Biomanufacturing
3D Printing Vasculature in a Polyethylene Glycol Nanofiber Granular Scaffold
Lauren G. Porter (she/her/hers)
4th Year Biomedical Engineering Undergraduate
University of Virginia
Springfield, Virginia, United States
Natasha L. Claxton
Graduate Student
University of Virginia
Charlottesville, Virginia, United States
Julia Tumbic (she/her/hers)
Graduate student
University of Virginia, United States
M. Greg Grewal
Graduate Student
University of Virginia, United States
Chris Highley
Assistant Professor
University of Virginia, United States
According to the Organ Procurement and Transplantation Network, there are currently over 100,000 people on the organ or tissue transplant waiting list in the United States alone (1). Unfortunately, organ transplantation is limited by donor shortage (2).
Towards addressing the unmet need for transplants, researchers have aimed to engineer artificial tissues and organs. Polymer hydrogel scaffolds have been widely researched as a basis for manufacturing engineered organs and tissues (3). These are engineered to contain needed biochemicals and cells, but important challenges remain. Vascularization of these hydrogel constructs is among the most critical of these. In vivo, native tissues are supplied with blood by a network of vessels, but most in vitro tissue models lack the hierarchical vascular networks seen in vivo.
Nanofibers will be created by electrospinning a 7 wt% PEG-thiol, 10 wt% PEG-norbornene, and 2.05 vol% thiolated arginine-glycine-aspartate (RGD) peptide solution. The fibers will be photocrosslinked, hydrated, and homogenized to produce fiber lengths of roughly 100 µm. The fibers will then be centrifuged to create a jammed granular hydrogel (Figure 1).
100 µL of the PEG hydrogel will be further hydrated in 1 mL of human umbilical vein endothelial cell (HUVEC) media overnight before cell experiments. The next day 500,000 HUVECs and 100,000 NIH 3T3 fibroblasts will be mixed into the hydrogel, and the resulting construct will be loaded into a custom-made polydimethylsiloxane (PDMS) culture device that is also compatible with our printing process.
For use as the 3D printing ink, 15 wt% gelatin microparticles will be prepared via batch emulsification in light mineral oil with 2% Span-80. The microgels will be jammed via centrifugation and loaded into a 100 µL syringe.
Gelatin ink will then be 3D printed into the granular hydrogel as a filament roughly 100 µm in diameter and 8 mm in length. To covalently stabilize the material around the printed channel, excess norbornenes will be photocrosslinked, and the construct incubated at 37 ℃ overnight, melting the gelatin and creating a perfusable channel (Figure 2). The following day, the media with melted gelatin will be aspirated off and 1 mL of fresh HUVEC media with added vascular endothelial growth factor (VEGF) will be added to media wells to drive the formation of vascular structures within the construct.
I hypothesize that, ultimately, vasculature will form as the media with VEGF flows throughout the channel. Cells adjacent to the channel will line the space left by the gelatin filament, and cells within the surrounding material will organize into a microvascular network surrounding a perfusable channel lined by cells. In the work described here, I focus on establishing the printing process in this new material system and characterizing the formation of a cell lined channel once gelatin is removed.
Using live/dead (Calcein AM/ethidium homodimer-1) staining, initial cell culture experiments showed approximately 85% cell viability at a 24-hour time point. This indicates that PEG nanofibers are a cyto-compatible support network. Creation of channels with diameters of roughly 200 µm was possible, as visualized with widefield imaging.
Ongoing work looks to create channels with smaller diameters by modifying needle size, printer extrusion rate, and feed rate. I also intend to print and seed more complex gelatin configurations including branching and reconnecting designs. I aim to expand upon existing rheological testing of the nanofibers (4) and to complete novel rheological testing on the gelatin ink to completely characterize the viscoelastic properties of both materials. Finally, we plan to complete supplemental cell culture experiments at longer timepoints to further study cell behavior within our PEG scaffold towards developing a microvascular network.
In conclusion, within this novel PEG nanofiber-based granular hydrogel, it is possible to define channels for vasculature by 3D printing. These vessels can be printed into materials that can be engineered to include endothelial cell lines at high viability. I expect it will be possible to use 3D printed gelatin as a “map” for where cells will form vasculature, allowing for the design of reproducible, precise, highly customizable vascular networks.