3D printed O₂-generating scaffolds enhance osteoprogenitor- and type H vessel recruitment during bone healing

Oxygen (O₂)-delivering tissue substitutes have shown tremendous potential for enhancing tissue regeneration, maturation, and healing. As O₂ is both a metabolite and powerful signaling molecule, providing controlled delivery is crucial for optimizing its beneficial effects in the treatment of critical-sized injuries. Here, we report the design and fabrication of 3D-printed, biodegradable, O₂-generating bone scaffold comprising calcium peroxide (CPO) that once hydrolytically activated, provides long-term generation of oxygen at a controlled, concentration-dependent manner, and polycaprolactone (PCL), a hydrophobic polymer that regulate the interaction of CPO with water, preventing burst release of O₂ at early time points. When anoxic conditions were simulated in vitro, CPO-PCL scaffolds maintained the survival and proliferation of human adipose-derived stem/stromal cells (hASCs) relative to PCL-only controls. We assessed the in vivo osteogenic efficacy of hASC-seeded CPO-PCL scaffolds implanted in a non-healing critical-sized 4-mm calvarial defects in nude mice for 8 weeks. Even without exogenous osteoinductive factors, CPO-PCL scaffolds demonstrated increased new bone volume compared to PCL-only scaffolds as verified by both microcomputed tomography analysis and histological assessments. Lastly, we employed a quantitative 3D lightsheet microscopy platform to determine that O₂-generating scaffolds had similar vascular volumes with slightly higher presence of CD31^hiEmcn^hi pro-osteogenic, type H vessels and increased number of Osterix⁺ skeletal progenitor cells relative to PCL-only scaffolds. In summary, 3D-printed O₂ generating CPO-PCL scaffolds with tunable O₂ release rates provide a facile, customizable strategy for effectively treating, craniofacial bone defects. STATEMENT OF SIGNIFICANCE: Oxygen(O₂)-delivering bone substitutes show promise in defect repair applications by supplying O₂ to the cells within or around the graft, improving cell survivability and enhancing bone matrix mineralization. A novel O₂-generating bone scaffold has been 3D printed for the first-time which ensures patient and defect specificity. 3D printed calcium peroxide-polycaprolactone (CPO-PCL) bone scaffold provides uninterrupted O₂ supply for 22 days allowing cell survival in deprived O₂ and nutrient conditions. For the first time, O₂-driven bone regenerative environment in mice calvaria has been captured by light-sheet imaging which illuminates abundance of Osterix+ cells, angiogenesis at a single cell resolution indicating active site of bone remodeling and growth in the presence of O₂.