Which molecules of the initial phase of wound healing may be used as markers for early detection of skin damage?

Which molecules of the initial phase of wound healing may be used as markers for early detection of skin damage? L.H. Cornelissen October 2004 BMTE 04.53 Promotor: prof.dr.ir. F.P.T. Baaijens Coach: dr.ir. C.W.J. Oomens Eindhoven University of Technology Department of Biomedical Engineering Section Materials Technology Division Biomechanics and Tissue Engineering

Contents Abbreviations 3 1 Introduction 4 2 Wound healing 5 2.1 Acute wound healing.............................. 5 2.2 Chronic wounds................................. 7 3 Growth factors and cytokines in wound healing 9 3.1 Role of growth factors and cytokines..................... 9 3.2 Time scales of growth factors and cytokines................. 15 3.3 Effect of growth factors and cytokines related to pressure ulcers...... 16 4 Ions in wound healing 18 4.1 Role of Ions................................... 18 4.2 Time scales of ions............................... 19 4.3 Effect of ions related to pressure ulcers.................... 20 5 Theoretical models of wound healing 21 6 Conclusion and aim 23 Bibliography 26 A Expression and function of growth factors and cytokines 36 2

Abbreviations BMP CTGF EGF FGF GM-CSF GRO HGF IGF IL IP KGF MCP MIP MMP MSP NGF PDGF TGF TIMP TNF VEGF Bone morphogenetic protein Connective tissue growth factor Epidermal growth factor Fibroblast growth factor Granulocyte-macrophage colony stimulating factor Growth related oncogene Hepatocyte growth factor Insulin-like growth factor Interleukin Interferon-γ-induced protein Keratinocyte growth factor Macrophage chemoattractant protein Macrophage inflammatory protein Matrix metalloproteinase Macrophage stimulated protein Nerve growth factor Platelet derived growth factor Transforming growth factor Tissue inhibitor of metalloproteinase Tumor necrosis factor Vascular endothelial growth factor 3

Chapter 1 Introduction Patients who are subjected to sustained mechanical loads such as pressure, shear, or friction, may develop pressure ulcers. These ulcers appear predominantly in patients who are bedridden or wheelchair bound. The positive predictive value of the risk score lists, now used to assess the risk for an individual patient to develop pressure ulcers, is very small, meaning that of all patients at risk, only a small number of patients really develop pressure ulcers. Besides, a number of patients is not identified as at risk. 103 It is expected that an objective measure to assess epidermal damage is much more useful in the prevention of pressure ulcers. Directly after damage is applied to the epidermis, the wound healing process is initiated. This process is regulated by cytokines and growth factors that attract neutrophils and macrophages to the wound site. Other actions of these signalling molecules are the regulation of proliferation, migration and differentiation of the keratinocytes in the epidermis and the fibroblasts in the dermis. Depending on the time scales that these molecules are secreted, the cells that secrete them and the site where they act, these molecules may be useful in the assessment of skin damage. Therefore, this literature report discusses in more detail those markers that are involved in the wound healing cascade. Some of these may be used to assess damage to the skin in an early phase, even before the damage is visible with the eye. These markers may also be used to assess the risk for an individual patient to develop superficial pressure ulcers. 4

Chapter 2 Wound healing Wound healing is a complex biological process that requires the interaction of different tissue structures and a large number of resident and infiltrating cells such as immune cells, keratinocytes, fibroblasts, and endothelial cells. In this chapter, the general healing process and subsequently, deficiencies of the healing process leading to chronic wounds are discussed. 2.1 Acute wound healing Wound healing involves a number of different processes that must be performed to repair the damaged tissue and can last two years. An optimal environment for the natural wound healing process is warm, moist and non-toxic. A reduced temperature can delay wound healing, since it may inhibit cell activity. 97 The normal healing process is generally divided in three overlapping processes: (1) hemostasis and inflammation, (2) proliferation and repair, and (3) remodelling. Figure 2.1 shows the course of the different phases and points out the time scales of the different invading cells. Hemostasis and inflammation The inflammatory response usually extends to the fourth day of healing and the most obvious result is the removal of the debris and necrotic tissue from the injured area and the local reduction of infection. 20 Hemostasis precedes the inflammation 124 ; platelets are released from the blood vessels and a fibrin clot is formed that covers the wound and brings wound edges together. This blood clot serves as a matrix for invading cells and as a reservoir of growth factors and cytokines. 121 These initiate a cascade of reactions which eventually results in vasodilatation of the existing blood vessels and an increased permeability for neutrophils, monocytes and other white blood cells. Monocytes rapidly differentiate into macrophages after migration from the vasculature to the wound site. Neutrophils arrive approximately 24 hours after injury and their main role is phagocytosis and wound debridement. They are a source of cytokines, which are one of the first signals to activate fibroblasts and keratinocytes. 76 Two or three days after injury, macrophages enter the wound space to digest the fibrin clot. Their antimicrobial function is carried out by phagocytosis and the generation of reactive radicals. 87 The major contribution 5

Wound healing Figure 2.1: Migration of immune cell populations to the wound site with respect to days post wounding. This migration correlates well with the different phases of wound healing. 124 of macrophages to wound healing is the release of growth factors and cytokines that attract fibroblasts and regulate their proliferation. The growth factors and cytokines also regulate collagen synthesis. Proliferation and repair This phase starts with the migration and proliferation of keratinocytes. 51 They arise from the periphery of the wound and from epidermal appendages like hair follicles. After day 4, a hypertrophic neo-epidermis has formed over the wound. 33 Migration of the keratinocytes is accompanied by degradation of the extracellular matrix through metalloproteinases (MMPs) that are secreted by keratinocytes. The activation of the MMPs is partly regulated by an extracellular calcium gradient, which is also required for the migration of the keratinocytes. 46 The rate of migration is dependent on the tissue oxygen tension and the humidity of the environment. Once the migrating edges have united, the rate of epithelial proliferation falls to three to four times the normal rate and the epidermal cells reassume their original morphology and function. 20 In a later stage, dermal fibroblasts are triggered to secrete a loose extracellular matrix, which contains large quantities of fibronectin. This binds cells to the matrix, promoting further cell adherence and the provision of a support mechanism for the deposition and orientation of the collagen fibrils. Once a collagen-rich matrix has developed, the fibroblasts are down-regulated and collagen production is reduced. 20 Macrophages, blood vessels and fibroblasts can migrate through this new formed extracellular matrix. Fibroblasts transform into myofibroblasts, which are involved in wound contraction. 51 Together with the formation of the matrix, angiogenesis occurs to provide the granulation tissue with new blood vessels to supply the wound with nutrients and oxygen. The loops of the newly formed capillaries give the tissue its red appearance. 97 Remodelling Remodelling of the immature tissue commences simultaneously with granulation tissue formation, but is often regarded as the third phase of wound healing. During this phase, granular tissue changes into a mature scar, which is less cellular and less vascular than the granulation tissue. The decrease in cellularity might be due to migration of the cells out of the wound site or to apoptosis. What mechanisms cause apoptosis are not 6

Wound healing clearly understood, but oxidative stress, nitric oxide, calcium, proteases, nucleases, and mitochondria are considered to be important mediators. 20 2.2 Chronic wounds A chronic wound is defined as any interruption of the continuity of the body s tissue that requires prolonged time to heal, does not heal or recurs. Skin ulcers, like diabetic ulcers, venous ulcers and pressure ulcers are the most common types of chronic wounds. Chronic wounds become heavily colonized with bacteria as a result of hypoxia, poor blood flow, and the prolonged period of time that the wound is open. 34 It is also found that these ulcers contain excessive reactive oxygen species that further damage the cells and the healing tissue. 119 Processes seen in acute wound healing (inflammation, proliferation and remodelling) are also found in chronic wounds 125 The events of early wound healing reflect a fine-balanced environment in which proteolytic activity and matrix synthesis occur under tight regulation. This balance is lost in chronic wounds. 110 Fluid obtained from chronic wounds will block cellular proliferation and angiogenesis 17, 29 whereas fluid from acute wounds stimulate in vitro proliferation of fibroblasts, keratinocytes and endothelial cells. 58 Differences between acute and chronic wounds are summarized in table 2.1. Table 2.1: Differences between acute and chronic wounds 125 Wound characteristic Acute wound Chronic wound healing primary intention secondary intention tissue loss none / minimal extensive edge apposed no apposition stage of repair sequential simultaneous clot formation / hemostasis present absent matrix deposition minimal extensive angiogenesis minimal extensive epithelialization minimal extensive bacteria absent to minimal present to excessive phagocytosis minimal increased to extensive contraction minimal increased scar minimal extensive fluid exudate transudate remodelling minimal extensive In an attempt to heal chronic wounds, different strategies are tried, including wound bed preparation, application of growth factors, application of bioengineered skin, gene therapy, and stem cell therapy. 34 These treatments are only partially successful and are generally limited to nonspecific and generalized attempts to reduce the conditions that initially led to propagate the injury. 77 Several differences in the molecular environments of acute and chronic wounds may play a role in the pathophysiology of chronic wounds. Mast et al. 77 formulated a hypothesis that integrates observed cytokine profiles in the molecular pathophysiology of chronic wounds (figure 2.2). 7

Wound healing Figure 2.2: Hypothesized model for chronic wound pathophysiology. 77 Chronic wounds are characterized by one or more inflammatory stimuli such as repeated trauma and ischemia, which eventually lead to a broken skin barrier and colonization of bacteria. An excessive infiltration of neutrophils is often seen in chronic wounds 27, 28, but also macrophages enter the wound site. Neutrophils and macrophages secrete proinflammatory cytokines like IL-1β and TNF-α. Neutrophils release enzymes that are capable of destroying important healing factors such as PDGF and TGF-β. 126 The cytokines increase production of MMPs and inhibit the production of TIMPs (tissue inhibitor of metalloproteinase). The elevated MMP activity degrades the extracellular matrix 84 and growth factors, which further limits the wound healing cascade. Entry to the repair phase is impaired. 77 Strategies to prevent or heal chronic ulcers need to be focussed on the down-regulation of the neutrophil infiltration and they should also inhibit or neutralize the host of destructive proteases released from these powerful neutrophils. 27 8

Chapter 3 Growth factors and cytokines in wound healing The repair process is initiated immediately after injury by the release of various growth factors and cytokines. The expression and function of these molecules is discussed in this section. It is not exactly known how all growth factors and cytokines influence each other. Stadelmann et al. 110 schematized the role of a number of growth factors and cytokines in the wound healing process (figure 3.1). The main functions of the different growth factors are schematized in Appendix A, just like the release of the growth factors and cytokines by different cells. 3.1 Role of growth factors and cytokines Platelet derived growth factor (PDGF) PDGF is released in large amounts from platelets 21, immediately on wounding. 51 Macrophages, fibroblasts and endothelial cells are also able to secrete PDGF. 31 This cytokine is chemotactic to various cells, such as neutrophils, monocytes, macrophages and fibroblasts. 21 Furthermore, it enhances proliferation of fibroblasts in an early stage of wound healing and it stimulates these cells to produce the extracellular matrix. 49 PDGF can activate monocytes to mature into macrophages that secrete a number of other growth factors and cytokines. 116 In a later stage, PDGF stimulates fibroblasts to contract collagen matrices and induces its transition into myofibroblasts. 121 It is found that levels of PDGF in non-healing ulcers are strongly reduced 108 and overexpression of PDGF results in hypertrophic scars. 83 Insulin-like growth factor (IGF) Two different forms of IGF are produced by wound fibroblasts 21 and IGF is also found in platelets from which it is released during clotting. 7 It is a potent stimulator of mitogenesis and survival of many different cell types. 121 IGF alone has minimal effects on wound healing, but dermal and epidermal synthesis is increased significantly when IGF is applied together with PDGF. 72 9

Growth factors and cytokines in wound healing Figure 3.1: Wound healing is regulated by a number of complex processes, many of which are mediated by growth factors or cytokines. 110 Vascular endothelial growth factor (VEGF) Expression of the VEGF gene is strongly induced after cutaneous injury, with keratinocytes and macrophages as the main producers. 15 Several growth factors including KGF, EGF, TGF-α and HGF have been shown to stimulate the production of VEGF by keratinocytes in culture. 35 It is suggested that VEGF stimulates angiogenesis, since its receptors are found on blood vessels in granulation tissue. 39 Impaired wound healing is associated with a reduced expression of VEGF 35 and overexpression of VEGF accelerated healing. 14 Treatment of ischemic wounds with VEGF improves wound healing. 22 Fibroblast growth factor (FGF) Upregulation of FGF after injury is found in keratinocytes, macrophages, endothelial 21, 121 cells and fibroblasts. Some functions of FGF are stimulation of angiogenesis and regulation of migration and differentiation of the target cells. Most types of FGF, like FGF1 and FGF2 (also named afgf and bfgf respectively), have a broad mitogenic function. They stimulate proliferation of various cells including keratinocytes and fibroblasts. FGF7, also known as keratinocyte growth factor (KGF), does not have a broad spectrum but is specific for epithelial cells. 121 The most potent stimulator of KGF expression was PDGF 120, but KGF induction is also found by IL-1β, TNF-α, and IL- 6. 13 Werner et al. 120 suggested a model for regulation of KGF expression during wound healing (figure 3.2). Experiments 122 demonstrated that highest levels of KGF mrna 10

Growth factors and cytokines in wound healing expression were found in the dermis. Marchese et al. 75 found a strong upregulation of KGF in acute human wounds. KGF not only stimulates proliferation and migration of epithelial cells, but also affects their differentiation. 120 It is demonstrated that reduced FGF expression can be correlated to impaired wound healing 108 Figure 3.2: Suggestion for the regulation of KGF expression during wound healing. 120 Epidermal growth factor (EGF) EGF is produced by epithelial cells 21 and lymphocytes 9. It stimulates mitosis of keratinocytes and fibroblasts and is therefore suspected to be involved in reepithalization and granulation tissue formation. 60 EGF is an attractant for fibroblasts and also stimulates migration and division of epithelial cells. The cells should be exposed for more than 4 hours to EGF to complete division. 7 Topical application of EGF accelerated wound closure of full-thickness wounds. 36 Transforming growth factor α (TGF-α) This growth factor belongs to the same family as EGF and has some overlapping functions. 121 Just like EGF, TGF-α stimulates mitosis of keratinocytes and fibroblasts and is therefore suspected to be involved in the early reepithalization and granulation tissue formation. 60 Both keratinocytes and hair follicle epithelial cells are identified as a source for TGF-α. 60 Transforming growth factor β (TGF-β) TGF-β is released from platelets in large amount immediately after injury, 2 resulting in a fast infiltration of neutrophils, macrophages and fibroblasts because of the chemoattractant properties of TGF-β. This growth factor is also synthesized by macrophages, lymphocytes, fibroblasts, bone cells and keratinocytes. 7 MCP-1 induces TGF-β release from fibroblasts. 51 Three different isoforms of TGF-β exist, which have overlapping functions. They are mitogenic for fibroblasts and inhibit proliferation of most other cells, including keratinocytes. In addition, TGF-βs are potent stimulators of the expression of extracellular matrix proteins and integrins. 93 It is suggested that TGF-β stimulates reepithalization and the formation of granulation tissue, since it was shown to stimulate angiogenesis, fibroblast proliferation, myofibroblast differentiation and matrix deposition. 93 It has a double-edged effect on fibroblast growth: it is inhibitory together 11

Growth factors and cytokines in wound healing with EGF and stimulating together with PDGF. 21 A study with TGF-β deficient mice 16 showed impaired late stage wound repair. The granulation tissue was much thinner and less vascular. Similar conclusions were drawn by Bonomo et al. 11, who applied TGF-β3 in a chronic wound model in rabbits and found that more granulation tissue was formed. The opposite was found in another animal study. 129 Chronic wounds in mice showed a significantly higher amount of TGF-β in the wound exudates than the amount in a control group. Hepatocyte growth factor (HGF) Expression of HGF in keratinocytes of wounded epidermis is strongly upregulated. This upregulation is also found in several cell types in the granulation tissue. 23 HGF stimulates migration and proliferation of keratinocytes 128 and it enhances production of metalloproteinases by keratinocytes. 30 Furthermore, HGF affects angiogenesis mediated via VEGF. 121 Macrophage stimulated protein (MSP) MSP is a liver-derived serum protein that regulates proliferation and differentiation of various cell types. Its receptor is present on macrophages and keratinocytes. 121 Skeel et al. 109 found that MSP stimulates macrophage pinocytosis and phagocytosis in vitro. It is therefore suggested that MSP enhances macrophage-dependent wound debridement. Nerve growth factor (NGF) The nerve growth factor plays a key role in the initiation and maintenance of inflammation in various organs and is therefore also suspected to play a role in wound healing. Studies showed that exogenous NGF indeed accelerate wound healing in normal and healing-impaired diabetic mice. 69 Expression of NGF at the wound site is found in granulation tissue fibroblasts and myofibroblasts 48, but also keratinocytes are able to synthesize and release NGF. 89 Upregulation of NGF is found after exposure to TGF-β. 18 The major function of NGF appears to be stimulation of nerve ingrowth, but NGF affects also other cell types in the skin. It stimulates proliferation and inhibits apoptosis of keratinocytes in vitro 89 and enhances proliferation and adherence on human dermal microvascular endothelial cells. 91 Furthermore, it is noted that NGF has a potent effect on fibroblast migration. 79 Bone morphogenetic protein (BMP) The expression of BMP-6 is observed in the regenerating epidermis at the wound edge as well as in fibroblasts of the granulation tissue. After wound closure, BMP-6 accumulated throughout the suprabasal layers, suggesting an inhibitory role of keratinocyte proliferation and/or induction of keratinocyte differentiation. 57 Connective tissue growth factor (CTGF) Vascular endothelial cells express CTGF, which stimulates proliferation and chemotaxis of fibroblasts. 12 Furthermore, CTGF is a potent inducer of extracellular matrix production and in these processes, it acts as a mediator of TGF-β. 38 Expression of CTGF during wound repair is found together with the ingrowth of granulation tissue. 54 Macrophage chemoattractant protein (MCP) From resident keratinocytes at the wound edge, MCP-1 is released on skin disruption. Keratinocytes of the hyperproliferative wound epidermis are pinpointed as 33, 123 the major source of MCP 56, but some endothelial cells and inflammatory cells in the 12

Growth factors and cytokines in wound healing granulation tissue also express MCP. Several cytokines, including TNF-α, IFN-γ, IL-1, and PDGF induce MCP production in fibroblasts. 25 The expression of MCP in keratinocytes in the wound egde can be inhibited by nitric oxide. 123 It is found that MCP-1 is a dominant monocyte chemoattractant during wound healing 33 and that MCP-1 is responsible for the lymphocyte recruitment in the initial period of healing. Furthermore, Gharaee-Kermani et al. 41 performed experiments from which was concluded that MCP-1 stimulated collagen synthesis and TGF-β expression in fibroblasts. Macrophage inflammatory protein α (MIP-α) MIP-α is produced by macrophages and has been shown to play a critical role in macrophage recruitment in a murine skin wound model. 56 It is not a direct angiogenic factor, but act as one by the attraction of macrophages, which are a source of angiogenic cytokines. 42 Tumor necrosis factor α (TNF-α) This growth factor is released primarily by mononuclear cells after stimulation by bacterial and matrix products, shortly after release of MCP-1. 51 It upregulates its own synthesis by macrophages 77 and stimulates macrophages to express other cytokines like IL-6, IL-8, GM-CSF, G-CSF, MCP-1 and IL-1. 51 TNF-α is implicated as a possible mediator of angiogenesis 22 and can activate monocytes to mature into macrophages. 51 Furthermore, TNF-α triggers the activation of metalloproteinases 47 and affects collagen synthesis. 77 The average level of TNF-α in chronic wounds is higher than in acute wounds. 129 The hypothesis that this excessive level is associated with impaired healing is tested by Wallace et al. 117 They concluded that because healing was initiated without a significant decline in the level of bioactive TNF-α, TNF-α-mediated events may not be the contributing factor to the impaired healing in chronic ulcers. Interleukin 1 (IL-1) IL-1 is stored in large amounts in the epidermis of intact skin and is released postwounding. 82 IL-1 is also released from disrupted endothelial cells, shortly after the release of MCP-1. 51 This cytokine is involved in many processes that are associated with inflammation and tissue repair like activation and chemotaxis of neutrophils and macrophages, proliferation of keratinocytes and fibroblasts, angiogenesis, matrix synthesis and collagen production. 21 IL-1 can stimulate macrophages to express other cytokines like IL-6, IL-8, GM-CSF, G-CSF, MCP-1 and autosecretion of IL-1. 51 An experimental human study 24 demonstrated that IL-1β stimulated migration of Langerhans cells away from the epidermis and that IL-1β is involved in local production of TNF-α. Levels of both IL-1α and IL-1β in the exudates of a chronic wound model were significantly higher than in 4, 129 those of the control group. Interleukin 4 (IL-4) IL-4 is secreted by mast cells, which are believed to stimulate a number of fibroblast activities such as migration, proliferation, and synthesis of the extracellular matrix via this secretion. 115 A late increase of IL-4 is found at the wound site, correlating with the 42, 127 down-regulate expression of several other cytokines. Interleukin 6 (IL-6) It appears that IL-6 is crucial for the quick start of the healing response, both via its mitogenic effects on wound edge keratinocytes and via its chemoattractive effect on neutrophils. 121 IL-6 is also known as a potent stimulator of fibroblast proliferation 46 and 13

Growth factors and cytokines in wound healing it is important in inhibiting extracellular matrix breakdown during proliferation. 99 Levels of IL-6 were two to four times higher in chronic wounds than the levels measured in late acute wounds. 77 In contrary to this, Werner et al. 121 reported that a complete lack of IL-6 caused impaired wound healing and excessive levels are associates with cutaneous scarring. Interleukin 8 (IL-8) It is expected that IL-8 is released from a preformed pool in keratinocytes or produced by wound cells like endothelial cells and fibroblasts. 111 The expression of IL-8 is triggered by IL-1 and TNF-α 42 and correlates strongly with neutrophil infiltration in the wound site. 33 In an in vitro a study of Iocono et al. 55 it is shown that IL-8 has an inhibitory effect on the proliferation of keratinocytes and contraction of collagen lattices by fibroblasts. They also found that high levels of IL-8 are associated with impaired wound healing. In contrast, Rennekampff et al. 92 observed a stimulating effect of IL-8 on keratinocyte proliferation in vitro. IL-8 was found to be a major chemoattractant for polymorphonuclear leukocytes. In in vivo experiments, topical application of IL-8 in human skin grafts in a mouse model stimulated reepithalization as a result of increased keratinocyte proliferation. 92 The diminished effect on wound contraction was consistent with the findings of Iocono 55. Interleukin 10 (IL-10) The anti-inflammatory cytokine IL-10 is thought to be involved in the limitation and termination of the inflammatory process. IL-10 regulates growth and/or differentiation of immune cells, keratinocytes, and endothelial cells 81 and inhibits infiltration of neutrophils as well as the expression of several cytokines. 100 An increased expression of IL-10 was shown to correlate with impaired wound healing. 70 Interferon-γ-induced protein 10 (IP-10) The expression of IP-10 after the initial wound healing can be correlated to the lymphocyte infiltration in the wound site. 33 Data from a study with IP-10 overexpressing mice 71 suggested that IP-10 is able to inhibit wound repair by disturbing the normal development of granulation tissue. Which is supported by the known inhibitory effect of IP-10 on angiogenesis. 6 Growth-related oncogene-α (GRO-α) GRO-α is expressed colocalized with IL-8 mrna expression in the superficial wound. Its expression results in infiltration of neutrophils in the wound site. 33 Granulocyte-macrophage colony stimulating factor (GM-CSF) GM-CSF is a cytokine that is mitogenic for keratinocytes 59 and stimulates migration and proliferation of endothelial cells. 19 Accelerated wound healing was observed in mice that overexpressed this cytokine as a result of increased keratinocyte proliferation. In addition, neovascularization and granulation tissue formation were enhanced. Since the expression of other cytokines such as TGF-β were changed, it is expected that GM-CSF stimulates wound repair indirectly. 74 14

Growth factors and cytokines in wound healing 3.2 Time scales of growth factors and cytokines Several studies are performed that relate cytokine and growth factor expression in wounds to time after injury. Kondo et al. 62 studied the expression of IL-8, MCP-1 and MIP-1α to test if these cytokines can be used to determine the wound age in human skin wounds. All the cytokines showed enhanced expression after 4 hours, but the peak was found approximately 1 day after injury. The strongest expression difference between groups with different wound ages were found for IL-8. In another study with human skin wounds 44, it was found that TNF-α, IL-6, and IL-1β showed slightly enhanced expression 15-20 min after injury. Marked expression of IL-1β was observed after 30-60 min, whereas a marked reaction of TNF-α and IL-6 was not found until 60-90 min. The expression of IL-1β persisted until 90 min in contrary to the expression of TNF-α and IL-6, which lasted up to 3 and 5 hours respectively. The expression of KGF was tested by Marchese et al. 75 They found increased KGF transcript levels in acute wounds 1 to 3 days after injury, compared with controls. The levels remained elevated 7-8 days after wounding, but to a lesser extent. MCP-1 expression was examined in a burn wound. 105 A significant increase in MCP-1 content was observed 1 day post-burn injury. This upregulation, which was more in young mice than in aged mice, persisted at 4 days post-burn injury. At this time, the same levels for young and aged mice were reached. Experiments from Engelhardt et al. 33 showed elevated levels of both IL-8 and GRO-α in the early wound healing. They also found early expression of MCP-1, but these levels were also elevated 2-3 weeks after wounding, whereas Gillitzer et al. 42 mention that MCP-1 expression is almost exclusively found during the first week after wounding. The pro-inflammatory cytokines IL-1β and TNFα were present in high levels during early normal wound healing, but there was a regular and consistent reduction in their levels as healing proceeds. 77 Ohshima et al. 86 measured levels of TNF-α and IL-1β and noted that the peak concentration was reached three hours after wounding. They also noted peak levels of IL-1α and IL-6 after 6 and 12 hours respectively. A rebound of all cytokine levels was found 72 hours after wounding. Henry et al. 51 graphically displayed the temporal expression profile of several cytokines (figure 3.3). Figure 3.3: Temporal expression profile of several cytokines throughout the wound healing timeline. 51 15

Growth factors and cytokines in wound healing 3.3 Effect of growth factors and cytokines related to pressure ulcers The expression pattern of growth factors and cytokines in de development of pressure ulcers is not known. But it is suspected that this pattern is different from that in acute wounds and treatment with growth factors or cytokines will mimic the normal healing situation and thus improve healing. Topical treatment of pressure ulcers with growth factors or cytokines is investigated in clinical trial studies. Bernabei et al. 8 tested the effect of application of NGF in a subject with two pressure ulcers. One ulcer was treated with NGF and the other received normal treatment. After 15 days, the ulcer treated with NGF was healing in contrary to the other ulcer that was unchanged. A more thorough investigation about topical application of NGF was performed by Landi et al. 63 who found a statistically greater reduction in pressure ulcer area after 6 weeks of treatment with NGF. In a randomized clinical trial, topical application of recombinant PDGF-BB produced an increase in the rate of wound closure compared with placebo controls. 96 Quantitative and objective measurements suggested that PDGF-BB accelerated the deposition of new granulation tissue within the entire wound bed. The effect of topical recombinant human IL-1β on pressure ulcers was examined by Robson et al. 94 They did not observe any acceleration of wound healing at three different doses. A positive effect may be noticed with application of a higher dosis. It was concluded that the application of IL-1β did not have any negative side-effects. Hirshberg et al. 52 concluded from their experimental study that topical application of TGF-β3 is safe and useful in the treatment of pressure ulcers. It is found that application results in a significant increased rate of wound healing at the earliest stages. The healing rate was not significantly different at the termination of the study. Robson et al. 95 compared wound closure of grade III or IV pressure ulcers treated with sequential GM-CSF and bfgf, both cytokines alone, and placebo controls. A significant number of patients receiving any cytokine therapy achieved more than 85% decrease in ulcer volume than placebo controlled patients after 35 days of treatment. Patients achieving bfgf treatment obtained the best results. The long-term outcome of this study 88 showed no significant difference amongst the percentages of patients healed across the four treatment groups at any of the follow-up visits, lasting over one year. The time to achieve healing was also not significantly different for the four groups, although there was a trend that the bfgf-treated group achieved faster healing. In another study, 32 it was reported that healing was observed when GM-CSF was injected locally around and into a pressure ulcer. Unfortunately, this study did not include controls to see if the healing was significantly faster. The presence of the angiogenic peptide VEGF was examined in several regions of pressure ulcers. 90 Biopsies were taken from the necrotic central part, the surrounding granulation zone and the adjacent skin. VEGF is found in every region and its strong expression suggests that this factor is not responsible for the poor healing response. Bonnefoy et al. 10 compared the production of cytokines IL-1, IL-6, and TNF in the serum of bedridden patients with and without pressure ulcers and control patients. The bedridden patients without pressure ulcers were at risk according to the Norton scale. The production of IL-1 and TNF did not change significantly between the two groups. In contrast, abnormal levels of IL-6 were observed in patients with pressure ulcers and 16

Growth factors and cytokines in wound healing this production was significantly different from patient without ulcers. There was no significant difference in IL-6 levels between the non-ulcer group and the controls. 17

Chapter 4 Ions in wound healing Chronic or non-healing wounds are potentially caused by deficiencies or imbalances of ionic concentration profiles. Little is known about the exact role of ions in wound healing, but experimental studies have shown that cadmium, zinc, copper, magnesium, iron, 64, 66 manganese, and calcium are necessary to varying extents in wound repair. The role of calcium is best investigated, a number of other ions is briefly discussed in this section. 4.1 Role of Ions Calcium Intracellular calcium is emphasized as an important signal transmitter for mitosis, (terminal) differentiation, and apoptosis. 50 Waves of calcium concentration gradients evoked by chemical or physical injury reflect changes in calcium influx from the extracellular environment and activation of calcium channels in plasma membranes that may be modulated by potassium and sodium. 68 Percutaneous calcium absorption through intact skin is low and much greater uptake is expected from wound sites where the epidermal barrier function is destroyed. Outward diffusion of calcium in intact skin is likely to be low and some calcium loss is expected in wound exudates and cellular debris associated with wounds. 68 Experiments have shown that disturbances in the calcium gradient caused by calcium channel blockers, tape stripping or skin injury lead to an earlier release of phospholipid secretions, differences in migration patterns and a block in terminal keratinocytes differentiation. 78 An increase in the extracellular calcium gradient leads to a flux of calcium into the cell, thus calcium can be seen as a messenger translating extracellular signals into cellular responses. 61 3, 85 Wounds are often treated with calcium alginate dressings to improve healing and topical calcium chloride was shown to increase granulation tissue formation. 80 These experimental studies suggest that calcium is important in wound management, and it is thought that calcium is modulated via hormones, vitamin D or various growth factors. The first event in wound healing is hemostasis, a process in which calcium is needed. Platelets in the wound site release a number of growth factors, enzymes and clotting factors, of which calcium is identified as Factor IV. 68 The synthesis of other clotting factors is promoted by calcium. PDGF is indicated to influence the calcium influx and evokes the 18

Ions in wound healing release of calcium from intracellular pools. An appropriate calcium concentration is required in the proliferative phase for the proliferation of keratinocytes. Excessive calcium urges keratinocytes to differentiate and then, the proliferation/migration is skipped. 68 Calcium is also necessary in neutrophil activation. 65 Cell proliferation is affected by TGF-α through blocking of the calcium channels. 50 Calcium plays an important role in activation of MMPs. 46 In the remodelling phase, TGF-α has been shown to control terminal differentiation via the extracellular calcium concentration. 26 The calcium amount should be increased to trigger the keratinocytes to differentiate. It is also believed that extracellular calcium triggers intracellular signals leading to apoptosis. 50 Excessive calcium leads to a decrease in proliferation and chemotactic response and is a possible cause of delayed healing. 98 High extracellular calcium levels result in an increased calcium influx, causing premature keratinization and maturation of the cells. Migration, which is essential for resurfacing the wound phase, is inhibited. 68 Zinc Zinc has been used for more than 3000 years in the treatment of skin wounds, but the exact role is still not totally clear. In it found that zinc is a constituent of several enzymes that have a central role in the reconstruction of the wound matrix. 67 Zinc concentrations in wound fluid are higher than in normal skin 113 and increase predominantly during the formation of granulation tissue and reepithalization. 43 Zinc also exerts an anti-inflammatory effect on phagocytotic cells, which is important to late healing and wound closure. It is also noted that zinc stimulates the proliferation of keratinocytes and fibroblasts. 114 A raised level of zinc in the early phases of wound healing is believed to upset calcium concentrations and retard healing. 65 Animal studies demonstrate that zinc deficiency is associated with an increased risk of delayed wound healing and the 37, 104 development of chronic wounds. Copper Zinc and copper can interact with each other, an excess of one of these ions can impair processes modulated by the other. Copper is necessary in the cross-linking of collagen fibres 67 and it also affects the maturation of the collagen fibres. 114 Furthermore, copper seems to stimulate the proliferation of keratinocytes and fibroblasts in a monolayer. 114 An experimental study showed that levels of copper were higher in wound fluid than those expected in plasma. 113 Magnesium An initially high magnesium concentration is harmful in hemostasis since magnesium inhibits platelet aggregation and promotes the release of anti-clotting factors. 68 An initially high concentration of magnesium was measured by Grzesiak et al. 45 who concluded that magnesium was needed for the adhesion of marophages and neutrophils to fibrinogen. 4.2 Time scales of ions An experimental study investigated the time scales of different ions involved in wound healing. 65 Full thickness skin wounds were made in rats and the amount of zinc, calcium, copper, and magnesium was measured in the healing period. A progressive increase in zinc, calcium, and magnesium was noticed 5 days postwounding, which coincides with 19

Ions in wound healing the maximal inflammation in the wound site and maximal proliferation in the epidermis and the dermis. The concentrations of magnesium and zinc were more or less the same in the first four days, in contrast to calcium, which was already remarkably increased at day 2. Levels of magnesium, zinc, and calcium were declined to normal by day 7. Copper concentrations were rather low at all stages in the wound healing process, but a marginally increased level of copper was found in the first two days. In contrary to this study, Gray 43 reported a initial decline in the bioavailable zinc in the serum and wound bed. The amount of zinc increases during late phases of wound healing. A decrease of extracellular calcium concentration in wound fluid in early stages of wound healing was noticed by Grzesiak et al. 45 They also measured the extracellular magnesium concentration, that increased in the early stages. As wound healing progressed, both magnesium and calcium concentrations returned to normal plasma levels. 4.3 Effect of ions related to pressure ulcers In a randomized trial, calcium alginate dressings have been shown to improve the healing of pressure ulcers. 102 After treatment, a 40% reduction in wound surface area was observed after four weeks in the treatment group and after eight weeks in the control group. Sequential treatment with calcium alginate dressings and hydrocolloid dressings also accelerated healing compared with treatment with the hydrocolloid dressing alone. 5 Gengenbacher et al. 40 compared biochemical nutritional parameters in acutely ill elderly with pressure ulcers and a control group of acutely ill elderly without ulcers. After one day admission, blood samples were collected to measure iron, zinc, and some other markers. The levels of both ions were significantly lower in patients with pressure ulcers than in patients without ulcers. The efficacy of oral zinc supplementation in the treatment of pressure ulcers is examined in different studies. No difference was found between men receiving zinc sulfate and those receiving a placebo (reviewed in Gray 43 ). It is not always safe to administer oral zinc sulfate. A quadriplegic man receiving zinc to promote the healing of pressure ulcers developed zinc-induced copper deficiency anemia. 112 A more recent study 53 evaluated the effect of administration of a mix of nutritional supplements with e.g. proteins, zinc and vitamines in patients with a hip-fracture on the development of pressure ulcers. The incidence of pressure ulcers in the placebo group was slightly, but not significantly higher than the incidence in the supplement group. A trend was observed towards later development of ulcers in the supplement group. They suggest that in order to prevent pressure ulcers, nutritional supplementation should be started in an earlier stage, probably before the critical event. 20

Chapter 5 Theoretical models of wound healing In literature, different models are found that describe wound healing. In this section, only models involving epidermal wound healing are discussed since the early phases of superficial pressure ulcers are expected to start in the epidermis. Savakis et al. 101 developed two different models of wound healing in which the epidermis is assumed to be a plane surface without thickness and that epidermal wound healing results from cell migration. The exact biological mechanism responsible for cell migration is not included. Mitosis, serving for epidermal thickening, is neglected, which is allowed since the epidermis is assumed to be without thickness. In the first model, a difference was made between injured and healthy tissue, depicted as a square with in the middle a circular wound. One element at the boundary of the wound was randomly chosen and treated as healthy tissue in the next time step, continuing until no wounded tissue was left. In the second model, an area in the mesh was pointed to be injured. The locations of the mesh that do not belong to the wound are occupied by an epidermal cell and wound locations are empty. The cells perform a random walk and when they reach an empty location, this location is filled, representing healing of that part of the wound. Another mathematical model 73 described the replacement of wounded epidermal tissue by a negative feedback mechanism in a one-dimensional model. Low oxygen levels attract macrophages which release different growth factors that promote angiogenesis. The newly formed blood vessels deliver oxygen and nutrients involved in the healing process. The raised oxygen level diminishes macrophage attraction and the process eventually stops. This model is based on diffusion equations of oxygen, growth factors and capillary density that together describe tissue regeneration. The results suggest that healing of a circular wound depends on the oxygen level within the wound site. A reaction-diffusion model was proposed by Sherratt et al. 106 The mitosis was represented by a diffusional migration of cells down the gradient of cell population density. The model solutions lack the typical wound healing phases (the lag phase followed by a linear phase) that characterize experimental studies. To improve this model, biochemical markers were included which can either activate or inhibit the healing response. 107 These markers were assumed to be produced in the epidermal cells. The increase of cell density was taken as a function of cell migration, natural loss, and the inhibiting or activating chemical concentration. These model solutions did include the typical healing 21

Theoretical models of wound healing response. It was suggested that biochemical autoregulation of mitosis can promote epidermal wound healing by providing a new population of cells in the advancing front. The model developed by Adam et al. 1 was kind of similar to that of Sherratt et al. 107, only the inhibitor was not included. They evaluated the critical size defect, which is defined as the smallest wound size that does not heal during lifetime. The value that was calculated was somewhat low, but of the right order of magnitude compared to observational data. Wearing et al. 118 extended the model of Sherratt et al. 107 by adding the role of KGF in epidermal wound healing. They defined wound healing as the migration and differentiation of cells at the wound edge to regenerate an epithelium across the wound surface. A one dimensional model was developed, representing a single row of epidermal cells. Since KGF is mainly expressed in the dermis and the receptors are found in keratinocytes in the epidermis, a dermal-epidermal interaction is included in the model. It was shown that the large up-regulation of KGF postwounding extends the KGF signalling range, but reduces epidermal regeneration. 22

Chapter 6 Conclusion and aim It is expected that an objective measure is much more useful in the prevention of pressure ulcers than the risk score lists now used in nursing care. The amount of growth factors, cytokines and ions that are produced after pressure injury may give an indication of the susceptibility of a patient to the development of pressure ulcers. Therefore, it has to be known whether the expressions of growth factors and cytokines or the ion concentrations are different in patients that are more susceptible for the development of pressure ulcers than in non-susceptible patients. It is also important to know whether the growth factors and cytokines are detectable at the skin surface and whether their measurements can be performed easily. The growth factors and cytokines that play a role in the late stages of wound healing such as EGF, TGF-α, BMP, CTGF, IL-4, IL-10, and IP-10 are no good markers for the early detection of the development of pressure ulcers. HGF, MSP, IGF, MIP-α, GM-CSF, and GRO-α are also omitted as marker since their contribution to wound healing is suspected to be small or their role is not well defined. Experiments showed that VEGF is present in different regions of pressure ulcers and it was concluded that VEGF did not seem to be responsible for the poor healing. 90 Therefore, VEGF is not seen as a possible indicator of early detection of pressure ulcers. A possible marker is PDGF, which is expressed directly after wounding. Its production is decreased in non-healing ulcers 108 and topical application of PDGF on pressure ulcers promotes the healing 96. Whether the decreased production of PDGF is really related to an increased susceptibility or just to the diminished amount of platelets that is involved in the development of pressure ulcers in comparison to acute skin injury has to be investigated. Release of KGF is related to the production of PDGF, since this growth factor is the main inducer of KGF secretion 120. A reduction of FGF, the family to which KGF belongs, is observed by impaired healing. 108 If this reduction comes solely by a reduced amount of PDGF, then PDGF suffices as a marker. Otherwise, the role of reduction in KGF in the development of pressure ulcers may also be related to the patients susceptibility. The expression of TGF-β is found early in the wound healing cascade, but its exact role in chronic ulcers is not yet clear. It is suggested that a chronic wound has a decreased level of TGF-β, 11 whereas the opposite, a significantly higher amount of TGF-β, is also found. 129 This may be due to differences in the experimental setup. Bonomo et al. 11 created ischemic wounds in rabbits and observed improved healing after application of 23

Conclusion and aim TGF-β whereas Zhou et al. 129 used TGF-β deficient mice and observed the wound closure. NGF is related to TGF-β in the same way as KGF is to PDGF. The release of NGF is induced by TGF-β. So if the reduction of NGF in pressure ulcers is only related to a disordered TGF-β release, then it is not necessary to take NGF as a marker together with TGF-β. A protein that is released upon wounding by keratinocytes is MCP. 33, 123 The production of MCP mrna is induced by other cytokines such as TNF-α and IL-1. 123 It is also noted that TNF-α and IL-1 are released after release of MCP. The levels of TNF-α and IL-1 are both increased in chronic wounds. 129 It is not found in literature if this is also true for MCP. It may be possible to find a relation between the release of these three factors such that one or two can be omitted for the determination of the susceptibility of patients to pressure ulcers. The expression of IL-8 is triggered by IL-1 and TNF-α and elevated levels are found in chronic wounds. Thus, this cytokine can probably be omitted. IL-6 is indicated to be important in the quick start of the healing response. 121 Different 77, 121 results are found for IL-6 expression in chronic wounds and a study with pressure ulcers noted elevated levels of IL-6. 10 It is not found in literature how expression of IL-6 is induced and this is important in making a decision whether this cytokine is a potential marker or not. It may also be possible to take ion concentrations as a measure for susceptibility to pressure ulcers. Changes in ion levels by oral supplementation seem to improve healing of pressure ulcers. 53 It was noted that a larger effect was expected when administration of the nutrients was in an earlier stage, even before the risk of pressure ulcers increased. Therefore, the measurement of some ion concentrations in the human body may give an indication of the nutritional state of the patient and therewith of the susceptibility of the patient to the development of pressure ulcers. To study the influence of cytokines, growth factors and ions on the development of skin damage, it would be useful to study first their behaviour in the normal dynamics of the epidermis. Therefore, a model will be developed that describes the interactions of several molecules in the normal renewal of the epidermis. Figure 6.1: Lay-out of the numerical model of the epidermal differentiation This model will consist of different layers (Figure 6.1): a layer in which the cells proliferate (stem cells, transit amplifying cells), a layer with differentiating cells and a layer 24