CME Wound Care

Wound Science - Delayed Healing in Normal and Diabetic Skin

Steve McClain, MD

Steve A. McClain, MD reviews impaired wound healing in the general population and in diabetics. Dr McClain also relates the anatomy of the skin and the factors important in the healing process. Dr McClain defines delayed healing, discusses animal research and identifies multiple factors that can cause delayed wound healing. Dr McClain also recognizes the role of collagenase inhibitors for wound healing.

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Goals and Objectives
  1. Identify normal wound healing principles
  2. Define delayed healing in normal and diabetic skin
  3. Recognize the role of collagenase inhibitors in wound healing
  4. Review the history of fungal ulcer
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  • CPME (Credits: 0.75)

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

  • Author
  • Steve McClain, MD

    Adjunct Professor Dermatology and Emergency Medicine
    Stony Brook, LI

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  • Lecture Transcript
  • Male Speaker: Speaker is Dr. Steven McClain who hails from High Ridge, Missouri, where he graduated from the University of Missouri and trained at University of Vermont with dermatopathology fellowship at New York University. He’s an associate professor of dermatology and emergency medicine at SUNY, which is Stony Brook with research interest in wound healing, malignant melanoma and pathology informatics. He practices dermatopathology and has his own lab called McClain Laboratories. Most recent work, “Thickness of Normal Toenails”. At this point, we are going to ask for the topic Wound Science, Delayed Healing in Normal and Diabetic Skin. Please welcome, Dr. Steven McClain.


    Steven McClain: Good afternoon. I am going to talk about delayed healing. They didn’t even start the timer here, so I got a few extra minutes it looks like. Okay. I have no financial interest. I want you to learn something about normal wound healing principles, be able to define delayed wound healing in normal and diabetic skin. I want to look at some diabetic healing in animals versus nonhealing ulcers versus necrobiosis lipoidica diabeticorum. I would urge you as trainees not to use the acronyms. Use, learn and own the diagnosis, necrobiosis lipoidica diabeticorum. You will wow any number of people when you learn that diagnosis. Explain doxycycline as a collagenase inhibitor under utilized property of the tetracyclines, describe the historical treatment for fungal ulcer and develop an approach to improve pathologic study in this abnormal healing. I put on five references. One is about the standard of care being 15 days to 50% healing by Ambroise Pare, 1553. All gangrene is infected by Frederic Mohs in 1941. I have a notion hospital gangrene is aggravated or produced by fungi by Henry Vandyke Carter in 1861. And some work on doxycycline as collagenase inhibitors. I wanted to let you know that there’s more than 200 clinical trials on tetracyclines in progress worldwide right now. Hundred fifty of which are in the US. This is available freely. In fact, all these references if you email me, I'll send them to you. Okay, here we go. I need your seatbelts on, your train tables up in your seats in their full upright position because we’re going to go quickly. Yes, I did all those things and I have an interest in wound healing. What we do in our laboratory is we make diagnosis with photographic proof and so I show pictures. I’d like to address my comments today to the podiatric surgeons in particular because the problem we’re going to talk about requires surgery. I’m going to ask you specifically at the end of the lecture -- will you invite pathology to the wound healing team? It’s pretty simple to do. You just send all your debridements and formalin to the path lab. Not the stuff you’re going to put in microculture but even the necrotic tissue. I want you to do that and just say, rule out infection. You can ask specifically if you want for a PAS and then Alcian blue and a gram strain. There’s five components in that set, plus the H&E. I’m going to show you some data that I don’t think is anywhere else in the literature because this hasn’t been talked about for about 70 years. But this is the stuff on top of an ulcer. Okay. This was scraped off. This is not biofilm. This is hard. This is a callus. It’s composed of cornified cells and all kinds of microbes. It’s not a biofilm. It’s a structure and it’s hard and it’s hard to get rid of. If we look at under the microscope, there’s parakeratosis. It’s layered. Look at it by gram stain. Look at all these layers. It looks like the geology of the Grand Canyon or something. You got blue layers, yellow layers, red layers, blue, purple, white. Every single one of those, that’s a structure being built up through a proliferation of cornified cells, infected cornified cells in my opinion.


    Here’s the layering. There’s a network. Notice this cleft down here. Look at these things. How about this? Budding yeast. This is Candida in a foot ulcer. If you treat the fungus in a Candida foot ulcer you know what happens? They improve. Healing is normal. Nonhealing is abnormal. It has a cause. I hope that you will adopt that mentality that nonhealing has a cause and you need to treat it. If you go after that cause and treat it, your care will improve. This is the animal studies I’m going to show you. These are rats, 6-millimeter punch biopsies. This is an acute diabetic model. Glucose is 400 to 600. This is the human. They don’t look anything alike. In fact, from this kind of work, all the wound researchers would say you need a moist wound healing environment and so on, except it doesn’t work in yeast. When you put these on and something like this white tissue happens, notice this looks like a sheet of skin has come peeled off of here. We’ll look at that in a minute. But notice this yellow structure here. It’s the same thing we just looked at microscopically. Much different than the rat wounds. Maceration, maceration is fungal. When you peel that stuff off and send it to the laboratory you can see the hyphae in here and globs of immature fungi. These yellow cheesy parts here. Okay. Nonhealing differs from delayed healing in diabetes mellitus. Human ulcers differ from animal research wounds. How? Well, none of this heals spontaneously or practically none. These all are chronic diabetes, not three weeks of diabetes in an animal. In my experience, now, I only have about 250 cases but 70% of them have fungi on them. Okay. Now, the other little known piece that doesn’t happen in the rats is most rats do not have 80% toe fungus like the diabetics. They already have this and many diabetics haven’t established fungal infection in the adjacent tissue, in the toenails. But you won’t find this in the literature. Fungus in ulcer, you won’t find it for practically since antibiotics came out in 1949. There is a paper on treating Candida when you culture Candida and they treated them and they got better. But the recent wound healing literature, for example, Dr. Tom Serena, no mention of fungus. We have to go back ways to find this. We could go back to Ambroise Pare and why would you go back to him? I supposed you can go back to him because he was a man of courage and conviction. You could go back to him because he was a barber surgeon. He was the leading surgeon in France at the time, probably all of Europe. His daddy was a barber surgeon. His uncle was a barber surgeon. His grandpa was a barber surgeon and two of his brothers were barber surgeons. He was not an MD. He was not a surgeon. But he came the best known surgeon in Europe in the 1500s. His description exactly that these callus ulcers are sorted, hollow, hard callus, round but not simple, complicated. The method to cure them if cured it can be. Okay. If there’s blood flow, this can be cured. Okay. Henry Vandyke Carter described mycetoma or the fungal disease of India 1860. A year later, he followed up with another paper on mycetoma and he has a note in there. I have a notion that hospital gangrene is caused or exacerbated by fungus, 1861. In 1874, he described something, the red fungus that grew out of these wounds. Okay. 1908, Leo Buerger of Mount Sinai Hospital in Manhattan describes thromboangiitis or thromboangiitis obliteran. Eight years later, he followed up with the paper and said, “I think all these are fungal.” Frederic Mohs of Mohs surgery, 1941, he said all gangrene was infected. I’ll show you some of his data.


    And finally, Kenneth Thomson in 1944 described venous leg ulcers as surely fungal. How did he know that? He did five cultures in every wound. Five. He cleaned them off, he scraped them down and then he took his samples. Five samples. Okay. The yield in cultures is low. It’s somewhere around 20% to 40% depending on your technique. Okay. Let's see. Let’s begin at the beginning. So we’re going to quickly go through wound healing in normal. There’s the epidermis. It has a cornified layer of granular zone. This part in the middle is the spinous zone and then there’s a basal layer. Inside there, there are nerve fibers, Langerhans cells, which are the immune presenting cells, Merkel cells, the pressure receptors, and melanocytes. In the dermis, there’s type 1 and type 2 collagen. There’s a superficial flexes and a deep flexes. The basement membrane is made up of type 4 collagen. All review for you I’m sure. But hair follicles, sebaceous lobules and the eccrine glands are all important in wound healing as we’ll talk about in a minute. In the pathology laboratory, in our laboratory, we photograph every specimen. This is how it looks if it comes out of formalin. This was where the Shay biopsy was made and then they did a re-excision. My job now is to make sure is the cancer out, are the margins clear, is there any cancer even present? We ink it blue on one side and kind of purple red on the other and then we slice it about every 3 millimeters. We’ll take a look at this particular slice in the next slide. Day 16, here’s re-epithelialization. This tongue comes down along here but this is fibrin and red blood cells. This is re-epithelialization proceeding from this side. But there’s red blood cells, there’s fibrin and here’s your early scar that’s forming right down here at the base. I don’t think that adds anything except you can see the tongue of re-epithelialization. These cells crawl out and proliferate out of the eccrine ducts and produce this new epidermis. Okay. Diabetic skin, and all skin heals from below. The epidermis is said to regenerate from hair follicles eccrine ducts. If those are completely destroyed, then it has to crawl in from the sides and that’s a slow process. Deep wounds where these structures are destroyed, it’s a problem. It’s a slower healing. Dermis or deep wound is delayed healing in many ways unless you cut it out and bring the edges together. The dermis is repaired. It’s patched by granulation tissue which produces scar. Granulation tissue is made up of three parts, endothelial cells, fibrocytes or fibroblasts and the extracellular matrix. These cells crawl into the fibrin platelets and that’s the first matrix. And soon they produce mucin or hyaluronic acid then they produce soluble collagen, goes outside the cell, cross links and become scar. The mesenchymal stem cells, I wrote a paper on this in 1996, the mesenchymal stem cells in deep wounds are in the subcutaneous fat. As long as there’s fats still present there, you can grow a new dermis, at least the scar at dermis. Let’s talk about impaired wound healing in the general population. Age is important, especially sex hormones, testosterone and estrogen. Nutritional deficiencies are important. Vitamins B, C and zinc and protein malnutrition all those things lead to delayed healing. Smoking is bad. I’ll just leave at that. Medications especially steroids delay healing. Now, there’s a local diseases that have impaired healing. Look at all these infections, MRSA, pseudomonas, tinea, candida, herpes, leprosy, mycetoma. Aldo Castellani decribed cases of diphtheria. There was foot ulcer with diphtheria. They had clinical diphtheria. Now, this is 120 years, 110 years ago. Clinical diphtheria with a foot ulcer. There’s a variety of organisms that can inhabit a wound or you can develop a chronic infection in an ulcer. Abscesses for the past hundreds of years require drainage.


    They don’t heal well. Those will break open again and again until the abscess is drained. Foreign body granulomas or suture granulomas don't heal well. Thermal burns, chemical burns, and all these probably the most common harsh chemical that is used in clinical practice and a deep phenol burn, that goes right through and that chemical stays and it burns, and it burns, and it burns. They're hard to wash out because it binds the tissue. Prior radiation is delayed, necrobiosis lipoidica diabeticorum and cancer. Cancer can present as a nonhealing sore. If you think it looks like cancer, it needs to be biopsy. In our state-- in this state there are plenty of malpractice attorneys who would fine you if you miss the cancer. OK. How do we define delayed healing? Let's look at 1553 Ambroise Pare versus 2012. Pare defined out the maltreatment or optimal healing. He promised a patient with an ulcer to expect 50% closure in 15 days. You guys make promises like that to your patients? No, right. But he had an educated patient. He educated the patient and he treated them. That is a heroic tale in someday may we can talk about over cool beverage tonight the story of Ambroise Pare. But 2012 we call it 50% closure in 30 days is delayed. Well, think about he's expecting closure by 50% in 15 days versus we're going to defined when we fail. Once a positive definition and the others are negative. Many of the Europeans studies were use 100% healing in 90 days if it's not healed by 90 days it's delayed. OK. How do you treat delayed healing in legals? Let's look at Ambroise Pare. He was the surgeon of four French Kings. He couldn't pay for his board examinations so he went in the army. Some familiar to any of you? He went in the army and he became one of the best knows surgeons. He was a bright, bright guy. But when he made that promise he put an obligation on the patient. You must do all that I ask including bed rest, including compression, dressings, including purgatives, he gave mercury they really clean you out on those days from stem to stern when they treated you. Metal salt fixation, we're talking lead, mercury, we're talking harsh metals. This will fixed tissue. OK. And his definition of good wound care was to débride all which does not belong whether it's on the foot. If it's not native to the foot, you should take it off. Get rid of it completely. And use the general antiseptic of red wine. Do you think there's tannins in there, oak tannins by any chance, vinegar and saffron dye, red dye. And red dyes were used for 500 years in treating ulcers. OK. And he said according to Galen some 1,500 years before medicine should remain and prolonged contact with the wound to allow their effect to occur. OK. Frederick Mohs said all gangrene is infected, 1941. His second paper was on partial amputations in gangrene, OK. His first one was on using chemosurgery with mapping, OK. He use metal salt fixation in general antiseptics. There were no antibiotics in those days and he expected a granulating base by day 10. There are neurovascular studies then either. If granulation did not occur, if the tissue did not respond, move straight to amputation, OK. Now by mapping he literally mapped out and when he would excise an ulcer, a cancer he would make a map and where it did go and he would keep treating it until it was completely treated. Here's a picture from his paper showing a gangrenous toe and I don't know if you can read this. It says level of amputation in 24 hours level of fixation. So he fixed it and he amputated it and then he applied fixed it again and some of that would fixed down to a certain level. Day 2, he excised it again. Day 3, he excised it again. So he would fix and then débride the next day. He would do this several days in row two, three, four. How ever many days in a row. He treated them every day.


    Then he applied his dye and let them heal, okay. Now, by mapping, he would trace out every gangrenous sinus. Because these things tend to have roots. They’ll track down long bone, tendon sheath and so on. Okay, Fred Mohs, 1941. Okay, now we’re going to get to the scientific part, I have 10 minutes left. Glucoses of 400 to 600 milligrams per deciliter. We would give the animals streptozotocin, and in 21 days, you’d see visible skin atrophy with that kind. The skin would lose 40% of its thickness in 21 days. These animals are starving. They can’t get glucose into their cells. Their cells are sick. Sick healing results in a thin, weak, gaping scars with little collagen. They also have elevated collagenases, especially MMP9, okay. These cells are less motile, less contractile. Okay, so here’s the results to repeat that. We use streptozotocin in three weeks before. We treated them only for 14 days. The first 14 days with either petrolatum or 1% doxycycline. Then the animals were sacrificed at day 28 and collagenases were assayed, collagen was assayed biochemically and then histopathologically. But the results were pretty clear. All the diabetic wounds, these acute ones, were 100% reepithelialized. Now, they weren’t perfect wounds, let’s take a look. The point is nonhealing has a cause, and in these noninfected diabetic young animals, all these would heal. Now let’s take a slide through that. This is at day 28. This is a trichrome, the epidermis is up here, hair follicles. All the red structures either epidermis, hair follicles or the skeletal muscle. The blue structures in collagen, so here’s the normal dermis and you can outline the wound right here in cross section. Okay. The diabetic wounds are gaping. They’re depressed. This is quite thin, and the collagen is only half of what it is in the normal wound. Put them on top of each other. These wounds average 1.1 in the normals. So it contracts from 6 millimeters down to 1 millimeter. There’s a lot of contraction in these wounds. But in the diabetics, 2.9 millimeters, almost three times as wide. Same data. The question is, can diabetic healing be modified with collagenase inhibiters? I’ll show you two of them in a minute. What about treatment? Can you treat diabetic healing? Well, we use 1% doxycycline. It went from 6 millimeters down 2.1, somewhere in between, okay. The collagenases were less in those and there was nearly as much collagen as in the normals. One of the things that happens is this thinness to the scar, these are weak scars. Look how thick this one is and this one. These fill up completely, but this one, the diabetics do not. This is untreated diabetes, okay. Collagenase inhibitors, you can use them topically, you can use them low dose. Low dose tetracyclines have an inhibitory effect, even if they’re in sub-antimicrobial levels or if you use a tetracycline that has no antimicrobial properties. There are substituted tetracyclines that will do that. Percumin, there is 10,000 papers last year on percumin, and wound healing and all kinds of stuff. This is probably going to be something good because percumin as it turns out, is almost exactly 1/2 of a doxycycline molecule. Instead of a tetracycline molecule it's a doxycycline molecule. Otherwise they’re quite similar. Okay, we’re going to look at atrophy of normal skin in diabetes mellitus. The collagenases were elevated, the nerves were atrophied, the hair is absent and I've got five minutes and 45 seconds. This is atrophic skin right next to a lesion, but I want to show you this cigarette paper wrinkling. The dermis becomes thin, the vessels become prominent. Epidermis becomes thin and the vessels again are prominent.


    These vessels have thickened basement membranes that are reduplicated. That’s one of the major pathologies. If we look at vascular disease and diabetes, it's either large vessel or atherosclerosis, arterial sclerosis or the microvascular disease and it affects all these organs. Tobacco makes all these worse. How about alcohol? Mild to moderate alcohol consumption has a positive protective affect on myocardial infarction, but not stroke. This is from the Journal Atherosclerosis one year ago. In microvascular disease, look at these odds ratios, 0.6, 0.6, 0.36. These are favorable ratios improved less retinopathy, less neuropathy, less macroalbumin. Now, what some people call moderate, two to five drinks a week, other people might call breakfast, so you turn an alcoholic loose and you don’t know how they’re going to take it. But in general, there’s no medical indication to say they absolutely can’t, okay. These guys will negotiate everything with you. Okay, alcohol pathology lowers intraocular pressure, help solve the microvascular but stroke is still a risk. Here are the vessels in the microangiopathy. Okay, let’s look at necrobiosis lipoidica diabeticorum. In here you see there’s an infiltrate. You can’t really see too much. There are granulomas in here. If we do a histiocyte stain, the granulomas tend to follow vessels. Like here’s one around the vessel. Here’s the vessel, here’s endothelial. But it’s also around this nerve up here. That’s the basic pathology in the inflammatory stage. Let’s look at the clinical because these sometimes ulcerate. There's usually a yellow zone in the center and it oftentimes becomes white, okay. But the more active phases are red at the periphery. There’s oftentimes striking atrophy and look at this microvascular disease. If you look in the retina and solve these vessels, you’d say that’s microangiopathy, okay. Microangiopathy, look at these on the leg, tremendous atrophy, okay. White centers. These all comes from a site. This is the New Zealand Dermatology Society, dermnet.com. It’s a free site and you can look at these images. They have a number of good images on diabetes. This was put together by a friend of mine, so I used it. Ulceration in necrobiosis lipoidica. Okay, so in summary, it's about three times more common in women. It’s a microvascular disease with granulomas arises in atrophic skin, almost exclusively in DM. It’s almost a reportable case. In fact, in 1965, someone did report a case of a necrobiosis lipoidica diabeticorum. If you see it in a patient who’s not diabetic, what should you do? Work them up for diabetes, absolutely. Sometimes that’s how they present as with necrobiosis. Slow healing to a white scar and the differential would be more for you. Let’s talk about neuropathy. I have two minutes left. Normal epidermis, this stick diabetic. Normal nerves, no nerves. There’s cutaneous atrophy and nerve fiber loss. Can it be treated? Well, this is one patient treated for a year. The epidermis became about twice normal thickness and there were never fibers again. So maybe there's hope for these, I don’t know. This is a tough battle. Same nerve fiber. Experimental denervation impairs wound healing and it doesn’t matter whether it's in ligaments, tendons. There’s delayed healing with denervation or neuropathy, even outside of diabetes. It seems to be a loss of substance P and calcitonin gene-related peptide, okay. I’m getting close to the end. Cellular proliferation is where the power to heal comes from. You have to repair the dermis by this proliferation. You have to allow that to heal. You have to get that to heal. Debridement, to manage this is important because it [indecipherable] [29:36] granulation tissue and bleeding brings fresh growth factors and new proliferation. Okay, these are general causes. I said full thickness injury burns, where there's specific causes. We mentioned cancer, foreign body. I’ll make a mention of cotton fibers because I don’t like cotton fibers. I don’t even like them in laboratory. I won’t even paint ink on the tissues with it because the cotton fibers stick to the tissue.


    I have a preference that you avoid cotton fibers, but there all kinds of polyester things that you can use to apply medicines with and not cotton tip applicators. A wooden stick is better than a cotton tip applicator. Debridement causes granulation tissue to grow anew, fibrin and new growth factors come to the healing site and send tissue to pathology. Any questions? I think we’re done.