Development of embedded bioprinting for fabricating zonally stratified articular cartilage

Embedded bioprinting enables direct deposition of bioinks in three dimensions inside a support bath with shear-thinning and self-healing capabilities, and it has been used to fabricate complex tissues and organs for several biomedical applications. In this study, a support bath comprising gelatin/alginate microparticles and oxidized alginate solution was developed and crosslinked in situ with carbonyl hydrazide-modified gelatin bioink via the Schiff base reaction. The numerical model of embedded printing was established to analyze the extrusion process and disturbance of the support bath. The process window (e.g., extrusion pressure, nozzle moving speed, nozzle size, and support bath composition) was established experimentally to ensure stable fiber formation. In addition, the compressive modulus of the printed construct has been reinforced due to the formation of interpenetrating polymer networks in the microparticles. Based on the process investigation, a zonally stratified artificial cartilage with a three-layered structure was designed: vertically printed fibers in the bottom, oblique fibers in the middle, and horizontally printed fibers in the superficial layer. The bioprinted cartilage supported cell survival, proliferation, and spreading, with the observed deposition of cartilage-specific proteins, offering a new strategy for developing tissue-engineered cartilage constructs with biological and histological relevance.