Nerve Growth Factor-Preconditioned Mesenchymal Stem Cell-Derived Exosome-Functionalized 3D-Printed Hierarchical Porous Scaffolds with Neuro-Promotive Properties for Enhancing Innervated Bone Regeneration

The essential role of the neural network in enhancing bone regeneration has often been overlooked in biomaterial design, leading to delayed or compromised bone healing. Engineered mesenchymal stem cells (MSCs)-derived exosomes are becoming increasingly recognized as potent cell-free agents for manipulating cellular behavior and improving therapeutic effectiveness. Herein, MSCs are stimulated with nerve growth factor (NGF) to regulate exosomal cargoes to improve neuro-promotive potential and facilitate innervated bone regeneration. In vitro cell experiments showed that the NGF-stimulated MSCs-derived exosomes (N-Exos) obviously improved the cellular function and neurotrophic effects of the neural cells, and consequently, the osteogenic potential of the osteo-reparative cells. Bioinformatic analysis by miRNA sequencing and pathway enrichment revealed that the beneficial effects of N-Exos may partly be ascribed to the NGF-elicited multicomponent exosomal miRNAs and the subsequent regulation and activation of the MAPK and PI3K-Akt signaling pathways. On this basis, N-Exos were delivered on the micropores of the 3D-printed hierarchical porous scaffold to accomplish the sustained release profile and extended bioavailability. In a rat model with a distal femoral defect, the N-Exos-functionalized hierarchical porous scaffold significantly induced neurovascular structure formation and innervated bone regeneration. This study provided a feasible strategy to modulate the functional cargoes of MSCs-derived exosomes to acquire desirable neuro-promotive and osteogenic potential. Furthermore, the developed N-Exos-functionalized hierarchical porous scaffold may represent a promising neurovascular-promotive bone reparative scaffold for clinical translation.