Formation of elastic cartilage in the shape of an auricle using 3D printing. Potential use for auricular reconstruction.

Abstract

Auricular reconstruction using 3D printing and tissue engineering techniques could be an alternative for reconstructing the ear in conditions such as microtia, a congenital disease characterized by the total or partial absence of the auricle. The use of costal hyaline cartilage is the standard procedure for surgical auricular reconstruction; however, side effects such as pneumothorax, chest cavity deformity, and respiratory problems have been reported. The use of 3D-printed structures in the shape of an auricle could offer an alternative for developing support structures that, in addition to simulating the shape of the ear, can be used to generate auricular elastic cartilage.

Objective: To develop a biocompatible 3D-printed structure in the shape of a human auricle, using microtic elastic cartilage, as a potential alternative for auricular reconstruction.

Materials and methods: Auricular chondrocytes were isolated from remnants of patients with microtia using enzymatic mechanotherapy techniques, after obtaining signed informed consent. Chondrocytes were expanded in vitro, and their cell viability was assessed. The chondrocytes were seeded onto three-dimensional polycaprolactin (PCL) scaffolds shaped like a human auricle, printed using CAD/CAM at the Wake Forest Institute for Regenerative Medicine, and implanted into athymic mice. The extracellular matrix (ECM) of the newly formed tissue was evaluated using histological techniques. The presence of elastin and type II collagen was confirmed by immunofluorescence. The shape and characteristics of the auricle were assessed to determine its potential for auricular reconstruction in patients with microtia. Statistical analyses were performed using one-tailed ANOVA and Student's t-test. A p-value < 0.05 was considered statistically significant.

Results: Chondrocytes were isolated from auricular remnants of microtia, expanded in vitro, and their chondral phenotype was confirmed during in vitro expansion and in the neotissue by PCR. The in vivo assay to evaluate tissue neoformation demonstrated that after four months of implantation, only the 3D PCL auricle seeded with auricular chondrocytes retained its shape, with the helix, lobule, tragus, and triangular fossa becoming visible. The skin of the implanted area adhered to the anatomy of the 3D auricle, and no necrosis was observed. Histological and semi-quantitative analysis using Masson and Verhoeff-Van Gieson stains showed that the extracellular matrix (ECM) formed by the chondrocytes was rich in proteoglycans (67%), collagen fibers (69%), and elastic fibers (40%). The chondrocytes were observed organizing into isogenic groups. Semi-quantitative protein analysis using immunofluorescence demonstrated that 70% of the neotissue expressed type II collagen and 60% elastin.

Conclusion: A three-dimensional auricle made of elastic cartilage was obtained, with potential applications for auricular reconstructions in pediatric patients using microtic tissue. This strategy would represent less risk to the patient, as well as fewer surgeries and complications in the future, due to the use of autologous cells.