Type I collagen self-assembles into three-dimensional (3D) fibrous networks. These dynamic viscoelasticmaterials can be remodeled in response to mechanical and chemical signals to form anisotropicnetworks, the structure of which influences tissue development, homeostasis, and disease progression.Conventional approaches for fabricating anisotropic networks of type I collagen are often limitedto unidirectional fiber alignment over small areas. Here, we describe a new approach for engineeringcell-laden networks of aligned type I collagen fibers using 3D microextrusion printing of a collagenMatrigel ink. We demonstrate hierarchical control of 3D-printed collagen with the ability to spatiallypattern collagen fiber alignment and geometry. Our data suggest that collagen alignment results froma combination of molecular crowding in the ink and shear and extensional flows present during 3Dprinting. We demonstrate that human breast cancer cells cultured on 3D-printed collagen constructsorient along the direction of collagen fiber alignment. We also demonstrate the ability to simultaneouslybioprint epithelial cell clusters and control the alignment and geometry of collagen fibers surroundingcells in the bioink. The resulting cell-laden constructs consist of epithelial cell clusters fully embeddedin aligned networks of collagen fibers. Such 3D-printed constructs can be used for studies ofdevelopmental biology, tissue engineering, and regenerative medicine.
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