| Background:Liver is the largest and most important gland in the body, which participates in almost every metabolism process of the body. In addition, the detoxification and phagocytic immune functions made it to be the most important barrier in human body.In the past two decades, liver diseases did more and more harm to human beings and thousands of patients suffered these diseases every year. It was usually accompanied with severe metabolic disorders, neurological complications in these patients with liver failure,and eventually leading to multiple organs failure. China is a county with the highest incidence of viral hepatitis and liver cancer in the world. There were more than millions of people died of liver cancer and more than a half of them were in our country.So far as I know, orthotopic transplantation remains to be the most important treatment to the end-stage liver failure. However, the demand number of donor liver for liver transplant was far more than the current number of available donor liver. In this context, a number of new methods such as the use of tissue engineering and regenerative medicine to achieve the function substitution were regarded as new choices to relieve the contradiction of donor deficiency. Although it was reported that there were much new progress in the regeneration of tissue and organ in vitro, but all failed because of the deficiency in whole blood vessel network, that made it hard to do perfect nutrition and gas exchanges, let alone to achieve organ transplantation. In recent years, with the development of decellularising technologies, the emergence of whole organ decellularized scaffold made it possible to regenerate new organs. These scaffolds retain most kinds of the natural extracellular matrix components,particularly retain the micro-architecture of the tissues and organs such as the complete vascular system, so as to achieve the exchange of substances and vascular anastomosis for the circulatory system. Currently, the research of artificial organs developed by decellularization technology has been related to the heart, liver, lung,kidney, and other organs, with some experimental results encouraging. However, as most of the thick solid organs were complex in structure, the decellularization process for whole organ especially the large animal whole liver is very complicated.Porcine liver was similar to human liver both in size and shape. Therefore, it was the most suitable scaffold for regenerating a new tisse regeneration liver in vitro.However, the decellularized efficiency was affected by the sort of decellularized reagent and perfusion rate due to the thick tissue in liver and the complicated change of tissue sucture in the decellulaising process. At present there was no consensus in the decellularized method. It was reported that lot of reagent and prolonged perfusion time were needed in the process, with the result of consume in material and lose of scaffold material such as sGAG and so on. So it was very essential to find the best method for decellularization of the liver in large animals.On the other hand, the liver structure and function are extremely complex, and it is very difficult to rebuild a liver in vitro. So it had litter progress in recent years. It has been a new hot topic that how to further expand the application scope of decellularized scaffold.As we all know, hepatocytes quickly reduce its function and growth rate when cultured outside the body. For example, cell synthesis and secretion of albumin, urea and other substances, cell transformation, drug metabolism, ammonia and free bilirubin P450 -dependent metabolic, glutamine synthetase (GS) et al were all descent.Other enzymes such as glucuronosyltransferase (UGT), glutathione S-transferase(GST), aminotransferase such as ALT, AST, tyrosine aminotransferase (TAT), lactate dehydrogenase (LDH), carbohydrate metabolism as well as the associated G-6-P are also reduced to different degrees. Therefore, in order to culture and stimulate the growth of hepatocytes in vitro, it was very importment how to simulate the growth microenvironment of hepatocytes.Decellularized tissue, organ scaffolds retained most of the extracellular matrix components, including specific combinations of kinds of collagen,glycosaminoglycan (S-GAG) and various other components of glycoproteins.Therefore, we could conclude that decellularized tissue, organ scaffolds were theoretically the most suitable material for cell growth in vitro. In recent years,decellularized liver scaffold is increasingly used in the research of liver tissue engineering. Many scientists have mastered the technology of making decellularized liver scaffold and start to try to reconstruct a liver tissue by utilizing the decellularized liver scaffold in vitro. It has been reported in recent years that some scientists succeeded in raising and culturing hepatocytes, endothelial cell and stem cells in the decellularized liver scaffold. Some recellularized tissue engineered livers were transplanted into the body of animals suffering from hepatic failure. However,as there were very complex and subtle spatial tructs in the liver, these hepatocytes and endothelial cells were more vulnerable to suffer the harm affected by fluid shear forces and other harmful factors. At this point, its cell shape and function were obviously degraded, coupled with de-differentiation of these cells. In summary, there was still little progress in the research in reconstruction of the tissue engineering tissue on the use of decellularized liver scaffold.In order to further expand the application scope of decellularized scaffold, we intended to make a sort of gel-like soluble material with certain hardness and shape.The content and ratio of collagens,and someactive materials were tested compared with type I collagen and decellularized heart scaffold. At last, cell cuturing experiments were used to testify their effect in cell proliferation and differentiation of hepatocytes. In future, we would transform it into kinds of gel-like product with different three-dimensional structures, and finally let it mixed with cell to treat animal models with liver failure by minimally invasive surgery.Chapter Ⅰ: Optimization of the decellularising protocol for porcine liver scaffold and the following identification of components Objective: Make progress in preparing for the decellularized porcine liver scaffold and get an optimized preparation scheme. The structural properties and tissue compatibility of the scaffold were all tested for the use of large animal decellularized liver scaffold in vitro in future.Methods: Porcine cadaveric livers were regarded as the research objects. Solution of SDS as the detergent, urokinase soultuin and EDTA were used successively to handle the porcine cadaveric livers. After the initial treatment of cadaveric liver, a silicone tube (19#) was inserted into the portal vein of the liver and a peristaltic pump was connected to the tube. First all of, 500ml saline soultion with 100000IU/L urokinase saline was perfused into the portal vein at a speed of 10ml/min, and then put intoa-80℃ refrigerator for storage more than 48h. About 3000ml solution I (saline solution with 0.2g/L EDTA) was perfused with a speed of 40ml/min. About 15L solution II (saline solution with 5g/L SDS) was perfused with a speed of 20ml/min.About 200ml solution Ⅲ(saline solution with 0.01g/L DNase) was perfused and the liver was then stored at 4℃ for 2h. About 200ml solution IV(saline solution with 0.01 % peracetic acid) was perfused and stored at 4 ℃ for 3h. Finally, about 10L saline solution with 1% mycillin was perfused for about 8h. HE staining , PAS staining,Masson staining and electron microscopy were used to test the decellularized effect,DNA analysis kit was used to test the residual amount of DNA debris. SDS-PAGE and LC-MS/MS were used to determine the retain status of collagen and other active components of the scaffold. Blyscan Assay Kit was used to measure the S-GAG content on the scaffold.Results: In this study, 25 porcine livers were decellularized and succeed for 20 times,with a success rate of 80%. Portal duct of different levels were gradually visible when the contrast media (meglumine diatrizoate) was perfused through the portal vein,revealing no significant leakage of the contrast agent. Some examinations such as HE,Masson’s staining confirmed no significant residual cell structure in the scaffold,showing a lot of fibrous structure and intact ducts. The examination of DNA concentration showed that there was 7.411± 0.88 ng DNA residual on the lyophilized porcine decellularised scaffold. Purple substance residues could be seen in PAS staining, confirming polysaccharide glycogen and other kinds of polysaccharides.Scanning electron microscopy also revealed a lot of reticulated and tubiform structure preparation into a fiber network structure and tubular structure. Immunofluorescence stainings revealed a large number of I, IV, elastin, laminin and fibronectin materials in the scaffold. Besides these inherent proteins in the scaffold, LC-MS/MS experiment also showed a variety of of other polysaccharides. The result of sGAG kit test showed about 23.766± 0.423ug sGAG per mg lyophilized porcine decellularised liver scaffold, which was significantly more than that in lyophilized porcine decellularised heart scaffold (23.766±0.423ug/mg vs 14.519±0.328 ug/mg3,P<0.05,n=3).Conclusion: It was feasible to prepare the cadaveric decellularized liver scaffold by using the combined reagent solutions with chemical detergents and urokinase. In this method, cellular components could be effectively removed and most extracellular matrix components could be retained, with good biocompatibility. We could conclud from this study that this scaffold might be used as an alternative liver tissue engineering scaffolds.Chapter Ⅱ: Preparation for hepatocyte-specific culture medium by use of porcine decellular:ized scaffoldObjective: Hepatocytes quickly reduce its function and growth rate when cultured outside the body. Therefore,it was very importment to reconstruct a mimic growth micro-environment in vitro to cultivate and amplify hepatocytes. Decellularized tissue scaffold retained most extracellular components of organs matrix, including specific combinations of kinds of collagen, S-GAG components and growth factors. Therefore,the decellularized scaffold is theoretically the most suitable scaffold for culturing of cells. This study was going to make a sort of hepatocyte-specific culture medium from decellularized porcine liver scaffold. In order to certify the hepatocyte-specific function of this culture medium, we compare it with other culture medium and type I collagen for the difference in proliferation and function of hepatocytes.Methods: Decellularized cadaveric porcine liver scaffold was regarded as the experimental group and the decellularized heart scaffold and type I collagen regarded as control groups. Urokinase solution, SDS solution and DNA enzyme solution were successively used on liver, heart tissue slices for decellularization treatment. It gradually became a gel-like solution after these treatments of freeze-drying,crushing and being digested by pepsin-Hcl solution for 48h. Whereafter, SDS-PAGE and sGAG measured experiments were used to compare the differences of compositions among decellularized porcine liver scaffold culture medium, heart scaffold culture medium and type I collagen culture medium. Circular dichroism was used to validate the activity of collagen protein. Finally, C3 A cells were used as the tool to compare the specificity in cell proliferation, morphology and function during the culture period.Results: The decellularising process of porcine liver, heart tissue sections were completed in about 36h. These scaffold gradually become a white and viscous solution when digested by hydrochloric acid solution containing pepsin after 48h. The pH and osmotic pressure of the solution were adjusted by NaOH solution and 10PBS and following that the solution was ready for the use of cell culturing. The sGAG content of the solubilized liver matrix was determined to be 14.287± 0.127ug per mg of matrix, which was greater than that of the solubilized heart matrix, which was 12.373±0.125 ug per mg of matrix(P<0.05,T-Test). However, no GAG content was present in Type I collagen. In the test of cell apoptosis by Annexin V kit, there was about 94.16% normal C3A cells when cultured in the decellularized liver culture medium in the fifth day, more than that when cultured in the decellularized heart culture medium. In addition, there were less apoptosis cells cultured in the decellularized liver culture medium than that in the heart culture medium and type I collagen culture medium, no matter in the earlier or later culturing period. In testing the synthetic ability of C3A cells, we found that the amount of albumin and urea secreted by C3A cells was higher in the decellularized liver culture medium than in the other two groups (P<0.05, n=3) . On day 5 of culture, Albumin, G6PD and CYP3A4 mRNA levels were measured. There was no sifnificant difference in the expression of these three genes between the decellularized heart culture medium and type I collagen culture medium. However, the expression level of C3A cells were significantly higher than that in the decellularized heart culture medium and type I collagen culture medium (P<0.05, n=3)Conclusion: Most components of collagens and polysaccharide were reserved in the culture medium after digestion of the decellularized liver scaffold. This medium could enhance the proliferation ability and function of C3A cells when cultured in the decellularized liver culture medium. We could conclude that this culture medium made from decellularized liver scaffold was suitable to be used in the proliferation of C3 A hepatocytes in vitro. |
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