3D-printed advanced scaffold armed with exosomes derived from human skeletal stem cell identified by single-cell RNA sequencing enhances osteochondral regeneration

Osteochondral defects (OCDs) pose a significant clinical challenge due to their limited self-repair capacity. The complex structure and distinct biological properties of articular cartilage and subchondral bone further complicate regeneration.In this study, we introduce a novel osteochondral regeneration strategy leveraging single-cell RNA sequencing (ScRNA-seq) to identify a unique population of skeletal stem cells (SSCs) derived from the infrapatellar fat pad (IFP). These SSCs exhibit high differentiation potential and robust chondrogenic capacity. Using flow cytometry, we isolated SSCs and extracted their exosomes (Exos), which were subsequently combined with hydrogels to develop a novel bioink. Employing 3D printing technology, we fabricated an innovative hydrogel scaffold designed to adapted to the defective areas enhance OCD repair.In a rat OCD model, the 3D-printed hydrogel scaffold loaded with SSC-derived Exos (SSC-Exos) demonstrated exceptional osteochondral regeneration, facilitating synchronous repair of both cartilage and subchondral bone. In vitro experiments revealed that SSC-Exos significantly enhanced the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Importantly, SSC-Exos derived from the IFP exhibited superior cartilage regeneration capabilities compared to Exos from adipose-derived mesenchymal stem cells (ADSC-Exos). High-throughput sequencing further elucidated the critical role of the microRNA-214-3p (miR-214-3p)/jagged canonical Notch ligand 2 (JAG2) axis in SSC-Exos-mediated cartilage regeneration. Collectively, the 3D-printed hydrogel scaffold loaded with SSC-Exos represents an innovative and effective strategy for OCD repair, with potential for clinical translation.