Cartilage tissue engineering by extrusion bioprinting utilizing porous hyaluronic acid microgel bioinks

3D bioprinting offers an excellent opportunity to provide tissue-engineered cartilage to microtia patients. However, hydrogel-based bioinks are hindered by their dense and cell-restrictive environment, impairing tissue development and ultimately leading to mechanical failure of large scaffoldsin vivo. Granular hydrogels, made of annealed microgels, offer a superior alternative to conventional bioinks, with their improved porosity and modularity. We have evaluated the ability of enzymatically crosslinked hyaluronic acid (HA) microgel bioinks to form mature cartilagein vivo. Microgel bioinks were formed by mechanically sizing bulk HA-tyramine hydrogels through meshes with aperture diameters of 40, 100 or 500µm. Annealing of the microgels was achieved by crosslinking residual tyramines. Secondary crosslinked scaffolds were stable in solution and showed tunable porosity from 9% to 21%. Bioinks showed excellent rheological properties and were used to print different objects. Printing precision was found to be directly correlated to microgel size. As a proof of concept, freeform reversible embedding of suspended hydrogels printing with gelation triggered directly in the bath was performed to demonstrate the versatility of the method. The granular hydrogels support the homogeneous development of mature cartilage-like tissuesin vitrowith mechanical stiffening up to 200 kPa after 63 d. After 6 weeks ofin vivoimplantation, small-diameter microgels formed stable constructs with low immunogenicity and continuous tissue maturation. Conversely, increasing the microgel size resulted in increased inflammatory response, with limited stabilityin vivo. This study reports the development of new microgel bioinks for cartilage tissue biofabrication and offers insights into the foreign body reaction towards porous scaffolds implantation.