Exosome-Functionalized Ceramic Bone Substitute Promotes Critical-Sized Bone Defect Repair in Rats

Ceramic biomaterials are promising alternatives to bone autografts. However, limited bioactivity affects their performance. Therefore, bioactive molecules and cells are often added to enhance their performance. Exosomes have emerged as cell-secreted vesicles, delivering proteins, lipids, and nucleic acids in a paracrine/endocrine fashion. We studied two complementary aspects required for exosome activity/therapy using purified exosomes: first, the intracellular uptake of labeled exosomes and second, the influence of delivered exosomes on cell behavior. Origin-specific differences in the characteristics of purified exosomes, quantification of time-dependent intracellular uptake of PKH-26-labeled exosomes by mesenchymal stem cells (MSCs) and preosteoblasts, and influence on cell behavior were evaluated. Furthermore, exosomes from osteoblasts and MSCs cultured under normal and osteogenic environments were isolated. There is little data available on the concentration and dose of exosomes required for bone regeneration. Therefore, equal amounts of quantified exosomes were implanted in vivo in rat tibia critical defects using a calcium sulfate-nano-hydroxyapatite nanocement (NC) bone filler as the carrier. Bone regeneration was quantified using micro-computed tomography and histology. Along with inducing early maturation and mineral deposition by primary preosteoblasts in vitro, exosome treatment also demonstrated a positive effect on bone mineralization in vivo. Our study concludes that providing a local delivery of exosomes loaded onto a slowly resorbing NC bone filler can provide a potential alternate to autografts as a bone substitute.