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Biofabrication of a shape-stable auricular structure for the reconstruction of ear deformities

Bioengineering of the human auricle remains a significant challenge, where the complex and unique shape, the generation of high-quality neocartilage, and shape preservation are key factors. Future regenerative medicine–based approaches for auricular cartilage reconstruction will benefit from a smart combination of various strategies. Their approach to the fabrication of an ear-shaped construct uses hybrid bioprinting techniques, a recently identified progenitor cell population previously validated biomaterials, and a smart scaffold design. Specifically, they generated a 3D-printed polycaprolactone (PCL) scaffold via fused deposition modeling, photocrosslinked a human auricular cartilage progenitor cell–laden gelatin methacryloyl (gelMA) hydrogel within the scaffold, and cultured the bioengineered structure in vitro in chondrogenic media for 30 days. Their results show that the fabrication process maintains the viability and chondrogenic phenotype of the cells, that the compressive properties of the combined PCL and gelMA hybrid auricular constructs are similar to native auricular cartilage, and that biofabricated hybrid auricular structures exhibit excellent shape fidelity compared with the 3D digital model along with deposition of cartilage-like matrix in both peripheral and central areas of the auricular structure. Their strategy affords an anatomically enhanced auricular structure with appropriate mechanical properties, ensures adequate preservation of the auricular shape during a dynamic in vitro culture period, and enables chondrogenically potent progenitor cells to produce abundant cartilage-like matrix throughout the auricular construct. The combination of smart scaffold design with 3D bioprinting and cartilage progenitor cells holds promise for the development of clinically translatable regenerative medicine strategies for auricular reconstruction.



Regenerative medicine (RM) is a promising strategy for the future treatment of auricular cartilage defects and congenital malformations. It typically applies a combination of cells, materials, and bioactive factors to engineer a new tissue or stimulate the regeneration of native tissues. Current surgical strategies for auricular reconstruction use autologous costal cartilage for shaping the implant framework, the generation of neocartilage in the laboratory would obviate the need for a large harvest site and thus reduce associated morbidity. In addition, RM techniques have the potential to further mimic the structural and functional complexity of native tissue. Compared to the rigid costal cartilage framework or the synthetic alternative porous polyethylene, the engineered auricular implant should ideally exhibit biochemical and mechanical properties that are more similar to the native elastic cartilage. The first clinical trial with tissue-engineered ear-shaped constructs implanted in five children presents encouraging preliminary outcomes.



Nevertheless, engineered auricular constructs still face a number of challenges before they become viable alternatives for currently applied reconstructive strategies. The human auricle presents a complex structure that is difficult to fabricate and maintain. Firstly, its unique shape requires a patient-specific approach while highlighting the anatomical details to ensure an aesthetically satisfactory result after implantation under the cranial skin. Secondly, the maintenance of that shape should be ensured for a lifetime, requiring excellent cellular performance and a long-term balance between stiffness and flexibility. This means that cartilage matrix deposition should be abundant and appropriately organized to properly mimic the native tissue's microscopic anatomy and biomechanical properties. However, especially during the first stages of tissue development and maturation, these properties are inferior to the native situation, as well as deformation and collapse are frequently reported in long-term in vivo studies evaluating tissue-engineered ear-shaped constructs. Necrosis due to nutrient limitation and inferior mechanical integrity of the developing neo-tissue may be contributing factors to this deformation and collapse. Third, the production of a large structure requires a significant number of autologous cells. Native chondrocytes lose their chondrogenic phenotype upon repeated expansion and subsequently produce a more fibrocartilage-like matrix. Mesenchymal stromal cells (MSCs) are readily expandable but may favor hypertrophic differentiation and the endochondral ossification pathway, resulting in a mineralized matrix that may lead to rigidity and implant extrusion. These challenges taken together indicate that auricular cartilage reconstruction using RM-based approaches would benefit from customized patient-specific shapes with adequate reinforcement, potent regenerative cells, and improved tissue quality before translation to daily clinical practice would be suitable. An additional challenge in the application of a tissue-engineered cartilage implant for the reconstruction of microtia is that patients have limited cranial skin available to sufficiently cover the implant. The high mechanical integrity of the implant is required to withstand the contractive forces of the overlying skin.


Biofabrication-based RM uses additive manufacturing technology with cells and supporting materials as building blocks to create living structures, with the goal to recapitulate and restore functions of native tissues. Through a computer-aided design and computer-aided manufacturing (CAD/CAM) process, patient-specific and customizable shapes can be generated. The technology's ability to deposit multiple materials with high control over the structural organization allows the fabrication of complex external and internal architectures. Reinforcing scaffolds can be combined with cell-laden hydrogels to create hybrid constructs with improved performance on a biochemical and biomechanical level compared with traditional tissue engineering strategies. In regard to cell type, cartilage progenitor cells hold great potential as an alternative to chondrocytes or MSCs. These cells can be harvested through a small biopsy from the patient's normal external ear or cartilage remnants on the affected side and subsequently expanded to high cell numbers while maintaining a potent chondrogenic differentiation capacity.


The way toward clinical application of engineered cartilage may well be a combination of various strategies. In view of this, their study combined smart scaffold design with a progenitor cell population for the biofabrication of an auricular cartilage structure. For the first time, they applied a population of novel human auricular cartilage progenitor cells (AuCPCs) in bioprinting with the objective to fabricate an ear-shaped construct. They evaluated cellular performance after the printing process and determined an appropriate reinforcing scaffold design to support the mechanical properties of the developing cartilage. They designed the auricular shape to match current surgical strategies to emphasize the native anatomical details and coprinted AuCPC-laden gelatin methacryloyl (gelMA) hydrogel with an ear-shaped, reinforcing polycaprolactone (PCL) framework. These dual-printed auricular constructs were cultured in vitro and subsequently assessed on shape fidelity and biochemical composition. The hybrid fabrication of a mechanically reinforced and anatomically emphasized structure in combination with chondrogenically potent AuCPCs provides an interesting avenue for the development of clinically translatable cartilage RM strategies1.


I.A. Otto, P.E. Capendale, J.P. Garcia, M. de Ruijter, R.F.M. van Doremalen, M. Castilho, T. Lawson, M.W. Grinstaff, C.C. Breugem, M. Kon, R. Levato, J. Malda,

Biofabrication of a shape-stable auricular structure for the reconstruction of ear deformities,

Materials Today Bio,

Volume 9,

2021,

100094,

ISSN 2590-0064,

https://doi.org/10.1016/j.mtbio.2021.100094.