Preliminary experimental study of tissue-engineered urethral reconstruction using oral keratinocytes seeded on BAMG

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.

After enzymatic treatment of a small segment of rabbit buccal mucosa (1.0 x 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.

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.

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.