Controlling cell adhesion to polymer gels

Learn about our recent advances aimed at converting non-cell adhesve hydrogels into cell adhesive substrates for the support of cells in Regenerative Medicine/Tissue Engineering and as new tools for Synthetic Biology:

Hydrogels are perhaps the closest materials to the extracellular matrix (ECM) but they are unusually non-adhesive to human cells. Whilst the reasons for this lack of adhesion are not well understood we consider that the many causes of this lack of adhesion are the diffuse nature of the swollen interface in water, the high mobility of the polymer chains and the changes in ordering of water in this diffuse interface.

Despite many attempts there appears to be very little correlation between surface energy and cell adhesion to hydrogels. Some increase in cell adhesion can be achieved by adding charge but in our experience with hydrogels these effects are limited.

The most effective methods are:

  1. Addition of cell adhesive peptides
  2. Using block conetwork architecture
  3. The addition of alkyl amine functionality; lysine-like strategy

1: Cell adhesive peptides

sulphonate_protected_guanidineThe use of polymer functionalised with peptides that bind to cell surface receptos, integrins, is now outine in tissue engineering1. The RGD sequence is most commonly used, but we realised that protection of the arginine residue during polymerisation would offer some advantages: the presence of arginine restricts solvent choice, at higher pH there are several side reactions and arginine would severely limit the use of controlled radical polymerisations sucn as ATRP and RAFT. The solution is to protect the guanidine group with an aryl sulphonate then to remove the protecting group post-polymerisation with glutathione-S-transferase: a mild strategy that does not degrade (meth)acrylate and other esters2.

Above right: A bromosulphonate protected cell-adhesion promoting monomer.

fibroblasts_on_gel

Above: human dermal fibroblasts attached to a poly(glycerol monomethacrylate) hydrogel functionalised with GRGDS monomer after deprotection.

Block conetworks

conetwork_gelBlock conetworks can be produced in which one block is hydrophobic. Manipulation of the polymer composition, block length, cross-link density and phase morphology can be used to produce excellent substrates for cell adhesion that out-perform tissue culture plastic for many cells. Polour materials are produced by allowing the polymer to precipitate from the reaction mixture during polymerisation. Although much remains to be explained on th ebehaviour of these materials it is clear that optimisation of the formulations can be a powerful route for controlling cell adhesion – several series of materials are available with similar chemical structure but very different behaviour as cell culture substrates3-5. Several of the materials can be formulated as cell adhesive coatings-offering a significant and cost effective alternative to conventional treatments.

Above right: a schematic of an amphiphilic conetwork. Blue is hydrophilic, black hydrophobic

cells_on_conetworks

Above: Scanning electron micrographs showing human fibroblasts on conventional hydrogel (left) and on a porous conetwork (right)

 

gel_structure_developmentAbove: Nano and meso structure development

Alkyl amines

alkyl_aminated_hydrogelThe reactions of the alkyl chain of lysine in wound healing are essential for proper repair of tissue. These reactions are mediated by a variety of enzymes and include key collagen cross-linking reactions between lysine and glutamate (catalysed by transglutamasees) and modification of lysine to alysine (catalysed by lysyl oxidase) prior to formation of Shift’s base-like cross links. Our recent observations showed that the addition of monomer units that mimic lysine (i.e. monomers containing a primary amine attached to a 3-6 carbon alkyl chain) produce enormous changes in the ability of hydrogels to support cells6,7. Out current hypothesis is that the effect is derived from enzymatic modification of the lysine-like groups and covalent atachment of the extracellular proteins that mediate cell adhesion. In our most recent work we show that non-cell adhesive conetworks can be modified in this way and we show that macrophages are not activated by these treatments.

Above right: A typical segment of an alkyl aminated poly(glycerol monomethacrylate) hydrogel.

cells_on_aminated_hydrogel

Above: A549 epithelial cells attached to an alkyl aminates conetwork: poly(glycerol methacrylate-l-butylmetharylate) modified with a hexyl amine moiety.

Read more about these advances here

  1. Production and performance of biomaterials containing RGD-peptides” L. Perlin, S. MacNeil, S. Rimmer, Soft Matter, 4, 2331 (2008)
  2. Cell adhesive hydrogels synthesized by copolymerization of arg-protected Gly-Arg-Gly-Asp-Ser methacrylate monomers and enzymatic deprotection” L. Perlin, S. MacNeil, S.Rimmer Chem Comm 5951 (2008)
  3. 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)
  4. 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)
  5. Synthesis and properties of amphiphilic networks 2: A Differential scanning calorimetric study of poly(dodecyl methacrylate-stat-2,3 propandiol-1-methacrylate-stat-ethandiol dimethacrylate) networks and adhesion and spreading of dermal fibroblasts on these materials” R. Haigh, N. Fullwood, S. Rimmer, Biomaterials 23 3509 (2002)
  6. 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)
  7. Cytocompatibility of poly(1,2 propandiol methacrylate) copolymer hydrogels and conetworks with or without alkyl amine functionality” S. Rimmer, Stacy-Paul Wilshaw, P. Pickavance, E. Ingham Biomaterials 30 2468 (2009)
  8. Arginine-Glycine-Aspartic acid Functional Branched Semi-Interpenetrating Hydrogels“, R. Plenderleith, C J Pateman, C Rodenburg, John Haycock, Frederik Claeyssens, Chris Sammon and Stephen Rimmer, Soft Matter, 2015, Accepted Manuscript