Multiphoton excited fabricated nano and micro patterned extracellular matrix proteins direct cellular morphology.

We use multiphoton excited (MPE) photochemistry to fabricate patterned extracellular matrices (ECM) and to investigate the morphology of human dermal fibroblasts adhered to the resulting photocrosslinked linear structures of fibronectin (FN), fibrinogen (FG), and bovine serum albumin (BSA). These proteins were chosen to systematically investigate the roles of topography and ECM biochemistry on cell spreading, as fibroblasts bind directly to both FN and FG at RGD sites through known integrins, whereas BSA provides no comparable ECM cues for cell binding. MPE crosslinked patterns are created from parallel linear structures 600 nm in width, 200 microm in length, and spaced by either 10 or 40 microm. Immunofluorescence staining of FN and FG was used to assay the functionality of crosslinked proteins. The metrics of orientation, elongation, and cell perimeter were used to quantitate the resulting cellular behavior on the crosslinked protein patterns. These parameters all reflect statistical differences for cells on BSA, relative to the similar statistical behavior on fibronectin and fibrinogen. Cells on the BSA patterns are constrained by physical guidance and orientation between linear structures. In contrast, cells adhered on both FN and FG had a greater propensity to spread across adjacent structures, indicating the importance of cell matrix interactions. Focal adhesion staining of cells adhered to the protein structures revealed similar trends. These findings are consistent with our hypothesis that these crosslinked matrix protein structures are expected to direct cell adhesion and spreading and that the topography and ECM cues lead to different forms of guidance.