| Tissue engineering and regenerative medicine are the most promising techniques for treating the failure and decline of tissues and organs. Tissue engineering in urologic system has experienced rapid improvements since the rise. Overseas scholars have aleady developed tissue engineered bladder, ureters and urethra which proved useful for relevant tissue replacement. Beacause the complicated microenvironment for normal bladder transitional epithelial cell growing is not easy to duplicate in vitro, and as a mater of fact, the technique for expanding them in vitro plays a key role in bladder tissue engineering.BackgroundSeveral congenital or acquired urinary organs disorders, such as hypospadia, bladder and cloacal exstrophy, neurogenic bladder, chronic interstitial cystistis and cancer, may require reparation to improve or create it's function. Multiple techniques have been proposed for augmentation of small and noncompliant bladders. Small and large bowel, and more recently stomach have been used for this purpose. The use of bowel is associated with multiple short and/or long-term complications. Chronic infection, hyperchlo-remic metabolic acidosis, mucus secretion, stone formation, calcium phosphate metabolism and gastrointestinal motility disorders have been reported. Bladder perforation and malignancy have also been found many years after enterocystoplasty. The majority of enterocystoplasty complications result from differences in functional properties of the intestinal and bladder epithelia, and the interface between urine and intestinal mucosa.Alternative approaches have been developed to avoid complications related to the intestinal epithelium, including use of pedicled de-epithelialized intestinal and gastric grafts has been described. Furthermore, native tissues from nonpedicled free tissue grafts have been suggested, including skin, fascia,peritoneum, pericardium, seromuscular flap, de-epithelialized submucosal bowel and acellular tissue matrix grafts. Such free grafts have been used based on the hypothesis that the bladder has regenerative capabilities and the graft serves as a template for ingrowth of normal bladder tissue. In addition, synthetic biodegradable biomaterials have been tried, including polylactic acid, polyglycolic acid and polylatic/glycolic acid copolymer. Unfortunately none of these materials has been satisfactory. For these reasons a new approach have developed. Autologous urothelium and bladder smooth muscle cell, which are harvested by biopsy, separated into cellular components and expanded in in vitro cultures. After an adequate number of cells are available following 2 to 3 subsequent subcultures cells are seeded on a supporting scaffold. After cell adherence to the scaffold the cell scaffold patch is reimplanted into the body, and allowed to regenerate and differentiate into functional tissue. So new advances in cell biology, tissue and materials sciences, and engineering, may soon change the future of bladder reconstruction.Objective1. Presently the seeded and unseeded types of tissue engineering techniques are being investigated to promote tissue regeneration. Seeded tissue engineering techniques use biodegradable membranes that are seeded in vitro with primary cultured cells that have been established from a biopsy from host native tissue. This composite graft is then placed back in the host for continuation of the regenerative process. Unseeded tissue engineering techniques involve direct in vivo placement of an unseeded biodegradable material in the host that will then function as a scaffold to allow the natural process of regeneration to occur. But some studies examined the role of unseeded graft for bladder tissue engineering, although short-term results have been promising, long-term data have been poor. At more than 1 year of followup these unseeded graft augmented bladders lacked mature detrusor muscle layer formation, had increased collagen deposition with resultant fibrosis and, as a result, had decreased capacity and poorer compliance. For this reason autologous cell transplantation may be beneficial for successful creation of functional urological tissue for bladder augmentation and may be further useful for surgical reconstruction of the defective genitourinary tract. So the aim of first part was to establish the way for acquiring large quantites of well differentiated urothlial cells which will be used for bladder tissue engineering.2. Scaffold is another important role of tissue engineering techniques. An ideal Scaffold for bladder replacement should be biocompatible, able to incorporate itself into native tissue without inducing an adverse response, and should have adequate anatomic and functional properties. Work in our laboratory has focused on silk fibroin film as a scaffold for in vivo bladder tissue engineering. Silk fibroin is a natural protein gotten from silk. Because of its superperformance and biocompatibility, it can be used as soft-tissue compatible materials in biomedicine, silk fibroin have already used in suture, contact lenses, manual vein,wound dressing, dural substitute, cell culture matrix. There are no studies about silk fibroin film which serve as a scaffold for genitourinary reconstruction. So the aim of second part was to evaluate the cytocompatibility of Silk fibroin film with transitional cells in vitro and to discuss the possibility of construction of urologic tissue engineering organ.Methods1. bladder transitional epthelial cell culture in vitroWe obtained bladder transitional epthelial cell through using collagenase and trypsin. Primary cultures of urothelial cells were established at a cell density of 2×10~5 cells per ml in DMEM/F12 medium which contains serum and EGF. Once urothelial cells initially achieved 70% to 80% confluence they were harvested with 0.05% trypsin/ethylenediaminetetra-acetic acid and routinely passaged. All cultures were incubated in 5% carbon dioxide at 37C and fed with fresh media every 2 days. Morphology of the cells was observed. Urothelial cell specific protein (cytokeratin AE1/AE3) was identified by immunofluorescence.2. Cytocompatibility of Silk fibroin film with bladder transitional cells in vitroThe experiment was divided into 3 groups : non-cell group(black control), culture mediumgroup(negative control), and extract medium from Silk fibroin film group(experimental group). The cells were implanted in 96-hole-plank at 1×10~4/ml every hole. And every group had 5 holes. Then absorption coefficient were detected at the wave length of 490 nm by MTT assay. Then the cytotoxicity and cytocompatibility were evaluated by comparison of the numbers of absorption coefficient.Results1.We found that the system containing 5%serum and 10μg/L EGF was an appropriate medium for bladder transitional epthelial cell growing,primary cell not only survived but also had been growing in 3 days'culturion,after 7-8 days'passages.We also found that the cultured cell could be confirmed as bladder transitional epithelial cell through the way of immunofluorescence. The renatured cultured cells also were characterized by maintenancing cells'normal appearance and function after removing from liquid nitrogen. So we can concluded that DMEM/F12 system can serve as an effective medium for bladder transitional epthelial cell culturing and growing in vitro. 2. The actual absorption coefficient of experimental groups was 0.424±0.020,0.996±0.118 and 1.285±0.048 after 24, 72 and 120 hours. The actual absorption coefficient of negative control group was0.419±0.030,1.105±0.098 and 1.228±0.052. There was no significant difference between the experimental group and negative control(P> 0.05).Conclusions1. The culture technique for growing urothelial cell has been established. The culturing cells may be used in urethral const ruction of tissue engineering and then be used to treat severe hypospadia, neurogenic bladder, chronic interstitial cystistis and cance in the future.2.Silk fibroin film has good cytocompatibility with transitional cells and no cytotoxity. It can be used as scaffolds of urologic tissue engineering.
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