A 3D Bioprinted Spheroid-Laden dECM-Enriched Osteosarcoma Model for Enhanced Drug Testing and Therapeutic Discovery

Osteosarcoma (OS) remains highly lethal due to tumor heterogeneity, metastatic potential, and chemoresistance, motivating preclinical 3D models that better reproduce tumor architecture and ECM-driven drug response. This study developed a spheroid-laden, extrusion-bioprinted OS model by embedding MG-63 osteosarcoma spheroids into an OS-tailored bioink enriched with MG-63 cell-derived decellularized extracellular matrix (dECM). The authors first generated and validated MG-63-derived dECM via chemical decellularization with strong DNA reduction below accepted thresholds while retaining key ECM proteins (collagen I, fibronectin, laminin) and matrix components. Two related inks were formulated (GMA and GMA-d) using GelMA, methylcellulose, alginate, and LAP photoinitiator, with GMA-d additionally containing 0.5% (w/v) MG-63 dECM. The inks were characterized for water content, swelling, degradation/mass loss, UV-gelation kinetics, storage/loss moduli, and printability metrics (printability factor and filament width). Both inks supported rapid UV-induced gelation (~10 s), high water content (~89–91%), and stable constructs, with the dECM-enriched ink showing improved printability and comparable compressive modulus (~7.2 kPa). MG-63 spheroids (~197 µm) were incorporated into the inks and successfully bioprinted without nozzle clogging into trabecular-bone-inspired 5-layer meshes. Biological validation focused on doxorubicin (DOX) response versus 2D MG-63 monolayers and scaffold-free spheroids. The bioprinted spheroid model exhibited the greatest DOX chemoresistance (higher retained viability and spheroid area), alongside upregulation of OS-relevant markers (MMP9, VEGF, RUNX2) and constitutively elevated P-glycoprotein expression, supporting a more clinically relevant drug-resistance phenotype attributed to combined 3D tumor architecture and ECM-mimicking barrier effects.