Introduction: Cells respond to their environment, including biological signals (such as extracellular matrix proteins and growth factors) and other features such as topography and mechanical properties. Because of this, interpreting cell responses to different environments can be challenging, particularly if the signals within these environments are not well controlled. Electrospun fibre matrices have long been investigated as 3D cell culture materials, however non-specific protein adsorption to these materials is often a confounding factor when analysing the resulting cell behaviour. Interpreting cell responses to these materials is thus difficult due to the interconnectedness of different intracellular signalling pathways; in order to better understand the signals being sent to cells, “noise” from adsorbed proteins must be suppressed while still allowing the attachment of various signalling molecules of interest.
Materials & Methods: Variants of polystyrene (PS) and polycaprolactone (PCL) that bore initiating sites for atom transfer radical polymerisation (ATRP) were polymerised via RAFT and ring opening polymerisations. These polymers were electrospun and then grafted with polymer brushes via aqueous surface-initiated-ATRP, creating a bottlebrush polymer coating. A trimethylsilyl-protected alkyne-PEG monomer was synthesised and incorporated into the brush. This was then deprotected and various azide-functional molecules were then attached via a copper-mediated click reaction, for example cRGDfK(azide), an integrin-specific cyclic RGD. Mouse L929 fibroblasts and human primary mesenchymal stem cells were cultured on the grafted fibres for 24 hours in both the presence and absence of serum.
Adapted with permission from Rodda et al[1]. Copyright 2015, American Chemical Society.
Results: The grafting of fibres significantly reduced the adsorption of serum proteins to their surface, while also allowing covalent attachment of specific molecules via the alkyne functional group. In the presence of covalently clicked cell-adhesive peptides, cells adhered and spread, while in the absence of these peptides, cells were rounded and/or formed aggregates on the grafted fibres due to cell-cell interactions becoming dominant. This was the case even in serum-containing media where soluble proteins would typically adsorb onto the fibres and induce spreading via pathways other than those being targeted.
Discussion: Blocking experiments showed that cell-fibre adhesion could be controlled such that it occurred through a single type of integrin receptor. Similar results were seen on both PS and PCL-based materials, demonstrating that the modular design of the materials allows surface properties of these fibres to be changed independently of the bulk material. Further studies will examine the roles of different adhesion receptors in cellular development.
Conclusion: We have developed a family of electrospun fibre scaffolds that provide improved control over surface properties that are relevant to cells. In this case, these allowed receptor-specific adhesion of cells in complex solutions, where non-specific adhesion would typically be significant.
References:
[1] Rodda et al, Biomacromolecules, 2015, 16(7), pp 2109-18