Development of collagen microencapsulation-based strategy to cultivate human auricle chondrocytes for ear cartilage tissue engineering : from micro-tissues to complex shaped implantable constructs

Abstract

Among all plastic surgeries, auricle reconstruction is one of the most challenging one as it requires the fabrication of a patient-specific and complex shaped ear implant that could integrate with the tissues at the surgery site. The current gold standard for ear implant fabrication requires sculpting patient’s rib cartilage biopsies by the surgeon since autologous grafts are better for implant-host integration. Unfortunately, this approach often causes damage to the donor rib cartilage and presses huge demand on the surgeon’s aesthetic skills. This tedious and painful surgery is, however, unavoidable to many microtia patients as to restore their normal ear appearance and alleviate their psychological burden. Tissue engineering offers an excellent alternative for making the complex shaped ear cartilage in vitro without the need of rib biopsy. In this study, a bottom-up strategy was devised with optimization to generate an ear graft with auricular cartilage-like phenotype. Using collagen microencapsulation, auricular chondrocytes isolated from patient’s deformed ear remnants was able to generate three-dimensional microtissues with the ability to fuse into a larger tissue, and a cartilage-like phenotype could be achieved, though less mature compared with the native tissue. The microtissue fabrication method was therefore further modified by reducing collagen concentration or incorporation of hyaluronic acid in the collagen microencapsulation platform. Reducing collagen concentration was found to be effective in enhancing the cartilage phenotype with improved cartilage matrix deposition and better mechanical properties, and the best results appeared to be coming from cell aggregates without any collagen input. Although the microtissues still appeared to be immature in vitro, they could further mature in vivo and generate a matrix with composition and mechanical properties comparative to the native auricular cartilage, and was especially the case for microtissues with extremely low or no collagen input. Based on the observation that auricular chondrocyte aggregates seemed to be the best microtissue fabrication method, these aggregates were used to made an ear-shaped cartilage construct. In address to the difficulties in large scale chondrocyte aggregate production and handling through the previous hanging drop method which involve individual droplet dispense, a polydimethylsiloxane device with arrays of mini-sized wells was designed and fabricated. With the use of the device, large sum of cell aggregates could be made with a single cell solution addition step, and the microtissues did not obviously differ from the previous hanging drop protocol, with even slightly better cell viability probably due to milder handling procedure. Finally, using the auricular chondrocytes aggregates and moulding them in an ear mould made with assistance from computer modelling and additive manufacturing, an ear-shaped construct with cartilaginous matrix was made. While more studies might be needed to explore the possibilities of maturating the engineered cartilage tissue in vitro, maintaining their phenotype and dimension in vivo, as well as to improve the design of the well array device or the microtissue fabrication protocol to enable a more effective large-scale production, this study could serve as the basis for fabricating full-size clinically applicable ear implant. (490 words)