Preliminary Experimental Study of Tissue-Engineered Urethral Reconstruction Using Oral Keratinocytes Seeded on BAMG

Abstract

Introduction: This study investigates the feasibility of replacing urinary epithelial cells with oral keratinocytes seeded on a bladder acellular matrix graft (BAMG) to reconstruct a tissue-engineered urethra. Materials and Methods: After enzymatic treatment of a small segment of rabbit buccal mucosa (1.0 × 0.4 cm), the epidermis and dermis were separated mechanically. Oral keratinocytes were isolated from the epidermis and seeded onto a culture dish with a feeder layer of 3T3 mouse fibroblasts inhibited by mitomycin (i3T3) or on a culture dish without i3T3, respectively. These cells were expanded and applied to sterilized BAMG to obtain a tissue-engineered mucosa. Oral keratinocytes were assessed by growth curve, morphology and immunofluorescence staining. The tissue-engineered mucosa was assessed using morphology, immunohistochemistry staining and scanning electron microscopy. Results: Oral keratinocytes seeded onto a feeder layer of i3T3 had finer morphous and better amplification capability, and could be passaged for 8 generations. Oral keratinocytes that were cultured without i3T3 could only be subcultured 2 generations before ageing. Passage 2 oral keratinocytes cultured with i3T3 were expanded and seeded onto sterilized BAMG to obtain a tissue-engineered mucosa for urethral reconstruction. The compound graft was assessed using HE staining and scanning electron microscopy. Oral keratinocytes had good compatibility with BAMG. Conclusions: Rabbit oral keratinocytes of can be cultured in vitro and attain magnitude in quantity. Oral keratinocytes also had good compatibility with BAMG and the compound graft achieved could become a new choice in urethral reconstruction.