GEORGE W. CHERRY, MARGARET N. A. HUGHES, ANDREW N. KINGSNORTH, AND FRANK W. ARNOLD

Modern advances in molecular biological research techniques have increased the gap between our understanding of the mechanisms of wound healing and the clinical application of this knowledge, as pointed out by Peacock in 1983. A number of factors besides the expansion of new knowledge have contributed to this distance, although probably the most important is that, in healthy individuals, tissue repair after acute injury is thought to occur at a maximum rate and is not really impaired unless adversely affected by localized or systemic conditions (Table 1) 0.

 Recent clinical studies, designed to demonstrate increased healing of acute wounds with therapeutic agents resulting from advances in basic research, such as growth factors and, in particular, epidermal growth factor, have yielded conflicting results. In addition, the clinical significance of improving the healing of acute donor site skin graft wounds by 1 or 2 days is debatable.

However, with regard to chronic wounds there has been a major improvement, over the past decade, in decreasing the gap between basic wound healing research and clinical application. An augmentation in the healing rate of these wounds would not only be beneficial to the patients but also to health care budgets. Chronic leg ulceration alone in the United Kingdom has been estimated to cost £300 million to £600 million per annum - similar to the cost of tobacco-related diseases.

There has been a renewed interest in recent years into the pathophysiology of venous ulcers, resulting in knowledge of the role of leakage of fibrinogen and other blood substances across the capillaries due to chronic venous hypertension (Fig. 1) 0. These pathological changes have been shown to be accompanied by an impairment of the microcirculation in patients with venous leg ulcers. Similar microcirculation impairment has been seen in patients with type I insulin-dependent diabetes, in which healing also is defective.

Another example where basic research has finally been utilized clinically is that of the practice of maintaining a moist wound environment under occlusive dressings to enhance healing. An increase in healing compared to wounds left exposed to air was demonstrated in animal studies in 1962 and a year later in humans. Because of the improved healing of skin wounds that occurs with occlusive dressings, the latter are now used as controls in experimental and clinical studies to test the efficacy of growth factors and other healing promoters (Fig. 2) 1.

There is a lack of standardized clinical definitions to describe the status of both acute and chronic wounds. This can be a problem in the dissemination of the results of clinical wound healing studies. In order to overcome this problem in chronic wounds, Knighton has established a classification of non-healing wounds, based on up to 20 clinical and wound status parameters, to generate what is termed a wound severity index (Table 2) 1. His assessment of wounds includes a number of variables ranging from the size, depth, extent of undermining, bone exposure, arterial blood supply, and duration of the lesion.

To a number of patients the most important perception of healing is not necessarily acceleration of healing, but the appearance of the resultant scar. For the burn patient, in addition to the cosmesis of the scar, the functional recovery of the injured tissue is of great importance. This goal has been accomplished for a number of years by using pressure garments to manage excess scar tissue formation in these patients. Over the past decade, research workers in Australia, the British Isles, and the United States have shown that covering hypertrophic scars with a silastic gel sheet results in both clinical and elastometrical improvement of the scars, without exerting pressure. The mode of action of this therapy in preventing hypertrophic scar formation is still not known, but studies by Quinn and colleagues have ruled out any pressure effect, temperature alteration, oxygen tension change, or occlusion as a factor.

Thus it appears that the gap between basic research and clinical utilization in wound healing is narrowing, particularly in the treatment of chronic wounds. This is evident in the amount of research that is being carried out currently on wound dressings and healing promoting agents, such as growth factors. The aim of this chapter is to review the basic science of wound healing, as well as to illustrate how this research is affecting the clinical management of wounds.

In order to describe the complex cascade of events that follows injury, it is convenient to look at this process as a number of overlapping phases: inflammation, formation of granulation tissue with angiogenesis, and scar formation (extracellular matrix remodelling) (Figs. 3 and 4) 2,3.

Injury to tissue leads to loss of structural integrity, instigating the coagulation cascade to prevent localized haemorrhage. In skin, mucosa, and gut especially, injury is also complicated by the invasion of micro-organisms. These events play an important role in initiating the defence and repair mechanisms by sealing off severed vessels and transferring blood constituents, circulating cells, and bioactive substances to the site of the wound (Fig. 5) 4. This transferral and the ensuing defence processes constitute the early aspect of wound healing, commonly referred to as the inflammatory phase.

It has been stated by Peacock and others that the inflammatory reaction in soft tissue, which begins literally seconds after injury, is the same whether caused by a surgeon's sterile blade or by invading bacteria after a street injury. Qualitatively, inflammation is the same, but it is likely to be more prolonged in the latter case. More specifically, the mechanism of leucocyte adhesion to the vascular wall after injury followed by diapedesis, a major part of the inflammatory response, is essentially the same in all wounds whether resulting from surgery or trauma.

The inflammatory phase is triggered by two classes of mediators (soluble signal factors): those controlling vessel permeability and those attracting or trapping cells. The clinical signs of inflammation are caused by changes in blood vessels - with dilatation leading to erythema, and endothelial cell separation allowing plasma extravasation, producing localized swelling. There are overlapping stages but, in general, the order of arrival at the wound site from an intravascular space is thought to occur in the following sequence: plasma with soluble components and cellular constituents, first platelets, then neutrophils, followed by monocytes and lymphocytes. The migration of epithelial cells to resurface the injured tissue begins during this phase, mediated by the above events.

Alterations in microvascular permeability after injury allow both fluid and plasma components to pass to the tissue. Vasoactive amines and peptides (including histamine from mast cells, serotonin from platelets, and bradykinin from neutrophils) cause the reversible opening of junctions between endothelial cells and allow the passage of neutrophils and monocytes.

Hageman factor (factor XII), a plasma glycoprotein, is activated by adsorption on to fibrillar collagen, leading to the generation of bradykinin and initiation of the complement cascade. The complement system is composed of 20 interacting soluble proteins in the serum and extracellular fluid, which can be activated by IgM and IgG antibodies bound to antigens on the surface of micro- organisms or by bacterial lipopolysaccharides. Large quantities of IgM or IgG antibodies lead to complement fixation by the classical pathway, whereas endotoxin released from bacteria and small quantities of IgG antibody enhance the activation process by the alternate pathway. These proteins are the substances responsible for the acute inflammatory reaction. IgM can lyse Gram-negative bacteria and neutralize viruses. The C5 to C9 factors of complement combine to form a large protein complex that mediates lysis of bacterial cell walls. Complement factors also opsonize invaders (coat their antigen with antibody), making them recognizable to phagocytic cells. The factor C5a is also chemotactic, attracting polymorphonuclear cells, neutrophils, to the site. The complement component C3b binds to specific receptor proteins on phagocytic cells and to microbial cell walls and enhances the ability of the phagocytes to bind, ingest, and destroy micro-organisms.

The earliest circulating cell or cell fragment detected in the injury site is the platelet. Platelets contain three types of organelles involved in haemostasis and initiation of the inflammatory phase.

1.a-Granules, which contain adhesive glycoproteins such as fibrinogen, von Willebrand factor, fibronectin, thrombospondin, and also growth factors - platelet-derived growth factor (PDGF), transforming growth factors a and b (TGF-a and TGF-b), and platelet factor 4.

2.The ‘dense body", the main storage site of serotonin, also contains adenine nucleotide, calcium, and pyrophosphates.

3.Lysosomes, containing neutral and acid hydrolysases, elastase, collagenase, antitrypsin, and a 2; macroglobulin.

The above substances are released when the platelets are activated by various factors. When injury occurs, contact is made between platelets and insoluble components of the subendothelial matrix, particularly collagen, promoting the release of the a-granule contents which then trigger the coagulation process. The activation of platelets is enhanced by some of the complement factors and by bacterial lipopolysaccharides. The latter produce a 50-fold increase in the amount of serotonin released.

Activated platelets become sticky and aggregate to form a plug that temporarily occludes small vessels. Both damaged platelets and tissues release thrombokinase, which converts prothrombin to thrombin, and this in turn ensures the conversion of soluble fibrinogen to insoluble fibrin. The release of serotonin and adenine nucleotides contained in the dense bodies of the platelets induce the aggregation of platelets, which interact with the fibrin network to form a clot which is stronger and more durable than the initial platelet plug. If the clot is allowed to dehydrate, it transforms to a dry eschar covering the wound.

Other substances released by the a-granules, such as platelet derived growth factor (PDGF), and by the dense body, such as cyclic adenosine monophosphate (cAMP) are chemotactic for neutrophils; both transforming growth factor-b (TGF-b) and PDGF are chemotactic for macrophages, while TGF-a and TGF-b are angiogenic factors. The role of platelet-derived growth factors in enhancing both experimental and clinical wound healing has been highlighted recently in a number of publications, and is elaborated in more detail below.

Accumulation of neutrophils

Adhesion

Interaction between damaged tissue and serum releases the complement factor C3, and the C3e fragment of this provokes the release of neutrophils from the bone marrow. At the same time, circulating leucocytes near the wound site, particularly neutrophils, cease to flow and adhere to the endothelium. It has been shown in vitro that adherence is enhanced by inflammatory mediators, such as C5a (the fifth component of complement), platelet-activating factor, and leukotriene. There is a very fast initial response, with onset of adherence as early as 30 s after injury and with a maximum response at 2 min.

The binding of leucocytes to endothelium results from the interaction of complementary receptors in both cell types. Their expression is enhanced by cytokines and bacterial lipopolysaccharide. Physical factors, such as haemodynamic shear stress, also influence adherence. This first stage of adherence is critical. While there is some evidence that some wounds can heal without the presence of neutrophils, patients with leucocyte adhesion deficiency, lacking an essential glycoprotein, are unable to mobilize neutrophils or monocytes, and exhibit decreased pus formation and impaired wound healing.

Diapedesis

Vasopermeability factors act on actin microfilaments inside the endothelial cells and effect the reversible opening of junctions so that neutrophils are able to pass between the endothelial cells to the extravascular space. It is suggested that the secretion of elastase and other enymes by the neutrophils enables them to degrade elastin and components of the endothelial basement membrane.

Molecules released by platelets following disruption of the blood vessels, e.g. kallikrein (an enzyme that leads to the formation of vasodilating peptides) and fibrinopeptides, diffuse to the site of the wound and set up a concentration gradient of chemotactic factors which attract the neutrophils that have traversed the endothelium through the extracellular space to the injury site.

At the site the neutrophils form the first line of defence against the invading micro-organisms. The neutrophils phagocytose bacteria, then kill the ingested cells by the production of microbiocidal substances - oxygen metabolites such as hydroxyl radicals, hydrogen peroxide, and the superoxide ion. Release of some of these substances to the outside of the cell may also lead to tissue damage and prolong the inflammatory phase. Some bacteria may be killed by non-oxidative mechanisms, but these are not defined in vivo. If bacterial contamination is low, the density of neutrophils declines, but if numbers of micro-organisms persist, the bacterial lipopolysacharides continue to promote the arrival of further neutrophils. The neutrophils are unable to regenerate their enzymes and so themselves decay after phagocytosis.

The macrophage is indispensable in the degradation of injured tissue debris and in the reparative phase of wound healing (Fig. 6) 5. If the macrophages are inhibited, wound healing is radically impaired.

Normal tissues contain very few macrophages, but, in response to chemotactic factors released after injury, circulating monocytes are attracted to the site of injury several hours after the first neutrophils arrive. Endothelial cells in wounded tissue also play a role in this process, and have been shown to regulate the preferential adhesion of monocytes and lymphocytes to endothelium.

At the injury site, monocytes differentiate into macrophages. One of the signals promoting this differentiation is the binding of fibronectin to surface receptors on monocytes, which induces the activation of the receptors for phagocytosis. Macrophages develop functional complement receptors and undertake similar operations to the neutrophils. However, further interactions with the interferons, and subsequently with bacterial or viral products, induce further differentiation into a fully activated phenotype. Interferons enhance endocytosis and phagocytosis and modulate the surface receptor functions of newly migrated macrophages. Ingestion of bacteria by endocytosis triggers the primary oxygenase which converts molecular oxygen to the superoxide, which then reacts to produce hydrogen peroxide and hydroxyl radicals required for microbiocidal activity. Oxygen is essential. If the partial pressure of oxygen falls below 30 mmHg, macrophages are inactivated; their phagocytosing potential is reduced. The relationship between oxygen pressure and healing has been shown to be linear, explaining the beneficial role of oxygen pressure in repair.

The activated macrophage is the major effector cell for degrading and removing damaged connective tissue components, collagen, elastin, and proteoglycans. Initial degradation takes place extracellularly - up to several millimetres from the macrophage. Collagen and other fragments are then ingested and degraded by the cathepsin enzymes and other peptides. In contrast to neutrophils, macrophages can continue to synthesize the necessary enzymes, thus persisting for a longer time. They also phagocytose the decaying neutrophils.

Apart from their role in debridement, macrophages secrete chemotactic factors which bring additional inflammatory cells to the wound site. Macrophages also produce prostaglandins, which are strongly vasodilatory and affect the permeability properties of microvessels. The macrophages act after the amines and kinins, and are produced on demand, prolonging the inflammatory phase. Prostaglandins also augment the adenyl cyclase activity in T lymphocytes, which accelerates the mitosis of other cells.

The angiogenesis stimulated in the early phase of wound healing has been shown to be related to the presence of macrophages. Increased levels of lactate production, up to 15-fold, have been found in wounded tissue, and have caused macrophages to produce and release angiogenic substances. The macrophages also produce growth factors, such as platelet-derived growth factor (PDGF), transforming-growth factor-b (TGF-b), and fibroblast growth factor (FGF), which are necessary for the initiation and propagation of granulation tissue. In this way the macrophages mediate the transition from the initial inflammatory response to the early repair phase of wound healing.

B lymphocytes may be absent from the wound site. However, helper T cells are activated following injury, when they recognize any foreign antigen on the surface of antigen-presenting cells, e.g. Langerhans cell in skin, and certain types of macrophage.

The T lymphocytes migrate into the wound along with the macrophages. Advances in the past 5 years have helped to elucidate the role of the T cell in wound healing. Monoclonal antibody staining has permitted the identification of sets and subsets of lymphocytes, and cell culture and biochemical studies have identified and characterized some of the lymphokines, molecular messengers secreted by lymphocytes, which influence other cells, particularly macrophages and fibroblasts (Fig. 6) 5. Thus, lymphocytes can produce macrophage chemotactic factor (MCF), macrophage inhibiting factor (MIF) regulating movement, macrophage activating factor (MAF), and interleukin-2 (IL-2) which enables the T cells to proliferate by an autocrine mechanism. TGF-b, produced by the a-granules of platelets, is chemotactic for both fibroblasts and macrophages, and g-interferon (g-IFN) modulates the surface receptor function of newly migrated macrophages and enhances their phagocytic activity, and also activates macrophage oxidative metabolism and antimicrobial activity. T lymphocytes also produce colony stimulating factors (CSF). These are glycoproteins that act on neutrophils and macrophages through specific receptors which have recently been identified - granulocyte-CSF, macrophage-CSF, granulocyte/macrophage-CSF, and interleukin-3 (IL-3).

The colony stimulating factors are very potent, being effective at very low concentrations (pg/ml). They are involved in the stimulation of proliferation, and of the commitment of the monocyte to differentiation and maturation. They stimulate the function of phagocytosis, and the production by macrophages of substances such as prostaglandins, tumour necrosis factor (TNF), g-IFN, and further colony stimulating factors. As quantities are very small, it is not known whether all cells are able to produce colony stimulating factors. They are induced in vivo by the presence of micro-organisms. Colony stimulating factors are currently in clinical use for the treatment of neutropenia, both congenital and induced by cancer therapy. It has been suggested that there could be a prophylactic role for them in abdominal and genitourinary surgery, where infections are common.

Macrophages and lymphocytes have been shown to be present from day 1 in wounds, although lymphocytes are fewer in number than macrophages. In a study on human wounds by Martin and colleagues, macrophages peaked between 3 and 6 days and lymphocytes between 8 and 14 days. Thus they persist into the early repair phase of wound healing. Both macrophages and lymphocytes disappear from mature wounds by an unknown mechanism, but in abnormal scars both persist long afterwards. In hypertrophic scars, macrophages and lymphocyte levels have been found to be very high 4 to 5 months after wounding, and lymphocytes were still present at 40 per cent of the high level after 2 years. It has been suggested that control of lymphocytes might be a useful approach to control of scarring. It is of interest that minoxidil, a drug that has been shown in vitro to inhibit collagen lattice contraction, has been shown to inhibit DNA synthesis and leucocyte migration inhibition factor (LIF) production by T lymphocytes.

Epithelial cells are important in the inflammatory phase as well as in the later repair aspect of wound healing. In partial thickness wounds, epithelial cells migrate from the edges of the wound and from the epithelial linings of hair follicles, sebaceous glands, and sweat glands and begin to proliferate. In full thickness wounds, only the epithelial cells at the edges of the wound are available to migrate, because of the destruction of dermal appendages, and closure takes longer. In sutured surgical wounds epithelial migration begins within the first 24 h of injury and may be completed as early as 72 h in healthy individuals.

Closure of the wound is not the only function of epithelial cells in the inflammatory phase (Fig. 4) 3. The development of techniques in molecular biology has led to unequivocal identification of many cytokines. Keratinocytes have been shown to produce the granulocyte/macrophage colony stimulating factor and interleukin-3 (IL-3) or multicolony stimulating factor (GM-CSF), as well as the growth factors TGF-a, TGF-b, and TNF-a.

Keratinocytes also produce interleukin-1 (IL-1) which stimulates fibroblast proliferation and enhances the production of type I and III collagen mRNA and of an angiogenic factor. Thus they help to prepare and promote the next phase of wound healing. They also produce IL-6, which induces in the liver the synthesis of proteins, some of which act to terminate the inflammatory phase.

By definition, chronic ulcers have a deficit in epithelialization. This could arise through reduced cell proliferation, or excess cell loss. Early studies of mitotic frequency at the edge of superficial ulcers failed to show any difference between those which healed expeditiously with treatment and those which did not. It is therefore probable that the surface extracellular matrix of such wounds governs the process of wound closure by forming an environment which may be either permissive of, or prohibitive for, epithelial cell adhesion and migration. The nature of these interactions remains relatively unexplored.

Various chemotactic, growth, and activating factors produced in the inflammatory phase are concerned in the initiation and development of granulation tissue which lasts from about day 4 to day 21 after wounding. Granulation tissue comprises a loose matrix of fibrin, fibronectin, collagen, and glycosaminoglycans, particularly hyaluronic acid, containing macrophages, fibroblasts, and ingrowing blood vessels. In deep wounds, granulation tissue serves as a scaffold for new tissue ingrowth. In incisional wounds during this phase the wound begins to gain tensile strength, although it is during this early period that wound dehiscence and evisceration most frequently occur.

In the initial phase after wounding, fibroblasts migrate into the wound site 24 h after injury. During this phase of healing (4 to 21 days) the fibroblasts are activated and undergo a burst of proliferative and synthetic activity, initially producing high amounts of fibronectin, and then synthesizing the other protein components of the extracellular matrix, including collagen and elastin, and glycosaminoglycans. The fibroblasts align themselves along the wound axis and form cell to cell links, which contribute to the contraction of the wound.

There has been much discussion about the type and origin of fibroblasts that appear in the wound. These fibroblasts have characteristics in between those of normal resting fibroblasts and smooth muscle cells. This altered phenotype, which has been called the ‘myofibroblast" is more mobile and more contractile than the inactivated fibroblast, and disappears on the completion of wound healing. Early distinctions between fibroblasts and myofibroblasts were based on ultrastructural criteria, but immunochemical analyses have, more recently, led to identification of subspecies of myofibroblasts based on permutations of expression of vimentin, desmin, and a smooth muscle actin. It has now been shown that smooth muscle cells in culture can reversibly modulate from contractile to synthetic cells, i.e. the reverse of the myofibroblast development, and this may reflect changes occurring in vivo. In addition, it has been demonstrated that smooth muscle genes can be switched on transiently in certain circumstances by other non-muscle cells, including macrophages and some epithelial cells. It is still not known what controls the change in phenotype.

Complex factors influence the behaviour of the fibroblasts in the formation of granulation tissue. Migration is promoted by TGF-b (produced by platelets and keratinocytes). Proliferation is promoted by thrombin, by serotonin (produced by platelets), by interleukin-1 (IL-1) produced by keratinocytes, by fibroblast growth factor from macrophages, and by epidermal growth factor. Synthetic activity is promoted by IL-1 and factor XIII for collagen, and by thrombin, epidermal growth factor, and TGF-b for fibronectin, while lysyloxidase activity is augmented by serotonin. Some remodelling of the extracellular matrix may take place at this stage. Degradative activity, which is also necessary for remodelling, is enabled by the promotion of collagenase synthesis by prostaglandin E2;.

Research into factors influencing angiogenesis has been directed at means of inhibiting new vessel growth in regard to tumour metastasis or, in the case of wound repair, means of stimulating angiogenesis to enhance healing.

Hypoxia following injury, if not so severe as to lead to tissue death associated with ischaemia, acts as a major stimulus for angiogenesis, which is required for restoration of blood flow. Along with fibroblast proliferation, neovascularization is a common feature of granulation tissue in the early phase of healing. One stimulus for new vessel growth in fibroblast growth factor, while other angiogenic factors, such as those secreted by macrophages and other cells, also contribute to the neovascularization. The growth of vessels in surgical wounds starts from capillary loops a few days after surgery, and vascularization may be complete in 6 to 7 days. In burns, development is later and may be complete in 12 to 16 days. The secondary wound (reopened and resutured) revascularizes at a significantly faster rate than a control wound which has not been reopened and resutured (Fig. 7(a,b)) 6. This aspect of the importance of angiogenesis in wound healing has been observed in other types of wounds where differences in regional vascularity and healing are directly related.

The endothelial migration seen in granulation tissue is supported by the increased fibronectin in this tissue. Mitotic activity leads to the formation of capillary buds which sprout from blood vessels adjacent to the wound and extend into the wound space. There is a gradual establishment of flow. Endothelial cell proliferation is stimulated by a low wound Po 2; in the early stages, but growth of vessels is later enhanced by a high wound Po2; which is also essential for the synthesis of collagen necessary for the complete formation of the vessels. The pattern of vascular growth is probably the same in the healing of skin, muscle, and intestinal wounds. In fractured bones, vessel growth can be stimulated by repeated muscle contraction which increases bone blood flow, while vascularization is reduced by immobilization.

Modulation of angiogenesis is currently a very active area of research; inhibition of vessel growth in tumours and its promotion in wounds are both appealing therapeutic strategies. Progress has been hampered by difficulty in quantifying the dynamic process of neovascularization without interfering with it. Laser-Doppler flow measurements have been found to correlate with vessel counts in experimental wounds, and may offer a non-invasive and repeatable method. However, conventional laser-doppler techniques show large variations between readings even at adjacent points. Recently, a scanning laser-doppler device has been developed, which overcomes this difficulty and which allows the imaging of blood flow in surface wounds. It has been used to study the evolution of blood flow over the time in experimental and clinical wounds.

Wound closure by contraction, the inward movement of the edges of the injured tissue, is a normal part of the healing process. However, in some wounds, such as full thickness freeze injury, contraction does not occur. Wound contraction begins between days 8 and 10 after injury (Fig. 3) 2. It is controlled both by the fibroblasts and by the extracellular matrix, and is due to the fibroblasts applying tension to the surrounding tissue matrix. In vivo it has been demonstrated that with contraction there is constant centrifugal tension. The rate of contraction has been shown to be constant for animals of a particular species or strain, and independent of the shape of wounds. However, there is marked interspecies variation. Contraction makes a much greater contribution to closure of full thickness wounds in rats than in man, which adds to the difficulty of extrapolating from experimental studies to the clinical situation.

Collagen synthesis plays an important role in the early stages of healing and the formation of the granulation matrix. Production of collagen remains a major process in wound repair for several weeks after wound closure, and the collagen continues to undergo remodelling for 2 years or more until the injured tissue is finally restored.

Collagen is the major component by weight of the extracellular matrix of the skin, accounting for about 60 to 80 per cent of the dry weight of the tissue. There are known to be at least 13 different genetically distinct collagen type (Table 3) 2, six of which occur in human skin.

The extracellular matrix of tissues is composed of various polysaccharides and proteins and their complexes. These are secreted by cells in situ and different amounts and types are assembled to form diversely organized structures related to the functions of theparticular tissue. The matric not only serves as a support, but has a role influencing the behaviour of the cells in contact with it, affecting their development, migration, proliferation, shape, and metabolism, all of which are important with regard to wound healing.

The polysaccharides are glycosaminoglycans - long, unbranched chains of disaccharide repeating units. They fold with wide curvature in a random fashion and absorb large amounts of water, filling much of the extracellular space. Proteoglycans are formed by the combination of a number of glycosaminoglycan chains with a protein, and may contain up to 95 per cent (w/w) carbohydrate. Glycoproteins, on the other hand, are composed of short, branched oligosaccharide chains, containing from 1 to 60 per cent carbohydrate. The proteins of the extracellular matrix are principally structural proteins such as collagen and elastin, and adhesion proteins such as fibronectin and laminin.

All collagen molecules are composed of three polypeptide a-chains, with a left-handed triple-helix configuration. The chains have about 1000 amino acids and have a distinctive amino acid composition of 33 per cent glycine and 20 per cent of the imino acids, proline and hydroxyproline, with a particular repeating trimeric sequence of glycine - X - Y, where either X or Y is often proline.

The synthesis of collagen involves a progression in the combination of amino acids to form chains which associate to form molecules, and then association to form fibrils which aggregate into fibres or bundles. Fibroblasts are the major cell type to synthesize collagen. The first stages of synthesis take place intracellularly, to produce procollagen molecules which undergo activation stages in the extracellular space (Fig. 8) 7.

In the nucleus the genes are activated and there is translation of mRNAs, specific for single polypeptide chains. The mRNAs pass into the cytoplasm and are translated on the ribosomes of the endoplasmic reticulum, the three polypeptide chains being synthesized simultaneously (Fig. 8) 7. The three a-chains may be identical (as in type III collagen), or a hybrid molecule consisting of two identical chains and a different third chain (as in type I), or three different chains (as in type VI). The molecule is a triple-chain molecule by the time it is detached from the ribosomes. Thesmall, regularly spaced glycine residues situated in the central area of the chains allow them to pack tightly together to form the triple helix. This is the preprocollagen molecule. The molecules then undergo post-translational modifications, principally the hydroxylation of a large number of the proline and lysine residues by the enzymes lysyl hydroxylase and 3- and 4-prolyl hydroxylase. Hydroxylation is a rate-limiting step for collagen secretion, and appears to be tightly regulated by tissue levels of oxygen and lactate. The non-enzymatic glycosylation of some of the hydroxylysine residues also takes place at this stage. The hydroxy groups of hydroxyproline residues form interchain hydrogen bonds, which contribute to the stabilization of the triple-stranded helix. The 4-hydroxyproline moieties stabilize the collagen triple helix at physiological temperature (if not hydroxylated it unwinds above 24 C). The hydroxylysine-saccharide units are also factors in the proper subunit alignment and the subsequent assembly of fibres. This precursor procollagen molecule has extension non-helical peptides of 15 to 20 amino acids in non-collagenous sequences at both ends of the chains. These propeptides contain both intra- and intermolecular disulphide bonds, giving a globular form and probably serving as the starting point for the rapid triple-helix formation. They also prevent intracellular formation of large collagen fibres. These post-translational modifications result in the formation of the procollagen molecule, which is then transported to the Golgi apparatus, enclosed in vesicles, and taken via the microtubules to the cell surface.

The processing of the procollagen to collagen fibres takes place in the extracellular space. The first step is the activation of the molecule by the cleavage of amino- and carboxy-peptide ends by amino- and carboxyl-propeptidases (Fig. 9) 8. Lack of, or defects in, one of these enzymes results in defective fibres, e.g. type VIII Ehlers-Danlos disease, dermatosporaxis in calves lacking the aminoprotease. The sequence of charge and hydrophilic amino acids in the collage molecule is such that it allows self-assembly of collagen into fibres in vitro, possibly because the helical portions allow electrostatic interaction with adjacent collagen molecules, but the in-vivo process is considered to be more complex. The e-amino group of certain lysine and hydroxylysine residues is converted to aldehyde by the extracellular enzyme lysyl oxidase. The aldehydes react to form covalent bonds between the short, non-helical end of the collagen molecules, thus cementing the overlaps. The polymerization of many molecules in a staggered arrangement gives rise to the typical periodicity of 60 nm seen in electron microscope sections (Fig. 10) 9 and this arrangement maximizes the tensile strength of the structure. At this stage type III fibrils have diameters of 40 to 60 nm and type I 100 to 500 nm. The size of the collagen fibrils may depend on the order of cleavage of the non-helical domains, cleavage of the carboxy-terminal first resulting in thin fibrils and of the amino-terminal first leading to thick fibrils.

The build-up of the propeptides released in the transformation of procollagen to collagen inhibits collagen synthesis and thus provides a feedback for switching off the process of synthesis. The failure of this feedback system may be a contributory factor in excessive scarring.

The aggregates of collagen molecules formed in the extracellular space then undergo cross-linking. The extent and types of cross-links, or ratios of types, vary from tissue to tissue, with age, and in disease. In skin, the collagen produced after injury is initially stabilized by cross-links derived from hydroxyallysine. In normal wound healing this changes to cross-links derived from the modified amino acid allysine, but this change does not occur in hypertrophic scars. With time, these cross-links change to ‘mature", more stable cross-links, which have not been completely characterized.

In skin the major mature cross-link may be hydroxyaldohistidine. In most other connective tissues a hydroxypyridinium cross-link is predominant. The greater the number of cross-links, the stiffer a tissue will be, although stiffness may also be influenced by the type of cross-link. In normal bone, for example, the hydroxypyridinium cross-link occurs with a frequency of 0.24 moles per mole of collagen.

Abnormal numbers or types of cross-links can lead to malformation of tissue. The hydroxypyridinium cross-link prevalent in bone also occurs in hypertrophic scars, but is not found in normal skin or in normal mature scar tissue. Increased hydroxylation of lysine residues can occur in hyperglycaemic states, and affects the types of cross-links as well as the number. In one study of diabetic patients, the numbers of five different types of cross-link, were shown to increase between three- and six-fold. Clinical assessment of hand contracture was associated with an increased number of cross-links, which was also correlated with the duration of diabetes and with skin changes.

The organization of the collagen in tissues is also influenced by the kinds and amounts of non-collagenous macromolecules that the cells secrete along with the collagen. In addition, the fibroblasts have a mechanical role in the assembly, crawling over the collagen, pulling and compacting it. The final architecture of the collagen network is related to its function. Thus collagen fibres in the papillary dermis are aligned in thin bundles almost perpendicular to the basement membrane and they hold up the dermal papillae. Some of the fibres glide into the loops of the anchoring fibrils attached to the basement membrane. In the reticular dermis the thick, undulating bundles are nearly parallel to the epidermal plane, and are connected by interlacing fibres, allowing the tissue to resist stress in all directions. In the hypodermis interlacing collagen fibres surround the adipocytes.

At one time it was suggested that there might be different phenotypes of fibroblast, each synthesizing a particular type of collagen, but it has been shown that at least types I, III, and VI can be all synthesized by the same cells.

Types I and III, the interstitial collagens, are the major types of collagen in skin, the rod-shaped molecules providing its tensile strength. It is suggested that the fibrils are hybrids of types I and III, with type I present throughout the body of the fibril and type III round the periphery, and that type III plays a role in the regulation of fibril diameter. It has been possible to control fibre size in vitro by changing the ratio of type III to type I collagen, a higher proportion of type III producing thinner bundles. The ratios of type I to type III in normal human skin vary with age. In a 15-week fetus the ratio is 0.8 to 1 and this increases to 3.6 to 1 by 3 months after birth. Determinations in adult skin indicate I to III ratios of between 3.5 and 6 to 1. In healing wounds, the percentage of type III collagen in the early stages is higher than in normal skin, and as healing progresses there is a change to a greater predominance of type I collagen, mirroring the changes during fetal development. There is a linear decrease in the total amount and density of collagen with age, and the amount of skin collagen is lower in females than males.

Ratios of the amounts of type I to type III collagen also vary in disease, having been determined as only 2:1 in hypertrophic scar, but 19:1 in keloid. Levels of type III collagen are raised in the nodules and contractures of patients with Dupuytren's disease. In diabetic patients the type III:I ratio is also high, and finger contraction occurs in some cases. Insulin treatment causes expression of type III collagen in the mesangial matrix. In Ehlers - Danlos, type IV disease, no type III collagen is produced, and the skin is very thin.

Type IV collagen is a major component of the basement membrane in the dermal-epidermal junction to which basal keratinocytes attach preferentially. It is also produced by endothelial cells and forms an essential element of the microvascular wall. The triple-helix conformation is interrupted by non-collagenous segments lacking the Gly - X - Y repeat sequence. The procollagen molecules are not cleaved after secretion, they retain their propeptides and therefore their globular regions. The carboxy-terminal globular domains of pairs of molecules associate ‘head to head", and further lateral associations between amino terminals, stabilized by disulphide bonds, allow the formation of a sheet-like polygonal mesh. The higher carbohydrate content, with disaccharides of glucose and galactose attached to the hydroxylysine residues, also contributes to the formation of the mesh. Several layers of such sheets are eventually joined together by covalent bonds, making it more flexible than types I and III.

Type V collagen is ubiquitous and interfaces between the cell surface and the surrounding matrix. It has a chain length similar to that of the interstitial collagens, but an amino acid content more like that of the basement membrane collagens. It does not contain any disulphide bonds.

Improved techniques have shown type VI collagen to be more abundant in skin and other tissues than was previously thought. In cultured fibroblasts the ratio of amounts of I:VI mRNA was 3:1, indicating that type VI could be more abundant than type III, and the expression in cultured cells reflected that in skin sections. Type VI collagen is a heterotrimer, composed of three different a-chains, which have unusually large globular domains at the ends of the polypeptides. It has a high cysteine content and is highly disulphide bonded. The chain length is short.

Type VII collagen is a homopolymer that aggregates in bundles of various diameters and degrees of curvature. It has a longer chain length of 467 nm or more (750 nm has been measured). Discontinuities within the triple-helical domain allow the molecules to be flexible. Type VII collagen is the predominant component of the anchoring fibrils (Fig. 11) 10, localized in the sub-basal lamina of the dermal–epidermal junction. The carboxylate terminals of the fibrils insert into the lamina densa and may extend into the dermis. These fibrils are the strongest mechanism for the adherence of the epidermis to the dermis, stronger than the attachment effected by the hemidesmosomes. They are absent or reduced in the skin of patients with recessive or dominant dystrophic epidermolysis bullosa; this leads to easy blistering.

In the extracellular space, procollagenase is transformed to collagenase which cleaves the helical portion of the collagen molecule, causing the fragments to unwind. The single-stranded polypeptides are then susceptible to degradation by extracellular proteinases and peptidases, or undergo endocytosis and are degraded by intracellular enzymes. Some procollagen may be retained and degraded in the cell rather than secreted. There is some evidence for this, but the mechanisms are not understood. Mature collagen may also be absorbed by phagocytosis. Mature banded collagen found in vacuoles in the cytoplasm of fibroblasts musthave come from the extracellular space, because it is only there that such collagen is formed. If it were from the intracellular synthetic pathway, it would be in the form of procollagen. The peak levels of collagen phagocytic activity have been shown to correspond with the period when a change of configuration and fibre orientation was occurring.

There is a relationship between the stability of the collagen triple helix and the degree of intracellular degradation of collagen. Underhydroxylated collagen is non-helical at 37 C and its secretion rate is only one-tenth that of fully hydroxylated collagen. Non-helical collagen is degraded in the lysosomes. Degradation produces free hydroxyproline, which is a measure of collagen degradation because proline hydroxylation occurs after translation.

In health, the synthesis and degradation of collagen is finely regulated to maintain the optimal level of collagen. Imbalance is associated with disease states, such as scleroderma, as well as alterations in wound healing. In scleroderma, in-vitro studies indicate that collagen accumulation is due to enhanced synthesis rather than any other process. On the other hand, excessive collagen degradation is responsible for the imbalance in recessive dystrophic epidermolysis bullosa. Skin from friction blisters in these patients produced high concentrations of collagenase in organ culture, and a radiommunoassay study found that this reflected the in-vivo pathology, the concentration of collagenase being ten-fold in normal skin. Increased collagenolysis may occur with more normal levels of collagenase if the collagen is abnormal, e.g. collagen synthesized under hyperglycaemic conditions is more susceptible to degradation. Imbalance may also be due to defective regulation of cellular growth, leading to low collagen levels, as in focus dermal hypoplasia. Lesions show the absence of dermal connective tissue, the epidermis apposing on to fat tissue, separated by a thin layer of reticulate fibres. Measurement of the proliferative capacity of fibroblasts from adjacent skin in culture indicated a population doubling time twice as long as normal and a saturation density only one-fifth that of controls. The rate of collagen synthesis was normal.

The failure of regulation also causes a problem in the formation of keloids, when the synthesis and breakdown are both stimulated, but the former to a greater extent, thus leading to imbalance and overgrowth. The remodelling of scar tissue also requires the degradation and synthesis of collagen.

In the adult, the normal repair of wounds occurs by the formation of granulation tissue and its organization to a scar. Scar is a dynamic, metabolically active tissue. Precise regulation of collagen metabolism during the repair process is exerted by cytokines (see below) and by the interactions of the extracellular matrix with fibroblasts. In vitro, fibroblasts in contact with collagen fibrils in a three-dimensional lattice show decreased production of types I and III collagen, but enhanced gene expression and activation of collagenase. Experimental studies on collagen deposition in wounds show some variation between species and some differences that may be due to the particular experimental techniques. Some studies in mice showed little collagen in the granulation tissue after 1 week, but another study in rats showed the presence of some collagen from day 1. Recent work on rat wounds indicated that type III collagen was synthesized within 10 h after injury. Collagen synthesis is maximal between 14 and 21 days, although increased collagen deposition may be provoked to occur earlier by electric stimulation. After 21 days the rate of synthesis and the volume density of collagen in the wound return to the normal level. However, the tensile strength of the tissue continues to increase for a considerable time, up to 60 days or even 1 year. Wound tensile strength is a physical measurement which reflects the degree of intermolecular cross-linking, rather than collagen biosynthesis. Its increase is due to the formation of further cross-links and a change to more stable forms which lead to reorientation of the direction of the bundles. Breaking of the tissue occurs by disruption of the connection between fibres in the bundles and by slipping of molecules one over the other in the fibre. Between 3 weeks and 2 months after the injury, cellularity decreases, as does fibroblast cytoplasm volume.

In contrast to the healing by scar formation in the adult, the healing of fetal wounds up to the early third trimester of gestation proceeds without scar formation. Collagen is deposited more quickly in the fetal wound than in the adult, but is rapidly organized and is not excessive. It is thought that this might be due to glycosaminoglycans, which are also deposited. The nature and ratios of the glycosaminoglycans, which affect the cross-linking of collagen fibrils and the migration of fibroblasts, vary in different stages of wound healing. Hyaluronic acid, in particular, the content of which is high in the fetal wound matrix and which is found wherever there is tissue regeneration or repair, has been shown in vitro to facilitate the movement of fibroblasts. While hyaluronic acid is present only in the early stages in adult wounds, it is present throughout the process in fetal healing and the wound is closed by mesenchymal ingrowth on to the hyaluronate-enriched matrix. An in-vitro study of the activity of a hyaluronic acid stimulating factor in sheep detected levels ten-fold higher in the fetal serum than in the serum of normal adults. In animal models there is variation in the open-wound healing response in the fetus of different species - wounds in sheep contracting, while those in the rabbit and monkey heal without contraction, as in the human.

The relevance of reduced scar formation in fetal wound healing and the potential application to clinical healing has been high-lighted in recent work by Ferguson and colleagues. These workers have stated that fetal wound healing is characterized by a reduced growth factor profile and have demonstrated that neutralizing TGF-b activity by antibodies results in decreased wound scarring in adult rats. While this method is not applicable in the clinic, it clearly demonstrates that the modulation of scarring may be an attainable goal.

One of the problems with scar tissue is that it tends to remain somewhat weaker and more brittle than previously unwounded tissue. A second problem is that it tends to contract abnormally. A third problem in certain people is that of overhealing, leading to hypertrophic scars or keloids (Figs. 12 and 13) 11,12. Some clinical differences between keloids and hypertrophic scars are listed in Table 4 3. While some researchers consider that there is spontaneous regression of hypertrophic scars eventually, others maintain that many such scars persist indefinitely. Biochemical differences between hypertrophic scar, keloid, and normal skin have also been characterized. Keloids have a greatly increased proportion of type I to type III collagen compared with normal skin, whereas in hypertrophic scars the ratio is lower than normal. Glycosaminoglycan contents are also abnormal in hypertrophic scars. One study determined the level of hyaluronic acid in hypertrophic scar to be less than half that of normal skin, while the level of chondrotin 4-sulphate was six times higher, as was also the case in granulation tissue. Either the chondroitin sulphate continues to be formed in the hypertrophic scar or it is not removed.

One reason for the imperfect characteristics of scar tissue is that the newly formed collagen pattern in a wound is abnormal, and that rapid intermolecular cross-linking while fibres are being formed leads to their being irreversibly fixed. The use of scanning electron microscopy to study collagen morphology led to the hypothesis that if the cross-linking could be temporarily delayed or slowed, a more nearly physiological collagen pattern would have time to develop.

Attempts to delay or inhibit the cross-linking of collagen have met with some success, e.g. b-aminoproprionitrile and other nitriles inhibit lysyl oxidase, the enzyme that deaminates the lysyl e-amino group to aldehyde, an early step in the formation of intermolecular cross-links. d-Penicillamine reacts with aldehyde groups and so blocks cross-linking of newly formed collagen, but it may also make existing cross-links more labile. Improvement has also been effected using corticosteroids which inhibit fibroblast migration and proliferation and collagen synthesis. Compression has also been of benefit in some cases.

As described in the introduction, silastic gel is being used in the treatment of abnormal scars and is now licensed for use in the United States. g-Interferon, which suppresses collagen synthesis in vitro and in animal models in vivo, leads to improvement in keloids in a small group of patients. An in-vitro study has shown that calcium antagonists reduce extracellular matrix component synthesis in a connective tissue equivalent. Cryosurgical treatment has also led to considerable improvement in one group of 45 patients. Increased understanding of the regulation of scarring has led to the suggestion that the inhibition of helper T cells by cyclosporin or compounds with similar properties should be investigated. A further approach to the control of different phases of wound healing, including hypertrophic scar and keloid formation, is that of influencing cell-matrix adhesion by using monoclonal antibodies or synthetic peptides.

A wound is a very complex biological system and detailed studies of repair processes and the factors that influence them require both in-vivo and in-vitro models. An in-vitro model is more of a closed system, allowing the isolation of a tissue and investigation of modulation without the complication of other factors. While in-vitro models are only partial, and cannot be extrapolated directly, they do allow perceptions which may be valid in-vivo.

One in-vitro model that has proved useful is the hydrated collagen lattice. An acidic type I collagen solution polymerizes in vitro when raised to physiological pH and ionic strength, forming a three-dimensional gel. If fibroblasts are incorporated into the polymerizing solution, they organize and align collagen fibrils and compact them, causing the lattice to contract. Such a lattice provides a simple model for connective tissue, and has been used as an in-vitro model for connective tissue contraction and scar contracture in wound healing. The degree and speed of contraction of the lattice are related to the cell density, to the concentration of collagen and of serum, and also to the type of cell involved, and to the presence of drugs or other factors. This model has been used recently by the authors to investigate the effect of different concentrations of minoxidil on the contraction of type I collagen lattices. In these studies minoxidil was found to inhibit the contraction of lattices in a dose-dependent manner (Fig. 14) 13. Inhibition was evident in 24 h and was reversible to an extent depending on the length of time of exposure to the particular minoxidil dose. Washing out the minoxidil after 2 days for a dose of 4 mmol (800 &mgr;g/ml) or after 6 days for 2 mmol, led to the resumption of contraction (Fig. 15) 14. Visualization of cells with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium bromide), which is metabolized to a blue formazan by the mitochondria of living cells, showed that the inhibition during the first 48 h was not due to fibroblast death. However, prolonged exposure beyond the 2 or 6 days, respectively, led to cellular death. Resumption of contraction with exchange to normal medium coincided with the return of rounded cells to an elongate morphology and to proliferation (Fig. 16) 15. A lower dose of 0.5 mmol slowed contraction but did not affect morphology (as observed in the light microscope) nor cell proliferation.

The finding that minoxidil inhibits lattice contraction as well as fibroblast proliferation in vitro, and that the inhibition of contraction is reversible, supports the suggestion that it might be of therapeutic significance in cases of fibrotic disease and excessive scar formation.

Another study of the collagen lattice model showed the contraction of type III collagen lattices to be faster and to a greater degree than type I hydrated collagen lattices with identical numbers of cells. This could well be correlated with the high type III:I ratios in patients with Dupuytren's contraction and diabetics, certain of whom suffer hand contracture. In another in-vitro study using hydrated collagen lattices, fibroblasts from patients with Dupuytren's disease showed a consistently higher contractility than normal skin fibroblasts.

Growth factors are messenger peptides secreted from a number of cell types integral to the repair mechanism. In general, growth factors are mitogens and chemoattractants. They show a remarkable specificity for cell types, acting selectively to induce cells into the wound environment in an orderly sequence, beginning with neutrophils, followed by macrophages, fibroblasts, and endothelial cells. As previously stated, the functions of these cells in wound healing include removal of cellular debris, leading to the formation of granulation tissue and its replacement with collagen and subsequently, mature scar tissue. Growth factors are also released from blood on coagulation and subsequently are contributed by macrophages and probably by fibroblasts.

To date, the following growth factors have been shown to play a pivotal role in wound healing:

1.Transforming growth factor beta (TGF-b) is a potent stimulator of the synthesis of matric proteins, such as collagen and fibronectin, and of glycosaminoglycans. It is present in high concentration in blood platelets and is released instantaneously into the wound at the site of injury. Multiple forms of TGF-b and the TGF-b receptor exist; these may generate a complex and diverse range of interacting signals on matrix, mesenchyme, and endothelial cells, some of which are inhibitory and some of which are stimulatory.

2.Platelet-derived growth factor (PDGF) has a more restricted target-cell specificity in comparison with other growth factors, but it is a major serum mitogen, also released from the a-granules of platelets, inducing fibroblast proliferation, matrix production, and maturation of connective tissue. Its particular attributes are effects on cell surface (cytoskeleton) motility, mainly directed at smooth muscle cells and fibroblasts.

3.Basic fibroblast growth factor (b-FGF) has its main stimulatory effect on the growth and differentiated function of fibroblasts and on the proliferation of vascular smooth muscle cells and endothelial cells. Therefore it has a major function as an ‘angiogenesis peptide".

4.Epidermal growth factor (EGF) and its homologue, transforming growth factor alpha (TGF-a), generally stimulate cell proliferation by binding to the EGF receptor in a variety of tissue types. They are also released from a granules of platelets.

Numerous other growth factors and cytokines have been proved to be, or are likely to be present in wounds. These include insulin-like growth factor (IGF), platelet derived endothelial cell growth factor (PDECGF), vascular endothelial growth factor (VEGF), heparin binding epidermal growth factor (HG-EGF), and granulocyte monocyte colony stimulating factor (GMCFS). Their role(s) are still uncertain. The situation is further confused by the fact that many growth factors, notably TGF-b, PDGF, and IGF exist as multiple ‘isoforms". Growth factor activity is also modulated by association with binding proteins, and depends upon the availability and type of receptor(s) present. Thus, for example, IGF1 is transferred from blood and local sites of production to its cellular targets via a sequence of binding proteins, whose affinities are modulated by the pH of the wound environment. Alterations in the levels of binding proteins, and elevations of IGF antagonists have been found in situations associated with defective repair, including diabetes, malnutrition, uraemia, and jaundice.

The function of growth factor receptors and of post-receptor signalling pathways is also important. In vitro, high levels of several growth factors have been shown to reduce (down-regulate) expression of the relevant receptor at the cell surface. As a result, increasing levels of growth factor are ultimately associated with decreased cellular responses.

The pattern of growth factor expression in human wounds is being described by the techniques of immunoassay of wound fluid and of immunohistochemistry of biopsies. Unfortunately, these methods recognize antigenic determinants on the growth factor molecules, not their biological activity. Thus, for example, chronic venous ulcers have been found by Ferguson and colleagues to contain high levels of growth factors, including FGF, PDGF, and TGF-b, predominantly distributed in the fibrin cuffs around the blood vessels. It is not known whether these growth factors are biologically active, and if so whether they are sequestered or able to diffuse, or whether the appropriate receptors are expressed on adjacent wound cells.

Thrombin, formed from the clotting cascade, stimulates the release of growth factors from a-granules of platelets. TGF-b, PDGF, and EGF are released locally and are chemotactic for both macrophages and fibroblasts. Macrophages play a critical role in subsequent events because they secrete a large repertoire of peptides involved in wound-healing mechanisms. These peptides include TGF-b, PDGF, b-FGF, EGF, TGF-a, and tumour necrosis factor (TNF). Phenotypic changes occur in macrophages as the wound matures, as evidenced by variation in their abilities to secrete peptides under basal or stimulated conditions. This modification in synthetic activity presumably leads to differential production of matrix, collagen, or ground substance at a particular time in the maturation of the wound. In addition, growth factors such as b-FGF have a strong influence on cellular movement within the wound, stimulating chemotaxis followed by synthetic activity when cells are positioned to deliver their products. PDGF may be a multifunctional ‘first signal" peptide at the site of injury, being the first messenger stimulating fibroblast proliferation.

Extracellular matrix proteins provide a spatial organization that strongly influences the proliferation, differentiation, shape, and migration of cells. Growth factors modify the extracellular matrix and, by so doing, modify cell-surface matrix receptors and cellular receptors which, in turn, affect synthetic activity and the ultimate speed of wound repair. In the resting state, the matrix acts as a reservoir for inactive growth factors which are liberated or solubilized at the time of injury and are then free to transduce intracellular signals for replication and matrix synthesis. Experimentally, it can be shown that the action of TGF-b depends on the type of substratum in which it is acting, since in a non-retracting fibrin lattice, which restricts TGF-b mobility, matrix and collagen synthesis is impaired. In fact, TGF-b itself influences stromal formation because it regulates the splicing pattern of the ground-substance molecule fibronectin–mRNA precursor in granulation tissue.

Two latent proenzymes (metalloproteinases) have an important influence on extracellular matrix structure and function. These metalloproteinases are synthesized by fibroblasts and are characterized as collagenases (which degrade interstitial collagens) and stromalysin, (which degrades basement membrane proteins, including type IV collagen). Angiogensis can only take place once the surrounding basement membrane has been degraded, allowing angiogenic cells to migrate and proliferate in the wound. Furthermore, the proteolytic activity of these metalloproteinases is regulated by endogenous tissue inhibitors of metalloproteinases. b-FGF has been shown to be a regulator of collagenase synthesis, thus affecting tissue remodelling during angiogenesis. In vitro, TGF-b increases the production of tissue inhibitors of metallo-proteinases.

Using simple chemotactic assays, TGF-b and PDGF can be shown to influence movement of monocytes at physiological concentrations. b-FGF, TGF-b, and PDGF are chemotactic for fibroblasts. b-FGF is mitogenic and chemotactic for endothelial cells in vitro, reaffirming its critical role in angiogenesis and granulation tissue formation. In culture, many peptide growth factors are both mitogenic to fibroblasts and also increase their synthetic activity in a differential manner. In respect of synthetic activity, TGF-b appears to be the most active, stimulating the transcription of mRNA for both procollagen and matrix proteins, and increasing the production of these proteins in a number of cell lines in vitro. However, it is likely that many of the actions observed in vitro are not strictly analogous to the compartmentalization of function within the wound microenvironment.

The implantation of a subcutaneous wound chamber represents an experimental form of injury that allows the examination of the organization of granulation tissue, excluding the process of re-epithelialization. A specific number of parameters such as cellularity, collagen content, DNA content, and matrix protein synthesis are used to assess this healing process. After a few days, wound fluid from such implanted chambers contains EGF/TGF-a, TGF-b, and PDGF-like activity. Activated monocytes from wound chambers have increased transcript numbers of the mRNA species TGF-a, TGF-b, and PDGF.

Subcutaneous implantation of a polyvinyl alcohol sponge provokes an inflammatory reaction within the interstices of the sponge, which accelerates accumulation of cellular elements. Small quantities of EGF incorporated into pellets embedded within the sponge, or injected daily into the sponge in an albumin carrier, increase cellularity, nucleic acid content, collagen, and glycosaminoglycan content in the granulation tissue. The synthetic effects are dose-dependent, and the observed accumulation of hydroxyproline is increased significantly by as little as 1 &mgr;g/day EGF.

In the wire mesh-type Hunt-Schilling wound chamber implanted subcutaneously in rats, exogenous TGF-b as a single injection soon after implantation increases the deposition ofcollagen. TNF has no effect on its own, although it inhibits the effects of TGF-b while not influencing those of PDGF. These experiments demonstrate the importance of the interaction between peptides within a wound.

A cylindrical chamber produced from polytetrafluoroethylene (PTFE) allows granulation tissue to grow along the lumen. Cellularity can be increased two- to six-fold by a single injection into the chamber lumen of small concentrations (100–400 &mgr;g) of PDGF, TGF-b, and b-FGF in a collagen gel. The half-life of iodinated peptides within the gel is in the region of 20 h and the most potent peptide is TGF-b

Hunt and colleagues are using miniaturized porous chambers in human studies. Subcutaneous implantation can be performed under local anaesthesia, and is only slightly more traumatic than the insertion of an intravenous cannula. These ‘human wound models" have been used to investigate the effects of tissue hypoxia, underperfusion during and after surgery, and parenteral nutrition before operation, and on rates of collagen synthesis. Eventually, they may be applied to determine variations in intrinsic healing potential, to predict wound failure and dissect its mechanisms, and to select patients for experimental treatments.

The first clue to the involvement of growth factors in wound healing became apparent from experiments using topical application of EGF to a standardized back wound in mice. Enhanced wound closure was observed both in control and sialectomized animals receiving EGF, indicating that the local delivery of wound-healing factors accelerated healing. EGF incorporated into multilamellar liposomes to prolong release, but not when given as a single dose, doubles tensile strength in skin wounds of rats at 7 days after wounding. A single dose of 2 &mgr;g TGF-b in a collagen vehicle increases tensile strength by 51 per cent at 9 days, whereas PDGF in saline does not produce any significant effect on breaking strength. The acceleration of the healing process is accompanied by increased infiltration into the wound of mononuclear cells and fibroblasts and by collagen deposition. The beneficial effects of collagen as a vehicle for peptide growth factors in these models is demonstrated by the fact that 2 &mgr;g of PDGF in a collagen suspension doubles the strength of wounds at 5 days, an effect which is still apparent at 40 days. Histological analysis of the wounds treated with these growth factors demonstrated an in-vivo chemotactic response of macrophages and fibroblasts and an increase in type I collagen. With both TGF-b and PDGF a dose–response curve is apparent. Wounds treated with both peptides demonstrate an augmented cellular infiltrate and, in the case of PDGF, increased staining with a monoclonal antibody procollagen type I. Recombinant b-FGF 400 &mgr;g, infiltrated into healing incisional wounds on the third day post-wounding, increased breaking strength assessed on days 5, 6, and 7 by 39 per cent. However, this peptide had no effect on wound collagen content, although histological examination showed better organization and maturation in b-FGF treated wounds. Effects are presumably due to earlier accumulation of fibroblasts and/or collagen cross-linking. Local TNF increases wound disruption strength by one-half in incisional wounds in mice in a narrow dose range (50–500 &mgr;g). Outside this range, its effect on wound strength appears to be inhibitory and associated with a dense inflammatory infiltrate.

The depth of wounding has an important bearing on whether its repair can be accelerated by exogenous application of peptide growth factors. For instance, in full-thickness skin incisions in pigs treated twice daily with EGF for 14 days, no benefit was observed in treated animals. However, in partial-thickness dermal wounds, EGF and TGF-b increased epitheliazation, cellularity, and thickness of the dermis, presumably through generation of epithelium from dermal appendages. In an excisional skin model, increased staining for collagen matrix protein mRNA after treatment with exogenous TGF-b has been found. The staining for TGF-b mRNA itself was also increased, indicating that TGF-b was auto-stimulatory, accounting, at least in part, for the persistent effects of single doses of this peptide.

Uncontaminated surgical wounds in healthy patients heal by primary intent simply with the support of wound sutures. It is unlikely that, in this situation, growth factors will be a useful adjunct. The greatest therapeutic benefit of these peptides is likely to be derived in situations where impaired healing results in increased morbidity or mortality for patients. In animals, a wound-healing deficit can be defined artificially and the impairment in healing can be successfully ameliorated by growth factors.

However, impaired acute healing in animal models bears an indeterminate relationship to the common human problems of chronic wound healing failure. In essence, it has not yet proved possible to create good analogues of venous, arteripathic, or decubitus ulcers, if only because laboratory animals do not live long enough to achieve the tissue changes seen in some of these conditions in man.

The wound-healing deficit induced by the use of corticosteroids is well documented. In steroid-sensitive animals, treatment causes a prolonged monocytopenia, preventing macrophage migration into the wound and thus diminishing this essential element of the wound-healing cascade. The in-vitro effects of steroid treatment are to depress fibroblast proliferation and inhibit procollagen and matrix protein synthesis. A single local application of TGF-b (10–40 pmol/wound) in a collagen suspension in the rat reverses a 50 per cent wound-healing deficit resulting from methylprednisolone treatment. PDGF in the same model fails to reverse the wound-healing deficit, but does increase fibroblast numbers in the wound. However, these fibroblasts lack enhanced expression of procollagen type I. Wound macrophages remain absent from both PDGF and TGF-b treated wounds. In steroid-treated pigs, local applications of exogenous TGF-b reverse the depression of matrix protein synthesis, procollagen mRNA, and TGF-b mRNA. In methylprednisolone-treated rats, daily injection of 5 &mgr;g EGF in an albumin carrier into a polyvinyl alcohol sponge restores collagen and matrix protein levels to normal.

Local irradiation impairs wound healing by depleting dermal fibroblasts and decreasing the proliferative potential of endothelium, whereas total body irradiation depresses bone marrow-derived elements, virtually eliminating wound macrophages. A single application of 2 to 10 &mgr;g of PDGF in a collagen vehicle partially reverses the surface irradiation wound-healing deficit in the skin of rats 7 days after creation of the wound. PDGF does not reverse the wound-healing deficit seen with total body irradiation. These studies support the hypothesis that PDGF requires the presence of activated wound macrophages for activity in vivo.

Cytotoxic treatment decreases circulating white cells and impairs the formation of granulation tissue in a wound chamber. Adriamycin treatment reduces the level of TGF-b and PDGF-like activity in aliquots of wound fluid removed from wound chambers. Injection of TGF-b into the wound chamber returns granulation tissue formation to a normal level. Incisional wound healing is impaired by Adriamycin treatment, and the strength of wounds can be returned to normal by a single intra-incisional application of 2 &mgr;g of TGF-b or 50 mg TNF in a collagen vehicle.

Neovascularization is impaired in wound healing in diabetics. In implanted Hunt-Schilling wound chambers in diabetic rats, PDGF restores granulation tissue formation and angiogenesis to normal. The influx of connective tissue cells, DNA synthesis, and collagen deposition are increased, effects that are augmented by the addition of insulin to PDGF. In diabetic mice, 0.5 &mgr;g b-FGF applied locally to an open wound once a day increases granulation tissue thickness, infiltrated cells, capillary number, and tissue strength of full-thickness punch biopsy wounds. Moreover, in diabetic rats, the wound-healing deficit is improved in a differential manner by b-FGF and TGF-b: collagen synthesis in polyvinyl alcohol sponges increases 136 per cent at day 9 by a single application of 2 &mgr;g TGF-b but tensile strength is unaltered, whereas b-FGF suppresses collagen synthesis and increases cellularity. EGF induces a selective increase in the synthesis of type I collagen, whereas insulin returns collagen activity to normal and causes temporary inhibition of proteolytic activity directed primarily at type I collagen.

The maturation of intestinal wounds differ from the maturation of skin wounds, and is mainly reflected in the rate of accumulation of collagen. Fibroblasts are present in low numbers in intestinal submucosa and collagen is generated by smooth muscle cells. TGF-b augments collagen production in smooth muscle cells by 100 per cent and non-collagen proteins by 40 per cent in vitro. EGF has no effect on collagen synthesis in smooth muscle, indicating that modulation of wound repair in intestine differs from that in skin. The gain in strength of intestinal wounds is far more rapid than in skin whether assessed by measurement of anastomotic bursting strength or breaking strength of linear enterotomy wounds. Continuous intraperitoneal delivery of 0.5 &mgr;g EGF/kg.day increases by 20 per cent the tensile strength of linear enterotomy wounds in stomach, ileum, and colon at 5 days. This is accompanied by an increase in cellularity. In the rabbit stomach, topical application of TGF-b (0.1–2 &mgr;g per wound) to partial thickness (excluding mucosa) longitudinal wounds, accelerates wound breaking strength by 4 days. PDGF (10 &mgr;g/wound) does not enhance gastric wound strength but increases cellular influx 2.9-fold, whereas TGF-b does not affect cellularity.

Clinical trials are awaiting a clear definition of the efficacy of individual growth factors. In anticipation of clinical application of growth factors, the United States Food and Drug Administration has discussed guidelines to be adopted prior to phrase 1 clinical studies. The first clinical trials with EGF were negative, possibly due to lack of purity before recombinant DNA techniques were available. Daily application of 5 &mgr;g EGF to suction blisters on the anterior abdominal wall failed to improve healing, nor did local EGF affect epithelial healing after penetrating keratoplasty. Alternative reasons for this negative effect have been investigated in the alkali-burned cornea of rats, showing the effect to be due to a lack of adherence of regenerated epithelium to underlying stroma. EGF has no effect on endothelial cell or stromal cell regeneration, and hence there is no adherence of epithelium to underlying stroma due to the absence of a basement membrane. Further developments will have to address such problems and it is likely that a wound-healing cocktail will contain a number of peptides to stimulate various elements of the wound-healing cascade. A clinical trial using skin-graft donor sites has demonstrated that 10 &mgr;g EGf/ml applied in an antibiotic cream at each wound dressing reduces the length of time to healing by 1.5 days.

As mentioned previously, platelets contain numerous growth factors, which can be released by incubation with thrombin. Knighton has reported that topical application of autologous platelet lysate may accelerate the healing of chronic wounds. These findings remain to be confirmed by further studies.

A large number of phase I and II clinical trials of single growth factors are in now progress. Most are being carried out in chronic venous, decubitus, or neuropathic-diabetic ulcers (Table 5) 4. Some encouraging preliminary results have been reported for diabetic and decubitus ulcers.

‘A material which, when applied to the surface of a wound, provides and maintains an environment in which healing can take place at the maximum rate."

Thomas (1986)

Wound dressings have been utilized since the beginning of time, and some of the dressings used today in plugging and concealing wounds, such as lint and cotton wool, would not seem out of place to practitioners of medicine in early civilizations. In the classification of wound-healing products, these dressings have been referred to as passive. New dressings, such as polymeric films, polymeric foams, particulate and fibrous polymers, hydrogels, and hydrocolloids, have been classified as interactive dressings, providing a microenvironment which is conducive to healing. One of the hydrocolloid dressings (DuoDerm/Granuflex), to which a considerable amount of clinical and experimental research has been devoted, provides a wound-healing environment that, in addition to improving healing, also stimulates angiogenesis (Figs. 17, 18) 16,17). The third part of this classification is that of active products which actively stimulate healing beyond that of the normal biological maximum.

One of the worries that clinicians have in using some of the new occlusive dressings in the treatment of chronic wounds is that, because of their impermeability to oxygen, this will lead to an environment that will increase wound infection. A recent review of more than 103 published papers, comparing the clinical infection rate between conventional and occlusive dressings, found a clinical infection rate of 2.08 per cent with wounds treated with occlusion compared to 5.37 per cent with conventional dressings. One of the reasons for this finding might be the ability of some occlusive hydrocolloid dressings to increase wound angiogenesis and local blood supply, thus providing an environment not conducive to bacterial growth (Figs. 2 and 17(c)) 1,16.

Biological wound coverage other than by conventional skin-grafting techniques has gained prominence with the use of cultured epidermal autografts and allografts. The latter grafts, although initially thought not to be rejected by the host, have since been shown to be replaced rapidly by host keratinocytes, thus acting as a temporary wound coverage but significantly stimulating the healing process.

In the future, dressings that deliver specific growth factors, pharmacological agents to stimulate healing, or serve as transducers to provide physical forces to the wound (such as electrical stimulation, ultrasound, or other stimuli) will be available to manage chronic wounds. These are being evaluated currently in a number of clinical and experimental studies.

At the time of writing, the explosive increase in our understanding of tissue repair and its defects is just beginning to be translated into practical strategies. This burst in wound healing knowledge is reflected in the newly established Wound Healing organizations in the United States and Europe ‘The Wound Healing Society" for the former and the ‘European Tissue Repair Society".

It is important to define realistic clinical objectives. In acute wound healing, the major problems are wound failure (including anastomotic leakage, hernia recurrence, and some types of fracture non-union) and over-healing in its many guises (adhesions, strictures, contractures, and hypertrophic and keloid scarring). Although technical errors are a major and preventable cause in some cases, intrinsic defects in wound healing potential are also important. Human wound healing models as described in this chapter may help to predict repair failure and permit the selection of patients for new therapies.

In chronic wounds the main aims will be to induce healing in patients where this was previously impossible, to accelerate it, and to prevent recurrence.

Several important developments will be required to realize the promise of growth factors and other new agents. Local pharmacology of wounds is in its infancy; new delivery systems will be required to apply these agents precisely to the site and at the time they are needed. The plethora of new agents will mandate careful investigation to determine which ones are optimal for which clinical situation. Finally, the design of clinical trials, including ethical issues, recruitment criteria, and choice of endpoints, remains a serious problem. Successful resolution of these issues promises finally to close the gap between basic research and clinical application.

Lydon MJ, et al. Dissolution of wound coagulum and promotion of granulation tissue under DuoDERM t; m;. WOUNDS 1989; 1: 95–106.