Facile Osteoimmunomodulatory Nanoparticles Augment 3D-Printed Scaffold-Mediated Bone Regeneration
Regenerating critical-sized long bone defects poses substantial challenges due to limitations of autografts and processed allografts. Biomaterial scaffolds offer versatile alternatives, yet their effectiveness is often constrained by their limited innate osteoinductivity. While growth factors and cells can enhance osteoinduction, the inclusion of biologics in biomaterial scaffolds creates regulatory challenges for clinical translation. To address this, here we describe three-dimensional (3D) printed polycaprolactone (PCL) scaffolds for temporally controlled delivery of osteoimmunomodulatory amorphous calcium phosphate-chitosan nanoparticles (ACPC-NP).  In vitro, the ACPC-NP exhibit concentration dependent effects on osteoblasts, monocytes, and osteoclasts. At increasing concentrations up to 500 µg/ml, these nanoparticles stimulate osteogenesis, modulate M2/M1 macrophage polarization, and inhibit osteoclast maturation and activity. Leveraging these concentration-dependent effects in vivo through temporally controlled release of ACPC-NP from 3D-printed PCL scaffolds, we observe the complete regeneration and the restoration of biomechanical strength of critically sized radial defects in rats. Such healing is absent in defects implanted with bare PCL scaffolds or those loaded with calcium-phosphate microparticles. The tunable osteoimmunomodulation by the NP underscore the translational potential of this technology to yield structurally sound and functionally robust bone regeneration outcomes. Overall design: Three types of scaffolds were fabricated using 3D printing techniques, including PCL scaffolds containing  amorphous calcium phosphate chitosan nanoparticles (ACPC-NP), calcium phosphate microparticles (CaP), and bare PCL. We then set out to test their effects in vivo using a rat radius critical-sized bone defect model. To gain mechanistic insights on the osteogenic capacity of the ACPC-NP scaffold compared to CaP and PCL scaffolds, bulk RNA sequencing was performed on mRNA from cells released from the bone tissues extending 1mm proximal and distal to the scaffolds at weeks 2 and 6 post-implantation.