23Principles of Wound Healing

Haemostasis

Haemostasis occurs immediately following an injury.⁵ To prevent exsanguination, vasoconstriction occurs and platelets undergo activation, adhesion and aggregation at the site of injury. Platelets become activated when exposed to extravascular collagen (such as type I collagen), which they detect via specific integrin receptors, cell surface receptors that mediate a cell’s interactions with the extracellular matrix. Once in contact with collagen, platelets release the soluble mediators (growth factors and cyclic AMP) and adhesive glycoproteins, which signal them to become sticky and aggregate. The key glycoproteins released from the platelet alpha granules include fibrinogen, fibronectin, thrombospondin, and von Willebrand factor. As platelet aggregation proceeds, clotting factors are released resulting in the deposition of a fibrin clot at the site of injury. The fibrin clot serves as a provisional matrix.⁶ the aggregated platelets become trapped in the fibrin web and provide the bulk of the clot (Figure 23.2). Their membranes provide a surface on which inactive clotting enzyme proteases are bound, become activated and accelerate the clotting cascade.

Growth factors are also released from the platelet alpha granules, and include platelet derived growth factor (PDGF), transforming growth factor beta (TGF-β), transforming growth factor alpha (TGF-α), basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), and vascular endothelial growth factor (VEGF). Major growth factor families are presented in Table 23.1. Neutrophils and monocytes are then recruited by PDGF and TGF-β from the vasculature to initiate the inflammatory response. A breakdown fragment generated from complement, C5a, and a bacterial waste product, f-Met-Leu-Phe, also provide additional chemotactic signals for the recruitment of neutrophils to the site of injury. Meanwhile, endothelial cells are activated by VEGF, TGF-α and bFGF to initiate angiogenesis. Fibroblasts are then activated and recruited by PDGF to migrate to the wound site and begin production of collagen and glycosaminoglycans, proteins in the extracellular matrix which facilitate cellular migration and interactions with the matrix supporting framework. Thus, the healing process begins with hemostasis, platelet deposition at the site of injury, and interactions of soluble mediators and growth factors with the extracellular matrix to set the stage for subsequent healing events.^1,2,7

Inflammation

Inflammation, the next stage of wound healing occurs within the first 24 hours after injury and can last for up to 2 weeks in normal wounds and significantly longer in chronic non-healing wounds (Figure 23.3). Mast cells release granules filled with enzymes, histamine and other active amines, which are responsible for the characteristic signs of inflammation, the rubor (redness), calor (heat), tumor (swelling) and dolor (pain) around the wound site. Neutrophils, monocytes, and macrophages are the key cells during the inflammatory phase. They cleanse the wound of infection and debris and release soluble mediators such as proinflammatory cytokines (including IL-1, IL-6, IL-8, and TNF-α), and growth factors (such as PDGF, TGF-β, TGF-α, IGF-1, and FGF) that are involved in the recruitment and activation of fibroblasts and epithelial cells in preparation for the next phase in healing. Cytokines that play important roles in regulating inflammation in wound healing are described in Table 23.2.

In addition to the growth factors and cytokines, a third important group of small regulatory proteins, listed in Table 23.3, has been identified, and are collectively named chemokines, from a contraction of chemoattractive cytokine(s).^8,9,10 the structural and functional similarities among chemokines were not initially appreciated, and this has led to an idiosyncratic nomenclature consisting of many acronyms that were based on their biological functions, (e.g., monocyte chemo-attractant protein-1 (MCP-1), macrophage inflammatory protein-1, MIP-1), their source for isolation (platelet factor-4, PF-4) or their biochemical properties (interferon-inducible protein of 10 kD a (IP-10), or regulated upon activation normal T-cell expressed and secreted, RANTES). As their biochemical properties were established, it was recognized that the approximately 40 chemokines could be grouped into four major classes based on the pattern of cysteine residues located near the N-terminus. In fact, there has been a recent trend to re-establish a more organized nomenclature system based on these four major classes. In general, chemokines have two primary functions: 1) they regulate the trafficking of leukocyte populations during normal health and development, and 2) they direct the recruitment and activation of neutrophils, lymphocytes, macro-phages, eosinophils and basophils during inflammation.

Proliferative phase

The milestones during the proliferative phase include replacement of the provisional fibrin matrix with a new matrix of collagen fibers, proteoglycans, and fibronectin to restore the structure and function to the tissue. Another important event in healing is angiogenesis, the in-growth of new capillaries to replace the previously damaged vessels and restore circulation. Other significant events in this phase of healing are the formation of granulation tissue and epithelialization. Fibroblasts are the key cells in the proliferative phase of healing.

Summary of acute wound healing

There are four phases of wound healing:

• Haemostasis – establishes the fibrin provisional wound matrix and platelets provide initial release of cytokines and growth factors in the wound.
• Inflammation – mediated by neutrophils and macrophages which remove bacteria and denatured matrix components that retard healing, and are the second source of growth factors and cytokines. Prolonged, elevated inflammation retards healing due to excessive levels of proteases and reactive oxygen that destroy essential factors.
• Proliferation – fibroblasts, supported by new capillaries, proliferate and synthesize disorganized ECM. Basal epithelial cells proliferate and migrate over the granulation tissue to close the wound surface.
• Remodelling – fibroblast and capillary density decreases, and initial scar tissue is removed and replaced by ECM that is more similar to normal skin. ECM remodelling is the result of the balanced, regulated activity of proteases.

Cellular functions during the different phases of wound healing are regulated by key cytokines, chemokines and growth factors. Cell actions are also influenced by interaction with components of the ECM through their integrin receptors and adhesion molecules. MMPs produced by epidermal cells, fibroblasts and vascular endothelial cells assist in migration of the cells, while proteolytic enzymes produced by neutrophils and macrophages remove denatured ECM components and assist in remodelling of initial scar tissue.

Normal and pathological responses to injury

Pathological responses to injury can result in non-healing wounds (ulcers), inadequately healing wounds (dehiscence), or in excessively healing wounds (hypertrophic scars and keloids). Normal repair is the response that re-establishes a functional equilibrium between scar formation and scar remodelling, and is the typical response that most humans experience following injury. The pathological responses to tissue injury stand in sharp contrast to the normal repair response. In excessive healing there is too much deposition of connective tissue that results in altered structure, and thus, loss of function. Fibrosis, strictures, adhesions, keloids, hypertrophic scars and contractures are examples of excessive healing. Contraction is part of the normal process of healing but if excessive, it becomes pathologic and is known as a contracture. Deficient healing is the opposite of fibrosis. It occurs when there is insufficient deposition of connective tissue matrix and the tissue is weakened to the point where scars fall apart under minimal tension. Chronic non-healing ulcers are examples of severely deficient healing.

Biochemical differences in the molecular environments of healing and chronic wounds

The healing process in chronic wounds is generally prolonged, incomplete and uncoordinated, resulting in a poor anatomic and functional outcome. Chronic, non-healing ulcers are a prime clinical example of the importance of the wound cytokine profile and the critical balance necessary for normal healing to proceed. Since cytokines, growth factors, proteases, and endocrine hormones play key roles in regulating acute wound healing, it is reasonable to hypothesize that alterations in the actions of these molecules could contribute to the failure of wounds to heal normally. Several methods are used to assess differences in molecular environments of healing and chronic wounds. Messenger ribonucleic acid (mRNA) and protein levels can be measured in homogenates of wound biopsies. The proteins in wounds can be immunolocalized in histological sections of biopsies. Wound fluids collected from acute surgical wounds and chronic skin ulcers are used to analyze the molecular environment of healing and chronic wounds. From these studies, several important concepts have emerged from the molecular analyses of acute and chronic wound environments.

The first major concept to emerge from analysis of wound fluids is that the molecular environments of chronic wounds have reduced mitogenic activity compared to the environments of acute wounds.⁴ Fluids collected from acute mastectomy wounds when added to cultures of normal human skin fibroblasts, keratinocytes or vascular endothelial cells, consistently stimulated DNA synthesis of the cultured cells. In contrast, addition of fluids collected from chronic leg ulcers typically did not stimulate DNA synthesis of the cells in culture. Also, when acute and chronic wound fluids were combined the mitotic activity of acute wound fluids was inhibited. Similar results were reported by several groups of investigators who also found that acute wound fluids promoted DNA synthesis while chronic wound fluids did not stimulate cell proliferation.^18,19,20

The second major concept to emerge from wound fluid analysis is the elevated levels of pro-inflammatory cytokines observed in chronic wounds as compared to the molecular environment of acute wounds. The ratios of two key inflammatory cytokines, TNFα and IL-1 β, and their natural inhibitors, P55 and IL-1 receptor antagonist, in mastectomy fluids were significantly higher in mastectomy wound fluids than in chronic wound fluids. Trengove and colleagues also reported high levels of the inflammatory cytokines IL-1, IL-6 and TNFα in fluids collected from venous ulcers of patients admitted to the hospital.²¹ More importantly, levels of the cytokines significantly decreased in fluids collected two weeks after the chronic ulcers had begun to heal. Harris and colleagues also found cytokine levels were generally higher in wound fluids from non-healing ulcers than healing ulcers.²⁰ These data suggest that chronic wounds typically have elevated levels of pro-inflammatory cytokines, and that the molecular environment changes to a less pro-inflammatory cytokine environment as chronic wounds begin to heal.

The third concept that emerged from wound fluid analysis was the elevated levels of protease activity in chronic wounds compared to acute wounds.^4,22,23 For example, the average level of protease activity in mastectomy fluids determined using the general MMP substrate, Azocoll, was low (0.75µg collagenase equivalents/ml, n = 20) with a range of 0.1 to 1.3µg collagenase equivalents/ml.²⁴ This suggests that protease activity is tightly controlled during the early phase of wound healing. In contrast, the average level of protease activity in chronic wound fluids (87µg collagenase equivalents/ml, n = 32) was approximately 116-fold higher (p<0.05) than in mastectomy fluids. Also, the range of protease activity in chronic wound fluids is rather large (from 1 to 584µg collagenase equivalents/ml). More importantly, the levels of protease activity decrease in chronic venous ulcers two weeks after the ulcers begin to heal.²⁴ yager and colleagues also found 10-fold higher levels of MMP-2 protein, 25-fold higher levels of MMP-9 protein, and 10-fold higher collagenase activity in fluids from pressure ulcers compared to surgical wound fluids using gelatin zymography and cleavage of a radioactive collagen substrate.²⁵ o ther studies using immunohistochemical localization observed elevated levels of MMPs in granulation tissue of pressure ulcers along with elevated levels of neutrophil elastase and cathepsin-G.²⁶ TIMP-1 levels were found to be decreased while MMP-2 and MMP-9 levels were increased in fluids from chronic venous ulcers compared to mastectomy wound fluids.²⁷ Recently, L dwig and colleagues reported that the ratio of active MMP-9/ti MP-1 was closely correlated with healing outcome of pressure ulcers treated by a variety of protocols (Figure 23.6).²⁸

It is interesting to note that the major collagenase found in non-healing chronic pressure ulcers was MMP-8, the neutrophilderived collagenase. Thus, the persistent influx of neutrophils releasing MMP-8 and elastase appears to be a major underlying mechanism resulting in tissue and growth factor destruction and thus impaired healing. This suggests that chronic inflammation must be decreased if pressure ulcers are to heal.

Other classes of proteases also appear to be elevated in chronic wound fluids. It has been reported that fluids from skin graft donor sites or breast surgery patients contained intact α1-antitrypsin, a potent inhibitor of serine proteases, very low levels of neutrophil elastase activity, and intact fibronectin.²⁹ In contrast, fluids from the chronic venous ulcers contained degraded 1-antitrypsin, and 10-fold to 40-fold higher levels of neutrophil elastase activity, and degraded fibronectin. Chronic leg ulcers were also found to contain elevated MMP-2 and MMP-9, and that fibronectin degradation in chronic wounds was dependent on the relative levels of elastase, α1-proteinase inhibitor, and α2-macroglobulin. ^30,31

Besides being implicated in degrading essential extracellular matrix components like fibronectin, proteases in chronic wound fluids also have been reported to degrade exogenous growth factors in vitro such as EGF, TGF-α, or PDGF.^1,24,32,33 In contrast, exogenous growth factors were stable in acute surgical wound fluids in vitro. Supporting this general concept of increased degradation of endogenous growth factors by proteases in chronic wounds, the average immunoreactive levels of some growth factors such as EGF, TGF-β and PDGF were found to be lower in chronic wound fluids than in acute wound fluids while PDGF-AB, TGF-α and IGF-1 were not lower.^32,34

In general, these results suggest that many chronic wounds contain elevated MMP and neutrophil elastase activities. The physiological implications of these data are that elevated protease activities in some chronic wounds may directly contribute to the failure of wounds to heal by degrading proteins which are necessary for wound healing such as extracellular matrix proteins, growth factors, their receptors and protease inhibitors. Interestingly, Steed and colleagues³⁵ reported that extensive debridement of diabetic foot ulcers improved healing in patients treated with placebo or with recombinant human Pd GF (Figure 23.7). It is likely that frequent sharp debridement of diabetic ulcers helps to convert the detrimental molecular environment of a chronic wound into a pseudoacute wound molecular environment.

Biological differences in the response of chronic wound cells to growth factors

The biochemical analyses of healing and chronic wound fluids and biopsies have suggested that there are important molecular differences in the wound environments. However, these data only indicate part of the picture. The other essential component is the capacity of the wound cells to respond to cytokines and growth factors. Interesting new data are emerging which suggest that fibroblasts in skin ulcers which have failed to heal for many years may not be capable of responding to growth factors and divide as fibroblasts in healing wounds. Ågren and colleagues³⁶ reported that fibroblasts from chronic venous leg ulcers grew to lower density than fibroblasts from acute wounds from uninjured dermis. Also, fibroblasts from venous leg ulcers that had been present greater than three years grew more slowly and responded more poorly to PDGF than fibroblasts from venous ulcers that had been present for less than three years. These results suggest that fibroblasts in ulcers of long duration may approach senescence and have a decreased response to exogenous growth factors.

Role of endocrine hormones in the regulation of wound healing

Classical endocrine hormones are molecules that are synthesized by specialized tissue and secreted into the blood stream which are then carried to distant target tissue where they interact with specific cellular receptor proteins and influence the expression of genes that ultimately regulate the physiological actions of the target cell. It has been known for decades that alterations in endocrine hormones can alter wound healing. Diabetic patients frequently develop chronic wounds due to multiple direct and indirect effects of the inadequate insulin action on wound healing. Patients receiving anti-inflammatory glucocorticoids for extended periods are also at risk of developing impaired wound healing due to the direct suppression of collagen synthesis in fibroblasts and the extended suppression of inflammatory cell function. The association of oestrogen with healing was recently reported by Ashcroft and colleagues³⁷ when they observed that healing of skin biopsy sites in healthy, postmenopausal women was significantly slower than in healthy premenopausal women. Molecular analyses of the wound sites indicated that TGF-β protein and mRNA levels were dramatically reduced in postmenopausal women in comparison to sites from premenopausal women. However, the rate of healing of wounds in postmenopausal women taking oestrogen replacement therapy occurred as rapidly as in premenopausal women. Furthermore, molecular analyses of wounds in postmenopausal women treated with oestrogen replacement therapy demonstrated elevated levels of TGF-β protein and mRNA that were similar to levels in wounds from premenopausal women. Aging was also associated with elevated levels of MMPs and decreased levels of TIMPs in skin wounds, which were reversed by oestrogen treatment.^38,39 The beneficial effects of oestrogen on wound healing could be achieved with topical oestrogen and were also observed in healthy aged men.⁴⁰ These data indicate the significant interactions that can occur between endocrine hormones and growth factors in the regulation of wound healing.

Molecular basis of chronic non- healing wounds

Conditions that promote chronic wounds are repeated trauma, foreign bodies, pressure necrosis, infection, ischemia, and tissue hypoxia. These wounds share a chronic inflammatory state characterized by an increased number of neutrophils, macro-phages, and lymphocytes which produce inflammatory cytokines, such as TNF-α, IL-1 and IL-6. In vitro studies have shown that TNF- α and IL-1 increase expression of MMPs and down-regulate expression of TIMP in a variety of cells including macrophages, fibroblasts, keratinocytes, and endothelial cells. All MMPs are synthesized as inactive proenzymes, and they are activated by proteolytic cleavage of the pro-MMP. Serine proteases, such as plasmin, as well as the membrane type MMPs can activate MMPs. Another serine protease, neutrophil elastase, is also present in increased concentrations in chronic wounds, and is very important in directly destroying extracellular matrix components and in destroying the TIMPs, which indirectly increases the destructive activity of MMPs.^4,22,25,33 Thus, the general molecular profile that appears in various types of chronic ulcers is (1) increased levels of inflammatory cytokines, which leads to (2) increased levels of proteases and decreased levels of protease inhibitors, which (3) degrade molecules that are essential for healing, including growth factors, their receptors and ECM proteins, which (4) prevent wounds from healing normally. Nwomeh and colleagues²³ further describe this common pathway in chronic wounds as a self-perpetuating environment in which chronic inflammation produces elevated levels of reactive oxygen species and degradative enzymes that eventually exceed their beneficial actions of destroying bacterial and debriding the wound bed and produce destructive effects that help to establish a chronic wound.

Based on these biochemical analyses of the molecular environments of acute and chronic human wounds, it is possible to propose a general model of differences between healing and chronic wounds. As shown in Figure 23.8, the molecular environment of healing wounds promotes mitosis of cells, has low levels of inflammatory cytokines, low levels of proteases and high levels of growth factors and cells capable of rapid division. In contrast, the molecular environments of chronic wounds generally have the opposite characteristics, i.e., the molecular environment does not promote mitosis of cells, has elevated levels of inflammatory cytokines, has high levels of proteases and low levels of growth factors and cells that are approaching senescence.^41,24,21 If these general concepts are correct, then it may be possible to develop new treatment strategies which would re-establish in chronic wounds the balance of cytokines, growth factors, proteases, their natural inhibitors and competent cells found in healing wounds.

Chronic venous stasis ulcers

Mechanisms involved in the creation and perpetuation of chronic wounds are varied and depend on the individual wounds. In general, the inability of chronic venous stasis ulcers to heal appears to be related to impairment in wound epithelialization. The wound edges show hyperproliferative epidermis under microscopy, even though further immunohistochemical studies revealed optimal conditions for keratinocyte recruitment, proliferation, and differentiation. The extracellular matrix and the expression of integrin receptors by keratinocytes that allow them to translocate play an important regulatory role in epithelialization. After receiving the signal to migrate, epidermal cells begin by disassembling their attachments from basement membrane and neighboring cells. They then travel over a provisional matrix containing fibrinogen, fibronectin, vitronectin, and tenascin and stop when they encounter laminin. During this process, keratinocytes are producing fibronectin, and continue to do so until the epithelial cells contact, at which time they again begin manufacturing laminin to regenerate the basement membrane.

There is evidence that the interaction between the integrin receptors on keratinocytes with the ECM will transform resting cells to a migratory phenotype. Integral in this transformation is the alteration in the pattern of integrin receptors expressed. After epithelialization is completed, integrin expression reverts back to the resting pattern. To further complicate this process, growth factors are involved in mediating keratinocyte activation, integrin expression, and in alterations in the matrix. Growth factors are able to differentially affect these processes. For example, TGF-β is able to promote epithelial migration while inhibiting proliferation. Although TGF-β induces the necessary integrin expression for migration, the cells behind those at the leading edge have little proliferative ability and so epithelial coverage of the wound is inhibited. Some chronic wounds may be deficient in TGF-β and its receptor.⁴²

Pressure ulcers

Chronic wounds have also been demonstrated to have elevated matrix degrading enzymes and decreased levels of inhibitors for these enzymes. Pressure ulcers, unlike chronic venous stasis ulcers, appear to have difficulty in healing related to impairment of ECM production. Studies have indicated that neutrophil elastase present in chronic wounds can degrade peptide growth factors and is responsible for degrading fibronectin. Pressure ulcers have also shown an increase in matrix metalloproteinases and in plasminogen activators in tissue. Chronic wound fluids demonstrate increased levels of gelatinases MMP-2 and MMP-9. Levels of MMP-1 and MMP-8 were also found to be higher in pressure ulcers and in venous stasis ulcers than in acute healing wounds. In addition, several of the endogenous proteinase inhibitors were shown to be decreased in chronic wounds. Proteinase inhibitors serve a regulatory role in matrix degradation by containing the matrix-degrading enzymes. Factors that promote MMP production or activation could counteract the effectiveness of proteinase inhibitors, for example the destruction of TIMP by neutrophil elastase. The tissue inhibitor level to MMP ratio may indicate an imbalance which contributes to the wound chronicity.

Bacterial biofilms in chronic wounds

Bacterial biofilms are well known in other medical specialities to cause a variety of chronic pathologies including periodontal disease, cystic fibrosis, chronic otitis media and osteomyelitis and prosthetic graft infection.⁵² Biofilms are characterized by an exopolymeric matrix of polysaccharides, proteins and DNA synthesized by the multiple bacterial species (polymicrobial) comprising the biofilm community. Bacteria (and fungi) contained within the biofilm matrix are highly tolerant to killing phagocytic inflammatory cells (neutrophils and macrophages), antibodies, and exogenous antibiotics, antiseptics and disinfectants. Several factors contribute to the increased tolerance of bacteria in biofilms to these agents, including reduced penetration of large proteins (antibodies) into the dense exopolymeric matrix, binding of oppositely charged molecules like antibiotics or cationic heavy metal ions (silver ion) by negatively charged components of the exopolymeric matrix, or neutralization of highly reactive chemicals like hypochlorous acid (bleach) by reaction with molecules comprising the exopolymeric matrix. Also, some bacteria in mature biofilms become metabolically quiescent and these ‘persister cells’ are therefore highly resistant to antibiotics that disrupt bacterial metabolism. These factors contribute to make biofilms extremely difficult to kill and clear from chronic wounds. Furthermore, components of the biofilm matrix and products produced by bacteria in the biofilm stimulate chronic inflammation, which leads to persistently elevated levels of molecules like proteases and reactive oxygen species that kill wound cells and damage proteins that are essential for healing.

Assessment of the ‘bioburden’ of wounds has traditionally relied upon relatively simple microbiology laboratory techniques that typically provide information on major bacterial and fungal species in swabs or biopsies that can grow under the nutritional and environmental conditions provided in the lab. These assessments of bacteria and fungi in wound samples have unquestionably generated important data that have been used for decades to help select therapeutic regimens for patients and their wounds. However, multiple publications have pointed out that, in many patients, measurements of total bacterial bioburden (expressed as colony forming units per gram of tissue biopsy or 0-4+ levels of bacterial growth) alone do not correlate well with the failure of wounds to heal. As shown in Figure 23.9, this led to the concept of ‘critical colonization’ or ‘occult infection’ to explain the discrepancy, because there was an apparent link between microbial bioburden in these wounds and the impaired healing in the wounds. However, it was not clear what aspect of the relatively low total bioburden was ‘critical’ to impairing healing. More thorough evaluation of these ‘standard’ clinical microbiology assays led to the realization that these assays are inherently limited by the rather poor ability to culture or identify most of the bacterial and fungal species that are actually present in an individual chronic wound. In other words, standard clinical microbiology assays only culture planktonic bacterial and fungal species that are able (capable) of growing on agar media plates supplemented with general nutrients in air at 37ºC. Thus, it is reasonable to assume that a more complete picture of different bacterial species (aerobes, facultative anaerobes, and obligate anaerobes) and fungal species in a particular wound should improve the ability to assess the microbial bioburden on individual wounds and to indicate what therapeutic strategies would be optimal for each wound. Fortunately, in the last few years sophisticated laboratory research techniques have been developed that allow a more complete assessment of bacterial bioburden. Specifically, these techniques demonstrated that a high percentage (~60%) of chronic skin wounds have extensive bacterial biofilms.⁵³ Using sophisticated polymerase chain reaction (PCR) techniques Dowd et al⁵⁴ reported that the bacterial and fungal complexity of chronic wound samples was much greater than previously thought. In fact, on average, approximately 60% of the bacterial species present in chronic pressure ulcers and around 30% of those present in diabetic ulcers were strict anaerobic bacteria, and many bacterial species were present that had never been reported in cultures of chronic wounds. These data suggest that many of the bacteria present in biofilms in a chronic wound may never be successfully cultured in the standard clinical micro-biology laboratory due to obligate cooperation with other bacteria that create unique environmental conditions in a polymicrobial community of bacteria in biofilms. A second major concept recently reported by Wolcott and colleagues⁵⁵ showed that mature biofilms are rapidly re-established in chronic wounds following surgical debridement, on the time frame of 24 to 72 hours. This indicates that sharp debridement opens a time-dependent therapeutic window to prevent the re-establishment of mature biofilms that are highly tolerant to host inflammatory response or to exogenous antimicrobial agents.

The clinical principle that should guide ‘biofilm-based wound care’ is to reduce plank-tonic and biofilm bacterial burdens by the most appropriate and effective means (surgical debridement, curettage, irrigation, etc), then follow the debridement by covering the wound with an effective bacterial barrier dressing, of which there are many types, including dressings with microbicidal metal ions (silver), quaternary amines, or occlusive films.⁵⁶