Polymer micropatterning induces spatially organised fibronectin nanonetworks for efficient integrin/growth factor interaction

Abstract

Event Abstract Back to Event Polymer micropatterning induces spatially organised fibronectin nanonetworks for efficient integrin/growth factor interaction Annie Zhe Cheng1, 2, Nikolaj Gadegaard1, Matthew Dalby2* and Manuel Salmeron-Sanchez1* 1 University of Glasgow, Biomedical Engineering, United Kingdom 2 University of Glasgow, Centre for Cell Engineering, United Kingdom Introduction: The study of material-protein interactions is important in the development of biomaterials for tissue engineering. We previously showed that poly(ethyl acrylate) (PEA) induces the organisation of fibronectin (FN) into nanonetworks in a process known as material-driven fibrillogenesis[1] and exposes specific structural domains of FN for growth factor (GF) binding[2]. In addition, the incorporation of microscale features is interesting in biomaterials as it allows the mimicking of in vivo systems, whereby cells thrive in a microscale environment[3]. Here we use a simple photolithographic procedure to fabricate micropatterns of PEA, allowing material-driven fibrillogenesis of FN to occur in microscale regions. The synergistic relationship between FN nanonetworks and growth factors is also investigated with respect to mesenchymal stem cell (MSC) response, in particular adhesion, migration, and differentiation. Materials and Methods: Photolithography (PL): PEA is spin-coated onto glass substrates, which are subsequently coated with photoresist and patterned with a photomask via exposure to UV light. Oxygen plasma is used to etch PEA and expose glass for PEG-silane grafting, yielding a non-fouling background. Residual photoresist is removed to expose the underlying PEA for protein coating. Surface characterisation: Micropatterned surfaces coated with FN and BMP-2 on PEA are characterised by SEM, AFM, immunogold staining, and immunofluorescence to verify successfu protein patterning on the substrates. Cell culture: MSCs are seeded on micropatterned, PEA-modified surfaces coated with FN and/or BMP-2 for up to 4 weeks to study adhesion, migration, and differentiation. Results and Discussion: PEA is patterned on 100 μm × 100 μm square islands using PL. FN is coated onto these patterns and immunostaining of FN is carried out to visualise the resulting FN microdomains. The interaction between FN and PEA is confirmed using AFM, which reveals FN nanonetworks on the microdomains. Additionally, BMP-2 localisation is visualised by immunogold labelling. When MSCs are cultured on micropatterned, protein-modified surfaces, they selectively adhere to the FN microdomains while avoiding the non-fouling background. The osteogenic potential of these micropatterned surfaces is investigated. With the incorporation of BMP-2 within the FN nanonetworks, MSC differentiation into osteoblasts is observed on the microscale islands, indicating the ability to spatially control MSC differentiation using bioactive cues including chemical interfaces and GF signalling. Conclusions: We demonstrate a simple method of patterning microdomains of PEA using PL and subsequently coating the microdomains with FN and BMP-2 to facilitate synergistic interactions and influence MSC fate. This approach can be utilized with different shapes, dimensions, and order, and can also be downsized to yield much smaller feature sizes, enabling the precise design of bioactive surfaces to promote osteogenesis in MSCs. Medical Research Council; James Watt Nanofabrication Centre