Disclosure: Dr. Parenteau and Dr. Young are principals of Parenteau BioConsultants, Inc. This article represents a synopsis of some findings from a report series published and sold by the firm.
The scientific understanding of wound healing has advanced significantly over the last 20 years. In the last 5 to 10 years alone, the biology of inflammation, repair, and regeneration has been advanced by the tremendous effort to understand factors and signaling mechanisms involved in inflammation, angiogenesis, and cell proliferation—particularly as they relate to cancer progression1–3 and cardiovascular disease.4–6 While cutaneous healing research has certainly contributed to this body of information,7,8 product development and clinical practice has not benefitted from recent advances in the scientific understanding of these processes as much as it could have benefitted. An extensive review and analysis of the available biological data regarding the biological mechanisms and the relationships between biological processes that could impact wound repair was undertaken.9 Presented is a synopsis of findings regarding the role of injury and innate immunity and how each has the potential to impact healing and the practice of wound care.
The difficulty in dissecting cutaneous healing has always been compounded by the fact that the biological processes are being carried out in an environment where physical factors can have almost as much impact as physiological factors. There is no doubt that practically speaking, the management of the physical environment, (ie, a moist healing environment, control of wound exudate, and control of bacterial contamination), has an impact on healing. For normal acute healing, this management may be all that is necessary to enable an optimum outcome. However, chronic wound conditions like a chronic venous ulcer or a hard to heal diabetic foot ulcer can be biologically and physiologically far removed from the norm. While environmental conditions are still a factor, they are more likely to be superseded by underlying pathological processes in these cases. To significantly improve healing in chronic wounds requires going beyond the physical environment to address the biological mechanisms affected by the pathological causes of tissue failure and ultimately, the inadequate response mechanisms that undermine the ability to heal. In the chronic wound, the interaction and relationship of the biological processes of repair and regeneration are more likely to be the final arbiters of success for any treatment regimen or therapeutic product.
Although the response mechanisms of the chronic wound may be altered,10–15 the native processes and biological relationships between them still serve as the foundation for cellular behavior and interaction. This synopsis focuses on the biological mechanisms underlying the acute injury and innate immune responses. The analysis draws from available knowledge of injury and inflammatory signaling and cell response. The mechanisms concerned with injury and inflammation and how they impact the biological goals of wound repair are examined. Examples are given to illustrate ways in which a biological perspective can affect how one might view and ultimately treat wounds in different situations.
The Connection of Injury and Inflammation
In an acute wound, there is both an injury response and an innate immune or inflammatory response. They are connected but distinct processes based on the biological mechanisms that govern them and their primary purpose. The goals of the injury response are hemostasis and communication of the injury, while the goal of the inflammatory response is protection and removal of pathogens through the process of innate immunity. Specifically, the inflammatory response is designed to neutralize bacterial pathogens and remove them along with necrotic tissue and debris, and then resolve.
Both injury and inflammation work secondarily to prepare for and initiate the early regenerative and repair responses evidenced by the formation of granulation tissue and the activation of epidermal migration through the stimulation of factors such as TNF-a7,16 and IL-8.17 However, repair is not the primary purpose of either process. This suggests that mechanistically, one should be able to stimulate an injury response while limiting inflammation and experience inflammation without an acute injury response. While an injury response without inflammation is harder to observe, there are examples where an inflammatory response is active in the absence of an obvious injury response.1,18–20 One might also postulate that it should be possible to activate a repair response without the full participation of either injury or inflammation. How could that impact wound healing? To understand the connection, goal, dependency, and impact of these processes, the biological mechanisms underlying injury and inflammation and their connection to repair were examined.
The Injury Response
The injury response is communicated by blood exposure to extravascular tissue cells. Factor VII from the blood forms an active proteolytic complex with tissue factor (TF) present on the surface of extravascular cells, such as the epidermal keratinocyte. This initiates the extrinsic clotting cascade that leads to fibrin clot formation and hemostasis. The components of the clotting cascade, principally thrombin followed by Factor Xa (activated) and the Factor VIIa-TF complex, also activate a family of protease-activated receptors (PAR) on the surface of epidermal keratinocytes, dermal fibroblasts, endothelial cells, and inflammatory leukocytes.21–25 Both TF and PAR expression is constitutive in the keratinocyte and fibroblast.26–28 Also, TF and PAR are induced in the endothelial cells, leukocytes, and some suggest the platelets, upon injury.23,29 Protease-activated receptors are self-activated by proteolytic cleavage of their extracellular domain.6 Their activation is therefore dependent on the amount and types of proteases in the environment.30 Wound proteases capable of PAR activation are contributed by infiltrating neutrophils,31 resident mast cells, bacterial contaminants, and damaged cell contents in addition to thrombin—a major component of the clotting cascade and primary activator of PAR-1 present on endothelial cells and fibroblasts.6 Protease-activated receptor signaling initiates a change in phenotype or activation of the cells that leads to the production of inflammatory mediators and growth factors that will be important in the advance of inflammation and healing.32–35 The more prolonged the activation of the extrinsic clotting cascade, cell damage, or bacterial contamination, the more prolonged the cellular activation through PAR signaling (Figure 1). The PAR receptors activate a number of G-protein pathways36 and communicate the nature and extent of the injury while facilitating vascular permeability, platelet aggregation, cell survival, and factor expression. Protease-activated receptor signaling initiates the expression of inflammatory cytokines like interleukins 1 and 8, causes changes in vascular permeability as well as TF expression on endothelial cells and platelets, upregulates the expression of genes that protect cells from apoptosis, inflammatory mediators, and growth factors associated with granulation such as platelet-derived growth factor (PDGF) and connective tissue growth factor (CTGF, CCN2)32 (Table 1). Inflammatory cytokines work cooperatively with the PAR system by upregulating PAR expression on the cell surface.
Broadly speaking, the injury response of cells in the wound environment encompasses:
• Early cytokine production in most cells that foster the recruitment of inflammatory cells
• The production of apoptosis inhibitors
• Cell surface changes that foster adhesion, migration, and different cell/matrix interaction
• Platelet aggregation and release of contents
• Endothelial shape and permeability changes that enable leukocyte extravasation.
Another major component of the injury response is the activation of platelets. Platelets are active participants in the clotting cascade. Platelets alone are sufficient to achieve hemostasis even in the absence of fibrinogen.37 Under normal circumstances, platelets enable the extrinsic clotting cascade, serving as the primary catalytic surface for the generation of thrombin and delivery of clotting factors.38 The release of platelet contents within the wound not only ensures an adequate supply of clotting factors but also growth factors and signaling molecules. Platelet contents and their affects rapidly change the tissue environment from a homeostatic one to an activated one, directly and indirectly through the clotting cascade and PAR signaling.29 Platelet factors support both the biochemical and cellular changes that are necessary for attracting and sustaining an adequate inflammatory response and set the stage for healing. A few of the important factors supplied by the platelets are platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-b), vascular endothelial growth factor (VEGF), and thrombospondin-1, all of which contribute in some way to wound granulation.
The Acute Inflammatory Process
Once hemostasis is established and the injury communicated through protease activation of PAR, the inflammatory response must kill and eliminate infectious agents, remove debris, and prepare for and activate the processes of repair. Two “initiating” cytokines principally mediate early response: transforming growth factor alpha (TNF-a) and interleukin-1 (IL-1). Although they are distinct molecular entities, they result in similar and overlapping effects. Both lead to broad responses mediated by the action of transcriptional regulator nuclear factor-kappa B (NF-kB). Both are released almost immediately by resident macrophages and by the epidermis, which is a particularly rich source of IL-1a. TNF-a and IL-1 initiate a change in phenotype in most cell types of the wound that will be important for the processes of early healing. Both factors can act directly on the cells that produce them and also in a paracrine manner, eg, IL-1 released from the injured epidermis can act on inflammatory cells and the dermal fibroblasts in addition to the epidermal cells. The changes in phenotype mediated through NF-kB include facilitating matrix degradation, cell recruitment, and cell proliferation in most cell types of the wound (Table 1). Both TNF-a and IL-1 signaling will stimulate the production of chemokines that rapidly attract neutrophils. After initial release of cytokine stores, a continued supply of these mediators in the wound is provided by cells in the wound environment and is dependent on mechanisms, such as PAR signaling. Thus, the injury response communicated through PAR activation plays a role in the extent and duration of the inflammatory response (Figure 1).
The Innate Immune Response
Neutrophils are attracted to the wound site by the chemotactic molecular gradients formed by the release of chemokines and also by microbial peptides.39,40 NF-kB, which plays a central role in inflammatory signaling is regulated not only by TNF-a and IL-1 and their associated receptors but also by the activation of toll-like receptors (TLR) that can be activated by both necrotic cells and by lipopolysaccharides (LPS) from bacterial cell walls.41–43 Therefore, injury, contamination, and inflammation are linked not only by PAR but also by NF-kB. Just as PAR (and associated G-protein signaling) constitutes a signaling system for the activation of the injury component of the response, NF-kB constitutes the signaling system for the inflammatory component of the first phase of healing.
While activation of acute injury and inflammation responses are important to initiate healing, their resolution is important to progress to repair. The inability to adequately resolve one or both responses is a significant factor in chronic wound pathology. Macrophages, or activated monocytes recruited from the blood stream are an important cellular component to the resolution of inflammation. Indeed, studies have empirically established the presence and activity of macrophages to be an important component to positive healing.13,44,45 While neutrophils are attracted to the wound within minutes, macrophages reach their highest number in the normal acute human wound on day 2.17 Macrophages release a number of cytokines and growth factors into the wound site while limiting the proteolytic activity of neutrophil elastase. However, macrophages interact with neutrophils in additional ways that are important to the resolution of acute inflammation. Macrophages bind neutrophils and cause neutrophil apoptosis in conjunction with surface-bound TNF-a.46–48 Phagocytosis of the apoptotic neutrophil by the macrophage then results in suppression of interleukin and prostaglandin production while increasing the production of TGF-b, a potent anti-inflammatory factor that effectively limits further release of pro-inflammatory cytokines. Macrophages prepare the area for wound granulation through:
• Inflammatory cell clearance
• Debris removal through phagocytosis
• Initiation of granulation through the support of angiogenesis and fibroblast chemotaxis.
Inflammatory cell clearance may be one of the most significant contributions of the macrophage since angiogenesis and fibroblast chemotaxis are also supported through the production of cytokines and factors produced by multiple sources in the wound environment (Table 1). The timing and degree of neutrophil and macrophage activity will depend on the degree of contamination as well as the physiological and hormonal status of the individual.45 In elderly patients, there is a natural decline in the adrenal estrogenic precursor dehydroepiandrosterone (DHEA). Estrogen hormones normally dampen the inflammatory response and support additional aspects of healing in part, by limiting the activity of macrophage migration inhibitory factor (MIF).49 The decline of DHEA and estrogen leads to increased MIF, resulting in enhanced inflammation and poorer healing.50 In elderly men, the effects of increased MIF are exacerbated by the presence of testosterone.51
Injury and Inflammation and Their Relationship to Wound Granulation
From a biological perspective, the granulation response can be characterized as the first phase of a regeneration response. The formation of granulation tissue essentially represents the regeneration response of the vasculature and supporting connective tissue. Its appearance is considered a hallmark of a good healing response which, in essence, indicates that the injury/inflammation response was mounted effectively and has progressed effectively, resulting in the regeneration response that physically manifests as granulation tissue. Forming granulation tissue involves the proliferation of endothelial cells, sprouting from existing microvessels, the recruitment of perivascular cells and the concomitant recruitment of fibroblasts and their associated matrix biosynthesis to support the developing vasculature. Under normal circumstances, granulation “matures” or resolves and many of the new primitive vessels recede while the remaining mature to functional vessels. While there are many factors that either promote or inhibit wound granulation, the vascular endothelial growth factor (VEGF) is considered the principal factor. Most activated cells in the wound environment contribute VEGF: platelets, fibroblasts, epithelial cells, and macrophages, in addition to the endothelial cells themselves. Vascular endothelial growth factor is connected to the extrinsic clotting cascade and thus to the injury response, by TF signaling. It has been reported that signaling via the cytoplasmic domain of TF causes upregulation of VEGF.52 Vascular endothelial growth factor expression can in turn increase tissue factor expression on the cell surface.53–55 Vascular endothelial growth factor is also a component of the platelet releasate—another major component of the injury response. Inflammation can also work through hypoxia-inducible factor signaling to increase levels of VEGF expression.56,57 NF-kB signaling through activation of both inflammatory cytokines (IL-1 and TNF-a) as well as injury-related signaling (LPS and necrotic cell contents via TLR) is responsible for the upregulation of the cytokines that will initiate repair.41 Therefore, injury and inflammation mechanisms each contribute to the overall response that leads to granulation and the beginnings of repair.
While the acute response provides insight into the mechanisms that govern these fundamental processes, from a biological perspective, a chronic wound is not an acute wound that fails to heal even though it contains elements of the injury, inflammation, and repair. The question is whether knowledge of mechanisms of the acute response can lead to better therapies for the chronic condition.
Exacerbating, Assisting, or Limiting Injury and Inflammation Responses in the Chronic Wound
As previously mentioned, the chronic wound has components of injury and inflammation but both are present in a significantly different way. For example, if we view platelets as the native way to affect rapid change in the tissue milieu toward one that supports both inflammatory and early repair processes, then it may appear that the application of platelet extracts should be a highly effective and desirable. However, clinical results using platelet extracts to stimulate healing have been far from definitive.58,59 One reason may be that the chronic wound already exists in a state of concomitant activation, repair, and resolution—a state significantly different than the homeostatic environment that the platelets normally activate upon injury. In this particular case, the addition of platelets or platelet extracts may not be adding anything significantly different to what is already being contributed by the elements already present in the wound environment.
Sharp debridement, which is often indicated in these chronic conditions may serve 2 purposes; removal of tissue embroiled in a chronic battle between inflammation, repair, and resolution (manifesting as fibrotic tissue), and the activation of a native acute response in the surrounding unaffected or minimally affected tissue while still able to mount a normal response. The effectiveness of debridement in re-establishing normal healing may therefore be dependent on the ability to activate tissue that is marginally compromised by underlying disease pathology and/or chronic trauma. This may explain why debridement is generally advised in the treatment of chronic skin wounds60 but that its effectiveness in ultimately leading to healing can differ.61 The biological processes of injury and inflammation also suggest that removal without significant acute injury may yield a different biological result than debridement does. In cases where sharp debridement may be contraindicated, platelet extracts may serve as a mechanism to enhance the response in the absence of an ability to mount a sufficient native injury response.
Since inflammation naturally precedes wound granulation and contributes factors that assist the repair process, it is reasonable to conclude that factors that promote inflammation may be beneficial in fostering wound granulation and ultimately healing. Chitin or chitosan is one example of a material that can stimulate inflammatory cell infiltration and thus, it is postulated, enhance wound healing by promoting granulation.62 The inflammatory effects of chitosan are not entirely unlike those elicited from bacterial cell walls, which also contain the polysaccharides present in chitin. Recent experimental evidence indicates that chitosan activates macrophages through interaction with the mannose receptor.63 This results in upregulation of macrophage inflammatory protein-2 (MIP-2), which bacteria can upregulate, hypoxia, and hyaluronan fragments. Macrophage inflammatory protein-2 is chemotactic for neutrophils and macrophages therefore chitosan can be considered to be a pro-inflammatory agent. By virtue of recruiting higher numbers of inflammatory cells, it may also make a greater contribution to the following granulation response, postulated to be through the increased delivery of PDGF and TGF-b.63 However, from the perspective of biological mechanism, could chitosan be anticipated to promote granulation in the chronic wound? Given chitosan’s method of action, a greater benefit might be seen in conditions where monocyte (the circulating precursor to the wound macrophage) activation is diminished like the diabetic foot ulcer but might be ineffective or even counter-productive in situations of chronic inflammation where macrophage activation is not limiting, like a chronic venous ulcer or a pressure sore.
In the natural situation of an acute wound, injury, inflammation, regeneration, and repair are interconnected on many levels and naturally follow. It is known that in the chronic conditions, the relationship between these processes is short-circuited, inadequate, and stalled by the inability to resolve or remove certain pathological or damaging factors. In the examples of debridement and chitosan, we dealt with the approach of rekindling the native sequence from the point of injury and inflammation. The next question is whether one can therapeutically administer a regenerative or repair stimulus without them and be effective.
There are 2 ways researchers have approached the delivery of such stimuli: first, by the administration of growth factors, and second, the use of cells and tissues. In the case of growth factors, as in the above discussion on platelet releasates, working with biological mechanisms requires that the pathological condition indicate a need for the biological purpose of the factor in question. That is, administering the growth factor at the right time and under the right circumstance appears pivotal. For example, putting aside possible issues of VEGF isoform action and the challenge of promoting functional vessel formation, generally speaking, VEGF might be anticipated to play a greater role in conditions where vascularization is limited over and above what might be stimulated through debridement alone as in the diabetic environment. In the case of keratinocyte growth factor (KGF), a factor not specifically associated with repair, but rather with the support of regeneration and mechanisms of tissue renewal, one could expect that it will be effective in protection against epithelial breakdown, such as the case is with mucositis as a result of radiation therapy (Palifermin recombinant human KGF, Amgen, Thousand Oaks, Calif), but not effective in repair processes like stimulation of epidermal migration in a chronic wound, both of which have been documented to be the case in prospective human clinical studies (Repifermin KGF-2, Human Genome Sciences, Rockville, Md).
In cell and tissue therapy, the issue is compounded by the fact that the therapy itself will mount a response to conditions in the wound bed. Will a bioengineered tissue construct perform better on a wound bed prepared by sharp or enzymatic debridement? Will the choice be different for a diabetic ulcer and a venous ulcer? It is of interest to note that the clinical protocol used in a pivotal clinical trial using a bioengineered skin construct (Graftskin, Organogenesis) for the treatment of chronic venous ulcers employed minimal debridement64 yet a stimulation of wound granulation was often described and was sometimes dramatic in long standing wounds.65 This suggests that regenerative therapies can act in the absence of a significant injury or inflammatory response on the part of the patient. It is important to note that when using an interactive living therapy although a patient may not mount a response, it does not mean that the cells of the construct do not. Indeed, one benefit of an interactive therapy is that it can. The therapy may re-establish a normal injury and inflammation cascade within the wound that is supplied by the therapy, rather than through a specific alteration of a pathological process present in the patient. Whether the effectiveness of the living therapy centers on a response that extends back to injury and inflammation or whether its principal mode of action is focused on regenerative stimuli remains to be determined. It appears that the patient response does not require a native rekindling of injury and inflammation. Further discussion of the intricacies of cell therapy response and the biology of different chronic wounds extends beyond the scope of the present synopsis but nevertheless, it is important to note that the condition of the wound bed, as in any other therapies, will still play a significant role in the activity and function of an interactive therapy.66 Questions regarding wound bed preparation, companion therapy, and the biological composition and state of the product should be addressed the best way possible with the information available to ensure the product will be used most effective manner.
This synopsis establishes that injury and inflammation can be viewed as 2 distinct biological processes, both of which should be taken into account when devising a strategy for healing or the use of therapeutic products. The major components of the injury response are the proteases of the environment and extrinsic clotting cascade, which communicate the extent and duration of the injury through a system of protease-activated receptors. Mechanisms of innate immunity lead to leukocyte infiltration, the removal of pathogens debris, and the production of cytokines and growth factors that will initiate repair and regeneration. Platelets associated with the injury response, as well as inflammatory cells and cells of the tissue, primarily contribute to the propagation and maintenance of the inflammatory response and secondarily to the initiation of repair evidenced by wound granulation. However, inflammation is distinct from repair. The development of granulation tissue represents the regenerative response of the vasculature and supporting connective tissue. The different types of chronic wounds exhibit altered states of injury, inflammation and repair while biological mechanisms governing these processes may be more or less intact depending on the condition. One might ponder if mounting an acute injury response can overcome stalled chronic inflammation or if it will exacerbate it. Can chronic inflammation be overcome by specifically targeting the repair response? One can better target available therapeutic options and design more effective alternatives by devising a therapeutic approach based on biological mechanisms at work or conversely, at fault, in a given situation. While knowledge of the acute response can serve as a guide to understanding how a therapeutic strategy might assist or deter native mechanisms, it will also be important to define in greater detail the biological relationships underlying these responses in different types of chronic wounds.