BACKGROUND: Adipose tissue-derived stem cells receive a high attention in tissue engineering research. Adipose tissue-derived stem cells lack of specific surface marker, there is no effective purified method. Purified adipose is a simple method to elevate purify of stem cells. OBJECTIVE:To analyze how to purify adipose tissues before primary culture of adipose tissue-derived stem cells. DESIGN, TIME AND SETTING: The controlled animal experiment was performed at the Shanghai Animal Center of Experimental Medicine of Shanghai Sixth People’s Hospital between December 2007 and March 2008. MATERIALS: Four-week old Sprague Dawley rats were used for obtaining adipose tissues from the inguinal groove. METHODS: Adipose tissues from rat inguinal groove were dissected to educe superficial blood vessel and blood vessel branches. Both blood vessel inside and elliptic nodal tissues surrounding blood vessels were excised. MAIN OUTCOME MEASURES: Stained elliptic nodal tissues stained by Hematoxylin-Eosin were observed with a microscope to make sure what kind of tissues they are. The purified adipose tissues and unpurified adipose tissues were stained by Hematoxylin-Eosin. The differences in their tissue construction were observed using the microscope. RESULTS: Elliptic nodal tissues stained by Hematoxylin-Eosin were proved to be lymphatic tissues. The tissue construction of purified adipose tissues was pure, and the cellular component was simple. Conversely, the tissue construction of unpurified adipose tissues was complicated, and cells were various with complicated components. CONCLUSION: The component of adipose tissues used to primary cultured adipose tissue-derived stem cells is complicated. As resection of superficial blood vessel, skin and muscle tissues, blood vessel inside tissues and lymphatic tissues should also be excised.

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.

Stress urinary incontinence is one of the most common diseases in urinary system.At present,the major methods for treating stress urinary incontinence include medication,physico-behavior therapy and operation.However,for various reasons,the current methods do not yield satisfactory results.As a newly emerging technique,tissue engineering provides a new concept and method to treat stress urinary incontinence.The application of tissue engineering in the treatment of stress urinary incontinence is reviewed in this article.

Objective To explore the method of culture of rat adipose-derived stem cells(ADSCs) and differentiation induction into myoblasts. Methods Adipose tissues were obtained from SD rats,and were isolated by enzyme digestion and cultured into ADSCs.The expression of surface antigen CD90,CD105 and CD34 was detected by immunofluorescence and flow cytometry.ADSCs of the second passage with logarithmic growth were obtained,and culture media containing 5-azacytidine(5-aza) and basic culture media were employed for cells in induction group and control group,respectively.The induction lasted for 7 d,14 d,21 d,28 d and 35 d,respectively.Cell growth and cell morphology were observed by inverted phase contrast microscope,and immunofluorescence and flow cytometry were utilized to detect the expression of myoblast specific antigens desmin and myosin. Results ADSCs were successfully isolated and cultured,and were identified to be stem cells.On the 28th day of induction,cells in induction group displayed "swirl" morpholgy,and multinucleation was observed.It was revealed by immunofluorescence and flow cytometry that the highest expression rates of desmin and myosin were 52.57% and 50.04%,respectively on the 28th day of induction,while there was no expression before induction and in control group. Conclusion ADSCs can be isolated and cultured from rat adipose tissues,and can further differentiate into myoblasts after induction by culture media with 5-aza.The expression of myoblast specific antigen is the highest on the 28th day of induction.

Multilocus sequence typing (MLST) is a molecular genotyping method based on nucleotide sequencing. The procedure of this method characterizes isolates of bacterial species using the DNA sequencing of multiple housekeeping genes(usually seven). For each housekeeping gene, the different sequences present within a bacterial species are assigned as distinct alleles.For each isolate, the alleles at each of the loci define the allelic profile or sequence type (ST). MLST has the advantages of being robust (based on genetic data) and electronically portable to generate data that allow rapid and global comparisons between different laboratories. In this paper, the principle, method, data analysis, application, advantages and flaws of MLST are introduced.

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.

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 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.