MARCO CONCEPTUAL DE LA INVESTIGACIÓN

3.1 INTRODUCTION

Collagen is a major constituent of the extracellular matrix in vivo. It is known to act as a substratum for cells in tissue and regulate many cell activities including adhesion, morphology, growth and differentiation. The function and morphology of fibroblasts have been shown to be influenced by the extracellular matrix (Kleinman et ai, 1981). Type 1 collagen is the natural substratum for fibroblasts in vivo (Kleinman et ai, 1981).

The attachment and growth of anchorage-dependent cells such as fibroblasts, is a multistep process that involves cell contact with the substrate, spreading and replication. Cell attachment has been shown to be a very important process (Vasiliev, 1985), since fibroblasts cannot spread until after they are attached. Cell attachment of cells to their substratum is passive, but spreading is an active process (Grinnell, 1978). Cells attach initially with a rounded shape, and then spread radially, characterized by circumferential actin organization. This involves the reorganization of the cell’s cytoskeleton and a change of morphology, resulting in the projection of filopodia. With time, polarized cell spreading occurs characterized by the formation of focal adhesion plaques and stress fibres (Grinnell, 1978). Cell attachment and spreading has been shown to be dependent on a number of incubation conditions, including temperature (Klebe, 1975).

Human fibroblasts, a cell strain of limited lifespan (Hayflick & Moorhead, 1961), attach and spread on tissue culture plastic or denatured collagen substrata without the addition of other factors such as fibronectin (Grinnell & Minter, 1978). They secrete sufficient fibronectin to spread on tissue culture substrata or dried collagen gels in the absence of added fibronectin (Grinnell & Feld, 1979). Cell lines (unlimited lifespan) such as 3T3 fibroblasts, however, generally require cold insoluble globulin (CIG) (plasma form of fibronectin) to be previously adsorbed onto the substratum (tissue culture plastic or denatured collagen substrata) for the cells to spread (Grinnell & Hays, 1978).

Collagen matrices can be prepared in a variety of forms for cell culture. For example, dishes can be coated with collagen. The collagen substrate will have a low pH, since the dissolution of

collagen in acetic acid during preparation is required. If the pH is adjusted to physiological pH, the collagen comes out of solution and a gel forms (Bell et ai, 1981b), This gel forms the basis of a "living skin equivalent", which can be used as a three dimensional substrate for cell grovyth (Bell et ai, 1981b), However, the effect of any pH variation of the substrate, although within an acidic range, on cell spreading has not previously been studied. The density of collagen in the substrate film may also influence the extent of cell spreading.

Fibroblasts, and their interactions with collagen, play important roles in wound healing. Native collagen fibrils are stabilized by lysine-derived intermolecular cross-links, which are formed by a mechanism that involves the formation of aldehydes from lysine and hydroxylysine side-chains. Additional cross-links can, however, be introduced into collagen by a number of reagents including aldehydes, isocyanates, alkyl and aryl halides, imidoesters, carbodiimides, N-substituted maleimides and acylating compounds (Wold, 1972), It has been reported that cell spreading was enhanced in skin fibroblasts that were cultured on a collagen gel in which the intermolecular cross-linking had been increased by incubation in vitro (Nishikawa et ai, 1987),

It might therefore be expected that cell spreading on my modified collagen preparations would be enhanced compared to cell spreading on native, untreated collagen. Previous studies have shovm that the spreading of 3T3 mouse fibroblasts on films of malondialdehyde-treated collagen was enhanced, by comparison with spreading on native collagen, and that the modified substrate was substantially cross-linked by the aldehyde (Davies et ai, unpublished work).

The spreading response of both 3T3 and human skin fibroblasts on aldehyde-treated collagens will be studied: 3T3 fibroblasts will be studied since some data already exists (Davies et ai,

unpublished work) and human skin fibroblasts since these could be assumed to be more representative than 3T3 cells of the behaviour of human skin fibroblasts in contact with modified collagens in vivo.

The technique of immunofluorescence microscopy will be employed to characterize the distribution of various cytoskeletal components (actin, a-actinin, vinculin and vimentin) during the fibroblast spreading process. Extracellular matrix molecules have previously been shown to bind to integrins on the cell surface which connect with actin microfilaments within the cell, resulting in cell shape changes (Hynes, 1987; Ruoslahti & Pierschbacher, 1987), Since vinculin and a-actinin are

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actin-binding proteins which interlink integrins with microfilaments it is expected that their distributions change during spreading. Vimentin, an intermediate filament, will also be studied.

3.2 METHODS

3.2.1 Cell Culture

Human fibroblast cultures were established from the outgrowth of skin biopsies obtained from healthy volunteers. The human fibroblasts were kindly provided by Ms. T. Slade (Guy’s Hospital, London). A cell line of 3T3 fibroblasts from Swiss mouse embryos were also used. All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Life Technologies Limited, Uxbridge, Middlesex), supplemented with glutamine (4 mM), penicillin and streptomycin (200 lU) (Flow Laboratories, Irvine, Scotland) and 10 % foetal calf serum (Gibco, Life Technologies Limited, Uxbridge, Middlesex). They were grown on plastic tissue culture dishes (Lux) in an IR 500 Automatic CO; Incubator (Flow Laboratories, Irvine, Scotland), providing a moist atmosphere of 9.2 % CO; at 37°C.

Confluent monolayers were passaged by trypsinization (0.05 % trypsin and 0.02 % EDTA in Special Salt Solution) (Flow Laboratories, Irvine, Scotland). All cells were used before passage number 15, and were grown to near confluency prior to each spreading experiment.

Cells were frozen, for storage, in freezing media containing 10 % DMSO, which prevents the formation of ice-crystals in the cells, that would result in damage to the intracellular organelles (McIntyre et al, 1974). The freezing media also contained 20 % foetal calf serum instead of the normal 10 %. Cells were lowered into liquid nitrogen at a rate of I®C per minute and stored till required.

3.2.2 Preparation of Collagen Solution

Calf skin collagen (Calbiochem Novabiochem (UK) LTD., Nottingham) was dissolved in 0.15 M acetic acid, providing a stock solution of 100 pg protein/ml. Distilled water was added to give a final collagen concentration of 50 pg/ml. The collagen solution was incubated at 37°C for 20 hours.

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