3d-Printed Ceramic Scaffolds Functionalized With Exosomes Derived From Mineralized Osteoblasts: A Cell-Free Preclinical Approach

In regenerative medicine, the quest for innovative strategies to address the multifaceted challenge of bone tissue repair has shifted towards acellular approaches. Exosomes, nanoscale vesicles enriched with bioactive molecules, have emerged as promising candidates for tissue regeneration without relying on living cells. This study explores the therapeutic potential of exosomes derived from mineralized osteoblasts (MOBs), central players in bone formation. MOB-derived exosomes (MOB-Exos) were isolated from osteoblasts cultured in osteogenic medium for 21 days and integrated into 3D-printed gyroid scaffolds composed of hydroxyapatite (HA) and tricalcium phosphate (TCP). Characterization of MOB-Exos by nanoparticle tracking analysis confirmed their size, morphology, and concentration, while proteomic profiling revealed a cargo rich in bone-associated proteins. In vitro , MOB-Exos enhanced osteogenic differentiation of rabbit bone marrow mesenchymal stromal cells (rBMSCs), promoting mineralization and upregulating bone-related gene expression even in the absence of osteogenic supplements. In vivo , their osteoconductive and osteoinductive capabilities were evaluated using two rabbit calvarial defect models and a heterotopic mouse model, respectively. While MOB-Exos did not significantly enhance bone formation in the rabbit calvarial defects, their functionalization onto ceramic scaffolds in the heterotopic mouse model demonstrated robust osteoinductive activity, as evidenced by µCT and histological analysis. This study highlights the potential of acellular, exosome-functionalized 3D-printed scaffolds to bridge the gap between cellular and non-cellular therapies for bone regeneration. By leveraging the bioactivity of MOB-Exos, our approach presents a compelling alternative to traditional cell-based strategies, offering a scalable, clinically relevant pathway to bone repair and regeneration.