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

Abstract

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.

High‐Resolution Patterning of Hydrogels in Three Dimensions using Direct‐Write Photofabrication for Cell Guidance

“…Although the studies reported here focus on the use of BSA-based microstructures modified via avidin-biotin interactions, the Shear group and others have also successfully employed MPE techniques to crosslink other bioactive proteins, including various enzymes, while retaining substantial activity. [14][15][21][22][23][24] Additionally, the Shear group has previously demonstrated the feasibility for creating metalized, conductive protein-based microstructures, [23] materials potentially useful in applications such as nerve regeneration, where a combination of electrical stimulation, chemical gradients, and physical guidance may be required. [18] …”

“…[20] By scanning the laser focus relative to reagent solution, it is possible to create structures with arbitrary 3D morphology and low-to submicrometer features. [14][15][21][22][23][24][25][26][27] After fabrication, protein guidance paths are decorated with laminin-derived peptides, using avidin-biotin linkages, to promote cell adhesion and migration.…”

Two‐Photon Polymerization of Biocompatible Photopolymers for Microstructured 3D Biointerfaces

“…ECM proteins (like fibrinogen, fibronectin, collagen) as biocompatible scaffold materials were microstructured via singlet oxygen crosslinking using sensitizing dyes [70] even inside living cells. [71,72] Catalytically active microstructures have been made by 2PP via Rose Bengale crosslinking using enzymes as building material. [73,74] One of the most serious drawbacks of the 2PP technology is the inherent serial, voxel-by-voxel character of the fabrication process resulting in extended processing times.…”

A thermoresponsive, microtextured substrate for cell sheet engineering with defined structural organization

“…Micropatterning has been widely used as a method of controlling cell morphology, spatial arrangement, and function [27,28] by imparting various types of cell-scale guidance cues by controlling regions of adhesion [25,[29][30][31], topology [32][33][34][35][36][37], and "direct writing" of scaffolds and cells [38][39][40]. A common method used in many of these approaches involves a set of techniques known as soft lithography [41].…”