Engineering plasma-polymerised surfaces for synergistic integrin/growth factor signalling to promote mesenchymal stem cell adhesion and differentiation

摘要

Introduction: The effective presentation of proteins and growth factors (GFs) is a requirement in synthetic materials for tissue engineering. Fibronectin (FN), an extracellular matrix protein that regulates cell adhesion, binds GFs via specific structural domains, exhibiting synergistic effects on cells. Polymers have the ability to facilitate conformational changes in protein structure, in turn altering protein-GF interactions. Here we used an inductively coupled plasma system to modify 2D surfaces with thin coatings of poly(ethyl acrylate) (PEA), a polymer known to induce FN organisation into nanonetworks by exposing its integrin and GF-binding domains. The efficiency of plasma polymerisation as well as FN adsorption and interaction with bone morphogenetic protein-2 (BMP-2) on PEA-coated surfaces were evaluated. Human mesenchymal stem cell (hMSC) adhesion and differentiation were assessed at the protein-GF interface to determine the overall efficiency of the system. Materials and Methods: Plasma polymerisation: A custom-built inductively coupled plasma chamber was used to deposit EA onto glass coverslips via plasma polymerisation. Spin-coated PEA surfaces were used as controls. Surface characterisation: Surfaces coated with plasma-polymerised PEA (pPEA), FN, and BMP-2 were characterised by XPS, WCA, AFM, immunogold staining, and ELISA to verify elemental composition, wettability, protein adsorption, and FN/BMP-2 binding. Cell culture: hMSCs were seeded on PEA-modified surfaces coated with FN and/or BMP-2 for up to 4 weeks to study adhesion, integrin/GF receptor co-localisation, and differentiation. Results and Discussion: The successful coating of pPEA was confirmed by XPS, where carbon moieties corresponding to those in PEA were observed. Increasing water contact angles on pPEA-coated surfaces indicated changes in surface chemistry. AFM revealed differences in the conformation of FN adsorbed on thin pPEA coatings compared with that on spin-coated PEA. BMP-2 localisation was visualised by immunogold labelling, and ELISA for BMP-2 showed comparable FN/BMP-2 binding between surfaces modified with spin-coated PEA and pPEA, an evidence of efficient protein/GF presentation. hMSC adhesion and spreading were enhanced on surfaces coated with pPEA and FN compared with controls. Integrin/GF receptor co-localisation was observed, suggesting that changes in FN conformation on pPEA led to increased interactions with BMP-2 with the potential to promote differentiation. Conclusions: Plasma polymerisation results in thin PEA coatings on 2D surfaces, which enables the effective presentation of FN and low doses of GFs for synergistic interactions. Consequently, hMSC fate can be directed by such surfaces. The originality of this study lies in expanding the utility of PEA and similar polymers as unique materials that trigger FN organisation into nanonetworks. These materials have the potential to be applied as coatings in biodegradable systems.