Electron Spin Resonance Probe Incorporation into Bioinks Permits Longitudinal Oxygen Imaging of Bioprinted Constructs

The feasibility of the cointegration of 4D EPR imaging and 4D bioprinting has been demonstrated. The proof-of-concept experiments, which were conducted using oxygen probes loaded into GelMA, lay the foundation for a broad range of applications, such as bioprinting with many types of bioinks loaded with diverse varieties of molecular spin probes.

Lithium octa-n-butoxy-phthalocyanine (LiNc-BuO) probes have been introduced into gelatin methacrylate (GelMA) bioink. GelMA is a cross-linkable hydrogel, and LiNc-BuO is an oxygen-sensitive compound that permits longitudinal oximetric measurements. The effects of the oxygen probe on printability have been evaluated. A digital light processing (DLP) bioprinter was built in the laboratory. Bioprinting protocols have been developed that consider the optical properties of the GelMA/LiNc-BuO composites. Acellular and cell-laden constructs have been printed and imaged. The post-printing effect of residual photoinitiator on oxygen depletion has been investigated.

Bioprinting is an additive manufacturing technology analogous to 3D printing. Instead of plastic or resin, cell-laden hydrogels are used to produce a construct of the intended biological structure. Over time, cells transform this construct into a functioning tissue or organ. The process of printing followed by tissue maturation is referred to as 4D bioprinting. The fourth dimension is temporal. Failure to provide living cells with sufficient amounts of oxygen at any point along the developmental timeline may jeopardize the bioprinting goals. Even transient hypoxia may alter cells' differentiation and proliferation or trigger apoptosis. Electron paramagnetic resonance (EPR) imaging modality is proposed to permit 4D monitoring of oxygen within bioprinted structures. Procedures: Lithium octa-n-butoxy-phthalocyanine (LiNc-BuO) probes have been introduced into gelatin methacrylate (GelMA) bioink. GelMA is a cross-linkable hydrogel, and LiNc-BuO is an oxygen-sensitive compound that permits longitudinal oximetric measurements. The effects of the oxygen probe on printability have been evaluated. A digital light processing (DLP) bioprinter was built in the laboratory. Bioprinting protocols have been developed that consider the optical properties of the GelMA/LiNc-BuO composites. Acellular and cell-laden constructs have been printed and imaged. The post-printing effect of residual photoinitiator on oxygen depletion has been investigated.

Models have been successfully printed using a lab-built bioprinter. Rapid scan EPR images reflective of the expected oxygen concentration levels have been acquired. An unreported problem of oxygen depletion in bioprinted constructs by the residual photoinitiator has been documented. EPR imaging is proposed as a control method for its removal. The oxygen consumption rates by HEK293T cells within a bioprinted cylinder have been imaged and quantified. Conclusions: The feasibility of the cointegration of 4D EPR imaging and 4D bioprinting has been demonstrated. The proof-of-concept experiments, which were conducted using oxygen probes loaded into GelMA, lay the foundation for a broad range of applications, such as bioprinting with many types of bioinks loaded with diverse varieties of molecular spin probes.