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Updated: Oct 3, 2025
Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model
Published on: April 1, 2019
A two-stage in vivo approach for implanting a 3D printed tissue-engineered tracheal replacement graft: A proof of
Lidia Frejo1, Todd Goldstein2, Pooja Swami2
1The Feinstein Institutes for Medical Research, Manhasset, NY, USA; Division of Pediatric Otolaryngology, Steven and Alexandra Cohen Children's Medical Center, New Hyde Park, NY, USA.
Objectives:
To optimize a 3D printed tissue-engineered tracheal construct using a combined in vitro and a two-stage in vivo technique.
Methods:
A 3D-CAD (Computer-aided Design) template was created; rabbit chondrocytes were harvested and cultured. A Makerbot Replicator™ 2x was used to print a polycaprolactone (PCL) scaffold which was then combined with a bio-ink and the previously harvested chondrocytes. In vitro: Cell viability was performed by live/dead assay using Calcein A/Ethidium. Gene expression was performed using quantitative real-time PCR for the following genes: Collagen Type I and type II, Sox-9, and Aggrecan. In vivo: Surgical implantation occurred in two stages: 1) Index procedure: construct was implanted within a pocket in the strap muscles for 21 days and, 2) Final surgery: construct with vascularized pedicle was rotated into a segmental tracheal defect for 3 or 6 weeks. Following euthanasia, the construct and native trachea were explanted and evaluated.
Results:
In vitro: After 14 days in culture the constructs showed >80% viable cells. Collagen type II and sox-9 were overexpressed in the construct from day 2 and by day 14 all genes were overexpressed when compared to chondrocytes in monolayer.
In Vivo:
By day 21 (immediately before the rotation), cartilage formation could be seen surrounding all the constructs. Mature cartilage was observed in the grafts after 6 or 9 weeks in vivo.
Conclusion:
This two-stage approach for implanting a 3D printed tissue-engineered tracheal replacement construct has been optimized to yield a high-quality, printable segment with cellular growth and viability both in vitro and in vivo.
