Transcribed by Albert Cheng 9/17/14

[General Pathology] [Bone Wound Healing and Osseointegration – by Dr. Ricci]

Slide 1 – Bone Wound Healing & Osseointegration[Dr. Ricci] My name is John Ricci, I’m from biomaterials. In having some conversations with Joan Phelan, we decided it would be a good idea if you guys got a little bit of not only wound healing but bone wound healing and osseointegration since you do get some training in undergrad in implantology.

Slide 2 – ObjectivesEssentially, what we’re gonna look at today is really a couple of learning objectives here. I want you to understand the way bone wound healing occurs and see how that works. Bone responds to implants, osseointegration as it’s refer to with dental implants, is really just wound healing with a foreign body present. Since this is not normal tissue, it does fall into the category of pathology. Also, I’m going to talk a little bit about biomaterials…about how we modify implant surfaces in order to influence the healing process, which we pretty much routinely do with implants. First off we need to understand the process of bone wound healing. If you have Ron Craig’s lecture, what you’re essentially doing is you’re growing new tissues from existing live tissue. Ron probably explained how wound healing occurs in soft tissues. In bone, pretty much is what we call primary bone formation. There’s different type of bone formation. There’s endochondral bone formation that occurs in long bones during fractures where you have a cartilage intermediate. That’s not the way bone heals in small defects such as periodontal defects…drill hole…tooth sockets. In craniofacial bones, most bone wound healing is what we call primary bone repair where the bone actually forms from existing vital bone. You damaged the bone and that damaged bone generates some new bones. I’m going to show you how that process works. And then we’re going to talk about what happens when that process occurs while there’s an implant present.

Slide 3 – Tooth Socket ModelSo if you look at a tooth socket model, it’s actually a very good model to use as an example of primary bone repair because alveolar bone tends to be very active. It remodels and heals very quickly. How the bone responds to tooth extraction is a very exuberant response. Bone heals very nicely in that situation. But there’s also this competition that occurs there between bone healing in the socket and the soft tissue from the gingiva. So if you see an extraction socket, after a couple of months, what you will see there is the…not only will there be a slight invagination there where some soft tissue has grown into the socket, the bone isn’t exactly leveled there. You will also see some loss of dimensions of the alveolar ridge. The width and height of the alveolar ridge will start to change over time. Once you lose the teeth, loading of the teeth is what really keeps the alveolar ridge healthy and once you lose those teeth, you start losing the alveolar ridge too. The alveolar ridge remodels. And so there’s this competition there between the soft tissues migrating in from the gingiva and bone healing that occurs from the walls of the defect

Slide 4 – Picture courtesy of Yeo-Eun YoonIf you look at that early on, after a day or so, it’s not very much to look at. This is the tooth socket filled with blood clot. And of course, what you want to do with a tooth socket is basically maintain that blood clot at the socket. The last thing you want to do is lose that clot because as you’ll see, that is essentially the new template for new bone formation. If you look at the same site…this is basically a dog model…if you look at the site 3 weeks later…it’s a very different situation.

Slide 5 – Bone Repair: Primary Bone FormationAnd this is probably the most important slide in the presentation. You can see this is the structure of alveolar bone. Alveolar bone is relatively dense cancellous bone. It’s dense spongy bone, which supports the teeth. You can see the original walls of the socket are roughly right here. You can see where the original outline of the tooth was. You can also see this is new bone. You can see it’s characteristically very different than the surrounding bone. This is matured lamellar cancellous bone which means layered cancellous bone…it’s matured structure. This is woven bone. This is very very immature bone…very new….only 3 weeks old. And just as a rule of thumbs in a dog model, you can pretty much take anything…depends on who you read…but in a dog model, if you multiply the time period by about 2-2.5 times, that’s approximately what you would get in a healthy human. If this is 3 weeks in a dog model, it’s probably 6-8 weeks in a human. You can see that the bone is directional. It originates from the damaged walls of the tooth socket. You can actually see the spots where the bone has grown inward from the tooth socket and it’s still growing inward. You can see it has not reach the center yet. This is soft tissue. This soft tissue has migrated in from the gingiva, which has healed by 3 weeks over that wound. So that’s a very good example of how early bone formation looks like in a socket. It would look the same way if you had a drilled hole. You would see something very similar at 3 weeks…especially if that drilled hole is open to the outside world. Now you can prevent that soft tissue ingrowth by putting guided tissue regeneration membrane over that socket or you can also fill that socket with a graft material and you can maintain the bone better that way.

Slide 6 – Bone RepairAs I mentioned, there’s 2 different types of bone repair. The type you just saw was direction formation of new tissues by activation of existing differentiated cells. These are existing bone cells that are adjacent to the site. A socket is a great example of that because you got all the walls around that socket…consists of live vital bone and so you can get very good bone ingrowth from that by activating those cells in that socket. This is basically a secondary union model. There’s no such thing as primary union in bone unless you’re working with long bone. If Ron Craig went over the different types of skin union, you can never really approximate the edges of bone very well and get primary union. You’ve always got a defect you’ve gotta fill especially in something like a socket. So it’s a secondary union model. Regeneration as oppose to repair, is what I mentioned earlier where you get endochondral bone formation and that’s what happens in long bone fractures where you get a cartilage intermediate that has to vascularize and cartilage gets replaced by mineralized tissue. That’s not what happens here.

Slide 7 – Healing by secondary union (skin)So this is really a secondary union model. In skin, it’s refer to as secondary union. In bone, it’s pretty much the standard in any kind of a defect.

Slide 8 – Wound Healing and Bone RepairNow in wound healing, you’ve got 3 phases. Most tissues you can break this down into 3 phases. You’ve got an inflammatory phase. Some people refer to that as a resolution or wound cleansing. You’ve got a proliferative phase, which is where the real action occurs, or the tissue repair part happens. And the remodeling phase, which in some tissues is more of a finite event…in bone, it’s pretty much continuous because bone is always turning over at a slow rate. So what happens is you repair/remodel the bone back into a more mature structure and then that remodeling never really stops in bone. Bone is pretty much a continually remodeling tissue. The inflammatory phase is the most important phase for getting wound healing started. And because what happens during inflammatory phase is you’ve got a blood clot…bleeding is no accident…of course any surgical site soon as you damage any tissue, you get bleeding. We tend to think of blood as in the tissue but it’s not…blood is in blood vessels. What’s in tissue is generally carefully regulated filtrate of blood. Once you release whole blood into tissues, it causes a lot of things to happen.

Slide 9 – Wound Healing: Inflammatory Phase (days to weeks)It really stimulates the wound healing response because you’re releasing serum, PMNs, macrophages, fibroblasts, platelets…you’re releasing all kinds of things into the tissue that aren’t normally there. Macrophages begin wound debridement. During this phase, PMNs go after bugs that are there. This results in these cells producing cytokines and growth factors; especially platelets produced PDGF, VEGF, FGF, and TGF-beta. These are all chemo attractant that actually cause cells to migrate into the site and stimulate proliferation of cells. These are essentially recruiting factors for bringing new tissues into the site. Slide 10 – Who wins the cell race to the wound site?Who wins the race? Well the 1st cells to get these wound sites are usually neutrophils depending on the site…it’s usually neutrophils…probably a few macrophages will get there first. Those are cells on roller skates. Pretty much compared to anything else, a fibroblast if you can get it to move in a straight line will move about 1mm a day which is pretty hard to get to migrate in a straight line. If you get neutrophils moving, they will do that in a matter of a couple hours and osteoblasts are somewhat slower than fibroblasts. Osteoblasts and bone cells really don’t migrate into the site until somewhat later. So you’ve gotta try to keep fibrous tissue out of these sites if you can.

Slide 11 – Lymphocytes Homing at Wound SitesSo this is an interesting little video. [Plays video} That actually shows a wound that’s done in a zebra fish larvae fin by a little pinprick. These fins are clear so you can see through them. What you can see is a venule down here and you’ll see blood flowing through the venule. And you’ll see macrophages that are actually rolling along the vessel walls. They sense things from the damaged tissue that are released and they know where to exit the blood vessels because of that. You can see these cells migrating to the wound

site. Keep in mind, this wound site is not even a big enough wound site to penetrate your blood vessels…there’s no bleeding involved here. All there is is a little bit of damage of a few cells…that’s enough to attract these WBCs to the site and these fibroblasts which we pointed out here haven’t even begun to move yet as far as the healing response. In this video, 15 seconds represents 60 minutes worth of migration. So those cells are moving quite fast to be able to move that distance in a matter of a few seconds.

Slide 12 – Wound Healing: Proliferative Phase (days to months)So now the proliferative phase…that’s the phase where you take that clot and convert it into granulation tissue. Granulation is an immature connective tissue with a lot of BVs and you still have some growth factors and cytokines being expressed but during that proliferative phase is when primary bone formation begins. And in different animal models and humans, this occurs at different times.

Slide 13 – Granulation Tissue versus GranulomaOne thing that I want you to be aware of is when I talk about granulation tissue, I am not talking about granuloma. They’re 2 different things. Granulation tissue is a temporary connective tissue that is largely new BVs, fibroblasts, immune cells and those sorts of things. It’s a conversion from a clot, essentially the RBCs are removed and fibrin is replace by loose collagenous tissue and that’s the beginning of the healing process. Granuloma is something different. Since this is a pathology course, I figured I’d mentioned that. That’s a chronic inflammatory lesion. So just keep that in mind. They’re not the same.

Slide 14 – Wound Healing: Proliferative Phase (days to months)The proliferative phase is really… the granulation tissue is the key to bone formation. We use to be taught that granulation tissue had to be removed before bone formed in the site. In essence, granulation tissue is not really replaced by bone. It’s actually used as a template by bone. There’s beautiful histology that actually shows the collagen from granulation tissue being actually pulled into osteoid…seem to be used to make new woven bone. So it is actually the template for new woven bone. And of course the woven bone remodels back to a more mature structure. It is the template for new bone formation which is why they always tell you in an extraction socket, don’t remove the clot…make sure you get a stable clot formation. Don’t do anything to compromise that later.

Slide 15 – Bone Repair: Primary Bone FormationSo primary bone formation begins during this proliferative phase as the formation of those very thin trabeculae…those very thin spongy cancellous bone structures that you saw in the earlier slides. That’s woven bone and it uses the granulation tissue as a matrix. This can be seen at less than a week in mice. Mice are very fast at making bone. Dogs and rabbit start producing at about a week. You’ll see that histologically in some slides I’ll show you. It probably takes 2 – 2.5-3 weeks to get started in human and again that’s a healthy human. A 70-year-old diabetic is going to be different in terms of getting the healing response to occur.

Slide 16 – Early bone formation near implants

If you look at bone response around a dental implant, this is a electron micrograph of…this is called backscattering imaging…and all you can see here is an implant that has a metal beaded supported surface implant and this is bone trabeculae. This site was probably prepared roughly to where this dotted line is. It was drilled to that site and the implant was place there. These are the original bone trabeculae and these new little thin ones are new bone trabeculae of woven bone. You can tell it’s woven bone because there’s a lot more cells. These little black dots are where the cells are. This is lamellar bone. The reason you can see that is because the cells are lined up in rows. This is only 8 days post-op in a rabbit. This is the beginning of new bone formation. You can actually see the bone growing into this porous metal structure here, which is the beginning of bone fixation.

Slide 17 – Early bone formation is directionalThis early bone formation is directional only because the site contains the clots and granulation tissue and there are still growth factors and things present there that take this population of pre-osteoblasts that actually grow off of the damaged bone trabeculae and they directionally grow into the wound site because of the things contained in the wound site. And those pre-osteoblasts whether they’re growing along the surface or into a granulation tissue tell the cells behind them to create bone by appositional bone growth.

Slide 18 – Bone Formation – Matrix ProteinsThe way they do that is by these pre-osteoblasts producing things like osteopontin, bone sialoprotein, osteocalcin and different bone marker proteins that stimulate the formation and remodeling of new bone behind them.

Slide 19 – What stimulates preosteoblasts to Form New Woven Bone?What stimulates those pre-osteoblasts to form the new woven bone while the environment is what does this. It’s a chemotactic gradient of components from the damaged cells and serum component and cytokines and growth factors that cause this growth. There’s recent evidence that indicates that macrophages for instant can produce bone morphogenetic proteins, which are bone marker proteins that actually stimulate the differentiation of new bone. We knew that macrophages could produce osteopontin but it was only about 10-12 years ago that we found BMPs can also be produced by macrophages. Macrophages are not always the bad players. We think they are because we see them at wound sites. They can actually mark the matrix for bone deposition

Slide 20 - De novo alveolar bone formation adjacent to endosseous implants. A model study in the dogThis is a beautiful paper from 2003 that was actually presented here by Tord Berglundh at an implant meeting. It’s an interesting paper because this is a dental implant but it’s a funny-designed dental implants because basically it has a healing channel built into it. It has this little spiral cut channel built into the system that produces a 400-micron healing channel and you see this is drilled to this diameter and so these threads are what holds the implants in place. But this little chamber is left emptied and now you can look at this during time and you can see bone ingrowth into that chamber. You can watch the process of bone formation and this is the only histology paper that I’ve ever seen where it has 2

hour histology…which isn’t much to look at…2 hour histology is basically just a blood clot with a few bubbles in it. You can see that here’s a couple of ways of repairing this and different stains that are used. Here’s your original bone and this is just blood clot at 2 hours. Slide 21 – Berglundh et al., 2003 – 4 daysBy 4 day that blood clot, however, is really now granulation tissue. Now keep in mind this is only a 400-micron wound. This is less than ½ of mm of defect. So things happen a lot faster in this small defect than they would with 3 or 4 mm size defect. But nonetheless, if you look closely at this tissue that forms here, most of the RBCs are gone from the clot and you’ve got lots of vascular sinusoids that are forming in here. These are new BVs…you can see them all over the place. This is a going to be a mixture of macrophages and different types of white cells and fibroblast that are creating a loose collagenous tissue in this site.

Slide 22 - Berglundh et al., 2003 – 1 weekIf you look at a week, depending on the preparation…the first preparation is a demineralized preparation…doesn’t really show the bone all that well compared to the implants because the implant isn’t there. Here you can see the implant and you can see the original trabecular bone and even at 1 week, you can see the how new bone…that’s these blue lines here…these very thin woven bone lines are forming from the surface of the damaged trabecular bone. You can see them actually growing off of the surface. And in this case, they’re actually growing along the implant surface as well. Now, I look at this and say…jeez this is 1 week…that’s a lot of bone for 1 week in a dog. This is also what’s known as selective histology in the field. They actually picked the best thread out of the entire implant. You’ll notice most of the thread don’t have bone formation and this one does. So they picked that particular one to show. But there is some bone formation in most of these threads, it’s just not a lot. That’s quite a bit of bone for 1 week but bone does form at 1 week in dogs and that is equivalent of about 2-2.5 weeks in a human

Slide 23 – Berglundh et al., 2003 – 2 weeksIf you look at this later, at 2weeks, you’ll notice the bone trabeculae have now thickened. They’re not as thin as they were originally. This again is the demineralized preparation so it looks a little different but nonetheless, bone is starting to get a little more robust. If you look at the interface between the existing bone and the implant thread, you can see new bone forming here at the interface. There was probably some resorption of the old bone that occurred too. There is some resorption of the existing bone when new bone formation occurs.

Slide 24 – Berglundh et al., 2003 – 4 weeks (L), 6 weeks (R)And this is at 4 weeks and 6 weeks. 4 weeks, you got very thick trabeculae now. These are starting to remodeled back to lamellar bone structure. Again, this is a small defect. It takes longer in larger defects. And by 6 weeks, it’s actually pretty hard to tell what’s old bone from what’s new bone in this particular preparation. So you’ve actually remodeled that bone back into something more like a lamellar structure. And at 6 weeks, that’s probably 15-16 weeks in a healthy human. In this small defect, you would see something like that.

Slide 25 – What surfaces work best for protein and cell attachment?Now let’s talk a little bit about implants. What implant surfaces work best for osseointegration? We found empirically over the year that hydrophilic surfaces work very well and roughened surfaces work very well. We’ll talk about why in a little bit. We’ll talk more about the roughening than the hydrophilic part. Hydrophilicity just means it’s a very wettable surface. You will never see implants made out of Teflon. Teflon is very hydrophobic. Water doesn’t stick to it, neither does proteins very well that’s why they make non-stick pans out of Teflon. Hydrophilic surface like Ti because titanium once it’s exposed to air or an oxidizing acid, which is routinely done to treat titanium surfaces, you produce titanium oxide. Ti02 is not only extremely stable as a surface but it’s extremely hydrophilic so it binds proteins very well. Cells actually never see Ti during the osseointegration process. What they see is a layer of adsorbed proteins that stick to that surface immediately upon implantation of the implants…within the nanosecond of that implant being placed…that implant is coated with a layer of complement factors, albumin, and all kinds of things that are present in blood. Blood proteins play a role in that process too. They stick to that surface. So that’s really what the cell sees. It’s not really titanium. They see proteins. And actually if you use a hydrophobic surface, proteins will stick to a hydrophobic surface, they just don’t stick well and they actually change the configuration of the protein and cells don’t stick as well to proteins on hydrophobic surfaces. They do on hydrophilic surfaces but we won’t get into that today. Why does micro texturing work? That’s a different story

Slide 26 – Surface Texturing and Osseointegration (Microstructure)I spent 16 years in the orthopedic field and there are 2 different types of hardware in orthopedics. There’s stuff like dental implants but they’re total joint replacement…much bigger that are roughened because they’re expected to stay in the body for a very long time. They’re expected to integrate. There’s another type of hardware that you don’t want to integrate in orthopedics and that’s fracture fixation devices. Things like bone plates and bone screws…intermedullary rods. You want to be able to take those out later. Well you’ll never see a textured/roughened bone plate cause you would never get it out. What they do is mirror polish bone plate, screws, and intermedullary rods. What we found is when you mirror polish a surface, it forms a fibrous capsule. If you texture a surface, it integrates with bone. So the question that goes way back to when I use to be at hospital for joint diseases was why does a textured surface osseointegrate and smooth surface fibrointegrate.

Slide 27 – Surface Texturing and OsseointegrationAnd you can see that actually in a couple of pictures from a very old publication. This was a smooth surface metal implant and you can see fibrous tissue. Blue is bone. No bone interaction with this surface whatsoever. But this is a electron micrograph of a textured metal surface and you can see direct bone integration of this surface. This is osseointegration. It’s not complete. There are areas with soft tissues…black area where there’s some marrow.

Slide 28 - Surface Texturing and Osseointegration (“Machined surface”)

So empirically over time, the surfaces we evolved into using for implants started out with machined surfaces. I’m sure somebody at some point early on in the implant field polished an implant because machinist like to make their things look pretty. And so somebody probably machined an implant and polished it and probably found out very quickly that that doesn’t work for bone integration because if you look at the original Branemark patents on dental implants, they actually specified the size of the machine marks that have to be on the surfaces of their implants because they recognize the importance of those machine marks. We have since found out that those machine marks are not really optimal for osseointegration. So we’ve developed other ways of texturing surfaces. This is a blast textured produced by a modification like sand blasting but a different type of blasting method. This is an acid-etched surface. Acid-etch came about because people sand blasted the surface and sand stuck to the surface and you can’t dissolve sand very easily so what you do is dissolve a little bit of the metal to release the sand. SO you acid etch the surface to clean it. This has led to other surfaces that are more evolved.

Slide 29 – Osseointegration: Recent MicrotexturesThis is a combination of sand blasted acid etched surface called the SLA surface. This is a RBT surface. This was developed here. This is a surface produced by using a blast medium that’s soluble…you don’t have to dissolve the metal to get rid of it. It’s an acid sensitive blast medium. It’s an apatite…it’s like bone mineral…very sensitive to pH so you can literally put this stuff in vinegar and it would dissolve the blast medium. This is a very common surface on dental implants. This is not…an anodic oxidation surface produced by oxidizing a surface while currents are passing through it, which produce these little volcanoes on the surface. Only 1 implant system has that.

Slide 30 – Surface TexturingSurface texturing works on a lot of levels. The first level it works on is the simplest to remember because basically what happens is you’re increasing the surface area when you texture a surface and so if you think the whole protein attachment process, you’re going to attach more proteins to a textured surface than you do to a smooth surface. That’s one reason it works. If you just think about sheer forces and tissue stability in a surface, it works much better if you got a textured surface. You’re much less likely to slip a tissue off that surface on a textured surface than you are on a smooth surface. The other reason is that a clot sticks better to a textured surface than it does to a smooth surface. There have been studies that have shown that…we’ve seen that. We’ve did implantable chamber studies and when we look at smooth surfaces, we found that the clot didn’t stick to the surface, it actually pulls away during healing and you wind up with space and it ends up filling with fibrous tissue.

Slide 31 – Osseointegration: Molecular ModelsOne of the other things that happen is you get enhanced platelet adhesion. Platelets stick better to textured surfaces than they do to smooth surfaces. And since platelets make a lot of things necessary for that inflammatory phase of wound healing, you do get better tissue formation on a textured surface because platelets stick better to the surface. You’ll notice there’s a bunch of references here from a book called “Bone Engineering” from

2000. It’s a very good book. We know a lot more about the process now. We know which genes are turned on now but the process was outlined in the book. It was pretty much dead on.

Slide 32 – Mixed Topograph Textured Surface: Are They Optimal?Most of the surfaces we use are the roughened surface called mix topography surfaces that are a combination of structures. Usually what we found that works the best is if you superimposed small features over larger waviness surface like 1-3 micron features onto about a 6-10 micron overall surface texture seems to work well. That seems to be a common denominator amongst dental implant surfaces. The debate on what constitute an optimal surface has been going on for a very long time since 1991. There’s still no consensus as to what the best surfaces are.

Slide 33 - Systematic study of osteoblast and fibroblast response to roughness by means of surface-morphology gradientsOne of the things that surfaces do and this is probably the simplest way to look at this…if you look at osteoblasts and fibroblasts growing on a smooth surface versus a rough surface…you’ll notice that a smooth surface, the cells spread out on the smooth surface. On the rough surface, the cells are much less spread. It may seem like a relatively minor thing but cell spreading is a way of triggering cell proliferation. When cells spread, they produce a lot of attachment plaques or hemidesmosomes that are actually cell signaling events…there are cell signaling proteins that are a part of those attachment plaques and there’s some very old work from 1978 that showed that if you limit cell spreading, you can limit cell proliferation. And so that’s essentially what rough surfaces do because the projections on the surface limit the way cell send out processes and so they limit spreading.

Slide 34 - In vitro studies of cell response to surface microtexture: Cells on controlled microtexturesWe did a series of study a couple of years back looking at…started with regular microtexture surfaces…you can see these colonies of cells growing on this roughened surface are much smaller than the colony of cells growing on the smooth surface. They all started with the same # of cells and they just grow faster on smooth surfaces. We have a tendency to think of smooth surfaces because we grow cells all the time as being normal. Smooth is not normal in the body. Nothing is smooth about the body in terms of structure. Cells on smooth surface are not a normal situation. Cells behave actually badly on smooth surface. They don’t behave normally at all. We did a whole series of study that looked at surfaces that were made using chip technology and found that for instance using a micro-channel surface like this , we can limit cell spreading a lot and have the colonies formed directionally instead of radially. And you can actually cut cell spreading drastically using surface like this. We found there was a optimal groove size on these surfaces of about 6-12 microns that you can actually use to inhibit fibrous encapsulation. And so really what these cells do…these roughened surfaces do 2 things. We found that they do inhibit fibrous encapsulation but they also increase the differentiation of bone cells to a point where they produce bone better on textured surfaces than they do on

smooth surfaces. So you’re not only decreasing fibrous tissue formation, you’re increasing bone formation.

Slide 35 – Human Bronchial Epithelial (HBE) Cells We also found that epithelial cells respond to these textured surfaces as well. Like if you grew epithelial cells on a smooth surface and they hit these grooved surfaces, the cell stop and grow along the surfaces instead of crossing and this only works in a specific size range. This is all based on something called contact guidance.

Slide 36 – Contact Guidance – How Cells Respond to Surface MicrostructureContact guidance goes back to 1907. The first guy that grew neurons in culture was a guy R.G. Harrison. Round that time period between late 1800s early 1900s was the beginnings of the cell culture revolution and it didn’t start out that way. It actually started out with the Frankenstein revolution. People were actually trying to keep tissue alive outside of the body. They found that if they put tissue slices in glass culture plates and they left them there long enough, cells would actually migrate from the tissue out on to the glass. That was the beginning of cell culture. R.G. Harrison did that will frog spinal ganglia in frog lymph. You couldn’t buy cell culture medium in 1907. He actually observed neurons growing out of live nerve cells. He also observed that if there were scratches in the surface of the glass plates and since we re-used glass culture plates which is why “in-vitro” means in glass. The cells will actually migrate along scratches. So small features if they’re in the right size range will influence the way cells migrate and that’s essentially what we’re doing with textured surfaces. It’s called contact guidance. That term wasn’t coined until the 50s by Paul Weiss and Beatrice Garber.

Slide 37 – Mesenchymal Cell Attachment and Shape Versus DifferentiationJudah Folkman work showed that if you limited cell spreading, you could enhance the differentiation and prevent proliferation. If the cells spread, they proliferated. If you limit attachment too much or if you prevent it entirely, the cells die and that’s apoptosis. Judah Folkman observations were correct but his explanations of why this happened were completely wrong. Lot of this has to do with the cell cytoskeleton, which they didn’t even really know about then. Donald Ingber has put a lot of that together. This is a very deep paper on cellular mechanotransduction…how cells sense changes in ECM…they sense mechanical loading and that’s based on the cell’s cytoskeleton as well. Ingber’s work actually explains Folkman and Moscona’s observation from 1978.

Slide 38 – Micro- and Nanostructure Control of Cell Attachment and SpreadingIf you’re looking at what we’re doing with surfaces, what we’re doing is we’re taking a cell that would normally flattened and spread on a smooth surface that would proliferate rapidly and by limiting its spreading in a groove or a surface with a lot of little projections, you limit the way it makes attachment plaques and so you limit the growth of the cell that way. And cells do 1 of 3 things normally. They either proliferate, differentiate, or they undergo apoptosis. They do all 3 during the wound healing process. But they only do 1 thing at a time. If they’re proliferating, they’re not making tissue. What you want your cells to do is you want them to proliferate at the beginning of the wound healing process, then you want them to differentiate and start making tissue

Slide 39 – Implant SurfacesSo if you look at implant surfaces that are out there, there are 4 different groups of surfaces…well there’s really 3 different groups…passivation is a separate category. By far the most common surface is a subtractive surface. Most of the surfaces that are on most implants are subtractive surface meaning you’re removing a little bit of the surface in order to texture it. There are a few additive surfaces and a few special cases. Passivation is just something we do to all surfaces…all Ti surfaces just to make sure there’s a nice stable consistent coating of oxide. We basically put Ti implants in nitric acid for a short period of time. Nitric acid is a oxidizing acid. It does not etch the surface. It just produces a oxide layer and just make sure it makes a nice stable oxide layer on the surface. That’s all I’m going to say about passivation.

Slide 40 – Machined SurfaceThe first subtractive surface was the machine surface, the original Branemark implants. Removing materials in order to roughen this surface and was not really optimal but it worked to a degree.

Slide 41 – Subtractive Surfaces – BlastingWe then went to blast-textured surfaces where you put a blast medium in an airstream and you blast the surface …you gouge little bits out of the surface. Again the only problem with that is the early blast medium actually stuck in the metal surface and then you had to etch them out. Aluminum oxide use to be a very common blast medium and it’s been pretty much replaced by RBM (resorbable blast medium) which I mentioned earlier is a blast medium that is soluble. You can get it off the surface easier. Aluminum oxide is essentially ruby. You can’t dissolve that.

Slide 42 – Blast Texturing (MTX Surface)Some examples…blast texturing…this is the MTX surface from Zimmer. This is what it looks like at different magnification levels. It’s an gouge/roughened surface. The other major type of subtractive surface around is called the acid-etched surface.

Slide 43 – Subtractive Surfaces – Acid EtchedAgain that came about because people trying to clean the blast medium debris off of the blasted surfaces but here you’re basically etching along grain boundaries by using different types of acid. You use hydrochloric acid, sulfuric acid, phosphoric acid, and hydrofluoric acid. There’s a number of different one that do different things basically in terms of what the structure size that they make. And some of them are combinations

Slide 44 – Blast Texturing/Acid EtchFor instance, the SLA surface is a combination of sand blasted surfaces and acid etch. It’s a very good surface for osseointegration.

Slide 45 – Dual Acid EtchThe OsseoTite surface is a dual acid etch surface. These 2 different acids in succession and it produces a lot of fine structures…there’s not a lot of big microstructure on the

surface but it works nonetheless. So again most of the surfaces you see are going to be the subtractive surfaces on most implants.

Slide 46 – Special Cases – Anodic Oxidation (related to passivation)Some special cases… the anodic oxidation is a weird surface again. You’re oxidizing the surface and you’re also passing a current through it and what it does is…this is actually a coating…this is more of an additive surface than anything because you’re actually adding material to this. You can actually flake this surface off with instrumentation. There’s actually brittle oxide coating on the surface.

Slide 47 – Anodic OxidationIt’s a very strange looking surface. This is what it looks like. You see a lot of little volcanoes with holes in them because this produces a gas during the oxidation process and that gas has to come out somewhere…so it comes out through these little volcanoes. And this is really only used on the Nobel (TiUnite) surfaces…pretty much the only major company that makes these.

Slide 48 – Special Cases – Laser Microtexturing (Subtractive/Additive)We develop a surface here at NYU some years ago. It’s a lasered microtexture. It’s based on what’s called large area masking technique. It’s not as simple as it’s showned here. Mask is actually back in the laser before the beam actually coalesces…you can use UV lasers. They’re very short wavelengths. It’s called a cold laser. It breaks bonds without producing a lot of heat. You can use that to shape metal surfaces. And we produced a micro group surface that was based on the cell culture studies I showed you earlier

Slide 49 – Laser Microtexturing It looks like this. This is the surface. It’s actually used on the collar of dental implants as a way of preventing epithelial migration and getting fibrous tissue and bone attachment. Its used on BioHorizon implants.

Slide 50 – BioHorizons Laser-LokThis is what it looks like now. That was a previous generation surface. It’s a very highly micro nano rough surface but it’s also very organized. It’s actually the only organized surface…it’s not randomly textured. The whole idea behind this is this surface is to prevent bone loss and soft tissue recession around implants which is a very common thing.

Slide 51 – Clinical Cases: Dr. Cary ShapoffThis is older style Branemark implant that probably the bone level and soft tissue started up here somewhere and over time, you see this bone loss around the implants. It’s not necessarily killer depending on where your lip line is but yet eventually removes a lot of your alveolar ridge and the soft tissue go away with it.

Slide 52 – Picture of teethSo you can end up with a situation like this where your soft tissue probably started out up here and you end up with some metal exposed. It’s not a very esthetic way of doing

dentistry…dental implants…we’ve gotten around that largely with these implants. We had one of our investigator who did the 1st placement of these implants (Cary Shapoff). And he’s up in Connecticut. He’s actually one of the board examiners for the perio societySlide 53 – Clinical Cases: Dr. Cary ShapoffWe told Cary this was our first prototype we’ve ever placed in a human in 2000 and this has a laser machine collar and we told Cary as part of our clinical protocol, put this someplace invisible because it’s the first one we’re placing in a human. We don’t want it visible because we don’t know what’s going to happen to it. So of course he put it in the central incisor with a high smile line but this is the way the implant looks after 6 months. This is 3, 8, and 11+ years. It’s out 14 years and still has the same bone attached to the collar part of the implant, which is a good thing. That doesn’t happen on most implants. We’re getting to a point now…we’re getting into much more sophisticated surfaces…they’re produced using different methods than the older style blasting and acid etching.

Slide 54 - How Does Implant Surface Microtexture Influence Hard and Soft Tissue Interfaces?How does an implant surface affect hard and soft tissues. Well again the surface we use are hydrophilic on a nano-structural level. And so they do bind proteins very well. You will never see…that’s why pretty much all dental implants are either Ti alloy or commercially pure Ti and so they bind proteins very well. The microstructure we use are…you have to remember cells are not really smart on an individual level…to a cell anything outside the cell is ECM and so we’re slowly starting to come around to the idea that if we use certain roughness and certain size structures…we’re essentially creating surfaces and people are now engineering surfaces like this to look more like what cells normally see as ECM which is essentially a network of proteins outside. It’s certainly not a smooth surface. It’s a nano-roughened surface. So we’re starting to use microstructure as form of ECM to control cells. Again this happens through control of cell attachment plaque formation and cell shape and those sorts of things.

Slide 55 - How Does Implant Surface Microtexture Influence the Hard Tissue InterfaceIf you look at the tissue interface, the current microtexture we currently use…osseointegrate through a combination of suppression of early fibrous capsule formation because they prevent fibroblasts from spreading and proliferating to form a capsule. And they enhance bone cell differentiation. You’ll find bone cells on textured surfaces respond differently to circulating growth factors (vit D) than they do to cells on smooth surfaces. Textured surfaces work in a couple of ways. They inhibit fibrous encapsulation and they enhance bone differentiation. We empirically arrive at a set of surfaces that work reasonably well. We can probably get more consistent integration using controlled topography, which is what we’re starting to do now.

Slide 56 – How well do current implant surfaces work?There are actually very few bad surfaces left. There was just a 5-part paper that just came out comparing 60 different types of implants. What is surprising is that there are still a few surfaces that aren’t cleaned properly…still have some blast medium embedded on the surface. Most of the surfaces around especially the major companies osseointegrate

very well. So osseointegration has become very consistent among dental implants. The question is can you speed it up by using some specialize surfaces. The same cannot be said for epithelial and fibrous tissue attachment. That’s where the action is these days in trying to get a good soft tissue seal around dental implants because dental implants are very unusual implants as implants go. It crosses the boundary between tissue and the oral cavity. That doesn’t happen in a total hip replacement. Just about every medical device is totally contained in the tissue. With a dental implant, one end is in the oral cavity and one end is in the tissue. Unless you get a good skin seal around that, you’re eventually going to up with peri-implantitis…very much like periodontitis…you’re going to get bone loss around your implant. So most of the research in the implant field is centered around trying to get soft tissue to attach to surfaces and to get a good skin seal to prevent the bone loss around implants.

Slide 57 - Bone Repair and Osseointegration: How to Optimize Bone Formation, Limit Soft Tissue Formation, and Limit Bone Resorption!If you look at what you need to do…this is the last slide…to get good bone formation around implants…first thing you do is protect the clot. If you’re just removing the tooth, make sure that clot stays there because that’s going to be the predecessor of the granulation tissue and new bone that’s going to form there. If it’s even surrounding an implant, the first thing you’re going to get at the interface is a clot. Whenever you prepare a site for implants, it is done very slowly. You drill very slowly…very slow RPM…lot of time with irrigated drilling systems…the idea behind that is to prevent heating during drilling. Friction from drilling can cause local heating and can kill cells. If you kill the cells in the adjacent bone, the bone has to grow from farther away because remember that bone growth comes from existing live cells. If you kill the cells, you don’t get the bone growth. So you want to limit tissue damage from heating. You can use this process called guided tissue regeneration where you put a membrane over the site to keep the soft tissue out while the bone grows. It gives the bone a head start to prevent the fibrous tissue from mechanically growing into the site. And on implants, at least, you can use implants surfaces to attach bone and in some cases now attach soft tissue. This uses the soft tissue to seal the bone from the oral environment…keeps bugs from getting down into the interface…causing inflammatory response…causing peri-implantitis. And lastly, you want to limit any inflammation whether it’s from infection or debris or anything around that. Inflammation leads indirectly to bone loss because inflammation causes the formation of inflammatory cytokines, which actually cause indirectly the product of local osteoclasts, which will cause bone loss, which is how periodontal disease works. Periodontal disease, you’re not losing bone because of the bugs that are there, you’re losing bone because your body is responding to the bugs and so anything that causes inflammation especially chronic inflammation will cause bone less. If anybody has any questions, fire away. Thank you [Student question – inaudible] It doesn’t release oxide. It actually releases a gas during oxide production. It’s just produces an odd texture surface. No what they do is put a current through it while they’re oxidizing it and it produces a gas and that has to come out somewhere so it makes these little volcanoes. It’s a funny looking surface but it doesn’t release anything. [student question] Contact guidance is strong on most cells…the exception being various types of cells like macrophages and PMNs…the cells that migrate very quickly are less likely to be influenced by small

microstructure than fibroblast. Fibroblasts and bone cells respond very well to microstructure. Any mesenchymal derived cells work very well and epithelial cells and neurons are all very sensitive to any kind of contact guidance