ObjectiveTo assess the feasibility of seeding adipose-derived stem cells (ADSCs) onto bladder acellular matrix grafts (BAMGs) for bladder reconstruction in a rabbit model.MethodsAutologous ADSCs were isolated,expanded and identified by flow cytometry.In the experimental group,ADSCs were seeded onto BAMGS for reconstructing bladder defects in 12 male rabbits.Unseeded BAMGs were used for bladder reconstruction in the control group of 12 rabbits.Cystography was performed at 24 weeks after grafts implantation.Following cystography,the animals were scarified and grafts were harvested； H&E and immunohistochemical staining were performed with cytokeratin AE1/AE3,smooth muscle α-actin and S-100 markers.ResultsFlow cytometry demonstrated that the ADSCs expressed CD90,CD44,CD105,CD166 and CD34,but not CD45 or CD106.The cells demonstrated good biocompatibility with BAMGs.At 24 weeks,in the experimental group,the reconstructed bladders reached a mean volume of (94.68 ± 3.31 )％ of the precystectomy bladder capacity.Complete regeneration of smooth muscle and nerve tissue was evident.Regenerated SMCs,urothelium and nerve cells stained positively for α-smooth muscle actin,AE1/AE3 and S100.In the control group,the mean bladder volume was (69.33 ± 5.05 )％ of the pre-cystectomy volume.Histologically,the control group was characterized by multi-layered urothelium without evidence for organized muscle or nerve tissue.Conclusion The tissue engineering bladder constructed by ADSCs and BAMG can be used as an ideal biomaterial to replace and repair the bladder.

Objective To study the diagnostic value of 3-dimensional CT reconstruction in posterior urethral strictures or ankylurethria.Methods Thirty patients with strictures or ankylurethria of posterior urethra caused by pelvic fracture underwent helical CT scan and 3-dimensional reconstruction of the urethral canal as well as radiographic urethrography before and post open urethral reconstruction to observe the urethral anatomy,the length and position of the urethral strictures,the depth of periurethral scar.Results The mean stricture or ankylurethria length measured by radiographic urethrography was 4.0 cm (range from 1.0 cm to 7.0 cm),and the correlation coefficient of stricture or ankylurethria length was 0.92,21 (70%) patients were diagnosed accurately by radiographic urethrography.The mean stricture or ankylurethria length measured by 3-dimensional CT reconstruction was 4.3 cm (range from 1.2 cm to 7.6 cm),and the correlation coefficient of stricture or ankylurethria length was 0.96,there were 28(93%) patients diagnosed accurately by 3-dimensional CT reconstruction.The mean stricture or ankylurethria length measured by open urethral reconstruction was 4.2 cm (range from 1.5 cm to 7.5 cm).Five patients with urethrorectal fistula were also diagnosed accurately by 3-dimensional CT reconstruction rather than by radiographic urethrography.Conclusions 3-dimensional CT reconstruction of the urethral canal can accurately evaluate the urethral anatomy,the length and position of the urethral strictures,as well as the depth of periurethral scar after crush injury and provide useful information for operation that may not be provided by radiographic urethrography.3-dimensional CT reconstruction may become the most valuable means for detecting posterior urethra strictures or ankylurethria with urethrorectal fistula.

Objective To investigate the feasibility of using small intestinal submucosa (SIS) graft for the repair of anterior urethral strictures. Methods From June 2009 to August 2010, 18 men (mean age, 38 yrs) with anterior urethral strictures underwent urethroplasty using a four-layer SIS as an onlay patch graft. SIS was used to augment the urethral caliber at the stricture site. The mean stricture length was 4.6 cm (range 3.5 to 7 cm). The pre-operative mean maximal flow rate was 3.8 ml/s (range 1.5 to 5.5 ml/s). The required SIS grafts (4 to 7.5 cm long and 2 cm wide) were positioned into the urethrotomy defect and were spread-fixed to the corpora cavernosa using 5-0 polyglactin interrupted sutures. Two apices of the graft were sutured to the proximal and distal apices of the urethrotomy with 5-0 polyglactin interrupted stitches. The margins of the opened urethra were sutured to the SIS patch with 5-0 polyglactin running sutures. Results The mean follow-up period was 10 mon. (range 6-18 mon.). No postoperative complication, such as infection or rejection related to the use of heterologous graft material was observed. Seventeen patients voided well postoperatively with the mean peak urine flow of 25.4 ml/s (14-44 ml/s). Cystoscopy revealed that at four weeks and six weeks, the SIS graft was well distinguishable from the normal surrounding tissue; and at 16 weeks, the urothelium was regenerated and the biomaterial was not distinguishable from the normal surrounding tissue. The squamosal epithelium was seen in the histological examination of the grafts. The remaining one patient with failed hypospadias developed a slight urethral narrowing at five months post-operatively and needed sound dilatations. Conclusions SIS matrix appears to be a safe and effective reconstructive material in selected urethral reconstructions.

ObjectiveTo improve the recognition of lichen sclerosus (LS) involving the anterior urethral strictures and to investigate the outcome of urethroplasty using free mucosal grafts for the treatment of urethral strictures caused by LS. MethodsFrom January 2007 to December 2010,36 patients with anterior urethral strictures caused by LS were treated using oral mucosal grafts or colonic urethroplasty.The mean age was 41 years (range,27 -75) and the mean anterior urethral stricture length was 11.5 cm (range,5.0 -20.0 cm).Of the 36 patients,27 patients underwent dorsal lingual mucosal graft or combined buccal mucosal graft urethrography.Eight patients underwent colonic mucosal urethrography according to the length and seriousness of urethral strictures,and the remaining patient underwent anterior urethral split.Biopsies were taken from the glans,urethral meatus and urethra before urethroplasty. ResultsThe urethral silicone stent was removed 21 d post-operatively and all the patients voided well.An epithelial-stromal lesion characterized by hyperkeratosis,thinned epithelium and diffuse perivascular lymphocyte infiltrate was seen upon histological examination of the biopsied areas.The mean follow-up was 22 ( range,6 - 50) months post-operatively.Meatal stenosis developed in 2 patients undergoing oral mucosas urethroplasty and 1 patient with colonic urethroplasty,the patients voided very well after re-operation.The other patients voided well and the urinary peak flow rates ranged from 17.2 to 47.0 mL/s ( mean,23.4). ConclusionsFree mucosal grafts urethroplasty can obtain good results for the treatment of urethral strictures caused by LS.But there is a risk of recurrence of urethral stricture and closing follow-up is required,especially for meatal stenosis.

ObjectiveTo investigate the long-term outcome of urethral reconstruction using colonic mucosa grafts for the treatment of long-segment,complex urethral strictures and to identify clinical factors that impact long-term outcomes. MethodsForty-six patients underwent colonic mucosal graft urethroplasty from October 2000 to September 2009 were retrospectively reviewed.The mean age was 39 years ( range,17 -70).The patients underwent an average of 2.7 prior unsuccessful repairs and the mean length of urethral strictures was 15.2 cm (range,10.0 to 20.0).The voiding status of all patients was evaluated postoperatively.Some of the paitents underwent uroflowmetry.urethrography and urethroscopy.Successful repair was defined as voiding well with urinary peak flow greater than 15 ml/s without the need for any post-operative procedures,such as dilatation. ResultsUrethral reconstruction was done with colonic mucosa grafts 11.0 -21.0 cm long (mean 15.4).One patient was lost to follow-up.Mean follow-up in the remaining cases was 62 months ( range 20 - 120 ).Complications related to urethroplasty developed in four patients (8.9％).Of these patients,meatal stenosis developed in three patients at 3,8 and 24 months respectively.Anastomotic site stricture occurred at the neourethra and proximal urethra in one patient at 29 months.In another two patients,recurrent strictures unrelated to urethroplasty were found. ConclusionsColonic mucosa graft urethroplasty could be an effective technique for the treatment of complex urethral strictures or panurethral strictures.The factors that impact long-term outcomes are meatal stenosis and stenosis at the anastomosis.

Objective To investigate the efficacy and safety of using combined lingual mucosa and buccal mucosa onlay grafts or foreskin flap urethroptasty for the treatment of long or multi-seg-ment urethral strictures. Methods Seven patients with long and 4 cases with multi segment urethral strictures(range 10 to 15 cm,mean 12)underwent substitution urethroplasty using combined lingual mucosa and buccal mucosa onlay grafts or foreskin flap urethroplasty.The patients'age ranged 24 to 56,mean 32 and the course of disease was from 6 to 96 months.Of the 11 patients 7 underwent com-bined lingual mucosa and buccal mucosa grafts urethroptasty,4 patients underwent combined lingual mucosa graft and foreskin flap Urethroplasty. Results The patients were followed up 5-1 2(mean 10)months postoperatively. Meatal stenosis developed 3 months postoperatively in 1 patient who un-derwent combined lingual mucosa and foreskin flap urethroplasty.The patient could void well after re-operation.The other patients could void well and the peak flow rate ranged from 2 1 to 3 6 ml/s(mean 26.8 ml/s). Conclusions Combined lingual mucosa and buccal mucosa onlay grafts or foreskin flap substitution urethroplasty may have the advantage of easier harvest,less trauma.It could be a good U- rethral substitution technique for the treatment of long or multi-segment urethral stricture.

Objective To investigate the feasibility of replacing urinary epithelial cells with oral keratinocytes by being seeded on bladder acellular matrix graft(BAMG). Methods Twenty-four male rabbits were randomly divided into 2 groups:experimental group and control group.A length of 2.0 cm and width of 0.8 cm penile urethral mucosal defect was induced in the anterior urethra 2.0 cm awav from the urinary meatus in all the rabbits.Oral keratinocytes were isolated from a small buecal mucosa(1.0 cm×0.4 cm)of the 12 rabbits in experimental group and seeded onto a culture dish with a feeder layer of 3T3 mouse fibroblasts inhibited by mitomycin(i3T3).Passage 2 oraI keratinocytes cuItured with i3T3 were expanded and seeded onto sterilized BAMG(2.2 cm×1.0 cm)to repair the de-fects of the urethra.Urethroplasty was performed with BAMG with no cell seeding in the controlgroup.Catheter examination and retrograde urethI ography was done in 1,2 and 6 months after graft-ing.The urethral grafts were harvested and analyzed by histological staining. Results Oral kerati-nocytes seeded onto a feeder layer of i3T3 had better morphous and amplification capability.The corn-patibility of the compound graft was assessed by HE staining and scanning electron microscope.Twelve rabbits in the experimental group could void without difficulty.Catheter examination and ret-rograde urethrogram revealed that a wide urethral caliber was maintained with no sign of strictures and fistula formation.Histological examination showed the grafted urethra was covered with smooth mu- cosa with no strictures at 1,2 and 6 months.The oral keratinocytes of the graft still existed even 6 months after grafting.On contrast,gross and urethrogram demonstrated urethral stricture in the con-trol group.And inflammatory reactions could be found in the area of the stricture through light micro-scope. Conclusions Oral keratinocytes have good compatibility with BAMG and the compound graft could be used for urethral reconstruction in the animal model.

Objective To investigate and assess the best seeding method for constructing three dimensional urethral tissue in vitro. Methods High speed agitation decellular method was used for preparing the porcine acellular corporous spongiosum matrix (ACSM). Before seeding, the matrix was sterilized via soaking compound iodine solution. Rabbit tongue epithelial cells and cavernosal smooth muscle cells were isolated and cultured. Three different groups of seeding method was used in this study. Group A (sandwich seeding group): The smooth muscle cells and epithelial cells were seeded onto the different side of ACSM by static method. Group B (injection seeding group): The smooth muscle cells were injected into the scaffold. Then, epithelial cells were seeded onto the urethral surface of ACSM by static method. Group C (agitation seeding group): The smooth muscle cells were seeded into the scaffold by agitation method. Then, epithelial cells were seeded onto the urethral surface of ACSM by static method. After being seeded, all matrixes were cultured in vitro for 14 d. HE and immunoassay staining were used to examine the results of seeding. Results Looser matrix was obtained after using high speed agitation decellular method. Compound iodine solution could not only sterilize efficiently but also reserve the original structure of biomaterial. An intact epithelial cellular layer onto the surface of scaffold could be observed in HE staining section after 14 d culturing in vitro.Few smooth muscle cells could be found in big space of biomaterial in group A. In group B, smooth muscle cells were restrained in some regions of the matrix. Smooth muscle cells were well distributed into the scaffold in group C. Conclusions After using high speed agitation decellular method, an ideal matrix with three dimensional structure can be obtained. Combined with agitated seeding method, three dimensional urethral tissue can be constructed.

Objective To investigate the feasibility of constructing tissue engineered corpora cavernosa smooth muscle by seeding human umbilical artery smooth muscle cells (HUASMCs) in acel-lular collagen matrices.Methods Acellular corporal collagen matrices (ACCM) were obtained from the penis of adult rabbits by a cell removal procedure.HUASMCs were isolated from human umbilical cords through explant techniques and cultured in vitro.Subsequently,HUASMCs were seeded to ACCM and cultured in vitro.After that,the seeded ACCMs were implanted subcutaneously in 9 BALB/C athymic mice.Animals were killed 10,20 and 40 days after implantation.The implants were retrieved and morphological examinations were performed to evaluate characteristics of the engineered tissues.Additionally,organ bath studies were performed to address the contractility of the engineered tissues.Results The deeellularization process successfully extracted all cellular components; colla-gen fibers maintained their original porous morphology and structure.ACCM could be reseeded with cultured HUASMCs in vitro,and HUASMCs had the potential of attachment and proliferation on the three-dimensional ACCM scaffolds.Histologic analyses of the explants from all time points demon-strated a progressive regeneration of corpus cavernosum smooth muscle,with structures very similar to those of the native corpus cavernosum,The maximum contraction force induced by phenylephrine and electrical stimulation was (3.64+0.18)g and (2.50+0.21)g.Conclusion HUASMCs can be seeded on 3-dimensional ACCM scaffolds and will develop a tissue similar to that of the native corpus eavernosum smooth muscle.

Objective To evaluate combined buccal mucosa and lingual mucosa grafts for urethroplasty in a dog model. Methods Seven female mongrel dogs were selected.After a segment of proximal urethra mucosa (4 cm×1 cm) was excised and onlayed,urethroplasty was performed by using the combined free buccal mucosa (2 cm×1 cm)graft which had been harvested from the inferior cheek and free lingual mucosa graft(2 cm×1 cm)harvested from the inferior lateral surface of the tongue.A 12 F urethral catheter was kept for 7 d.Retrograde urethrography was done and urethra diameter was calibrated with a 10 F catheter before animals were sacrificed at week 12.Then the grafted areas excised and evaluated grossly and histopathologically. Results All dogs survived during the procedure and there was no tongue or bueeal complications.One dog developed a severe urethral stricture at the proximal anastomosis site.The remaining 6 dogs voided spontaneously with no difficulty.Retrograde urethrography showed that no stricture or fistula formed.The combined buccal mucosa graft and lingual mucosa graft shortened from a mean (SD) of 4.00(0.15)to 3.75(0.23)cm (statistically.significant,P＜0.05).No stricture was found in the connection of the buccaI mucosa and lingual mucosa grafts.Histological examination showed that the combined buccal mucosa and lingual mucosa grafts were well-incorporated into the urethral walls and covered by a keratinized squamous epithelium.Neovascularization was evident beneath the grafts. Conclusion Combined buccal mucosa graft and lingual mucosa graft could be an option for urethral substitution.