Glycan‐Mediated Grafting of Extracellular Matrix Proteins for Stable Biofunctionalization of Polystyrene Surfaces

ABSTRACT Adherent cells are highly sensitive to the physical and biochemical properties of their microenvironment, particularly the extracellular matrix (ECM), which regulates cell adhesion, signaling, and overall behavior. Cells also actively modify and remodel the ECM, creating a continuous interaction comparable to a dialogue. Consequently, artificial cell microenvironments are used to influence adherent cell behavior. However, these environments must be carefully modulated to enhance communication with cells. To this end, bio‐functionalization of cell culture substrates has been developed to improve interactions between adherent cells and their microenvironment. To optimize cell–biomaterial surface interactions, various protein grafting techniques can be employed, including random grafting via amine groups, semi‐oriented grafting via thiol groups, and glycosylation‐based grafting. This study specifically focuses on the glycosylation‐based grafting method, which creates a natural spacer between the substrate and the immobilized protein. We introduce a novel glycan‐based surface functionalization approach using two ECM adhesion proteins commonly used in biomaterials: fibronectin (Fn), a fibrillar protein with low glycosylation (5% w/w), and vitronectin (Vn), a globular protein with high glycosylation (30% w/w). Both proteins are highly purified from human blood plasma to preserve their native state and bioactivity. We analyzed the effects of glycan‐based grafting on the conformation and bioactivity of these proteins. Given their essential roles in ECM, human pre‐osteoblastic STRO‐1⁺A cells are cultured on the bio‐functionalized surfaces, and their early‐stage behavior is compared for both Fn and Vn. Our results demonstrate that glycosylation‐based grafting significantly influences the conformation and bioactivity of Fn and Vn. Cell adhesion, viability, and morphology are assessed, revealing that this grafting method enhances cell–material interactions, making it a promising strategy for improving the performance of biomaterials in biomedical applications.