Long-Term Morphological and Microarchitectural Stability of Tissue-Engineered, Patient-Specific Auricles In Vivo

Abstract

Current techniques for autologous auricular reconstruction produce substandard ear morphologies with high levels of donor-site morbidity, whereas alloplastic implants demonstrate poor biocompatibility. Tissue engineering, in combination with noninvasive digital photogrammetry and computer-assisted design/computer-aided manufacturing technology, offers an alternative method of auricular reconstruction. Using this method, patient-specific ears composed of collagen scaffolds and auricular chondrocytes have generated auricular cartilage with great fidelity following 3 months of subcutaneous implantation, however, this short time frame may not portend long-term tissue stability. We hypothesized that constructs developed using this technique would undergo continued auricular cartilage maturation without degradation during long-term (6 month) implantation. Full-sized, juvenile human ear constructs were injection molded from high-density collagen hydrogels encapsulating juvenile bovine auricular chondrocytes and implanted subcutaneously on the backs of nude rats for 6 months. Upon explantation, constructs retained overall patient morphology and displayed no evidence of tissue necrosis. Limited contraction occurred in vivo, however, no significant change in size was observed beyond 1 month. Constructs at 6 months showed distinct auricular cartilage microstructure, featuring a self-assembled perichondrial layer, a proteoglycan-rich bulk, and rounded cellular lacunae. Verhoeff's staining also revealed a developing elastin network comparable to native tissue. Biochemical measurements for DNA, glycosaminoglycan, and hydroxyproline content and mechanical properties of aggregate modulus and hydraulic permeability showed engineered tissue to be similar to native cartilage at 6 months. Patient-specific auricular constructs demonstrated long-term stability and increased cartilage tissue development during extended implantation, and offer a potential tissue-engineered solution for the future of auricular reconstructions.

FIG. 1.

Ex vivo gross analysis of injection molded ear before implantation (A) and explanted following 6 months in vivo (B) displaying maintained anatomic fidelity. Scale bar = 1 cm. Color images available online at www.liebertpub.com/tea

FIG. 2.

Ex vivo horizontal cross-section through the center of an ear construct 6 months after implantation displaying healthy, living tissue through full thickness. Scale bar = 1 cm. Color images available online at www.liebertpub.com/tea

FIG. 3.

Analysis of explanted construct length and width. Both the length (A) and width (B) of the postharvest constructs displayed no change following 1 month in vivo, but the dimensions of all explanted constructs decreased compared to preimplantation 0-month constructs n = 3–5, p < 0.05.

FIG. 4.

Histological staining of constructs harvested after 1 (A, E, I), 3 (B, F, J) and 6 months (C, G, K) compared to native bovine auricular cartilage (D, H, L). Safranin O staining (with Fast Green counterstain) (A–D) displayed increasing proteoglycan deposition and development of cellular lacunae. Picrosirius Red staining (E–H) displayed the formation of bundled collagen fibers forming a PC layer. Verhoeff's stain (I–L) indicates increasing development of EF in the tissue bulk. Microstructure of both 3- and 6-month constructs appears similar to native tissue (scale bar = 100 μm). EF, elastin fibers; PC, perichondrial. Color images available online at www.liebertpub.com/tea

FIG. 5.

Biochemical analysis of constructs and native auricular cartilage normalized to dry weight. DNA (A) rapidly decreased after 1 month in vivo, but steadily rose to approach native amounts. Both GAG (B) and hydroxyproline (C) composition increased in vivo, reaching similar values to native cartilage content by 3 months (n = 4–6). %, Significant difference from 0 month; #, significant difference from 6 month; *, significant difference from native (p < 0.05). GAG, glycosaminoglycan. Color images available online at www.liebertpub.com/tea

FIG. 6.

Mechanical properties of ear constructs and native auricular cartilage. The aggregate modulus of implanted ears were significantly higher after 3 months than the preimplanted constructs and no difference existed between 3 months, 6 months, and native tissue (A). The hydraulic permeability decreased significantly after both 1 and 3 months in vivo (B) (n = 4–6). %, Significant difference from 0 month; &, significant difference from 1 month (p < 0.05). Color images available online at www.liebertpub.com/tea