Multiphoton based biofabrication of 3D protein micro-structures and micro-patterns : voxel and cell matrix niche studies

Two-photon excitation photochemical crosslinking is an efficient and rapid approach to freely fabricate 2D/3D protein structures with resolution up to nanoscale. In this work, naturally occurring protein bovine serum albumin (BSA) was photochemically crosslinked using infrared laser in the presence of a photosensitizer rose Bengal to form micropillar arrays with submicron structures. Excellent 3D resolution in defining the shape of micro-pillar structures, either pyramidal or conical, was achieved by precisely controlling various scanning parameters and solution concentrations. The morphology of the protein structures fabricated was evaluated by multi-photon confocal microscopy and scanning electron microscopy. Protein voxels including lines, spots, and micropillars are fabricated. Laser power, exposure time, z-position, protein and photosensitizer concentrations, but not scanning speed, are important controlling parameters. A lateral fabrication resolution of ≈200nm is demonstrated in 2D line voxels. 3D spot voxels are ellipsoids with 400nm lateral and 1.5 μm axial dimensions. Fibroblasts survived and expressed cell matrix adhesion molecules such as integrin alpha v and paxillin on micropillar arrays without any other protein coating. Moreover, they exhibited physiological functions by expressing extracellular matrix (ECM) such as collagen and fibronectin. They showed a more “3D” morphology comparing with that in 2D monolayer cultures and exhibited physiological functions such as matrix deposition. Multiple extracellular matrices such as fibronectin and laminin can be included during the crosslinking fabrication process so as to further functionalize the BSA microstructures and micropatterns with bioactive signals and thus engineer complex cellular niches or microenvironments. hMSCs seeded on protein micropatterns with different topographical features, ECM components or stiffness differentially expressed 3D matrix adhesions, which were identified by triple-colocalization of paxillin, fibronecin and integrin ...