(J-363) 3D Bioprinting of Tissue Engineered Vascular Graft with Collagen-Based Bioink

Introduction:: Congenital heart disease is the leading cause of newborn motility and requires reconstructive surgery for affected patients. The development of an effective vascular graft composed of natural material and growth potential for in-vivo transplantation is in great demand for congenital defect repair. However, current vascular grafts either employ synthetic polymers susceptible to thromboembolism, stenosis, and infection or involve an extended culture period (8 weeks) for cell remodeling, hindering mass production and rapid deployment. Three-dimensional (3D) bioprinting is a novel tissue engineering technology that allows precise deposition of cells and extracellular matrix (ECM) materials, including natural ECM components such as type 1 collagen. Previously we have developed a rapid collagen gelation technique that can produce various 3D cell-laden structures using low-concentration collagen and physiologically relevant cell density. Therefore, we propose to combine this technique with extrusion-based embedded 3D bioprinting to manufacture biocompatible tissue-engineered vascular graft (TEVG) composed of natural ECM. We hypothesize that collagen-based TEVG with high printing fidelity and resolution can be successfully 3D printed in a high throughput manner. The resulting TEVG retains its structural integrity and features a smooth surface topography, allowing for endothelialization and perfusion. The TEVG also exhibits sufficient initial mechanical durability, indicating its potential use for in vivo implantation in the future.


Materials and Methods:: A novel granular slurry-based support bath that promotes rapid collagen gelation, termed RapidBath, was created for extrusion-based embedded 3D bioprinting. The rheological behavior of the support bath was evaluated. Both acellular and cell-rich bioinks were prepared using either acid-solubilized type I rat tail collagen or low-concentration pre-neutralized collagen incorporating human primary vascular smooth muscle cells (VSMCs) at a final concentration of 10⁶ cells/mL. Tube models with various aspect ratios were designed in SolidWorks, exported as a stereolithography file, then sliced and printed in a layer-by-layer manner using a pneumatic bioprinter Cellink BioX6 within the support bath. The printed structure was allowed to gel, extracted, and washed with PBS, and its geometric accuracy was evaluated. The acellular TEVG was tuned via dehydration and crosslinking to modulate its mechanical property. The cell-rich TEVG was cultured in the TEVG medium that promotes ECM deposition for up to five weeks. The structural integrity of the printed TEVG was evaluated by the perfusion of food dye through its lumen. Suture retention strength and rupture pressure were evaluated to assess the biomechanical robustness of the bioprinted TEVG. VSMCs markers MYH11 and α-SMA staining was used to confirm the presence of VSMCs, and KI67 positive cell quantification was used to verify VSMCs proliferation.

Results, Conclusions, and Discussions:: In this study, we developed a novel granular support bath, RapidBath, for collagen bioprinting. We characterized the mechanical properties of the bath, with the resulting support bath exhibiting a yield stress fluid behavior, as demonstrated by the storage (G’) and loss (G’’) moduli. Using this support bath, we successfully printed tubes at various aspect ratios using either acellular or cell-rich collagen bioink in a free-standing way, and extracted them from the bath easily after short gelation periods (within minutes). The printed TEVG was highly versatile with various wall thicknesses that could be tuned by printing with different combinations of extrusion settings and nozzle types. We were able to achieve a TEVG with a minimum inner diameter of 1.5mm, a maximum length of 3cm, and wall thicknesses ranging from 0.2 to 0.5mm, which was suitable for rat abdominal aortic implantation. The printed structure closely matched the original computer-designed object, indicating the combination of rapid collagen gelation technique and embedded 3D bioprinting locks the extruded bioink at the desired location. For TEVG composed of acellular bioink, post-printing modifications enhanced mechanical properties including suture retention and rupture pressure compared to that of TEVG direct after printing. TEVG containing SMCs cultured in TEVG medium for 5 weeks also exhibited high cell viability, maintenance of VSMC properties, increased collagen production and mechanical strength. The results demonstrated that our bioprinted TEVG holds great potential for rat implantation.



This study presents a 3D bioprinting technique for the rapid and precise fabrication of TEVGs using biocompatible natural ECM material and physiologically relevant cell densities. The resulting TEVG exhibited high geometric accuracy and mechanical robustness, making them suitable for in vivo rat implantation. Future work will involve endothelializing the printed TEVGs and characterizing their in vivo performance in rats. Overall, the study provides promising results for meeting the clinical demands for functional and durable vascular grafts.




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