HI-FI Tissue Engineering: Custom 3D-Printed Cages with Anatomic Elements Prevent Loss of Volume and Topographic Detail of Engineered Auricular Cartilage In Vivo

PURPOSE: Autologous reconstruction of the ear, whether for microtia or acquired deformity, is a complex procedure with substantial donor site morbidity and frequent suboptimal aesthetic outcomes. An engineered auricular scaffold would obviate donor morbidity and provide improved aesthetic outcomes. Currently, clinical translation of tissue-engineered auricles is complicated by the significant contraction and loss of topography that occurs during maturation of the cell-seeded hydrogel into elastic cartilage. Previously, we demonstrated that a 3D-printed biodegradable cage significantly mitigated contraction of simple disc-shaped collagen hydrogels seeded with human auricular chondrocytes (HAuCs) in vivo without impeding the development of elastic cartilage. Herein we fabricate cages to invest chondrocyte-collagen hydrogels with more intricate “anatomic” topographic features.

 

METHODS: Custom external cages were designed with a geometric element representative of the helical rim using SolidWorks (Dassault Systèmes, Vélizy-Villacoublay, France), then 3D-printed using polylactic acid (PLA) on a 5^th generation MakerBot printer (MakerBot, New York, NY). Using auricular cartilage from freshly slaughtered 1-3d old calves, bovine auricular chondrocytes (BAuCs) were harvested and expanded to passage 3. The chondrocytes were then encapsulated at a density of 25 million cells per mL into type I collagen hydrogels with high fidelity contour matching to the cages. The hydrogels, either protected or unprotected by the PLA cages, were implanted into nude rats and explanted after 3 months.

 

RESULTS: After 3 months in vivo, all constructs developed a glossy white cartilaginous appearance, similar to native auricular cartilage. Histologic analysis demonstrated development of an organized perichondrium composed of collagen, a rich proteoglycan matrix, cellular lacunae, and a dense elastin fibrin network by safranin-O and Verhoeff’s stain. Biochemical analysis confirmed similar amounts of proteoglycan and hydroxyproline content in the constructs when compared to native auricular cartilage. Cage-protected constructs contracted significantly less than unprotected constructs on base area comparison (14.33% vs. 56%, p = 0.0023), retained volume (213.4mm³ vs. 117.2mm³, p = 0.0290), and maintenance of the topographic “helical rim” feature compared to unprotected constructs. Constructs were imaged via computed tomography with an Inveon Pre-clinical MicroPET/CT/SPECT (CTI/Siemens, Knoxville, TN), then digitally reconstructed with Imaris (Bitplane, Belfast, UK). Preservation of the “helical rim” feature was evaluated subjectively by gross examination and objectively by measuring the angle between the rim and base of the constructs. There was significantly more flattening  of the helical rim element in the unprotected constructs versus caged ones (197.7° vs. 151.8 °, p = 0.0445).

 

CONCLUSIONS: We have shown that custom contour matched 3D-printed biocompatible/biodegradable external cages significantly mitigate contraction and maintain the complex topography of BAuC constructs without impeding the formation of mature elastic cartilage. This technique can be used to create custom cages that contour to any form, enabling the fabrication of engineered autologous cartilage tailored to individual patient anatomy, without the significant contraction and loss of topography that has thus far impeded translation of this technology to the clinic.