Improving the biocompatibility of photo-crosslinkable poly(2-oxazoline)-based hydrogels for stem cell research

Poly(2-oxazoline) (POx)-based hydrogels were developed as a biocompatible, tunable alternative to poly(ethylene glycol) (PEG)-based materials for cell-instructive biomaterials. Using cationic ring-opening polymerization, photo-crosslinkable POx macromonomers were synthesized and processed into hydrogels with adjustable cross-linking density, stiffness and swelling behavior. The intrinsic “stealth” character of poly(2-ethyl-2-oxazoline) (PEtOx) was systematically modified through incorporation of monomers bearing methyl ester functionalities (MestOx) to enable the subsequent covalent coupling of integrin-binding motifs like the RGD-peptide sequence to the formed hydrogels, transforming the inert matrix into a bioactive scaffold. The RGD motif describes the core-sequence of three-amino acids with an Arginine-Glycine-Aspartic acid sequence, found in many ECM proteins like fibronectin, vitronectin or fibrinogen. Human mesenchymal stem cells (huMSCs) cultured on these RGD-functionalized POx-based hydrogels exhibited enhanced adhesion, spreading, proliferation and osteogenic differentiation compared with non-functionalized controls. Increasing RGD content correlated positively with focal adhesion formation and alkaline phosphatase activity, while softer hydrogel formulations further supported cell proliferation. These findings demonstrate that RGD-modified POx-based hydrogels provide a versatile, cytocompatible and tunable platform for the engineering of defined microenvironments in stem cell research and regenerative medicine.