Phase 2(i): Migration and proliferation 1 Epithelialisation

Epithelial cells, specifically kératinocytes in the skin, migrate across the wound a few hours after injury. Rapid re-epithelialisation and closure o f the wound is vital, as this can reduce infection and fatalities (Kirsner and Eaglstein, 1993; Clark, 1995). Approximately two days post-injury the epithelial cells begin to proliferate and move inwards from the wound margin (Krawczyk, 1971), resulting in the formation o f a new epithelium. Epithelial cells migration occurs in several ways, including MM? activation (formation o f a migratory path), changes in the basement membrane zone (normally present laminin and collagen type IV, which promote cell adhesion, disappear), the presence o f a moist environment, chemotactic factors (eg: TGFp) and, or contact guidance (Kirsner and Eaglstein, 1993; Clark, 1995).

1.4.3.2 Granulation tissue formation

Granulation tissue formation begins around 4 days post-injury and consists o f macrophages, fibroblasts, blood vessels and loose connective tissue (Kirsner and Eaglstein, 1993; Ehrlich and Krummel, 1996; Mutsaers et al. 1997). The provisional

fibrin clot, made o f fibronectin and f i b r i n acts as a contact guidance framework and

encourages granulation tissue formation. It also ensures efficient cell mobility, through the presence o f hyaluronic acid (Toole, 1991), provides a store for cytokines (Nathan and Spom, 1991), and also has the ability to transmit signals directly to the cells, through integrin receptors (Damsky and Werb, 1992).

Constant secretion o f cytokines by macrophages activates fibroblasts to further release cytokines and synthesise, deposit and remodel new extracellular matrix (Welch et al. 1990). The extracellular matrix in turn regulates fibroblast activity, controlling their ability to synthesise, deposit and remodel the extracellular matrix (Grinnell, 1994). This

dynamic interaction o f fibroblasts and extracellular matrix is termed fibroplasia, and

evolves during granulation tissue development (Clark, 1995).

1.4.3.3 Fibroplasia

Fibroblasts initially penetrate the fibrin clot present in the wound. The clot consists mainly o f fibrin, however also contains fibronectin and vitronectin adhesive proteins. It is predominantly through use o f fibronectin, and to a lesser extent through fibrin and vitronectin that the fibroblasts migrate through the wound. Fibroblasts attach to these

adhesive proteins (ligands) through special cell surface receptors called integrins

(Ruoslahti, 1991; Hynes, 1992, Stuiver and O’Toole, 1995) (refer to section 1.6.7). As well as attachment, cells must also be able to facilitate a proteolytic response, in order to enzymatically degrade the fibrin clot and extracellular matrix, thereby creating a pathway for migration. Possible contenders for this task include matrix metalloproteinases and the plasminogen-derived enzyme, plasmin (Birkedal-Hansen, 1995; Basbaum and Werb, 1996; Martin, 1997), which are themselves regulated by enzyme specific inhibitors (Birkedal-Hansen, 1995; Basbaum and Werb, 1996).

1.4.3.4 Extracellular matrix synthesis

After fibroblast migration ceases the migratory phenotype o f the fibroblast changes to a proliferative-profibrotic phenotype, where initially fibronectin and later, collagen synthesis dominates. This process has been linked to the increased expression o f the cytokine, TGFp by these cells (Clark et al. 1995; Shah et al. 1995; Martin, 1997). It is known that TGFp is a potent stimulator o f fibronectin (Ignotz and Massague, 1986; Shah et al. 1995; Martin, 1997; Sarkissian and Lafyatis, 1998) and collagen (Kirsner and Eaglstein, 1993; Shah et al. 1995) production. Once the wound defect is replaced, the expanding fibroblast population stops proliferating and regresses, and ECM remodelling commences (Grinnell, 1994). However, the level o f TGFp expression does not decrease (Clark et al. 1995). Regression o f wound fibroblasts, probably myofibroblasts, is thought to occur through programmed cell death (apoptosis), and is probably the mechanism responsible for the evolution o f granulation tissue to scar (Desmouliere et al. 1995). This down-regulation o f fibroblast proliferation and collagen synthesis has also been linked to the cytokine gamma-interferon (Duncan and Berman, 1985; Granstein et al. 1987; Pittet et al. 1994). Furthermore, it has been suggested that such a decrease may also be caused by the collagen matrix itself (Grinnell, 1994; Clark et al. 1995), but not by fibrin or fibronectin

matrix proteins (Clark et al. 1995). It appears that mechanical organisation o f the collagen tissue can regulate ECM synthesis and remodelling, as well as fibroblast proliferation (Grinnell, 1994). Experiments performed using untethered versus tethered collagen lattices have shown great variation in ECM synthesis and fibroblast proliferation. It appears that in the absence o f tension (untethered gels) cell DNA synthesis falls dramatically and fibroblasts move into Go (growth arrest) o f the cell cycle, hence causing cell regression. Collagen synthesis also declines in the absence o f tension and this is accompanied by an increased release o f collagenase, compared to the tethered gel counterparts (Grinnell, 1994). Hence, as the collagen tissue begins to relax during the transition fi*om granulation to scar tissue (a form o f stress-relaxation), cell proliferation and collagen biosynthesis declines.

1.4.3.5 Collagen synthesis

Collagen is the primary scar tissue constituent, accounting for 25% o f total body protein (Bell, 1979). 90% o f the total body collagen content is present as mature type I collagen (Asmussen and SoUner, 1993). Synthesis and secretion o f this fibrous protein by fibroblasts occurs in several stages (Tahery and Lee, 1989; Asmussen and SoUner, 1993). StructuraUy coUagen consists o f 3 polypeptide chains which are brought together in the rough endoplasmic reticulum to form a procoUagen triple heUcal molecule. The terminal end o f the procoUagen is cleaved extraceUularly and yields the precursor tropocoUagen. Lysyl oxidase crosslinks the tropocoUagen and forms collagen, which aUows for subsequent wound closure and scar formation (Tahery and Lee, 1989). Type III coUagen is initiaUy deposited at the wound site. This form predominates in granulation tissue and is graduaUy replaced by type I coUagen during the later stages o f repair. It is the type I collagen, its packing and crosslinking, which provides the increased tensUe strength seen in later repair tissues (Clark, 1995; Wong et al. 1996).