(178u) Self-Oxygenating Biomaterials for Regenerative Medicine

Oxygen supply is essential for the long-term viability and function of engineered scaffolds in vitro and in vivo.^1,2 Achieving integration with the host blood supply as the primary source of oxygen might take up to a few weeks in vivo. Therefore, cells that are encapsulated in 3D scaffolds during this process are prone to oxygen deprivation, damage, cellular dysfunction, and ultimately hypoxia-induced necrosis. Materials that can generate oxygen include solid peroxides, liquid peroxides, and fluorinated compounds can be used as oxygen carriers. However, liquid peroxides and fluorinated reagents do not have high oxygen content. While effective, solid peroxides have a risk of uncontrollable burst release of oxygen which can be damaging to surrounding tissues. One approach to control the release of oxygen is to limit the rate of exposure of the solid peroxide to water using a hydrophobic barrier around it.^3,4 Here we developed novel self-oxygenatingbiomaterials, which contain calcium peroxide (CaO₂) and polycaprolactone (PCL), that substitute the host oxygen supply via hydrolytic degradation. We have demonstrated the feasibility of our scaffolds in vitro and their regenerative capability in vivo.

Materials and Methods: We generated self-oxygenating scaffolds by reinforcing microparticles that contained the cargo for oxygen release. The microparticles were synthesized by encapsulating CaO₂ within a hydrophobic PCL phase through emulsification. We fabricated the self-oxygenating scaffolds using 5-10% (w/v) gelatin-based hydrogel matrix and 10-15 (w/v) CaO₂-loaded PCL microparticles. We carried out swelling, degradation, high-resolution SEM, and dynamic mechanical analyses for the self-oxygenating scaffolds. The PCL content was modified in the microparticles to evaluate how the hydrophobic barrier alters the oxygen release kinetics of the scaffold. The PCL concentrations ranged between 5-20% (w/v) PCL. The in vitro cytocompatibility of the self-oxygenating scaffolds was studied using different cell types including fibroblasts, myoblasts, primary cardiac fibroblasts, stem cells, or osteoblasts. Cellular response to these scaffolds was characterized through metabolic activity, lactate dehydrogenase activity, and apoptosis assays, and pH measurements. Finally, the self-oxygenating scaffolds were used to regenerate critical size cranial defects in vivo using a rat model.

Results and Discussion: We have demonstrated the controlled and sustained release of oxygen initially in in vitro platforms. We successfully tuned the oxygen release kinetics by varying the amounts of CaO₂ and PCL. We were able to increase the amount of dissolved oxygen by increasing CaO₂ loading. We also have tuned the material properties such as swelling, degradation, and mechanical properties of the scaffolds. To characterize the in vitro cellular response to our biodegradable self-oxygenating scaffolds, we 3D-encapsulated fibroblasts, myoblasts, primary cardiac fibroblasts, stem cells, or osteoblasts in the scaffolds. The in vitro results showed enhanced cell survival, proliferation, and function under hypoxia. During continuous oxygen release, the 3D constructs maintained a stable tissue culture environment between pH 8 to 9. The in vivo rat model demonstrated that the self-oxygenating scaffolds regenerate a bone volume that was comparable to the native bone and yielded higher than 90% regeneration in critical size cranial defects. In addition, in vivo bone remodeling and vascularization was confirmed through immunohistochemistry for tartrate-resistant acid phosphatase (TRAP) and vascular endothelial growth factor (VEGF).

Conclusions: We have developed a wide range of self-oxygenating scaffolds with distinct oxygen release kinetics. These scaffolds produced oxygen consistently both in the short-term and long-term under hypoxia. Material properties including compressive moduli, swelling, and degradation were tuned and controlled by varying the concentration of CaO₂ in the composite scaffolds. Our biomaterials were also tailorable to diverse cell types. The in vitro results support that the self-oxygenating component provides functional benefits for cell cultures. The metabolic activity and apoptosis behaviors were controlled by the self-oxygenating scaffolds. Moreover, we achieved stable cellular microenvironments without causing major fluctuations in pH. We anticipate that our scaffolds will be easily modified and customized to obtain a wide range of oxygen release kinetics and biological properties for different applications in regenerative medicine. Our self-oxygenating technology, along with efforts to improve vascularization approaches, has the potential to advance the in vivoperformance of 3D tissue scaffolds.

References: ¹Nguyen, M.A., Camci-Unal, G., Trends in Biotechnology, 38(2): 178-190, 2020. ²Suvarnapathaki, S., Wu, X., Lantigua, D., Nguyen, M.A., Camci-Unal, G., Nature Asia Materials, 11(1): 1-18, 2019. ³Suvarnapathaki, S., Nguyen, M.A., Goulopoulos, A., Lantigua, D., Camci-Unal, G., Biomaterials Science, 9, 2519-2532, 2021. ⁴Suvarnapathaki, S., Wu, X., Zhang, T., Nguyen, M.A., Goulopoulos, A.A., Wu, B., Camci-Unal, G., Bioactive Materials, 13, 64-81, 2022.