Transcribed by Amy Maya Craniofacial Biology – Crown Development by Harvey Wishe

4.17.14

1- Intro slide y’all Okay we might as well get started (this is literally the first sentence he says every single time). The next 16 hours will be devoted to the formation of the crown and the mature enamel and dentin, and then after you'll receive hours dealing with formation of the root, the PDL, alveolar bone and what they look like. After that we do pulp, eruption and shedding and we conclude with some miscellaneous types of topics. 2- Nanci TC Fig 5-1 Primary Band 7th Ed. The development of the dentition is really a good system to use to study development. It’s an example of what occurs overall in general development process. Many of the pathways that we’ll talk about involve an interaction between epithelium and mesenchyme and we refer to that as induction. The rest of the body induction also occurs. There is no such thing as one event occurring all by itself. (Cleans glasses with his tie) Guys if you need to clean your glasses, your tie makes a good whipey cloth. Ah that’s better. What you're seeing on the screen is actually the development of your mandible and maxilla and this purply checker box going all the way around is the primary epithelial band. So what happens is that on day 28 the buccopharyngeal membrane ruptures. Prior to that the stomadeum or primitive oral cavity was completely separate from the rest of the but, once that membrane ruptures you now establish a continuity between the oral cavity and the rest of the gut and roughly 2 weeks later you find the primary epithelial band which gives rise to your dental lamina which gives rise to dentition. This band has horseshoe or Cshaped arrangement as you look at each particular chart. Once you get to the point of your dental lamina, then we take off the rest of the development of the dentition. I’ll briefly mention what's going on. 3- Nanci TC Fig 5.2 Primary Band 7th Ed. Before we do that look at the pictures at the bottom. Here is your oral epithelium, which is your primary epithelial band. You’ll see these arrows in here and they're pointing to certain cells that are undergoing mitosis so when that happens the epithelium band thickens in this direction (up/down) and expands in this direction as well (side to side). Then finally in the last diagram you can see there has been a downward or a migration of the epithelial cells into the underlying CT, which is the mesenchyme. The epithelium, being epithelium, is sitting on a basement membrane, which is this purple pink color. All epithelium sits on a basement membrane. The top 2 pictures are showing you the developing head and this rectangle is enlarged to show you you're getting migration of epithelium into underlying CT The formation of enamel is Amelogenesis, the formation of dentin is dentinogenesis, I also use the term odontogenesis. Dr. Craig uses it a bit different than I do. So in normally development you get primary and secondary dentition. There are 20 primary teeth and 32 secondary teeth, whether or not you actually get 32 is another

Transcribed by Amy Maya

4.17.14

story. There isn’t enough room in the oral cavity to accommodate the other teeth especially the third molar, alias, your wisdom teeth. 4- BB Table 6-1 Stages of Tooth Development This is a summary showing you various stages that are taking place and we will go over this in more detail so the first picture is showing the oral epithelium, the band giving rise to this little projection down into the underlying mesenchyme. This is actually the beginning of, the initiation of tooth development. This is occurring through induction where you have an interaction between epithelium and mesenchyme. This little projection into the underlying mesenchyme continues so you end up with a structure that looks like this, known as the dental lamina. This occurs strictly through mitosis and proliferation. With continued proliferation the dental lamina blossoms out a bit and gives a tooth bud. So this is the Bud Stage of development. With continued mitosis, growth, differentiation, morphogenesis, the bud changes to the Cap Stage. I don’t care for this picture as a representation of the cap because it looks similar to the picture down here, so a more accurate representation of the cap would be something like that and to me the cap looks like a French beret. Once you get to the cap stage you can identify different layers that are present and we will go over those shortly as well. Then with continued proliferation, differentiation and morphogenesis, this caps stage continues to undergo growth and these prongs get deeper and this diagram of mine is supposed to represent this bell stage picture (4th down), which is a Bell stage. Then once the bell stage takes place you get the formation of various dental tissues. Again the process of induction and proliferation take place and I don’t know how well you can see it but this pink represents your dental papilla, the future pulp, this greenish is the dentin and this blue area is the formation of the enamel and you’ll see the word apposition, which means to add on so dentin is being formed, added on. By the way, dentin forms before enamel. If dentin doesn't form, you will not get any enamel at all. Finally the last diagram is showing you some eruption and this takes place when the root is forming. So you first get the crown formed and then the root begins to develop and as it develops the tooth is being pushed into the oral cavity, the eruption phase. And eventually this tooth will project into the oral cavity and you establish a clinical crown and anatomical crown. The clinical crown is what you actually see when you look at the tooth, but the anatomical crown is much deeper. So from this point out is your clinical crown but the enamel extends down to here so that is your anatomical crown (from the CEJ). If we were doing a study of your first molar and your mandibular molar was extracted for whatever reason, the maxillary molar would be lost, in a sense, and would look for its mate. In order to do that it continues to erupt further into the oral cavity. One tooth is missing and the other tries to compensate and find the missing tooth so then the anatomical crown could be the true clinical crown depending on how much eruption takes place. 5- A&C Fig 5-1 Tooth Development 3rd Ed. This is a series of diagrams from a different book. That’s your oral epithelium, the projection into the underlying CT is the dental lamina, and then the little rounder part is the tooth bud. The shape of the tooth bud changes and this is something like I drew which is the cap stage. We are establishing the enamel organ which gives

Transcribed by Amy Maya

4.17.14

rise to the enamel of the tooth. You can see this little area under the bud, this little area under the cap stage (orange area), which is your dental papilla which gives rise to pulp, your odontoblasts which form dentin. Here we are in the bell stage and these prongs are getting much longer and when we get past this bell stage, the dental papilla becomes vascular in nature and once it becomes vascular you can now call this area the pulp. There is also a little projection (C, on the left) it is coming off the dental lamina, and this gives rise to successional lamina, which gives rise to your permanent tooth bud or onlagger, which is the same thing as a bud and disappears in the bell stage. As we go to D, the developing enamel organ is getting larger and larger as well as the dental papilla and surrounding this enamel organ, this violet color area will give rise to the dental sac or dental follicle which forms alveolar bone, PDL and cementum. You see this lighter staining tissue, and that is the formation of your dentin so here you're having apposition, which includes formation and the laying down of dentin. Then in E you see a dark area on top of the dentin, much better seen in F, this is the formation of the enamel. And finally G is showing you the tooth is erupting and at the same time you're having formation of the root. 6- Timeline of Human Tooth Development This is a timeline of human tooth development. You don’t have to memorize these numbers (holla) but it gives you an idea of what takes place. There isn’t much time between events occurring. So two weeks after the buccopharyngeal membrane ruptures, there's around 42 days and then you see the bud stage with various teeth, they left out the cap stage, but that occurs then the bell, then you’re beginning to form various tissues. 7- Nanci TG Fid. 5-3 Lamina 7th Ed. In terms of initiation of tooth development, depends what period of time we are looking at. Initially the epithelium controls the development of the tooth. For the first maybe 12 days or so. Afterward it switches to the mesenchyme, which becomes the controller of the type of tooth that forms. So lets say we’re at day 6 and you take molar epithelium and mix it with incisor mesenchyme, the epithelium is the controlling agent so you get formation of a molar. You could take some incisor epithelium mix it with molar mesenchyme, since it’s the early period the epithelium still controls, and you get an incisor. But after 12 days the epithelium loses its potential to control anything. When we say controlling the epithelium or mesenchyme is responsible for determining the type of tooth, the shape of tooth, etc. Other experiment such as taking mesenchyme from the first arch, in the early period (first 12 days), and combining with epithelium, you get formation of an enamel organ. So this gives additional proof that in the first 12 days it is the epithelium that’s the controlling agent. Most of the work has been done with a mouse. They volunteer for the project (lol). There are a number of genes called Homeobox genes, and probably Dr. SJ made reference to that in his general embryology lectures. There are a number of factors that play a role in development. So one thing I mentioned back in bone are bone

Transcribed by Amy Maya

4.17.14

morphogenic proteins (BMPs) and there's a whole host of them playing a role in overall development of the body and BMPs are osteoinductive, dentin-inductive and cartilage-inductive. So it has an important role causing hard tissues to develop. There are 90+ genes in the oral epithelium and the mesenchyme associated with the development of the tooth and there are 12 different transcription factors so you can tell that this is not just a simple matter, its rather complex in nature. There’s the hedgehog gene, the Shh gene that is needed for initiation of the formation of dentition. If you look at birds many moons ago, they had teeth. They don’t have teeth anymore. They haven’t had teeth for at least 100,000 years, what happened? So the bird oral epithelium does have the ability to form an enamel organ but it lost its ability to do that. Experiments have been done where scientists have managed to form a tooth and the favorite spot to try that out is in the eye (way gross). They take epithelium, insert it into the eye, take mesenchyme, put it into the eye (bird torture yo), and you get formation of a tooth, not necessarily complete, in the eye. So all these genes and factors play a role in the formation. Big difference between us and the mouse is that we have 2 sets of dentition and the mouse only has one. There will be a whole series of stages from bud to cap to bell to the actual formation of tissues. Another 2 genes I would imagine Dr. SJ mentioned also, the Pax genes, they play a role in the formation of the teeth as well. As we look at this picture this should look familiar (left pic) because we had a session dealing with sections through the head. Here's your nasal septum, oral cavity, and these projections represent lateral palatine shelves or plates which are part of the maxillary bone. Initially this tongue was sitting way above the nasal septum and the shelves couldn’t fuse because the tongue was in the way so the mandible and the tongue had to recede, drop out of the nasal cavity, then the 2 palatine shelves could rotate upward and eventually grow towards each other and fuse. When the 2 fused together as well as with the nasal septum that’s when you get the hard palate. Without the nasal septum involvement, and just the shelves are fusing together, you get the soft palate and uvula. This diagram (left bottom) shows something else. You see a projection coming off the oral epithelium extending into the mesenchyme, that is the dental lamina. Here’s a higher power (right picture) it hasn’t quite rounded out to form a nice bud. Underneath is condensation of a lot of cells. Condensation mean to come together, this is formation of dental papilla. There is condensation over here and here as well forming dental sac or follicle. This picture also shows you the vestibular lamina. As the dental lamina is forming you get another ingrowth of oral epithelium into underlying mesenchyme. That would be this region right here and this is the vestibular lamina, also called the lip furrow band, and eventually the cells in this region disintegrate so this is the structure initially, and after loss of cells you now have a space, the vestibular groove which forms the vestibule of oral cavity. 8- A&C Fig 5-4 Tooth Bud Good picture. Oral epithelium, dental lamina and now at bottom of the dental lamina it’s more rounded like you just blew up the balloon, that’s the tooth bud. Same thing

Transcribed by Amy Maya on the right cartoon, this accumulation is condensation of cells representing mesenchyme and that forms the future dental papilla. 9- G&H Plate 13-4 Fig 1a Dental Lamina

4.17.14

Here’s another nice picture. Epithelium, dental lamina, underlying mesenchyme, and this hasn’t blossomed out yet but that’s where the tooth bud will form. 10- G&H Plate 13-4 Fig 1b Tooth Bud Now we’re beginning to see a change where the bottom of the dental lamina is swelling out because of increased proliferation of mitosis so its blossoming out in all different directions. You can see this area here (around DP) there's lots of mesenchyme cells together as opposed to this region (to the right), this is your condensation to form the dental papilla (DP). This area, although not labeled, the other condensation to form the dental sac or follicle. You might be able to see a little line upon which the epithelial cells are resting which is where you find the basement membrane because all epithelium rests on a basement membrane which thickens to become a structure called Membrana Preformativa which becomes the future DEJ. 11- Nanci TC Fig. 5-11 Early Cap Stage 7th Ed Now we switch from a bud to a cap, and again you're having proliferation. This picture shows unequal proliferation taking place so here's one prong and there’s the other prong. At this point in the cap stage we can identify the following layers, the outer enamel epithelium (OEE), the inner enamel epithelium (IEE), and the stellate reticulum (SR). Everything has a specific function. There's a region here where the outer and inner enamel epi (OEE and IEE) comes together forming a loop like structure which will be the cervical loop which is important to root formation. You see a nice condensation of mesenchyme cells which is dental papilla forming. 12- Nanci TC Fig. 5-13 Cap Stage 7th Ed This is a little more advanced in terms of prong formation but still it resembles a hat. I want you to notice the cells in the outer enamel epithelium aren’t really that big they’re kind of short whereas the cells of the inner enamel epi are much bigger and this is the layer (IEE) that forms the ameloblasts, which eventually forms enamel. So the bud stage, the cap stage and the bell stage, we’re talking about the formation of the enamel organ. Right over here is the dental papilla and on the outside is formation of dental follicle, all mesenchyme. The head region developed from neural crest, which came from the developing ectoderm. 13- G&H Plate 13-4 Fig 2 Cap Stage Here’s a very nice picture (just gorgeous, flawless, this picture woke up like this) showing us again the Cap stage. And you can see the IEE cells versus the OEE cells. At this point, we can go into the functions of these few layers and fill in as we go along.

Transcribed by Amy Maya

4.17.14

Inner enamel epi will become ameloblasts and produce enamel. During this stage you have blood vessels in the dental papilla and via those vessels you have nutrients and oxygen passing into the cells of the IEE. In a similar fashion, waste products and CO2 pass back into the dental papilla. But once dentin forms, you are putting up a cement wall. You could be the mesenchyme (that’s always been my dream omg), I’ll be the inner enamel epithelium, and there's a cement wall between us, we can't contact each other. Nutrients, oxygen, waste products, CO2, can't pass back and forth, so what would happen under those circumstances? (Silence) You're a little tired. The inner enamel epithelium cells would die, but all is not lost because there is a savior, the outer enamel epithelium (OEE). Initially the OEE looks like this and then begins to change and the straight line of cells becomes invaginated which increases surface area. In each little indentation is a capillary network established so now the vascularity is gone for the dental papilla because dentin is in the way but these capillaries that are developing externally to the OEE can now deliver oxygen and nutrients through the OEE into the stellate reticulum. In addition we can have the opposite flow of traffic from the stellate reticulum to the vessels outside of the OEE, we get rid of waste products and CO2. But whatever passes in is able to supply the inner enamel epi cells so that’s how the IEE cells are able to exist. You get an alternate source of nutrition and removal of waste products. Then we have this stellate reticulum (SR). If you see a better picture of it, it’ll almost look like mesenchyme. But this is all referred to as the enamel organ, which came from oral epithelium, it came from ectoderm. The cells in the stellate reticulum are spread apart but contact each other by desmosomes. In addition you’ll have these cells contacting the OEE by desmosomes as well as the IEE. 14- A&C Fig 5-5 Cap Stage This is a diagrammatic version of your cap stage, dental papilla, this is your dental sac or follicle, this is the OEE (blue), that’s the IEE and here's the cervical loop, you can see the cells are spread out and tightly bound together. 15- DVD Enamel Knot Another nice picture, definitely cap stage. This is the OEE, that’s the IEE, there's your SR, the stellate reticulum, and underneath is the dental papilla. And now we have this outgrowth from the IEE called the Enamel Knot. You can have an actual cord trailing off and the cord is the Enamel Cord and you get a knot on top of it. What the enamel does is it controls the type of tooth that forms. And again this is all epithelial in nature so it’s believed to be some sort of organizational center, which directs say cusp of formation and other goodies. 16- Nanci TC Fig 5-14 Early Bell 7th ed. Now the prongs are getting longer so it’s beginning to look like a bell stage, it has the same layers 17- BB Bell Stage 2nd Ed. This is diagrammatic but it shows you development of the permanent tooth bud or onlagger meaning a young stage forms something else. Coming off the dental

Transcribed by Amy Maya

4.17.14

lamina is another lamina called the successional dental lamina which gives rise to the permanent dentition. So that’s one thing that makes its appearance in the bell stage of development. 18- A&C Fig 5-6 Bell Stage 3rd Ed. The second thing that makes its appearance is this layer of cells, which are flattened cells that are more or less perpendicular to the IEE cells. This is the Stratum Intermedium, if you don’t have this Stratum Intermedium (SI) you won't have enamel formation. So the SI helps control the flow of fluid, contributes certain enzymes necessary to enamel formation. So there are two major differences between bell and cap – formation of permanent tooth onlagger, and formation of stratum intermedium. This picture shows good representation of these epithelial cells spreading out There are a lot of molecules, which tend to attract water. So you find additional fluid present in the stellate reticulum, so besides passing back and forth the respiratory gasses, the stellate reticulum’s main function is acting as a shock absorber, a cushion for the underlying IEE cells. 19- G&H Plate 13-4 Fig 3 Bell Stage 5th Ed. Just another picture again showing the various layers. You can see the spaciousness now in the stellate reticulum. There's lots of GAG molecules in here which attract fluid, and if you think back to basic tissues, we discussed hyaline cartilage which has lots of GAG molecules and the hyaline cartilage that served as the articulating end of the bone, the fluid in there could be released into the synovial cavity cushioning the joint. So we’re seeing a similar function in the stellate reticulum. 20- DVD – Bell/Early Crown In terms of the dental lamina, it doesn't last forever it has a certain lifespan usually 4-5 years because it is during the later period development you get the formation of the molars. So the first molar (student sneezes, Wishe blesses them) develops around 16-20 weeks of development, the second molar is after birth during the first year and finally the third molar develops 4-5 years after birth. And that is consistent with the lifespan of the dental lamina. All sorts of things can go wrong, the jaws aren’t large enough, the oral cavity doesn't have enough room for 12 permanent molars. The third molar is normally the problem with being crowded out so it can become impacted which leads to problems and most people have it extracted because it can lean into the 2nd molar, cause dental breakdown, resorption, etc. Some people are missing teeth and usually those are the 3rd or 2nd molars. So in this picture, this is past the bell stage because this little purple in here is the beginning of dentin formation, so this is Bell/Early Crown. 21- Nanci TC Fig 8-6 Dentin Formation 7th Ed. Some additional pictures. What you see in blue is dentin forming. The dentin here (top) is much thicker than lower down. The dentin and the enamel are laid down, the coronal or occlusal portion of the tooth and it extends down to the cervical region and eventually dentin continues during root formation. The dentin being a

Transcribed by Amy Maya hard tissue will become calcified, and in terms of calcification it is 70% hydroxyapatite. 22- A&C Fig. 5-2 Tooth Development

4.17.14

Here is a diagrammatic representation of the mandible. This groove is the vestibule, this is the tongue, and here are tooth buds, which are developing off the primary epithelial band. As you look at this side (left) this looks like a cap, this is a bud, this is a bell, here’s a later bell and a later bell, and you notice this little projecting which is the permanent tooth bud forming. 23- BB Fig. 6-27 Successional Lamina 2nd Ed. The same thing is being shown here. This is formation of our primary tooth, and this is formation of the secondary tooth. 24- BB Fig. 6-28 Primary and Secondary Teeth 2nd Ed. X-ray here, you see crowns forming underneath, that is the appearance of the secondary dentition. Here's a cast showing the same thing, the teeth down here are primary, and the teeth buried are the secondary teeth. 25- A&C Fig 5-1 Tooth Development 3rd Ed. This is what we saw before, just as a review. 26- BB Fig 6-25 Tooth Eruption 2nd Ed Here's a series of pictures that show you the formation of dentin, of enamel, as you look from diagram A to B the crown has moved closer to the oral epithelium. Go to this diagram C it looks like crown is pushing through oral epithelium. We haven’t discussed the structure called reduced enamel epithelium, but it’s to protect the covering over the crown and it forms from the remnants of the OEE, the stellate reticulum, and the IEE so those various layers all come together, fuse as a CT covering protecting the crown from being eaten away by enzymes released in the actual CT. but it also releases its own enzymes to digest the pathway through the oral epithelium and eventually erupt into the oral cavity. (LOLtastic picture of a dog drinking a mimosa) This is our breaking point. Boy this tastes good... I just want another mimosa. So we’ll finish this on Monday I believe and we’ll start mature enamel formation.

Transcribed by Amy Maya

4.17.14

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