| Hepatopathy is one of the most serious global diseases that harm human health. Now,liver transplantation and hepatocyte transplantation are feasible choices to treat liver diseases. However, procedures of liver transplantation are limited by the shortage of donor organs, high cost, and lifelong dependence on immunosuppression. Hepatocytes transplantation will bring the organism a strong post-transplant immune rejection, which makes transplantation ultimately fail. Therefore, it is urgent to find new techniques and methods for the above problems. The development of tissue engineering brought new hope for the therapy of liver diseases.Tissue engineering, as an interdisciplinary science, which purpose is constructing and replacing human tissue and organ, has made great progress in the skins, cartilage, nerve, blood vessel in the past 20 years.The development of liver tissue engineering is fast recently; meanwhile in this field great breakthroughs had been made. However, many problems and challenges are existed, for example, how to improve the vascularization of reconstructed liver tissue and to avoid host immunologic rejection after in vivo transplantation.This study used collagen/Matrigel and alginate microcapsules as liver tissue engineering scaffolds respectively. Based on this, we promoted the vascularization of engineered liver tissue through adding the vascular endothelial cells in tissue engineered hepatocyte/vascular endothelial cell sheets. Our results demonstrated that vascular endothelial cells could promote the hepatocyte viability and function. In other hand, based on the merit of avoiding immunological rejection, we prepared microencapsules as another tissue engineering material. Then we transplanted the co-encapsulated hepatocyte and vascular endothelial cell into rats to treat hepatic failure, vascular endothelial cell has improved the biological function of microencapsulated hepatocytes.The main study contents from the reconstruction of the 3D tissue to the evaluation of the microencapsulated hepatocytes include the following five parts:1. Construction of 3D engineered hepatocyte sheet based on collagen/Matrigel in vitroIn this part, we firstly isolated the rat primary hepatocyte by the improved collagenase digestion, which were cultured in the collagen/Matrigel environment for the construction of engineered hepatocyte sheet. Compared with the collagen sandwich structure, hepatocytes in the engineered hepatocyte sheet grow more aggregative and express the more actin, E-cadherin and albumin by assessing the cell morphology, cell-cell conjunction and secretion of albumin. These results show the 3D collagen/Matrigel environment is good for the growth and function of hepatocytes. The in vitro reconstruction of the 3D liver tissue played important roles in the treatment of liver disease and the relative basic study.2. Construction of 3D co-cultured the hepatocyte/vascular endothelial cell system based on collagen/Matrigel in vitroIn this part, we co-cultured the hepatocyte and vascular endothelial cell in the collagen/Matrigel environment and constructed the hepatocyte/vascular endothelial cell sheet. By assessing the cell morphology, H.E. staining, immunofluorescence and secretion of albumin, we found the vascular endothelial cells in hepatocyte/vascular endothelial cell sheet migrate together to form a vessel like structure. The albumin secretion of hepatocyte/vascular endothelial cell sheet was higher than the hepatocyte sheet, after 1, 3, 5, 7, 14 days culture in vitro.3. Preparation of microencapsule and evaluation of their biological functionIn this part , we used barium ion to substitute calcium ion to form Ba–alginate–polylysine–alginate(BPA) microencapsules based on the alginate–polylysine–alginate(APA) microencapsules. And then we tested the mechanical strength of APA microencapsules and BPA microencapsules with mechanical shaking experiments, and the osmotic pressure of two types mincroencapsules was measured by long-time culture in simulated body fluids. The results showed that BPA microencapsules have higher mechanical strength and long-time stability than APA microcapsules. We measured the transplantation strength of the microencapsules by transplanting the microencapsules into the muscle of rats. The results showed the APA and BPA microencapsules have little fibrosis, while APA microencapsules were out of shape and more swelled than BPA, BPA microencapsules were still spherical. After that, we measured the biocompatibility of the microencapsules by transplanting the microencapsules into the peritoneal cavity. After being transplanted for 7,14,28,56 days, the recovery was up to 90%, the recovered two types of microcapsules were intact and less fibrously adhesive.4. In vitro evaluation of microencapsulated hepatocytesWe prepared the BPA microencapsulated hepatocytes and evaluated the function and viability of the hepatocytes in microencapsules by the secretion detection of Live/Dead staining and albumin secretion. The results showed the microencapsules kept the integrity morphous in the process of culture. After being cultured for 7 days, the secretion of albumin of BPA microencapsulated hepatocytes is higher than the alone cultured hepatocytes, and most hepatocytes keep viable. Therefore, the BPA microencapsules we prepared are a suitable material for hepatocyte growth.5. Co-transplantation of encapsulated hepatocyte and vascular endothelial cell for the treatment of fulminate hepatic failureWe transplanted the empty microencapsules, isolated hepatocytes, microcapsulated hepatocytes, co-encapsulated hepatocytes and vascular endothelial cell intraperitoneally into rat models of fulminate hepatic failure induced by D-aminogalactose (D-gal). After 1d,3d,7d,2w,3w,4w transplantation, we calculated the mortality and assessed the ALT,AST,ALB in the serum of the rat model using the spectrophotometer, evaluated the integrality and recovery of microencapsules, and then carried out H.E. staining. The results showed that after 4w transplantation, the function of liver in co-encapsulated hepatocytes and vascular endothelial cell group was significantly higher than that in microencapsulated hepatocytes group, while such function in microencapsulated hepatocytes group also higher than that in isolated hepatocytes group. The recovered microencapsules were intact and less fibrously adhesive, which confirms the BPA microencapsules are suitable for the hepatocyte transplantation. Additionally, the microencapsules have good immunoprotection to protect the hepatocytes from immunological rejection and maintain the function of hepatocytes.In conclusion, this study firstly constructed engineered collagen/Matrigel hepatocyte sheet in vitro, then evaluated the collagen/Matrigel environment made for the hepatocytes viable and function. After that, we investigate the interaction between the hepatocyte and vascular endothelial cell, as well as the promotion of the vascular endothelial cell to the function of hepatocyte. Based on this, we prepared the BPA microencapsules with immunoisolation, and evaluated the biological function of the BPA microencapsulated hepatocytes. Subsequently, we co-transplanted the encapsulated hepatocyte and vascular endothelial cell for the treatment of fulminate hepatic failure, because the microencapsule can avoid the immunological rejection, co-encapsulated cells improved the liver function of fulminate hepatic failure, it brings new approach and theoretical basis for the treatment of fulminate hepatic failure. |
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