Microtissue-Based Bioink as A Chondrocyte Micro-Shelter for DLP Bioprinting

Background: Bioprinting specific tissues with robust viability is a grand challenge, requiring a delicate balance between a densely cellular distribution and hydrogel network crosslinking density. Microtissues composed of tissue-specific mesenchymal stem cells and extracellular matrix (ECM) particles provide an alternative scheme for realizing biomimetic cell density and microenvironment. Nevertheless, due to their instability during manufacturing, scarce efforts have been made to date to assemble them using rapid prototyping methods. Here, we introduce a novel microtissue bioink with well printability and cellular viability maintenance for digital light processing (DLP) bioprinting. Generally, the microtissue bioink was prepared by crosslinking acellular matrix microparticles and methacrylated gelatin (GelMA) hydrogel with a specific proportion. Our microtissue composite bioink for DLP printing not only enables accurate assembly of organ building blocks but also provides a three-dimensional shelter to ensure printed cells' viability. Methods We first determined chondrocyte and stem cell characteristics of microtia chondrocytes extracted from abandoned microtia-tissue samples. Cartilage acellular matrix (CAM) was decellularized from porcine ear cartilage tissue, then lyophilized, quickly frozen, and ground into microparticles. The microtia chondrocytes were seeded on CAM microcarriers to construct cartilage microtissues. We then prepared bioink for DLP bioprinting by mixing the microtissues with GelMA solution, then cultured the constructs in vitro for 20 days and in vivo for 12 weeks, respectively. Results When CAM particle size ranges from 0µm to 150µm, the microtissue bioink exhibits desired mechanical properties and had no significant sediment in a 3 hour time window. DLP bioprinting of cartilage microtissues mixed with GelMA can produce auricular constructs with high elasticity, high printing accuracy and low swelling ratio. A large amount of extracellular matrix deposition can be achieved after in vitro culture, and mature cartilage regeneration can be observed after subcutaneous implantation in nude mice for 12 weeks. Conclusion Cartilage microtissue bioinks have good biocompatibility and mechanical properties, the chondrocytes in printed ears showed obvious advantages in cell proliferation in vitro and auricular cartilage regeneration in vivo, which sheds new light on the optimization of bioink and brings novel hope for the treatment of microtia patients.