Construction Of Tissue Engineering Bladder By Murine Marrow Mesenchymal Stromal Cells And Human Acellular Amniotic Membrane

PartⅠThe studies on separation and cultivation of rat marrow mesenchymal stromal cells (MSCs) and their differentiation into smooth muscle cells by the supernatant of homogenized bladders in vitro.Objective: To investigate separation, cultivation and purification of murine marrow mesenchymal stromal cells(MSCs) in vitro and its differentiation into smooth muscle cells by the supernatant of homogenized bladder so as to contribute to the seed cells of bladder tissue engineering.Methods: MSCs were collected from degermed femurs, tibias and sternum of 4 to 6-week-old SD rat by flushing the shaft with buffer (D-Hank's, PH 7.2) using a syringe with a No. 26 G needle. Cells were cultivated in culture flask and re-fed every 2-3 days (L-DMEM with 10% FBS and 100u/mL penicillin-streptomycin). When 95% fusion, cells were digested with 0.125% trypsogen and 0.02% EDTA 2 min and passaged. After successive isolation, purification, subculture and proliferation, the morphology was observed with phase contrast microscope. The morphologic characteristics of MSCs were studied by HE staining and scanning electron microscope (SEM). The growth curve was tested by MTT assay. The 4th MSCs were cultured in DMEM with 1% DMSO 8 hours before changing the supernatant of homogenized bladders to induce differentiation. Morphology of cells was observed and immunocytochemistry was performed to detect the expression of specificα-SMA.Results: The components of primarily cultured MSCs were very complex. The marrow cells were round in the beginning and a few cells adhered to flask at 1-2 days, which were in irregular shape such as fusiform, polygon and so on. After changing half of the medium, the cell clones began to proliferate immediately. About 7-9 days later, cells might overgrow the bottom of culture flask and reached over 95% fusion. The cells arranged regularly as a whirlpool. Generated cells stuck on the wall more quickly than primary cells. From the 2nd hour, they began to adhered and completely adhered within 24h. The cell morphology was more uniform and all the cells arranged more regularly. Cells could spread the full flask bottom for 6 or 7 days. The HE staining result showed that the cell body was fusiform, and some of them had processes. The big and deep staining basophilia nucleus were in the middle of the cell body. Under SEM we could find three kinds of cell morphologies: the most were the large flattened cells and there were a few spindle-shaped and globe cells. On the cells surface we could see a large amount of short and thick microvillus. The growth curve of P2, P4, P6 were quite similar and the cells biological characters kept stable. The result of growth curve showed that cell growth phase was composed of latency phase, logarithmic phase. After induction, MSCs demonstrated smooth muscle cell-like morphology and a hill-and-valley growth pattern as well as expressed the specificα-SMA. Its differentiation rate was (45.6±3.5)% vs control group's(3.8±0.77)% ,there was significant deviation between the two groups.Conclusion: 1.In this part, a simple new method which was used for the separation, purification and cultivation in vitro of MSC from SD rat bone marrow by total marrow culture associating with adhering to wall has been established. The MSCs could proliferate immediately and keep their biological character stable in vitro.2. The cells were noted to have a large expansive potential and a fusiform morphology. The growth curves of P2, P4, P6 were quite similar and the cell biological character kept stable.3. MSCs could be induced into smooth muscle cells by the supernatant of homogenized bladders in vitro, thus providing sufficient amount of seeds cells to bladder tissue engineering. Part II: Research the characteristic and preparation of human acellular amniotic membrane graft for tissue engineering applicationsObjective:To investigate the preparation method of human acellular amniotic membrane(HAAM) and evaluate the feasibility of using HAAM as biomaterial scaffold to construct tissue engineering bladder.Methods:Human amniotic membranes were decellularized by the method of enzyme digestion and eradicator washing. The human acellular amniotic membranes (HAAM) were then examined by HE staing and electron microscope to confirm no cell elements remained. Murine marrow mesenchymal stromal cells were implanted in 96-hole-plank and cultured by HAAM extract liquid or normal culture medium. The cytotoxcity of HAAM was tested through MTT assay.6-8 pieces of HAAM were overlapped with fiber trend and exposured under 200W light for 3 hours to increase its thickness and tension.Results:The HAAM was white and translucent, and the thickness was about 0.1mm. It was lubricous and had good elasticity and toughness. There were no cell elements remained under the examination of optical microscope and electron microscope. Many hole structure and remain figure were seen and rich collagen were observed by scanning electron microscope in HAAM. The cytotoxcity score was I, which indicated that HAAM had a good biocompatibility. The thickness of cross-link HAAM was about 0.6-0.8 mm and its tension was increased.Conclusion:HAAM can be obtained by enzyme digestion and eradicator washing method. This scaffold has good elasticity and toughness, on which the seeds can adhibit and metabolize. The HAAM has good biocompatibility. The cross-link HAAM is thick and tough to endure the suturation and drag. HAAM can be used as ideal biomaterial scaffold of bladder tissue engineering.Part III: Construction the tissue engineering bladder and replacement experiment for ratObjective: To investigate the feasibility of bladder repair and replacement by tissue engineering bladder through the construction of murine tissue engineering bladder by marrow mesenchymal stromal cells(MSCs) and human acellular amniotic membrane graft(HAAMG).Methods: The MSCs of the rats were implanted in HAAM and cultured for about 5 days. Then the tissue engineering bladders were harvested. The cells were observed with phase contrast microscope and the tissue engineering bladders were observed by HE staining and scanning electron microscope.30 rats were divided into 3 groups randomly and marked with A, B and C. There were 12 animals in group A and B, but 6 animals in group C. Hemicystectomies were performed in all 3 groups. The 12 defects of group A were repaired with the tissue engineering bladders, group B with HAAM grafts and the last 6 defects of group C were sutured directly. The rats underwent postoperative assessment of bladder volume and cystography after 2,4,8 weeks. The animal bladders were obtained after that, through which the tissue regeneration was examined.Results: There was no evident cell proliferation on the first and second day of coculture of HAAM and MSCs. The cells began to stick to HAAM on the third day and the number of adherent cells increased with the time. On the fifth day of coculture, by HE stain, it could be seen that MSCs adhered to amnion. The cells bodies were bigger, fusiform and uniform. Under scanning electron microscope, there were scattered cells adhered to the surface of material. At 2,4,8 weeks after surgery, the bladder volumes of the group A and B were different significantly as compared with that of the group C(p<0.01), but there is no significant difference between the group A and B(p>0.05 ).The Cystography indicated the morphology of the bladder was normal in A and B group at 8 weeks, but became small in C group. The tissue of the anastomoses area grew well after 2 week in A and B group. Portion of HAAM was absorbed after 2 weeks. The tissue structure of the replacement was somewhat the same as that of the normal one after 4,8 weeks. Through the pathological examination, there were lots of inflammatory cells in the replacement tissue in A and B group after 2 weeks, and these inflammatory cells vanished 8 weeks later. The musculature could be observed in A group after 2 weeks and increased along with the time, but little in B group. The epithelial cells could be observed in both A and B group after 2 weeks and increased with the time.Conclusion: After transplantation with MSCs/HAAM, epithelial cells and smooth muscle cells can regenerated in the graft, and the graft can be absorbed quickly. The tissue engineering bladder constructed by MSCs and HAAM can be used as an ideal biomaterial to replace and repair the bladder。...