Experimental Study Of Tissue Compatibility Of Acellularized Liver Scaffold And Co-culturing Test With Cells In Vitro

China is a big country of liver diseases,so the patients with end-stage liver failure are very common.However, liver transplantation is still the only radical trement for end-stage liver diseases.Nevertheless because of the shortage of organ source and the long-term use of anti-rejection drugs after surgery,liver transplantation can not meet the clinical need, many patients who need liver transplantation were dying in the process of waiting for the liver donor.Bioartificial liver system has been used as an effective means for acute liver failure (ALF) treatment, also working as a temporary liver substitution while waiting for the liver or in the recovery phase of the liver after liver replacement therapy.However,being limited by the negative factors of technology, bio-safety and so on,the existing artificial liver system remained to be further improved. In short, the existing liver replacement therapy have failed to meet the clinical needs,and finding a safe,economical and effective alternative method is very urgent.Tissue engineering is an innovative way to build a transplanted liver tissue.The major task is to build a artificial liver by compounding of a large number of liver cells (or liver-like cells), endothelial cells and other cell material with the scaffold.The aim is to construct a tissue engineering liver for the patients whose liver has lost its morphology, structure and function,to abide by the use of biological and engineering principles.Therefore,it is of a great significance for liver tissue engineering research to alleviate the existing problem of lacking suitble treatment for liver failure.Because of the complex function and physiological structure of liver.Liver tissue engineering has more slashing demanding on seeding cells and the scaffold material.The perfect scaffold material should have enough surface area and space for cell mass to attach and proliferation,having the capability for shaping specific structures.In addition, the scaffold must be bio-compatible,degradable and most importantly having the function of exchange for Nutrition and oxygen.However, comparing with the complicated liver structure,the previously used synthetic scaffolds are all obviously too simple to meet the nutritional requirements for high-density growth of liver cells,let alone the imitation of the comlex structure and the micro-environment of liver.Therefore,it is urgent to find a kind of scaffold that can provide a three-dimensional micro-environment,which utmostly imitating the vivo environment liver cells lives. Only in this way can we ensure the number and function of liver cells.The emergence of decellularized scaffold breaks through the limitation of traditional scaffolds which have simple structure.Decellularization was a process of removing all the cells from organs and leaving ECM and three-dimensional structure without being damaged.It is usually accopmlished by means of chemical or physical methods.Decellularized scaffold have been become a hot research in tissue engineering in recent years.Decellularized liver scaffolds have a great deal of advantages compared with previous liver scaffolds.First,it retained the three-dimensional network structure of liver which provides the necessary micro-environment for cell growth and prolification.In addition it also retained the most part of the liver extracellular matrix, including collagen,elastic fibers, proteoglycans and other components,which play an important role not only in the liver cell adhesion,growth and other biological behaviors,but also in the process of interacting with the surrounding cells for cell differentiation,migration and interaction by providing signals between cells.Second, the retained blood vessels network of the scaffold resolve the problem of nutrition input and waste discharges,that is very necessary to hepatocyte growth and make the organization culture of liver cells as possible.Third,because extracellular matrix components are all highly conserved between different species,extracellular matrix scaffold has good bio-compatibility and low immunogenicity.Finally,there may be some active components left in the extracellular matrix which will be benefit for the cell growth, proliferation and differentiation. So far,decellularized scaffolds have been successfully used in the study in cardiovascular, ureter, bladder, liver tissues and other organs.On the other hand, tissue engineering liver constructed by decellularized scaffold must be transplanted into allogeneic body to play a role in the final form.The compatibility test is one of the importment criteriaits Whether or not decellularized liver scaffold can be tansplanted into the allogeneic recipient.It will cause severe immune response and serious inflammation if the cellular components can not be completely removed from the tissue,and result in the degradation of tissue and calcification.However, even though the most highly immunogenic substances, such as histocompatibility (MHC) I and II-like protein had been removed from the scaffold,a small amount of cellular debris and lipid composition left in the scaffold still can cause immune rejection and lead to calcification.So far,there are few relevant reports concerning the allogeneic histocompatibility of decellularized liver scaffold.Therefore,it is of important significance to carry out the research of making tissue engineering liver with decellularized scaffold and simultaneously testing the tissue compatibility of scaffold. In previous work, our group has successfully made decellularized scaffold by chemical methods.On the basis, we have combined the scaffold that preserved the pipeline structure after the decellularization into the Circulation devices constructed by ourself and co-culturing with the liver tumor cell-lines C3 A in vitro.Experiments result confirmed that the cells could adhere to the scaffolds closely and be able to keep certain hepatecytes activity and function for a long time even in the circulating state.It is proved once again that the acellular scaffold has good biocompatibility and reserve the pipeline stucture that stimulated the Intrahepatic structure of the liver at its Maximum.In addition, the Allograft implantation test result showed that the acellular scaffold has been comletely decellularized and resulted to minor minimal inflammation and good histocompatibility.The test made a basis for the further use of acellular liver scaffolds to construct a tissue engineering liver that can be transplanted.ChapterⅠ:Circulating co-culturing test by use of decellularized scaffold in vitro.Objective:In order to further construct the experimental basis for further liver tissue reconstruction,we performed the circulating perfusion co-culturing test by use of decellularized whole liver scaffold with C3A cells in vitro,and doing the function assay and histological examination on time.Methods:SD rats as the subjects,making whole liver decellularized scaffold by use of chemical detergents.Cannulated the portal vein and perfused sequentially withl%TritonX100 and 1%SDS for about 4h,and then perfused with phosphate buffered saline to dilute SDS residue.The pipe structure of the hepatic vein, bile duct and hepatic artery were ligated with silk thread l.The acellular scaffold were sterilized through oxygen acid (0.1%) and rinsed repeatedly with PBS solution.Injected about 5mlL C3A cells (about 106/ml) to the scaffold through the portal vein by using of Penicillium needle with a volume of 1ml.Then,the scaffold was connected to the self-construcred device (with incubators, oxygenator, peristaltic pumps, gas cans and mixed media, etc.),with the inlet connected to the preserved portal vein structure in vitro.The culturing period lasted for 10 days.The culture medium was changed for a half every 2 days.We insisted on regularly check of the functional status and whether there was pollution in the circulatory system.Finally, the specimens was dehydrated by paraformaldehyde and embedded in paraffin,HE staining and the biopsy tissue were made to show the cell growth and distribution. RESULTS:We constructed the circulation culture system with scaffolds successfully and made 11 times of culturing tests.Among them,culturing pollution were found in two times in the training process.The main reasons might be for the improper operation of fluid changing in the building process and training process and so on.Pollution does not appeared in other tests,among which the result of 5 times were not very well.A large number of cell debris were observed in the medium cultured in the 3th day,and the biochemical liver function tests showed poor cell growth and function state,the pathological examination showed Most cells fall from the scaffold and a large number of cell debris with a small amount of residual liver cells existed.The reasons leading to the cell damage maybe the unreasonable oxygen supply,too fast perfusion speed causing the shearing forces and other reasons.Afterwards,through further exploration and improvement of culturing methods,the results of circular culturing of C3A cells had developed greatly.Conclusion:Perfusion with chemical detergents is a repeatable method to make decellularized scaffold.The circulating perfusion culture is also a feasible method to construct the tissue engineering liver with acellular scaffold, but it needs further test to explore.Chapter II Local reaction experiment by allograft implantation of acellular liver scaffoldObjective:To evaluate the biocompatibility of acellular liver scaffold made by chemical detergent perfusion,to make experimental basis for the further use of scaffold to reconstruct the tissue engineering liver and the following transplantation in vivo. Methods:SD rats decellularized scaffold as the research object. Experimental group scaffold was prepared by the chemical detergents in the light of experiment 1, and the positive control group was prepared except for omitting the perfusion process of SDS.After the decellularization, the sterilizing procenture was acomplished by use of peracetic acid (0.1%) (as the same method I)and thoroughly washed by repeated perfusion with PBS solution for 10 times, At last the scaffolds were immersed in PBS solution for preservation in-20℃refrigerator. Before the experiment the frozen scaffold tissue were cut into slices with a diameter of about 1cm and 1mm thick. Select 5d,14d,21d,28d,35d as the five different implanting periods. There were three Ba Bi Xi mice for each implanted period in contrast, the positive control group with three mice. All animals were anesthetized by diethylether.The scaffold slices were embedded in the place between the skin and muscles of the mice back.Gross observation and histological evaluation such as tissue inflammation degree, with or without blood vessel formation,and the condition of fibrous capsule formation and so on.Rating According to the degree of inflammatory reaction and capsule formation Results:The scaffold in the experimental group were completely decellularized.The scafffold looked translucent white and no obvious residual cellular components by microscopy; Positive control group remained opaque and some remnants of cell debris could be seen by microscope. The experimental biology results of the positive control group showed visible local swelling, purulen in the 5th day after surgery. No deaths occured in the experimental group animals during the experiment and the surgery scar healed well, without swelling,ulceration and other symptoms of infection.The histological result of the positive control group shows a large number of neutrophil infiltration.But,no serious inflammatory reaction and no calcificationin were observed in the experimental group after surgery. Tissue reaction results of the assessment were with one accord with the histocompatibility criteria (qualified) Conclusion: Chemical decellularized method can effectively remove the cellular components of the liver and retain low immunogenicity of the extracellular matrix components. So, the acellular liver scaffolds has a satisfying tissue compatibility and can be seem as a candidate for liver tissue engineering scaffolds.