| Reconstruction of the total external ear remains one of the most difficult challenges in reconstructive surgery. Although the auricle represents only a minority of the total body surface area, it is one of the most complex three-dimensional structures of the external body. The ability to construct a fully satisfactory complete external ear has been an elusive goal for many years.Auricular reconstruction methods includes:prosthetics and reconstructive surgery. A continuous debate over the two major surgery materials alloplastic prosthesis implantation versus autologous cartilage grafts is still ongoing.The advantages of alloplastic implants, such as silicone or polypropylene , include widespread availability,consistent predetermined shape, and shortened operation time. However, risks of infection, extrusion,biocompatibility, and uncertain long-term durability are major concerns. Autologous costal cartilage is the most commonly used and preferred material for total external ear reconstruction. Nevertheless, technical skills of the surgeon are of paramount importance. It also has the disadvantages of being less consistent, extensive operation time, and donor site morbidity.With the advances both in surgical technique and biotechnology, tissue engineering cartilage became the hot topic of the auricular reconstruction field. Although investigators have demonstrated intricate three-dimensional tissue engineered structures in the shape of a human ear with excellent initial cosmetic detail, the architecture could not be maintained over time.The goal of auricle reconstruction is not only to produce neocartilageous tissue, but also to create a detailed three-dimensional frame work that can be maintained over a long period of time when possible.In fact, so far no perfect materials have been found to substitute the shapely elastic cartilage normally present in the ear.Therefore,new system to fabricate tissue engineering auricular cartilage is necessary.In this article, a new concept for auricle framework manufacturing is presented. We described polyurethane frameworks created by FDM and investigated the feasibility of the feasibility of cartilage regeneration in vitro and in vivo culture.The content of this study includes four parts summurized as following.1. Fabrication of auricular polyurethane scaffolds and assessment of mechanical properities.The fabricating process of the tissue-engineered auricle scaffolds made of polyurethane is investigated based on FDM rapid prototyping machine.The forming parameters of the scaffold are selected through analyzing their influences on the formability of the scaffold. By building models with different types of mesh generation, the mechanics properties and the pore size of the scaffold is tested, and the relationships between mesh generation and mechanics properties of the scaffold is established. Finally, the auricle scaffold with suitable mechanics properties and pore size is fabricated, which provides foundation for the following biological experiments.2. Biological experimentsCartilage from the ears of newzealand white rabbits was dissected and minced. Cartilage fragments were digested with 0.25% trypsin and 0.2%(w/v) type II collagenase (Sigma). The chondrocytes were expanded in vitro for two passages to get adequate cell numbers for seeding. While chondrocytes were cultured,morphology were observed through microscope; chondrocytes growth curve was depicted.The results showed that chondrocytes could be isolated successfully with collagenaseⅡ.In this study,the constructs were divided into two groups:the experiment group and the control group. An aliquot of chondrocyte suspension was mixed with Pluronic F-127 or chondrocytes/H-DMEM suspension seeded on the scaffolds.After 4,8 weeks, Cell attachment, proliferation and morphology were then studied using phase-contrast light microscopy and scanning electron microscopy. The specimens were also processed hard tissue sections. Sections were cut and stained with hematoxylin and eosin(HE), safranin O/fast green , toluidine blue staining and immunohistochemically stained for type II collagen . Athymic male mice and newzealand white rabbits were obtained. They received auricular constructs or sheet constructs.After 4,8,12 weeks follow-up, the architectures of the ear shape were evaluated and the constructs were obtained. The implants were harvested for histological analysis. The specimens were processed and identified,the methods were the same as that of vitro culture.The results showed as following.1. We fabricated well-defined elastic polyurethane auricular frameworks. The frameworks had precise profile of auricular three-dimensional structures and enough mechanical property. SEMs of the microstructure of cell–polymer constructs in vitro culture showed that the cells are attached and spreaded after 1 week and lots of extracellular matrix formation after 4 weeks.The results of vivo studies showed that: ear architecture maintained refined architecture after 4,8 and 12 weeks follow-up; HE staining of Chondrocyte- polyurethane constructs showed typical lacunae formation,but chondrocytes were relatively small and irregular in distribution compared to those of native cartilage. Light microscopic images of staining of explant section showed that cartilage-like tissue morphology was evident with typical lacunae formation. Immunohistochemically stained for type II collagen exhibit a little weaker staining comparing to the native cartilage.Conclusions:We described a novel method to demonstrated the feasibility of three dimensional polyurethane framework fabricated by FDM to create complete precise auricular cartilage. Polyurethane-chondrocyte construct supported chondrocyte adhesion, proliferation and formation of new auricular cartilage in vitro and vivo study. Challenges do remain. Further experiments is underway to optimize the porisity and fibers'diameter of the framework to get larger mass and the right elastic property of engineering cartilage.
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