Multifunctional bilayer scaffolds integrating melt electrowriting fibers and drug-loaded microspheres promote posteochondral regeneration through PI3K–AKT–mediated chondro-osteogenic signaling

Osteochondral defects (OCDs) remain challenging to repair due to the complex structural and biological heterogeneity of the cartilage-bone interface. Here, we developed a bilayer microsphere-scaffolds composite by integrating melt electrowriting (MEW) scaffolds with two types of functionalized hydrogel microspheres (HMPs). The upper layer consisted of disulfide-containing poly ethylene glycol diacrylate (HB-PBHE)/thiolated hyaluronic acid (SH-HA) microspheres encapsulating kartogenin (KGN) liposomes (HM@PHK) to promote chondrogenesis, while the lower layer was formed by GelMA/nano-hydroxyapatite (nHAP) microspheres loaded with curcumin (Cur) liposomes (HM@GMAC) to provide osteoinductive, antioxidative, and anti-inflammatory cues. This bilayer construct exhibited uniform architecture, favorable mechanics, and controlled drug release. In vitro, the system promoted bone marrow-derived mesenchymal stem cells (BMSCs) migration, chondrogenic and osteogenic differentiation, and attenuated oxidative stress-induced apoptosis while modulating macrophage polarization. Transcriptomic analysis revealed that both layers activated the PI3K-AKT pathway, with the upper phase regulating the PI3K-AKT-SOX9 axis for chondrogenesis and the lower phase promoting osteogenesis and cytoprotection via PI3K-AKT-RUNX2/BAX signaling. In vivo implantation in rat femoral condyle defects demonstrated superior cartilage-bone reconstruction. Collectively, this multifunctional bilayer microsphere-scaffolds system provides stratified regulation of osteochondral repair through coordinated structural support, bioactive delivery, and immunomodulation, offering a clinically translatable approach to osteochondral defect regeneration.