Optically Micro-fabricated Linear and Freeform 3-D Extracellular Matrix Scaffolds for Tissue Engineering

This work was aimed at advancing multi-photon excited, freeform fabrication technology with nano-scale and sub-micron precision as an enabler for tissue engineers to investigate cellular response to a biomimetic, bio-active extracellular matrix. We demonstrated that sub-micron and micron scale Collagen and Fibronectin structures can be fabricated via multi-photon excited photochemistry using a modified Benzophenone dimer and Rose Bengal while maintaining the biomimetic ECM structures’ bioactivity. We confirmed that three-photon excitation produces significantly smaller features at comparable excitation wavelengths as a consideration to better approach focal adhesion size. Bioactivity of MPE cross-linked FN and Collagens I and II was established via immunofluorescence and fibroblast adhesion. Additionally, the relative rates of degradation in these cross-linked matrices are consistent with the known activities of these enzymes. Morphology measurements of fibroblasts grown on these proteins include log(Area), Perimeter, Area/Perimeter2 were considered as proxies for cell response. Fibroblast perimeters are statistically different when associated with the Collagen I microenvironment. Among fibroblasts grown on MPE structures of Collagen I, Fibronectin, BSA and the BSA Monolayer, the stress fiber distributions on Collagen I (all fiber lengths) are highly significantly different (p \u3c 1x10-4) than the distribution of stress fibers of cells on BSA Lines. This suggests contact guidance only for cells on BSA Lines but yet a combination of contact guidance and chemical signaling (RGD) with cells on Collagen I Lines. This supports additional overall orientation findings based on fibroblasts’ fitted ellipse major axis direction for Collagens I, II and Fibronectin. Stress fiber distribution on BSA Monolayer differed significantly from those on BSA structures (p = 0.01). This underscores the effects of pure contact guidance alone provided by the BSA fibers compared to the combined contact guidance and ECM cues provided by the FN, and collagen structures. A method similar to rapid prototyping or three-dimensional printing was accomplished to resolve cellular response at the submicron level by fabricating biomimetic, bioactive extracellular matrices in a freeform three-dimensional (3D) manner. To the best of our knowledge, simultaneous 3D spatial and chemical control of collagen scaffold synthesis at the micrometer and sub-micrometer size scales has not been fully demonstrated