Converging laser-induced forward transfer and melt electrowriting for biofabrication of reinforced cartilage constructs

Microtissue-based strategies have gained significant attention for the fabrication of cartilage grafts. Their spatial organization within three-dimensional constructs plays a crucial role in directing tissue formation and maintaining the immediate mechanical stability of the printed structure. Melt-electrowritten (MEW) fibrous scaffolds have been widely used to reinforce cell-laden hydrogels, while also guiding microtissue fusion and self-organization within the constructs. However, current bioprinting methods used for positioning microtissues or spheroids within these structures are limited by insufficient control over spheroid deposition, low throughput, and technical challenges, such as nozzle-clogging. In this study, we leveraged laser-induced forward transfer (LIFT) to print articular cartilage progenitor cell (ACPC) spheroids of two different sizes (Ø ∼80 and 150μm) into fibrous polycaprolactone scaffolds. We investigated the effects of laser energy, spheroid size, and concentration in the bioink to identify the key parameters for controlled deposition. Furthermore, we assessed print fidelity, post-print spheroid viability, and chondrogenic differentiation capacity. The deposition rate of the spheroids was studied to maximize transfer efficiency, and the resulting optimal parameters were subsequently applied to place the spheroids within the MEW meshes. However, the spheroid transfer efficiency remained limited, not due to shortcomings in the printing process, but because uniform encapsulation becomes challenging when working with discrete and larger entities, such as spheroids. While single cells benefit from homogeneous suspension, enabling random encapsulation, spheroids require precise targeting to be successfully transferred. To address this challenge, an AI-based imaging analysis system was employed, and the amount of bioink on the donor slide was reduced to improve the transfer of larger spheroids further. Here, we demonstrate for the first time the successful convergence between LIFT and MEW for the deposition of ACPC spheroids into reinforcing meshes as the next step towards automated production of tissue constructs.