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

Molecular Imaging and Biology https://doi.org/10.1007/s11307-023-01871-0 RESEARCH ARTICLE Electron Spin Resonance Probe Incorporation into Bioinks Permits Longitudinal Oxygen Imaging of Bioprinted Constructs Sajad Sarvari1,2 · Duncan McGee3 · Ryan O’Connell2,4 · Oxana Tseytlin2,4 · Andrey A. Bobko2,4 · Mark Tseytlin2,4,5 Received: 5 May 2023 / Revised: 27 October 2023 / Accepted: 30 October 2023 © The Author(s) 2023 Abstract Purpose 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. Results 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 proofof-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. Keywords Bioprinting · Oximetry · EPR Imaging · Rapid Scan EPR · GelMA · LiNc-BuO · Photo-Crosslinking · Hypoxia · Microfluidics · Bioink Duncan McGee is an equally contributing primary author. * Mark Tseytlin 1 2 Department of Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, WV, USA In Vivo Multifunctional Magnetic Resonance Center at Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV, USA 3 Department of Chemical and Biomedical Engineering, West Virginia University, Morgantown, WV, USA 4 Department of Biochemistry and Molecular Medicine, West Virginia University, Morgantown, WV, USA 5 West Virginia University Cancer Institute, Morgantown, WV, USA 13 Vol.:(0123456789) Molecular Imaging and Biology Introduction Bioprinting is an additive manufacturing technology intended for the engineering of functional human tissues and organs [1, 2]. Human-relevant models are created that expand our knowledge of the underlying pathological mechanisms in diseases [3–7]. The pharmaceutical industry and academia use bioprinting with human cells to screen drugs, including for personalized care [5, 7, 8]. Bioprinting promises to bridge the translational gap between pre-clinical findings and successful clinical trials since, often, the drugs that exhibited positive results in animal models fail in clinical settings. Another emerging application of this technology is organ transplantation [9]. Bioprinted organs made using the patient’s own cells will eventually solve the problems of donor shortage and immune rejection [10]. Treatments of burns and other skin injuries are expected to be one of the first clinical applications of bioprinting [3, 4, 6, 11]. The recently passed US Congress ‘FDA Modernization Act 2.0’ encourages using alternatives to animals for drug testing [12]. Bioprinting is such an alternative. Bioprinting is analogous to the widely used 3D printing technology that manufactures solid structures with pre-defined geometrical and mechanical properties [2] from plastics, ceramics, and metals. Similarly, bioprinting forms gel-like solid biological structures from bioinks that consist of hydrogels, cells, cell media, and cross-linking agents. A wide variety of biological composites have been developed that are tailored to a specific tissue or organ of interest [1]. During the post-printing process, cells undergo modifications, such as differentiation, proliferation, and junction formation. The evolution and maturation of the printed cell-laden constructs are referred to as four-dimensional (4D) bioprinting [13, 14], where the fourth dimension is time. The major challenge related to 4D bioprinting is consistent and reliable delivery of nutrients to and removal of waste products from the bioprinted tissues. Adequate tissue oxygenation is most critical for attaining bioprinting goals. Even transient hypoxia may not only affect cell development but also trigger apoptosis. To overcome the problem of oxygen ( O 2) deficiency in thick (> 1 cm) prints due to underdeveloped vasculature, several strategies have been implemented to supply cells with O2 during neovascularization. Chemical (peroxides [15, 16]) and biological photosynthesis (algae [3, 17, 18]) approaches have been utilized toward this goal. Introducing such oxygen-generating materials into bioinks has been evaluated as a solution to the intermittent hypoxia problem [19–21]. Oxygen generation and delivery must be optimized to ensure long-term bioprinted construct progression to the functional tissue or organ. Towards 13 this goal, the 4D bioprinting technology has to be supplemented with 4D oximetry ( O 2 mapping in 3D space and time). Several imaging modalities can be considered for this purpose. Given the tissue size of centimeters, the utility of traditional optical methods is questionable. Light scattering and absorption by biological substances limit 3D optical imaging depth to several millimeters at best. Oxygen-detecting techniques of positron emission tomography (PET) and magnetic resonance imaging (MRI) can be used as they are not limited by penetration depth. However, both modalities have practical limitations in the context of longitudinal 4D oxygen imaging. PET requires the intr (...truncated)