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Lecture Hall Wound Care | Treatment

The Science and Clinical Application of Bi-Layered Living Cell Therapy

John Steinberg
John Steinberg, DPM
Assistant Professor
Department of Plastic Surgery
Georgetown University School of Medicine
Washington, DC
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Lecture Transcription


Present e-learning systems


Hello, and welcome to this Present Online Educational Program. My name is John Steinberg; I’m an Assistant Professor in the Department of Plastic Surgery at Georgetown University’s School of Medicine in Washington, D.C. I’m presenting on the Science and Clinical Application of Bi-Layered Living Cell Therapy.

So this talk certainly will be product-focused, to some degree, but I want to take a very critical look at the evidence-based literature that is out there. I want to show and discuss where Apligraf fits in with the other technologies and the other bio-engineered alternative tissues that are available to us for patients with problem wounds. And I also want to look, very critically at techniques and clinical outcomes for utilizing Bi-Layered Living Cell Therapy.


Production of this PRESENT Lecture was made possible by a generous grant from Organogenesis.


I want to take a step back here for a second and look at the big picture. This is a journal article that I was co-author with Chris Adinger and others that was published in 2006 in Plastics and Reconstructive Surgery Journal. The real gist of this paper basically points out that wound care and complicated diabetic foot wounds, including partial amputation wounds, has undergone a significant change in practice dynamic. It used to be that if a patient lost a significant portion of their foot, or had a large planter wound site, the only plausible, and feasible long-term way to provide these individuals with healing ad ability to return to weight-bearing was aggressive free-flap management and aggressive surgical intervention. This placed patients in intensive care units afterwards, and placed them at significant surgical risk, and while this is still a technique that is utilized and is appropriate for many patients, for the most part we have undergone a significant shift in the approach to these patients being now mostly outpatient-based, utilizing advanced technologies including negative pressure and bio-engineered alternative tissues and growth factors, and improved topical wound care that has been very recently made available to us. So the overall, uh, uh, direction that we’re going with wound care now has certainly a surgical root, but has a significant outpatient component now with the increasing ability to deliver advanced care outside of the hospital.


The epidemiology of amputation and the high-risk patient with Diabetes is certainly a very staggering public health phenomenon at this point. There are greater than sixty percent of all amputations involve Diabetes in the United States; and most importantly, as far as both points, I would look at the second where approximately 84 or, depending on which literature source you quote, up to 88% of lower extremity amputations are preceded by a foot ulceration.

This may seem like just another statistic at the first glance, but in reality, if you really critically look at this, 84% of lower extremity amputations potentially could be prevented with more appropriate, more advanced, and a greater ability to heal these diabetic foot ulcerations. So, the the real synopsis here is that most diabetic foot amputations are not he two o’clock in the morning abscess that is resultant from stepping on a nail from someone with Diabetes. The majority of diabetic foot amputations in this country involve chronic, non-healing diabetic foot ulcers that eventually progress, deepen, become osteomyeletic, infected, and possibly ischemic, therefore costing this person, ultimately, their limb.


To get even more general about things, there are basically two reasons why someone with an ulceration would lose their leg. The foot on the left shows a deep ulcer; if this ulcer continues to deepen, it will provide a portal for deep infection, and indeed, eventually, a portal for exposure of bone, and therefore Osteomyelitis, becoming a surgical disease requiring at least a partial foot amputation.

The photo on the right; however, you say ‘well, that’s a healthy granular or a nice-looking wound, what’s to worry about?’ Even in the best of circumstances, and even perhaps in the non-diabetic patient, this is a large wound requiring a high oxygen flow that has a high propensity for infection, because of this large area of exposed tissue. The longer you leave this, the longer you let this lie, despite the healthy appearance of it, the greater the risk for infection and also the greater the risk that eventually the blood supply will not be able to meet the increase in oxygen demands of this wound, and you will therefore have local ischemia, and eventually, perhaps, have necrosis and possible limb loss.

So even the healthy wound, if it’s a large wound or a concerning wound, or if you’re dealing with an impaired population, such as Diabetes, uh, even a healthy-appearing wound can be in need of significant assistance for healing.


You can see at the top that I’ve underlined the word ‘early’ twice, it is a key point that we’ve learnt in recent years, that we need to identify problem wounds early, in other words, we need to differentiate healing wounds from non-healing wounds, and we need to transition to advanced therapy early in those wounds that are non-healing. Probably the best literature reference I can cite for you is Peter Sheehan, et al, in Diabetes Care 2003, where he really studied this wound-healing trajectory and with that the healing potential of wounds, and was able to, at a very early period, decipher which wounds were going to eventually heal versus which wounds were not going to eventually heal based on that initial wound trajectory, even just in the first few weeks. So, the the reality of having to wait months before we can label something a chronic or a non-healing wound is really no longer a reality.

So, on the left hand side, you see good wound healing, certainly good standard practice, something that should be employed in the three- to four-week range; you’re going to take your basic history and assessment for these patients. You’re going to debride the wound on an aggressive weekly basis. Provide a warm, moist environment via dressings, and you’re going to provide the best possible offloading of pressure while you continue to use topical care.

If that therapy does not produce 10 to 15% healing in the wound site on a weekly basis by the time you hit the three- to four-week mark, it’s probably time to consider changing gears and moving to the right hand side of the screen, to the so-called ‘Advanced Wound Care’ side.

This is where you’re going to attempt different interventions, attempt different therapies; again, the whole point of this slide, is that we’re well aware now of these advanced technologies, what we’re becoming aware of is the need to transition to these technologies earlier, rather than later, so that these technologies can have a better effective rate, and have an overall limb salvage and wound closure rate.


Vince Falanga and many others have really studied and published now on the science of wound bed preparation. Now, it sounds like a very basic principle, but the reality is that this is something that we really have been able to define and describe, and practice appropriate wound bed preparation. And this is key, especially if we’re going to be talking here about Apligraf and bi-layered cell therapy. If you have not properly prepared that wound bed to receive an advanced technology, then you have just wasted time and effort and resources with the new technologies. A properly appeared wound … prepared wound bed, is going to be a clean, viable wound bed, not a culture-negative wound bed, but a clean, viable wound bed that does not show gross signs of infection. You’re going to have controlled bacterial surface contamination; you’re going to have a minimal amount of exudate, so that you don’t destroy a graft or other product that you’re putting on the wound bed. You want to have a Major Metalaprotodase Protease imbalance and again, culture is I put a question mark next to that. I really do not practice or promote a negative culture prior to grafting or advanced wound therapies, uh, but it is important that clinically you document that the wound shows no acute signs of infection.


Topical antibacterials or antimicrobials are a very significant part of wound bed preparation. The slow-release silver technology and others have very much changed the dynamic of how we maintain wound beds and how we prepare these wound beds for bio-engineered and alternative tissue grafting, and clearly, have in some cases, made this topical wound care more consistent and less labor-intensive, via this controlled, slow-release dressing material that can be left on for a longer period of time.


We like to utilize the terminology now, ‘Bioengineered Alternative Tissues’ or B.A.T. This really is a change of gear from where we used to label these as ‘skin substitutes’ or ‘bioengineered skin”; I really do not favor that type of terminology, even for Apligraf and Dermagraft, because I just feel that these, give a wrong impression of where and when to utilize this technology.

The whole point of bioengineering or actively intervening with problem wounds began with Procurine and then later developed into Regranex being a topically applied growth factor, a major advancement from where we used to be in wounds, of just providing an appropriate wound environment. We were able to truly interact with the wound bed at this point, and we’re doing so through some of the products that I have listed here. By no means is this a complete list of each of these categories, but I’ve just picked out some of the more commonly used, uh, common names.

The middle category, the BioActive Wound Adjuncts, are the scaffold products; these are non-living, generally acellular products, uh, that are utilized in usually deeper wounds to bridge across these wound margins, provide a surface area for angiogenesis to occur, and try to facilitate and make this a more rapid and predictable process for a wound bed to fill in.

The final category, obviously we’re working on some training, the Living Cell Therapy category is the most advanced and the most high-tech, and there really are only two members here, Apligraf being the bi-layered component containing the human fibroblasts and human keratinocytes that are both living, and the Dermagraft, containing the living human fibroblasts that are seeded unto a vicryl mesh.


Now, as much as we may want to leave things like fibroblasts and cellular proliferation to, uh, scientists sitting behind a bench in a laboratory, the reality is that as physicians, we really have an increasing need to understand where all this fits in because of the role that we’re now seeing and the products that are now available, in order to make an appropriate choice for what’s appropriate at a particular wound-healing stage. The fibroblast really has a significant function in the wound base, as we all remember learning in the basic science of Biology; fibroblasts are key to everything from cellular migration and proliferation, all the way through to angiogenesis and protease release, so the significant factors here for down the chain promoting growth factors and linkages and tensile strength between cells.


Moving more specifically now to an overview for Apligraf or Bi-Layered Living Cell Therapy, essentially here you have an epidermis and a dermal component to this graft component. This is derived from a neonatal foreskin cell bank that is now a rapidly proliferating bank, where these cells are then derived into a fresh culture medium, and once formed into this graft material in combination with the bovine collagen, have a ten-day shelf life once shipped out from the facility. F.D.A. approved this in March of 1998 for venous leg ulceration after an extensive trial was conducted, and the F.D.A. approved that June of 2000 for diabetic foot ulceration.


Looking at the four components of Bi-Layered Living Cell Therapy, this tissue graft contains at its surface a cornified layer. These have been exposed to air during the manufacturing process to give a basket-weave pattern of keratinocytes. The epidermal layer itself is obviously the viable and living human keratinocyte layer. The dermal layer of the graft is human fibroblasts, and then this is combined with the extracellular bovine collagen matrix for this protein matrix.

Apligraf itself does not contain cells that would stimulate a significant immune response, so this would include the absence of melanocytes, macrophages, blood cells and sweat glands. Bi-Layered Living Cell Therapy has been used successfully and safely in over 150,000 applications since its F.D.A. approval.


If you break this the science down here, there are basically three benefits of Bi-Layered Living Cell Therapy. First is the physical benefit of applying these living, viable fibroblasts and keratinocytes unto the wound site, or cell therapy. We are deriving growth factors here, cytokines, and delivering a form of natural antibiotic to the wound site.

The barrier function gives some level of immediate physical protection, uh, through the physical coverage of the graft, and certainly a biologic barrier, as well.

And then, finally, the dermal matrix, giving just that substrate or that protein base for cellular migration on top of the wound site.


This is a brief schematic of the manufacturing process for the Bi-Layered Living Cell Therapy. Essentially, the fibroblasts and connective tissue proteins are combined into this dermal matrix material; they are then combined with the human epidermal keratinocytes, and formed into a bi-layer therapy sheet.


At the top is a drawing, representing on the left Apligraf, on the right, human skin; similar type on the bottom portion of the slide showing on the left, Apligraf under the microscope, and on the right, human skin under the microscope.

Obviously, particularly looking at the microscopic images, it’s quite a scientific achievement that’s been made here of producing what appears to be, scientifically, skin through a laboratory process; however, I would caution you about this, as amusing as these slides are, that you really should stay away from the concept of thinking and treating this as if it were skin. Uh … I truly believe that bi-layer therapy is basically a mechanism to deliver a sheet of growth factors to the wound site, and if you think of this as as a convenient form of delivering these grown factors to via these living cells, I think you’ll utilize the graft at the appropriate time. If you think of it strictly as skin, and you’re looking and depending on this for structure and strength, I think you’re going to be grossly disappointed, and you’ll probably utilize this at the wrong time in the wrong wounds, because really, Bi-Layered Cell Therapy, for the most part, after a week or two on the wound site, has become a gelatinous matrix; it’s a healthy, viable gelatinous matrix, but it no longer has the appearance of skin.


This slide just shows some of the level of science that goes into this product; in the center you can see an electron microscope image showing a fibroblast seeded into that extracellular matrix that we talked about. There’s a lot going on here with these grafts and the extracellular component of the graft; but again, the key benefits for us are the growth factors, essentially, that are produced by these living fibroblasts and the living keratinocytes.

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Some of the basic characteristics of Bi-Layered Living Cell Therapy … again, these are neonatal cells. Keep in mind, they’re not fetal cells; this is not from aborted tissue, these are children that have been born and their circumcised tissue was utilized for the graft material into forming these large cell banks.

These are highly proliferative, young, healthy neonatal cells. They have a high rate of replication; they are obviously non-diabetic cells, and this is one of the reasons why this type of tissue can work so much better in some of our patients with Diabetes than would an autogenous skin graft. We’re using this for different reasons that we would a skin graft in the first place, but also, we have the benefit of of this being healthy, young cells rather than transplanting or transporting one’s non-healthy cells from one part of the body to another.

These cells produce matrix protein, a strong array of cytokines, they do produce an antimicrobial peptide that has some level of antibiotic effect on the wound site, and these have been shown to persist and respond to the wound environment quite well in the diabetic foot, as well as the venous leg ulceration.


One question that I commonly get asked is why does Bi-Layered Cell Therapy not come pre-fenestrated. Well, this set of slides should pretty much answer that question for you, because if the product at the time of manufacturing were fenestrated at the facility and then placed into the FedEx box and shipped off to your clinic or hospital, it would heal itself by the time it arrived at your facility. So, essentially, there is no way to pre-fenestrate this, you need to do this at the time of application, and you’re doing this for a couple of different reasons; you’re doing this to promote cellular replication and repair and … growth factor secretion from these injured cells, and you’re also doing this to fenestrate it physically to allow the hematoma or saroma materials to progress through the graft, rather than accumulate below the graft.


Bi-Layered Cell Therapy through the fibroblasts and keratinocytes is known, at this point at least, to produce 46 cytokines or growth factors that are involved in tissue development and wound healing. These extend from vascular endothelial growth factor to platelet-derived growth factor. And, indeed, there is believed to be a strong interaction between the two layers of this graft, being the keratinocytes and the fibroblast layer, where you derive a differentiated structure that produces cytokines that wouldn’t be expressed in either of these alone. There is a synergistic effect that seems to take place.


This slide goes back to my point of thinking of Bi-Layered Cell Therapy as a big sheet of growth factors. These healthy cells are secreting as labeled here, fibroblast growth factor, the insulin-like growth factor, interleukins, transforming growth factor, vascular endothelial growth factor, and the list goes on and on. Some of these are derived from the keratinocytes, some of these are derived from the fibroblasts, and all of these are present and continue to be produced by the graft after it’s applied unto the wound site.


This slide represents some data published in 2000 in the Journal of Wounds. In fact shows in vivo data for Bi-Layered Cell Therapy, and shows the increase in growth factor production in relative units on the left hand side, and days extended down at the bottom layer. This increase in production is what we expect and hope for when we apply this unto a problem wound site, from this delivery of the graft unto a viable vascular bed, and the injury that we cause at the time of application through fenestrating the graft.


Some basic information with regards to Bi-Layered Cell Therapy and and looking at some of the scientific studies that have been performed on graft material itself, and just looking and realizing that this certainly we understand the role of angiogenesis and wound healing, and this is a product that should help us significantly with the angiogenesis because of the presence of particularly vascular endothelial growth factor and fibroblast growth factor.


Matrix synthesis is another key activity that we need represented in the human wound site, and that we’d like to be able to deliver to a problem non-healing wound, and this certainly can be delivered by transforming growth factor Beta, and you can see here the presence of transforming growth factor Beta, as illustrated in normal skin, granulation tissue, and Apligraf itself.


In just the past couple of years, we’ve been learning the importance of controlling Matrix-metallo proteases. These are breakdown products in wounds, and often times, heavily-exiodated wounds continue to form this yellow slough material, um, despite aggressive debridements, can be the wound type that is being plagued by excess wound breakdown or Matrix-metallo protease presence. The normal regulatory products are the TIMPs or the Tissue Inhibitors of Metallo Protease. This is present and demonstrated here in the Bi-Layered Living Cell Therapy product, and it certainly would assist in controlling this in the wound bed when it’s delivered and applied in the appropriate timing.


While there’s only a small amount of data on this, and ability to research into it, there clearly is an antibiotic peptide that is produced by Bi-Layered Living Cell Therapy when it’s applied unto the wound base. This would explain some of the ability to control wound bed contamination, despite an exclusive dressing that is often times utilized with these graph materials for up to one week after application.


There is a lot that is known about the mechanism of action of bioengineered alternative tissue or Bi-Layered Living Cell Therapy on the wound base. In particular, obviously we understand and have talked about already the delivery of young, healthy, active fibroblasts and keratinocytes to the wound site; the fact that these cells will, in general, persist on the wound bed for up to six weeks. They, themselves, will produce new matrix material in cytokine and growth factors, and will also stimulate the recruitment of other cell types, including the potential for stem cells, in these non-healing, non-viable wound sites.


I’d look to look for the next few slides looking at venous leg ulceration, utilizing the bioengineered alternative tissue, and the evidence-based clinical trial data that we have available.


To cut right to the chase here, the summary of some of the most important bullet points from this F.D.A. pivotal trial demonstrated that for ulcers venous leg ulcers that were present for greater than a one year duration that were enrolled in this clinical trial, in comparison to a control group.

At eight weeks, the Apligraf group was three times more effective for frequency of complete closure. At six months, the active group was more than twice as effective for frequency of complete closure. And at all times throughout the study, the active group was superior for complete wound closure.


This bar graph summarizes the randomized control trial looking at venous leg ulcerations, comparing a control or compression group to the active or Apligraf group with compression. Looking obviously at bullet points here for the data at four, eight, 12 and 24 weeks, at 24 weeks seeing, uh, uh, 57% healing in the active group versus 40% healing in the control group.


This bar graph being the carve-out of patients from that same study that were greater than one year of duration with their venous leg ulceration. Obviously the data here much more substantial; I think the point here is, even in the hardest to heal wounds, this is where that change in wound dynamics and delivery of the right biology to the wound base through this active bioengineered alternative tissue, can make all the difference. 47% healing in the active Bi-Layered Living Cell group, versus 19% healing in the compression group alone.


This is a post-marketing study that was published in Dermatology Surgery 2001 by Harold Brown, looking at the outcomes in two different centers of 33 patients with venous 50 venous leg ulcerations that were present for greater than a year duration. These are all patients that arterially intact but venous. The graft was applied after being fenestrated; they all received compression therapy. Healing time was 33 to 51 days, and the median healing rate in this patient group was 74%.


The next few slides are going to look at the F.D.A. pivotal trial and evidence-based data in regard to diabetic foot ulceration, and Bi-Layered Living Cell Technology.


We’ll look real briefly at the molecular growth factor and cellular abnormalities that are present in diabetic foot wounds. They’re obviously quite expansive, and most of them are biologically-derived: the growth factor and cytokine deficiencies that are well-known, endothelial cell dysfunction within the wound site is well published; neuropathy, obviously, in almost 100% of these individuals; arterial occlusive disease being a significant factor for [inaudible]; their decreased ability to provide angiogenesis at the wound base, and the abnormalities in fibroblast production and the extracellular matrix all compound into a impaired and non-healing wound site in these compromised diabetic patients.


The F.D.A. pivotal trail for Apligraf in diabetic foot ulcers was designed as a prospective, randomized, controlled study that looked at Apligraf in comparison to a conventional therapy alone, which was debridement, saline dressings and off-loading of the wound site. 208 patients were enrolled, all with diabetic foot ulcerations, most of which had Type II Diabetes, and the study excluded patients who at in a one-week period were able to heal greater than 30% as a control group.


The summary shown here in terms of those 208 patients, the statistical significance was very strong, looking at 56% of the patients enrolled in the active group progressing to complete wound closure, versus only 39% progressing to complete closure in the control group.


The data in this bar graph may be even more important than that of the previous bar graph, because this looks at time to wound closure. As we discussed earlier, the longer a diabetic foot wound is open, the more likely it is to become infected and/or ischemic. In the active group, represented by the green bar here, the wounds that did heal, healed in 65 days, versus in the control group, in the conventional therapy group, uh, the wounds that did heal took 90 days to heal. So not only did this active heal more wounds, but it healed them on a quicker timetable by upwards of one month. This is a whole another month that potentially we could avoid infection, ischemia and amputation in this patient group.


Logic and, I guess common sense, would tell you that if these wounds are closing quicker, then you’re going to have lower incidents of infection, and that is what is proven here, it’s looking at the active group, represented by the green bar, having a 2.7% rate of Osteomyelitis and obviously likely leading to an amputation, versus the control group having a much higher rate of Osteomyelitis, being over 10%.


Again, a similar point here; if we have deep infection and Osteomyelitis, this is going to be a surgical disease or an amputation-requiring disease, the active group having a 6% rate of amputation, versus the control group having a greater than 15% rate of amputation.


This is, uh, some much newer data, a post-marketing study by Dove and Sheehan, published in October of 2005. This is an open-label multi-center study looking at Apligraf in the treatment of diabetic foot ulcerations.


Some of the key objectives of this study were to obviously evaluate the safety and efficacy of Apligraf in patients with diabetic foot ulcerations who had failed with conservative therapy, but also to look at the actual incidence of complete wound healing by week 12, and it should be noted that complete wound healing in this study, was defined as full epithelialization of the wound with absence of drainage.


In addition to basic wound care and documentation, these patients received Apligraf at the baseline visit, as well as at the six-week visit after treatment, an additional three months of safety evaluation were also completed.


To be included in the study, patients had to have either Type 1 or Type 2 Diabetes; as illustrated here, age 18 to 80 were included with moderate control of Hemoglobin A1C required between 6% and 12%. The ulcer had to be at least two weeks in duration, and the size of the ulcer ranging from one to 16 cm2.


The key exclusionary criteria for individuals in this study were if they had active Charcot, if they had Osteomyelitis, obviously infected target ulcers, or if there was any evidence of skin cancer.


Demographics for the study: 50 patients were enrolled, of which 68% were male, 62% were Caucasian, age range was 25 to 79, with almost equal groups here of Type 1 versus Type 2 Diabetes.


The ulcer duration had a range of two to 312 weeks in duration, for non-healing, and the ulcer size ranging between one cm2 and 108 cm2.


The two determinants of efficacy as end points were: number one, the incidence of complete wound healing by week 12, and also documenting the time to complete wound healing of the actual target ulceration.


End results for this study: at week 12, 70% of the 50 patients were healed, and at the three-month post-treatment follow-up, 76% of these individuals were healed.


The time to complete wound closure, again, as defined by complete epithelialization and absence of drainage at the site, was 45 days.


This slide, shows a very important data point for this study; it’s something that we’ve certainly batted back around clinically with questions as to how many Apligrafs how many applications are necessary for wound healing. This study proved again that the majority of wounds will heal with just one application, most likely because of that six-week duration of cell survivability in the wound base; so 84% healed with one application and another the remaining 16% that did heal required two applications.


In summary here for this study, at week 12, 70% of the patients had achieved complete wound closure with the Bi-Layered Living tissue graft. The median time to closure was 45 days for those that did heal, and 84% received just one application of the Apligraf material. Four patients had treatment-related adverse events, none of which were serious, and the results of this study further support the safety and efficacy for diabetic foot ulcerations.


At this point, I want to take you through some clinical slides that will demonstrate wound bed preparation, and the clinical application of this technology in your patients.


This slide shows a wound site being debrided at the time immediately prior to the application of the Bi-Layered Living Cell Therapy. Essentially, I try to do my aggressive surgical debridement one week prior to the Apligraf application, and then on the day of application we’re just doing a curettage or a basic … uh … uh … wound debridement, not stimulating a huge amount of bleeding, so as not to create a hematoma or saroma underneath the graft. If your debridement on the day of application does stimulate a significant amount of bleeding, then you should use some topical epinephrine, or, if available, topical Thrombin in combination with pressure to control this bleeding prior to applying the product.


After your debridement, a surgical prep with something such as hibocleanse can and often times is performed using saline to aggressively rinse that field free from any of the scrub solution, after that you’re going to open and inspect the packaging, make sure that the graft is in the appropriate color range, and does not progress outside of the acceptable pH, which would be a bright pink or purple color, or a yellowish appearance to the Agar medium.


There are certainly several techniques as far as details of how to elevate, fenestrate and prepare the graft. One, as per illustrated here, is to put a little bit of saline on top of the graft, use some moistened cotton tip applicators to twirl the edge of the graft, gently lifting that from the transwell or the membrane that separates it from the Agar, and then grasping with your fingers or a forcep. An alternative method would be to fill the well with saline and apply a four-by-four; the graft will generally stick to this four-by-four and can be lifted out as a single piece.


With the graft now placed top-side down, or upside down, the onto the moistened four-by-four. I generally will fenestrate this with a number 15 blade, literally placing around a hundred holds into the graft if utilizing the entire piece. Uh … the alternative method would be, if you’re in the operating room and you have available to you a 1.5 to one split skin-graft measure card, and meshing hardware, then certainly this can be run through the graft mesher, to facilitate speed of application.


Once meshed or fenestrated, the graft is then carried on its four-by-four to the wound area, then applying directly unto the wound so that the graft is back into its proper orientation, with the epidermal side facing surface and the dermal side facing the wound base itself. This will allow the appropriate revascularization or angiogenesis to take effect and therefore keep these graft cells viable from the underlying wound bed blood flow.


Once you’ve applied the Apligraf to the wound, make sure that it’s intimately adherent to the wound base, that’s there’s no pockets or elevated areas from the wound base, again, this is a living cell product, if it’s not in contact with the blood supply, it’s not going to be viable when you take your dressing off. You can suture this, as pictured on the left hand side, you can staple this as pictured on the right hand side, you can sterry strip it, you can use Fibron glue, or you can simply use a compressor dressing. This is completely surgeon choice. Occasionally, the anatomy will dictate that you do secure it, but for the most part, a compressor dressing would be sufficient.

One item that I would point out is on the right hand side; there’s really no need to trim the Apligraf to fit the wound base. If you need to cut it ahead of time to apply to multiple wound sites, then that certainly does make sense, but just leave the excess, if you’re not going to use it elsewhere, just leave the excess as an overhang, it’s going to peel off simply at the one-week post visit.


Everyone seems to get very anxious of about what type of dressing to use over Apligraf. There are a few basic principles that I try to stick to, and that is that you want it to be something that’s non-adherent; you want it preferably to be something antimicrobial but certainly not cytotoxic. I will often time use products that have topical silver in them to maintain a clean environment underneath my dressing which I’m usually going to leave on for a week. Or you may even consider using negative pressure or vac therapy over this, but stick to the basics of non-adherent, moist and compressive dressing, with antimicrobial if possible.


After you’ve applied your primary dressing, generally a secondary compressive dressing is applied to give some security to the area, to prevent any slippage of the draft and its overlying primary dressing, and also to immobilize the area.


If the wound site is an area that requires it, you certainly will continue and optimize your off-loading for these patients, preferably using one of the devices from the back row, being a removable cast walker device, a splint total contact cast, crow-walker, something that immobilizes the foot at the ankle to prevent the aquinous and forefoot pressures that can result from a tight, tendo-Achilles.


I generally advocate compression therapy for both venous leg ulcer applications, as well as diabetic foot ulcer applications, partly because a nice, compressive, multi-layered dressing is going to assist in preventing slippage of your underlying bandaging and Apligraf, as well.


We generally see our patients who have received Apligraf back on a once-a-week basis. The primary dressing is often times left in place and not disturbed, but if there is any concern or question, then you go ahead and take down the complete layers, and again, that’s the reason why we want to use something that’s non-adherent. If you did use sutures or staples, you can certainly take them out at one week. You just want to inspect the periphery, make sure there’s no obvious signs of friction, no significant changes to the peripheral tissues that would make you have any concern, and really, you’re going to resist the temptation to do anything more than just re-apply your dressing at the one-week mark, and in reality, the less you do to these grafts after application, probably the better most of them do.


I’m showing you these photos for those of you who may not have used Apligraf or Bi-Layered Living Tissue before, that at the one-week mark, you can have some pretty distinct appearances, and sometimes surprising appearances, that don’t necessarily mean you’ve had a negative result. Each of these is photos post-application. On the top left is an immediately post-application prior to dressing being applied, but on the top right and bottom center, you see wounds that are one-week out from application. And you can see, these look as if they are in need of debridement; you’ve got this yellow, somewhat [inaudible] film over the wound base. This is hydrated viable graft with living cells mixed in with all this tissue, and the less you debride and disturb this tissue, the better off you are, so remove the staples as you can see in these wound sites, apply a non-adherent dressing and see the patient back in another week. There’s really not much to do here.


In contrast, venous leg ulcer wounds often times have a more immediate granular and viable appearance, perhaps because of all the different depths and different biology of these wound types, but they are some post-application photographs after utilizing Apligraf in a venous leg ulcer setting, times two.


Looking now at a patient who received a guillotine-type transmetatarsal amputation because of a chronic and ischemic planter flat. This individual elected not to go back to the operating room to have a revision amputation or a major surgical reconstruction such as a free-clap, but instead wanted outpatient management, and what you’re seeing here is one, three and four weeks after Apligraf was applied; this was utilizing two side-by-side segments, and securing with staples with weekly dressing changes.


You can see now on the left hand photo, at six weeks Apligraf was re-applied to the wound site. It was debrided, prepped, and graft reapplied with a new compressive dressing. Here looking at the photo of the right hand side, this is at the ten-week mark, a full month after the second Apligraf was applied.


This is at about the 12-week mark, a total of two applications of Apligraf with continued weekly compressive dressing changes. Obviously, a rather lengthy healing course, but starting off with such a large wound, certainly a significant outcome. And the fact is, here, that we’ve regenerated these tissues through this bioengineered tissue, and we have now a full thickness closure over these bony aspects that should allow us to progress into an appropriate insert and expect weight-bearing with hopefully a minimal chance of recurring ulcer.


Looking now at a patient who presents in the Emergency Room with obviously a gross infection, abscess, and also involving an Osteomyelitis to the first Ray.


This patient was managed aggressively with surgical debridement on multiple occasions, brought back to the operating room for repeat debridement until we were at the point of complete disarticulation of the first metatarsal, and you can see, with loss of almost the entire dorsal soft tissues down to the level of the extensor tendons, out to the fifth metatarsal level. This large wound site was initially managed with aggressive combination IV antibotics, hyperbaric oxygen and negative pressure therapy, and after repeat surgical debridements, got to the viable wound base that you see on the right hand side, we began to regenerate this over the problematic deep exposed tissues and filling in these defects utilizing bioengineered … Bi-Layered Living tissue.


You can see here debriding the wound on the left hand photo in preparation for Apligraf, and then two segments of each graft is 7.5 centimeters across, utilizing the two segments here to fill this entire defect, secure with staples after fenestrating, then a compressive dressing after that.


Looking at the same patient, on the left hand side, three weeks post application, and then on the right hand side, we’re at five weeks post application with weekly dressings, showing significant healthy granulation coming up to the surface of the skin now, with decreasing wound surface area. A second graft was applied at this point, because, again, we expect about four to six weeks’ worth of cell viability from each graft, and it’s time now to re-apply. We’ve gone to the second graft, you can see approximately six weeks after that second graft was applied; we’ve had rapid and complete epithelialization of the wound site.


Another case example of utilizing the Bi-Layered Living Tissue technology in a patient whose status post an aggressive I and D, wound site is now healthy and granular, infection has been cleared, the graft has been applied and utilized to build this wound up to the surface of the skin, and in this case was utilized up to the point of epithelialization and closure.


Another case example of utilizing Apligraf to fill in a void from a partial fourth and fifth Ray amputation; here you can see the graft fenestrated, secured and maintained down to the wound base with a compressive bolster-type dressing. two applications were required and eight weeks for healing, but you can see the end result of closure and epithelialization.


Looking here at a very aggressive limb-salvage case. This is a 70-year-old gentleman who was transferred to our institution after being previously told and scheduled for a Below-Knee Amputation. He refused that amputation and we were able to intervene.


The problem list for this individual is very extensive, and certainly amputation would not be wrong for this individual, but if you’re going to attempt limb-salvage, then you certainly need to realize what you’re up against, and the problem list for this individual involves poorly controlled history of Diabetes, multiple wound sites on the same extremity, severe arterial disease that needs to certainly be dealt with before we can intervene, Osteomyelitis [inaudible] Resistant [inaudible] and an existing contra lateral limb loss.


After revascularization was attained, this patient was brought into the operating room for surgical debridement. You can see here the posterior Achilles wound being aggressively debrided down to the level of the viable healthy tendon, and now we have bleeding, healthy tissues underlying that necrotic tissue.


As far as the foot’s concerned, this transmetatarsal amputation was revised to a Choparts amputation; this left us with a healthy, viable planter flap that was utilized to cover his exposed bony aspects and the remaining portions of the foot. An onligraft of the discarded material was utilized and meshed for the interior aspect of the ankle, and a … uh … a drain was placed post-closure.


Looking at the posterior Achilles site on the left hand slide set, top and bottom, you can see that initially this was managed with a matrix-type product for deep coverage, and shortly thereafter, on 9/15, the Bi-Layered Living Cell or Apligraf technology was applied to the posterior Achilles for wound closure.


This was a rapid effect from Apligraf, and you can see, focusing on the 9/22 top left photo, being the Achilles, we had rapid epithelialization, and really a graft-take, almost like what you would expect from a [inaudible] skin graft in this type of a setting. This is something that we do see on occasion, but certainly is not the norm. Usually we see Apligraf with a staged, slower pattern of healing over a period of four to six weeks; in this case, that was achieved in about a week with almost immediate graft-take and epithelialization over this wound base.


Another example here, where living bioengineered alternative tissues can be considered. A 52-year-old female with obviously an aggressive Charcot rocker bottom foot-type, but in her particular case this central photo shows a deep, probing ulceration down to mid foot Osteomyelitis. She was brought in-patient, the wound was excised through a large planter incision, the bone was repeatedly debrided in the operating room until we got the negative biopsies to where we had rid the Osteomyelitis, then the mid foot wedge could be performed and attempted arthrodecis utilizing external fixation, and eventual closure of her wound site, which was complicated with dehissance that was countered with bioengineered alternative tissue and negative pressure therapy.


Looking at the 15-week clinical photo and the radiographic six-month photo showing a planter-grade foot with a closed wound site and a viable extremity for ambulation.


We’ve covered a lot of ground in this lecture, and looked at a lot of o the evidence-based literature that leads us to understand when and how to utilize some of these technologies in wound-healing. One thing that we have not discussed a whole lot of is how to prevent these wounds from coming back once you’ve healed them. This, certainly in my opinion, can be sometimes harder than the actual wound-healing itself because of the high frequency of recurrence and because of the reality that the bioengineered alternative tissue, and even the living tissue technologies don’t necessarily treat the etiology of this wound site. So we need to recognize the etiology, if this can be treated appropriately with shoes and inserts and accommodated long-term, then by all means that is our pathway most commonly chosen; but if shoes and inserts, or other accommodative devices are not going to be sufficient to provide long-term off-loading for these individuals, then they probably need to undergo some type of a prophylactic or curative type surgery to treat the actual bony tenderness or other tissue abnormality that cause the ulcer in the first place.


I hope that you benefitted from this discussion, looking at the evidence, looking at the clinical application and looking at some of the case examples of when and where and how to use Bi-Layered Living Tissue Technology. This is a field that clearly has just started to enter its prime, I think we’re going to see significant continued advances in wound-healing and bioengineering that will give us more and more tools to utilize for these individuals, but at this point, I hope that I’ve given you a better ability to utilize what we currently have, uh, at our fingertips.

Thank you very much.


Production of this PRESENT Lecture was made possible by a generous grant from Organogenesis.

Dr Steinberg discusses the use of Apligraf, the only currently available FDA
approved bi-layered cell therapy. He reviews in detail the current literature available
to support its use in wound healing and where this product fits with other available wound healing modalities.
Dr Steinberg additionally critically reviews the techniques and clinical outcomes of bi-layered cell therapy.
Goals and Objectives
After participating in this activity, the viewer should be better able to:

1. Appreciate the epidemiology of ulceration and the importance of limb salvage.
2. Discuss the basic biology of various living cell therapy modalities.
3. Understand the medical evidence supporting the use of Apligraf living cell technology.
4. Appropriately debride and apply Apligraf to various wound sites.

Estimated time to complete this activity is 59 minutes.
Target Audience
Physicians, diabetes educators, and other health care professionals who treat patients with diabetes.
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The Science and Clinical Application of Bi-Layered Living Cell Therapy
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The Science and Clinical Application of Bi-Layered Living Cell Therapy
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Disclosure Information
The Science and Clinical Application of Bi-Layered Living Cell Therapy
It is the policy of PRESENT e-Learning Systems and it's accreditors to insure balance, independence, objectivity and scientific rigor in all individually sponsored or jointly sponsored educational programs. All faculty participating in any PRESENT e-Learning Systems programs are expected to disclose to the program audience any real or apparent conflict(s) of interest that may have a direct bearing on the subject matter of the continuing education program. This pertains to relationships with pharmaceutical companies, biomedical device manufacturers, or other corporations whose products or services are related to the subject matter of the presentation topic. The intent of this policy is not to prevent a speaker with a potential conflict of interest from making a presentation. It is merely intended that any potential conflict should be identified openly so that the listeners may form their own judgments about the presentation with the full disclosure of the facts.
John Steinberg, DPM has no relevant financial relationship(s) to disclose.
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