Custom 3D-Printed External Biocompatible Cage Mitigates Contraction During Maturation of Human Auricular Cartilage Scaffolds
Jaime L. Bernstein, New York1, Benjamin P. Cohen, MS2, Julia Jin, BS1, Yoshiko Toyoda1, Alice Harper, BA1, Lawrence J. Bonassar, PhD2, Jason A. Spector, MD1.
1Weill Cornell Medical College, New York, NY, USA, 2Cornell University, Ithaca, NY, USA.
BACKGROUND: In previous work we successfully fabricated full-scale human ear scaffolds that are histologically, biochemically, and biomechanically indistinguishable from native human auricular cartilage. Despite these successes, obstacles remain prior to clinical translation of our auricular cartilage scaffold—specifically we must address the contraction and loss of intricate topographic features that occurs during their in vivo implantation/maturation phase. It is known that tension forces generated by skin and other surrounding tissues contribute to the contraction and loss of topography of an implanted collagen hydrogel. In order to mitigate these undesirable effects, we fabricated a custom designed 3D-printed external cage to shield our scaffolds from naturally occurring external forces and implanted the "caged" constructs in vivo.
METHODS: Human Auricular Chondrocytes were isolated from otoplasty specimens and encapsulated (25 million cells/mL) into 8mm disc type I collagen hydrogels (10 mg/ml). Custom external cages were designed using a combination of Spaceclaim and Sketch-up, which allowed us to create custom stereolithography or STL files with precise measurements and high-fidelity contour matching to the hydrogels. The STL files were then optimized for 3D printing utilizing a makerbot replicator 5th generation and Fortus 250mc and subsequently printed with biocompatible polylactic acid (PLA) with a resolution of 100 microns. The hydrogels were placed into the cages and implanted into the dorsum of nude mice and explanted after 1 and 3 months in vivo.
RESULTS: The external PLA cages were able to maintain their shape and strength after 3 months in vivo, providing the protection the hydrogels needed for undisturbed formation of neocartilage. The discs maintained in the external cages contracted on average only 4.69% in diameter, which is significantly less contraction than unshielded human auricular cartilage constructs, which contract at least 25% in diameter (p<0.0001). Within the cage, the chondrocyte-collagen discs developed a shiny white cartilage-like appearance, similar to native auricular cartilage. Histologically, the cartilage produced from the discs was indistinguishable from native auricular cartilage. Staining with picrosirius red demonstrated organized collagen fibers forming a perichondrial layer. Safranin-O stain revealed a rich proteoglycan matrix with cellular lacunaue, and a dense elastin fiber network was seen with Verhoeff's stain. Mechanical and biochemical testing are pending to further characterize the human auricular cartilage formed in vivo.
CONCLUSIONS: We have shown that a custom 3D-printed external cage comprised of a biocompatible material, can be used to mitigate contraction of our auricular chondrocyte scaffolds without compromising the formation of elastic neocartilage. We believe the same technique can be applied to create cages that faithfully conform to the contour of full-scale auricular scaffolds to minimize contracture, a major impediment to the translation of ear tissue engineering. Further, because the cages are 3D printed, patient specific data will allow for rapid prototyping of patient specific cages.
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