Integrated studies of the interactions of macrophages with synthetic hydrogels

In this project a glycerol methacrylate hydrogel surface was synthesised, which acted as a biomaterial. This biomaterial was used as a surface on which fibroblast and macrophage type cells were grown. The hydrogels were synthesised from glycerol methacrylate, a hydrophilic monomer, cross-linked with glycerol dimethacrylate using chiefly thermal polymerisation methods. The hydrogels were shown to have similar water contents of around 75-85% mass and similar physical structures. Polystyrene latex particles were also synthesised using emulsion polymerisation. Latex particles were synthesised in 3 sizes: 100 nm, 250 nm and 720 nm. These particles were produced easily and in large quantities in the three sizes and with good control. The particles were incorporated into the hydrogel structures to give three sets of hydrogels with surfaces of varying roughnesses but identical surface chemistry. The surfaces were analysed with electron microscopy and white light interferometry to gain an understanding of the roughness and appearance of the surfaces. These latex hydrogels were optimised for cell culture and shown to be non-cytotoxic by the culture of 3T3 fibroblast cells on their surfaces. Normal human dermal fibroblast cells were also grown on the hydrogels and were shown to have survived on the surface for at least 48 hours with some evidence of proliferation. A simple staining and imaging method using Giemsa nuclear stain, PicoSirius red cellular collagen stain and upright inverted light microscopy was demonstrated. Optimisation of the hydrogels for the culture of THP-1 macrophage like cells was demonstrated and ruled out epifluorescence imaging for these materials due to excessive autofluorescence generated by the hydrogels. It was shown that it was possible to induce differentiation of the THP-1 cells using PMA and for the cells to adhere to the hydrogel surface, however an experimental attempt to investigate to what extent the hydrogel surfaces caused an activation of the macrophages was inconclusive. Using ELISA it was shown that the detection of very small levels of inflammatory cytokines was a possible. Alongside these investigations, a set of water swollen core-shell particles was synthesised from a protected monomer in water using emulsion polymerisation. These particles were shown to swell with water on the removal of a protecting acetonide group. The particles increased in size from around 100-300 nm to 5-6 µm. This water swelling is a key indication of hydrogel function. The core shell particles were shown to be able to adsorb protein molecules (lysozyme, albumin and fibrinogen) onto their surfaces and the change of surface charge, measured by zeta potential was shown. Higher adsorbed protein concentration had a more marked effect on the elevation of charge on the particles and the particles with a smaller shell diameter showed the largest change in zeta potential with adsorbed proteins. Total protein content adsorbed to the particles was measured using the BCA assay. The protein adsorption showed that these particles may have potential used in a biological context and could be investigated further in the area of drug or biomolecule uptake and release.