Extracellular matrix (hMatrix) derived from osteogenically enhanced mesenchymal stem cells (OEhMSCs) can be deposited onto scaffolds in a process referred to as "bioconditioning". While bioconditioned gelatin scaffolds have been shown to enhance stem cell retention and significantly accelerate repair of critical-sized bone defects, there is a need for alternative biofabrication methods that have greater clinical potential in bone regenerative therapies. Injectable delivery vehicles are desirable because of their ability to space-fill defects and form a continuous interface with existing bone. Herein, we report gelatin methacrylate (GelMA) microspheres fabricated using a custom 3D-printed coaxial flow focusing device. We demonstrate that OEhMSC attachment to the hMatrix coating stimulates the secretion of osteogenic and angiogenic factors and upregulates BMP-2 gene expression. OEhMSCs delivered with microspheres coated with hMatrix enhanced bone regeneration in a murine femoral defect model compared to bare microspheres. OEhMSCs administered with microspheres coated with hMatrix also supported bone regeneration comparable to that induced by the current gold standard of BMP2 delivered from a collagen sponge in a murine calvarial defect model. These results indicate the hMatrix/OEhMSC composite microspheres are effective vectors for delivery of stem cells for bone regeneration. While a versatile space-filling delivery method has many benefits, customized, patientspecific 3-D grafts can also allow for good tissue integration with an increased level of sophistication from control over the hierarchical structure. Additive manufacturing is a promising method for producing these customized three-dimensional bioactive constructs for regenerative medicine. While thermoplastics and metal implants have been successfully used for permanent, non-degradable craniomaxillofacial implants in adults, these materials lack bioactive characteristics to degrade and allow for neotissue formation that can grow with young patients. Therefore, we also demonstrate a workflow for a 3D bioprinted osteoinductive bone graft using an innovative hydrogel ink conjuncted with the bioconditioning process to direct osteogenic differentiation of human mesenchymal stem cells (hMSCs). Specifically, we printed freestanding bone graft geometries using a Nanoengineered Ionic-Covalent Entanglement (NICE) bioink. Bioconditioning NICE with a matrix deposited by osteo-enhanced hMSCs (iP-hMatrix) derived from human induced pluripotent cells resulted in a significant increase in mineralization and deposition of collagens VI and XII that contribute to the osteogenic activity of iP-hMatrix. Bioconditioned NICE increased the expression of osteogenic marker genes, including bone morphogenic protein-2, osteocalcin and osteopontin. These results suggest that bioconditioning NICE scaffolds using iP-hMatrix enhances osteogenic potential of the scaffold and can be used for craniomaxillofacial bone grafts with precise geometries.
