Objective To investigate the effect of vaginal reconstruction with autologous buccal micro-mucosa graft. Methods From March 2007 and April 2008, 10 patients with absence of vagina were treated, aged 18-31 years (mean 26 years). Nine of them were congenital absence of vagina, and the remaining one was vaginal stenosis after vaginal reconstruction.They all exhibited normal secondary sexual characteristics, normal hormonal levels and 46, XX karyotype. Their abdominal ultrasounography revealed the normal ovaries and tubes but absence of the uterus or small rudimentary horns. However the one with vaginal stenosis had normal uterus. The buccal mucosa graft was minced into 0.5 mm in size and was transplanted to the cavity which was dissected between the bladder and the rectum. Results The operation was performed successfully in all cases. The operative time was about 1-2 hours and operative blood loss was 80-100 mL. Postoperative compl ication occurred in only one case for vaginal bleeding. The patient recovered and the wound healed well after immediate management. The others healed primarily without any compl ications. All cases were followed up for 4-16 months. The depth of neovagina which was formed was 6-10 cm and the width was about two fingers. The l ining was pink-colored and smooth, and was confirmed as nonkeratizing squamous stratified mucosa by histopathological examination. The donor sites healed uneventfully with no change in mouth opening. The perineal area was not disturbed. Four patients were married and satisfied with their sexual l ife without pain and bleeding. Conclusion Vaginal reconstruction with autologous buccal micro-mucosa graft is an easy, minimally invasive and useful method.
Objective To investigate a method of repairing hypospadias by combining buccal mucosal graft with scrotal flap and its therapeutic effect. Methods From March 2002 to December 2007, 42 patients with hypospadias underwent primary urethral reconstruction using buccal mucosal graft and scrotal flap. The patients ranged in age from 18 months to 18 years. There were 21 cases of penoscrotal type, 12 cases of scrotal type and 9 cases of perineal type. Among them,8 cases were at initial operation, and 34 cases suffered from the failure of hypospadias repair 6-19 months (average 10 months) after initial operation. During operation, the defect of urethra was 3-7 cm (average 4.2 cm) when the penis was straightened; the buccal mucosa (3.0 cm × 1.2 cm-7.0 cm × 1.5 cm) was transplanted to the tunica albuginea in the ventral aspect of the penis, and was paired with the scrotal flap (3.0 cm × 1.5 cm-7.0 cm × 1.5 cm) to repair urethra. Results The incision of 38 cases healed by first intention, and no compl ication occurred. At 7 days after operation, 4 cases had urinary fistula at either coronary sulcus or anastomotic stoma, one of which spontaneously closed 2 months after operation and the rest 3 recovered by repairing urinary fistula 6 months after operation. All patients were followed for 3-48 months (average 18 months). Urination was smooth, the reconstructed urethral opening was at the tip of glans peins without retraction and with apperance similar to the normal urethral opening. The appearance of penis and scrotum was satisfying, and the penis was straightened completely. Conclusion Combined buccal mucosal graft and scrotal flap, with considerable tissue for uretha tract reconstruction and low incidence rate of urethral stricture, is one of the effective methods to repair hypospadias.
Objective To investigate the feasibil ity of replacing urinary epithel ial 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 × 1 cm was randomly selected to observe the effect of acellularization. The second passage oral mucosa cells cultured with 3T3 were appl ied to steril ized BAMG to obtain a issueengineered 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 steril ized BAMG to obtain a tissue-engineered mucosa. Good compatibil ity 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 biocompatibil ity with BAMG after the tissue engineered mucosa was cultured for 1 week. Conclusion Oral mucosa cells of rabbit can be cultured in vitro and attain magnitude quantities. Oral mucosa cell also have good biocompatibil ity with BAMG and the compound graft could be a new material for urethral reconstruction.
Objective To study the allograft effect of two kinds of tissue engineered oral mucosa lamina proprias on skin fullthickness wounds. Methods The cultured Wistar rat oral mucosa fibroblasts (OMF) were incorporated into collag en or chitosancollagen to construct the tissue engineered oral mucosa laminaproprias, and then the OMFs were labeled with BrdU. The fullthickness round skin defects were made with a round knife (diameter, 0.8 cm) on the backs of 36 Wistar rats (2125 weeks old), which were divided into 2 experimental groups: the fibroblastpopulated collagen lattices (FPCL) group (grafted by FPCLs) and the fibroblastpopulated chitosan collagen lattices (FPCCL) group (grafted by FPCCLs), and the control group (only covered with gauges). All the wounds were observed by the naked eyes or the light microscope, and were measured 4, 7, 14, and 21 days postoperatively. Results There were no infection during the wound healing period. At 7 days after the grafting, the wounds in the 3 groups were covered by scab and/or gauze; at 14 days, the gauze and scab on the wounds in the three groups were all replaced by the new epidermis naturally except one scab each in the FPCCL group and the control groups,which was replaced at 17 days.All the centers of the new epidermis were measurable as the pink red points. At 21 days, all the new skins were smooth without hairs, and their color was similar to the normal one. At 4, 7, and 14 days,there was an indication that the wound diameters became significantly smaller in the three groups; but after the 14th day, there was no significant indication of this kind. At 7 days, the wound diameter in the FPCL group was significantly smaller than that in the FPCCL group and the control group (Plt;0.01). Under the lightmicroscope, at 4 days postoperatively, the decayed tissue on the surfaces of the recipient wounds in the FPCL group and the FPCCL group was separated from the lower granular tissue in which there were many inflammatory cells, fibroblasts, and new vessels. There was a similar-phenomenon in the control group. Each skin wound in the three groups was only partly keratinocyted at 7 days postoperativel y. The recipient wounds were wholly keratinocyted with when rete ridges observed at 14 and 21 days, but in the control group the wounds were keratinocyted with no rete ridges. Fibers in the new dermis were thin. The OMFs with Brdu appeared in the granular tissue and new dermis at 4, 7, 14, and 21 days postoperatively, which could be illustr ated by the immunohistochemical staining. The positive OMFs and the granular tissue joined in the repair of the skin defe cts without any allergic reaction during the period of the wound healing. Conclusion The oral mucosa fibroblasts as the new seed cells can join i n the repair of the skin defects effectively and feasibly. The fibroblastpopul ated collagen lattices and the fibroblastpopulated chitosan collagen lat tices can repair skin defects effectively and feasibly, too. And the quality of the new skins was better in the two experimental groups than in the control group.
Objective To investigate the growth of the tissue engineered mucosa after its heterotransplantation. Methods The epithelial cells and fibroblasts were isolated from a postoperative tissue of the 3month patient with labialcleft. The epithelial cells and fibroblasts were separately seeded on the polylactic／glycolic acid copolymer membrane, and then they were exposed to the air-liquid interface. Seven volunteer patients, whose traumatic beds were repaired with the tissue engineered oral mucosa. The biopsy tissue from one of the seven patients was observed under light icroscope 18 and 30 days after transplantation, respectively. Results The tissue engineered oral mucosa having 5-6 layers anticytoeratin staining positively cells in the epithelial layer and 3-7 layers anti-Cytoeratin staining negatively cells in the subepithelial layer grew well after the he terotransplantation. No differ ence could be found between the transplanting and normal areas. At 18 days, the epithelial layer and lamina propria grew well and the fibroblasts were found; at 30 days, collagen was obviously observed. The structures in both the transplanting and the normal areas were similar. Conclusion The tissue engineered oral mucosa can grow well after the heterotransplantation.
Objective To explore the feasibility of one-stage repair of hypospadias using the meatalbased flap overlapping with buccal mucosal graft. Methods From March 2002 to May 2004, 21 patients with hypospadias were treated with the meatal-based flap overlapping with buccal mucosal graft. Their ages ranged from 14 months to 8 years. The procedure were as follows:urethralplate at proximal corona was cut to correct glandular tilt and chordee; the buccal mucosa taking from inner cheek was then fixed on tunica albuginea of ventral shaft with suture; and the meatalbased flap was rotated distally and overlaid with buccal mucosal graft to repair urethra.Results All patients were followed up 318 months (7 months on average). A cosmetic glans and a vertically oriented, normal appearing slit meatus were achieved. Two patients had fistulas on lateral corona. Fistula spontaneously healed in 1 case and the other one was repaired after 6months. Conclusion The technique of meatal-based flap overlapping with buccal mucosal graft can completely correct glandular tilt and chordee, prove good cosmetic and functional glans and meatus.
Objective To investigate the possibility of culturing human oral keratinocyte using autologous serum in order to provide theoretical and technical foundation for clinical application of tissue engineering oral mucosa epithelium.Methods The human oral keratinocytes were cultured by the medium containing different concentrations of autologous serum(10%,20%,30%)and fetalbovine serum (10%), respectively. The growth conditions for the cell and the mucosa epithelium in the groups were observed, the cell growth curves were drawn, and the population doubling time (PDT) was counted. Results The results showed that the human oral keratinocyte could proliferate well in the medium containing autologous serum or fetal bovine serum. The differences in the 24hour clone rate and PDT were not significant. Both the area and the thickness of the cultured oral epithelium increased with the increase of the autologous serum concentration, and the difference between autologous serum and fetal bovine serum was significant, especially with the medium containing 20% autologous serum( P＜0.05) . The human nature of the cultured epithelium was demonstrated by the immunofluorescent mouse anti-HLA antigen. Conclusion The autologous serum can replace the fetal bovine serum to culture the oral keratinocyte well, and the cultured oral mucosa epithelium can be better differentiated in the autologous serum than in the fetal bovine serum.