4D EPR Oximetry Imaging for 4D Bioprinting and Tissue Engineering

TL;DR: Researchers develop 4D electron paramagnetic resonance (EPR) oximetry imaging for 4D bioprinting, enabling non-invasive, quantitative oxygen mapping in 3D bioprint volumes over prolonged maturation times, with applications in tissue engineering and bioprinting technologies.

Abstract: Bioprinting, akin to 3D printing, employs cell-laden hydrogels such as GelMA (Gelatine Methacrylate) and Alginate instead of plastic or resin to create biological structures. The printing process followed by subsequent tissue maturation is known as 4D bioprinting, where the fourth dimension is time. Ensuring adequate and consistent oxygen, O2, supply within 3D bioprint volume over the entire maturation period process is crucial, as even transient hypoxia can impact cellular behavior and phenotypes. However, printing of fully developed vasculature remains an unresolved technical challenge. Several approaches to chemically (peroxides) or biologically (photosynthesis) generate oxygen during bioprint maturation have been proposed. However, a comprehensive evaluation and optimization of these methods is not currently possible due to the lack of quantitative longitudinal O2 imaging modality. 4D electron paramagnetic resonance imaging (EPRI) is demonstrated to be the method of choice to solve the problem of even bioprint oxygenation in 3D volume and over a prolonged maturation time. EPRI permits longitudinal quantitative noninvasive O2 mapping using incorporate in bioinks probes. Two types of probes have been utilized: water soluble triarylmehyl (trityls) stable radicals and solid lithium octa-nbutoxy-phthalocyanine (LiNc-BuO) particles. Incorporation of these materials in bioinks modifies its chemical and optical properties. For example, LiNc-BuO absorbing light affects photopolymerization. Also, bioprinting process affects the probes. Trityls chemically react with the formed intermediate radicals. Part of the presented work was dedicated to understanding and optimization of the bioprinting process that includes the addition of oxygen probes into the commonly used GelMa and alginate bioinks. Bioprinting was done using locally developed digital light processing (DLP) and extrusion bioprinters. 4D oximetry was performed using a locally built EPRI instrument. Both acellular and cell-laden constructs were successfully printed and imaged. Experiments revealed a previously unreported phenomenon of oxygen depletion due to the presence residual photoinitiator. Oxygen consumption rates by HEK293T cells within printed structures were quantified. 4D EPRI mapping of changing oxygen levels within a murine macrophage-laden alginate constructs have been demonstrated using freeform reversible embedding of suspended hydrogels (FRESH) methodology. Oxygen consumption rates were computed by analyzing consecutive EPR images in the time dimension. Given the incorporation of the EPR probes into the alginate bioink, proper control experiments evaluating the temporal EPR probe stability and EPR signal intensity in acellular bioink were conducted. In conclusion, we've shown the feasibility of integrating 4D EPR with bioprinting, paving the way for diverse applications using various bioinks and molecular spin probes. These findings are foundational for advancing bioprinting technologies and understanding oxygen dynamics in tissue engineering.