Dual-orientation of collagen fibers to guide cell alignment in 3D-printed constructs

Natural tissue comprises fibrous proteins with complex fiber alignment patterns. Here, we develop a reproducible method to fabricate biomimetic scaffolds with patterned fiber alignment along two independent orientations. While extrusion-based approaches are commonly used to align fibrous polymers in a single orientation parallel to the direction of flow, we hypothesized that extrusion-based 3D printing could be utilized to achieve more complex patterns of fiber alignment. Specifically, we show that control of lateral spreading of a printed filament can induce fiber alignment that is either parallel or perpendicular to the flow direction. Theoretical prediction of the printing parameters that control fiber orientation was experimentally validated using a collagen biomaterial ink. The velocity ratio of the printhead movement relative to the ink extrusion rate was found to dictate collagen fiber alignment, allowing for the informed fabrication of collagen scaffolds with prescribed patterns of fiber alignment. For example, controlled variation of the ink extrusion rate during a single print resulted in scaffolds with specified regions of both parallel and perpendicular collagen fiber alignment. Human corneal mesenchymal stromal cells seeded onto the printed scaffolds adopted a spread morphology that aligned with the underlying collagen fiber patterns. This technique worked well for filaments either printed onto a printbed in air or extruded within a support bath using embedded 3D printing, enabling the fabrication of 3D structures with aligned collagen fibers. Taken together, this work demonstrates a theoretical and experimental framework to achieve the reproducible fabrication of 3D printed structures with controlled collagen fiber patterns that guide cellular alignment. Statement of Significance Natural tissues contain collagen fibers aligned in multiple directions, which are essential for guiding cell behavior; however, most existing fabrication methods can achieve only unidirectional fiber alignment. Here, we introduce an extrusion-based 3D printing strategy that enables precise control over collagen fiber orientation in both parallel and perpendicular directions. This allows multidirectional collagen fiber alignment patterned spatially within a single construct, thereby guiding corneal mesenchymal stromal cells to align multidirectionally. This approach works when printing in air or with embedded printing in a support bath. Thus, this strategy can enable the fabrication of complex 3D scaffolds that mimic the anisotropic architecture of native tissues.