Tissue Engineering Of Ureter With Human Adipose Derived Stem Cells And Biodegradable PLA/collagen Scaffold

Background and ObjectivesUreteral injuries often resulted in ureteral defect or stricture, and lead to secondaryurinary tract obstruction, infection, urinary extravasation, and urinoma, etc, whichdeeply impaired the wellbeing and life quality of the patients. Under the condition ofureteral major injuries or large defect, the commonly used material for ureteralreplacement is gastrointestinal tract. However, replacement with gastrointestinal isassociated with lots of complications including urinary calculi, chronic infections,intestinal adhesions, etc. Some researchers have tried the synthetic materials orallotransplantation to reconstructing the ureter with disappointing effect. Therefore,the treatment of ureteral injuries and defects has been an urgent problem to be solved.The development of tissue engineering offers a new way for reconstructing theureteral injuries and defects. The two key factors of tissue engineering the ureter arescaffold and seeding cell. In this research, we took the isolated human adipose derivedstem cells (ADSCs) as the seeding cells. In addition, we constructed a new typeureteral scaffold with PLA/collagen biomaterial in that providing a new method forureteral tissue reconstruction.The objectives of our research were as follows:1. To explore the method of isolation, culture and characterization of humanADSCs, in order to provide practical foundation for the research about using thehuman ADSCs as the seeded cells.2. To test the possibility of transdifferentiation of human ADSCs into urothelialcells, in order to provide new resource of seeding cells for ureteral engineering.3. To construct a new type of tissue engineering ureteral scaffold and to testwhether the scaffold is good for the growth and proliferation of human ADSCs.4. The seeded scaffold was implanted into the body of athymic mice to evaluatethe biocompatibility. Besides, the growth and the expression of urothelial markers from the seeded cells within the scaffold were also assessed for in vivo study, in orderto evaluate the feasible of ureteral tissue engineering with the novel ureteral scaffold.Methods1. Samples of human subcutaneous adipose tissues were obtained from patientsundergoing liposuction procedures. Enzyme digestion was used to primary isolate thehuman ADSCs. Flow cytometry assays were used to confirm the phenotype. Thedifferentiation potential was initially testified by adipogenic and osteogenicdifferentiation protocol.2. HADSCs were indirectly cocultured with urothelial cells in a transwellcoculture system for urothelial differentiation. Then, the differentiation was evaluatedby detecting urothelial lineage markers (cytokeratin-18and uroplakin2. throughRT-PCR, western blot, and immunofluorescence.3. A new type of PLA/collagen biodegradable ureteral scaffold was constructedthrough solvent evolution and electrospinning method. Then, the human ADSCs werecultured with the constructed ureteral scaffold in vitro. Whether the scaffold was goodfor the growth and proliferation of human ADSCs was evaluated by scanning electronmicroscopy (SEM), hematoxylin and eosin staining, LIVE/DEAD, and MTT analysis.4. The scaffolds, which were pretreated by seeding the urothelial differentiatedhuman ADSCs, were then implanted subcutaneously into4-week-old female athymicmice in the back.14days later, the grafted scaffold was harvested and evaluated bycell tracking, H&E staining, immunofluorescence.Results1. The human ADSCs isolated by Enzyme digestion displayed fusiform shapeand adherent to the bottom of the cell flask, observing under the inverted opticmicroscope. Flow cytometry demonstrated the isolated cells positive expression ofmesenchymal stem cell markers (CD29, CD44, CD90, and CD105), negativeexpression of hemopoietic stem cell marker CD31and histocompatibility antigensHLA-DR. After14days adipogenic induction, the formation of intracellularmicrodroplets was noted and stained positive for Oil Red O. After21days osteogenicinduction, the presence of calcium deposits in cultures was stained with Alizarin Red S.2. After cocultured with urothelial cells for7days, the morphology of the humanADSCs changed into urothelial-like. After14days coculture, the induced cellsdisplayed up-regulation of urothelial markers (CK-18, UP2) both in mRNA andprotein level testified by RT-PCR and Western blot. In addition, about40%-50%induced cells express urothelial markers (CK-18, UP2) according toimmunofluorescence.3. Three dimensional structure was observed on outer surface of PLA/collagen Rureteral scaffold according to SEM. The scaffold was constructed by fibers about3m(3.44±0.94m) in diameter and the pole size between the fibers about10m (10.54±3.18m). After the cell seeded onto the ureteral scaffold and cultured over a period of3days, the cells displayed a spreading appearance and uniform distributed on thescaffold, according to the SEM. MTT assays confirmed that the cells seeded onto thescaffold showed continual proliferation according to the measurement in1,3,5,7days after cell seeding. After cell seeding onto the scaffold for7days, the cells spreadevenly on the surface of scaffold and some of cells infiltrating into the inside of thescaffolds, according to the H&E staining. More than90%of the cells grown on thescaffold were alive according to Live/dead analysis.4. All the16surgical animals lived normally during the14days follow-upobservations after the novel cell seeded ureteral scaffold implantation in vivo. Inaddition, the wound healing was fine and no infection and inflammation was observed.The differentiated human ADSCs survived, infiltrated into the grafts and maintainedspecific urothelial markers after14days implantation in the body of athymic mice.Conclusions1. Enzyme digestion method was feasible for isolation of human ADSCs. Theisolated cells expressed mesenchymal markers and possess the good proliferation anddifferentiation potential.2. The human ADSCs can be induced into urothelial lineage differentiation byindirect coculture method. The differentiated human ADSCs changed their morphology and expressed urothelial markers.3. The novel ureteral scaffold, constructed with PLA/collagen, was suitable forgrowth and proliferation of human ADSCs.4. For in vivo study, it was demonstrated that the novel seeded ureteral scaffoldhad good biological function. The differentiated human ADSCs could survive, growand maintain differentiation marker after implantation with the scaffold. This researchprovided a new way for ureteral reconstruction and tissue engineering.