Multiscale Anisotropic Tissue Biofabrication via Bulk Acoustic Patterning of Cells and Functional Additives in Hybrid Bioinks

Recapitulation of the microstructural organization of cellular and extracellular components found in natural tissues is an important but challenging feat for tissue engineering, which demands innovation across both process and material fronts. In this work, a highly versatile ultrasound-assisted biofabrication (UAB) approach is demonstrated that utilizes radiation forces generated by superimposing ultrasonic bulk acoustic waves to rapidly organize arrays of cells and other biomaterial additives within single and multilayered hydrogel constructs. UAB is used in conjunction with a novel hybrid bioink system, comprising of cartilage-forming cells (human adipose-derived stem cells or chondrocytes) and additives to promote cell adhesion (collagen microaggregates or polycaprolactone microfibers) encapsulated within gelatin methacryloyl (GelMA) hydrogels, to fabricate cartilaginous tissue constructs featuring bulk anisotropy. The hybrid matrices fabricated under the appropriate synergistic thermo-reversible and photocrosslinking conditions demonstrate enhanced mechanical stiffness, stretchability, strength, construct shape fidelity and aligned encapsulated cell morphology and collagen II secretion in long-term culture. Hybridization of UAB is also shown with extrusion and stereolithography printing to fabricate constructs featuring 3D perfusable channels for vasculature combined with a crisscross or circumferential organization of cells and adhesive bioadditives, which is relevant for further translation of UAB toward complex physiological-scale biomimetic tissue fabrication.