Board Review Wound Care

Confronting Complexity: The Role of HC-HA/PTX3 in Orchestrating Regenerative Healing

Eric Martin, DO, MS

Eric Martin, DO, MS discusses the concept of fetal scarless healing and how it can be translated into healing of a chronic wound. Dr Martin also discusses the role of HC-HA/PTX3 and the use of umbilical cord/amniotic membrane to achieve wound healing.

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Goals and Objectives
  1. Discuss the concept of fetal scarless healing in the adult model and understand the effects of how amniotic membrane allograft and umbilical cord contribute to orchestrating a regenerative homeostatic repariation process of the chronic wound
  2. Understand the basic science of regenerative wound healing using amniotic membrane allograft-orchestrating a regenerative homeostatic environment
  3. Understand the basic science of the chronic wound and how it is differentiated from the acute wound
  4. Understand the effects of heavy chain hyaluronic acid pentraxin 3 and its ability to reduce inflammation in the chronic wound and stimulate the proliferative phase through immunomodulation
  5. Understand the concept of fetal scarless healing and how that can be translated to healing in the adult environment
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  • CPME (Credits: 0.75)

    PRESENT eLearning Systems, LLC is approved by the Council on Podiatric Medical Education as a provider of continuing education in podiatric medicine.

    PRESENT eLearning Systems, LLC has approved this activity for a maximum of 0.75 continuing education contact hours.

    Release Date: 03/16/2018 Expiration Date: 12/31/2018

  • Author
  • Eric Martin, DO, MS

    COL, MC, USA
    Chief, Department of Vascular Surgery
    Chief, Department of Wound Care Medicine
    DDEAMC Fort Gordon, GA

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    Eric Martin has nothing to disclose.

  • Lecture Transcript
  • Robert Frykberg: Be right on time. We have a new speaker to Desert Foot who’s going to be talking on the use of amniotic membrane again but also with umbilical cord. The title is Confronting Complexity, which we all deal with, Orchestrating Regenerative Healing with the Amniotic Membrane and Umbilical Cord. For this we’ve asked Dr. Eric Martin to come to speak. Dr. Martin got his DO degree from the University of Michigan, School of Osteopathic Medicine. And is now the chairman of the Wound Care Clinic, I believe, Department at the Dwight D. Eisenhower Medical Center and around Augusta, Georgia. Okay, so let’s welcome Dr. Martin.


    Robert Frykberg: Okay.


    Eric Martin: Good morning. Thank you Dr. Frykberg for allowing me to address the audience here at Desert Foot. As Dr. Frykberg said, my name is Eric Martin and I’m an active duty military surgeon. I have no financial interest, my one disclosure. Here are the four learning objectives we’re going to be talking about today. The first is to understand the basic science of agenda of wound healing using amniotic membrane allograft specifically umbilical cord and orchestrating a regenerative homeostatic environment. Second would be to understand the basic science of chronic wounds and how it’s differentiated from the acute wound. Thirdly, to understand the effect of heavy chain-hyaluronic acid pentraxin 3 and its ability to modulate the inflammatory response within a chronic wound. And then five to understand the concept of what it’s meant by fetal scarless healing. I’m from Augusta, Georgia. Not much really happens in Augusta except for best the one week out of the year. And for those of you who are avid golf fans, you know that we’re kind of famous for the Masters for first week in April. I also work at a hospital called Eisenhower Army Medical Center in which I head up the Department of Vascular Surgery, and about six months ago I got put in charge of the Department of Wound Care. Like most surgeons, during my general surgery training, I had very little formal training on wound care. Pretty much what I was thought was that if the wound was wet, make it dry. But if the wound was dry, find a way to make it wet, more like maybe make it moist. Something about wound vacs, black sponges, white sponges, granulation tissue. And always don’t forget about good surgical debridement. About two years ago during one of our journal clubs with our residents, I was introduced to regenerative medicine and my life has kind of changed since then. Today, I’m going to be talking a little bit about terms such as cellular apoptosis, immunomodulation, biologically bankrupt wounds, and orchestrating regenerative homeostasis. For some of us in the audience who may not be familiar of this, you may ask where does this all begun? For those of us who are Judeo-Christians, it begun at the Old Testament with the book of Genesis. Adam, Eve, the Garden of Eden, a snake and oh yes, that forbidden fruit, the apple, and the subsequent fall from grace. For those of us in regenerative medicine the picture is a little bit different, it begins with fetal scarless healing. Understand that that’s the heart of regenerative medicine, and basically that fetal tissue as we know decrease inflammation in the absence of scar formation. Basically, what we know is that amniotic membrane and umbilical cord is derived from fetal tissue. That these have unique biological characteristics and they also serve to immunomodulate the inflammatory response within the chronic wound. What we know about this tissue that it has innate regenerative capabilities, both that can be preserved as well as transplanted. When we talk about fetal scarless healing, the father of fetal scarless healing known as Michael Harrison published this report back in 2011 a case study that he did in which he explanted a fetus 26 weeks gestation and resected a large neck mass. He then took the fetus, transplanted it back in utero. And what you can see here is the result four months and 18 months post gestation. What this kind of signifies is the regenerative powers of a fetal tissue and the ability to heal in almost a scarless environment. When we talk about the generative properties of fetal tissue, another example would be looking at a sheep fetus model. Here you can see on the very top to the right, this is an example of feet of sheep skin allograft that was used, and in the left side, we look at adult sheep skin allograft. And what you can see is that when the sheep was delivered post gestation, what you can see is that on the right side here, you can see that there’s almost healed scarlessly compared to the left side where you see evidence of some scar and fibrosis where the adult sheep skin allograft was used.


    And we’ve talked a lot about amniotic membrane and we know that it’s the innermost lining of the amniotic sac. We know that it also lines the outside of the umbilical cord. If you take a look below which you’ll see histologically is that there’s a lot of similarity between the umbilical cord as well as the amniotic membrane except the umbilical cord is just much thicker, keeping in mind that the umbilical cord serves as the conduit between mother and fetus. These next two slides are crucial. If there’s nothing else you take home from my talk, I would hope that this would be one of the key slides. This is kind of the meat and potatoes of what I’m going to talk about, which is understanding the biology and the pathogenesis of the chronic wound. Realize that these wounds are biologically bankrupt. And if we look at this as a disease process and then treat this fairly aggressively with multiple modalities, we have a very good chance of healing these wounds. What we know, first of all, that these have a prolonged inflammatory phase. Compared to the acute wound that goes through the inflammatory phase, proliferative phase or modelling, pretty much free. We also know that there’s defective remodeling in the extracellular metrics. Such things as glycosaminoglycans, proteoglycans, hyaluronic acid, all become depleted. These wounds have a tendency to fail to reepithelialize along with decreased growth factors. We also know that there’s impaired dermal fibroblast, endothelial cells and keratinocyte proliferation, which is going to affect the amount of collagen synthesis within the wound bed, angiogenesis as well as reepithelialization. We know also that there’s an increase in foreign inflammatory cytokines, IL-6, TNF-alpha for example and that we have increased levels of these matrix metalloproteinases that further degrade the structural integrity of the wound. The final point I really want to illustrate to everybody, I know that it’s been talked about by other speakers is the importance of understanding about the bioburden of the chronic wound, and this was a lesson that I had to learn too. Unlike most of the speakers up here, I’ve really only been in wound care for about a year and a half, two years at most. It was a very steep learning curve from me when talking some of these more chronic complex wounds. And what is so important is to make sure that we do very good debridement both with sharp debridement. I also happen to use ultrasonic debridement as well in addressing that biofilm that is present, the glycocalyx that forms within these wounds that kind of serves as the force field that makes these bacteria more recalcitrant to just dozing with oral systemic or parenteral antimicrobial therapy. Taking a look a little bit more at the inflammatory phase which is what I talked about initially, we know that inflammation occurs. It’s a natural process within any wound. And we know that there are some proinflammatory cytokines that are presents such as TNF-alpha IL-1, IL-6. We also know that within the first 24 hours, neutrophils are the predominant white blood cell that infiltrate into wound bed. We didn’t know that within the next 48 hours, the next 24 to 48 hours, the macrophage comes in and helps to engulf and clean up the wound bed. The problem that occurs is that in this chronic wound, this inflammatory phase becomes prolonged. There’s a perturbation that occurs within this process. And what ends up happening is that these cells become dysfunctional. What happens is that, the wound becomes infiltrated with neutrophils and there’s excessive phagocytic activity within the chronic wounds. The consequence of this is that this leads to an increase in debris and exudate within the wound bed. That macrophage which typically is going to kind of the service like the bulldozer to help clean up the wound and help move the wound onto the proliferative phase, that also becomes dysfunctional. And so what happens is this macrophage within the chronic wound essence becomes senescent. Now we have to tackle this problem, how are we going to modulate this inflammatory response? The way we could do this is through the use of the umbilical cord amniotic membrane. And when this has been placed into wounds, this is what has been shown clinically. What we know is that it helps to inhibit further infiltration of neutrophils and it engages in cellular apoptosis or program cell of the already existing PMNs within the wound bed. Secondly, what it does too is that it causes phenotypic polarization of the macrophage such that the M1 macrophage that now is senescent within the wound bed turns out and becomes an M2 macrophage which allows the wound bed to start clearing out the debris and move on to proliferative phase. The question in so many masters is what’s so unique about the umbilical cord? Well what has been found within the umbilical cord is something known as the specific effector protein. Heavy chain-hyaluronic acid/PTX3 or something called pentraxin 3. And it’s this specific protein found within the umbilical cord in abundance compared with just amniotic membrane layer alone that allows for these events to occur that helps to down regulate, if you will, this inflammatory process.


    This slide is a little bit busy. But basically what it ends up showing is that when the heavy chain-hyaluronic acid pentraxin 3 is placed into wound beds, there’s an increased level of both PMN and M1 macrophage cellular apoptosis. On the right of the slides if you look, when heavy chain-hyaluronic acid pentraxin 3 is placed into a wound bed, what you can see is that there is a paucity of key lymphocytes that are present compared to hyaluronic acid alone or the control. The bottom two graphs, B and C, what they also show then is that there’s also immunomodulation of proinflammatory cytokines such as interferon gamma and IL-2 by heavy chain-hyaluronic acid pentraxin 3. We also know that in addition to its immunomodulatory effects, it also has effects on changing the inflammatory environment within a wound bed, such that it up regulates anti-inflammatory cytokines such as IL-10 compared to hyaluronic acid or control, and it down regulates proinflammatory cytokines such as IL-12 and IL-23. Alright, so some of you maybe asking, what is this heavy chain-hyaluronic acid pentraxin 3? What we know is that hyaluronic acid is derived and contained within the amniotic membrane matrix. The heavy chain comes from inter-alpha inhibitor in the amniotic matrix, and pentraxin 3 is an aggregating protein from the amniotic matrix. Basically the specific direct effects this effector protein is threefold. One, again, it down regulates proinflammatory cytokines, it helps to increase program cell death of PMNs and it decreases cellular infiltration of both PMNs as well as M1 macrophages. The end result of this is that it’s going to decrease information modulating these effects. It’s going to help to enhance regenerative healing and suppress scar formation and fibrosis. This is just the chart taking a comparison of the heavy chain-hyaluronic acid pentraxin 3 from umbilical cord, comparing it to hyaluronic acid alone. And if you look very clearly you can see that there are a lot of very positive effects when heavy chain hyaluronic acid is placed into a wound bed compared to just hyaluronic acid alone. It is very important to understand too that process is product. And it’s the cryopreservation process that use and helps preserve the viable state at extremely low temperatures of this tissue. And the significant advantage is that it’s going to preserve the viability as well as decreased tissue damage and preserve most importantly the effector proteins and molecules within this umbilical cord amniotic membrane. If you take a look below here at this example using a steak as a metaphor, if you were to take a steak and freeze it, put it in the freezer, we thought out two months later, it’s still a steak, looks like a steak and will taste like a steak. If we’re to take that same steak and if you would dehydrate it, make it into jerky, you see that it completely changes its morphology. And what the concern is here is preserving these effector proteins that are present, and again this is cryopreservation process that seems to do this. Alright, so this is just kind of a cartoon picture of looking at what is this heavy chain-hyaluronic acid pentraxin 3. Basically, if you look at the little red flowers that’s kind of like the pentraxin 3. The branches, if you will, the black, that’s the hyaluronic acid. The blue leaves, these are the heavy chains. And what we know, again through this cryopreservation process is that we want to increase the patency and the longevity of this product within the wound bed. And basically, we want to maintain the structural integrity and the productivity of this heavy chain-hyaluronic acid pentraxin 3. Alright, I’m not going to go over several case studies, talk about three of them and highlight a little bit about the chronic wounds and what I’m healing. The first person that came to me was a 46-years-old active duty Navy Seal who worked at Fort Bragg, North Carolina, he was actually involved in the bin Laden raid several years prior. He’s no longer in operative with the team. He now serves in a support role. He had recalcitrant onychomycosis of the right first digit and after he failed the oral systemic therapy with an antifungal, he had his nail removed. Coincidentally, he had been complaining of a six-month history of bilateral calf cramping when running, consistent with what would be a vasculogenic claudication picture. His wife was also a very active and involved in martial arts and was a Robertson instructor and they would often go out and runs together. And he was subsequently falling behind her when they were going out for 5 and 10K runs.


    He had no risk factors for PAD other than nicotine dependence. No diabetes, no hypertension, no hyperlipidemia. Initial imaging studies that were performed was a CT angiogram, and what it showed was bilateral thrombotic occlusion of his tibioperoneal vessels along with the popliteal artery behind his knees as well as the infrageniculate popliteal artery. He was initially seen, like I said, at Fort Bragg where the vascular surgeon there had recommended amputation of his right leg. He was devastated by this, being 46 years old and a Navy Seal, otherwise, very, very healthy male. He was referred to a second opinion. The initial diagnosis that the surgeon had was that he had advanced atherosclerosis. When I had seen this gentleman, something really did not fit right with that picture. And after further workup and assessment, what I had diagnosed him with was a condition known as thromboangiitis obliterans or Buerger’s disease. For those of you in the audience who may not be familiar with this disease or something you may have read about at medical school, basically this is a non-atherosclerotic, proinflammatory, prothrombotic vasculitis that affects arteries as well as veins. And it can often result in distal ischemia that can result in tissue loss or gangrene. The treatment for this, there’s no real good medical treatment. There are no real good surgical options for this. But basically, the mainstay of therapy is cessation of all nicotine products. We obtain the CTA, we’ll take a look at this. Then, we’ll take a look also at his digital subtraction angiogram. So you can see here in the middle of the aorta, the bifurcation to the common iliacs, bifurcation to the hypos, and the externals now into the common femoral. And all that anatomy looks pretty good. If we can run that one more time please, that would be great. Again, focusing on right here, you can see his aorta, widely patent, very healthy. Common iliac artery is right here. External and hypogastric is all normal, common femoral artery, bifurcation to the SFA and profunda. Everything so far are looking great. So then I took him and I took him to our angio suite and performed a percutaneous intervention, catheter-based intervention, and did a right lower extremity arteriogram. You can see here that abrupt occlusion of the above knee popliteal artery just across Hunter’s canal. No main vessels really below the reconstituted peroneal, which has diffused collaterals. And this is his lateral foot shot. And again, if you can also run through this one more time that’d be great. So again, SFA, profunda, SFA stops right there. Again, collateral is coming off. Here, again, just robust colletarals. There’s the reconstituted peroneal. AT and PT are occluded. Again, maybe flow here through a plantar vessel but not direct inline flow with the AT, I’m sorry, with the PT or the AT. I made the decision not to bypass him. The reason why is because the only vessel that I can see as a good list of target was his peroneal. And as we all know, that doesn’t provide inline flow to the foot. I do believe in angiosomes and that would not actually provide direct supply to that area. He did not want a big surgery with the resection of his fibula in order to a distal bypass. As a result of this, I opted for another treatment option and this was what was presented to me. You can see that he had significant infection. I also did TCOMs on him. And at the region of his foot, his TcO2 was 30 millimeters of mercury. With an O2 stimulation challenge, he rose to about 100. I did decide to also treat him with hyperbaric oxygen. But concomitantly, I also had to address this infection. Maybe you can get an appreciation here of this significant forefoot edema. This had been going on for approximately two, two-and-a-half months before he made it to my attention. This here, people thought was actually exposed nail. This actually was distal phalanx right here. What I ended up doing is taking him to surgery and ended up resecting out the distal phalanx and doing a significant debridement. What I ended up doing next then was taking a burrito wrap of this umbilical cord, amniotic membrane and wrapped it circumferentially around the toe. And then I also injected a combination of DHA-CaM and Amniox Flo [Phonetic] into the soft tissues. What ended up happening two weeks later was you can see, a significant improvement here, looking at this picture. We then re-debrided him again. Put on a combination of both umbilical cord as well as DHA-CaM, and basically you can see the improvement here. And this was about three weeks with him completing his chamber dives. I then turned him over to my wound care nurses.


    They further treated him with some collagen-based products like Prisma. He eventually closed and returned back to act of duty and jump status without any problems. This was another case that I had, a 62-year-old female who underwent a prophylactic bilateral mastectomy. She had no history of breast cancer. However, she did have a family history of a sister having breast cancer. Her genetic screening came back positive, being BRCA2. Her wound complications following her mastectomy was that she developed bilateral skin dehiscence, bilateral [Indecipherable] [20:32], myonecrosis of the right pectoralis major muscle, and she had significant purulence and exudation from the wound bed when it finally made it to my attention about 6 weeks afterwards. In addition to all this, she also had an axillary cutaneous fistula from the lymphatic leak on the left side, where the resection was carried back to much into the axilla and ended up unfortunately transecting some lymph vessels. This is how she initially have presented. There was some necrotic tissue here that was resected that wasn’t evident. I took her. This is her right breast, ended up doing a resection. All of this was done actually under local anesthetic done in our clinic. She tolerated this very well. And what you can see me doing is laying down a piece of umbilical cord, amniotic membrane. In addition to this, I also used a cocktail of products and combined with DHA-CaM as well as porcine bladder xenograft and I make a paste on top. And then what I ended up doing was taking a piece of porcine bladder xenograph, which is one of their thicker sheets. I ended up kind of sealing or incasing all this product into place serving as a biological band aid. Here now, this is me resecting out the left portion of her breast. What I want to stress here is that after doing sharp debridement, both of these wounds received extensive debridement using an ultrasonic debrider prior to placing in this product. This was a partial closure down below. And then, again, I packed this wound with, again, a combination cocktail of both DHA-CaM, Amniox Flo, as well as porcine bladder xenograft and then I patched up the area. This is pretty much what she looked like after her first operation. She came back a week later. I re-operated on her again. Pretty much did the same surgery that you see here. Except this time, she allowed me to resect more of this scar tissue on this left side. She was very, very resistant to allowing me to do any extensive surgery here. The other thing that I forgot to mention that I want to stress is that in her left axilla where she had this significant axillary leak, I ended up combining porcine bladder xenograft as well as some Amniox Flo, and that ended up causing stoppage of this leak. And I used about 3 grams of the product which packed up in that left axilla. This was her then approximately three weeks after I had initially seen her on the left side. I was able to actually get finally a delayed primary closure. I’ve built up enough tissue in her wound bed. I was able to reconstruct or, if you would, regenerate or get good granulation tissue over that right pectoralis major muscle. Because when I initially saw her, what happened was that I was looking at exposed rib cage and intercostal muscle where the infection had eaten away some of her pectoral major muscle. And so what ended up happening, I was able to finally get a delayed primary closure on her. And you can see a month later when she returned to clinic, she looks a lot better. Just by accident, I happened to see her one week ago when she came and now for treatment of some of her varicose veins. And what was most impressive was that a lot of this induration and scar tissue up here was completely resolved and this had flattened out very, very nicely, and both of these look even more impressive than they did here on this film. The final wound to talk about with you all is this presacral region wound. This was a 75-year-old male with invasive bladder cancer. He underwent a cystoprostatectomy back in February of 2014. That surgery weren’t uncomplicated. He was discharged from the hospital approximately five days following this operation. And he came back four months later with what appeared to be a small bowel obstruction. He was initially treated conservatively, being made NPO, given an NG tube, Foley catheter, and correction of any electrolyte perturbations. However, after about three days of conservative care, he did not make any progression and he was taken for exploratory surgery. This postoperative course was complicated by multiple, multiple problems. He had an anastomotic leak three days after his initial resection that required him to go back to the operating room for further resection. He developed a ventral incisional hernia. He developed an enteroatmospheric fistula. He had severe visceral protein malnutrition with an albumin of 0.7.


    He also had a radial artery thrombosis from residents trying to stick an A-line in him several times. And unfortunately, when trying to place a chest tube when he was hypotensive, he developed an iatrogenic pneumothorax, requiring tube thoracostomy. The story becomes even more complicated. I initially saw this patient 10 weeks after his prolonged hospitalization, of which mostly was spent in the ICU. Because there was so much attention placed upon his abdominal wound, he initially presented to us with a stage 1 sacral wound. And over the time, and this was all admittedly out of neglect iatrogenesis, he developed a stage 4 sacral wound. And unfortunately, this had become infected. At the time that I had received him when I did my initial cultures, he had three gram-negative polymicrobial organisms, to include, E. coli, Klebsiella, as well as Enterococcus faecalis. His bone culture from the sacrum grew out Peptostreptococcus. What I did with him is I took him to surgery. And this is what I was looking at initially. This picture here actually shows further exposed bone that may not be evident, and this was after the initial debridement. Again, I want to again stress as several other speakers have, the importance of good debridement in these chronic wounds and the importance to make sure that you decrease this bioburden specifically the biofilm that is present. Again, I do this with the combination of both sharp debridement as well using an ultrasonic debrider. The initial application that included me laying down many pieces of the umbilical cord, amniotic membrane allograft. I then put a combination paste using DHA-CaM as well as an ACell powder. And then I used a thick ACell sheet to cover the wound, which I’ll show here in the next purpose. The reason why I do this is because, in some way, I need to protect this product. And what I’m trying to protect is the umbilical cord amniotic membrane as well as the DHA-CaM product. What I’m trying to do is offload this wound, and you’ll see what I’m talking about. This wound that you’re seeing right here now is approximately a week after I initially got him from that presentation. What again this is showing is this porcine bladder xenograft, and what I use as a thick hernia sheet to kind of protect the wound. Again, what this is depicting here is me sowing in the umbilical cord amniotic membrane. Once I sow this in, I also end up fenestrating these sheets. And the reason why I fenestrate these sheets is because these wounds tend to leak a lot. And what I want to have a good coaptation between the wound bed and the product itself. To allow the fluid to leak to out, that’s why I fenestrate these. Then, what you end up seeing is me laying down product here and then finally placing in, like I said, a protective sheet, if you will, to offload. Then, I put like a Mepitel Mepilex dressing over this. Now you can see that this has made significant improvement. Remember, I got this patient on the 8th of August, and now, this is about six weeks later. And what you can see, again, is now I’m going in for like my third or fourth treatment here again with the same repeated process, laying down the umbilical cord, placing down the DHA-CaM paste. One of the thing that I also need to point out, the other thing that I end up doing for a lot of these wounds is what I’m doing is I’m also injecting into the soft tissues around here. The reason why I end up injecting into the soft tissues, and again, I use a cocktail of product, is because what I’m interested in this paracrine and autocrine cell signaling. And there has been several studies that have come out that has been showing that using certain products, you’re going to increase recruitment of mesenchymal stem cells into the wound bed. And so what I’m looking at doing is by increasing the surface area of product within the wound, I’m hoping to bring more of the endogenous mesenchymal stem cells from the subcutaneous tissue as well as the bone marrow into this wound bed to help accelerate the granulation process. This is now looking at the wound on 10/1. This is like nine weeks after I initially received the patient. Again, this is just another bedside application of some amniotic membrane down below. Then, finally, this is placement, again, of the Mepilex Mepitel dressing. Again, this is kind of just to depict what was has happened. Basically, we still got the patient on the, again, the 8th of August. And now, we’re looking at the end of October. You can see that there’s significant improvement here. You can see that all of this here used to be how big the wound used to be, and this is how much tissue now has regrown, not to mention, re-epithelialized. I did take the patient on the 3rd of November for a skin graft. That graft took successfully and we’re able to close this would. He had one week in the hospital with the application of a wound vac and the patient did very, very well. This concludes my talk. These are some of the pictures. If you guys don’t remember, freedom isn’t free. These are some of the infiltration techniques that the US military has taught me for a night time job that I do, additional to my job at Eisenhower, working with US operation, Special Operations Command. This concludes my talk. Thank you for your time.