Exosome-packaged ILK from healthy female serum stimulate osteogenesis via the MAPK signaling pathway

The pathogenesis of osteoporosis (OP) is multifactorial, with impaired osteogenic differentiation of bone marrow stromal cells (BMSCs) serving as a fundamental pathological basis. Enhancing osteogenesis is a key strategy for OP therapy. Exosomes are recognized as promising cell-free therapeutic agents. Here, we demonstrate that serum exosomes derived from healthy females (Exo-HF) exhibit superior pro-osteogenic potential and identify underlying mechanisms through proteomics and RNA sequencing. Exo-HF was isolated and characterized. Subsequently, we established an ovariectomized (OVX) mouse model to evaluate the in vivo therapeutic efficacy of Exo-HF. A series of in vitro functional assays was conducted to assess the impact of Exo-HF on osteogenesis and proliferation of BMSCs from postmenopausal osteoporosis (PMOP) patients. The candidate molecule integrin-linked kinase (ILK), identified through proteomic analysis as a major mediator of Exo-HF function, was validated using a specific inhibitor (QLT0267), lentivirus, and AAV9 vectors. Furthermore, RNA sequencing (RNA-seq) analysis was conducted to explore the signaling pathways involved in ILK-mediated osteogenesis. Finally, a comparative study of peptide versus aptamer modification was performed for systemic bone targeting. Exo-HF exhibits cross-species therapeutic effects by significantly improving bone microstructure and bone strength in OVX mice. Exo-HF also enhanced the osteogenesis of PMOP-derived BMSCs. Proteomics analysis identified that Exo-HF was enriched with ILK. Functionally, blocking ILK with targeted inhibitors partially attenuated the osteogenic activity of Exo-HF both in vitro and in vivo, whereas overexpression of ILK promoted osteogenesis both in vitro and in vivo. RNA-seq linked this process to the MAPK signaling pathway, and MAPK inhibition abolished ILK-induced osteogenesis. Preliminary systemic tracking revealed that, compared to the peptide strategy, the Apt-19s modification exhibited superior bone-targeting efficiency. Exo-HF serves as a safe and efficient vehicle for OP treatment. Furthermore, the robust osteogenic stimulation by Exo-HF is centrally driven by the ILK/MAPK signaling axis. While ILK acts as a major, but not exclusive, mediator in this process, other exosomal proteins may also exert synergistic effects. Apt-19s modification successfully achieved systemic bone targeting, paving the way for non-invasive clinical applications.