Integrated studies of the interactions of macrophages with synthetic hydrogels

Dr Stephen Rimmer (PI) Polymer and Biomaterials Chemistry Laboratories, Dept. of Chemistry, University of Sheffield, S3 7HF ()

Professor Eileen Ingham (CI) Institute of Molecular and Cellular Biology University of Leeds, LS2 9JT ()

Professor Jenny Southgate (CI) Department of Biology, University of York, YO10 5YW ()

Despite many years of work on the performance of polymer materials in biological systems there is still a scarcity of data from well designed experiments examining the materials variables in a systematic manner. This unsatisfactory situation results from observations that several interdependent variables often should be considered. For many materials this means synthesizing model materials and carrying out large numbers of well designed experiments. Also, until recently it was often difficult to fully characterise the required sets of polymers. However, new developments in polymer synthesis, characterisation and advances in tools for biological studies now make these powerful integrated studies much more accessible. An area in which a full and systematic study would be of enormous benefit in the design of new medical devices and scaffolds for in vivo tissue engineering is the formation of a framework to predict the response of macrophages in contact with materials. In the long term, materials that control macrophage phenotype could revolutionise the medical devices industry. In this programme, we will use a hydrogel platform, which we have previously shown to have low toxicity and does not illicit an immune response.

The project will be designed to address the effect of a wide variety of variables, which at various times have been only partially addressed by previous workers in the field. The disparate nature of the literature in this area has lead to a great deal of confusion and no knowledge of how the various materials variables are interconnected. Also, there is an absence of literature aimed at examining how materials structure and properties might affect the development of either an inflammatory (M1) or alternatively activated (M2) macrophage phenotype. The materials design strategy will produce sets of materials that address the variables, shown in table 1, using methacrylate hydrogels, produced by radical polymerisation. The manner in which these variables can be addressed using this hydrogel platform is shown in table 1.

Set number / Variable / Hydrogel parameter
1 / Swelling (water content) / Crosslink density and back bone hydrophilicity
2 / Water structure / Crosslink density and back bone hydrophilicity
3 / Porosity / Addition of post-polymerisation porogens
4 / Modulus (stiffness) / Crosslink density
5 / Roughness-1 / Addition of colloidal particles
7 / Roughness-2 / Phase separation
8 / Molecular architecture / Design of polymerizaton mixture
9 / Interfacial chemistry-1 / Addition of amines to epoxy monomers
10 / Interfacial chemistry-2 / Addition of acid comonomers
11 / Interfacial chemistry-3 / Addition of peptide-monomers

Swelling has long been recognised as a factor affecting macrophage activation, however, swelling is related to other factors, especially water structure, porosity and modulus, and nothing is known of the inter-relationship of these variables in terms of macrophage activation. In this project the student will carefully design sets of experiments to differentiate these factors and allow determination of the relative importance of each and then compare these results to materials parameters found in natural systems. Roughness is potentially an important parameter for macrophage activation. However, examination of this aspect is at a very early stage and there is no data on the effects of surface roughness combined with surface modulus, which are clearly factors which should be considered together. Two strategies could be used here: 1) the addition of polymer nanoparticles (produced by emulsion polymerisation), of defined size, to the polymerisation mixtures and 2) allowing the reaction mixtures to phase separate from the reaction mixtures during polymerisation. There are also early indications in the literature pointing to significant chemical structure effects in macrophage activation and this project will aim to systematically probe these effects using a reactive hydrogel platform. The literature appears to be surprisingly contradictory and only partial experiments appear to have been carried in this area and the aim will be to produce unambiguous data comparing key biochemical functionalties in respect of their effects on macrophages. In one set of materials the effects of polymer architecture will be investigated using synthetic techniques previously optimised at Sheffield.1,2 On the other hand chemical functionality can be investigated by using our previously published use of epoxides to add amine functionality3, copolymerisation with acid functional monomers4 or coupling of peptides to activated esters.5 Efforts will concentrate on Arg-Gly-Asp (RGD) based peptide functionalities, which are known to facilitate macrophage adhesion and have recently been implicated in macrophage activation. The effects of the various parameters on macrophage phenotype and gene expression will be determined by culture of the polymers with both murine peritoneal macrophages (screening) and human peripheral blood derived macrophages (validation). Macrophage phenotype will be determined by the expression of surface markers (eg. CD 80; CD163) using immunofluorescence and cytokine production by ELISA. Gene expression for markers of inflammatory (TNF-; IL-1 etc) and alternatively activated macrophages (eg. arginase) will be determined by RT PCR and RTqPCR.

References

1) "Synthesis and properties of amphiphilic networks 3: Preparation and characterization of block conetworks of poly(butyl methacrylate-block-(2,3 propandiol-1-methacrylate-stat-ethandiol dimethacrylate))" S. Rimmer, M.J. German, J. Maughan, Y. Sun, N. Fullwood, J. Ebdon, S. MacNeil, Biomaterials, 26 2219 (2005)

2) “Culture of dermal fibroblasts and protein adsorption on block conetworks of poly(butyl methacrylate-block-(2,3 propandiol-1-methacrylate-stat-ethandiol dimethacrylate))” Y. Sun , J. Collett , N.J. Fullwood , S. Mac Neil, S. Rimmer, Biomaterials 28 661 (2007)

3) “Epithelialization of hydrogels achieved by amine surface modification and co-culture with stromal cells” S. Rimmer, C. Johnson, B. Zhao, J. Collier, L. Gilmore, S. Sabnis, P. Wyman, C. Sammon, N.J. Fullwood, S. MacNeil Biomaterials, 28 5319 (2007)

4) “Polymethacrylate networks as substrates for cell culture” Y Sun, J. Maughan, R. Haigh, S.A. Hopkins, P. Wyman, C. Johnson, N.J. Fullwood, J. Ebdon, S. MacNeil, S. Rimmer Macromol Symp, 256 137 (2007)

5) “Highly branched Poly-(N-isopropylacrylamide)s with Arginine-Glycine-Aspartic acid (RGD) or COOH chain ends that form sub-micron stimulus responsive particles above the critical solution temperature” S. Rimmer, S. Carter, R. Rutkaite, J. W.Haycock, L. Swanson Soft Matter, 3 971 (2007)