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It was the aim o f this study to understand and control certain cellular processes involved in wound contraction and consequent scarring, in order to reduce or increase healing, as required. For this, three potential areas were investigated: cell-matrix attachment, growth factor influence and photodynamic therapy. The first two were to understand possible key stages o f the process and their role, whilst the third was a potential intervention. Each is known to effect the healing process to varying degrees in vitro, with the latter also having been used in vivo.

1.10.1 Interruption o f cell-matrix interactions

Previous studies have suggested that contractile force generation is correlated with attachment, extension o f cell processes and migration o f cells through the collagen matrix (Stopak and Harris, 1982; Ehrlich and Rajaratnam, 1990; Delvoye et al. 1991; Andujar et al. 1992; Grinnell, 1994; Sheetz et al. 1994; Eastwood et al. 1996; Talas et al. 1997). Also intracellular signalling following integrin-ligand binding is thought to occur (Roeckel and Krieg, 1994; Langholz et al. 1995; Stuiver and O’Toole, 1995), mediating inside-out and outside-in signalling between the cytoskeleton and the ECM (Adams and Watt, 1993; Stuiver and O’Toole, 1995) and inducing contraction (Machesky and Hall, 1997; Rosenfeldt et al. 1998; Parizi et al. 1999 (In press)). It therefore seems likely that cell- matrix attachment is critical to contraction, and so its mechanism is an important potential target for the regulation o f collagen contraction in tissue repair or in tissue engineering.

The initial stage o f this study was to investigate cell-matrix interactions during the early stages o f contraction. Conducting this phase o f research provided insight into the factors involved in the process, and when they came into play. The collagen matrix used in this

part o f the study was a highly porous sponge. This represented the dermis, was used in

combination with human dermal fibroblasts. Interruption o f cell-matrix interaction was achieved by adding blocking antibodies, synthetic peptides or by removing cell-binding ligands from the serum used in the system. Consequent changes in forces generated in matrices were measured and analysed using the CFM.

1.10.2 Influence o f T G Fpi

Manipulation o f wound healing using growth factors, in particular TGFpl has been well documented (Reed et al. 1994; Chegini, 1997; Martin, 1997). It has been reported that TGFpl stimulates contraction through fibroblast differentiation (into myofibroblasts) and upregulation o f a-SMA expression by such cells (Desmouliere et al. 1993). TGFp also increases the deposition o f ECM proteins such as fibronectin and collagen (Sarkissian and Lafyatis, 1998), by increasing synthesis and decreasing breakdown (Desmouliere and Gabbiani, 1994; Reed et al. 1994; Chegini, 1997), and that this can lead to the development o f hypertrophic scars and other fibrotic disorders (Desmouliere and Gabbiani, 1994). The suggestion that TGFp plays an important role in both fibroblast differentiation (change from fibroblast to myofibroblast) and in fibrosis makes it a key growth factor that could possibly mediate the extent o f granulation tissue contraction

(Grinnell, 1994). Thus, the second stage o f the study was to observe at which stage

this growth factor exerted its effect, as well as to characterise changes in rate and magnitude o f force generation caused by TG Fpi. Changes in morphology and receptor expression were also investigated, to identify the possible mechanism o f action TGFpl has on the process o f contraction.

1.10.3 Effect o f photodynamic therapy

Lasers have been used in cutaneous surgery for many years, the argon laser was initially employed and later replaced by the carbon dioxide laser (Sauer and Hall, 1996). The latter has been used for a wide range o f surgery techniques, including incisions, excisions (of skin cancers), mobilisation of, and grating o f skin flaps in reconstructive surgery. The carbon dioxide laser has also been used to level out raised scars (keloids) and hypertrophic scarring, generating re-epithelialisation and healing, though with limited success. Stemming from the early staged use o f lasers for such surgery, a subsequent and expansive array o f varied laser types has appeared (Sauer and Hall, 1996). Photodynamic therapy is the most recent addition to the ever increasing list o f laser treatments. To date it has been used extensively for the treatment o f oral, skin and gut cancers, however holds potential for use in cutaneous surgery (Sauer and Hall, 1996). Especially for reducing or eliminating comestically and anatomically disfiguring keloid and hypertrophic scars.

PDT treatment involves irradiating the tumour tissue, which has been pre-treated with a photosensitiser (Bishop et al. 1993). PDT treatment selectively destroys the tumour cells, leaving the extracellular matrix unaffected, and allows for normal healing to commence. Taking this concept o f removal o f abnormally functioning cells and allowing a consequential influx of healthy normal cells (repair), effect o f PDT on cell-matrix attachment during early stage contraction was addressed. Manipulation o f such processes using PDT could provide a critical point at which the process o f contraction may be regulated. Optimal light and drug dosage to minimise cell death was achieved by the use o f viability assays. Changes in contractile force generation was analysed using the conventional free floating gel method and the quantitative CFM.