A sandwich model for engineering cartilage with acellular cartilage sheets and chondrocytes

Acellular cartilage can provide a native extracellular matrix for cartilage engineering. However, it is difficult for cells to migrate into acellular cartilage because of its non-porous structure. The aim of this study is to establish a sandwich model for engineering cartilage with acellular cartilage sheets and chondrocytes. Cartilage from adult pig ear was cut into a circular cylinder with a diameter of approximately 6 mm and freeze-sectioned at thicknesses of 10 μm and 30 μm. The sheets were then decellularized and lyophilized. Chondrocytes isolated from newborn pig ear were expanded for 2 passages. The acellular sheets and chondrocytes were then stacked layer-by-layer, in a sandwich model, and cultured in dishes. After 4 weeks of cultivation, the constructs were then either maintained in culture for another 12 weeks or implanted subcutaneously in nude mouse. Histological analysis showed that cells were completely removed from cartilage sheets after decellularization. By re-seeding cells and stacking 20 layers of sheets together, a cylinder-shaped cell sheet was achieved. Cartilage-like tissues formed after 4 weeks of culture. Histological analyses showed the formation of cartilage with a typical lacunar structure. Cartilage formation was more efficient with 10 μm-thick sheets than with 30 μm sheets. Mature cartilage was achieved after 12 weeks of implantation, which was demonstrated by histology and confirmed by Safranin O, Toluidine blue and anti-type II collagen antibody staining. Furthermore, we achieved cartilage with a designed shape by pre-shaping the sheets prior to implantation. These results indicate that the sandwich model could be a useful model for engineering cartilage in vitro and in vivo.