3D Bioprinting of Human Chondrocyte-Laden Tracheal Construct Using the Freeform Reversible Embedding of Suspended Hydrogel Technique

Y. Zhu¹, C. Stark¹, S. Lee¹, S. Yajima¹, L. Lin¹, M. Vergel¹, A. Venkatesh¹, L. Doan¹, C. Wu¹, S. Elde¹, Y. Woo², N. S. Lui^1
1Stanford University, Stanford, California ²Stanford University School of Medicine, Stanford, California

Purpose: Long-segment tracheal defects are associated with significant morbidity and mortality. Complex reconstruction may be performed but outcomes remain poor. Tracheal transplantation, however, may be a viable therapeutic option but donor organs are limited. The objective was to evaluate feasibility of 3D bioprinting cell-laden tracheal constructs for in vivo implantation.

Methods: 5% (w/v) Gelatin methacryloyl and 8% (w/v) gelatin type A (GelMA/Gel-A) bioink was prepared containing 0.5% (w/v) lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and loaded into standard syringes for extrusion-based 3D bioprinting. Freeform reversible embedding of suspended hydrogel sacrificial support bath was also prepared according to our previously established protocol with 0.5% (w/v) LAP incorporated. Human chondrocytes at 10 million cells/mL concentration were mixed in the bioink for cellularized construct printing. Bioprinting and UV cross-linking parameters were optimized based on single filament resolution using light microscopy; construct mechanical properties such as Young’s modulus and suture retention force using a 7-0 polypropylene suture were assessed using a tensile tester; cell viability was evaluated via the live/dead assay. Cellularized printed constructs were further matured in vitro for 4 weeks (n=7) and in vivo in RNU nude rats behind the ears for 2 weeks (n=5). Matured constructs were harvested for further mechanical testing.

Results: The average single filament diameters printed with the speed of 0.5, 1, and 2 mm/s were 264.4+/-27.4, 263.2+/-52.7, and 258.4+/-71.8um under 30psi; and 312.9+/-71.1, 330+/-67.1, and 256.4+/-57.4um under 35psi (Fig.A). Chondrocyte viability at 30psi vs. 35psi was 58.7+/-3.7% vs. 60.6+/-5.3% (Fig.B). Extrusion pressure of 30psi and print speed of 1mm/s were selected for the remainder of the study. Chondrocyte viabilities were similar after 10 vs. 60 seconds of UV cross-linking at 62.9+/-3.5% vs. 61.7+/-2.6% but significantly decreased after 300 and 600 seconds of UV cross-linking to 53.0+/-4.2% and 44.8+/-4.1% (Fig.C). Average Young’s moduli of printed constructs in the ASTM geometry (Fig.D-F) after 10, 60, 300, and 600 seconds of UV were 42.7+/-2.8kPa, 127.3+/-11.1kPa, 806.3+/-613.7kPa, and 492.3+/-370.3kPa (Fig.G). Suture retention force increased from 7.7+/-2.5mN to 22.9+/-1.1mN, 112.1+/-27.1mN, and 162.7+/-13.2mN after 5, 60, 300, and 600 seconds of UV (Fig.H). 60 seconds of UV cross-linking duration was selected to allow for maximal cell viability. Compression modulus of constructs matured in vitro (Fig.I) vs. in vivo (Fig.J) was 14.0+/-2.9kPa vs. 10.9+/-4.9kPa. Suture retention force post in vitro vs. in vivo maturation was 8.4+/-3.3mN vs. 546.2+/-75.6mN.

Conclusion: 3D-bioprinted chondrocyte-laden tracheal constructs demonstrated appropriate cellular viability and mechanical properties to allow for surgical manipulation. In vivo implantation of the 3D-bioprinted trachea will be performed to further evaluate construct function and histology. This may represent a feasible therapeutic option for patients who require complex tracheal reconstruction.

Identify the source of the funding for this research project: Departmental Funding