(595f) Continuous Chaotic Bioprinting of Pre-Vascularized Tissue Constructs

Hollow and perfusable conduits are ubiquitous in living beings. In human tissues, the dimensions of these conduits range from dozens of millimeters to a few microns and enable crucial functions. Examples are the intestines, esophagus, pharynx, external auditory meatus, non-lactating mammary ducts, and arteries. At the extreme microscale, hollow microchannels play key roles in tissue homeostasis; they are a keystone in the survival of thick tissues by providing them with nutrients and oxygen.

The biofabrication of tissue models containing hollow channels is crucial for mimicking and studying biological processes, but it becomes challenging when the tube diameter reaches the microscale. In this study, we introduce the use of continuous chaotic printing—the use of chaotic flows generated by static mixers to produce multilayer architectures—for fabricating scaffolds containing multiple hollow channels. By coextruding a fugitive and a permanent biomaterial through a printhead containing a Kenics static mixer (KSM), we produced continuous filaments approximately 1 mm in diameter comprising intercalated empty channels at a high throughput (i.e., up to 1.5 m of fiber per minute). The quantity and diameter of hollow channels in the printed filament were modulated simply by changing the number of Kenics elements in the printhead. The versatility of this system was demonstrated using different sets of fugitive and permanent materials.

In a first set of experiments, we coextruded an aqueous solution of pluronic acid F-127 along with an alginate aqueous solution through printheads containing 3, 4, 5, or 6 Kenics static mixer (KSM) elements. A calcium chloride bath was used to cross-link the alginate and obtain solid filaments. The aqueous solution of pluronic acid left the filament during incubation, creating hollow channels within the printed filament. Perfusion of dyes through the filaments fabricated by this simple technique confirmed that the channels were hollow. The chaotically printed filaments were immersed in liquid nitrogen and dehydrated in a freeze-dryer for 12 h for scanning electron microscopy (SEM) analysis. SEM micrographs confirmed that the number of hollow channels in the filaments was increased just by increasing the number of KSM elements in the printhead. The average widths of the channels were 85 and 22 µm when the filaments were printed using printheads with 3 or 6 KSM elements, respectively.

In a second set of experiments, we used an aqueous solution of hydroxyethyl cellulose as the fugitive material, whereas alginate, or a blend of gelatin-methacryloyl (GelMA) and alginate, was used as the permanent material. We bioprinted filaments containing C2C12 cells in the permanent material in order to verify whether the process was cell-friendly. Cells surrounded by empty channels exhibited a higher metabolic activity and viability compared to their counterparts printed in solid filaments.

In addition, we bioprinted a mammary ductal carcinoma model using MDA-231-MB cells. We were able to culture these constructs in a continuously perfused system for 2 weeks. We show results from immunostaining experiments that demonstrate the expression of key tumor markers (i.e., the proliferation biomarker KI67 and hypoxia inducible factor 1 alpha [HIF1-α]) during this culture period. Overall, we anticipate that continuous chaotic bioprinting will enable the recapitulation of a more complex and wider variety of biological models and scenarios, including the facile fabrication of multi-cellular thick tissues embedded with blood capillaries.