Chris Brodie, PhD discusses how Low Intensity Pulse ultrasound works on a molecular level. Dr Brodie shows it can be successfully used as an adjunct to bone healing in patients who would otherwise have difficulty healing.
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Release Date: 03/16/2018 Expiration Date: 12/31/2018
Chris Brodie, PhD
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Male Speaker: Our next speaker is Dr. Chris Brodie, who has a PhD in molecular biology from the University of Minnesota, postdoctorate degree at Duke. He is a full time scientist for Bioventus. He is going to be talking about Low-Intensity Pulse Ultrasound, How Does It Work. So please welcome, Dr. Brodie. Here he is.
Chris Brodie: Thanks. Yes. I’m not a clinician. I’m going to ask you to bear with me while I take a little bit of a deeper dive into some of the mechanisms behind the technology that actually Dr. Dr. Schoenhaus talked about yesterday afternoon, which is the use of ultrasound as an adjunct treatment for bone healing. I’m going to start with my disclosures. I am an employee of a company that manufactures a device that emits this LIPUS signal, this low-intensity pulse ultrasound signal. But I have a couple of important learning objectives that I hope you’ll take away from this morning. I hope you’ll understand how the fact that we’re using ultrasound, it affects how you use the device in your practice. Know how the mediators of this response are actually affecting the clinical presentation and therefore guiding how you’re going to use it in patients. There are several aspects of fracture healing that are impacted by the use of this device. Then, lastly, I hope that you’ll take away with this some ability to discuss the mechanistic basis. In other words, how does it work? How do we think that this low-intensity ultrasound is actually changing the clinical course of a patient with a difficult fracture? In the US, this device, this EXOGEN device has two primary indications. It’s the only device or drug that’s indicated for the acceleration of fracture healing. Right now, if you want to make a fracture heal more quickly, this is the only FDA-approved way to do it. It’s also indicated for the treatment of established nonunions for all bones except skull and spine. In terms of safety, there are no known contraindications for this device, which is good. There are certain patient populations that we didn’t study though and those are listed here. I’m not going to talk about the clinical data today. But I am going to mention them as an overview because it provides context for the mechanistic discussion. For the two types of indicated acute fractures, the heal rate in level one studies that we’ve seen is 38% faster than comparison treatment with a sham device, the placebo device. That’s for radiographic and clinical healing and that’s pretty meaningful. For the established nonunions, these include atrophic, oligotrophic, hypertrophic nonunions, we see an 86% heal rate overall. Again, I’m not going to go into the clinical data but this is setting up the clinical relevance of this therapy. I hope now you’ll take a dive with me down in the mechanism. So how does it work? This is a low-intensity ultrasound signal and ultrasound is just a high frequency sound wave that you can’t hear. But it’s a pressure wave. It’s a physical wave, it’s not electromagnetic wave. Because of that, the transducer has to touch the skin. This is a treatment that patients use at home. It’s 20 minutes once a day. When we say low intensity, we’re comparing it to a physiotherapy ultrasound and I know that many of you work with physios in your practice. They use a form of therapeutic ultrasound that’s much higher intensity. As a matter of fact, it heats up the tissue. It’s how its thoughts will work. By heating up the tissue, you’re promoting local blood flow. Therefore, you’re getting improved healing. This is a low-intensity signal. It’s about two orders of magnitude, less intense in the physio ultrasound. There’s no thermal component to it. it doesn’t heat up the tissue. It’s noncavitating. There’s no effect on screw torque and this is the reason why it’s compatible with all forms of fixation. The one thing that we’ll say though is that you can’t treat through metal so it doesn’t go through the metal. So if you have a patient with a plated fracture, you need to aim the ultrasound from some other aspect of the limb. The signal actually penetrates soft tissue very effectively. You can see once it gets in to the dense cortical bone, it transmits efficiently. The depth of penetration is substantial so particularly working in the foot and ankle, that’s never going to be a consideration for you. However, the width of the signal is relevant. So you should understand that with little overlying soft tissue coverage, the effective treatment area is not much larger than the size of the transducer itself.
But to answer this question of how it works, we first collaborated with a group at the Mayo Clinic to try to actually measure this physical motion caused by this physical sound wave. This is an osteotomy on a cadaver arm. We bounced a laser beam off of one side of the bone, turn on the ultrasound and then watch the shift in the laser frequency, the Doppler shift. That can infer a physical dimension of movement. What we found is that contrary to one of the early hypotheses about how ultrasound is working, it’s not Wolff’s law. It’s too small. So Wolff’s law premises motion, micro motion. This is nanomotion. It’s less than a nanometer of displacement within mineralized tissues, which is a lot smaller than we thought. Given that, how are cells detecting this motion because this is much smaller than an individual cell. So, we went looking for a cell surface receptor. This is what we found. This is a CartoDEM, an integrin receptor sits on a cell surface. It binds the extracellular matrix. When it gets activated by the ultrasound, it responds by clumping together to form a focal adhesion. That’s what they call these structures. You can actually watch this process happen in real time. So, in an unstimulated osteoblast, you see the green lines around the cell, these are stress fibers. These are actin filaments. The green label is actin in this image. At the corners of these cells, you can see these orange streaks, those are the focal adhesions. If you treat this osteoblast with ultrasound, you see not only an increase in focal adhesion formation but a reorganization of the actin cytoskeleton so now the cell looks like this. So this is a demonstration of one of the biological effects of using this ultrasound. Downstream of this change is a lot of different signal transduction cascades. We’ve mapped these out. We’ve shown that they culminate in a transcription factor binding to the promoter region of a gene that encodes the COX-2 enzyme. COX-2 is this enzyme that produces prostaglandin E2. We heard about PGE2 yesterday. It’s a key element in fracture repair. We know that it’s essential for fracture healing. If you eliminate COX-2 in an animal model, you get impaired healing. You get the same thing in a patient if you put them on NSAIDS. Okay, you slow down the healing process. Now, we know that in animals, the response is stronger, but it’s really profound. Here in this histology image, so the bone is colored, this orangeous [phonetic] red, this connective tissue is colored blue. You can see compared with the group that got the COX-2 inhibitor, you just have much more profound healing in the absence of this COX-2 inhibitor. So we know it’s important and we know furthermore that one of the theories about why older fractures don’t heal as well is because they have reduced expression of COX-2. In other words, that early inflammatory phase of fracture repair is impaired in older people, in older animals as well. The effect of ultrasound on a fracture is not massive. This is not a sledgehammer therapy. You can see that the peak callus volume is actually not greater under ultrasound treatment than it is under a sham treatment. The peak bone mineral density is actually no greater. But you’re getting there faster in these animal models. So that’s what you see here. The area between the orange lines is unmineralized callus. So you’re just accelerating this process. If you look at the callus, genes that are being expressed, you see upregulation not just of one or two but of many that are involved in various process of repair. So ostrix and Cbfa1 are involved in the differentiation of stem cells than on osteogenic lineage as opposed to a chondrogenic or lipogenic lineage. We also see increases in several BMPs. Not just BMP2, which is when you use it in the form of infuse, it’s millions of fold greater than the endogenous concentration. Here, it’s about eight to 12 fold higher than the indigenous concentration. We also see increases in growth factor. So FGF2 and VEGF, vascular endothelial growth factor, are both essential for driving the revascularization of bone. As speakers have mentioned previously at the conference, every fracture is associated with a little bit of necrosis. So every fracture is associated with a little bit of a requirement to revascularize. The extent to which you can do that effectively is a metric that shows how effective your healing is going to be.
We also see an increase in alkaline phosphatase in collagen 10. So we have a lot of different molecules, and cells, and processes that are enhanced by this ultrasound. I’ve talked about the gene expression studies. Integrants are on a number of different cell types and we’ve demonstrated an effect of ultrasound on these cell types. Then there are these key processes in the overall process of fracture repair that are upregulated or augmented using the ultrasound. I want to focus on three of these, angiogenesis, progenitor cell recruitment and mineralization. The first is angiogenesis. Now, this work was done by a foot and ankle ortho here in New Jersey named Sheldon Lin. What he was studying is in a diabetic animal model of fracture repair, what’s the relationship between expression of this growth factor, VEGF, the ability to regrow new blood vessels and can you impact these two variables with or without ultrasound. What he found is for diabetic animals relative to nondiabetic controls, the level of VEGF growth factor and the number of new blood vessels in the fracture callus were both about cut in half. But if you then treated those animals with the ultrasound, he was able to normalize the levels of the growth factor, the number of new blood vessels and also the rate of healing. In this case, even though we saw this morning that diabetes is really complicated, you guys are seeing these complications all the time. In terms of fracture repair, you get a snapshot of what’s wrong and how to fix it just by looking at this ability to revascularize in this animal model. He went on to further show in another study that you can visualize this increased vasculature by using Power Doppler imaging. And these changes are more profound earlier on in the fracture healing than later on. So day seven, you see a big difference between LIPUS treated and control treated, less of a difference by day 11. The chart showing those differences is here. And so, angiogenesis is one of the parameters, one of the processes that is augmented with ultrasound. The second one is progenitor cell recruitment. The best way of studying this was actually a paper that came out a couple years ago and it’s both pretty cool and pretty gross, because it uses something called a parabiotic mouse model. And what you do there is you take two mice that are genetically identical, except one of them carries a gene that constitutively expresses green fluorescent protein in all of its cells and tissues. You couple their circulatory systems together and then induce a fracture in the wild type animal. You can treat it either with ultrasound or with a sham device. What they found is that in the animals that were treated with the ultrasound, they got a lot more of these green fluorescent cells that colonize the fracture area. So you’re looking at two columns here. One is labeled DAPI, that’s just a cell stain for the nuclei. That just shows you where the cells are. The other is GFP. That shows you where the cells are that came from the partner in that parabiotic couple that expresses GFP. In other words, as a result of ultrasound treatment, you’re able to pull down more of these circulating cells into the fracture site. We could see again that early on, you see the most profound changes less so later on. Furthermore, this increase in GFP positive progenitors was associated with an increase in alkaline phosphatase. You’re pulling down more of these circulating cells, and as a result, you’re getting more expression of the enzyme that actually makes bone. Second thing I want to talk about with mineralization and this was in effect of this alkaline phosphatase expression, they went on to show similar effect in a pair of studies that came out with very similar results. So, both of them using human cells, one periosteal cells and the other cells taken from a hematoma. These are primary human cells in vitro. They found very similar results. Relative to unstimulated cells, cells that were stimulated with the ultrasound showed much greater mineralization in vitro. The net result of this, you can see in a study by Theresa Freeman who’s actually based in Philadelphia, Thomas Jefferson. You can see on the control side, this is a micro-CT. You can see it’s mineralizing well but you’ve got none of those four cortices that have been fully bridged. Whereas on the LIPUS treated side, you’ve got bridging of two, maybe three cortices. Furthermore, you got enhanced screen modeling, particularly of that distal segment.
These are images that are thresholded based on the density of the bone. So the original dense cortical bone appears red, the newly deposited trabecular bone appears green. You can see that not only is mineralization enhanced but remodeling is enhanced, you’re just progressing to a completed healing stage much faster. The remodeling is particularly important when you posit the use of ultrasound for the treatment of nonunion, particularly atrophic nonunions. If you’re going to say that you can use a non-invasive stimulus to treat an atrophic nonunion, you got to somehow explain how you’re able to get rid of that dead and sclerotic bone. The fact that we know that osteoclasts have integrin receptors that they respond to ultrasound and that they show increased TRAP staining. So the enzyme that actually degrades bone and remodels bone in the presence of ultrasound helps explain that finding. Last thing I want to say about mineralization is that the bottom line here is that you’re getting greater biomechanical strength. This was shown in a study where they took four groups of animals. They’re trying to find out when is the right time to initiate ultrasound treatment. The first group of animals was treated during the first week then left alone until they were euthanized about three and a half weeks later. Second group of animals treated just during the second week. Third group of animals treated just during the third week. Then the last group of animals was treated throughout the healing process. What they found is that all the animals had higher biomechanical strength relative to controls. But the greatest increase in biomechanical strength was seen when they initiated treatment at the start right after the fracture was induced and continued it until they’ve acquired full radiographic healing. This is demonstration that this enhanced mineralization is actually also giving you enhanced biomechanical strength. Here’s our proposed mechanism of action. We believe that the ultrasound comes through activates integrin receptors leading to a clustering that sends a signal to the nucleus, that gives us a pulse of COX-2 that produces prostaglandin E2 that is acting in an autocrine and a paracrine fashion. We know that it’s binding to the EP24 receptor. That kicks off a second wave of gene expression and that’s where you get the increases in growth factors, BMPs, alkaline phosphatase and all the rest. But, one of the knocks about ultrasound historically has been, well, there’s not enough evidence about how it works. So we propose to test this mechanism of action. We do that by or we did that by knocking out the COX to gene. Now remember that I said that we’ve mapped out these signal transduction processes all the way to the promoter region of the COX-2 gene. If we’re right and COX-2 is essential for this process, if we’re right about the mechanism of action, then knocking out COX-2 should make all the rest of the processes go away. So this is the test that we undertook. In normal mice, in wild type mouse, relative to an untreated control or LIPUS-treated animal, you see much faster healing. So the area between the black lines, again, is unmineralized callus. When we compared that to an animal that had a full knockout of the COX-2 gene, first of all, they heal more slowly. Second of all, there’s no effect to the LIPUS device. In other words, in the absence of COX-2, there does not seem to be an effect of the ultrasound. When we look in the callus at all these factors that had increased previously, we found that they all remain baseline. What's interesting however is that a full knockout of the COX-2 gene is not what we have in patients that are on NSAIDS. So they’ve done the studies as well where you look at animals that have one functional COX-2 gene. In other words, they have half the amount. Instead of two, they’ve only got one copy. They heal more slowly but ultrasound can still6 augment that healing process. Same results as if you take a wild type animal, put them on NSAIDS, remember I showed that histology slide where they were healing much more slowly but you can rescue that slow healing deficit in the presence of ultrasound. How does LIPUS work? To conclude here, we believe that it involves a physical displacement that is on the order of nanometer, so extremely small. We think it involves integrants and focal adhesions. We believe it upregulates COX-2, BMPs, VEGF and other factors that are critical for repair. We’ve seen enhancement of these key processes. The ones that seemed to be most relevant in a clinical sense are angiogenesis, progenitor cell recruitment and mineralization. The mechanism of action supports the clinical findings.
I mean, this is sort of the take home message and the reason why I, as a scientist, in coming out to talk to you guys is that understanding how it works at a mechanistic level helps you identify which patients are the ones that are most likely to get a benefit of using the device. It’s not your healthy patients. They may heal faster, most likely will heal faster. But the medical necessity is in the presence of patients that are compromised for healing. That’s where the real sweet spot is and that’s where I think the greatest effect of ultrasound lies in a clinical sense. The talk that I gave normally takes about 45 minutes. I apologize if it was rushed tonight or this morning. We've a ton of collaborators around the world, so I want to thank you for your attention very much.