Fibrillar Bundles as Fibrous Filler Materials for Attaining Cell Anisotropy in Bioprinting

Cellular alignment is essential for the function of anisotropic tissues such as skeletal muscle, tendon, cardiac, or neuronal tissues, where cell polarization governs mechanical integrity and signal transduction. However, engineering 3D tissue constructs with anisotropic extracellular microenvironments remains challenging, especially in larger constructs, which are commonly fabricated using extrusion-based bioprinting of cell-laden hydrogels, also known as bioinks. Here, a new class of bioprintable fibrous filler materials, fibrillar bundles, is presented that can be incorporated into bioinks and harness shear forces during extrusion bioprinting to achieve in situ alignment without the need for additional processing steps. These fibril bundles consist of multiple submicrometer fibrils fused into a larger bundle. They support robust cell adhesion and effectively promote polarization and alignment across multiple cell types. When incorporated into bioinks and printed with muscle cells, the fibrillar bundles enhance cellular alignment, and quantitative analysis confirms the directional growth of multinuclear myotubes and their morphological maturation. This approach offers a scalable and integrative solution for inducing anisotropy within 3D biofabricated tissues, holding promise for applications in muscle tissue engineering and beyond.