Biomedical Engineering Center for Translational Research 2007 Conference and Expo Friday, May 4, 2007. University of Wisconsin-Madison, College of Engineering. Engineering Centers Building. Morning Agenda: Focus on Translational Research. Prof. Ray Vanderby, PhD; Lee Kaplan, MD; Hirohito Kobayashi, Phd; Mon-Ju Wu, MS; Patricia Keely, PhD; and Paolo Provensano, PhD. Acoustoelastic Analysis of Ultrasound Waves to Determine In Vivo Tissue Strains and Material Properties 00:00 Speaker: Prof. Robert Radwin, Chair of the Department of Biomedical Engineering. So, Wally's work is in the biomedical imaging area. The next area in our department and the next speaker is from the biomechanics area. Professor Ray Vanderby is a professor in the biomedical engineering department and also in orthopedics and rehabilitation. And he directs the orthopedics research laboratories. Ray has been with our department from the very beginning. He is one of the co-founders, the co-conspirators, that helped found our department, and he is the associate chair for the graduate program. Ray is particularly interested in orthopedic tissues, bone ligaments, tendons, cartilages. His research focuses on wound healing or regeneration of these tissues via tissue engineering. And this includes equal parts of functional characterization, the biomechanics, microstructural morphology and composition, and the relevant biological sciences, cell and molecular biology and histology. In addition, Ray is interested in ultrasonic wave propagation in these tissues for clinically relevant characterization in knee reconstruction and fracture fixation, and in the biomechanics of the spine. Today Ray will tell you about some of his innovations in the ultrasound area in orthopedics. 01:42 Speaker: Professor Ray Vanderby. Thank you, Rob. Here we go. 02:08 Well, first of all, I want to thank the Coulter Foundation for letting us explore something that would otherwise be just kind of a scratch we wanted to itch, just an idea that we had and a place where we wanted to go. And I want to thank all of the people who have contributed to this as well, and that would include certainly, here I've bolded, Hiro Kobayashi, because he's really the theoretical guru in terms of mechanics and mechanics of materials and mechanics of ultrasound wave propagation that has driven this. Lee Kaplan is my collaborator in orthopedics for the clinical applications. Mon-Ju Wu, a student. Patty Keely has been helping us, and Paolo Provensano, in terms of some things that we've done that have taken us over into the cancer research area, and I'll show you that as well. So, what are they trying to do, you know? Acoustoelasticity. What is it? Have you ever heard of that term before? Most people haven't. When I thought about it more, basically, we're just trying to do biomimicry -- we're doing things already exist in nature. If you look at bats, if you look at cetaceans -- porpoises, whales -- they find things, and they know what they are just by the sound waves they put out either at sonic or ultrasonic levels and they can distinguish one thing from another. That's all we're really trying to do here is identify things with sound waves. Wally is picking up things based on their water content and the signal they get back with MR. We're getting information back based on their mechanical properties. So, it's another way of identifying, distinguishing things. We get into showing a little bit about what a propagating wave is. We have that for a dilational wave to propagate, there's certainly more than just the dilational waves that take place. For it to propagate through a medium, we get compression and then expansion behind it. We get that as it propagates through, it propagates through at the speed of sound. The speed of sound is related to the stiffness of the material and so we have this interrelationship of information that we get. Since it's related to the stiffness in the material, we can get compressibility of the material; it has to be compressible in order for this to occur. And we can get the stiffness of the material and the modulus. And you start gaining that kind of information because it's related to sound waves. And compressible materials won't hold sound waves, and so we had to analyze these as compressible materials. When a tissue deforms, two things happen. This is what acoustoelasticity is-it's a form of elastic analysis that's been around since the '50s, but it hadn't been applied in this fashion, and so it considers that if there's a change of geometry that takes place when a tissue is deformed and it considers that the acoustic characteristics due to stiffness changes and stress occur. And an example of the phenomenon- the guitar and the violin string- we get a change in geometry, a change in acoustic characteristics as the material is loaded. To show you or illustrate what we're looking at and how we go through it is if we look at a tendon for example, and the tendon is loaded here and not there, if we look at the signal in both cases. First of all, in ultrasound, a signal will go in, and it will be reflected back. In the loaded case, we're going to reflect back more signal because when this starts to load, it's going to get stiffer. Since it's stiffer compared with the material next to it, it reflects back more waves. So we get more reflected back here than before. We use that as part of the information from our ultrasound signal. Besides that, once it passes through and then comes back, so we have the wave that goes through and then starts to reflect back from the far surface- we get reflection here, reflection there- the time it takes to get back reflects how the material has deformed, and the it also reflects the fact that it's going to propagate at different velocities due to the fact that this is stiffer from loading than this one. So this one won't be back by the time this one is back. So we get two bits of information for every signal we put in there: We get the time it takes to go across the signal twice and we get the stiffness information related to the reflection coefficient. With this, we can start to say something about the geometry. With this, we can say something about the stiffness of the material. And if we gather that information over just a loads-if we just take it and load it and unload it-we can take that information and parcel it out into different levels of loading, we have actually a lot of information, and we can characterize the stiffness as it changes over a period of time, and we can characterize the strain or the stress in the material through that entire period of time. So the project overview was then to quantify tissue material properties using this theoretical concept and to evaluate in vivo strains in materials. Here's maybe a busy slide- maybe too busy- but sort of how we do it. So, if we're stretching a ligament, and we put an ultrasound wave in. First of all, we know what wave we're putting in. This is the way we take it into the frequency domain, and we see what the peak is, and then we get a reflected wave back. The reflected wave is different. If we look at the ratio of those, that's the reflection coefficient. That's one of the things that we get back. The other thing that we get back is the travel time from the front surface to the back surface. Those are the two things that we get back for every state of loading. Here's the real experiment, and this shows real data coming back out of it. But we see that the signal changes and gets amplified as a function of loading in the tissue, and this is what we're using for information. We see the travel time between those, which is twice the distance because it had to go over and back in the material. This is a porcine tendon that's being loaded in a bath, and we're just pulling on it. This is an isolated test just to show proof of concept. These are pretty ugly, but this is the acoustoelastic equations, and you go, "Wow, that's a lot of stuff to deal with," but if you think about it in 1D, it's really not very much of a problem. This is the old wave equation that you saw in freshman physics probably, except that this is no longer a constant. We have the deformation that takes place as a function of position, the acceleration here, the mass there, and then this changes as a function of strain, so this is nonlinear and this is the stress. So, it just includes more terms; all we're doing is putting more terms into a standard, simple wave equation. We have to extract those coefficients from the inside that describe the material property. So, it's an inverse, boundary value of problems, is what we're solve. We can do the same thing if we want to look at things in a different matter. That was if we were pulling on a tendon, but what if we just want to do palpate? What if we're trying to find material properties say in a tumor? And so, we just want to push on it once, see what it feels like and then gather that information. We can do that same sort of thing with a different boundary value problem, but we can then shoot at a tumor. We can bring back the material properties of the tumor, instead of using Wally's technique, which is a visual-optical technique to try to identify one tumor from another, we get a signal that is a mechanical signal, and see if that will help us distinguish one type of tumor, one type of material, from another. This is compression. We have an artificial material here (??) to represent surrounding tissue. We have a tumor here that we took out of mice, given to us by Patty Healy. You can see that as we compress it again, the size of the signal changes, and then we get changes in how long it takes to propagate, partially because the tumor is getting thinner and partially because it's getting stiffer as we load it. Most tissues have this very nonlinear behavior. Here's some results for two different tumors, These are tumors from mice, and they're models for the two most common breast cancer tumors. So, these are the signature patterns for those based on our ultrasound signals. First of all, this is a normalized Young's modulus, the modulus of the material as a function of strain. So we have one that's very nonlinear, one that's not so nonlinear- normalized information. If we test it in our testing machine, and take a mechanical signal, that's what this is- this red. This is what we estimate that to be with our ultrasound signal alone, taking no other information than the ultrasound signal. Here's a different tumor. These are the two most common types of breast cancer tumors. Both of them are malignant, but one versus another is more aggressive, and there would be different treatment patterns, and so it's something that you would perhaps want to know before you would do the treatment of the tumor. Here's the other one- the black line is the testing machine data, the blue line is our ultrasound data for seven tumors that we test. From this, you can see that, number one, we can estimate the mechanical behavior quite well, and we call that our stiffness gradient index method of characterizing it. We can also distinguish one type from another with very high specificity and very high sensitivity. We can tell this tumor from that tumor type very clearly. So we call that our "acoustoelastic biopsy," because we biopsied a tissue with just the acoustic signals to distinguish one from another with this characterization. Those were isolated tumors. We tested those in the laboratory. We're trying to do in vivo tumors, but we're doing them in mice. Those are tiny tumors to be working on. Here's in situ data from porcine tendons. So that means that we have the leg, and the skin, and everything there. We're pulling on the tendon in our test machine, but then we're shooting through the skin and the fat and everything to get the data, and then take that data back out. The red line is force versus strain in the tissue, and this is our machine data. Here's seven specimens, and this is what we predict the strain to be in the tissue. So, it's a pretty good representation. We're off a little bit, but we think we could even correct for that based on certain parameters in the material. This is what we would get if we went through the same type of analysis but didn't consider the fact that the material properties change as we stretch them, so that's the acoustoelastic effect that we're including in that other people don't use, when they do this type of analysis. I think if you use the other type of analysis, it would be more difficult to really understand what was going on in the strains in vivo, and here we think we got a pretty good idea of what's going on in vivo. This kind of information would be very valuable for orthopedic surgeons, for diagnostics for evaluating whether something is partially torn or not torn, the loads that are on there, if you have something that's reconstructed to see if it's being used right, for rehabilitation to see how things are being loaded. So we think there are a lot of applications for this kind of information as well. This is just physical functional loading information, but we think it's valuable. So, we built a little, sort of (?) Goldberg device that we can use for testing. We're moving that into a laboratory. It's only a 1D system; it's not 2D. Just point and shoot. We're trying to develop our algorithms of 2D right now, but we have this 1D system that we're moving into the lab. So, we adapted the AE system- this is sort of in summary of what we've done- we released the compressibility constraint, and then we could do this analysis. We can get the wave dependent velocity and reflection coefficients. We can get nonlinear material behavior. We've developed the applications. We've got material properties, our acoustoelastic biopsy to distinguish different types of tissues, and we can get strain information from our tissues as well. Thank you. Kristyn Masters: Designing an alternative aneurysm occlusion device: Background: Cerebral aneurysm occurs in about 5 percent of people. Causes could be congenital, from head trauma, high blood pressure, infection, tumors, etc. "However, when they do fail, it is a serious, serious problem. The consequences of rupture would be hemhorrage in the brain or a stroke or death." Current method for treating: Coil embolization, or coiling, is common treatment. Delivered via an intravascular catheter, shape-memory coils are deployed within the aneurysm, and coils "fill up" aneurysm with "ball" of wire. "It doesn't seem like a great way to block out the aneurysm, but the idea is that you fill it with this very tight bundle of wire that you just keep pushing in there and the hope is that this bundle of wire is going to instigate this wound-healing response or a scar formation around around the aneurysm that will sort of wall this part off from the blood vessel and no longer have a risk of rupture." Problems, however, is coils compact, or as blood is flowing past, coils get pushed up into the aneurysm and pushes it out. That could lead to increased risk of rupture or enlargement. Also unpredictable wound healing; how long is it going to take the scar formation to occur. There's been very little study of how that happens. Finally, it's a very long procedure time-could spend up to eight hours putting coils in. "It's a very imprecise procedure in that they're just putting coils in and imaging while they're doing this in order to decide when the aneurysm is essentially full of coils." Kristyn's project is to shorten time to insert coil and make safer. Idea originated with Dr. Montakar?? "His idea was that he just wants to shorten the time that it takes for surgeons to occlude the aneurysm." In addition, make the occlusion better and safer. His idea was to have a single coil inside a shell. The doctor looked for an expert on shape memory. Start out with intravascular delivery. NiTi coil (Wendy Crone expert) and to put straightened coil in an expandable bioactive polymer shell. Deliver via catheter; coil exits from catheter, expands the shell-essentially a balloon that surrounds it and expands, and fills the aneurysm. "This coil, as it's exiting from the catheter, expands the balloon to then create sort of instant contact with the surrounding aneurysm shell." Advantages: "The polymeric shell now is a barrier to blood flow." It's more elastic than coils and likelihood of being pushed up is less. Tests on prototypes show that. Also decreased risk of enlargements and rupture. "There's a precise delivery of a single coil, and probably one of the most important things to our surgeon is that this only involves putting in a single coil. It could dramatically, by orders of magnitude, decrease the time to occlude these aneurysms." "We also discussed making this polymeric shell bioactive in order to not just simply be this intert surface where maybe scar formation just sort of happens around it because it's there, but rather a material that will actively stimulate wound healing and scar formation so that perhaps we can get this, aside from just putting the device in, get this walled off biologically from the body now over a faster time frame." Design: Coil is NiTi shape memory alloy wire (in Wendy's lab), trained to remember to a specific shape when above phase transformation temperature. Their phase transformation temperature is somewhat below normal body temperature. "When wire is cool, you can straighten it out very, very easily and that facilitates putting it into a very small catheter. And then when it warms up, it wants to coil, but it can't coil because it's in that very narrow-diameter catheter." Same method for current delivery. So when it's pushed out into aneurysm it assumes coil shape. These materials already are used in aneurysm treatment. Kristyn's lab is focusing on the shell. Constraints: Had to be biocompatible and hemocompatible (which greatly reduces your biomaterial options; there are few polymers that are truly hemocompatible), highly elastic, had to be able to make into thin films or capsules (balloons) and ideally, contain element that actively stimulates wound healing process. "So, when we started thinking about this, we realized that there wasn't a good existing material to meet these criteria." So they had to do their own thing from scratch. Decided to use a polymer that was kind of hemocompatible-but not as much as they'd like for this application-and to make it more so. They went with polyurethanes (used widely in medical devices, easy to process, fairly elastic, biocompatible, but not great hemocompatability). Solution was to incororate an anti thrombotic molecule into the polyurethane polymer backbone. They selected hyaluronic acid, which stimulates wound healing, among many other properties. Kristyn calls it a wonder molecule. They made a variety of these polyurethane hyaluronic acid copolymers. Materials were extremely easy to process into thin films (the balloon part), they has good mechanical properties, with around 1,500 percent elongation ("By changing the amount of HA that we incorporate into these materials, we can dramatically change the mechanical properties of these materials as well"), it's dramatically hemocompatible (thrombogenic) (platelets won't adhere to) ("We found that even at our lowest HA incorporation, we get complete elimination of platelet adhesion to the materials, so these are highly hemocompatible"), and has promising wound healing properties. They have put endothelial cells onto the materials; hylauronic acid (a molecule often directly recognized by cells) helps cells adhere. "A lot of times there's a tradeoff between having something have no platelets to adhere to it and having cells adhere to it." So more of a chance of having cells adhere to the material without having platelets adhere. "We have had endothelial cells adhere and proliferate on these materials." Currently testing the whole thing in in vitro models of aneurysms they constructed out of silicone. Because of this work, other projects have emerged: "I'm very excited about the materials that we've developed." Polyurethane-HA copolymers can be used for a lot more than just the shell of these aneurysm occlusion devices. They show promise for lots of applications, like using native biomolecules to impart bioactivity, fabricate in physical forms for other applicaotions, etc. "You can really tailor the physical properties and the biological properties on your materials based on how much of the HA you put in them." And again, materials with this protein structure are much more likely to be hemocompatible ... and other applications. Bill Murphy: Collabs with Ben Graf and Mark Markel. Biologically active coatings on resorbable orthopedic implants. Problem is permanent, or bioresorbable, screw fixation during ACL reconstruction. 30,000 to 40,000 of these surgeries are done annually. However, five to 10 percent of cases require revision procedure and there is a long (six months) rehab after surgery. So people lose a lot of work time, there's a lot of pain. "One of the key limitations is that bone tendon healing in the tunnels that are formed during this process doesn't occur as it's expected to occur." Now, in ACL reconstruction, tendon graft (like a hamstring) is snaked in between two tunnels in the bone, so a tunnel in the femur and a tunnel in the tibia, and it's fixed into places via either metal (commonly) or bioresorbable (more recently) (materials that go away with time and are replaced with tissue) screws. "The issue is that this is what happens; so, after a period of time, after the implant has been placed, you end up with this rather large tunnel that formed during surgery. And the tunnel actually expands quite signifigantly over time. So there's a 60 percent increase in tunnel area at three years without fixation, and when you fix it with a screw you actually get an even larger increase in that tunnel area. So this is a significant problem. It really decreases the stability of the bone here and slows healing." Recent research looked at adding a growth factor in a "sponge." "And they pack that sponge into the defect site and they've seen quite good healing. The bone fills in, the bone-tendon interface looks quite nice as well." Bill thinks if they can deliver this kind of a growth factor (in this case, bone morphogenetic protein 2, or BMP2) "if we could deliver it locally and actually deliver it from some of these fixation screws, we could get the best of both worlds: mechanical fixation but also a biological effect over time." They grew a coating on a bioresorbable screw. They inserted drug molecules into the coating. "As the coating is resorbed, it releases over time locally in the defect site and then we get both those healing qualities." Screw gets resorbed over time. Started with Smith & Nephew (partner) bone screws. Screws are bioresorbable polymer material; Bill's group set about coating them. Used a widely used, published process; is a room-temperature process. Incubate resorbable materials in mSBF (modified simulated body fluid). As they're incubated, a calcium phosphate mineral forms, forming a coating on the materials. "They key here is that it's a room-temperature process so we can then incorporate biologics into the process without blowing them apart. Typically when you make a coating-particularly a ceramic coating on an orthopedic implant-it's done at very high temperatures that would really deactivate essentially any biological things that need to be incorporated." Can do this easily because there are not high temperatures required, like with ceramic coatings. If you look at the surface of one of the coatings under electron microscope, it looks like polymer; zooming in, there's a platelike mineral that's formed which is similar in structure and composition to bone. "We can control the properties of these coatings." Can also form on thin films of resorbable polymer materials. Next step in the process was to incoroprate a drug and release it from these coatings. Chose BMP2. Incorporated full-length BMP2 protein into these coatings, and release them over time (about a month); they released in an almost linear fashion (gradually over time). "So, quite a nice release profile and sort of what we were hoping for." Important for them to check does the protein remain bioactive? They cultured stem cells and looked at effect of protein on their differentiation; they show that when stem cells are exposed to the BMP2 incorporated into these coatings, they show an enhanced level of alkaline phosophase (?) activity--a hallmark of bone differentiation. Then they placed the materials into a sheep model of ACL reconstruction; sheep model is nice because size scale is similar to human implants. Looking at sheep tibia, can see bone tunnel drilled and a metal interference screw incorporated into it. Then in another image, theirs, same type of tunnel but with their screw pinning the tendon in place. These animal studies are underway to look at the effects of these coatings on healings-to look at both mechanics and ability of the tendons to stay in place, and the healing process adjacent to material. Commmercialization outcomes: First, research contract with smith&nephew, a key player in bioresorbable fixation products market with about 12.5 percent market share. Indirectly, Bill founded a startup a couple of years ago called Tissue Regeneration Systems; using similar kinds of technologies in spine applications. Another direction they've been heading with Coulter funding: They would like to be able to incorporate anything they want into a coating. To do this, they need a "tag," a localized protein. Looked at a natural protein that binds very strongly to the minerals that they grow, hydroxyapatite minerals. The protein is osteocalcin, binding to matrix. High affinity interaction. Also have an engineered growth factor they can derive from BMP2, a peptide sequence that retains much of the activity of BMP2. These two components, the tag that incorporates molecule into a mineral, and the active portion-they essentially paste them together, synthesize it into a new molecule that they can incorporate into these coatings. The proof of concept is a broad-based technology. Their coatings: In fluorescent micrographs of molecules bound to their coatings (they engineered a series of peptide sequences that can interact strongly or weakly with their hydroxyapatite materials). If they increase affinity of molecule for coating, have increasingly high fluorescence in cases of high levels of binding. Really interesting: Actually engineered a molecule that doesn't come off the surface. "The affinity is so strong that it's essentially linked to the surface." Applications in orthopedics area, like paint a growth factor on the surface of a material. Could dip-coat or paint-coat any orthopedic implant that has hydroxyapatite incorporated into it, which is a large number of implants. "The molecule we engineered to bind to the surface remains active, so it remains able to push stem cells to differentiate into bone cells, so we maintain the activity of the molecule." Dave Beebe: Collabs Carol Diamond (peds) and Ben Moga: Non-electric disposable drug delivery device for hemophilia. Problem: Increase in the development and use of large-molecule drugs much more rapidly than the rate at which small-molecule drugs are being used. Need for sustained delivery. Large molecule drugs can't easily be taken orally, like in pills. "So there is and will continue to be a growing need to the ability to deliver drugs cheaply and safely through the skin to avoid GI tract problems. And particularly, there's a need for sustained delivery." Current status of that is a simple inexpensive syringe or expensive ($5K) catheter-based infusion devices. Carol via video: Hemophilia: Inherited disorder in which the blood fails to clot normally because of a deficiency or abnormality or one of the clotting factors. Manifest by spontaneous, severe bleeding, particularly at the joints. If this happens, they need an infusion of factor VIII or factor IX. Administration of factors is challenging, particularly for small children. Example, young child is outside and starts crying and parents notice he has a red, hot swollen knee. The parents have the factor at home. But they have to go to the ER and wait for two hours to be seen. But then the ER staff have to mix the factor, find a vein and now it's four, five and six hours later. Child is traumatized, family is traumatized, staff is traumatized and lots of resources have been used. Patients go through experiences like this two or three times before they decide the want an in-dwelling catheter placed. It's a surgical procedure that requires anethesia and it does invite infection. "What's so exciting about this drug-delivery device (Beebe's), is that first of all, it'll allow a family to administer a drug immediately, in their home, safely. It is something that can be used in a prophylactic manner-so, at regular intervals, preventively for a child. It's something even for an older child-if they knew they were going to play in a rigorous task like a soccer game, they could infuse at home with this device, without intervenous access, without an in-dwelling catheter. This will dramatically improve the patient's quality of life." Now Dave: Worldwide factor VIII (eight) market $1.7 billion. Factor VIII deficiency; during an bleeding episode, it takes lots of time and people to inject factor. Other alternative is in-dwelling device. Dave; Administer drug immediately, safely in their palm. Dave's solution: Cross between drug pum? and patch, can be capable of sustained delivery, disposable, about size of a poker chip. "It can be configured to be microneedles or a single needle can be used." Goal was cheap and disposable, so stay away from traditional engineering with electronics, pumps and valves, etc. Basic technology: A number of years ago, we developed this method of using stimuli-responsive hydrogels to make, for example, valves. Hydrogels change their shape in response to temperature or pH changes. A former student of his capitalized on this to make a non-electronic pump. "It's a membrane-based device that has a flexible membrane, a drum reservoir, if you will, on the bottom and the top we have these swellable hydrogels. So then just by triggering the response of these hydrogels by exposing them to a change in pH, for example, in the solution, would then expand the volume of these hydrogels and simply push the drug on top. The nice thing about this is that by controlling the chemistry and the geometry of the gels we can control what the profile of delivery is like, whether it happens in five minutes or 24 hours. In theory, we can actually make specific profiles." "So this is a platform technology." They've demonstrated that this can be used for closed-loop delivery, which is really the holy grail of drug delivery. They're starting with an open-loop system. There's not much in the low-cost sustained delivery market-the space they're interested in capturing. His competition is mini transdermal pumps, IV injection, infusion pumps, needle/syringe, needleless injections, inhalers. Would position in the mini transddermal pumps market. Summary: It's cheap, disposable, sustained delivery device, initially designed to treat hemophilia episodes. "Again, there's a growing market for devices that can use sustained delivery on large-protein drugs, so we envision that this might be technology licensed widely to pharmaceutical companies." Status: Benchtop prototype testing is ongoing; animal (pigs) baseline data is complete. An animal study will begin this summer on a bolus delivery prototype. Next step would be to design chemistry and geometry for sustained delivery device. Then they could license, partner with pharmaceutical company or use as startup. Copyright 2007 The Board of Regents of the University of Wisconsin System Last modified: 26 July 2007. Created: 26 July 2007.