[A preliminary experimental study on urethral reconstruction using tissue engineered oral mucosa].
OBJECTIVE
To investigate the feasibility of replacing urinary epithelial cells with oral mucosa cell to reconstruct tissue engineered urethra by being seeded on bladder acellular matrix graft (BAMG).


METHODS
Eighteen male New Zealand rabbits, aged 10 weeks, weighing 0.3-0.5 kg, were used in this study. Oral mucosa cell of 12 rabbits were isolated and seeded onto a culture dish with a feeder layer of 3T3 and a culture dish without 3T3, respectively. The morphologic change and growth condition of oral mucosa cells were observed by inverted phase contrast microscope after 2 days of seeding. The quantity of oral mucosa cells was counted using cell counting meter; the cell growth curve was drawn and the immunofluorescence staining with broad-spectrum keratin antibody was carried out. The bladders taken from the rest 6 rabbits were decelluled to make BAMG and the tissue of 1 cm x 1 cm was randomly selected to observe the effect of acellularization. The second passage oral mucosa cells cultured with 3T3 were applied to sterilized BAMG to obtain a tissue-engineered mucosa. The tissue-engineered mucosa was assessed using HE staining and scanning electron microscope after being cultured for 1 week.


RESULTS
Oral mucosa cells seeded onto a feeder layer of 3T3 could be passaged for 7 or 8 generations with homogeneous forms and full function. Oral mucosa cells cultured without 3T3 could only be subcultured for 2 generations before aging and had multiple shapes and different sizes. Oral mucosa cells cultured by the two methods both started logarithmic growth on the 8th day and reached the peak value on the 14th day, which was indicated by the cell growth curve. However, more cells could be obtained through oral mucosa cells cultured with 3T3 than those cultured without 3T3. Oral mucosa cells manifestated green colour fluorescence cultured with or without 3T3. After the cells were removed, the BAMG presented as a porous membrane. The HE staining showed that the effect of acellularization was good and there were no cells at BAMG. The second passage oral mucosa cells cultured with 3T3 were expanded and seeded onto sterilized BAMG to obtain a tissue-engineered mucosa. Good compatibility of the compound graft was assessed using HE staining and scanning electron microscope. HE staining and scanning electron microscope showed that oral mucosa cells had good biocompatibility with BAMG after the tissue engineered mucosa was cultured for 1 week.


CONCLUSIONS
Oral mucosa cells of rabbit can be cultured in vitro and attain magnitude quantities. Oral mucosa cell also have good biocompatibility with BAMG and the compound graft could be a new material for urethral reconstruction.
Li
Chao
C
Department of Urology, Sixth People's Hospital, Jiaotong University of Shanghai, Shanghai 200233, PR China.
Xu
Yuemin
Y
Song
Lujie
L
Cui
Lei
L
Yin
Shuo
S
D000818:Animals; D018929:Cell Culture Techniques; D017690:Cell Transplantation; D002478:Cells, Cultured; D005109:Extracellular Matrix; D008297:Male; D009061:Mouth Mucosa / Q000166:cytology / Q000637:transplantation*; D009092:Mucous Membrane / Q000166:cytology; D011817:Rabbits; D023822:Tissue Engineering / Q000379:methods*; D014521:Urethra / Q000601:surgery*; D001743:Urinary Bladder