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Biomanufacturing

Biomanufacturing - Poster Session E

Poster M12 - Engineering Small Diameter Vascular Networks with Hollow Fibers Generated Using a 3D-Printed Coaxial Extrusion System

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  According to the American Transplant association, 104,000 people are currently on the waiting list to receive an organ transplant in the US. Modular tissue engineering seeks to bridge the gap by creating functional tissues that mimic organ structure using cells, biomaterials, and advanced manufacturing techniques. Since organs like the liver and kidney are highly vascularized, it's essential to produce hollow structures of different sizes to accurately model these vessels. While capillaries can be generated by angiogenic processes, formation of small arterioles and venules remains challenging. In particular, procedures for rapidly forming vessels with sub-millimeter diameters are needed to facilitate rapid assembly of vascularized modular tissues.

In this work, we expand on a previously reported method of generating hollow fiber structures using chitosan and glycosaminoglycans (GAG), by forming stable polyelectrolyte complex membranes using a custom built coaxial flow system. This method involves flowing a chitosan solution shell with a GAG solution core to create an insoluble complex hollow fiber. The diameter and wall thickness of the resulting fibers are dependent on parameters such as solution flow ratios, composition, and polymer molecular weights. This study aims to: (i) generate small diameter, hollow fiber structures suitable for submillimeter vessel development, and (ii) characterize the effects flow and reaction conditions on the fiber characteristics.

Materials and

Methods:  A single-piece coaxial extruder consisting of separate flow paths for the chitosan and GAG phases was designed using NX CAD software. The extruder was printed using clear resin with a Formlab 3+ printer at 50 𝜇m resolution without internal supports, and the prints were thoroughly rinsed with isopropyl alcohol and UV cured for 10 minutes. The extruder core and shell inlets were connected to separate syringe pumps for GAG and chitosan solutions respectively. The extruder was oriented vertically over a 10 petri dish containing 0.6 wt% chitosan solution.

The shell fluid (0.6 wt% chitosan in 1 wt% acetic acid) was perfused at 13 mL/min for preliminary experiments. The core GAG solutions tested consisted either 1 wt% Hyaluronic Acid (HA, 1800 kDa) 1.5 wt% HA,1 wt% HA + 2 wt% Chondroitin Sulfate (CS, 30-40 kDA), or 1.5 wt% HA + 2 wt% CS. All GAGs were dissolved in a HEPES-Sorbitol buffer and were extruded at flow rates ranging from 0.250-1.25 mL/min. The resulting continuous hollow fibers were transferred to a bath of 0.9 wt% saline for initial washing, after which they were stabilized by dipping in 0.1% sodium polygalacturonate (PGA). Fibers were imaged, and their diameters measured using ToupView software, and the resulting data analyzed using SigmaPlot. Scanning electron microscopy (SEM) imaging was used to characterize the fiber microstructure.

Results, Conclusions, and Discussions: Utilizing the Formlabs resin printer allowed the production of a single-piece extruder that effectively separated the reservoirs and flow paths of the two solutions, reducing clogging and backflow issues seen in earlier versions.

Hollow fibers were successfully created using the outlined testing conditions. Previous work indicated that a flow rate ratio of at least 10:1 between the chitosan and GAG phases should be maintained to ensure continuous membrane formation. Fibers created from 1.5 wt%HA + 1 wt% CS showed average diameters of 1885, 1070, and 775 microns under flow rates of 1.250, 0.750, and 0.250 mL/min respectively, with similar trends being observed with fibers created from 1 wt% HA and 1wt% HA + 2wt% CS. These initial results demonstrated that the hollow fiber diameter could be reliably controlled using the GAG solution flow rate. Repeated extrusions under the same conditions are ongoing to confirm consistency of dimensions.

SEM imaging of 1 wt% HA fibers showed that they had continuous internal surfaces suitable for cell attachment. It is anticipated that inclusion of cells in the the GAG phase and type I collagen in the chitosan phase may incorporate these elements into the hollow structure. Cell studies are ongoing and the results will be reported. Chitosan-GAG fibers have a variety of applications in modular tissue engineering, such as the creation of blood vessels and other hollow structures. The use of a coaxial extruder can also facilitate the inclusion of this technique in more complex bioprinting systems to achieve more complex geometries.

Acknowledgements (Optional):