There are several growth factors and cytokines involved in wound healing with each having a specific individual role as shown in Table 6.1 [3, 4]. Epidermal growth factors (EGF) are secreted by platelets and macrophages, and the upregulation of EGF contributes to faster re-epithelialization after an acute injury. Human EGF is a low-molecular weight polypeptide (6045 Da) with 53 amino acid residues
6 The Role of Growth Factors, Cytokines,
Nutrition and Matrix Metalloproteinases in
Wound Healing
and 3 intramolecular disulfide bonds. It stimulates cell growth proliferation and differentiation by binding to an EGF receptor, and increases the expression of K6 and K16, which are involved in the proliferative signalling pathway and is implicated in keratinocyte migration, fibroblast function and the formation of granulation tissue [5]. The vital function of EGF is in the control of normal dermal wound healing, on a cellular and molecular level. However, in a chronic wound environment, the formation and release of EGF is minimised or unable to perform its metabolic functions and is readily degraded, which affects the normal healing process. It has been shown that exogenous EGF supplementation, via topical application, increased epithelialization leading to a shortened healing time in nonhealing ulcers [6, 7]. It has therefore been suggested that sustained delivery of EGF from the wound dressing matrix may be useful for the treatment of chronic wounds. The EGF family consists of four proteins:
EGF, transforming growth factor-a (TGF-a), heparin-binding EGF and amphiregulin.
The topical application of EGF has been shown to decrease wound-healing time for partial-thickness burn wounds. EGF are topically applied as a gel, based on cellulose, and are commercially available. Regen-D®, manufactured by Bharat Biotech, is a recombinant human epidermal growth factor (rhEGF) gel indicated for the treatment of burns. The clinical safety and efficacy of rhEGF in accelerating the healing of diabetic foot ulcers (DFU) has also been demonstrated [6].
Fibroblast growth factor (FGF) comprise a group of 22 structurally related growth factors. The FGF family has a broad mitogenic spectrum and stimulates proliferation of various cells of mesodermal, ectodermal and also endodermal origin. FGF also regulate the migration and differentiation of fibroblasts and keratinocytes. FGF-1 and FGF-2 are shown to stimulate angiogenesis and are present in the early stages of wound healing. Expression of FGF-2 was found to be upregulated after injury in normal wounds but not in chronic wounds. The local application of the FGF family of growth factors has been shown to stimulate tissue repair. KGF is produced by M426 lung fibroblasts with a molecular weight of 26−28 kDa, it is also designated as FGF-7 as it belongs to the FGF family and promotes the growth of cells known as keratinocytes, cells that secrete a protein called keratin.
Wound Healing Table 6.1 Major growth factors and cytokines that participate in wound
healing in both acute and chronic wounds
Growth factors
Cells Acute
wound
Function Chronic
wound EGF Platelets
Macrophages Fibroblasts
Increased
levels Re-epithelialization Decreased
levels
KGF Fibroblasts − Keratinocyte migration, differentiation
and proliferation −
FGF-2 Keratinocytes Mast cells Fibroblasts Endothelial cells Smooth muscle cells Chondrocytes
Increased
levels Granulation tissue formation Re-epithelialization
Matrix formation and remodelling
Decreased levels
TGF-β Platelets Keratinocytes Macrophages Lymphocytes Fibroblasts
Increased
levels Inflammation
Granulation tissue formation Re-epithelialization
Matrix formation and remodelling
Decreased levels
PDGF Platelets Keratinocytes Macrophages Endothelial cells Fibroblasts
Increased
levels Inflammation
Granulation tissue formation Re-epithelialization
Matrix formation and remodelling
Decreased levels
VEGF Platelets Neutrophils Keratinocytes Endothelial cells Smooth muscle cells Fibroblasts
Increased
levels Granulation tissue formation Decreased levels
IL-1 Neutrophils Monocytes Macrophages Keratinocytes
Increased
levels Inflammation Re-epithelialization
Increased levels
IL-6 Neutrophils Macrophages
Increased
levels Inflammation Re-epithelialization
Increased levels TNF-a Neutrophils
Macrophages
Increased
levels Inflammation Re-epithelialization
Increased levels IL: Interleukin
KGF: Keratinocyte growth factor PDGF: Platelet-derived growth factor TGF-β: Transforming growth factor-β TNF-a: Tumour necrosis factor-a VEGF: Vascular endothelial growth factor
Adapted from S. Barrientos, O. Stojadinovic, M.S. Golinko, H. Brem and M. Tomic-Canic, Wound Repair
& Regeneration, 2008, 16, 5, 585 [3]
PDGF is a growth factor secreted by platelets, which directs the sequential migration of neutrophils, fibroblasts and macrophages into the area of injured tissue. At the wound site, stimulation by PDGF results in the endogenous production of growth factor, ECM and collagen synthesis, and fibroblast proliferation. During the remodelling phase of wound healing PDGF takes part in the collagen crosslinking. The levels of PDGF have been found to be significantly low in nonhealing ulcers compared with acute wounds; hence, PDGF is clinically applied topically on chronic wounds and in orthopaedic surgery due to its ability to enhance the cellular response and contribute to collagen production, and also because it is a strong mitogenic factor for cells crucial to musculoskeletal tissue repair, including mesenchymal stem cells, osteogenic cells and tenocytes [8]. In fact, PDGF is the only growth factor approved by the US Food and Drug Administration (FDA) and the European Medicines Agency for the treatment of diabetic neuropathic foot ulcers. However, augmented PDGF production is involved in the pathogenesis of hypertrophic scars and keloids.
VEGF is produced by the endothelial cells, fibroblasts, smooth muscle cells, platelets, neutrophils and macrophages involved in wound healing. The primary role of VEGF in wound healing is to promote angiogenesis; the steps involved are vasodilation, basement membrane degradation, endothelial cell migration and proliferation which finally initiate capillary tube formation, the anastomosis of parallel capillary sprouts and new basement membrane formation [9]. VEGF plays a significant role in several of these processes. Capillary tubes growing into the wound remove metabolites and provide a conduit for nutrients and other mediators of the healing response. Figure 6.1 highlights the multiple synergistic stimulants to angiogenesis that may be generated if VEGF is administered to a wound. VEGF binds to the kinase insert domain receptor, also called VEGF-2, and stimulates nitric oxide synthase and cyclooxygenase activities, which further promote simultaneous vasodilation and vascular permeability.
Procoagulant factors, such as the von Willebrand factor, which mediates platelet adhesion and aggregation, are induced by VEGF in endothelial cells. Platelets also release VEGF and ultimately generate thrombin and fibrin. VEGF accelerates the secretion of MMP-1, MMP-2 and tissue inhibitors of metalloproteinases (TIMP) in endothelial cells. It also stimulates vascular muscle cells to express MMP-1, MMP-3 and MMP-9. The efficacy of VEGF in healing DFU has been studied by several groups. The topical application of VEGF may help to promote the repair of nonhealing wounds in arterial occlusive disease and diabetes due to its mitogenic, chemotactic and permeability effects.
Wound Healing
Coagulation cascade Thrombin Fibrin
NO TF vWF
bFGF NO PGI2
tPA MMP-2 MMP-1
Endothelial Cell
BM degradation Proliferation
VEFG
uPA
Plasminogen Plasmin
Fibrin gel Chemotaxis Vasodilation / ↑permeability
Figure 6.1 Demonstration of the potential role of VEGF in wound healing.
By stimulating the endothelial cell, multiple phases of the angiogenic cascade are enhanced. bFGF: basic fibroblast growth factor; BM: basement membrane;
NO: nitric oxide; PGI2: prostacyclin; TF: tissue factor; tPA: tissue-type plasminogen activator; uPA: urokinase-type plasminogen activator; and vWF:
von Willebrand factor. Reproduced with permission from P. Bao, A. Kodra, M. Tomic-Canic, M.S. Golinko, H.P. Ehrlich and H. Brem, Journal of Surgical
Research, 2009, 153, 2, 347. ©2009, Elsevier [9]
Transforming growth factors are a group of substances with different properties.
TGF-a stimulates the growth and migration of keratinocytes and fibroblasts in the affected area. TGF-β1 and TGF-β2 have been shown to promote the growth of new
blood vessels (angiogenesis), ensuring adequate blood supply to the healing wound.
TGF-β1 is important in inflammation, angiogenesis, re-epithelialization and connective tissue regeneration; increased expression is found during the onset of injury and is involved in the upregulation of the angiogenic growth factor VEGF. It facilitates the recruitment of additional inflammatory cells and augments macrophage-mediated tissue debridement. It also increases the proliferation of keratinocytes, particularly in the latter stages of wound healing, and during matrix formation in the remodelling phase, it is involved in collagen production. In addition, it is a promoter of TIMP-1 synthesis and is a potent inhibitor of MMP-1, MMP-3 and MMP-9 which inhibits collagen breakdown. Furthermore, it plays an active role in the development of hypertrophic and keloid scars, and induces and activates keloid fibroblasts when over-expressed, particularly in burn wounds.
Insulin-like growth factors (IGF) are proteins with a high sequence similarity to insulin. IGF-1 is a critical mediator of tissue repair; however, it plays a significantly less apparent role in chronic wounds. IGF-1 can stimulate the growth, proliferation and migration of fibroblasts, which are essential in the wound-healing process, and it has been shown that the expression of IGF-1 is increased in subcutaneous, incisional and burn wounds. The topical administration of IGF-1 was not successful and it has been suggested that the endogenous production of IGF-1, induced by growth hormone administration, may enhance wound healing [10].
IL-1 is a cytokine that participates in the regulation of immune responses, inflammatory reactions and haematopoiesis. It is synthesised by keratinocytes in response to an injury and is shown to stimulate fibroblast and keratinocyte growth and proliferation.
IL-6 is a pro-inflammatory cytokine which affects various processes such as the immune response, reproduction, bone metabolism and ageing. It is synthesised by mononuclear phagocytes, vascular endothelial cells, fibroblasts and other cells in response to trauma, burns, tissue damage and inflammation. It has been shown that IL-6 deficient transgenic mice displayed significantly delayed cutaneous wound healing compared with control animals. Tumour necrosis factor (TNF) is released by activated macrophages; TNF-a influences the synthesis of collagen, MMP and TIMP. IL-1 and TNF-a enhance collagenase secretion; however, chronic exposure is a contributing factor in connective tissue disease. TNF-a concentration is found to be 100-fold higher in chronic wounds and seems to modulate ECM degradation via the modulation of MMP and TIMP production.
Since the 1960s it has been known that a moist environment plays an important role in accelerating wound healing with less pain than a dry wound environment; a dry environment causes cells to dehydrate and die, scab or crust formation over the wound and also prevents the migration of cells and deters epithelialization. Chronic wounds are usually characterised by a high bacteria count, with the probability of
Wound Healing the presence of more than one bacterial strain. Drug-resistant organisms, which form biofilms, stimulate chronic inflammation. Bacteria have now developed mechanisms by which they can circumvent the effects of antibiotics, so the antibiotics that were effective no longer work, either at all, or as well as they did. Bacteria have become resistant to the drugs that would once have killed them, as they multiply so rapidly that mutations occur, which makes them less susceptible to antibiotics. In addition, the development of new antibiotics, with novel modes of action, is scare, with no new antibiotics developed over the last 25 years, making wound treatment more difficult as infection is detrimental to wound healing. Studies by Margolis and co- workers concluded that ulcer size (>2 cm2), duration (> 2 months) and ulcer depth were the three most important factors determining healing outcomes [11]. The size (area and depth), sepsis, arteriopathy and denervation classification system identifies ulcer size and the presence of arteriopathy as the most important factors associated with DFU healing [12]. In chronic wounds, there is a tendency for the inflammatory response to be altered beyond the normal duration. Pro-inflammatory cytokines, reactive oxygen species and proteolytic enzymes, such as certain MMP, elastases and plasmins, are produced to a greater extent as a result of reduced inhibitor release, such as TIMP. This inactivates growth factors and leads to ECM degradation and alteration of the wound pH, which leads to reduced tissue repair, cellular proliferation and angiogenesis. It has been reported that stress, depression and a hostile marital environment have a possible role in the modulation of MMP and in the expression of TIMP [13, 14], thereby delaying wound healing.