Multidirectional Control of Collagen Fiber Alignment for 3D Printing of Tissue

Collagen is a prevalent fibrous protein in the human body, and its anisotropic alignment influences tissues’ physical properties and cellular behavior. Traditional methods for aligning collagen fibers such as electrospinning, microfluidics, magnetic flow, and shear flow are limited to unidirectional alignment, failing to replicate the intricate alignment patterns of native fibrillar tissue. To overcome this, we developed an extrusion-based 3D printing strategy to achieve both parallel and perpendicular fiber alignment within different regions of the same print. We identified two dimensionless numbers: velocity ratio V* (printing speed to extrusion speed) and spreading ratio D* (printed filament diameter to needle diameter) that can be controlled through different printing parameters (flow rate, needle diameter). For V*<1, extrusion speed dominates, causing filament spreading (D*>1) and perpendicular fiber alignment. Conversely, when V*>1, printing speed prevails, leading to filament stretching with reduced spreading (D*≤1) and parallel fiber alignment. Corneal mesenchymal stromal cells seeded on the prints align with collagen fibers in both orientations. This alignment method is applicable to other fibrous biomaterials and can also be utilized for embedded 3D printing in a support bath. Overall, this work harnesses fluid mechanics to controllably align collagen fibers during 3D printing and guide cellular behavior.