Benjamin P. Cohen

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

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. Verhoeffs 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.

Optimizing cell sourcing for clinical translation of tissue engineered ears.

Background . Currently, the major impediment to clinical translation of our previously described platform for the fabrication of high fidelity, patient-specific tissue engineered ears is the development of a clinically optimal cell sourcing strategy. A limited autologous auricular chondrocyte (AuC) supply in conjunction with rapid chondrocyte de-differentiation during in vitro expansion currently makes clinical translation more challenging. Mesenchymal stem cells (MSCs) offer significant promise due to their inherent chondrogenic potential, and large availability through minimally invasive procedures. Herein, we demonstrate the promise of AuC/MSC co-culture to fabricate elastic cartilage using 50% fewer AuC than standard approaches. METHODS Bovine auricular chondrocytes (bAuC) and bovine MSC (bMSC) were encapsulated within 10 mg ml-1 type I collagen hydrogels in ratios of bAuC:bMSC 100:0, 50:50, and 0:100 at a density of 25 million cells ml-1 hydrogel. One mm thick collagen sheet gels were fabricated, and thereafter, 8 mm diameter discs were extracted using a biopsy punch. Discs were implanted subcutaneously in the dorsa of nude mice (NU/NU) and harvested after 1 and 3 months. RESULTS Gross analysis of explanted discs revealed bAuC:bMSC co-culture discs maintained their size and shape, and exhibited native auricular cartilage-like elasticity after 1 and 3 months of implantation. Co-culture discs developed into auricular cartilage, with viable chondrocytes within lacunae, copious proteoglycan and elastic fiber deposition, and a distinct perichondrial layer. Biochemical analysis confirmed that co-culture discs deposited critical cartilage molecular components more readily than did both bAuC and bMSC discs after 1 and 3 months, and proteoglycan content significantly increased between 1 and 3 months. CONCLUSION We have successfully demonstrated an innovative cell sourcing strategy that facilitates our efforts to achieve clinical translation of our high fidelity, patient-specific ears for auricular reconstruction utilizing only half of the requisite auricular chondrocytes to fabricate mature elastic cartilage.

Tissue engineering the human auricle by auricular chondrocyte-mesenchymal stem cell co-implantation

Children suffering from microtia have few options for auricular reconstruction. Tissue engineering approaches attempt to replicate the complex anatomy and structure of the ear with autologous cartilage but have been limited by access to clinically accessible cell sources. Here we present a full-scale, patient-based human ear generated by implantation of human auricular chondrocytes and human mesenchymal stem cells in a 1:1 ratio. Additional disc construct surrogates were generated with 1:0, 1:1, and 0:1 combinations of auricular chondrocytes and mesenchymal stem cells. After 3 months in vivo, monocellular auricular chondrocyte discs and 1:1 disc and ear constructs displayed bundled collagen fibers in a perichondrial layer, rich proteoglycan deposition, and elastin fiber network formation similar to native human auricular cartilage, with the protein composition and mechanical stiffness of native tissue. Full ear constructs with a 1:1 cell combination maintained gross ear structure and developed a cartilaginous appearance following implantation. These studies demonstrate the successful engineering of a patient-specific human auricle using exclusively human cell sources without extensive in vitro tissue culture prior to implantation, a critical step towards the clinical application of tissue engineering for auricular reconstruction.

Abstract 129: Fabrication of the First Full-Scale Human Auricular Chondrocyte Derived Ear Scaffold for Clinical Application

PURPOSE: Trauma-induced heterotopic ossification (tHO) is the aberrant growth of ectopic bone in soft tissue, which develops in patients following severe musculoskeletal trauma. Much of HO literature focuses on a related pathology known as fibrodysplasia ossificans progressiva (FOP), which is caused by a hyperactivating mutation in the type I bone morphogenetic protein receptor (T1-BMPR) ACVR1 (ACVR1 R206H). Consequently, emphasis has been placed on developing inhibitors with improved specificity for ACVR1. However, patients who develop tHO do not harbor known ACVR1 mutations, and it is unclear whether emphasis on ACVR1-specific inhibition is beneficial for the treatment of tHO. Here investigate whether any single T1-BMPR is required for tHO, or whether these receptors perform overlapping roles during tHO development. We further evaluate the efficacy of the BMP ligand trap, Alk3Fc, as a broad-spectrum inhibitor of T1-BMP receptors in the treatment and prevention tHO.

Abstract: Utilizing a Novel Cell Sourcing Strategy to Fabricate the First Full-Scale Tissue Engineered Human Ear Scaffold

INTRODUCTION: Previously, we fabricated patient-specific auricles using bovine auricular chondrocytes, which displayed effective permanence with structural, biochemical, and mechanical properties similar to native auricular cartilage after 6 months in vivo. However, autologous tissue donation generates a limited cell yield, and further expansion of donor cells can negatively impact chondrogenic capacity. To overcome this challenge, we sought to generate human auricular cartilage through the combined implantation of human auricular chondrocytes (hAuC) with human mesenchymal stem cells (hMSC) as a novel cell sourcing strategy in order to fabricate the first full-scale human ear.

Abstract 94: Patient-Specific Tissue Engineered Constructs for Ear Reconstruction

PURPOSE: The current gold standard for reconstruction of pediatric microtia is autologous costal cartilage reconstruction. This method is limited due to morbidity at the donor site, failure to match auricular cartilage properties, and difficulty accurately recreating the ear morphology of the individual patient. We have previously demonstrated the capacity to fabricate high fidelity patient-specific ear constructs using digital photogrammetry and CAD/CAM techniques, and that these constructs formed auricular cartilage when implanted in vivo for up to 3 months. We have now applied the same methods for constructs implanted in vivo for 6 months.