Bioprinting Human Vasculature Within a Collagen Gel

Angiogenesis, the formation of new blood vessels from pre-existing vasculature, facilitates wound healing, repair, and tissue regeneration. The use of cell-delivery approaches—such as cell-loaded scaffolds or scaffold-free systems—to induce angiogenesis is thereby a potent area of study in wound healing research. However, an intense angiogenic response in the wound site corresponds with a lag of recession of blood vessels during the remodeling phase of wound healing thereby being associated with a higher risk of scar formation and in more severe cases, keloid formation. Our objective is to investigate a bioprinted blood vessel model that enables precise and controlled regulation of the angiogenic process during wound healing.<br/><br/>This study explores the potential and application of bioprinting human blood vessels within collagen. We used free-form printing to create vascular structures with human umbilical vein endothelial cells (HUVEC) stained with green fluorescent protein (GFP). These cells were suspended in a 3% gelatin gel and extruded into a viscous collagen solution to give the researcher control over printing the blood vessels. Gelatin was incorporated into the gel to act as a sacrificial material by dispersing once the collagen gels, allowing the cells to attach to the nearest binding site of the collagen. Through repeated parameter testing of the pump, rheology of the gel, a proof-of-concept model using fibroblasts within the bioink, and cell vitality assay, coupled with the ultimate use of HUVEC cells, a synthetic model of human vasculature was printed within a collagen solution and then initiate the gelation process.<br/><br/>The experimental setup of which the HUVEC-gelatin structure in the collagen gel was achieved consisted of a extrusion syringe containing 2mL of the un-gelled HUVEC-gelatin suspension. The suspension was pre-chilled at 4°C for uniformed gelling and then extruded into a 6-well plate, which was also at 4 °C to ensure the collagen-media solution inside the wells stayed as a liquid upon extrusion of the HUVEC-gelatin gel. A singular line of the suspension was extruded onto each of the 3 wells. The well plate was subsequently incubated at 37°C and 5% CO2 to allow the gelation of collagen, meanwhile the release of HUVEC from gelatin gel. EVOS M7000 imaging microscope to assess whether the GFP-stained endothelial cells branched from the gelatin structure after one day. The finding that could demonstrate the gel/cell suspension’s ability to form structure akin to human vasculature. Additionally, the cell vitality assay—conducted by comparing HUVEC cells before extrusion, in the syringe, and after extrusion—provided data that further supports the possibility of synthesizing human vasculatures from HUVEC-gelatin structures within a collagen gel.<br/><br/>The current data and results gathered from the aforementioned experiments thus support this prospective method of synthesizing human vasculature from HUVEC-gelatin gel. This consequently provides promising results for the future plan to integrate these synthetic vasculatures into bioengineered skin grafts.