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.

OBJECTIVETo investigate effects of the variation of immune function in high humidity environment in different time, and lay a foundation for further study of the related mechanism.

METHODThirty SD rats were divided into 3 groups (n = 10): 20 day group, 40 day group in 90% relative humidity chamber and control group in normal relative humidity. Peripheral blood and spleens were collected to detect the levels of T lymphocyte subsets by Flow Cytometery.

RESULTSIn peripheral blood of the 20 day group rats, the CD3+ %, CD4+ %, CD8+ % and CD4+/CD8+ were 52.91 +/- 6.27, 37.80 +/- 4.11, 14.85 +/- 3.73 and 2.72 +/- 0.82 separately. Expect CD3+ %, they all had significant differences (P < 0.05). In addition, the data of the 40 day group rats showed no diversity in statistics. In spleen, CD8+ % of the 20 day group rats was 6.23 +/- 2.87 with significant differences (P < 0.05) and IgG, IgA and IgM did not change a lot in blood serum of the high humidity groups except C3 of the 20 days group (P < 0.05).

CONCLUSIONIn high humidity environment, the immune function of the rats increased in the initial stage. As time went on, the immune function gradually went to normal level through the self adjustment.

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 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.

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.

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.