The Role of Macrophages in Controlling the Adaptive Response to Injury: Regeneration Vs. Scarring

Abstract

This chapter will cover the apparent role macrophages play in orchestrating the adaptive response to injury. The chapter will first explore the differences in adaptive response to injury for fetal vs. adult wound healing. In addition, the differences in adaptive response between animals that regenerate vs. ones that heal more by scarring. This information will be used to propose a theory of how to control the adaptive response by controlling the macrophages response. Part of this theory will be what is the evolutionary change in macrophages that tips the scale between regeneration and scarring as well as what is different about the response of macrophages in fetal vs. adult wound healing. The body responds to changes (stimuli) with an adaptive response. Additional stimuli can be added to an injury to alter the response of macrophages to effect the overall adaptive response. The theory developed helped to explain why specific strategies to control the adaptive response are successful.

1. Introduction

This chapter will cover the apparent role macrophages play in orchestrating the adaptive response to injury. Although macrophages are potent innate immune cells injury triggers an inflammatory response and a non-specific foreign body response virtually independent of an immune response (unless antigens such as bacteria are introduced into the wound).

As with any stimuli, there is a normal adaptive response to injury. Macrophages play an important role in controlling this adaptive response as well as why there are differences between species or as we age. The goal of this chapter is to explore the role of macrophages in this adaptive response to develop a theory on how macrophages control the response.

The chapter will first explore the differences in adaptive response to injury for fetal vs. adult wound healing. In addition, the differences in adaptive response between animals that regenerate vs. ones that heal more by scarring. This information will be used to propose a theory of how to control the adaptive response by controlling the response of macrophages. Part of this theory will be what is the evolutionary change in macrophages that tips the scale between regeneration and scarring as well as what is different about the response of macrophages in fetal vs. adult wound healing. This theory will be used to explain why specific strategies to control the adaptive response are successful.

2. Adaptive response to injury

The body responds to changes (stimuli) with an adaptive response. The goal of this review is to understand how macrophages control the normal adaptive response, in this case for a skin injury, as well as how additional stimuli can be added to alter the macrophage response to control the overall adaptive response.

This section will look at the normal adaptive response to a skin injury as well as differences between animals that regenerate and those that do not. In addition, the difference in adult wound healing vs. fetal wound healing will be explored. Based on these observations a theory will be presented on how the macrophage causes these differences in adaptive response to injury.

2.1 Adaptive responses to injury

The adaptive response to injury can be regeneration, grow larger to fill the space (hypertrophy), or use scar tissue to fill the space [1, 2, 3, 4]. In many cases, it can be a combination of these responses. Although the adaptive response is seen at the tissue/organ level [3] it is first triggered at the cellular level [4], which coordinates the ultimate response [5].

2.1.1 Regeneration

Regeneration in this case is to recapitulate structure and function of tissue after an injury. An important issue is how good are humans at regeneration? Of the four tissue types (epithelium, muscle, nerve, and connective tissue) only epithelium is totally regenerative [2, 3]. The regenerative ability, however, is limited and requires assistance in some cases.

For each tissue type, we have had successes in small amounts, but not large areas. The biggest reason is the inability to get blood supply fast enough for a large area. Even in skin grafts, they do not get blood supply fast enough (a few weeks in many cases), but it is able to recover sufficiently to take (heal in) [6]. This inability to form blood vessels in graft substitutes has hampered the ability to create actual graft substitutes that heal in [2, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16]. It also hampers the ability to seed scaffolds to use in vivo [2, 6, 7, 8, 9, 14]. The cells in the middle do not get blood supply fast enough to survive [1, 2, 6].

2.1.2 Loss of regeneration

Limits on regenerative healing is driven by evolution [8, 17, 18]. Certain species still have the ability to regenerate large structures (epimorphic regeneration) [8]. In humans, the loss of regenerative ability is seen as we age [8]. In utero, healing is regenerative and scarless. Even newborns can regenerative digits that are accidently cut off [19]. The length that can regenerate, however, decreases over time [19].

The question is how much of this ability can we get back? Understanding why we probably lost this ability and what changes occur between fetal and adult wound healing can shed some light on how we may be able to better control the healing response. It is likely that the normal adaptive response is genetic (evolutionary). However, adding additional stimuli will create additional adaptive responses that can alter the overall adaptive response. It is also likely that the differences that occur due to age are not genetic, since the genes do not change over time.

Through evolution and natural selection, the scale has tipped toward scarring over regeneration in humans [20]. Not entirely, however, since we can regenerate blood as well as other tissues and organs to a limited degree (e.g. bone, smooth muscle, liver, and epithelial tissue) [21]. It is not that we really lost it, since virtually all tissue is turned over at some rate (days to years depending on the tissue); most tissue has at least limited regenerative ability [1, 2, 3, 5, 8, 22, 23]; and we can increase the rate of regeneration with things like growth factors, stem cells, and electrical stimulation [1, 2]. Typically tissue that heals quickly (higher mitotic rate) has the fastest turnover rate [3, 4]. For example, epithelium is the fastest growing tissue and has the quickest turnover rate [21]. Even in thick layers of epithelium like epidermis, it only takes about 30 days for a basal cell to move upward to form all the layers of stratified squamous epithelium [1, 3, 21].

Further, babies up until about 6 months can regenerate an amputated finger and in utero surgery is essentially scarless [8, 19, 22, 23]. So we have the machinery to regenerate, but scarring normally dominates; and even more so when healing time increases as in larger wounds, more inflammation, or less stem cells (as we get older or with certain diseases) [1, 2, 8]. So many strategies geared toward speeding up healing have the added benefit of reducing scarring [1, 2].

So what has tipped the scales from regeneration to repair or scarring? From an evolutionary standpoint it is probably related to speed of recovery [4, 18, 23]; possibly why scarring increases as healing rate decreases. It may be as simple as if a wound does not heal within a specified time frame, the wound contracts and scars. Also from an epimorphic regeneration standpoint it could be how quickly the skin can cover the wound. The only thing that seems to shift is the maximum length of healing time before scarring starts to replace regenerative healing. There also are individuals that have a more robust scarring than the average, which can lead to hypertrophic scarring for even small wounds.

Why is scarring an evolutionary advantage? In part, “survival of the fittest” (natural selection) is more about those who can reproduce vs. that only the strongest survive [18, 19, 22, 23]. Many animals can regenerate large parts of their body [18]. As the species and/or part gets larger (and more complicated) it takes longer to regenerate and in most higher animals it serves more as a defense mechanism than for healing [18]. For example, a lizard’s tail comes off easily and will distract the prey, but requires nine months to regenerate [18, 23]. The African Spiny Mouse (skin comes off) and the axolotl salamander are other good examples of larger animals regenerating [18, 23].

Although evolution and natural selection may have kept regeneration as a defense mechanism to help in survival, for many larger species, the length of time for regeneration and the metabolic demand probably made it an evolutionary disadvantage [3, 4, 18]. It could make an animal easier prey, reduce their ability to reproduce, and increase the risk of infection [18, 22]. To that end, many mammals will kill their young if they are injured or deformed and zoos often have to rescue babies who might be killed by their parents [24].

Some claim it is partly due to mammals being warm blooded and becoming terrestrial [18, 22]. Warm blooded, makes infection easier [23]. Terrestrial means that legs would need to be weight bearing during regeneration and could be easily damaged [18]. Also as animals got larger and warm blooded the length of survival time without food has diminished [18]. It has been shown that the shorter the time an animal can survive without food the less likely it can have regenerative healing [18].

This is all also consistent with the theory that the only evolutionary change is how quickly scarring starts replacing regenerative healing. This means that the normal adaptive response tips more toward scarring. In many cases, in today’s world, with better infection control and the reduction in the need for fast healing, it is probably beneficial to tip the scales more toward regenerative healing. Since waiting for an evolutionary change could take at least 50 generations, once the mutation starts showing up [18, 22], strategies to control the response are a better option.

It appears, therefore that the bioprocess to control is time to complete healing. There are two bioprocesses that affect the time to heal: the delay in healing due to inflammation (since healing is limited during inflammation) and the rate of healing in the repair phase.

2.1.3 Mammalian regenerative healing

To explore this concept of shortening the inflammation part of healing more it would be helpful to look at examples of regenerative healing in mammals (including fetal wound healing). There are very few cases in adult mammals that undergo true (epimorphic) regeneration [8]. These include deer antlers and the ear of a rabbit [8]. These require blastema formation and require the epidermis to seal the growing blastema [8]. A hole punched in the rabbit ear can undergo epimorphic regeneration where ears of dogs will scar vs. regenerate [8].

Most injured tissue in adult mammals heal by repair and scarring (going through the inflammation, repair, remodeling phases) [8, 17, 18]. In the case of small defects the tissue can expand (hypertrophy) without mitosis to fill the gap—skeletal muscle is one example [8]. Also part of repair and scarring is wound contraction to reduce the amount of hypertrophy necessary [1, 2].

Regeneration of the finger of an infant is like epimorphic regeneration, but is more outgrowth of the bone [9, 19]. Fetal wound healing is essentially scarless, but does not require a blastema [23]. Without a skin wound, some internal structures can heal scarlessly and do not have a blastema formed [8].

Although we have learned much about epimorphic regeneration, it is unlikely to be a useful strategy in adults [8]. Besides the examples of deer antlers and rabbit ears, the process has not been able to be recreated in mammals, even with similar tissue to those found in other animals [21]. This is because there are a few requirements that are difficult to recreate at the same time including: skin injury, epidermal tissue quickly covering the injury, enervation, low or no inflammation, and the ability to start repair quickly (local cells or recruited cells) [4, 8].

It is also probably both a size and complexity issue, which is why it is only found in small animals less complex than mammals [8]. This would be consistent with the theory that it is about time to heal (including the time with little healing due to inflammation) as the trigger for scarring. Even, if we could get it to work for large structures like digits or limbs, however, it is unlikely that the size would be an adult size; since it takes almost two decades for human digits and limbs to grow to adult size [23].

Without a blastema, regeneration of structures such as digits and limbs has a number of hurdles we have not solved yet including: shutting down the natural scarring response, revascularization, re-innervation, acceleration of healing, and duplicating the 3D architecture macroscopically and microscopically [6, 23]. These would be both growing in vitro as a graft substitute or in vivo with a degradable regenerative scaffold [2, 6, 7]. A graft substitute has the additional problem of attachment to and integrating with the host [2, 6, 7].

2.1.4 Fetal wound healing

To further explore the theory that it is the time to heal that is the trigger for scarring, it is helpful to look at fetal wound healing vs. adult wound healing. Again since wound healing in utero is scarless and the regenerative ability decreases over time, understanding the differences between adult and fetal wound healing can offer some potential control strategies [23]. Although there are many specific differences, between the two, it is possible they are mostly related to two phenomena that change over time: the ability for cells to do multiple things (due to stem cells) at the same time and the size of the injury [4, 8, 25]. Both affect the time to start healing and the length of time for healing to occur [2, 4, 25]. In fetal wounds there are more local stem cells and therefore do not require as much homing of stem cells from the circulation (primary source in adult wounds) [2, 4, 25]. It also is possible that there is no true scarless healing without epimorphic regeneration and scarless healing is just no detectable scar [2, 23]. Then the amount of scarring increases as we age [17, 23].

Again, time to heal is both the length of the inflammatory phase and the length of the repair phase. The ability to heal during the inflammatory phase (due to stem cells) reduces the normal delay in healing due to the inflammatory response. The size difference between the fetus and the adult leads to a shorter repair phase for the same type of injury. Both of these advantages in fetal wound healing decrease as we age, helping to explain the decrease in regeneration as we age.

2.1.5 Developing strategies to reduce scarring and increase regeneration

Based on the differences between fetal wound healing and adult wound healing as well as potential reasons some animals have more regenerative ability than others, some control strategies can be developed. Again, the theory is that the length of time to heal is the trigger to start the scarring process. This is both the delay in healing due to inflammation as well as the amount of tissue to be healed determining the length of the repair phase. For example, incision wounds can heal without visible scarring even in adults, if clean and protected from forces that will break newly formed tissue [2, 25]. The amount of cleaning up (foreign material, necrotic tissue, bleeding, etc.) determines both the amount of inflammation and the length of the inflammatory phase [2, 25].

Although there are many biochemical differences between animals that regenerate (or fetal wound healing) and adults, the main evolution change could be just the length of healing time required to trigger scarring. This can be the total time to heal and/or the time required before healing is complete. Therefore, shortening the inflammatory phase or the repair phase should reduce scarring. In addition, increasing the relative amount of regenerative healing vs. scarring during the inflammatory phase, which appears to occur in fetal wound healing, or repair phase should also reduce scarring.

Looking at the typical adaptive response to injury can shed some light on what the specific evolutionary change might be as well as ways to control the adaptive response. This means, which bioprocesses control the time to heal and how they do it.

2.2 The usual adaptive response

2.2.1 Healing response

Since regenerative epimorphic healing is not an option in adults and hypertrophy only occurs to a limited extent, then healing occurs mostly through a repair process. Healing of an injury normally has four phases: hemostasis, inflammation, repair, and remodeling. The first step (hemostasis) is essentially stopping the bleeding. This normally occurs via the blood clotting cascade and ends up with fibrinogen, a blood protein, being cleaved by thrombin to allow polymerization into a fibrin clot [2, 21, 25]. The next step is the inflammatory response, which is to clean up the wound [25]. This can be removal of foreign material, dead tissue, and or products of hemostasis including: red blood cells, edema, and fibrin clot [25]. Neutrophils and macrophages are responsible for this phase [2, 25].

In adults, typically, little healing occurs during the inflammatory phase. If any it is predominantly granulation tissue (repair tissue with inflammatory cells and well vascularized) and/or scarring to wall off the granulation tissue [2, 8].

Once the inflammation subsides it turns into the repair phase. In some cases, the inflammation never goes away and becomes chronic inflammation (common with non-degradable implants particularly ones that are fibrous scaffolds) [2, 8]. Therefore, there is not always a clear demarcation between the inflammation and repair phases. In the repair phase, new tissue is formed to fill the defect. It is a mix of granulation tissue and regenerated tissue (similar to the tissue being repaired). This requires a number of interrelated events. To form the extracellular matrix (ECM) repair, cells need to migrate into the wound space once cleaned up by inflammation. The cells need to produce the ECM scaffold in front of them to allow them to grow into the wound [2, 5]. The cells need enough oxygen to produce the ECM, which requires blood vessels to grow into the wound as well [2, 25]. The cells, usually fibroblasts, need the blood supply to be within 100 μm to provide enough oxygen via diffusion to produce the scaffold [2, 6]. This works in a centripetal fashion from the wound edges until the defect is repaired; shutting down probably due to contact inhibition [2, 5]. In most cases, the repair tissue gets remodeled (fourth phase) over time to be closer in structure and function to the normal tissue [2, 5, 6]. The closer the repair tissue is to the surrounding tissue the less remodeling needs to be done.

Due to underlying pathology, the defect may not completely heal. The most common problem, however, is the production of scar tissue, which reduces function [2, 21, 25]. Although not fully understood, it appears that the extent and time frame of the inflammatory phase is one of the most important factors to determine the extent of the scarring [2, 8, 23]. Also typically the more the repair tissue is granulation tissue vs. regenerated tissue, the more scarring will occur [2, 8].

There are a number of interrelated bioprocesses that determine the extent of scarring [5]. These include: the amount of inflammation, the length of the inflammatory phase, the length of the repair phase, and the activity of both macrophages and fibroblasts [2, 26]. Although, it is unclear, which bioprocesses are triggers for the other bioprocesses, the end result is less regenerative healing until wound closure (end of the repair phase), which leads to a higher percent of granulation tissue and increased wound contraction (producing scar tissue) to reduce the size of the wound and thus shorten the healing time [2, 3, 4, 5, 7, 25]. Many diseases are due to inflammation leading to scar tissue including: arteriosclerosis and cirrhosis of the liver [2, 5, 21].

Figure 1a shows granulation vs. regeneration tissue percentages (in the repair phase) as the amount or time of inflammation increases due to cleaning up the wound. It is likely that in some cases, particularly in small wounds, inflammation does not trigger granulation immediately.

Figure 1.

Cell and tissue changes during the repair phase of wound healing. a. As the amount and time of inflammation increases (mostly due to cleaning up of the wound), the higher percentage of healing is granulation tissue. Part of the theory proposed is that animals that are more regenerative would require more clean-up/inflammation before increasing the percentage of granulation tissue formed (green and red lines). b. As the amount and time of inflammation increases (mostly due to cleaning up of the wound), a higher and higher percent of macrophages are pro-inflammatory vs. pro-healing. Again, animals that are more regenerative would probably require more clean-up/inflammation before increasing the percentage of pro-inflammatory macrophages (green and red lines). c. The ratio of pro-inflammatory macrophages to pro-healing macrophages should control the ratio of myofibroblasts to fibroblasts, which in turn will control the ratio of granulation tissue to regenerative healing (a), which will control the ratio of scarring to healing (d).

2.2.2 Evolutionary control of the adaptive response

Although it is not known what the evolutionary change is to push toward scarring, it is unlikely to be a biological clock. The difference is probably the amount of wound contraction due to the fibroblast/myofibroblast transformation, but the actual genetic difference has not been identified [1, 2, 26]. It is likely that macrophages control this transformation as well as any mechanical stress present. Many claim that macrophages orchestrate all the healing responses [2, 26]. The adaptive response of macrophages can be to help in the healing process or create a chronic inflammatory response. In addition, macrophages present antigens to lymphocytes to start the immune response [2, 26].

Although there has been work on identifying different phenotypes of macrophages (what they do and produce) and what are the triggers to change phenotype, there still is much to learn about how to control the phenotype of macrophages as well as if there are evolutionary differences in activation of macrophages between animals that heal more by regeneration vs. those that heal more by scarring. One change is the ability to form a blastema, for complete regeneration. The change between animals that heal without a blastema would be different. It is possible that the evolutionary change is the amount of stimulus to increase the ratio of pro-inflammatory macrophages to pro-healing macrophages as shown in Figure 1b. This could be a conversion of phenotype of resident macrophages or a recruitment of different types of macrophages from the blood. Macrophages, even in adults, can clean up some debris, like red blood cells and dead tissue without triggering an inflammatory response [26]. Figure 1b shows how the percentage of each type of macrophage could change based on the sensitivity of macrophages to cleanup/inflammation.

It is also possible that a given macrophage can be on the continuum from pro-healing to pro-inflammatory in addition to the relative amount of macrophages that are pro-healing vs. pro-inflammatory. It is also possible that different types of macrophages recruit different types and or numbers of macrophages from the blood or that they can change phenotype after they get to the wound. All of these are still consistent with the theory. It is also likely that the more pro-inflammatory macrophages in the wound the longer the inflammatory process, since resolving the inflammation involves removal of the pro-inflammatory macrophages.

Lengthening the inflammation phase will increase the overall healing time, since little healing besides scarring occurs in the inflammatory phase. Much of the regenerative healing, during inflammation, could be due to recruited stem cells from the blood, which again is why fetal wounds heal more by regenerative healing due to the presence of more local stem cells.

In adult wounds, the type of tissue formed is mostly due to the fibroblast to myofibroblast transition (Figure 1c), which is most likely controlled by the pro-healing to pro-inflammatory ratio. Myofibroblasts are responsible for the granulation tissue that contracts and leads to scarring (Figure 1a and d) [2, 23].

So, the evolutionary change could be the amount of clean up macrophages can do in a wound without shifting to a pro-inflammatory phenotype where only scarring can occur until the inflammation is resolved. There also is a trigger based on the length of time to close the wound (dependent on the application). This probably is a big factor in why adult wound healing scars relative to fetal wound healing.

Part of the theory is that the fibroblast to myofibroblast ratio set by the phenotype ratio of macrophages lasts through most of the repair phase, although a macrophages phenotype will revert back to pro-healing (unless there is a chronic inflammation). This ratio does set the granulation tissue/regenerative healing (G/R) ratio, however, it is likely that the granulation tissue healing rate is faster than the regenerative healing rate; leading to an increase in the G/R ratio over time. Therefore the longer the wound is in the repair phase the more there is tissue that contracts and scars relative to tissue that has regenerative healing.

Understanding the evolutionary changes as well as the fetal to adult wound healing differences can help in selecting designs to control the adaptive response. It can be by controlling the triggers for the transitions for macrophages and/or fibroblasts as well as controlling the biological responses to these transitions.

Although the theory presented is most likely a simplistic view of the actual mechanisms, it will be used to justify the selection of control strategies. The goal is not to prove the validity of the theory, but to see whether control strategies selected based on the theory do lead to meeting the desired outcomes in animal and clinical studies.

3. Control strategies

Although the desired adaptive response can be different for different clinical presentations, the mechanisms for control are the same. The control strategies can be used in different ways to achieve the desired adaptive response. There are essentially two types of control strategies for wound healing, ones that control the inflammation phase and ones that control the repair phase.

3.1 Inflammation control

3.1.1 Causes of inflammation

Both the level of inflammation and the time to resolution can alter the adaptive response. Inflammation is the normal adaptive response to injury. In adults, it functions to prepare the wound for the repair phase. In the beginning, (acute inflammation) neutrophils and macrophages are recruited to clean up the wound from foreign debris, necrotic tissue and cells, and byproducts of hemostasis such as fibrin and free red blood cells.

The recruited cells produce cytokines to recruit cells and blood vessels for the repair phase [2, 3, 4, 21, 25]. The pathological response normally results when the inflammation becomes chronic and slows or delays the repair phase, resulting in scarring [1, 3, 4, 5, 7, 8, 9]. In addition, the cytokines and enzymes produced by the inflammatory cells can damage local healthy tissue (leading to more inflammation to clean it up) as well as produce systemic effects [1, 4, 21, 25, 26, 27]. Histamine and other substances produced by inflammatory cells can increase inflammation in other parts of the body [5, 25]. Although not well understood many inflammatory diseases such as autoimmune (including arthritis) or colitis are made worse (exacerbated) by increases in histamine levels from injury or foods that are eaten [1, 4, 5].

3.1.2 Inflammation control strategies

These are mostly centered around reducing the amount of material that has to be cleaned up. In many cases, skin wounds are debrided to remove foreign material and necrotic tissue. This helps to reduce the inflammatory response that would have been necessary to remove the foreign material and necrotic tissue as well as reduces the likelihood of an infection, which stimulates both an immune and inflammatory response [1, 2, 21, 25, 26]. Similarly, limiting the bleeding and swelling reduces the inflammatory response by reducing the amount of clean up required [1, 5, 25]. Also in some cases, once repair has begun too much movement at the healing site can lead to microtears and a new cycle of inflammation [1, 2, 25]. In soft tissue, stabilizing the healing parts of the wound with sutures, staples, glue, etc. can limit this [1, 2, 25].

In many cases, foreign materials are used to stabilize the wound or help in the repair phase [1, 21, 25]. Reducing inflammation in these cases is tied to reducing macrophage activation [1, 5, 21, 23, 25, 26, 28, 29, 30]. As long as the materials chosen do not leach out chemicals that directly or indirectly activate macrophages, then size of the foreign material is the most important determination of inflammation [1, 2, 21, 28]. Macrophages are most activated in the size range where the foreign material can be phagocytized, but either cannot be or it is not easily done [1, 21, 28, 29, 30, 31, 32]. This is normally in the 1–50 μm range [1, 2]. Below 1 μm the foreign material is easily removed and above 50 μm individual macrophages do not try to phagocytize or surround it [28, 29, 30, 31, 32]. Figure 2 shows the change in activation of macrophages when fiber diameter is below 60 μm [1, 2].

Figure 2.

Shows how size can affect the adaptive response of macrophages. As size of the fibers goes below 60 μm the response of macrophages increase in vivo. This suggests that the inflammatory response significantly increases for diameters (fibers and probably particles) below a certain threshold.

The immune response can also occur in the inflammatory phase by using antigenic materials as the foreign material (proteins typically) as well as increased by increasing the response of macrophages, since macrophages typically present the antigens to the lymphocytes [1, 21, 25]. So, control of both the immune response and foreign body response is tied to the activation of macrophages.

There can also be chemical and environmental changes to reduce the inflammatory response or enhance its efficiency. There are a number of anti-inflammatory drugs that work to either reduce the cellular response or the effect of what the cells produce. Environmental changes can also have an effect. Temperature can be used to slow bleeding or increase the efficiency of the clean-up. Oxygen can directly affect the activity of macrophages, with low oxygen (seen in the beginning of an injury) activating macrophages [1, 4, 5, 33, 34, 35, 36, 37].

3.2 Repair phase control

Assuming there is a defect, the speed in which new tissue fills the space determines the length of time it takes to repair the defect. In the case of skin, there is the connective tissue layer (dermis) and the epithelial layer (epidermis). The epidermis requires a vascularized layer to grow across, so the speed of dermal tissue formation controls it [2, 9, 38]. The dermal repair requires fibroblasts to grow into the defect [2, 9, 36, 38]. The fibroblasts require a scaffold to migrate into the wound [2, 9, 20]. The fibroblasts produce the collagen, but need oxygen from blood vessels to produce the collagen scaffold [2, 9, 16]. Therefore, fibroblasts, collagen, and the blood supply grow into the wound together [2]. The rate-limiting step is the blood vessel ingrowth (angiogenesis) [1, 37]. Speeding up fibroblast proliferation, migration, or ECM production is only effective if the angiogenic response is also enhanced. So controlling the angiogenic response controls the rate of dermal and epidermal repair [1, 25, 37]. If healing rate is distance the epidermal cells migrate inward toward the center of the wound, then it is both an epidermal healing rate plus a contraction rate [1, 2, 39].

To increase the rate of angiogenesis a scaffold can be used, to reduce the need for fibroblasts to produce ECM in order for blood vessels to grow in. [2, 25]. Fibroblasts still need to be within about 100 μm of a blood vessel to survive [2, 37]. There are different types of scaffolds that are used: different materials, different degradation profiles, different additives (cells, growth factors, etc.).

There are a number of strategies to increase the angiogenesis ingrowth rate into the defect. These include adding angiogenic growth factors or cell seeding with endothelial cells or endothelial progenitor cells [27, 35, 36]. Electrical stimulation and hyperbaric oxygen have also been used [1, 2].

4. Design selection

Previous sections have shown typical adaptive responses, why you want to control them, and general strategies of how to control the adaptive response. An important part of controlling the adaptive response is deciding the “best” strategy to do that. As it turns out there is hardly ever a best choice in design or for most things in life. In general, all you can do is select an option that has a reasonable probability of doing what you want it to do. In research, using the Scientific Method, we tend to think statistically better is good enough. It is critical to use the engineering design process vs. the Scientific Method to design a treatment.

In reality, the engineering design process uses the Scientific Method, but studies are design driven vs. hypothesis driven. In addition, there are other steps in the engineering design process, which do not have to be included when using the Scientific Method. Too often, based on previous studies, a treatment that is shown to be better than current treatments is tried clinically and although it proves the hypothesis, fails to meet the desired clinical outcome. Researchers try to come up with reasons why it did not work; besides that it was not designed to meet the desired clinical outcome.

The engineering design process is typically taught as a linear process (Figure 3). Normally, however, in order to design a clinical treatment there are many feedback loops. This is typically because clinical performance design constraints (desired outcomes) may change over time as well as the performance requirements of the treatment to meet the clinical performance design constraints are not always known. Also, most of the commercializability concerns require iterative processes as the design evolves.

Figure 3.

The engineering design process.

This section (4) will describe the linear design process and then how some of the feedback loops could work. The specific design will depend on the desired clinical outcome (which is one of the steps left out by the Scientific Method) for a given clinical presentation.

4.1 Engineering design process

The linear steps of the design process are shown in Figure 3 (without the parts in red). The Scientific Method part of the process does not occur until the testing phase (the last step), where studies are done to prove that the system meets the design constraints (design based experiments vs. hypotheses based). The other preceding steps are not required for the Scientific Method, but are essential to design a clinical treatment.

These steps also can be used to justify a research study that is suggesting a better design than current treatments. Without them, it is difficult to justify why a different treatment option should be looked at. First, there needs to be a clinical problem that is not being met by current treatment, otherwise why do we need other options. Then what success looks like (performance requirements) have to be the listed and quantified. It is usually helpful to have a minimum level of acceptable benefits and a maximum level of acceptable harms. Also, additional design constraints can be listed as “would like to” design constraints that can be additional desires or higher levels of benefits as well as lower levels of harms.

Then there is brainstorming to come up with potential approaches. With the “best” one selected to develop further based on meeting all of the performance requirements and a desirable mix of the “would like to” constraints. Then the selected approach is fully developed and tested to assure it meets the performance requirements.

4.2 The real world engineering design process for skin wounds

4.2.1 Modified design process

In practice, however, this process is not linear and needs to be implemented with more detail. The more detailed approach is outlined in Figure 3 (with the text in red added) and the non-linear elements are described in the next section (4.2.2). The main difference is to make sure the right level of design constraints are used for selection. First, the clinical performance design constraints must be selected. This is again generally functional recovery rate (on the business side they are called “value propositions” if they are better than current treatments). Then the requirements of the system/treatment needed to meet the clinical performance requirements need to be specified. Then brainstorming is done for approaches and the “best” approach is selected.

4.2.2 Iterative nature of the design process

Again, there are two main reasons that this is not a linear process in practice: (1) performance requirements may change over time particularly after testing to see if performance design constraints are actually predictive of the next level up design constraint (e.g., does meeting the system performance requirements assure meeting the clinical performance design constraints) and (2) many of the commercializability concerns require iterative processes as the design evolves.

In most cases, the feedback loop would be after testing to determine, if the design requirements can be met and are predictive of meeting the clinical performance requirements. This iterative process more closely approximates the Design Controls required for FDA regulatory approval.

Again many of the commercializability issues have to be determined in an iterative fashion and can be written as design constraints. Although they should be used to select approaches and specific designs, the commercializability is not really known until the design is complete. In general, they can be looked at as either value added (benefit) to each stakeholder or additional costs to each stakeholder. This risk/benefit analysis is also something typically left out of Scientific Method based studies.

5. Conclusion

The goal of this Chapter was to explain how macrophages can orchestrate the adaptive response to various stimuli, in order to understand the specific role of macrophages in controlling the adaptive response to injury as well as to suggest the introduction of additional stimuli to modify the response of macrophages in an attempt to get a more desired overall adaptive response. In many cases, the normal adaptive response to injury leads to a clinical outcome that falls short of the desired clinical outcome.

Although there has been much work on the different phenotypes of macrophages in vitro and in vivo [26, 40]. There does not seem to be an on and off switch between pro-inflammatory (M1) and pro-healing (M2a) macrophages [26, 40]. There probably is a continuum and likely that not all macrophages in a wound are in the same place [26, 40]. There is general consensus on what cytokine changes occur during healing of an injury (Table 1), however it has been suggested that instead of sorting out phenotypes and cytokine production it would be better to just look at functions (as was done in this review) [26, 40].

Cytokine	Production during repair from onset to the end
	Increasing	Decreasing
TNFα		X
IL-1β. IL-6		X
YM1		X
TGFβ	X
CD206	X

Table 1.

Changes in macrophage cytokine production during injury repair.

A theory was developed to help understand why the normal macrophage orchestrated adaptive response was different in different situations. This theory was also used to suggest why specific clinical strategies could have a significant effect on activation of macrophages and therefore clinical outcome.

To actually design a system based on this theory a specific application would have to be used. This is because different applications would have different desired clinical outcomes. Many of the references cited in this chapter go into more detail (including clinical studies): e.g. [1, 2, 41, 42, 43, 44].

Another important aspect of design for injury is that the desired clinical outcome is almost always the rate of return of function to a certain percent of what it was pre-injury. Although it was important to understand how sensitivity of macrophages to stimuli is probably the difference between animals that regenerate and those that do not, regeneration is hardly ever the actual clinical goal. This is mostly because, based on current technology, there are very few cases that we have the ability to regenerate the native structure.

A recent panel discussion at the Society for Biomaterials [45], asked the question “Is it better to regenerate in vitro (graft substitute) or in vivo (degradable regenerative scaffold).” Both sides gave some very good arguments to justify their approach to a particular wound/injury. Someone from the audience made a good point that it is application dependent and our abilities are different in each area.

However, the panel discussion question missed the point. Again, it is not about regeneration but functional recovery. In particular, with skin wounds, we are not able to make graft substitutes that work like skin grafts nor are we able to make degradable/regenerative scaffolds that completely regenerate skin. Although it may be possible someday, until we are able to, the design goal should not be to move closer to regeneration either in vitro or in vivo, but to achieve the desired functional recovery.

An important corollary to this is the importance of determining the minimum desired clinical benefits (functional recovery). This requires identifying the problem to solve and describing quantitatively what the minimum clinical outcome requirements are for solving the problem. Too often a researcher proves that a specific design is significantly (statistically at a high confidence level) better than current treatment in one or more desired clinical outcomes, but does not get the desired results clinically (even though it was statistically better than current treatments); and is uncertain why this happened. This is because, in design, it is only important if it allows a better than the minimum desired clinical outcome; better than current treatment is meaningless in design.

Acknowledgments

The concepts and research examples came from research and book chapters written with graduate students and collaborators (who were cited with the applicable references). Funding for this research has come from the CDC, NIH, NSF, and Biofisica.

Conflicts of interest

The author declares no conflict of interest.