Bioprinting of miRNA-Induced Spheroids for Vascularized, Heterocellular Bone Regeneration

Successful bone regeneration requires coupled osteogenic and vascular development; however, achieving simultaneous multicellular differentiation within engineered tissues remains challenging. Here, we developed a microRNA (miR)-guided spheroid platform to induce dual osteogenic and endothelial differentiation of human adipose-derived stem cells (hASCs) for vascularized bone regeneration. hASCs were transfected with miR-148b or miR-210 to promote osteogenic and vascular-associated phenotypes, respectively, and assembled into spheroids that were bioprinted within an nHA-containing GelMA microgel environment using aspiration-assisted bioprinting (AAB). The integrated platform combined miR-guided osteogenic and endothelial differentiation, spatially organized AAB-based spheroid assembly, and an nHA-containing GelMA microgel environment to support vascularized bone tissue regeneration. The engineered constructs maintained high cell viability (> 90%) and supported active cell spreading and migration within the microgel matrix, together with increased osteogenic and endothelial gene expression. To further verify their in vivo regenerative potential, the constructs were implanted into mouse critical-size calvarial defects, where those containing miR-transfected hASCs improved bone regeneration, achieving ~91% of defect closure, and promoted the formation of vessel-like CD31-positive structures compared to controls. Together, these findings demonstrate that combining miR-mediated dual-lineage differentiation with spatially organized spheroid assembly and a supportive microgel environment provides a promising strategy for vascularized bone tissue engineering.