Reconstruction Of Bladder Tissue With Biological Substitutes Using Tissue-engineering Technique

The bladder is exposed to a variety of possible injuries from the time the fetus develops. Aside from congenital abnormalities,individuals may also suffer from other disoders such as cancer, trauma, infection, inflammation, iatrogenic injuries. All this may lead to painful bladder damage or loss, requiring eventual bladder replacement or repair. Currently, gastrointestinal segments are commonly used as tissues for bladder replacement or repair. When gastrointestinal tissue is in contact with the urinary tract, multiple complications may ensue. So numerous investigators have attempted the application of alternative materials for bladder replacement or rapair. With the developing of the tissue engineering, investigators applicated cell transplantation and tissue regeneraion to repair bladders. Tissue engineering is an intercross field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function. Recently, the research of bladder tissue-engineering has made a progress in other countries, they have successfully repaired the bladder and ureter in an animal model. However, in our country, there is no research report of bladder tissue- engineering yet. This study evaluated the feasibility of growing tissue- engineered bladder in vivo in a nude mice model. The preliminary results were as follows: In the first part of the study, we evaluated the biocompatibility of polymer foams of PLGA by testing its cytotoxicity in vitro and studying the hostresponse by intramuscular implantation. The results showed that ploymer foams of PLGA had no significant toxicity to cultured cells and possessed virtuous biocompatibility in vivo. PLGA foams exhibited preferable physical characteristics indicating that it may be suitable for use as a bio-resorbable scaffold for regeneration of bladder tissue. In the second part of the study, we investigated the primary cultures and in vitro expansion of the bladder seeding cells. Fresh bladders were obtained from lethal-dose barbiture-sacrified New Zealand rabbits, urothelia layers and smooth musule layers of the bladders were mechanically separated and then chipped into small pieces. The urothelium fragments were digested with the mixture of the trypsin(0.125%) and EDTA(0.02%).The acquired cells were cultured in Keratinocyte-SFM supplemented with bovine pituitary extract(BPE) and EGF. The results showed the cells possessed a stable proliferation from the second passage to the sixth passage, implying that this method is ideal for the primary culture and subculture of the urothelium cells. The smooth muscle fragments were subjected to type II collagenase (0.02%) and the smooth musule cells were cultured in DMEM containing 10% fetal bovine serum (FBS). Cells grown to confluence were subcultured. The third passage cells were collected and expanded in rotary cell culture system (RCCS) on cytodex-3 micro-carriers. In comparison with the cells statically cultured in flasks, a quicker growth was observed on the micro-carriers in RCCS. This demonstrates that micro-carrier-based smooth muscle cell culture can yield a large quantity of cells within a short time. In the third part of the study, The PLGA foams were shaped into the form of a bladder. In vitro expanded smooth muscle cells and urothelium cells were concentrated to cellular suspensions and seeded onto inner and outer surfaces of each polymer matrix respectively in experimental groups. The polymer matrix without seeding cells served as control. After incubation in plate for 1-4days, the constructs were implanted into subcutaneous pockets of athymic mice under general anesthesia. The animals were sacrificed at different time intervals and specimens were extracted for gross and histological analysis. Gross inspection of the constructs found neo-tissue formation in shape of the original spherical architecture on20,40,60days, but the polymers without seeding cell were simply absorbed and little tissue...