Inflammatory Phase (Day 0-5)
The healing response is initiated at the moment of injury. Surgical or traumatic wounds disrupt the tissue architecture and cause haemorrhage. Initially, blood fills the wound defect and exposure of this blood to collagen in the wound leads to platelet degranulation and activation of Hageman factor [1]. This in turn sets into motion a number of biological amplification systems including the complement kinin and clotting cascades and plasmin generation. These serve to amplify the original injury signal and lead not only to clot formation, which unites the wound edges, but also to the accumulation of a number of mitogens and chemoattractants at the site of wounding [2].
Production of both kinins and prostaglandins leads to vasodilatation and increased small vessel permeability in the region of the wound [3]. This results in oedema in the area of the injury and is responsible for the pain and swelling which occurs early after injury. Within 6 h, circulating immune cells start to appear in the wound. Polymorphonuclear leucocytes (PMN) are the first blood leucocytes to enter the wound site. They initially appear in the wound shortly after injury and subsequently their numbers increase steadily, peaking at 24-48 h [4]. Their main function appears to be phagocytosis of the bacteria which have been introduced into the wound during injury. The presence of PMN in the wound following injury does not appear to be essential in order for normal
wound healing to take place [5, 6], with healing proceeding normally in their absence provided that bacterial contamination has notoccurred. In the absence of infection, PMN have a relatively short life span in the wound and their numbers decrease rapidly after the third day [7].
The next cellular, immune element to enter the wound are macrophages. These cells are derived from circulating monocytes by a combination of migration and chemotaxis. They first appear within 48-96 h post-injury and reach a peak around the third day postinjury [4]. These macrophages have a much longer life span than the PMN and persist in the wound until healing is complete. Their appearance is followed somewhat later by T lymphocytes, which appear in significant numbers around the fifth day postinjury, with peak numbers occurring about the seventh day after injury. In contrast to PMN, the presence and activation of both macrophages and lymphocytes in the wound is critical to the progress of the normal healing process [8, 9]. Macrophages just like neutrophils phagocytose and digest pathological organisms and tissue debris. In addition, macrophagesrelease a plethora of biologically active substances. Many of these substances facilitate the recruitment of additional inflammatory
cells and aid the macrophage in tissue decontamination and debridement; in addition growth factors and other substances are also released which are necessary for the initiation and propagation of granulation tissue formation. These intercellular transmitters are known collectively as cytokines.
Proliferative Phase (Day 3-14)
In the absence of significant infection or contamination the inflammatory phase is short, and after the wound has been successfully cleared of devitalized and unwanted material it gives way to the proliferative phase of healing. The proliferative phase is characterized by the formation of granulation tissue in the wound. Granulation tissue consists of a combination of cellular elements,
including fibroblasts and inflammatory cells, along with new capillaries embedded in a loose extra cellular matrix of collagen, fibronectin and hyaluronic acid. Fibroblasts first appear in significant numbers in the wound on the third day post-injury and achieve peak numbers around the seventh day [4]. This rapid expansion in the fibroblast population at the wound site occurs via a combination of proliferation and migration [10]. Fibroblasts are derived from local mesenchymal cells, particularly those associated with blood vessel adventitia [11], which are induced to proliferate and attracted into the wound by a combination of cytokines produced initially by platelets and subsequently by macrophages and lymphocytes (Table 1). Fibroblasts are the primary synthetic element in the repair process and are responsible for production of the majority of structural proteins used during tissue
reconstruction. In particular, fibroblasts produce large quantities of collagen, a family of triple-chain glycoproteins, which form the main constituent of the extracellular wound matrix and which are ultimately responsible for imparting tensile strength to the scar. Collagen is first detected in the wound around the third day post-injury [12, 13], and thereafter the levels increase rapidly for approximately 3 weeks. It then continues to accumulate at a more gradual pace for up to 3 months post wounding [10]. The collagen is initially deposited in a seemingly haphazard fashion and these individual collagen fibrils are subsequently reorganized, by cross -linking, into regularly aligned bundles oriented along the lines of stress in the healing wound. Fibroblasts are also responsible for the production of other matrix constituents including fibronectin, hyaluronic acid and the glycosaminoglycans [14]. The process of fibroblast proliferation and synthetic activity is known as fibroplasia.
capillary loops, through which blood begins to flow. New sprouts then extend from these loops to form a capillary plexus [15, 16]. The soluble factors responsible for angiogenesis remain incompletely defined. It appears that angiogenesis occurs by a combination of proliferation and migration. Putative mediators for endothelial cell growth and chemotaxis include cytokines
produced by platelets, macrophages and lymphocytes in the wound [17, 18], low oxygen tension [19], lactic acid [20] and biogenic amines [21]. Of the potential cytokine mediators of neovascularization basic fibroblast growth factor (bFGF), acidic FGF (aFGF), transforming growth factors -aand b(TGF-aand -b) and epidermal growth factor (EGF) have all been shown to be potent stimuli for new vessel formation [22-24]. FGF, in particular, has been shown to be a potent inducer of in vivo neovascularization [25, 26]
While these events are proceeding deep in the wound, restoration of epithelial integrity is taking place at the wound surface. Reepithelialization of the wound begins within a couple of hours of the injury. Epithelial cells, arising from either the wound margins or residual dermal epithelial appendages within the wound bed, begin to migrate under the scab and over the underlying viable
connective tissue. The epidermis immediately adjacent to the wound edge begins thickening within 24 h after injury. Marginal basal cells at the edge of the wound loose their firm attachment to the underlying dermis, enlarge and begin to migrate across the surface of the provisional matrix filling the wound. Fixed basal cells in a zone near the cut edge undergo a series of rapid mitotic divisions, and these cells appear to migrate by moving over one another in a leapfrog fashion until the defect is covered [27, 28]. Once the defect is bridged, the migrating epithelial cells loose their flattened appearance, become more columnar in shape and increase in mitotic activity. Layering of the epithelium is re-established and the surface layer eventually keratinized [29]. Reepithelialization is complete in less than 48 h in the case of approximated incised wounds, but may take substantially longer in the case of larger wounds where there is a significant tissue defect. If only the epithelium is damaged, such as occurs in split thickness skin graft donor sites, then repair consists primarily of re-epithelization with minimal or absent fibroplasia and granulation tissue formation. The stimuli for re-epithelization remain incompletely determined, but it appears that the process is mediated by a
combination of loss of contact inhibition, exposure of constituents of the extracellular matrix, particularly fibronectin [30], and by cytokines produced by immune mononuclear cells [31]. EGF, TGF-b, bFGF, platelet-derived growth factor (PDGF) and insulinlike growth factor-l(IGF-l) in particular, have been shown to promote epithelialization [32].
Maturation Phase (Day 7 to I Year)
Almost as soon as the extracellular matrix is laid down, its reorganization begins. Initially, the extracellular matrix is rich in fibronectin, which forms a provisional fibre network. This serves not only as a substratum for migration and ingrowth of cells, but also as a template for collagen deposition by fibroblasts [33]. There are also significant quantities of hyaluronic acid and large
molecular weight proteoglycans present, which contribute to the gel-like consistency of the extracellular matrix and aid cellular infiltration. Collagen rapidly becomes the predominant constituent of the matrix. The initially randomly distributed collagen fibres become cross -linked and aggregated into fibrillar bundles, which gradually provide the healing tissue with increasing stiffness and tensile strength [34]. After a 5-day lag period, which corresponds to early granulation tissue formation and a matrix largely composed of fibronectin and hyaluronic acid, there is a rapid increase in wound breaking strength due to collagen fibrogenesis. The subsequent rate of gain in wound tensile strength is slow, with the wound having gained only 20% of its final strength after 3
weeks. The final strength of the wound remains less than that of uninjured skin, with the maximum breaking strength of the scar reaching only 70% of that of the intact skin [34].
This gradual gain in tensile strength is due not only to continuing collagen deposition, but also to collagen remodelling, with formation of larger collagen bundles [35] and alteration of intermolecular crosslinking [36]. Collagen remodelling during scar formation is dependent on both continued collagen synthesis and collagen catabolism. The degradation of wound collagen is controlled by a variety of collagenase enzymes, and the net increase in wound collagen is determined by the balance of these opposing mechanisms. The high rate of collagen synthesis within the wound returns to normal tissue levels by 6-12 months [37], while active remodelling of the scar continues for up to 1 year after injury and indeed appears to continue at a very slow rate for life.
As remodelling progresses, there is a gradual reduction in the cellularity and vascularity of the reparative tissue which results in the formation of a relatively avascular and acellular collagen scar. Grossly this can be observed as a reduction in erythema associated with the earlier scar and some reduction in the scar volume, resulting in a pale thin scar. This is normally a desirable feature of
healing; however, in some cases shrinkage of the scar may give rise to an undesirable reduction in skin mobility resulting in contracture.
Wound contraction, i. e. inward movement of the wound edge, is a further important element in the healing process and should be distinguished from contracture. Sharply incised wounds without significant tissue loss, approximated early after injury, heal rapidly without the need for significant reduction in the wound volume. Such wounds are described as having healed by primary intention.
Large wounds, however, particularly those associated with significant tissue loss, heal by secondary intention, with granulation tissue gradually filling the defect and epithelization proceeding slowly from the wound edges. Contraction of the wound edges can lead to a significant reduction in the quantity of granulation tissue required to fill the wound defect and a reduction in the area
requiring reepithelization, with a consequent reduction in scar volume. Contraction is only undesirable where it leads to unacceptable tissue distortion and an unsatisfactory cosmetic result. Although contraction normally accounts for a larger part of overall wound closure in looseskinned animals, it still accounts for a significant proportion of the healing process in man, particularly
in areas where the skin is not tightly bound down to underlying structures, such as on the back, neck and forearms. Initially following injury, where the wound edges are not approximated, there is a slight retraction of the wound edges due to the release of normal elastic tension in the skin, with a resultant increase in wound volume. The wound area starts to decrease rapidly from the
third day onwards. While this is due in part to reepithelization, the main reason is an inward movement of the uninjured skin edges. Wound contraction usually begins around the fifth day postwounding and is complete by 12-15 days after wounding [38-40]. Fibroblasts within the wound appear to be responsible for providing the force for this contractile activity [41]. It was initially felt that specialized fibroblasts called myofibroblasts provided the motive force for wound contraction via a musclelike cell contraction [42-
More recent studies reveal that wound contraction occurs as a result of an interaction between fibroblast locomotion and collagen reorganization [41, 45]. The contraction is thought to be mediated via the attachment of collagen fibrils to cell surface receptors [46], with the resulting tractional forces generated by cell motility bringing the attached collagen fibrils closer together and
eventually compacting them [47].
The regulation of wound contraction remains poorly defined. Information regarding the effects of specific cytokines on contraction is limited and often conflicting. TGF-bhas been found to promote contraction even in the absence of serum [48, 49]; PDGF has also been found to either increase contraction [50] or have no effect [49], while both FGF and EGF have been found by different authors to either have no effect or cause a moderate enhancement of contraction [48-50].
Scar Formation
As mentioned previously, the process of wound healing is essentially similar in all tissues and is relatively independent of the mode of injury; however, slight variation in the relative contribution of the different elements to the overall result may occur. The final product of the healing process is a scar. This relatively avascular and acellular mass of collagen serves to restore tissue continuity,
strength and function. Delays in the healing process cause the prolonged presence of wounds, while abnormalities of the healingprocess may lead to abnormal scar formation. Successful completion of wound healing may not always yield the desired clinical result, particularly where the final cosmetic appearance of the scar is of primary importance.
