The Better to Ear You With: Bioengineering Full-scale Auricles Using 3D-printed External Scaffolds and Decellularized Cartilage Xenograft

PURPOSE: Faithful reconstruction of the human auricle is a notorious challenge for the plastic surgeon. While the gold standard remains harvest and shaping of autologous costal cartilage, such procedures obligate donor site morbidity, scarring, and prolonged operative time, often coupled with suboptimal aesthetic results and biomechanical properties that bear little semblance to the structure they intend to replicate. In response, we have endeavored to bioengineer neo-ears utilizing decellularized ovine costal cartilage as a biocompatible xenograft placed within a full-scale, 3D-printed human ear external scaffold to foster tissue growth that predictably mimics the size, shape, and biomechanical properties of the native human auricle. METHOD: Polylactic acid (PLA) ear scaffolds were fabricated to match the anatomy of an adult human ear using 3D photo capture and subsequent modeling. All scaffolds were printed on a 3D printer (Prusa i3 MK3S) and sterilized. Ovine costal cartilage was isolated and processed either through mincing (1 mm3) or zesting (<2 mm3) and decellularized in-house through our usual protocol. Decellularized cartilage was packed into the ear scaffolds and implanted subcutaneously on the dorsa of immunocompetent Sprague-Dawley rats. After 3 and 6 months in vivo, the constructs were explanted for gross, histologic, biochemical, and biomechanical analyses. RESULTS: Upon de-molding, both the minced and zested neo-ears maintained the size and contour complexities of the native human ear through 6 months in vivo. Massing of minced and zested ears, respectively, revealed a 1.45 ± 0.09 fold increase (p=0.05) and 1.11 ± 0.07 fold increase in construct mass with respect to preimplantation mass after 3 months in vivo. Zested ears implanted for 6 months in vivo revealed a 1.20 ± 0.18 fold increase in construct mass over time, while preliminary explantation of a minced ear revealed a 1.21 fold mass increase after 6 months in vivo. Micro-CT scanning revealed de-caged auricular explant volumes of 4,237.9 mm3 and 4,838 mm3 for zested and minced ears after 3 months in vivo, while zested and minced ears after 6 months in vivo exhibited volumes of 4,053.2 ± 256.7 mm3 and 3,987.7 mm3, respectively. H&E staining confirmed a mild inflammatory infiltrate and corresponding fibrovascular tissue ingrowth enveloping individual cartilage particles, while safranin-O staining revealed an expected depletion of glycosaminoglycans (GAG) secondary to the decellularization process. All constructs were pliable and resumed their native conformation when twisted or bent and detailed biomechanical studies are ongoing. CONCLUSIONS: Utilization of decellularized ovine xenograft has proven highly efficacious at generating neo-ears that maintain their size and shape after 6 months in vivo. The use of scaffolds as a means of protecting grafted material after implantation allows for fibrovascular tissue formation between cartilage particles that successfully resists contractile forces, thereby producing a nonimmunogenic, full-scale construct that strongly resembles the adult human ear. Such constructs have demonstrated mass increases secondary to neotissue formation as well as grossly favorable biomechanical properties and deformability. Multiple ears fabricated using the same in vivo bioreactor approach will be explanted over the ensuing several months to provide further insight into construct longevity.