High-Fidelity Tissue Engineering of Patient-Specific Auricles for Reconstruction of Pediatric Microtia and Other Auricular Deformities

Abstract

IntroductionAutologous techniques for the reconstruction of pediatric microtia often result in suboptimal aesthetic outcomes and morbidity at the costal cartilage donor site. We therefore sought to combine digital photogrammetry with CAD/CAM techniques to develop collagen type I hydrogel scaffolds and their respective molds that would precisely mimic the normal anatomy of the patient-specific external ear as well as recapitulate the complex biomechanical properties of native auricular elastic cartilage while avoiding the morbidity of traditional autologous reconstructions.MethodsThree-dimensional structures of normal pediatric ears were digitized and converted to virtual solids for mold design. Image-based synthetic reconstructions of these ears were fabricated from collagen type I hydrogels. Half were seeded with bovine auricular chondrocytes. Cellular and acellular constructs were implanted subcutaneously in the dorsa of nude rats and harvested after 1 and 3 months.ResultsGross inspection revealed that acellular implants had significantly decreased in size by 1 month. Cellular constructs retained their contour/projection from the animals' dorsa, even after 3 months. Post-harvest weight of cellular constructs was significantly greater than that of acellular constructs after 1 and 3 months. Safranin O-staining revealed that cellular constructs demonstrated evidence of a self-assembled perichondrial layer and copious neocartilage deposition. Verhoeff staining of 1 month cellular constructs revealed de novo elastic cartilage deposition, which was even more extensive and robust after 3 months. The equilibrium modulus and hydraulic permeability of cellular constructs were not significantly different from native bovine auricular cartilage after 3 months.ConclusionsWe have developed high-fidelity, biocompatible, patient-specific tissue-engineered constructs for auricular reconstruction which largely mimic the native auricle both biomechanically and histologically, even after an extended period of implantation. This strategy holds immense potential for durable patient-specific tissue-engineered anatomically proper auricular reconstructions in the future.

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

“…30,31 This collagen solution was immediately mixed with cells suspended in media and injected into ear molds as previously described. 25 Briefly, an 8 mL collagen-cell mixture, with a final collagen concentration of 10 mg/mL and a final cell concentration of 25 · 10 6 cells/ mL, 13 was injected into the molds and allowed to gel for 50 min at 37°C. After 50 min, the ear constructs were removed from the molds and cultured for 3-5 days in media composed of DMEM, 10% fetal bovine serum (Gemini Bio Products, Sacramento, CA), 100 mg/mL penicillin (Mediatech, Inc.), 100 mg/mL streptomycin (Mediatech, Inc.), 0.1 mM nonessential amino acids (Gibco, Grand Island, NY), 50 mg/mL ascorbic acid (Sigma-Aldrich, St. Louis, MO), and 0.4 mM l-proline (Sigma-Aldrich) before implantation.…”

“…25 Briefly, 10-week-old male athymic nude rats (RNU; Charles River, Wilmington, MA) were anesthetized through intraperitoneal injection of ketamine (80 mg/kg) and xylazine (8 mg/kg). The surgical sites were shaved and prepped and all animals received a subcutaneous injection of buprenorphine (0.1 mg/kg) and an intraperitoneal injection of cefazolin (11 mg/kg) before surgical manipulation.…”

“…Following 3 months in vivo, these constructs maintained detailed morphological characteristics and developed structural features and mechanical properties similar to native auricular cartilage. 25 Although our previous study demonstrated the potential of utilizing photogrammetric and CAD/CAM methods to produce patient-specific molds and constructs, the long-term stability (greater than 3 months) of the developing tissue in vivo has not been verified. In general, few studies utilizing complete human ear geometries have exceeded this time point, 3,6,10,12,23,26 and none of these featured a collagen scaffold.…”

“…Our group has previously applied digital photogrammetry and computer-assisted design/computer-aided manufacturing (CAD/CAM) techniques to fabricate molds replicating juvenile patient ears, a novel technique to recreate specific ear morphology. 25 Ear constructs were then produced by injection molding of auricular chondrocytes encapsulated within high-density type I collagen hydrogels, which were implanted in vivo without external stenting or internal synthetic components. Following 3 months in vivo, these constructs maintained detailed morphological characteristics and developed structural features and mechanical properties similar to native auricular cartilage.…”

Biomimicry, Biofabrication, and Biohybrid Systems: The Emergence and Evolution of Biological Design

“…Recently, Spector and co-workers used 3D extrusion bioprinting to print a cell-laden hydrogel construct in the shape of a human ear, demonstrating that advanced manufacturing approaches can be used to accurately manufacture patient-specific replacements for cartilage tissue. [80] Tissue engineering of vascularized tissue, with an ongoing challenge, has also been investigated and demonstrated in a range of tissue types. Taylor and co-workers demonstrated a seminal example of reverse engineering a macroscale vascularized organ by manufacturing a contracting heart in vitro.…”