Interest in developing tissue-engineered cornea has increased with the decrease in the supply of donor tissue. The aim of the present study was to investigate the feasibility and method of reconstructing corneal equivalents with porcine corneal acellular matrix as the scaffold in a dynamic culturing system. Applying the detergent Triton X-100 (1%) and a freeze-drying process, porcine corneas were decellularized and prepared as a scaffold, and hematoxylin-eosin staining and scanning electron microscopy showed no cells in the decellularized stroma. In order to measure the in vivo biocompatibility, part of the scaffold was transplanted into a pocket of rabbit corneal stroma and observed for 3 months. No sign of rejection were observed, and the acellular matrix gradually integrated in the rabbit cornea, indicating that the scaffold had good biocompatibility. To reconstruct a tissue-engineered cornea, cultured rabbit keratocytes were seeded into the scaffold. After 1 week of culture in a culturing vessel, rabbit epithelial and endothelial cells were seeded on both sides of the stroma, respectively. The reconstructed cornea consisted of three layers in histological structure: the epithelium, stoma and endothelium. Stratified epithelial cells formed on the surface, which were cytokeratin 3 positive in the cytoplasm; endothelial cell monolayers were located on the inner side, and pump-related aquaporin 1 was found in the cells. These results confirmed that the corneal acellular matrix can be used as a scaffold for tissue-engineered cornea, and a biological corneal equivalent can be reconstructed in a dynamic culturing system.
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