The Research Of Ureteral Tissue Engineering Based On The Decellularized Matrix

Ureter, as a retroperitoneal organ, is located in the deep of the body, therefore the incidence of ureteral injuries is low. In recent years, with the increasing use of endoscopic technique to diagnose and treat the diseases, the risks of ureteral injures were increasing. The classical surgical therapies, such as bladder flaps, transureteroureterostomy and renal autotransplantation could not achieve entirely functional recovery and they also might induce a series of clinical complications such as recurrent strictures, urinary leakage, even renal damages. However, with the widely used of the tissue engineering in the urinary system, it offers new opportunity for the repair and reconstruction of the ureter.Tissue engineering, combing the concept of the material science, life science and engineering is a multidisciplinary science. The ultimate purpose of the study was to construct the engineered ureter which having biological function in vitro, replaced the damaged ureter in vivo, and eventually achieved the goal of clinical cure. With the extensive and intensive studies of the tissue engineering in the urinary system, the study, constructing the engineered ureter, had many breakthroughs, but there not had ideal engineered ureter for the clinical treatment. One of the main causes was the scaffold, because the structure and composition of the extracellular matrix (ECM) of the ureter was complicated, and it was very difficult to completely simulate the ECM microenvironment at this stage in vitro. As the emergence and development of the decellularized technology, preparing the decellularized materials had become possible and had taken widespread attention.The unique feature of the decelluarized materials was that the cellular components had been removed and the structure and components were well preserved. So the decellularized matrix was an ideal scaffold for tissue engineering. The commonly prepared methods of the decellularized ureter were to use the physical stirring combining with the chemical detergents to achieve the decellularized effect, which always need consume a long time and could destroy the components of the decellularized ureter. In addition, the action of chemical detergents and stirring for a long time could destruct the structure of the decellularized matrix and then affect the construction of the engineered ureter. Therefore, exploring a fast and convenient decellularized program, which completely removed the cellular components and well preserved the structure and the composition of the ECM, could have important significance for the future ureteral reconstruction.Previous studies had reported that the ECM played an important role in regulating the stem cell differentiation. The study of DeQuach and his colleagues showed that skeletal muscle acellular matrix could promote the differentiation of muscle-derived stem cell into skeletal muscle cell and heart acellular matrix could also promote the differentiation of embryonic stem cell into cardiomyocyte. It had been reported that the hardness of acellular matrix could regulate the differentiation of mesenchymal stem cell. For example, stiffness substrates were beneficial to the osteogenic differentiation of mesenchymal stem cell, while soft substrates were beneficial to the adipocyte differentiation of mesenchymal stem cell. In addition, there were also many reports that some components of the ECM could promote the differentiation of stem cell. There was no reported that the components of decellularized ureter could promote the differentiation of stem cell.Based on the analysis of above issues, this study can be divided into three parts:Part 1:The perfusion preparation and conditions optimization of decellularized ureterObjective:To explore the feasibility of decellularized ureter prepared by perfusion system, and systematic and detailed evaluation of the prepared decellularized ureter. Methods:Using the lumen structure of the ureter itself, the decellularized ureter was prepared by perfusion system. At the same time, according to the different characteristics of chemical detergents, we set three different perfusion groups, SDS group, TritonX-100 group and combined group (TritonX-100+SDS). Results:HE staining and DAPI staining showed that there were no obvious nuclear materials in the combined group. DNA quantification and agarose gel electrophoresis were further confirmed this result. Masson's trichrome staining, Alcian Blue staining, collagen quantification and GAG quantification determined that the ultrastructure and the bioactive components of the combined group were well preserved. SEM showed that there was a large amount of pore structure on the surface of the decellularized ureter prepared by the combined group. Conclusion This study demonstrated that perfusion system could be used to prepare the decellularized ureter. We also selected an ideal solution to prepare the decellularized ureter. Meanwhile the decellularized ureter could be used in the ureteral reconstruction.Part 2:The biocompatibility evaluation and recellularization of the decellularized ureterObjective:The biocompatibility evaluation and recellularization for the prepared decellularized ureter. Methods:To evaluate the biocompatibility of the prepared decellularized ureter by AO/PI staining and MTT testing. The beagle bladder smooth muscle cells were grown in the surface of the decellularized ureter to observe the behavior of cells in the acellular matrix. Results:AO/PI staining and MTT testing showed that the smooth muscle cells could grow and proliferate in the extract of the decellularized ureter. HE staining. DAPI staining and PCNA staining suggested that the smooth muscle cells could adhere, grow, migrate and proliferate in the decellularized ureter. Conclusion:The prepared decellularized ureter had good biocompatibility and could be used as the scaffolds for ureteral tissue engineering.Part 3:The chemical composition of decellularized ureter induced the differentiation of adipose-derived stem cellObjective:To explore whether the chemical composition of decellularized ureter induced the differentiation of adipose-derived stem cell. Methods:The adipose-derived stem cell was seeded on the surface of coverglass paved the digestive of decellularized ureter to observe the differentiation of theadipose-derived stem cell. Results:Immunofluorescence staining showed the differentiation of adipose-derived stem cell into smooth muscle cell. Western Blot testing confirmed the expression of smooth muscle cell. Conclusion:The chemical composition of decellularized ureter could induce the differentiation of adipose-derived stem cell into smooth muscle cellAbove all, in this study, the perfusion system was introduced to prepare the decellularized ureter, and selected an ideal solution to prepare the decellularized ureter. Meanwhile, the prepared decellularized ureter was evaluated systematically, which confirmed that the cellular components were removed, and the three dimensional structure and the bioactive ingredients were well preserved. We also identified that the decellularized ureter had good biocompatibility and made the recellularization testing. The results demonstrated that the decellularized ureter could be used as the scaffold for ureteral tissue engineering. Further study showed that the chemical composition of decellularized ureter could induce the differentiation of adipose-derived stem cell into smooth muscle cell...