory means nothing is being secreted. There are 2 sub-listings under that: morphogenic and organizing or differentiating stage. During the morphogenic stage, the ameloblast is sort of like a low columnar cell. We can call this the apical part of the cell. This, the basal part of the cell, and enamel will be formed at the apical part of the ameloblast. So there’s nothing particularly unusual about the morphogenic stage. You have your Golgi and centrioles located up here, the mitochondria are uniformly distributed, and the nucleus tends to be more towards the center. Then we come to the organizing and differentiating stage, and now the cell becomes much taller, a longer simple columnar cell, nucleus is more towards the base, and the Golgi and centrioles shift position from the base towards the apex of the cell. And this has been called a reversal of polarity, a shifting of the position of the organelles. The cell is being set up for eventual enamel production. And the mitochondria are no longer evenly dispersed; they’re more towards the basal end of the cell. All epithelial cells sit on a basement membrane, and it turns out that the basement membrane thickens a bit and becomes your membrane pre-formativa, which represents the future DEJ. Then as we go a little further on in development, we’re still in the organizing or differentiating stage, and by now you should know that differentiation means to become more mature. The inner enamel epithelial cells release your bone morhpogenic proteins. And these proteins induce some of the mesenchyme cells in the dental papilla/pulp to become odontoblasts. Then, more bone morphogenic proteins are released, and the odontoblasts produce dentin. Once you get the initial formation of dentin, the dentin now has an inductive influence on the inner enamel epithelium, and the inner enamel epithelium completes this maturation and can now produce enamel matrix. So there are a lot of interactions between mesenchyme and epithelium, in terms of tooth formation. There are other interactions with the rest of the body as well involving those tissues, but we’re just concerned now with the oral cavity. So the organizing or differentiation stage, a lot goes on. Then we come to the secretory stage, and here’s where you’re dealing with mature ameloblasts, and its laying down/producing your organic matrix. And in order to do that, these ameloblasts have to have a full compliment of organelles like an RER, Golgi, mitochondria, etc. I might as well show you now the difference between an amelobalst and an odontoblast. You saw before a series that I drew, and this

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represents an odontoblast and its process. And so dentin is produced within the process, and deposited outside. So the process, the ondontoblastic process, actually becomes surrounded by dentinal tissue. In reality what we’re forming here happens to be your dental tubule. The wall for the tubule, and the structure in the middle of the tubule is the odontoblastic process. The cell body of the odontoblast is living. The cytoplasmic process, or extension, is living because it’s attached to a living cell. Since this is the arrangement in dentin, and the actual dental tissue is surrounding the process, dentin is now considered to be living tissue. If you look at enamel, here’s your ameloblast, and I’m gonna sort of exaggerate this a little bit, there’s your ameloblastic process, or Tomes enamel process, so enamel matrix is formed and deposited outside the process. But the process is not trapped in the organic matrix. SO you have a cell body, a process that’s living, but because of the lack of the trapping of the process, in the sense of being surrounded by your enamel matrix, enamel is not a living tissue. Big difference between the two of them. There are others, but that’s all I’m particularly concerned with at the moment.

So we’re up to actual formation of the enamel matrix, and then we have to mineralize the tissue. So we’re in the maturation stage, and that’s subdivided into a transitional phase, a maturation proper phase. The maturation proper phase takes up about 80% of the time for this particular maturation stage to occur. During the transitional phase, you’re having a certain amount of cell death occurring, irreversible injuries to the ameloblasts, apoptosis which means programmed cell death, but that only makes up a small part of the maturation phase. It’s like cleaning up the house a bit. Then in your maturation proper phase is when the tissue starts to mineralize. And probably about 90% of the protein is lost during this particular phase of development, leaving about 10% of organic material and water. By the time the tissue is completely mineralized, you only have 2% water, 2% protein, and the protein surrounds the enamel rods. Then we have protective phase, and a couple of slides before you saw something that looked like this. Here was your dentin, let’s call this the dentin, here’s where the enamel should have been but there was an enamel space. And surrounding this whole apparatus was the reduced enamel epithelium. Now the reduced enamel epithelium plays a role in two different phases, the last 2 phases. First we have the protective stage and the lytic stage. So this reduced enamel epithelium is actually surrounding and protecting the developing crown. Within your connective tissue you do have enzymes, which can digest and attack the enamel of the tooth. This particular layer prevents that from happening. When we move to the lytic stage, desmolytic stage, then the reduced enamel epithelium releases its own enzymes to digest the pathway through the CT so the tooth can erupt into the oral cavity.

Slide 24 – Fig 7-44 Enamel: AmeloblastsSo with any hard tissue the matrix that’s first formed is organic in nature, it’s

short lived, and then it disappears and is replaced by your actual HAP crystals. And for our purposes we can say the mineralization part of amelogenesis occurs in two stages: primary and secondary stage. This is just a picture showing you several ameloblasts, and these two stages switch back and forth, it’s called the modulation cycle. And the ameloblasts basically spend most of their time in this particular stage

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(cells on right), where you have your RER. Whereas 20% of the time is spent in this particular stage (left image) where you have fewer organelles. And look at the apex of the cells, they look different. So this is called a smooth-ended ameloblast, this is a ruffle-ended ameloblast. And by now when we use the word ruffle, you should begin to think of microvilli, increased surface area. And when you have the ruffled end type of cell, that’s when mineralization of the tissue actually begins.

Slide 25 – Enamel Proteins 1So the first part of amelogenesis was formation of organic matrix. The second

is mineralization. And when I spoke about primary mineralization, for our purposes there’s primary and secondary mineralization. With regard to primary mineralization, about 1/3 of the HAP crystals are laid down. And then the second stage of mineralization is when the remainder of the crystal formation is complete. But during the second stage, you’re not producing more crystals; you’re just increasing the size of the HAP crystals. In the clinic they may refer to things as primary, secondary, tertiary, quaternary mineralization. Primary mineralization is always laying down of the initial 1/3 of the crystal. Secondary, tertiary, and quaternary is used to indicate what part of the enamel is being mineralized. But for our purpose, primary and secondary is good enough.

Clinically speaking different things can happen. Not necessarily on a positive note. There could be genetic gene or protein problems, this could lead to tooth agnesis. The enamel that’s formed isn’t proper. You could get the formation of runty-type of teeth. Hypomineralized type of teeth. We’ll go into that a little bit later when I show you the pictures. Local and systemic factors are important in the development of the dentition. If you were to have some medication present in your body, either given to while you were a child or while you were developing in mom, things like tetracycline, nicotine, alcohol, as examples, we did that in the beginning of the course, this could affect the formation of the dentition as well. Anything that travels through the circulation could be a potential problem. Ameloblasts are young embryonic cells, and just like any other young embryonic cells, they tend to be sensitive to what’s going on tin the environment. If it’s a major disturbance, then you’re gonna get major defects in the formation of the dentition. If it’s a sort of minor disturbance, then you may get little brown lines, or little chalky areas in the enamel, indicating that something went on as the teeth were forming. The disease process could be a potential problem. As a young child you develop strep infection. Usually when you get infections of this nature, it will affect the dentition, and you’re gonna see disturbances in the teeth, like the presence of brown bands. Tetracycline we’ve already spent a lot of time on it. Whether it’s given to a child or a pregnant woman, as long as the teeth have to form, the enamel is going to come out stained, amber, light brown, to literally pitch black in coloration. And you cannot remove that stain, the only thing you can do is remove the crowns of all the teeth, and puts new crowns on. There’s no way to get rid of that. Fluoride is important. It helps protect the enamel. When we talk about enamel, one of the properties is that it is porous, it’s semi-permeable. So things can enter the enamel, like fluoride, to sort of like you’re shellacking the floor, you’re sealing the floor up, to protect the tooth against invasion by bacteria. The terms hypoplasia and hypocalcification are not the

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same. They have the prefix hypo. Hypo means less than. So hypoplasia indicates a problem in terms of formation of your matrix. If you have improper organic matrix being formed, then the actual form of the tooth is going to be defective. You may have small runty teeth. You could possibly have a compete absence of enamel. You could have pitting of the teeth. This can be caused by again, any of the conditions that we’ve briefly discussed. And if it happened to be like a strep infection, so you’ll get a brown band, get rid of the infection, then you get another strep infection later on, so you can have a repetition of the defect forming. And I’ll show that to you at the end of the enamel lecture. Hypocalcification, also known as hypomineralization, that’s where you get the normal formation of the organic matrix, but the tooth is not mineralized or calcified properly. And as such the harness of enamel, the really protective feature of enamel, is not fully realized. In terms of, actually, lets take a break.

Ok, I guess we might as well continue. Shh. So a few words about dentinogenesis. I also use as a synonym odontogenesis. Dr. Craig uses odontogenesis in a slightly different way than I do, but that’s OK. Like the ameloblasts, the odontoblasts have their own life cycle, it’s not quite as complex as with the ameloblasts itself where you have different phases, etc. In essence we’ve spoken a bit about the life cycle of the odontoblast as I described to you the interaction between the inner enamel epithelium and the mesenchyme cells of the dental papilla, alias pulp. So, this is definitely a case of interaction, induction, between two different things. And the odontoblast starts off as a smaller cell, and eventually it becomes a little bit larger, more simple columnar in shape, and it lines up on one side of the membrane pre-formativa, which will be the future DEJ. What we have to keep in mind here is that as the odontoblast produces dentin, the odontoblast keeps moving back into the pulp. The term recedes into the pulp is sometimes used as a descriptor. So as dentin forms, there’s no two ways about it, you’re going to wind up with less pulp tissue. Just like any other hard tissue, there are two phases in dentinogenesis. One is the formation of the organic matrix, and the second is the mineralization of the tissue. The first formed dentin is going to be uncalcified, and that’s going to be called pre-dentin. It’s always going to be lining the pulp interface if you will, and once again is similar to osteoid or cementeoid, and its important that the dentin is in good condition to maintain the integrity of the developing crown. If you have dentin in a weakened condition, then the enamel tends to not be able to withstand pressure brought upon it as well. So in essence the dentin is acting as a cushion for the overlying enamel. And there’s always a certain amount of pre-dentin that exists. When you’re first forming the tooth, there’ll be a greater amount of pre-dentin than when the teeth are actually completely formed. So in the end run, dentin turns out to be 70% HAP, and the other 30% is like 10% water, 20% organic matrix. So when we decalcify a tooth, so we can cut it up, stain it, and show it to you under a microscope, the dentin stays together because of the 20% organic material, unlike enamel which only has 2% organic material. As far as mineralization goes, there are quite a few different proteins in dentin, just as they are in enamel. That’s what the screen is presently showing you for enamel. One important protein is called phosphophoryn, alias dentin phosphoprotein. Its abbreviation is DPP. You will only find this protein in fully formed dentin. We haven’t talked about different types of

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dentin, let me just say a word to you about it now. There’s primary, secondary, and tertiary dentin, and on the primary dentin there are 2 subdivisions: mantle and circumpulpal. Mantle makes up a very small percentage. And your secondary regular dentin is also circumpulpal. And circumpulpal means it just goes around the pulp. So when it comes to this dentin phosphoprotein or phosphophoryn, whatever you want to call it, this is sort of like a genetic or phenotypic marker for your odontoblast. And you will only find it located in curcumpulpal dentin. You will not find it in mantle dentin, or pre-dentin. That’s why it’s considered to be a marker for your ondontoblast. And this particular protein plays a role in sort of setting up the lattice work for your HAP crystals. It tends to bind to calcium and therefore plays a role in HAP formation. You’ll find that the dentin in the root is a little bit less mineralized than the dentin in the crown. Another protein is dentin sialoprotein, and the function of that is a little questionable. But if you don’t have your dentin sialoprotein, then you’re gonna get abnormalities forming in terms of mineralization of the tissue, so obviously it plays an important role.

For both dentin and enamel, there has to be a flow of traffic of your calcium. Is it intercellular or intracellular? Well the odontoblast are held together by tight junctions, so the intercellular space between two odontoblasts is not very much, so it really is questionable if calcium passes into the intercellular space between the cells, and eventually into the dentin. So the concept of intracellular transport is more likely the explanation of how calcium enters the dentin. In terms of your mineralization we have 2 types: linear or globular mineralization.

Slide 26 – Blank Slide but Wishe draws several pictures on itThis illustrates linear mineralization. Nice and organized. But in terms of

globular mineralization. What happens is that you get literally globules of the dentin mineralizing. So where we have the stars, those areas are mineralized patch by patch, call it a globule. You could literally call it globular dentin. But areas in between are not mineralized. And those areas are referred to as interglobular dentin. Remember I showed you a slide before where I sort of drew this type of outline on the slide, so those little bumps really represented pieces of dentin that were mineralized. And the areas in between, the tissue is either not mineralized, or partially mineralized, etc. Eventually with the globular calcification, it should fill in, and this whole block of dentin should be mineralized, but that doesn’t always happen. So you’re gonna find this globular pattern of mineralization in the crown, and in the root.

Slide 27 – Enamel Proteins 1 (again)Now we work our way into your enamel. We’re gonna start off with a brief

look at different types of proteins. And we can break this up into 2 categories: amelogenin vs. nonamelogenin proteins. These in essence are not amelogenins, and so you should be familiar with certain terms here. Cause they will be used again in other lectures in the course, as well as next year. There are a lot of different proteins that exist initially in enamel, but in the end run when you look at a mature tooth, it’s only 2% protein, compared to 96% HAP, and 2% water. So some of these nonamelogenins, enamelin. And I’m just going to basically tell you how these

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proteins are involved. We’re not gonna go into any detail about structure. So this type of protein sort of supervises, controls the formation of your HAP crystals. And in the secondary mineralization phase, it helps the crystals elongate. The ameloblastin sort of acting as a little glue to keep the ameloblasts up against the enamel. Adherence if you will. Then we have this next protein, tuftelin, which is playing some sort of role in mineralization process. It’s a little bit up for grabs as to what exactly is the role. Now we have these degradation proteins, and the first one that if Dr. Craig hasn’t spoke about it, I think he did, but he’ll bring it up in one of his upcoming lectures on the PDL. Enamelysin, alias MMP20, and this protein breaks up enamelin and ameloblastin into fragments. And then these fragments will go into the formation of other proteins. Next one is a mouthful. Kalikrein4 is easier to say than the enamel matrix serine proteins, breaks up amelogenin into smaller units. Amelogenin is the initial protein that forms and then it gets rearranged, reformulated, if you will, into enamelin. Then there’s a whole bunch of novel proteins which play some sort of role in controlling the ameloblasts and their activities. So if you look at the book you’ll find a lot more proteins listed and they’re really in small amounts, but they play some sort of role in the mineralization/formation of the tissues.

Slide 28 – Enamel Proteins 2We find these two proteins, which probably make up most of the protein

nature of enamel, amelogenin and enamelin. Amelogenin is the more, I hate to use the word, primitive form of your enamel protein. It’s the earliest protein that’s actually formed, and it has a certain property. And there’s the certain property, thixotrophy, and that means the ability to flow. So going along with the adage, two things can’t occupy the same spot, as the HAP crystals are being brought into the area of the enamel, something has to disappear, and that’s your amelogenin. Enzymes are released by the ameloblasts and breaks down amelogenin. As it flows out, it’s really being pushed or squeezed out by the inflow of your HAP crystals. In terms of your sex chromosomes, X and Y, there’s different types of genes that play an important role. The X chromosome has amelogenes, the Y chromosome has amelogenes. And there was an article which dealt with this topic, it goes back a number of years, late 1990s, by a Dr. Slafkin (sp?) and it talks about sex enamel and forensic dentistry. So if you’re female, you have two X chromosomes, and each X chromosome has the same amelogenin. But if you’re male you have an X and a Y. The X chromosome has 1 type of gene, the Y chromosome has another type of gene. So what are we gonna do with this information? Sometimes there’s a tsunami, sometimes an earthquake, sometimes an airplane crash, it becomes difficult to identify the bodies. They can do DNA analysis, and if nothing else they should be able to determine if the body belonged to a male or female individual. Now dental records are important, but everybody does these records differently. People in this country vs. another country, even in different parts of the country. So it’s kind of hard to compare dental records from different places, so DNA is still a good way to identify the sex of an individual. In fact, the Romanov family who ruled in Russia, 1915 or so, were killed in 1918. They analyzed the DNA, and how they came to Prince Philip of England I’m not sure, but it turns out he has the same DNA basis. So

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he really is a descendent of the Romanov family, and this is all done by analysis of mitochondrial DNA, or other DNA sources. So we have amelogenin being produced, it breaks down through enzymes released by your ameloblasts, and the remnants of the amelogenin, which exists as small fragments, plus the enzymes, plus some other ingredients, go into making up the next protein, enamelin. And enamelin is a much larger protein than amelogenin. But amelogenin is more abundant that the enamelin.

Slide 29 – MineralizationThis we already spoke about. Stages of mineralization. And for our purposes,

know that primary mineralization really refers to laying down of approximately 1/3 of the HAP tissues, and the secondary mineralization is where the crystal formation enlarges. It’s not that more crystals are being deposited, it’s just that the pre-existing crystals get larger. And by the way, if you look at all the hard tissues, enamel does have the largest HAP crystals. Here we also spoke about hypoplasia, hypocalcification, it’s important that you know the difference.

Slide 30 – Fig 7-5 – Enamel Rod CrystalsThis is just a picture from an earlier version of your textbook, and it’s

showing you the HAP crystals. Although this is sort of a high magnification, you get the feeling that these crystals are somewhat large in nature.

Slide 31 – Fig 3-1 – Tissue ComparisonThis is a nice graph which came from a book we used to use many years ago.

It’s Obend’s (sp?) Oral Histology, comparing enamel, dentin, and bone. This part of the axis, the horizontal axis, is a time axis. The vertical axis is how much mineral is present in the tissue. So here’s your starting time for enamel, and if you follow the pathway of the enamel, it’s almost a straight line. There’s a little bend in the curve, but it’s basically a straight line. Primary, secondary mineralization. (Separates the two with a vertical line going from “days” to the top). Primary mineralization produces about 1/3 of the crystalline structure, and the other 2/3 are just an enlargement of the crystals, until a certain point when it begins to level off. You’ll notice the numbers listed in these columns differ a bit from what I’m telling you, but it also depends on how the calcium hydroxide was determined. There’s a dry and wet way, but in any case the mineral component is fairly large. So this says 92%, I say 96%, so we’re in that particular ballpark. Now as you look at dentin, the mineralization sort of shoots up, then begins to level off with a slight incline. Cause for that we’re using 70% mineral. Here it says 64.5%, we’re not gonna quibble about a few percentage points. And finally, with regard to the bone, again it’s a pretty sharp rise, and then it inclines up slightly, and bone is actually 65% mineral, so we’re dealing with something 96%, vs. 70%, vs. 65%. And you’ll notice at the zero point, as soon as the organic matrix begins to form, it’ll start to mineralize. Whereas with bone and dentin, there’s a delay period between matrix formation and calcification.

Slide 32 – Fig 3-12 Rod Direction

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What are the physical properties of enamel? Well number 1 it’s the hardest tissue in town because of the high concentration of HAP crystals, and the arrangement, the homogeneity of the arrangement of the crystals. Being hard, it’s protective, but keep in mind, anything so hard can fracture easily. Take a porcelain plate and drop it on the floor and it cracks in many pieces. So it’s not hard to understand that if you place some undue pressure placed on the tooth enamel, that it could actually fracture. And as I mentioned earlier, the underlying dentin has to be good condition, it has to be able to support the enamel and act as a nice shock absorber. The color of enamel is based on the crystalline structure and the homogeneity of the HAP crystal. In essence, if you took enamel off the tooth, you’d be able to see through it. So we talk about the color of enamel, the color is really a direct reflection of the underlying dentin in itself. Normal coloration of teeth is somewhat yellow to white in nature. If you’ve had endodontic work done, the pulp chamber and pulp canal have been cleaned out, then the color of the enamel may shift more towards the grey side. Then of course we have those temporary conditions, smokers, the nicotine stains the enamel. But the nicotine can be eliminated through a bleaching process. Tetracycline stains the enamel, but again you cannot get rid of the tetracycline staining. The enamel is set to be a semi-permeable, it acts like a semi-permeable membrane. Not fully permeable, but semi-permeable, meaning certain things can pass into the enamel. When a tooth erupts into the oral cavity, it’s not fully calcified. You will find within saliva, if you analyze it, the presence of calcium, and that calcium will pass through the enamel because it is a semi-permeable tissue and further calcify the tissue. And when the tooth has just erupted and become functional, it is not exactly fully mineralized. So you have an internal and external mineralization occurring. The thickness of enamel does vary. It’s thicker in your secondary dentition than the primary. It varies from incisor to molar. And it varies within one single tooth as well. So the thinnest part of the enamel is going to be towards the cervix of the tooth, where it sort of thins down to a sort of knife edge, and there are different arrangements which Dr. Craig will talk about. Perfect meet, enamel overlaps cementum, cementum overlaps enamel, and it’s even possible to get a gap between the two tissues. That would be the worst scenario because you now no longer have a hard protective tissue. So in the gap, you’re coming up on a softer tissue, which is easier for bacteria to perforate through that enamel.

Chemical properties we’ve already mentioned. 96% inorganic calcium HAP. 2% water and 2% organic matrix, which is in the form of a rod sheath. All the crystals and all the hard tissues look alike, the only thing that really differs is the size of the crystal. But enamel has the largest crystals. Besides calcium, you can also have items like magnesium, lead, fluoride, strontium, replacing the calcium and conferring hardness on the tissue. That normally doesn’t happen, but it can.

What we’re looking at now is the basic unit of enamel. That’s your enamel rod. The basic unit of dentin is your dentinal tubule. These rods start at the DEJ and extend towards the surface of the enamel. Now you really don’t see any true measurements for the rods per se, cause it all depends on what you consider the rod. The length of most of these enamel rods are going to be larger, or large in number, that can be accounted for the by thickness of the enamel. That’s because

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the rods are inclining occlusally. So up around this particular region (towards apex on the right tooth) the length of the rod far exceeds the width of the thickness of the enamel. And these rods in the cervical region of the tooth could slant downwards, and eventually straighten out and be equal in terms of a measurement to the thickness of the enamel. So lets say this particular rod, the length will equal the thickness of the enamel at this point in time. As we go upwards, or towards the oral cavity, the picture does change.

Gnarled enamel. Normally the enamel rods are sort of parallel, or they’re extending in a nice clear-cut direction. Certain parts of the enamel, particularly by the DEJ, more occlusally, in terms of position, you’re gonna find the enamel instead of looking like this, the rods are going in different directions. And when the rods are going in different directions, that’s known as gnarled enamel. Basically this is going to occur way up here towards the cusps.

Slide 33 – Fig 7-27 – Enamel Ameloblasts and Processes Rod & Interrod Formation Now here’s a picture which comes from your book. Your ameloblasts, enamel

processes, also known as Tomes’ enamel process, ameloblastic process comes off at an angle. When I drew it before, I put in a much larger angle to overemphasize it. And the process doesn’t really become incorporated in the tissue. As you look at this picture, it’s a bit misleading, and you’ll notice this is labeled the rod, interred substance. It’s a little harder to put things together from this type of picture, but when you look at the keyhole hypothesis, you tend to understand it a little bit better.

Slide 34 – Fig 7-55 – Enamel RodsNice picture of enamel rods comes from an earlier edition of the book. And

what you see, say from here to here, same thing applies here, you’re seeing cross sections which is sort of multi-sided if you will. Hexagonal shape. So that’s really a cross section through your ameloblasts, which play a role in the formation of the rods. If you look down here you’re getting a completely different cut. This is more of a longitudinal section.

Slide 35 – Fig 7-2 Enamel: Rod and Interrod EnamelSome SEMs again showing us your interrod substance, and the actual rod

itself. SO if you go with a new classification, this seems to stand out and point to what I mentioned before. That’s the shape of the rod, it’s rod-like, tube-like. And this would then be your interrod substance. But there’s nothing wrong with the way it was originally spoken, head body, and a tail. This is just a higher power of picture A.

Slide 36 – Fig 7-34 Enamel: Pits and Interrod EnamelNow here we have what we saw before, keyhole hypothesis. In fact, as you

look at this picture, it looks like a honeycomb where bees live and deposit the honey. So what you’re really seeing are your little rod-like structures, whether you want to say that this is a rod and the other part is interrod substance. This is higher power, this is the rod and the interrod space. Or this is the head, body, and tail. In any case the two components stand out because of the difference in arrangement of your crystalline structures.

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Slide 37 – Fig 7-7C Rod & Interrod Enamel CrystalsWe’re getting much more higher power, and again this is your image of the

rod and that’s the interrod substance.

Slide 38 – Fig 7-9 – Enamel: Rod SheathI put this on specifically not to show you the rod so much, but you see this

dark outline? That’s your rod sheath. That’s where you find your 2% protein.

Slide 39 – DVD – Gnarled EnamelHere’s a picture of gnarled enamel. This is your dentin (below), that’s your

enamel (top) and this region in here is your DEJ. And you’ll notice that it tends to be somewhat scalloped in nature. In your mature dentition, teeth are much larger, and the enamel has to stick to the dentin. If you had a junction like this (straight) that doesn’t give you much adhesive forces. But having the DEJ looking like that, it provides much more surface area. Same thing as when we talked about rete pegs, the CT papilla. So the two tissues are adhering in this fashion.

Slide 40 – Fig 8-1 – IncisorThis just shows you general layout of a typical incisor. There’s your enamel,

and the rest of the tissue happens to be the dentin. So dentin makes up the major component tissue of the tooth. And this thin layer out here is your cementum. There’s your apical foramen.

Slide 41 – Fig 7-53 – Enamel Hunter-Schreger BandsHunter-Schreger bands. A lot of structures in enamel happen to be hypo,

hypomineralized. Hunter-Shcreger bands once believed to be the imagination of some mad scientists. Obviously with the name Hunter and Schreger. And someone, this person probably took a light and shined the light on the microscope slide. All of a sudden these structures appear. When the light comes from the bottom you don’t see them. So it’s not really a figment of one’s imagination, but there’s an alternation of light and dark bands. And there’s some sort of difference in terms of mineralization within the tissue. So Hunter-Shcreger bands are there for purpose, they tend to minimize the risk for cleavage of your enamel in the axial direction of the tooth. So when masticatory forces are applied, in a certain fashion, you’re subjecting the enamel to a greater amount of wear and tear. And these do start at the DEJ, which would be here, and extend out to the free surface of the enamel.

Slide 42 – Hunter-Shcreger BandsHere’s another picture of your Hunter-Schreger bands.

Slide 43 – Fig 7-8 – Hunter-Shcreger BandsThis is a decalcified section. So you can see these bands plus other structures

that we’ll talk about in both ground section, and decalcified section. The fact that this picture is stained is that it means its decalcified picture. But how come there’s

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enamel? This has to be a young tooth where the enamel is developing, and the HAP just isn’t being completely extracted.

Slide 44 – Fig 3-15 – Lines of RetziusOk.

(flipped through the final few slides without saying anything about them)

Slide 45 – Dogs sleepingAnd that’s our break. To be continued. I guess Wednesday.

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25/26: crown development ii and enamel i-

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