Transcribed by Anam KhalidFriday, September 12th, 2014

[General Pathology] [Wound Healing] by [Dr. Craig]

[1] [General Pathology Wound healing, repair and regeneration][Dr. Craig] Okay, so good morning everyone. Can everyone hear me? Its alright? Okay, so weve had four hours on inflammation so today were going to explore one of the three sequelae of an inflammatory response and thats going to be wound healing. And I just have a brief outline of what well be covering this morning.

[2] [Wound Healing: Outline][Dr. Craig] So, the only thing you have to stay awake for is this part right here. So Im going to give you Im going to condense into like two slides everything you need to take away from todays presentation, right? So, sort of like principles of wound healing and Ill try to distill it into a couple easy-to-understand concepts. Now, I think well, I know, having influenced at least my profession, right? Perio regeneration, that sort of stuff But I think will have even more impact on your clinical lifetimes as we go forward. And then at the cellular level were going to talk about the healing of soft tissue wounds. And one of the themes that kind of like percolates through my lectures is this idea of evolution. And bone, calcified tissue, appeared later in vertebrate evolution than soft tissue, obviously. So were going to see that the principles that govern the healing of soft tissue wounds are going to be adapted to the healing of osseous fractures, the healing of bone wound healing. And I think Dr. Riccis going to follow up after me and talk a little bit about how extraction sockets heal and a little more in specific depth, but Im sort of going to lay the groundwork for him this morning. And well talk a little bit about some of the factors that your patients may present with that may influence the time course of healing or the outcome of wound healing and thats kind of important for us to know when we treat our family of patients. Talk a little bit about some of the very, very quickly, about some of the complications of wound healing. Youll get more of this, is my understanding, when the oral surgeons give their material. And then in the second hour, were going to spend a lot of time on regulation of wound healing at the molecular level. So all this stuff is going to be more or less on the cellular level. First hour, cellular level. And then second hour, more on the molecular level, why? Because as it turns out theres a lot of clinical applications that are influencing oral surgery and periodontal surgery, endodontic surgery that actually have come to the point of actually having commercially available materials that we can use in the surgical field that kind of erratically change the outcome of wound healing. So thats kind of like an outline of what were going to be talking about.

[3] [Overview of wound healing: underlying principles][Dr. Craig] So these next two slides are really the only two slides you have to stay awake for. So, if you look at wound healing, youll see, both in soft tissue and hard tissue wound healing, that wound healing can be looked upon as a series of cellular interactions and they are always initiated by the inflammatory response. So you have to have inflammation before you have wound healing which is kind of interesting. Wound healing was always, you know, during our evolution, associated with pathogens because that initial barrier was broached. Now, you know, evolution didnt kind of foresee the Langone Medical Center, you know, where they do surgeries really aseptically. But, kind of put in your mind that gee, these patients have healed too so there has to be other initiating factors for the inflammatory response. So the inflammatory response is really important. And the outcome of the inflammatory response, as we talked last time, could be, or the last series of lectures, chronic inflammation, repair, regeneration. Repair kind of like is the you dont really have the anatomy totally regenerated. You may or may not have function. The pathologic word for this is fibrosis that weve talked about a little bit in this lecture series. So, wound healing proceeds in a highly predictable series of events. In fact, those events are sort of lock step. There used to be an old computer language called Basic yes?

[Student]Going back to the first bullet point, you said the outcome can be chronic inflammation. Is it always or just sometimes?

[Dr. Craig]- So inflammation has three outcomes, right? You can resolve it, you get resolution. Right? Which wound healing follows. Or you can get fibrosis, right? Or you can get chronic inflammation. Chronic inflammatory response, like periodontitis.

[Student]So lets say chronic inflammation does happen, does that always follow after inflammation?

[Dr. Craig] No, no. Inflammation has three termini. Alright? Chronic inflammation is one of the three, okay? Where was I? So wound healing proceeds in a highly predictable order, sequence of events, sort of like a computer program that was written in the old basic language where you have to execute every line of script in order to get to the next, right? And if you fail to execute one of the sequential lines, you have to go all the way back to the beginning again. And that has clinical significance, well talk a little bit about that. This is real important. So many of the events that occur during wound healing also occur during the development of that tissue, okay? So folks in surgical specialties have been really interested in understanding the developmental biology of the tissue of interest. So, periodontists are really interested in how the periodontal connective tissue attachment forms during development. If youre able to actually supply to the wound healing environment some of those inductive factors, some of those cell populations, set up the wound healing environment, to mimic what occurred during development, you can radically change the outcome into maybe from fibrosis to perhaps regeneration and we talked about that in Craniofacial Biology course last spring. This is more on the idea of biomimetics, where you set up a wound healing environment to mimic what occurred during development. In fact, I think we have a department on the eighth floor thats called the department of biomaterials and biomimetics that reflects this idea. And then again, the last point is, since wound healing is so important for survival of the species, you can expect multiple overlapping regulatory mechanisms. In a word, its going to be fairly complex. So what Im going to try to do is Im going to distill down some of the complexities and still have it retain enough significance so we can see the clinical relevance.

[4] [Overview of wound healing: Cellular events][Dr. Craig] Okay, so when you think of a wound, think of an extraction, right? That extraction socket is going to fill with blood. The clotting cascade is going to be initiated. Fibrinogen is going to be converted to a fibrin network. You have a nice platelet plug in there. From the blood, fibronectin is going to decorate that fibrin. Alright? So now we have this fibrin stage, if you will. And if you can envision, well gee, you know, if I wanna heal this extraction socket, I need to replicate cells. I need more cells. This is a void. I need to fill it with cells. And where the cells are are going to be probably in the periphery of that wound or theyre going to be blood borne. So in order to understand wound healing we have to kind of understand cellular replication and were going to kind of go over that kind of quickly. I know Dr. Kinnally probably went over this in detail. And were going to talk a little bit about how most tissues that were clinically associating with will contain cells that you can kind of label as labile cells, stable cells, and permanent cell populations. And once a cell starts to replicate, it has to migrate out onto that fibrin scaffold, alright? So it has to migrate. So we also have to understand what are the factors that control cell migration, alright? And cells, most connective tissue cells, have an unusual kind of well, not unusual, but they have a characteristic phenotype. If theyre going through mitosis or theyre migrating, they tend not to be expressing that cassette, that set of genes thats appropriate for that tissue. So once you have cells migrate, you have to target them to the correct loci for them to sit down, either attach to the extracellular matrix thats there or make an extracellular matrix thats appropriate for them. And they turn on that set of genes thats appropriate for that tissue. So we also have to understand some of the factors that regulate differentiation. So were going to talk about these three cellular events, replication, migration and differentiation as they pertain to wound healing.

[5] [N/A][Dr. Craig] So heres a little simplified diagram on cell cycle. So, over here is a permanent cell. This is a cell thats been kicked out of the cell cycle. We believe its never going back in. Each year I give this talk that list of cells and the permanent cell compartment gets smaller and smaller. But for the time being, this could be like an odontoblast, alright? An odontoblast doesnt go back into the cell cycle but does express that set of genes that are appropriate for that tissue. Theres another cell population thats resident in connective tissues and thats kind of a stable cell maybe thats a periodontal ligament fibroblast. And under appropriate stimuli, it can be induced to enter into the cell cycle. Remember from Dr. Kinnallys lecture, she talked about the RB restriction site [?], right? So you can get into the cell cycle and you have to get past this restriction site to make sure that your DNA is ready to be replicated. If you pass the RB restriction site, then you go through the four components of the cell cycle. Cells that are cycling through mitosis are called labile cells. And once they get to this stage, theres a decision to be made, right? So they can either continue to cycle through the cell cycle and make more daughter cells or they can be kicked out of the cell cycle into either the stable cell population or the permanent cell population. So were going to talk about factors that act right here. These are called mitogens, as you know, right? And were going to talk about factors that work right here, were going to call these inductive factors. And as it turns out, theres a number of inductive factors that youll be exposed to when you get into the clinic or when youre in your clinical practices that we can actually make use of now. So whats the significance of all this?

[6] [Overview of wound healing: Clinical significance][Dr. Craig] So if you really understand, as a surgeon, if you really understand the factors that control cell replication, migration, and differentiation in your tissue of interest, then you have the ability to radically change the outcome of wound healing with therapeutic aims. So in my little area, the Golden Fleece has always been, well gee; I would love to be able to regenerate cementum, periodontal ligament, alveolar bone. And lo and behold, were able to do that because of understanding of what controls these cellular events. Okay. So now were going to start talking about wound healing at a cellular level.

[7] [Epidermal wound healing: Types][Dr. Craig] So when I took the analogous course during my dental school education, this was all I learned about wound healing. This one slide. Alright? And I was taught that you can have two kinds of soft tissue wounds, right? Epithelial or gingival wounds. You can have something called healing by primary union, another synonym is intention. And you can have healing by secondary union, or secondary intention. And the only difference between the two is in one, in healing by primary union

[8] [N/A][Dr. Craig] You have an incision, perhaps created by you and your Bard Parker blade. And this has created a nice incision through the epidermis and the dermis, right? And you have extravasation of blood into the area. Nice fibrin clot. Were going to talk about the succession of cells and the cellular events that occur. But this is a relatively short distance to heal, to bridge and heal. And so thats healing by primary intention. In your clinical lifetime, there might be a little suture up here holding everything in position, not a big defect to take care of. As opposed to healing by secondary intention, alright? Secondary intention, you dont have well-opposed wound margins. You cant really suture it, cant close it. And the events that occur during wound healing, those are identical to the events that occur during healing by primary intention. Theyre kind of delayed because this is a much larger defect that needs to be remodeled and filled in.

[9] [Epidermal wound healing: Cellular events][Dr. Craig] Okay. So, lets talk about soft tissue wound healing. So, again, I want to kind of drive home this idea: wound healing can be seen as a succession of cells and cellular events that populate that wound environment and sequentially convert it from wound, non-vital tissue, to whatever the outcome of wound healing may be. So I kind of think of it, perhaps, a little like a stage, alright? So initially, you extract that tooth and theres extravasation of blood into the area and you get fibrin, a nice three-dimensional fibrin scaffold thats polymerized. And that fibrin scaffold is decorated with fibronectin, as you know. And then onto that fibronectin scaffold comes out a series of cells, a series of players that come onto the stage and change the physical features of that stage. And once theyve done that, they go away and another succession of players come out and transform that stage and telologically you think, well, gee, you know, I have a wound and the tissue is directly modeled into the mature tissue and thats not the case, alright? What happens is a lot of the cells and signaling molecules that were present during development are present. So you always have a succession of cells and cellular events that mimic what occurred during development of that tissue. And thats basically what I just said at that point. So here are a couple some slides, cartoons, and then Ill show you some histology of what actually happens.

[10] [Dermal wound healing: days 1-2][Dr. Craig] Cartoons are nice because you can kind of discuss events separately but the events kind of meld together into a progression. So here you put your Bard Parker blade in here. Youve lacerated blood vessels. Now, blood is into the extracellular environment and external environment and it sees things that it shouldnt see. What is one of the things that is being exposed to blood and blood products that you dont see in the vasculature? When you see me you think of one word and that word is ? Collagen. So all of a sudden you see a lot of type I collagen. Type I collage exposed in this kind of environment, really good at precipitating the clotting cascade as youll learn later in this course. So weve got a nice blood clot that starts to form. We have fibrin thats being deposited. We have degranulation of platelets in the area, releasing all their platelet granules that we talked about. You have an inflammatory response that ensues. And the first cells that migrate out onto that fibronectin scaffold are polymorphonuclear leukocytes in acute inflammatory response. Theyre in there to start snarfin around, looking for bacteria, guided by chemokines and other chemotactic factors that are released, we learned earlier. And they debride that wound and make it basically sterile. What else happens? Well, fibroblastic cells that are in the area begin to proliferate and go through mitosis and migrate onto that now sterilized scaffold, hopefully, and they start to lay down a very primitive matrix, kind of shown over here. And that matrix reflects what this tissue looked like during early embryology so you have a lot of proteoglycans, lot of glycoproteins, type III collagen thats present, alright? The proteoglycan is there. It absorbs a lot of water, just like it did in development makes for a beautiful environment for cells to migrate through. Because of the proteoglycans and because of the inflammation thats in the area, edema, these wound margins begin to become elevated. If you lacerate your finger, thats one of the first things you see after about a day, you know the whole are is kind of swollen and elevated. Well talk a little bit about angiogenesis in the second hour but from these intact vessels, there are tip and stalk [?] cells that begin to migrate into this area. These stalk cells begin to form a lumen. Well talk a little bit about the control of that. And they form a vasculature. This vasculature is kind of immature. It doesnt have a lot of smooth muscle basement membrane periocytes around. So its kind of a fragile kind of vascularity but it does function to give nutrients and oxygen to the area. What else is happening? The epithelial cells, the cells in the basal cell area of the epithelium, they have cell adhesion molecules and youve just put a dividing line between them. So some of these cells begin to change, radically change their phenotype. Usually basal cells sit on the basement membrane and they divide to provide a squamous, keratinized epithelium. In this case but now because they dont have that cell thats touching them through cell-adhesion molecules theyre beginning to change their phenotype they become migratory, okay? They can become they start to migrate over this connective tissue bed. Now, epithelium always needs, it doesnt always have vasculature. So epithelium also has to have intact vital connective tissue. So its going to migrate over that vital connective tissue and you start to see that really really early on during the healing of a wound. What else? There are other cells that are present in this area called myofibroblasts and the myofibroblasts proliferate and they begin to bridge this area. And they all contract so you get some wound contracture. I should also mention, early on in the wound healing cascade, just when you have your fibrin clot here theres an enzyme thats released, a transglutaminase that covalently links fibrin, fibronectin, the remaining intact collagen fibrils that are in the site into a cohesive mass. So it stabilizes the fibrin clot. And I think thats everything Im supposed to tell you at an early stage of wound healing of a dermal wound. The next cells that come in are derived from monocytes

[11] [Dermal wound healing: days 3 onward][Dr. Craig] And the monocytes come in, turn into macrophages and/or dendritic cells. Were not going to talk about the dendritic cells. But the macrophages come and they begin to turn over this primitive embryonic-like extracellular matrix, alright? And behind them is another wave of fibroblastic cells that come into this wound healing environment and they start to lay down a matrix thats more associated with a mature tissue lots of type I collagen, right? Those kinds of extracellular matrix proteins, not as much proteoglycan. And once they start to show up, they lay down a lot of type I collagen. Some of these initial blood vessels start to retreat. The ones that are intact become established. They have established lumen with smooth muscle, basement membranes and periocytes. So they become a mature vasculature. What else? Oh, and we also have this epithelium thats migrating over the vital connective tissue and the two sheets finally reach out and join. They have cell adhesion molecules that tell them of this event. So they stop their invasiveness, their migratory behavior. They sit down on the basement membrane that they synthesized and they begin to differentiate into squamous epithelium. So, whoever is up there favors the dental profession. I firmly believe this because in the oral environment, if you have say you make an incision. You can calculate how long its going to take for that area to epithelialize. Right? Because this sheet of tissue under ideal circumstances will migrate at about a half a millimeter a day. Thats really fast. And it migrates that fast because oral environment is very very well supplied with vasculature. Why is it well supplied with vasculature? One of the reasons is that arguably, the human mouth is the filthiest place in the entire human body. Welcome to the dental profession. And because of that, wound healing proceeds really well in the oral environment, because of that wonderful vascularity. So, thats kind of a cute thing to remember. Half a millimeter a day, this sheet will migrate over the connective tissue. So, those of you who move onto oral surgery, perio, those sorts of things, take a big chunk of gingiva off the palate, you can kind of looking across its narrowest point, its going to heal at about a millimeter a day, two sheets, half millimeter. Was that the question? Why did you ask that? Were you looking at old exams? Okay. So one of the things about wound healing is that when you go into your practices, you know, a new patient comes in; they dont know you from before, usually, right? Youre here in Manhattan. Patients can go anywhere, to any periodontist they want. So one of the things that Ive kind of incorporated is when I have to take them through something that is sort of a traumatic experience for them, an extraction of a tooth, placement of a membrane if I can predict for them accurately, what theyre going to feel and what theyre going to experience and Im correct, that gains their confidence in me, right? So one of the neat things about knowing a little bit about wound healing is you can predict the outcome of wound healing for your patients and you dont have to be, you know, a complete presage to only a few things, like, you know, how long is it going to take for this extraction, how long is it going to take for my palate to heal? Those kinds of things are kind of neat practice management tools. But thats not part of this course. But anyways. So what does it actually look like? So all these steps kind of one thing I should say here is so a lot of times when that secondary wave of fibroblasts come in and lay down type I collagen, they tend to be a little exuberant. So a lot of times if you have an incision, say, on your epidermis and you look down there after wound healing is complete, if your eye is up here and youre looking down here, theres a lot of type I collagen thats laid down and you cant really see the vasculature that well through it. So it looks like a scar because it is a scar. So, basically what this cartoon is really showing is a little bit of fibrosis here. We dont have as much of a problem in the oral environment with scarring after incisions but its still a concern. But anyways.

[12] [Rodent dermal wound: 5-7 days][Dr. Craig] So this is a series of slides from a rat that gave its life for this course years ago. And what this is is this is an incision line, okay? Where a Bard Parker blade came in, dorsum of the rat. And at this point, I mean, you can still see all this red stuff all over the place. Thats extravasated red blood cells that havent been cleaned out by macrophages yet. The epithelium has already reunited, alright? Rats heal very very quickly. Theres something called delta rate of wound healing is proportional to an animals life span, up to a point. Polymorphonuclear leukocytes have already done their job, alright? Were looking at the first wave of macrophages that are coming in, cleaning up the area. If you go a little higher power, so were going to take this area down here, kind of turn it ninety degrees and go a little higher power.

[13] [Rodent dermal wound: 5-7 days][Dr. Craig] The epithelium has already become contiguous, closed off that space. You can kind of appreciate the red blood cells that are extravasated here. They have been cleaned out by macrophages. You can kind of see this rolling pattern of fibroblastic cells that are laying down a primitive mesenchyme, the initial mesenchyme thats laid down.

[14] [Rodent dermal wound: 5-7 days][Dr. Craig] A little later, 7 days down the road, this is not the worlds best piece of histology. Dr. Phelans lab did not do this. But anyways if you kind of look, this is becoming remodeled into a more mature tissue. And then you start to see these weird things here, okay?

[15] [Rodent dermal wound: 5-7 days][Dr. Craig] I think I have a higher power, yea theres some weird things here and around them are these multinucleated cells. Any idea what these things are? Seven days after a surgery any idea?

[Student]Osteoclasts?

[Dr. Craig]Well, these big guys are kind of like osteoclasts, right? Because these are mononuclear cells that are fused together to deal with something thats bigger than an individual macrophage. But whats this structure, do you have any idea what that structure is? Its a suture. Okay? Its a suture and these monocytes have come in, theyve fused into multinucleated formed body cells to try to resorb these guys, alright? So, the reason why I put this in is I kind of want to set in your mind that what we do in the wound healing environment, what we place in the wound healing environment can influence what is happening at a cellular and molecular level, okay?

[16] [Rodent dermal wound: 21 days][Dr. Craig] And 21 days, rats heal very, very quickly. Now you dont see as much blood vessels in the area at all. You see a lot of fibroblastic cells that are laying down an extracellular matrix.

[17] [Rodent dermal wound: 28 days][Dr. Craig] So this is 28 days. And this is a mature wound, an area of scarring in this rat. You dont see a lot of vascularity in the area. You do see these very, very long fibers that are made up of type I collagen and other extracellular matrix molecules. Not a lot of cells. The cells are present but these fibers are really [unintelligible]. So this is what a scar looks like 28 days after healing. So folks used to do this long time ago and its kind of illustrates a point.

[18] [Relationship of time since wounding with tensile strength and cell populations in a dermal incision wound model][Dr. Craig] So what they used to do is they used to do those wound healing models like I just showed you. And they would count the number of cells and in this experiment, they were looking at cells that look like fibroblasts and cells that look like macrophages, just by the morphology of the cell. And they would create the wound on day 0, right? And at various times post wounding they would do exactly what I just showed you on the slides. They would take some histology in the area and they would count the number of cells and before doing that they would put surgical forks across the wound and they would measure how much tension it took to open the wound, to dehist the wound. So on one axis they have plotted the tensile strength, how much force does it take to open up the wound and on this axis they have plotted the percent of cells. And what you basically see is, at this point you have your fibrin scaffold, right? And in come the macrophages that remodel, that initial scaffold. And after the macrophages, theres a wave of fibroblastic cells that come in and they lay down that primitive, first matrix. And then its a little hard on this but theres another peak of cells that come in, of macrophages that begin to turn over that embryonic, extracellular matrix. And then another wave of fibroblasts who come in and lay down that mature matrix. And as they lay down that mature matrix, the tensile strength of this wound gains a long time. So early on, not a lot of tensile strength, why? No type I collagen or very little amounts of type I collagen. Type I collagen is being synthesized in this second wave of fibroblasts. Okay. Questions on the healing of soft tissue wounds? Okay?

[19] [Healing of osseous fractures][Dr. Craig] So what Im going to try to present to you, the healing of osseous fracture is very, very similar, with the addition of this hydroxyapatite matrix. So again, theres going to be, as you guessed, a succession of cells and cellular events that are going to sequentially transform that fracture into a mature osseous tissue and the events that occur during healing of a bone are going to recapitulate or repeat the events that occurred during development. And as you remember from last year, what course, Basic Tissues maybe, bone forms by two mechanisms. Theres going to be a condensation of mesenchymal cells in the area. You may get expression, frank expression, of type II collagen with expression of cartilage or you may just get expression of the type II collagen genes without actual translation into the protein. However it forms, that primitive matrix is going to mineralize, right? So that becomes so-called woven bone. And then derivatives of monocytes are going to come in and transofmr that primitive woven bone into a mature, lamellar or Haversian bone. And the characteristic of Haversian bone are these units called osteons, right? Mature bone, Haversian bone, is characterized by a central vascular core around which are rings, concentric rings, of osteoblasts. So bone and vasculature, at least Haversian bone and the vasculature, are intimately associated.

[20] [Osseous healing: early events][Dr. Craig] So heres some cartoons again and then were going to show you some histology. So heres a so-called green-stick fracture of a long bone, right? What happens? Well, theres laceration of blood vessels. Theres extrasavation of blood. Blood sees type I collagen. Platelets adhere and degranulate. Fibrin is formed from fibrinogen. You get a fibrin network thats decorated with fibronectin. Transglutaminase is activated, covalently linking everything into a mass, right? The difference here is you have bone tissue, alright? And some of these osteocytes are going to have their vascular supply embarrassed. Okay? So youre going to have some necrotic bone. So thats one difference thats occurring here, the presence of a little bit of necrotic bone, right? Then, over time, cells from the periphery, right, are going to start to migrate in. You have angiogenesis, blood, vessel formation that comes into this area. Cells will come in and begin to differentiate into bone forming cells and depending upon how close you are to these invading, vascular twigs that come in, the amount of oxygenation you experience, you can either become like a cartilage kind of structure or you can form woven bone directly by condensation. So if youre close to the vasculature you tend to form a woven bone directly. You express collagen, which mineralizes with hydroxyapatite. If youre far from the vasculature, you tend to express type II collagen and form cartilage. Alright? In either event, this mineralizes and stabilizes this fracture into something called a primary callous. Monocytic cells start to sense the presence of this necrotic tissue and they begin to resorb this necrotic bone that turns out to be very important.

[21] [Osseous healing: late events][Dr. Craig] Theres a series of cells that come into this primary callous, this calcified primary callous, led by a group of osteoclasts and right behind the osteoclasts are vascular endothelial cells. And right behind the vascular endothelial cells are osteoblasts that lay down concentric rings of bone and transforms that woven bone matrix into mature Haversian bone. Okay? Very similar to the way bone formed during development. So what does this actually look like?

[22] [Osseous healing: 14 days][Dr. Craig] I dont have really good histology on bone fracture so Im going to have to find some better so this is a long bone of a probably the knowing Walter, probably the same rat who had a piece taken out of a long bone and at 14 days, this is whats occurring. You still have some extravasated blood in the area. You have a primitive bone matrix thats been laid down. These islands might even be cartilaginous. You cant tell by this staining.

[23] [Osseous healing: 28 days][Dr. Craig] Heres a little better picture of a primary callous. This animal was labeled with tritiated proline so everywhere its dark theres collagen thats being laid down. Its an autoradiograph that weve talked about in Craniofacial Biology. So this is what a primary callous looks like.

[24] [Factors that effect wound healing][Dr. Craig] Okay. Factors that affect wound healing. So this is kind of important for your patient. Size, type and location, right? So size. Healing by primary intention goes faster than secondary intention because theres less of a bridge that has to be remodeled. Hence, when youre planning a surgical procedure, you probably want to do a nice flap rather than doing an excision. Just kind of plant that in the back of your mind for your practices.

Locations real important. So the better supplied with vasculature, the faster wound healing will proceed. So, as I said, someone loves the dental profession because the surgeries I do, I certainly dont I dont do aseptic surgeries, I do clean surgeries, right? But theres certainly bacteria in the area and because my patients come in and the oral environment has such wonderful vascularity, wounds heal relatively quickly. We talked about how fast epithelium migrates in the oral environment. It certainly doesnt do that in non-oral sites. Perhaps, the most extreme would be feet. Diabetic foot ulcers are very difficult to heal due to the decrease in vascularity.

Presence of infection, or bacterial products. So, what happens? You do this beautiful extraction and the patient somehow gets the thing infected. Even though the mouth has this wonderful ability, innate immune response and wonderful ability to have vascularity in the area, somehow they get the thing infected. Well, if you have presence of bacteria, bacterial products, even endotoxin, wound healing stops, has to go all the way back to the beginning again. You have an acute inflammatory response and all the steps in the wound healing cascade have to be re-executed. So, one of the things that you want to do when youre in clinic, you want to be real clean. You want to make sure that you debride areas, you dont want to leave any endotoxin-containing material behind. You want to make certain you remove calculus from extraction sites that will come back and bother you, okay? So you need to have the area real debrided.

Movement of wound margins. So this was known by the Egyptians. The Egyptians actually had sutures when they needed to do surgery. They didnt have local anesthetics so it must have been kind of exciting. But they did have means of immobilizing wound margins. So, one of the things that you need to look for in healing of soft tissue wounds is you have to make sure if youre healing by primary intention, right, that the two wound margins dont move. Early on, during wound healing that extracellular matrix thats laid down, it resembles an embryonic mesenchyme, doesnt have a lot of tensile strength. So if you have movement in wound margins and the margins open up again, now youve induced an acute inflammatory response and you have to go all the way back to the beginning again. So one way around some of that is to artificially immobilize tissue using sutures, right? Primary callouses in fractures with casts and other mechanisms for immobilizations. To give more support to that area so that you can have that succession of cells and cellular events transform that wound from embryonic mesenchyme to a mature tissue.

I hate this category. Age. So you look in the literature and you look in your book, theres a strong feeling that as one ages, certain things occur and wounds dont heal as well in older folks, older patients as they do in younger. And perhaps, the most extreme example is something called Thompsons digit, alright? I dont know how this pediatrician figured this out but anyways. If there happens to be a traumatic episode to an infant, and I dont remember what the cutoff is but its really young, you know, like a couple months post-partum. And they lose their digit, theres a good chance the distal part of the digit that they can regenerate it. Okay? Thompsons digit or Thompsons finger. Now, I will bet you a sack of gold that if you lacerate the end of my finger, its not going to regenerate, alright? So thats probably the most extreme example I can think of. However, having said that, there have been studies of looking at implants success and failure. Youre hoping that the patient does a lot of stuff to come up with a successful implant and many, many outcome studies have looked at age, right? And no one, to my knowledge, has found age to be a factor as far as, say, implant success. It has just one example. So thats why I have a question mark here. And its sort of a hopeful question mark, as I age.

Anyways, presence of metabolic disease so, if you have a patient who has diabetes, right? Defect in insulin production or insulin signaling. Insulins real important. Integrates dietary intake with metabolic needs of a patient, right? It governs the flow of glucose and perhaps more importantly, amino acids into peripheral cells from the vasculature. You would expect that youre going to have problems with wound healing especially if that person is poorly controlled. Same thing with osteoporosis. If you have a patient who has a defect in mobilizing calcium from the gut into the vasculature. Some problem with vitamin D metabolism, what have you. If theres not a lot of hydroxyapatite, if theres not a lot of calcium thats coming in, then youre not going to be able to synthesize hydroxyapatite. So diabetes, osteoporosis tend to inhibit wound healing.

Medications. So heres our friend corticosteroids again. If one alters that acute inflammatory response, inhibits it so the patients nice and happy, well you may have some problems with infection. You may have some problems with how quickly wound healing proceeds.

And then finally, you dont see this in this ... well, maybe you do see this. But you dont see ascorbate deficiency that much in this country. But if you have a deficiency in ascorbate, you cant make collagen that has tensile strength. Or you have a defect in vitamin D metabolism or youre low you have a patient thats low on vitamin D, then they may not be able to heal osseous wounds as easily as one who does not.

[25] [Complications of wound healing][Dr. Craig] Were only going to go over four complications of wound healing and then were going to take a break. We already talked about wound dehiscence. So a wound dehiscence is a reopening of wound thats closed by primary intention, unintentionally, unexpectedly. Keloid formation. So this is tends to be genetically associated with some individuals that, when theyre wounded they tend to form these very, very hyperplastic scars called keloids. And well show a couple pictures of them. Contractures. Remember we talked about those myofibroblasts that come out and they contract wounds? Well, they can also contract and form adhesions with other tissues, deep tissues that lead to limitation of movement. Those of you who go into oral surgery, you will be dealing with this quite a bit with impacted third molars. And then finally, fracture non-union and pseudarthrosis. So what happens if you take a bone thats going through the events of wound healing and all of a sudden you start moving it again prematurely and you break that primary callous and you continuously do it, youll end up with a pseudarthrosis, alright? So just about anyone who rides horses or anyone who does long distance bicycling will probably be at a higher risk for fracture non-union. So when I was in graduate school I used to do long-distance bicycling and of course I got in a few automobile accidents and in one I fractured my clavicle. And since my wife told me that I had to get out of graduate school, I could not be a student the rest of my life, I decided to put the sling away and lift, you know, centrifuge rotors in and out so I could get my PhD. So I had this wonderful pseudoarthrosis, alright? Doesnt affect me too much but I cant wear strapless gowns, which is a problem, you know?

[26] [N/A][Dr. Craig] So, heres a keloid. This is lifted from your text. Exuberant scar formation from someone who took a ring off of this individual.

[27] [Histopathology: Keloid][Dr. Craig] So if you excise it, so heres the epithelium and heres the connective tissue and all you see is like, just scar formation, exuberant, long, fibers, connective tissue fibers, mostly made of type I collagen. Not a lot of vasculature, not a lot of cells.

[28] [Histopathology: Keloid][Dr. Craig] Higher power. Big, big, big fibers that are present, mostly type I collagen. A few fibroblasts taking care of these guys. This tends to be a genetic predisposition. A lot of African Americans will have this problem. Thank god it doesnt occur intra-orally. Okay, so why dont we stop now. Take like a nine-minute break. Come back at the hour and well finish up with factors that regulate wound healing. And Ill take questions if anyone has them up at the podium.

[29] [Regulation of wound healing][Dr. Craig] Okay, everyone. Welcome back. So lets now so now you should have kind of an idea of what the succession of cells and cellular events is that occurs during healing of a soft tissue wound, gingival wound, or healing of an osseous wound, alveolar bone, right, in our area. And you should have a real firm understanding that it doesnt occur like you think it would from wound to mature tissue but it actually recapitulates many of the events that occur during development. And so lets now shift from the clinical level and the cellular level and lets get a little more molecular because thats where all of the applications are going to come in your clinical lifetime. So, regulation of wound healing and this is kind of like just thinking about it. So, since events are highly predictable, there must be a high degree of regulation. And theres probably overlapping because wound healing is so important. The processes of cell proliferation, migration and differentiation need to be tightly regulated. So if you think about that proliferation, migration, differentiation, that really becomes a problem of how do cells communicate with one another during a process like wound healing and if you think about it, theres really only three ways that cells, that I know of, can converse, if you want to use that word. One is by expression of mediators that they put out into the extracellular matrix and if the other cell has the receptor for that then they can talk that way and that happens and were going to talk about cytokines in a little bit. We already mentioned that cells and you know that cells can reach out and touch one another through cell adhesion molecules and well talk just briefly about that. But the most important one is cell to matrix. As it turns out, cells can come into an environment, a stage, and they can construct something, synthesize an extracellular environment and then they leave. And then other cells come into that wound healing environment and either they can sense the protein and structure in that extracellular environment and it changes their phenotype OR they can resorb some of that extracellular matrix and release signaling molecules that were deposited there during development. And well have examples of both, alright? We already talked about enamel matrix proteins and fibronectin in the past but were going to kind of explore them a little more deeply.

[30] [Regulation of wound healing: soluble factors (cytokines)][Dr. Craig] So I noticed, its not as apparent as it used to be, but Ive noticed that when I kind of look through other peoples lecture notes and stuff, the word cytokine is sometimes used kind of liberally. And I kind of like a more discrete definition so were going to talk a little bit about cytokines and Michael Bishop here, that then was at the University of California, San Francisco, in the journal Cell, long time ago, came up with this definition and I like this definition. And since he got the Nobel Prize for fibroblast growth factor, I think thats good. So anyways, Michael Bishop, Dr. Bishop, finds a cytokine as a soluble, small, non-antibody thats released by living cells that in picomolar to nanomolar concentrations, real dilute, right, regulate host cell functions. Now why is that important? So if you were in the pharmaceutical industry, youd jump on this because theyre small proteins that you, through recombinant DNA technology can put into a vector, bacteria or a virus, drive its synthesis, right, and then you end up with [unintelligible] of a factor thats able to, in very small concentrations, regulate host cell functions. So were going to have some examples of that. So this is kind of an interesting definition.

[31] [Regulation of wound healing: soluble factors (cytokines)][Dr. Craig] So cytokines can act as endocrine, paracrine or autocrine factors. Ive noticed that the idea of endocrine is kind of getting corrupted. When I kind of went to school, you know, endocrine meant it was a factor, protein, or a lipid, or a lipid-byproduct that was being secreted from a gland, an organized structure, had to go through the blood and the receptor cell had to have a specific receptor for that basically hormone that changed its function. And now Im beginning to see in the literature the idea of glands being kind of released because things like fat tissue release things like leptin and so thats kind of endocrine. So the idea that it has to travel through the blood is still there. Paracrine means that a cell in an area, localized area, releases a signaling molecule and it goes off into the local extracellular matrix and another cell, different cell, different type of cell, if it has a receptor for that, will recognize, bind with it, recognize, theres a signal transduction event that occurs and gene expression changes or the phenotype of that cells. So thats paracrine. And then autocrine, you already know about IL-2. Whats the function of IL-2? So you know, a cell releases, a T-cell, right, releases during T-cell activation releases IL-2 out into the environment. It also has a receptor for IL-2. IL-2 comes back and tells it to undergo cell differentiation. Whats the reason for that? Did Dr. McCutcheon tell ya? No? Okay, well IL-2, obviously, is a very important signaling cytokine but thats sort of like the typical example given for autocrine factors. So they can act as endocrine, paracrine, or autocrine factors. Rarely does a single cytokine initiate the whole wound healing response or even a segment of the wound healing response. There seems to be a lot of redundancy. And whether a given cell responds to a cytokine really depends upon whether it expresses a receptor, whether its in the right stage differentiation, even the type of extracellular matrix that its sitting in can have an effect on whether it responds to a cytokine or not. And of course, frequently several cytokines are thought to act in concert to affect the cellular response. So in my little area of periodontal regeneration, periodontal wound healing, everyones been kind of looking for something that we can put in to a wound healing environment to make wound healing proceed faster or make wound healing proceed in a different fashion, right? So instead of getting a fibrosis, getting regeneration, those sorts of things. And that magic molecule hasnt been found yet but because theres been success, some success, I predict in your clinical lifetime this quest will continue and you will be offered more things to sue clinically. So were going to talk about two examples. The first one is platelet-derived growth factor. Oh, thats from the inflammatory response, right? Released by degranulating platelets. And a little bit about IGF-1. And were going to talk a bit about vascular endothelial growth factor because it regulates seems to be the prime regulator of angiogenesis. And youre going to see this in your practice.

[32] [N/A][Dr. Craig] So, there was a cell biologist with a name of Samuel Antoneedies [lol?] and Samuel Antoneedies was very interested in how fibroblasts regulate mitosis. And Sam Antoneedies was one of the first people to find that if you took a cell culture, human cell culture, and you added to these stable cells that are kind of sitting in the cell culture, you added some platelet-derived growth factor, because he thought, well, gee, you know, one of the first events that occurred during an inflammatory response is the release of platelet-derived growth factor, is really good as far as stimulating wound healing so maybe thats kind of like the initiating factor. Now what he found in cell culture was, if you added platelet-derived growth factor, this would recruit cells from the stable cell compartment into the cell cycle but they would be arrested at this RB restriction site, a retinoblastoma restriction site. So he said that platelet-derived growth factor is a competence factor because it recruits cells out of the stable cell cycle into the cell cycle, makes them competent but you need something else in order to make them progress through the cell cycle. And at the time he, you know, insulin gene is actually part of a family. So there is a member of that family, insulin-like growth factor 1, used to be called somatostatin, I believe. But IGF-1, if he added IGF-1, then IGF-1 would allow these cells to progress to the cell cycle so he called IGF-1 a progression factor. So theres a person named Sam Lynch who did his PhD with Antoneedies at Harvard and Dr. Lynch now, I believe, is with OsteoHealth [?]. Hes a periodontist. He took this idea and said, gee, you know, wouldnt it be wonderful if we could make oral wounds heal faster, right? And so, they started to try to market a preparation of platelet-derived growth factor and insulin-like growth factor, or a type of insulin-like growth factor to the dental profession. As it turns out, in vivo, right, if you add platelet-derived growth factor, wound healing will proceed actually faster, you dont need the progression factor because theres probably enough progression factor there. So, in our profession there is two ways of increasing platelet-derived growth factor in a site. So, this is a strategy of trying to get wound healing to proceed faster, right? So the two goals is make it go faster and change its direction. So this is the first. And this idea is that, well, gee, if platelet-derived growth factor functions here, what if I take a whole bunch of platelet-derived growth factor and throw it into the surgical site, alright? Will I kind of like make things go faster, alright? So I kind of think of this as the Saturday night philosophy, right? One beer was good. That means, six beers would be six times better, right? Theres no problem with that thought process at all. But thats one of the things that will be marketed to you. Okay? So theres two ways of increasing platelet-derived growth factor in a site. One is to, before the surgery, you take a venous sample of blood from your patient. You put it in a modified centrifuge called an elutriator and you get out an increased concentration of platelets. You can either give the rest of the blood back to the patient or just throw the blood away. So when the patient comes back in and also you collect it in an EDTA so you dont have calcium so theres not clotting. And at the time of surgery, you put in a little bit of prothrombin, you put a little bit of calcium and this platelet-rich plasma, PRP, undergoes a coagulation cascade so you get this jelly-like stuff which you can place into a site. You can basically increase the amount of platelet-derived growth factor by a factor of ten if you do that. So when you get on the clinic floor youre going to hear residents all talking about PRP and so now you kind of have the idea of what the strategy is. The second strategy is the Michael Bishop cytokine strategy. So as it turns out, platelet-derived growth factor, the gene has been isolated from humans so theres a human recombinant, PDGF, thats out there. Its marketed in a matrix, in my mind is blanking on the commercial name of it but you can buy it and you can place it in wound healing environments and it really speeds up it actually does appear to speed up the healing of wounds, especially in areas that youre kind of concerned about; furcations, those sorts of things that you want wound healing to go real fast because youre worried about infection and other things in that area. So those are the two ways that are commercially available to us as a profession of increasing an early factor, in this case, platelet-derived growth factor, to make wound healing progress faster.

[33] [Regulation of angiogenesis: VEGF][Dr. Craig] Forgive me for this slide, its terrible but its the best one I can find. I wanted to talk a little bit about vascular endothelial growth factor, alright? As it appears, at this point in time, VEGF, seems to be a prime cytokine thats regulating angiogenesis during wound healing. It also regulates angiogenesis during tumor formation, alright? And theres a very well, theres a decent discussion of VEGF in your textbook, Im just going to abstract from that. Those of you who want a little bit more in depth presentation, take a look at your textbook, its quite good. So, heres a little section through a small, perhaps, capillary and/or venule. So here are our endothelial cells that we know are really intelligent, right? Theyre the gatekeepers of the passage of anything from the extravascular space. Theyre sitting on a basement membrane and here is some periocytes that make a mature vessel, right? So depending upon the type of vessel, there might be some smooth muscle cells. And then theres an angiogenic stimulus, right? So we have an inflammatory response. Theres an embarrassment, profusion into the area. So, oxygen tension is going to go down and that is one of the prime effectors of VEGF expression, is decreased oxygen tension. And along with an inflammatory response, this intact piece of microvasculature starts to undergo some striking changes. So, the periocytes leave, the small muscle cells leave. Some of the cells under the influence of the VEGF start to become theres two types of cells here. So this is a transformation or an induction endothelial cells. And once they begin to go out into the extracellular matrix lead the way, theyre called the tip cell. And just behind them are cells that form continuity with the existing cell vascular wall and these are called stalk cells. And these cells begin to migrate and replicate out into the area of the wound, right? And as this is occurring, these cells are expressing MMPs, which are digesting the extracellular matrix. Already premade in the extracellular matrix is some VEGF, which is kind of nice, alright? Or also, TGF-B is present and these cells, under the influence of VEGF, will continue growing into the site. So in your text, theres something called notch. Okay, so theres an inhibitory protein thats present on these tip and stalk cells and as soon as they start to get into an area and oxygen tension starts to go up again because of the increased vascularity, notch starts to become which is on the cell surface binds to its ligand and there are several different ligands, theres five of them theres a protease action that occurs, clips off a piece of notch. That little fragment of notch goes to the nucleus and starts to turn off VEGF expression, okay? So its sort of self-limiting. Also in the extracellular matrix, you know that there are things called tissue inhibitors and matrix metalloproteinases. So these MMPs that are being expressed also get inhibited. So the system has its own way of turning off. But the stimuli for VEGF expression is, the best one is, hypoxia. Another inductor of VEGF is platelet-derived growth factor and transforming growth factor, okay? So a little bit about angiogenesis. Why do I have to know this, Dr. Craig? Well, its another mechanism for platelet-derived growth factor. Also, youre going to have patients in your practice, I have several already, who have been given a humanized antibody against VEGF called Avastin. So youll see it in patients who have had a primary breast carcinoma resected or primary adenocarcinoma of the colon, I believe those are the two areas that people are using Avastin, at this point in time. And its an antibody against VEGF. If you have an antibody against VEGF, youre not going to have angiogenesis. And you will not stimulate well, the tumor will be unable to stimulate blood formation, blood flow into its so it tends to inhibit secondary lesions. Okay? Now, its not good to fool Mother Nature. So you kind of think, well, gee, if I have patients on Avastin therapy and they tend to be, Ive heard, a fair amount of time after the primary resection and Im thinking about doing maybe a periodontal procedure for them, alright? Are there some problems associated with having a patient on Avastin? I had a friend I have to tell you a story. So I have a real close friend and he had colon cancer. But you know hes a tough guy, hes a dentist. You probably know him. Anyways, he goes back to the country every weekend and we practice together and I look and I said whats the matter with your hands? And he says you know, I have all these cuts because I work on my farm on the weekends. And I said but youre on Avastin. He goes yea, they dont heal, I dont know. So I said, well dont you like wear gloves? Nah, gloves. Men dont wear gloves, you know. So, hes practicing dentistry, it looked terrible.

[34] [Wound healing regulation: cell to cell interactions][Dr. Craig] Anyways, hes off of Avastin and okay, cell to cell interactions. So cells communicate, as you know, through several different kinds of cell adhesion molecules. Some folks tend to group them into cadherins, the IgG superfamily, of which antibody, you know, B and T cell receptors are one, selectins which weve talked a lot about in this course, alright? And integrins. Weve already talked a little bit about how endothelial cells reach out and touch one another. We also talked about endothelial cells can pull out of the vasculature with the appropriate class of innate immune cells to help with inflammation and weve already been through this so these little guys have cell adhesion molecules that will tell them when they actually are touching an epithelial cell and that radically changes their phenotype from one that has proliferation and migration over connective tissue to one that allows them to differentiate. So I kind of predict that there will probably monoclonal antibodies, similar to the humanized monoclonal antibody Avastin, that will also start to screw up this kind of signaling because cancers tend to carcinomas tend to invade connective tissue. Itd be nice if you could kind of alter that.

[35] [Wound healing regulation: cell to cell interactions][Dr. Craig] Lets finish up by talking about cell matrix interactions, which is the area that has the most application for dentistry. So cells can both respond to the extracellular matrix and also alter the extracellular matrix. And this allows cells to have a third mechanism of communicating with various cell populations. So weve already had, in the Basic Tissues course, we talked a little bit about fibronectin and well just reinforce that.

[37] [Wound healing regulation: cell-matrix interactions][Dr. Craig] So fibronectin is one of the factors that allows for directed cell migration. Fibronectin is immobilized on the fibrin substrate so a chemotactic factor thats immobilized instead of being soluble is it allows for chemotaxis but if its chemotaxis along a solid substrate, by definition its called haptotaxis. Weve come across that term every so often in your reading. Well talk a little bit about bone morphogenic protein too. I know that youve had some discussion about BMP-2 but its really making a big difference in my little area so well talk a little bit about that. And well finish up with something weve mentioned in Craniofacial Biology but now, perhaps, you get a better appreciation of something called enamel matrix derivative. And, so lets talk a little bit about fibronectin.

[38] [Wound healing regulation: Cell-matrix interactions- fibronectin][Dr. Craig] So this is a cartoon of a fibronectin molecule. Remember that it has two protein chains that are united by disulfide bonds. So this cartoon has only put in one chain of the fibronectin molecule and the fibronectin molecule has these redundant exons that code for functional domains, alright? So-called type I, II, and III domains. And if you look along the fibronectin molecule, it has certain affinities. So it has an affinity for type I collagen. It also has affinity for gelatin, alright? It has an affinity for fibrin, hence its name, fibronectin, binds to fibrin, right? Also has an affinity for proteoglycans that are being released in the embryonic or wound healing environment. And these type III domains are kind of interesting because they contain these guys here. It runs right along the molecule. These contain this amino acid triplet, RGD, remember from Basic Tissue? And as it turns out, there is a class of integrins that use the RGD sequence as their ligand. So if you happen to be a polymorphonuclear leukocyte, a neutrophil, and you want to get out onto that fibrin scaffold, alright, if you happen to be expressing the integrin thats specific for that RGD sequence, you can kind of attach, flow, attach, attach, attach, all the way through and guide you through the wound healing environment. So, thats an example of haptotaxis, directed cell migration along a solid substrate that occurs early during wound healing. So, people in the perio industry kind of knew this long time ago. So we used to be marketed with enormous amount of fibronectin that we were told to paint on root surfaces, paint on bone, paint all over, just pour it in there. And it realy doesnt have much effect. Theres so much fibronectin in patients serum, that adding more of it doesnt hasten anything and that product I havent seen in a while.

[39] [Wound healing regulation: cell-matrix interactionsbone morphogenic proteins][Dr. Craig] However, I have seen this product. So, there was a person at the University of California, in the 60s. He was an orthopedic surgeon and at the time there was this really interesting mouse. It was called the osteopetrotic mouse and if you broke bones on this little mouse bones never heal, right? You get these pseudoarthrosis. So that was observation number one. Observation number two is, you know, you have to get this devitalized bone here has to be remodeled by osteoclasts. And in the osteopetrotic mouse, they have osteoclasts but they cant resorb very well. So this necrotic tissue would stay there and then in the orthopedic world and also in the perio world, you know, we had defects, bony defects, that this wouldnt fill in. And so, orthopedic surgeons think very directly like periodontists. Oh, okay, so lets, if its mostly hydroxyapatite, lets get some devitalized hydroxyapatite, stuff it in the hole and see what happens and actually, it actually some of these grafts worked pretty well. That devitalized bone, dead bone, was replaced with mature Haversian bone. So this led folks at the University of California to start thinking about, gee, there must be something in here thats inductive of the bone formation cascade, right? So [unintelligible] began a twenty-year quest. So here we get lots and lots of bone and you get this by brute biochemistry. You get lots of bone and the first thing he did is he tried the hydroxyapatite. And he had this wonderful little model. He would take a little compartment, a little screened-in compartment, and he would put pieces of whatever he was interested in in the compartment. He would implant it into the sartorious muscle of a rabbit, I believe, and then he would sacrifice the animal at various times and these were demineralization chambers that he would use actually in histology so he processed them for histology. So if he took the intact bone, that was non-vital and made a powder of it, put it in the capsule, put it in the sartorious muscle of a rabbit and took it out at various times. He would see blood vessels coming in, early on, right? And he would see cells that would come in and would begin to resorb this necrotic bone. And he would see areas of woven bone formation. And then he would see cutting cones come in the osteoclasts followed by endothelial cells followed by concentric rings of osteoblasts. And then the little capsule would fill with the sartorious muscle would fill with mature lamellar bone. So, he knew that there was a factor in there. So he took out the mineral and it still worked. And then he took out type I collagen which is 90 percent of the matrix and it still worked, unfortunately. So then he had to go and kind of go through all of the other 90+ proteins that are present in the non-collagen matrix of bone. He got real close and then the genetics institute in Cambridge, Massachusetts kind of took the story from Marshall and they used recombinant DNA technology to pull out this protein. Marshall thought it was a single protein and he called it bone morphogenic protein. As it turns out, theres a whole family of proteins, which are now called bone morphogenic protein. Theyre part of a bigger family called a transforming growth factor family. So the genetics institute said this is great. And so, they made [unintelligible] of BMPs and as it turns out, if you put BMP-2 or BMP-7 in a little collagen matrix and put it in that chamber, put it in the sartorious muscle in the rabbit, youll precipitate the entire osteogenic cascade. So theyre interested in BMP-7. And thats where the story stopped because unfortunately the Food and Drug Administration said, wait a second, transforming growth factor family that means cancer, doesnt it? What if you put this inductive factor into a patient and you induced osteosarcoma? Not good. So, poor genetics institute in Cambridge had to go through decades, or at least two decades, proving that if you put this into a wound healing environment, youre not going to induce cancer. So, finally, a few years ago, I dont know how much money the genetics institute lost on this but theyre marketing this and there is a product out called Enfuse. Enfuse is a collagen gel and the BMP-7 is in a liquid form and you soak the collagen pad with BMP-7 and you can place it into a wound healing environment. Thats the good news. The bad news is its about $5,000, the last time I looked, for the little kit, alright? It must take nickels to make this, you know, but they put so much research into it, they have to make the money back. So that sort of nixes it for the dental profession but the orthopedic surgeons use it a lot and theyll use it for spinal fusions and those sorts of things where you can place your little collagen sponge in a spot that is osteogenic but perhaps has a defect or in a spot that doesnt have bone that you want to induce bone formation and it will precipitate the entire osteogenic cascade, okay? So heres another example of BMP, bone morphogenic protein, that was deposited in an extracellular matrix during development it seems as if just in case, that bone would fracture in the future, in comes cells derived from mononuclear cells, right, osteoclasts, resorbing that matrix, releasing the bone morphogenic protein, telling that site well, this is a site where you have to undergo an osteogenic cascade. Questions on BMPs? Okay.

[40] [Bone grafts: induction via BMPs][Dr. Craig] And I have some pictures here. This was actually done by Gloria. So this was a study by Dr. Tarno, who was an alum of NYU and was faculty member here when I was at Columbia. And Dennis came up with this really hes a clinician but he came up with this but he appreciates science and he came up with this really neat model, okay? And its called the bilateral maxillary sinus graft model. So, sometimes if patients dont have enough bone height in the maxillary sinus area, alright, premolar, molar, maxillary teeth, what you can do is a sinus lift. So you go in, you make a little window, youll learn about it. You lift the Schneiderian membrane that lines the maxillary sinus up, you stuff it with whatever the bone grafting material is. You cover it all up, you pray that the patient doesnt get an infection, doesnt have sinus problems. As you can tell, I dont do grafts. And then if it actually forms patients bone, then you go in and you can do your osteotomies to place your implants. So Dennis said, oh, thats kind of interesting. So what if I put one kind of grafting material on a patients right side and one on the left side and my first drill that goes in, instead of being the solid, 2 mm twist drill, I put in a Trufine [?], right? So a Trufine is like a hollow drill, right? Takes out a core and so thats what you see here. So he would put, you know, whatever grafting material X from company X in one sinus and grafting material Y from another and then they would compare and its a beautiful control, right? Anyway, so this is a grafting material from Germany from the Tuttegin [omg, idk] company and so you can still kind of see the thread marks form the Trufine and this is after about 4 months of healing. And all this pink stuff, this is mineralized tissue. And if you kind of go at higher view, you kind of get a look that gee, this kind of like looks different than this. And if you go higher power still, what youll see is that some of this bony material has cells in the lacunae and some of this material has osteons but theres like no cells. So this is the grafting material and during preparation of this material for histology, they use dehydrating agents. So, this is like an artifact. So these cells here are right up against this surface. These cells are right up against this surface. And do I have a higher power?

[41] [Bone grafts: induction via BMPs][Dr. Craig] Yea, okay. And so these cells are right up against this surface. And in these cells are these multinucleated giant cells, right? Theyre resorbing this matrix and during the process of resorption, theyre releasing in situ signaling molecules, some of which are members of the bone morphogenic protein gene family.

[42] [Bone grafts: induction via BMPs][Dr. Craig] and this is inducing the patients own lamellar bone to form in this site, right? So this is the opposite strategy. Instead of just adding something that occurs like a cytokine early in the wound healing cascade and seeing if you can get the events to go faster, this is perhaps a little more elegant?

[43] [Bone grafts: induction via BMPs][Dr. Craig] So this is taking a signaling molecule that we know is present, developmentally and during wound healing, then adding more amounts of it into a site that perhaps wouldnt have this kind of outcome if the inductive factor was not present. So youre going to see a lot more of that in your clinical lifetime. So finally, my favorite product, I use a lot of this. So Lars Hommestrome [?] at the Carolinksy Institute in Stockholm, Sweden was very interested in how roots form.

[44] [Induction of cementum: enamel matrix proteins][Dr. Craig] So heres a forming root and you remember theres Hertwigs epithelial root sheath here and the inner cell layer of Hertwigs epithelial root sheath epithelium are really intelligent little cells, right? So not only does Hertwigs epithelial root sheath, this little tip here, not only does it trace out the root formed but it also induces odontoblasts from the dental papilla to differentiate into odontoblastic processes. And these odontoblasts start to lay down their matrix. And as soon as they start to lay down their matrix, the inner cell layer of Hertwigs epithelial cell root sheath becomes starts to secrete a protein matrix. We used to call that intermediate cementum. And one of the components of intermediate cementum is amelogenin or amelogenin-like peptides. And as soon as that matrix is deposited, the inner cell layer, actually all of Hertwigs root sheath starts to dissociate from the matrix its just laid down. And some of these cells undergo apoptosis so the Hertwigs epithelial root sheath fenestrates and through those fenestrations, we believe cells from the dental follicle then attach to that surface, sense the presence of amelogenin, amelogenin-like peptides and start to synthesize cementum. So, as you know, from your course in the spring, theres a wonderful company whos no longer in business, BioAura [?], they would get from pig sources in slaughterhouses, they would get developing teeth and they would isolate from the enamel, what they called enamel matrix derivative, Emdogain, and then Lars said he just put the gain on there because it just sounded sexy, right? So enamel matrix derivative gain. And it was marketed to our profession. They had some problems. But now theyre taken over by Strohmmen [?] whos marketing it again. And what you can do is, in a periodontal defect, that wouldnt undergo regeneration normally, you can apply Emdogain and you get this wonderful inductive event. So this is from a study we did here and what we did is we made a window through the alveolar bone to access. This happens to be in a pig, a big pig. The root surfaces and we wanted to see if we could induce cementum formation on biomaterials that wouldnt normally support cementogenesis, things like gutta percha, all the crazy things you learn about in endodontics. So this is a controlled section. So we made preparations in the roots. So heres the preparation at high power. So all of this was in here. So were just looking right in and we covered the area over with a barrier membrane. And before closure we covered this whole area actually the whole site with Emdogain. And what you get and here it is in high power. So heres regeneration of alveolar bone, periodontal ligament, and heres regeneration of cementum. And the cementum is kind of interesting because it doesnt have any cells in it, or very very few. Its really hard to find any cells in this matrix and this matrix is really very, thick. And also, this matrix, a lot of times if you look at guided tissue regeneration, just using a barrier and we talked about this in craniofacial biology, the cementum that regenerates here is cellular. It has lots of cellular inclusions in it. And during histology, the cementum separates from the prepared dentin surface and you dont see this we think thats because amelogenin operates as an inductive factor during development that induces the first cementum to be laid down and thats acellular cementum. And then afterwards, depending upon the site, you can get cellular cementum laid down which may be a different tissue and we talked about that in the last course. So, those are two things actually three things so you have platelet-derived growth factor, you have BMPs, which will probably come down in price I cant imagine five thousand for very long and amelogenin and enamel-matrix derivative which, thank god, is not five thousand dollars a shot for use in practice to not only hasten wound healing but perhaps also change the outcome of wound healing for therapeutic aims.

[45] [Wound healing: Summary][Dr. Craig] So, summary. To summarize all this. So wound healing, if you been moderately awake during this time, you should have it firmly in your mind that wound healing is a series of cells, cellular events, signaling events that occur in a very defined sequence. If you interrupt that sequence, either by infection, mobility, what have you, you have to go all the way back to the beginning and do it again. Its really a series of cellular events that include replication, migration, and differentiation. Weve talked a little bit about how those three are controlled during wound healing. Many of the events that occur during wound healing repeat events that occur during development of the tissue so this idea of biomimetics, alrigtht? And basic understanding of wound healing will allow you, as a surgeon, to change the outcome of wound healing for therapeutic aims. Thats what I have for the wound healing. Questions on wound healing? Yes?

[Student]For the induced product, that promotes the osteogenic cascade, does it will it sip the process where you resorb the necrotic bone?

[Dr. Craig]No.

[Student]Okay, so it doesnt matter. .

[Dr. Craig]Yea. The past governor of New Jersey, the millionaire that got into the automobile accident and had to have all those fusions Jack Ricci and I dont know but we highly suspect, when he showed up in the operating room to have all of these fractures reduced, they used a lot of Enfuse. In fact, so much that he had to go back in and have sort of like an osseo-trim did you hear about this? Yeah, so it must have been an orthopedic surgeon in there saying, oh, its the governor plus, hes a millionaire. You know, who cares if its five thousand dollars a sponge. Im putting it everywhere. He actually had to have a reduction afterwards. So Jack says, ah, I cant prove it but you know, Id love to know from the company, how many boxes were used that day. Okay? Yeah. And, most of these things, the prices are going to come down. Avastin, originally, was extremely expensive. Now, youll see it more and more with your patients. As you did Dr. McCutcheon talk about monoclonal antibodies and humanized clonal antibodies with you? Yes. Thats real important and youre going to see a lot of it. Okay? And initially, as you can imagine, it takes an enormous amount of development to get these humanized monoclonal antibodies out into the market. So initially the price is very, very high. But then, theyre very efficacious so you know; the price will drop as the demand goes up, okay? You guys have a great weekend. Ill see you next, formally, in Diagnosis and Treatment of Oral Disease. 4