Current Concepts In Managing The Wound Microenvironment
Wound healing is a process that involves the stages of coagulation, inflammation, cell proliferation and repair of the matrix, epithelialization and remodeling of the scar tissue. These stages overlap and the entire process can last for months.1 During the post-injury coagulation phase, platelets initiate the wound healing process by releasing a number of soluble mediators including platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), fibroblast growth factor (FGF), and transforming growth factor-beta (TGF-beta). These growth factors rapidly diffuse from the wound and inflammatory cells are drawn to the area of the injury.1 The inflammatory phase is initiated by the blood clotting and platelet degranulation process. During this phase, there is significant vasodilatation, increased capillary permeability, complement activation as well as migration of polymorphonuclear leukocytes (PMN) and macrophages to the site of the wound. The neutrophils and macrophages engulf and destroy bacteria. They also release proteases, including elastase and collagenase, which degrade damaged extracellular matrix (ECM) components. Inflammation is largely regulated by a class of molecules called cytokines, which have powerful stimulatory and inhibitory actions on inflammatory cells.1 The final phase of wound healing involves a balanced process that degrades old ECM and synthesizes new ECM in order to remodel the scar that was formed during proliferation and repair. Among the most important cells are the fibroblasts, which, among other cells, produce matrix metalloproteinases (MMPs) that degrade the matrix and tissue inhibitors of metalloproteinases (TIMPs) that regulate the activity of the MMPs.2 In short, normal wound healing is a complex and finely tuned process that is mediated by growth factors and cytokines.2 In the chronic, non-healing wound, these factors change the environment so the normal phases of healing cannot be completed or regulated. This includes decreased amounts of growth factors; increased bacteria, decreased oxygen and senescent cells, which are unable to respond to growth factors.3,4 This upset in the balance of the wound bed causes an increase or decrease in the cellular expression of wound bed factors. This results in delayed healing.
What About The Role Of MMPs?
Much of the tissue degradation required in wound healing is performed by a specific group of proteolytic enzymes called matrix metalloproteinases (MMPs). The MMPs are a family of structurally related, protein degrading enzymes that require calcium ions for structural conformation and zinc ions in their active site for function. The MMP family of enzymes is capable of digesting almost all of the components of the ECM, including needed growth factors.5 These enzymes function optimally at neutral pH. They are usually produced in response to tissue injury and are not normally present in detectable levels in healing, non-injured tissue.6,7 In regard to MMP activity during the inflammatory phase of repair, one should not confuse this activity with the release of MMPs during the remodeling phase of healing. During the inflammatory phase, MMP activity assists with debridement and preparation for the migratory phase of healing. During the final phase of healing, MMPs assist with the remodeling of scar and tissue after wound closure. Macrophages, keratinocytes and fibroblasts may produce MMPs in the inflammatory phase.4 Researchers believe high protease levels in chronic wound fluid delay wound repair by degrading newly formed tissue and beneficial proteins such as growth factors and extracellular matrix proteins.8 Elevated MMP activity degrades the extracellular matrix, which interferes with cell migration and connective tissue deposition. Matrix metalloproteinases also degrade growth factors and their target cell receptors that further limit the progression of the wound healing cascade by eliminating the mediators of the cascade.9 An individual MMP may have one or several protein substrates that they can degrade. Certain MMPs are very specific. For example, collagenases can only degrade collagens.5 Collectively, MMPs are capable of degrading almost all of the components of the ECM. They are also secreted in an inactive form and must be activated by ECM via various mechanisms. The level of MMPs is controlled by several mechanisms including local secretion of endogenous enzyme inhibitors, which are referred to as tissue inhibitors of metalloproteinases (TIMPs). The same cells that produce MMPs can synthesize TIMPs. If the balance is disturbed, high levels of MMPs may result in excessive tissue degradation or destruction of other protein components in the ECM such as growth factors, cell surface receptors, and TIMPs.5 Tissue inhibitors of metalloproteinases act to block tissue destruction by MMPs as the inflammatory phases terminate and proceed into a proliferative phase. A balance between MMPs and TIMPs is necessary throughout normal wound healing for successful and optimal closure. The cellular interplay between these wound components mediates the repair process in a complex manner.4 Proteases can also degrade growth factors and cytokines essential for wound healing. Recently, measurements of MMPs and their natural inhibitors TIMPs from chronic wound fluid showed a close correlation between high ratios of TIMP/MMP-9 and healing of pressure ulcers.10 The elevated levels of inflammatory cytokines and proteases, along with low levels of mitogenic activity and poor response to cells in chronic wounds, led to the concept that clinicians must strive to rebalance the molecular environment of chronic wounds to levels one would see in acute healing wounds.1,11
Key Insights On Cytokines
Cytokines are intercellular communication chemicals produced by immune system cells. They deliver messages to immune system cells that result in migration of those cells to an area. They also result in the synthesis and release of enzymes from those cells in order to mount a destructive inflammatory response to foreign organisms or materials in the wound.5 Cytokines transmit messages related to bacterial defense and debridement during the inflammatory phase, and usually decrease after the inflammatory phase. They shift the balance of the wound equilibrium toward the destructive process and elevate degrading proteases. The cytokine environment of chronic wounds is abnormal and researchers have shown elevated levels of IL-1a, a pro-inflammatory cytokine, in chronic wounds.12 Trengove, et. al., found that the levels of other pro-inflammatory cytokines, IL-1b and TNF-a, were also significantly elevated in nonhealing wound fluids in comparison to fluids from healing wounds.13 Tumor necrosis factor-alpha (TNF-alpha) affects the deposition of connective tissue in wound healing due to the cytokine affects of the synthesis of collagen, MMP and TIMP. The TNF-alpha stimulates the synthesis of MMP while simultaneously decreasing synthesis of TIMPs.9 The levels of these cytokines decreased substantially as the wound healed. This indicated a correlation between nonhealing wounds and increased levels of pro-inflammatory cytokines.2
Where Growth Factors Come Into The Picture
Growth factors are small molecular weight proteins that transmit messages between cells and deliver messages to nonimmune cells that result in cell proliferation or synthesis of granulation tissue components. A single type of growth factor may have more than one biological role during healing. This may depend upon when it is released or the degree to which it binds.5 Growth factors are secreted by many cell types including platelets, neutrophils, macrophages, fibroblasts, epithelial cells and endothelial cells. Growth factors initiate an effect by binding to and activating specific high affinity receptor proteins on target cell surfaces. Their receptors provide growth factor specificity to their action. The actions of growth factors are vital to the many events in the healing cascade. The contributions of growth factors to wound healing include the formation of new blood vessels, protease production, wound remodeling, extracellular matrix production, epithelialization and fibroblast proliferation.14 Nonhealing wounds have smaller and fewer types of growth factors than healing wounds. One of the most important growth factors in wound healing is platelet-derived growth factor-BB (PDGF-BB). This growth factor has the ability to communicate to many cells at the same time. It directly stimulates angiogenesis through many activities, including the activation of endothelial cells and fibroblasts, and the stimulation of vascular proliferation, migration and new blood vessel formation. This growth factor also recruits smooth muscle cells and pericytes to stabilize newly formed vessels.15 Skin biopsies from the edge of foot ulcers in non-diabetic and diabetic subjects have shown increased expression of transforming growth factor (TGF) beta 3 in the epithelium. However, there was no increase in the expression of TGF-beta 1 and this could explain impaired healing. A lack of expression of insulin-like growth factor (IGF) 1 in diabetic skin and foot ulcers and dermal fibroblasts may also contribute to delayed wound healing. However, IGF-2 was highly expressed in normal and diabetic skin as well as in diabetic foot ulcers, particularly at the ulcer edge.1 Since growth factors are proteins, they are subject to degradation by the MMPs and other types of elevated proteases. If growth factors are degraded, communication among the various cells participating in the wound healing process stops and wound healing is delayed.5 For growth factors to be beneficial in wound healing, some minimal critical concentration of physiologically active hormone must be present in the wound. If growth factors are under-secreted or rapidly metabolized, wound healing will be retarded.16
Do Free Radicals Factor Into Chronic Ulcers?
Free radicals may be important in the pathogenesis of diabetes-related healing deficit. Researchers have shown that a protective membrane antioxidant agent significantly improves impaired wound healing in diabetic mice through the stimulation of angiogenesis.1 When polymorphonuclear neutrophils (PMNs) are recruited to the wound site and activated, they consume an increased amount of oxygen, which is converted into reactive oxygen species.17 Oxygen-derived free radicals have been implicated in causing venous ulcerations and the persistence of these ulcers. Scavenging such radicals with antioxidants expedites the healing of venous ulcers.18 We know that nitric oxide (NO) combines with hydroxyl free radicals to form peroxynitrate, a potent free radical, which causes tissue destruction. Nitric oxide overexpression in chronic venous ulcers may be involved directly or indirectly in the pathogenesis and delayed healing of chronic venous ulcers through its effects on vasculature, inflammation and collagen deposition.19Jude, et. al., found that diabetic patients with recurrent neuropathic and neuroischemic foot ulcers had significantly higher plasma NO levels in comparison to patients with nonrecurrent foot ulcers.2,20
What You Should Know About Angiogenesis
Angiogenesis is a dynamic process that primarily occurs during the proliferative stage of wound healing. It begins as a fibrin clot that is initially replaced by blood vessel-rich granulation tissue and subsequently replaced by collagenous scar with much less mature blood vessels.21 The primary stimuli for neovascularization of granulation tissue are vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). When one removes VEGF from wounds, there is nearly a complete absence of granulation tissue as VEGF receptors are almost exclusive to the endothelium.16,22 While VEGF has been associated with angiogenesis in numerous pathological situations, including tumor growth, proliferative retinopathy and rheumatoid arthritis, it is imperative for the growth of new vessels in the wound bed.22 The ECM is also critical for the growth and maintenance of normal vessel growth. It acts as both a scaffold support, through which endothelial cells may migrate, and a reservoir and modulator for growth factors to mediate intercellular signals. Researchers have also shown that the extracellular matrix alters signaling patterns for cell cycle progression and promotes coordinate changes in cytoskeleton and nucleus function.21
How To Differentiate Between Colonization And Infection
Some bacterial colonization can be beneficial to wound healing. However, critical or heavy bacterial colonization can upset the balance in the wound bed by causing an increased inflammatory response. This occurs via increased oxygen consumption, increased inflammatory mediators and a negative impact on platelets and macrophages. The bacterial colonization also causes delayed healing via the release of bacterial endotoxins that damage growth factors and increase levels of MMPs. Bacteria also break down tissue and cell debris to produce offensive wound odor. Infection also changes the pH of the wound, making it more acidic. Bacterial colonization is different from contamination in that it refers to proliferating organisms on the wound surface. The bacteria have adhered to the superficial tissues and have begun to form colonies. Colonization is also characterized by a lack of immune response from the host and is generally not believed to impact or interfere with the healing process. Wounds that contain nonviable tissue, like slough and/or eschar, offer a hospitable environment for colonization because the dead tissues provide a ready source of nutrients for the growing bacterial colonies.10,11,23 The current thinking is that bacterial toxins play a key role in delayed healing due to critical colonization. By definition, toxins are chemicals that produce a toxic result or adverse health effect. Therefore, bacterial toxins are chemicals produced by bacteria that enable them to cause an adverse health effect in their host. These toxins also may be determinants of bacterial virulence. Bacteria that produce potent toxins are more virulent or pathogenic than those that produce weaker toxins and are able to cause disease at lower concentrations.24 Bacterial infection is proliferating bacteria that is not only present on the surface of the wound or in nonviable wound tissue, but also is evident in healthy viable tissue. In these clinical scenarios, bacteria have invaded healthy tissue to such a depth and extent that they elicit an immune response from the host. Local clinical signs of tissue redness, pain, heat and swelling generally characterize this immune response along with an increase in exudate production or purulence. Bacterial infection delays and may even halt the healing process. The mechanism of this healing delay involves competition between host cells and bacterial cells for oxygen and nutrients, and increased host cell production of inflammatory cytokines and proteases in response to the bacteria and their associated toxins.10,11,23 Bacteria may stimulate a persisting inflammation that leads to the production of inflammatory mediators and proteolytic enzymes. This causes ECM degradation and inhibition of reepithelialization among other effects. Accordingly, one must control bacterial burden in order to facilitate healing or maximize the effectiveness of newer therapeutic techniques such as bioengineered skin or growth factors.1 Produced by infection and bacteria, biofilm creates a barrier between healthy tissue and any kind of wound care product one applies to the wound to increase healing. Biofilm also acts as a barrier to parenteral antibiotics. Biofilms are formed from communities of organisms including fungi, bacteria, yeast and protozoa. They are usually encased in an extracellular polysaccharide called glycocalyx that they themselves produce. This glycocalyx protects the bacteria from antibiotics and accounts for the persistence of the infection. Essentially, one may find biofilm on any environmental surface in which sufficient moisture is present. It develops most rapidly in flowing systems where adequate nutrients are available. Fragments of biofilm that slough off at intervals can spread the infection to distant locations within the body.
How Proper Debridement Can Aid In Wound Bed Preparation
Efficient debridement is an essential step in chronic wound management. The underlying pathogenic abnormalities in chronic wounds cause a continual build-up of necrotic tissue. Regular debridement is necessary to reduce the necrotic burden and achieve healthy granulation tissue. Debridement also reduces wound contamination and assists in reducing tissue destruction. Dead spaces that may otherwise harbor bacterial growth must be exposed during debridement. When it comes to surgical debridement, the experienced clinician must exercise caution in patients with compromised immunity in order to avoid creating large open wounds that may favor opportunistic infection. Surgical debridement is inappropriate for ulcers with insufficient vascular supply to allow healing, and one should use extreme caution with this form of debridement in patients who are on anticoagulants. Mechanical debridement is the use of a non-discriminatory physical force to remove necrotic tissue from the wound surface. It can damage healthy tissue and may be painful. Examples of mechanical debridement include wet-dry dressings, wound irrigation and whirlpool therapy. In regard to autolytic debridement, one can achieve this by providing a moist wound environment via occlusive or semi-occlusive dressings that promote phagocytic activity and the formation of granulation tissue. Enzymatic debridement relies on enzymes to break down necrotic tissue. This requires a prolonged period of enzyme activity with the appropriate wound bed moisture, pH and temperature. Bear in mind that enzymes are inactivated by heavy metals (silver, zinc).
Understanding The Potential Impact Of Glycemic Control And Vascular Status
Control of blood glucose is imperative in the healing of chronic wounds. Hyperglycemia results in leukocyte dysfunction and suppression of lymphocytes, high blood pressure, and impaired endothelial function. Improved blood glucose levels increase the immune defenses of patients. Accordingly, this is a key component of clinical care for infected diabetic foot ulcers. Chronically elevated blood glucose levels result in reduced leukocyte function and cell malnutrition.25 This causes poor wound healing by reducing glucose utilization of skin keratinocytes as well as skin proliferation and differentiation. Glycosolation of basic FGF-2 significantly reduces its activity and its ability to bind to tyrosine kinase receptor and activate signal transduction pathways.1 Wound healing can only take place if there is adequate tissue oxygenation. A well-vascularized wound bed provides nutrients and oxygen to sustain newly formed granulation tissue and maintains an active immunological response to microbial invasion. Oxygen is available in two forms. It is either bound to hemoglobin or dissolved in plasma. In chronic wounds and skin, unlike in active muscle, the oxygen dissolved in plasma can be adequate for healing, assuming that perfusion of the tissue itself is satisfactory. Decreased oxygen levels impair the ability of leukocytes to kill bacteria, lower production of collagen and reduce epithelialization. However, low oxygen tension coupled with adequate oxygen tension to heal stimulates the release of angiogenesis factor from macrophages.1
There are many factors that play into the potential of a wound to heal. After analyzing the components of chronic wounds, one must strive to facilitate an appropriate balance of cytokines, growth factors and MMPs in the wound microenvironment. Once one has addressed these issues, the clinician must then turn to the overall health status of the patient, making appropriate referrals and monitoring bioburden, vascular status and glycemic control in order to ensure the identification and correction of contributing systemic factors. Keeping all of these variables in check will give our patients the best opportunity to heal their wounds. Dr. Driver is Chief of the National Center for Limb Preservation at the Advocate Lutheran General Hospital in Niles, Ill. She is the Director of Clinical Research at the Center for Lower Extremity Ambulatory Research at the Dr. William M. Scholl College of Podiatric Medicine at Rosalind Franklin University of Medicine in Chicago. Dr. Jelinek is a Chief Resident at the North Chicago Veterans Affairs Medical Center and St. Joseph Hospital in Chicago. She is currently doing an extended rotation with Dr. Driver at the National Center for Limb Preservation. Editor’s note: For related articles, see “A New Approach To Using Growth Factors In Wound Healing” in the October 2003 issue of Podiatry Today or “Closing Difficult Wounds” in the March 2006 issue. Also check out the archives at www.podiatrytoday.com.
1. Schultz GS, Sibbald RG, Falanga V, Ayello EA, Dowsett C, Harding K, Romanelli M, Stacey MC, Teot L, Vanscheidt W. Wound bed preparation: a systematic approach to wound management. Wound Repair and Regeneration April 2003; 11(Supp2): S1-28.
2. Enoch S, Harding K, Wound Bed Preparation: The Science Behind the Removal of Barriers to Healing. Wounds 2003; 15(7): 213-229.
3. Stanley A, Osler T. Senescence and the healing rates of venous ulcers. Journal of Vascular Surgery June 2001; 33(6): 1206-1211.
4. Mulder GD, Vande Berg JS. Cellular senescence and matrix metalloproteinase activity in chronic wounds. Journal of the American Podiatric Association Jan 2002; 92(1): 34-37.
5. Ovington L. Overview of Matrix Metalloprotease Modulation and Growth Factor Protection in Wound Healing. Matrix Metalloprotease Modulation and Growth Factor Protection. Ostomy Wound Management June 2002; 48(6): 52-57.
6. Armstrong DG, Jude EB. The role of Matrix Metalloproteases in wound healing. Journal of the American Podiatric Medical Association Jan 2002; 92(1): 12-18.
7. Nwometh BC, Yager DR, Cohen IKC. Physiology of the chronic wound. Clinics in Plastic Surgery July 1998; 25(3): 341-356.
8. Cullen B. The Role of Regenerated Cellulose/Collagen in Chronic Wound Repair: Matrix Metalloprotease Modulation and Growth Factor Proection. Ostomy Wound Management June 2002; 48 (6): 8-13.
9. Mast B, Schultz G. Interactions of cytokines, growth factors and proteases in acute and chronic wounds. Wound Repair and Regeneration 1996; 4(4): 411-419.
10. Thompson PD. Immunology, microbiology and the recalcitrant wound. Ostomy/Wound Management 2000; 46(1Asuppl): 77S-82S.
11. McGuckin M, Goldman R, Bolton L, et al. The clinical relevance of microbiology in acute and chronic wounds. Advances in Skin Wound Care 2003; 16(1): 12-23.
12. Barone EJ, Yager DR, Pozez AL, et al. Interleukin-1 alpha and collagenase activity are elevated in chronic wounds. Plastic Reconstr Surgery 1998; 102(4): 1023-7.
13. Trengrove NJ, Bielefeldt-Ohmann H, Stacey MC. Mitogenic activity and cytokine levels in non-healing and healing chronic leg ulcers. Wound Repair Regeneration 2000;8(1): 13-25.
14. Falanga V, Shen J. Growth factors, signal transduction and cellular responses. In Cutaneous Wound Healing, Editor V. Falanga Martin Dunitz, London 2001.
15. Heldin CH, Westermark B. Mechanism of action and in vivo role of Platelet-derived Growth Factor. Professional Reviews Oct 1999; 79(4): 1283-1300.
16. Stadelmann W, Digenis A, Tobin G. Physiology and Healing Dynamics of Chronic Cutaneous Wounds. American Journal of Surgery August 1998; 176 (Supp 2A): 26S-38A.
17. Van den Berg AJ, Halkes S, Quarles van Ufford H, et al. A novel formulation of metal ions and citric acid reduces reactive oxygen species in vitro. Journal of Wound Care 2003; 12(10).
18. Salim AS. The role of oxygen-derived free radicals in the management of venous (varicose) ulceration: A new approach. World Journal of Surgery 1991; 15(2): 264-9.
19. Abd-El-Aleem SA, Ferguson MWJ, Appleton I, et al. Expression of nitric oxide synthase isoforms and arginase in normal human skin and chronic venous leg ulcers. Journal Pathology 2000:191(4): 434-42.
20. Jude EB, Tentolouris N, Appleton I, et al. Role of neuropathy and plasma nitric oxide in recurrent neuropathic and neuroischemic diabetic foot ulcers. Wound Rep Regen 2001; 9(5): 353-9.
21. Li J, Zhang Y, and Kirsner R. Angiogenesis in Wound Repair: Angiogenic Growth Factors and the Extracellular Matrix. Microscopy Research and Technique 2003; 60:107-114.
22. Nissen NN, Polverini PJ, Koch AE. Vascular Endothelial Growth Factor Mediates Angiogenic Activity during the Proliferative Phase of Wound Healing. Am Journal of Path 1998; 152(6): 1445-52.
23. Pollack SV, The wound healing process. Clin Dermatol 1984; 2:8-16.
24. Ovington L. Bacterial toxins and wound healing. Ostomy Wound Management 2003 Jul; 47(7Asuppl): 8-12Review.
25. Lobmann, R MD, Schultz G PhD, Lehnert H MD, Proteases and the Diabetic foot Syndrome: Mechanisms and Therapeutic Implications. Diabetes Care 2005; 28(2): 461-470.