Different ratios of bone marrow mesenchymal stem cells and chondrocytes used in tissue-engineered cartilage and its application for human ear-shaped substitutes in vitro.

The application of chondrocyte-based cartilage tissue engineering is limited because of the lack of autologous cartilage sources and chondrocyte dedifferentiation after in vitro expansion. Coculture of bone marrow mesenchymal stem cells (BMSCs) and chondrocytes has been a promising strategy for cartilage engineering as chondrocytes can provide a chondrogenic environment for BMSCs. However, there are no systematic comparison studies for engineered cartilage constructed using different mixing ratios of BMSCs and chondrocytes, and the most effective mixing ratio with the lowest number of chondrocytes is unknown. Here, we set a gradient of mixing ratios of BMSCs to chondrocytes for an in vitro coculture system and compared the shape retention and quality of the engineered cartilage using macroscopic and histological assays, glycosaminoglycan content assessment and immunohistochemical staining of type II collagen, biomechanical evaluation and hypertrophy-related gene expression analysis. The results showed that at least 30% chondrocytes were required to generate cartilage tissue with satisfactory shape and quality. Therefore, we preliminarily assessed the feasibility of engineering a human ear-shaped substitute using a coculture system with a 7:3 ratio of BMSCs to chondrocytes. After 8 weeks of in vitro culture, the precise architecture of the human ear-shaped construct was well maintained with the typical cartilaginous composition confirmed by histological assays.