Characterization of biologic properties of wound fluid collected during early stages of wound healing.

The clinical effects of occlusive dressings on wound healing are well documented. However, the underlying biologic mechanisms associated with moist healing are not well understood. Experimental studies and clinical experience have shown enhanced eschar and clot removal, re-epithelialization, and collagen synthesis under occlusion, suggesting the possibility of elevated activities of proteinases and other effectors, e.g., growth factors, in the moist wound environment. To gain an insight into the biology of early wounds under occlusion, we have carried out biologic and biochemical analyses on fluids from occluded full- and partial-thickness wounds. Metalloproteinase activities were detected in the wound fluid samples. When applied to cultured dermal fibroblasts, mitogenic activity was observed with fluids from full-thickness wounds. Wound fluid-stimulated accumulation of urokinase-type plasminogen activator by fibroblasts was also observed in a time-dependent manner. Stimulation of metalloproteinase accumulation by fibroblasts was also observed. We have further demonstrated the presence of platelet-derived growth factor-like and basic fibroblast growth factor-like factors in wound fluid by antibody neutralization of their biologic activities. Proteinase presence and proteinase stimulatory activity of wound fluid retained in the occluded wound may contribute to an enhanced proteolytic environment in these wounds in comparison to non-occluded "dry" wounds. The presence of growth factors and the potential abilities of proteinases to activate latent growth factors and generate chemotactic peptides through connective tissue breakdown may also contribute to the enhanced healing of occluded wounds.

Cell-substratum interactions: serum spreading factor.

We have prepared a fraction of commercial fetal calf serum whose major component comprises proteins of apparent molecular mass 65-80 kDa. These preparations elicit cell spreading responses in serum-free medium at concentrations similar to those reported for human vitronectin. In addition, we have identified (by a protein blotting technique) the 65-80 kDa component as the active species in our preparations. Cell spreading responses in the presence of our 'bovine vitronectin' preparations are similar to cell spreading on fetal calf serum. (In contrast, cell spreading on bovine fibronectin showed very different kinetics). Our results suggest that bovine vitronectin may be an important component of fetal calf serum involved in conditioning cell culture substrata.

Cellular interactions with synthetic polymer surfaces in culture.

The success of synthetic polymers as biomaterials depends upon their interfacial properties and resultant interactions with cells and biological fluids in vivo. A useful experimental approach to defining requirements for biocompatibility is to study the mechanisms by which synthetic substrata influence eukaryote cell behaviour in culture. Here we present an overview of the relationships between physical and chemical substratum properties and cell behaviour on a range of synthetic polymers. We relate our results to theories of in vivo tissue compatibility.

Substratum topography and cell traction on sulphuric acid treated bacteriological-grade plastic.

Treatment of bacteriological-grade plastic with concentrated sulphuric acid is a well known technique which increases the wettability of the surface and renders it suitable for eukaryotic cell adhesion. We have noticed that these substrata present a distinctive surface topography in the presence of a serum supplement under normal culture conditions. The adsorbed serum layer is comprised of fine furrows and ridges and the influence of adherent cells on this layer leads to minute tears and distortions in the direction of the corrugations. This provides a novel system for the investigation of cell spreading and locomotion by scanning electron microscopy.

Requirements for cell spreading on polyHEMA coated culture substrates.

We have investigated the topography of polyHEMA coated culture substrates by scanning electron microscopy, and quantitatively assessed their effect upon the spreading activity of mammalian cells. Results indicate a clear correlation between cell spreading activity and polymer film discontinuity. Preparation of polyHEMA films on modified tissue culture substrates has allowed direct investigation of the role of the underlying substrate in regulating cell spreading, and confirms that apparent modulation of cell spreading by polyHEMA reflects increasing expression of the coated surface. We have further employed a spinning technique by which films of precise thickness, down to 0.01 micron, may be produced on coverslips. All polyHEMA coatings prepared in this way are smooth and complete. They do not allow cell attachment at any thickness. We conclude that polyHEMA is non-adhesive for mammalian cells.

Vital DNA staining and cell sorting by flow microfluorometry.

A procedure has been investigated for sorting viable cells according to their DNA content. Cells are stained with the U.V. activated fluorochromes 4'6-diamidino-2-phenylindole (DAPI), Hoechst 33258 or Hoechst 33342, and sorted with a Fluorescence Activated Cell Sorter. Hoechst 33342 is a suitable vital stain for a variety of cell types. Hoechst 33258 and DAPI, however, are quantitative vital stains for CHO cells only. Cloning efficiency is unaffected by the sorting procedure, and these stains are not mutagenic at concentrations suitable for vital staining. Potential applications of this procedure to cell biology are discussed.