Reverse intravital 3D bioprinting for biomedical applications

Additive manufacturing, and more specifically 3D bioprinting, has transformed the landscape of tissue engineering by allowing the precise construction of biological tissues and organs through a layer-by-layer deposition of biomaterials and cells. Despite these advancements, traditional additive processes are often hindered by significant time inefficiencies and challenges in achieving high precision, particularly when fabricating large-scale structures with complex geometries and intricate internal features. These limitations become especially pronounced in the context of in vivo applications, where the need for rapid, precise, and minimally invasive fabrication methods is critical. To address these challenges, this thesis introduces Reverse Intravital 3D Bioprinting (iR3D Bioprinting), a novel subtractive manufacturing technology specifically designed for in vivo 3D bioprinting. Unlike conventional additive approaches, iR3D Bioprinting enables the creation of complex three-dimensional structures directly within living organisms by selectively removing material from a pre-formed hydrogel block. This subtractive process offers a unique solution to the limitations of additive manufacturing, providing a means to rapidly produce intricate structures with high precision in a biological context. The process begins with the formation of a 3D hydrogel block using photoresponsive polymers, such as polyethylene glycol (PEG) and hyaluronic acid (HA), which are conjugated with 7-carboxymethoxy-4-methylcoumarin (CMMC). These polymers are crosslinked under UV-visible (UV-Vis) light to form a stable and uniform hydrogel structure. Subsequently, multiphoton (MP) near-infrared (NIR) light is employed to selectively induce decrosslinking in targeted regions of the hydrogel, allowing for the precise removal of material. This approach enables the creation of hollow structures and intricate networks within the hydrogel, all within the living tissue environment. The potential of iR3D Bioprinting for in vivo applications is demonstrated through two tissue engineering applications. The first application involves the fabrication of hydrogel-based vascular bypasses within living murine models. By creating perfusable microfluidic networks, iR3D Bioprinting successfully redirects blood flow in predefined configurations, such as vein-vein, vein-artery, and artery-artery bypasses. This capability highlights the technology's precision and adaptability in manipulating biological systems, which is critical for advancing vascular tissue engineering. Additionally, the feasibility of iR3D Bioprinting for vascular applications was explored through the fabrication of bypasses on coronary arteries. In a murine model, the technology was used to create microfluidic networks adjacent to coronary arteries in the heart of a previously sacrificed mouse. Following the injection of CMMC-4armPEG polymer and UV-Vis crosslinking, multiphoton light was employed to form microchannels, which were successfully connected to the coronary artery through laser ablation. Perfusion tests with fluorescent tracer confirmed that both the coronary artery and the microfluidic network were effectively connected, demonstrating the potential of iR3D Bioprinting for performing bypass surgeries on coronary arteries. The second application of iR3D Bioprinting is focused on neural tissue engineering, specifically the guidance of axon growth from human cortical brain organoids (CBOs). In this context, the technology was used to fabricate hydrogel microchannels that effectively guide axonal outgrowth, facilitating the formation of neural connections both in vitro and in vivo within murine brains. This application underscores the potential of iR3D Bioprinting to support the regeneration and repair of neural tissues, offering promising avenues for future research in neuroregenerative medicine. In conclusion, iR3D Bioprinting represents a groundbreaking advancement in the field of in vivo 3D bioprintin