Primary vs. Secondary Healing

by Richard Mitchell, MD

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    00:01 Welcome.

    00:02 So we're just about through making a scar in tissues that cannot regenerate.

    00:07 The final stages.

    00:09 Now that we've laid down new blood vessels is to deposit and remodel the extracellular matrix.

    00:18 Here's where we are on our overall roadmap.

    00:21 Remember, we've talked about moderating acute inflammation, recruiting and activating macrophages, regenerating the parenchyma, if we can.

    00:29 And now these last two topics are when we cannot completely regenerate.

    00:35 So we have to have angiogenesis, granulation tissue.

    00:39 And now we're going to deposit and remodel the matrix.

    00:42 So let's get going.

    00:45 Here we are on our sequential curves.

    00:49 And really at the peak of the chronic inflammatory response, where the macrophages are peaking at three to five days after the original injury, read the green arrow.

    01:00 And at that point, we are starting to lay down granulation tissue, and that's going to peak at somewhere between seven and 10 days.

    01:07 And even with the very earliest stages of new vessel formation, we're also starting to lay down the matrix that's going to be a scar.

    01:18 We may also have some degree of regeneration.

    01:20 Remember, the macrophages are still cranking out growth factors.

    01:25 But when we're going to have scar, it's going to be mostly that fibrosis.

    01:30 Okay, let's get going.

    01:33 This is a transverse section of an animal preparation of a heart, where the heart in the upper left hand side had a myocardial infarct induced by tying a suture around the coronary artery.

    01:51 This is a five to 10 days.

    01:53 And the special stain here is a trichrome stain, where blue is collagen.

    02:00 And we can see in the area of the infarct on the upper left hand side that we do have some blue, but it's pretty blue's, and it's not well developed.

    02:10 Okay, that's a five to 10 days when we're at the peak of granulation tissue.

    02:15 If we wait four weeks, wow, we have quite a bit of dark peacock blue.

    02:21 And that blueness there represents the deposition of collagen.

    02:26 That's where we're going to go for the next few slides.

    02:29 We are over the course of four weeks on that provisional matrix of the newly formed blood vessels, the granulation tissue.

    02:35 We're going to lay down extracellular matrix, and then we're going to remodel it.

    02:40 So "Factor 5" We've had Factor 1: Interferon gamma.

    02:44 Factor 2: Interleukin-1 / TNF.

    02:47 Factor 3: Epidermal Growth Factor.

    02:49 Factor 4: the angiogenic factors, basic FGF, and VEGF.

    02:53 "Factor 5", in quotes, remember, is Transforming Growth Factor-beta.

    02:58 This is actually a pretty important one, and worth remembering, because it really does drive the final stages of scarring.

    03:06 So it's important for causing fibroblasts migration, chemotaxis, and to drive them to synthesize matrix.

    03:17 It also will cause the reduced expression of matrix metalloproteinases.

    03:24 So there are various ways that we can degrade matrix.

    03:26 Matrix metalloproteinases are a major pathway.

    03:29 There's several of the matrix metalloproteinases in excess of 20.

    03:33 And when they are chewing away, they're degrading matrix.

    03:36 But if we turn them off, if we turn off their synthesis, then we can get more matrix accumulate.

    03:43 So this is happening also because of TGF-beta.

    03:47 An important brake on the entire process, everything up to this point has been kind of go go, go, go, go go.

    03:53 Well, at some point, we have to turn it off and go back to just being whatever it was to begin with.

    03:59 And TGF-beta inhibits the inflammation.

    04:02 It's going to be a very important anti-inflammatory break on the entire process.

    04:08 TGF-beta is synthesized in an inactive form by activated macrophages and T cells.

    04:13 As well as smooth muscle cells and endothelial cells.

    04:17 Its importance, they're in an inactive form, it's actually secreted into the provisional stroma, and it's sitting there.

    04:25 And it doesn't do its thing until we have proteolysis, until we have enzymes, proteases elaborated that will digest it.

    04:32 So, that's a fairly nuanced way to have regulation of this particular activity.

    04:40 It's going to be an important mechanism by which we shut off inflammation, and promote scarring.

    04:46 So that's what you need to remember that TGF-beta does.

    04:53 After we'd lay down the scar, we just don't leave it there, but in fact, we remodeled that.

    04:58 So, let's look at our M1 macrophage again.

    05:01 It's providing a whole bunch of M1 macrophage cytokines such as epidermal growth factor, interleukin-1, tumor necrosis factor.

    05:09 Those will actually drive fibroblasts to make precursors procollagenases, prostromelysins, that when activated by plasmin and other activators, that will create matrix metalloproteinases, that degrade the matrix.

    05:26 That's part of the original, cleaning up the debris is to break down all that degenerated extracellular matrix.

    05:34 And that's what M1 macrophages do.

    05:37 Degrading the matrix.

    05:40 M2, mainly through the effects of TGF-beta turn off that process.

    05:44 So we don't make those matrix metalloproteinases.

    05:48 This will allow us to accumulate extra matrix.

    05:52 So part of what TGF-beta is doing, besides turning off inflammation is inhibiting the breakdown of matrix.

    05:59 And that's going to allow us to synthesize more.

    06:04 M2 macrophages also make tissue inhibitors of metalloproteinases.

    06:09 So, this is getting again into alphabet soup.

    06:12 But TIMPs are molecules that inhibit any activated matrix metalloproteinase.

    06:20 So not only do M2 macrophages through TGF-beta turn off fibroblasts synthesis of things that would be MMPs that are active matrix metalloproteinases that are active to cleave matrix.

    06:33 There are also banking inhibitors that will turn off any residual MMPs that might be out there.

    06:41 Okay.

    06:43 Steroids.

    06:44 And I mentioning this as a sidelight because steroids are frequently given by plastic surgeons and other people to limit scarring.

    06:51 How is that happening? Well, steroids, in fact, inhibit both M1 and M2 macrophages to some extent.

    07:00 That's part of what makes them so potent in terms of anti-inflammatory agents.

    07:05 But they turns out, they inhibit M2 macrophages much, much more.

    07:11 So they are more effective on that.

    07:12 So there is a relative decrease in the amount of TGF-beta.

    07:16 So, there will be a relative increase in the amount of matrix metalloproteinases.

    07:21 And a relative increase in the degradation of the matrix, because we are having more of an effect on M2.

    07:29 So all things considered, if we give steroids, we get less scarring, because there's reduced fibrosis because of less activation of M2, and that's why we give that.

    07:39 On the other hand, if we really want good scarring to occur, we have a big surgical incision, and we want it to heal up really nicely, we don't want to give steroids.

    07:49 And patients who are on steroids for autoimmune disease need to be very carefully monitored, and the steroids adjusted, so that we don't have this effect.

    08:00 Okay.

    08:03 So the general overall picture that you need to keep in your mind when we're talking about injury in non-regenerating tissues, or when there's a lot of damage, is if we're gonna have scarring, we're gonna have fibrosis.

    08:15 So the first panel there we see ourselves, we see an area of injury where cells have died.

    08:20 And we have lost some degree of matrix around that area, or some blood vessels, okay.

    08:27 We then recruit in neutrophils first and then macrophages so that we get the tissue breakdown and set the stage for the subsequent healing responses.

    08:36 In these tissues that cannot regenerate, we get angiogenesis, we get granulation tissue.

    08:40 So we're seeing the sprouting from those vessels into the area of injury.

    08:45 Being recruited those new vessels by the macrophages that are in there.

    08:50 And then on that provisional matrix, on those railroad tracks, we bring in fibroblasts that proliferate, migrate, and lay down new matrix.

    08:59 So we get ECM, extracellular matrix deposition.

    09:03 And it's a combination of increased synthesis, and decreased degradation because we are inhibiting matrix metalloproteinases.

    09:11 In a minute, we're also going to see the next stage.

    09:14 where that scar changes.

    09:15 It's not just always going to be that big lump of fibrosis in the middle, it's going to remodel.

    09:22 But this paradigm is extremely stereotypical.

    09:27 And it happens in any tissue that cannot completely regenerate.

    09:32 And it's going to be kind of a major underlying process that will occur in a lot of pathologies.

    09:38 So we will revisit this in other guises.

    09:41 But this is a very stereotypical response in any healing injury.

    09:47 Okay, so in Non-regenerating tissues: The principal mediators and scarring.

    09:53 First of all we have the tissue breakdown, and macrophage activation, and that's through interferon gamma.

    10:00 We get neutrophils, we get macrophages, we get lysosomal enzymes, we get reactive oxygen species.

    10:05 And then we get angiogenesis, driven by basic FGF, and VEGF.

    10:11 All the activities of by those two factors and more, but those two factors, mainly.

    10:16 Fibroblasts need to migrate and proliferate.

    10:19 This is going to be growth factors, epidermal growth factor, interleukin-1 and TNF.

    10:25 They're going to deposit matrix through increased synthesis of matrix and decrease degradation, and that's driven by TGF-beta.

    10:33 And then we're going to have inhibition of cellular activation.

    10:36 We're gonna put a brake on the entire process.

    10:39 And again, really importantly, that's TGF-beta.

    10:43 So on this one slide, we've kind of summarized the important factors that you need to kind of keep in mind.

    10:50 Let's remodel this scar.

    10:53 So those of you who may have had a surgical procedure, know that very early on of the procedure is kind of a red looking, angry thing.

    11:04 And over time, that scar gets smaller and smaller, and more white.

    11:11 Why does that happen? So again, we're looking at overlapping effects.

    11:16 And slightly different than what we've seen previously, but inflammation, acute and chronic, kind of occurs over a period of two to seven days after injury.

    11:26 Granulation tissue overlapping five to 10 days.

    11:29 And it kind of peaks in that period of time.

    11:32 And then the dotted line that's going up, that's collagen accumulation.

    11:39 And there will be remodeling.

    11:40 So the cells that are in there, that are making the matrix are actually as it turns out, modified smooth muscle cells.

    11:49 They are fibroblast like, but smooth muscle like.

    11:52 And over time, they can contract, and we get a smaller wound.

    11:59 And over time, we also release matrix metalloproteinases, and don't release tissue inhibitors and metalloproteinases.

    12:08 And we get some restructuring of that matrix.

    12:12 And over a period of weeks to months, that wound will contract.

    12:16 This has important ramifications not only in terms of making that scar on your abdomen smaller and less conspicuous, but let's say, I have a total burn on my entire hand, and it's scars.

    12:29 And then if I don't have regular physical therapy, that wound will contract.

    12:35 And instead of having a hand, I'll have kind of a claw.

    12:38 So wound contraction actually has ramifications.

    12:42 As we'll see, when we talk about the complications associated with wound healing.

    12:51 Then this is now wound healing kind of one on one.

    12:55 This is what we worry about a kind of the surgical level.

    13:01 So surgeons talk about healing by first intention and healing by secondary intention.

    13:07 Healing by first intention or by primary intent.

    13:10 We have a nice kind of circumscribed injury, such as an incision that we cut into the tissue, and then we sew back together.

    13:20 So we approximate the edges.

    13:22 Clean, not infected, all of that.

    13:25 But the injury that's going on there and then typical wound healing process will lead us to have all the usual things.

    13:34 We will get inflammatory mediators to get elaborated, we'll get a little bit of angiogenesis, we will grow into that area.

    13:41 So over time, we'll get granulation tissue right on schedule, seven to 10 days.

    13:46 And then in that area of injury, we will regenerate mostly, but we will lay down a little bit of scar.

    13:52 Okay, that's healing by first intent, where we don't have big areas of damage.

    13:58 And it's just following the usual processes that we talked about.

    14:02 And then there's healing by second intent.

    14:05 And this happens when we don't want to necessarily close it up.

    14:09 This will actually happen quite frequently.

    14:11 You'll see it on the wards when a patient has a large decubitus ulcer, for example.

    14:16 Where the tissue has not healed, and there's a big area of ulceration in the skin.

    14:21 You don't want to close that up because there will be bacteria and other things in there that will get infected.

    14:26 So what you do is every day, as a medical student, go in, pack the wound with sterile gauze, sometimes containing antibiotics.

    14:34 And then the next day, you come along, take out the gauze and debris the wound and you'll see granulation tissue in there, and slowly but surely, that big ulcer will eventually close in and you'll get skin over the surface of that.

    14:48 That's healing by second intent. And we do it all the time.

    14:52 But it's a much bigger process with much more inflammation, much more injury, and greater angiogenesis, and we count on eventually that getting smaller, and smaller, and smaller because of wound contraction.

    15:04 So, here we have this big open area of ulceration, decubitus ulcer with a big scab in it, which is just coagulated blood products.

    15:14 And we are now recruiting because of the injury, all the macrophages that are going to drive angiogenesis as you see them in the new capillaries.

    15:23 And you're going to begin the process of filling this in slowly but surely.

    15:27 And over time, you will have a big area of fibrosis that will eventually over time contract.

    15:33 Note to, on this last piece.

    15:37 See in the middle one where there was a new capillary sprout, that was necessary to maintain this highly metabolic scar activity? Once we have scar, scar is actually kind of low metabolic activity.

    15:49 It doesn't take much blood supply to maintain a scar because it's mostly extracellular matrix.

    15:56 So over time, not only does that wound contract and gets smaller because of the myofibroblast, but it also blanches.

    16:05 We don't need as much vasculature, so that remodels and that goes away.

    16:10 And that's why a wound over a longer period of time starts out very pink because of neovascularization, and it ends up very white because we don't need much blood vessel supply to keep that scar alive.

    16:21 So, long term scars, get wider and wider, paler and paler.

    16:26 And they get smaller and smaller, up to a point.

    16:30 Primary versus secondary healing.

    16:32 So primary healing, there's mild inflammatory infiltrate because there's not much damage.

    16:36 In secondary healing, healing by secondary intent, more intense inflammation because we have these big areas of injury.

    16:44 There is less granulation tissue, in the primary intent.

    16:47 There is greater volume of granulation tissue and therefore a greater amount of resulting scar.

    16:53 And in the primary wound, there is not much significant wound contraction.

    16:58 You really haven't had much damage, but there may be significant wound contraction that occurs in healing by secondary intent.

    17:06 So with that, we've kind of walked all the way through, from acute to chronic inflammation, to angiogenesis, to wound healing, and we are ready to talk about the complications of wound healing.

    About the Lecture

    The lecture Primary vs. Secondary Healing by Richard Mitchell, MD is from the course Acute and Chronic Inflammation.

    Included Quiz Questions

    1. 3-5 days
    2. 7-10 days
    3. 12-24 hours
    4. 14-21 days
    5. 1-3 days
    1. Collagen
    2. Elastin
    3. Fibrillin
    4. Fibrinogen
    5. Dynein
    1. It is responsible for fibroblast migration.
    2. It is produced mainly by neutrophils.
    3. It plays a major role during the first 12 hours after injury.
    4. It does not affect the production of the extracellular matrix.
    5. It inhibits B-cell lymphocytes.

    Author of lecture Primary vs. Secondary Healing

     Richard Mitchell, MD

    Richard Mitchell, MD

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