(261a) Biomaterials That Breathe for Regenerative Engineering

Introduction: Oxygen supply is essential for the long-term viability and function of engineered scaffolds in vitro and in vivo.^1,2 The integration with the host blood supply as the primary source of oxygen to cells requires four-to-five weeks in vivo. Therefore, cells that are encapsulated in 3D scaffolds during this process are prone to oxygen deprivation, cellular dysfunction, damage, and hypoxia-induced necrosis. Materials that can generate oxygen include solid peroxides, liquid peroxides, and fluorinated compounds as oxygen carriers. However, liquid peroxides and fluorinated reagents do not have high oxygen content. Solid peroxides pose a potential risk of uncontrollable burst of oxygen which can be damaging to surrounding tissues in vitro and in vivo. One strategy to control the release of oxygen is to limit the rate of exposure of the solid peroxide to the water content in the cellular microenvironment by using a hydrophobic barrier around it.^3,4 In this work, we developed novel oxygen generating biomaterials, which contain calcium peroxide (CaO₂) and polycaprolactone (PCL), that substitute the host oxygen supply via hydrolytic degradation. We have shown the feasibility of our scaffolds in vitro and their regenerative capability in vivo.

Materials and Methods: We reinforced the oxygen generating scaffolds with microparticles that contained the cargo for oxygen release. These microparticles were synthesized by encapsulating CaO₂ within a hydrophobic PCL phase through an emulsification process. We then fabricated the oxygen generating scaffolds using a 5% (w/v) gelatin-based hydrogel matrix and 13.5 (w/v) CaO₂-loaded PCL microparticles. Subsequently, we carried out swelling, degradation, high-resolution SEM, and dynamic mechanical analyses for the oxygen generating 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 analyzed ranged from 5-20% (w/v) PCL. The in vitro cytocompatibility of the oxygen generating scaffolds was studied using different cell types including fibroblasts, myoblasts, primary cardiac fibroblasts, stem cells, or osteoblasts in long term cultures up to 35 days. Cellular response to oxygen generating scaffolds was characterized through metabolic activity, lactate dehydrogenase activity, and apoptosis assays, and pH measurements. Finally, the oxygen generating scaffolds were used to regenerate critical size calvaria defects in a rat model.

Results and Discussion: We have shown controlled and sustained release of oxygen over 4 weeks in vitro. We successfully tuned the oxygen release kinetics by varying the PCL and CaO₂ concentrations. We were able to increase the amount of dissolved oxygen with increasing CaO₂ loading. We also have tuned the material properties such as swelling capacities, degradation, and mechanical properties of the scaffolds. To characterize the in vitro cellular response to our biodegradable oxygen generating scaffolds, we performed 3D-encapsulation experiments using fibroblasts, myoblasts, and primary cardiac fibroblasts, stem cells, or osteoblasts in these scaffolds. The in vitro results showed enhanced cell survival, proliferation, and function under hypoxic environments. 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 these 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 via immunohistochemistry for tartrate-resistant acid phosphatase (TRAP) and vascular endothelial growth factor (VEGF) staining.

Conclusions: We have developed a modular microparticle synthesis process using an emulsification technique that produced a wide range of oxygen generating scaffolds with distinct oxygen release kinetics. These scaffolds released oxygen consistently over 4 weeks with dissolved oxygen levels up to 35 days under hypoxia. Material properties such as compressive moduli, swelling, and degradation were tuned and controlled by varying the concentration of CaO₂ in the oxygen generating scaffolds. Our scaffolds were also tailorable to diverse cell types. The in vitro results support that the oxygen generating component provides functional benefits for long-term cell cultures. The metabolic activity and apoptotic activity were controlled by the oxygen generating scaffolds. In addition, we achieved stable cellular microenvironments without causing major fluctuations in pH. We anticipate that these scaffolds will be easily modified and customized to obtain a wide range of oxygen release kinetics and biological properties for different regenerative engineering applications. Our technology, along with efforts to improve vascularization strategies, has the potential to advance the in vivo clinical performance 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.