An innovative 4D printing approach for fabrication of anisotropic collagen scaffolds.

Collagen anisotropy is known to provide the essential topographical cues to guide tissue-specific cell function. Recent work has shown that extrusion-based printing using collagenous inks yield 3D scaffolds with high geometric precision and print fidelity. However, these scaffolds lack collagen anisotropy. In this study, extrusion-based 3D printing was combined with a magnetic alignment approach in an innovative 4D printing scheme to generate 3D collagen scaffolds with high degree of collagen anisotropy. Specifically, the 4D printing process parameters-collagen (Col):xanthan gum (XG) ratio (Col:XG; 1:1, 4:1, 9:1 v/v), streptavidin-coated magnetic particle concentration (SMP; 0, 0.2, 0.4 mg ml^-1), and print flow speed (2, 3 mm s^-1)-were modulated and the effects of these parameters on rheological properties, print fidelity, and collagen alignment were assessed. Further, the effects of collagen anisotropy on human mesenchymal stem cell (hMSC) morphology, orientation, metabolic activity, and ligamentous differentiation were investigated. Results showed that increasing the XG composition (Col:XG 1:1) enhanced ink viscosity and yielded scaffolds with good print fidelity but poor collagen alignment. On the other hand, use of inks with lower XG composition (Col:XG 4:1 and 9:1) together with 0.4 mg ml^-1SMP concentration yielded scaffolds with high degree of collagen alignment albeit with suboptimal print fidelity. Modulating the print flow speed conditions (2 mm s^-1) with 4:1 Col:XG inks and 0.4 mg ml^-1SMP resulted in improved print fidelity of the collagen scaffolds while retaining high level of collagen anisotropy. Cell studies revealed hMSCs orient uniformly on aligned collagen scaffolds. More importantly, collagen anisotropy was found to trigger tendon or ligament-like differentiation of hMSCs. Together, these results suggest that 4D printing is a viable strategy to generate anisotropic collagen scaffolds with significant potential for use in tendon and ligament tissue engineering applications.