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The aim of this Letter is to investigate the feasibility of isolated tracheal chondrocytes on a novel-type nanocomposite scaffold for application to cartilage regeneration. The scaffold was based on biocompatible N-carboxyethylchitosan containing hydroxyapatite nanoparticles (NCECS/nHA). NCECS/nHA nanocomposite scaffold was fabricated by vacuum freeze-drying. Tracheal cartilage segments from a 8-week-old New Zealand white rabbit were chopped into small 1 × 1 × 1 × mm3 blocks. Following type II collagenase digestion, chondrocytes at passage 3 were seeded onto three-dimensional porous NCECS/nHA scaffolds. These cell-scaffold constructs were precultured in the incubator for 5 days and then subcutaneously implanted into nude mice for in vivo culture. After 8 weeks, explanted constructs were harvested and dedicated for histomorphologic and biochemical assays. The pore size of NCECS/nHA nanocomposite scaffold ranged from 200 to 500 m. The porosity was 85 . The chondrocytes cultured in this scaffold produced cartilaginous matrices. Immunohistochemical staining for type II collagen revealed that chondrocytes exhibited dark brown cytoplasm. After 8-week in vivo culture, the neonatal cartilage-like tissue exhibited histomorphological and biochemical characteristics similar to that of mature hyaline cartilage. These results demonstrated that NCECS/nHA facilitated the adhesion and viability of chondrocytes, and therefore allowed the authors to make further improvement by applying tissue-engineering methods to regenerate tracheas for clinical use.