DLP bioprinting of cartilage organoid-laden bioinks yields high-fidelity auricular constructs with enhanced chondrogenesis.

To develop and validate a cartilage organoid (CO)-laden digital light processing (DLP) bioprinting strategy for auricular reconstruction and to compare its performance with conventional chondrocyte-laden prints. Rat bone-marrow stromal cells (BMSCs) were aggregated into spheroid and chondrogenically induced to form CO. Organoid construction and characterization included EdU proliferation assay, CD73/CD90 immunofluorescence (IF), and real-time quantitative polymerase chain reaction (qPCR) of chondrogenic genes. O-nitrobenzyl functionalized gelatin (GelNB)/ methacrylated hyaluronic acid (HAMA) bioinks were screened by gross morphology, tensile/ compressive mechanics, enzymatic degradability (0.1% collagenase II), and swelling, identifying 10% (w/v) GelNB + 1% (w/v) HAMA as the working bioink. Full-scale ears were DLP-printed from acellular, chondrocyte-laden, or CO-laden inks. In vitro, whole-mount Live/Dead imaging was performed at 7/14/21 days and qPCR quantified COL2A1, aggrecan (ACAN), COL10A1. In vivo, constructs were implanted subcutaneously in nude mice and analyzed at 4/8 weeks by Hematoxylin-eosin (H&E), Safranin O/Fast Green, ACAN and COL II IHC staining, and compressive Young's modulus measurement on implants. The 10% GelNB + 1% HAMA ink balanced print fidelity, mechanical robustness, and controlled degradability. Loading CO did not impair printability. During 21-day culture, viability remained high; at day 21, CO-laden ears showed higher COL2A1/ACAN and lower COL10A1 than chondrocyte-laden controls. After implantation, CO-laden explants exhibited more abundant lacuna-like cartilage morphology, stronger ACAN and COL II staining, increased EVG-positive elastic fiber-associated matrix, and a higher compressive Young's modulus at 8 weeks. CO-laden DLP bioprinting enhances chondrogenesis and mitigates hypertrophy versus chondrocyte-laden printing, offering an exploratory high-fidelity strategy for auricular cartilage-like tissue engineering rather than definitive mature elastic cartilage regeneration.