| BackgroundThe liver is one of the body’s most important organs responsible for the body to secrete bile, blood clotting, metabolism, swallowed or immune function, detoxification, regulating water and electrolytes, such as a series of complex physiological functions. Acute liver failure in the rapid progression of severe liver dysfunction caused severe liver dysfunction disease, seriously endanger human life. Various causes of acute liver failure, the conventional treatment fatality rate as high as60-80%. The effective treatment of acute liver failure in the clinical consist primarily of three types:orthotopic liver transplantation, bioartificial liver support system as well as liver cell transplantation. With the development of liver transplantation and immunosuppressant, liver transplantation has been the only effective treatment is the treatment of a variety of end-stage liver disease and acute liver failure. But because the liver organ transplant by the shortage of donor organs, surgical risk, high cost of treatment, the postoperative immune rejection leading to graft loss and lifelong need to take immunosuppressive therapy and other factors limit determines its still widely used in China certain difficulties. Liver cells cultured in vitro for the core build bioartificial liver support system is an ideal alternative treatments, but expensive bioreactor donor cell origin as well as the course of treatment may produce hemodynamicdisorders and other factors limit its wide range of clinical applications. Hepatocyte transplantation technique is simple, easy to operate, small patient trauma, a relatively low cost, reduced cell surface antigen after liver cells in vitro, can reduce immune rejection, transplanted hepatocytes can play a liver detoxification, syntheticspecial advantage. The relevant test studies have demonstrated that hepatocyte transplantation may be extended acute liver failure patients with donor waiting time and significantly improve the level of the patient’s liver function, because of the liver’s ability to regenerate and great potential, after accepting hepatocyte transplantation, some patients remaining liver wasregeneration, liver function can be restored to a normal level. Therefore, hepatocyte transplantation has been considered to be the end-stage liver disease and acute liver failure, a good prospects for the development of treatment methods. However, it needs to solve a lot of problems remain, including cell source and a high density of active liver cell culture. The quality and quantity of the liver cells are the critical issues in the study of hepatocyte transplantation, how easy access to high-density, high-quality, high activity of liver cells has become a successful liver cell transplantation important issues that need to be addressed. Macroporous microcarriers scaffold material as the core of the three-dimensional hepatocyte culture is expected to resolve this problem. The main use of the high density culture of the microcarrier suspension culture technology to achieve liver cells and amplification, the cell can achieve the clinical requirements of the application in the number and function. In recent years, research on microcarrier at the field of tissue engineering applications are increasing, mainly use the microcarrier suspension culture tissue engineering technology for the amplification of seed cells and as a transmission vector in vivo cell therapy. Compared to solid microcarrier, macroporous microcarrier has the advantage of provide a sufficient space and a larger area to grow and attachment. The microcarriers are favorable for nutrients into and metabolite eduction from cell, improve the metabolism of density and cell cultures. In the liver tissue engineering scaffolds, Galactosyl modification of various natural raw materials, and artificial polymers, asialoglycoprotein receptor between a unique molecular recognition function, optimize the liver cell specificity of the stent through the galactose group, and the surface of the liver cells in the polymer chain to improve the specific function of the liver cells. the researchers found that polymer materials suitable for chemical modification, such as surface modification and treatment can improve liver cells induced in the stent material on the adhesion and proliferation behavior. Galactosyl are the liver cell surface asialoglycoprotein receptor, liver cells are identified and produce the corresponding site-specific interactions. In tissue engineering scaffolds of the liver extracellular matrix based on the introduction of galactose, can induce and improve liver cells in the scaffold of extracellular matrix on the adhesion and proliferation. In our preliminary studies, we use the natural polymeric materials with good biocompatibility include silk fibroin, chitosan and Lactobionate as raw material, galactose was successfully grafted on the chitosan. Then a liver cell-specific SF/GC macroporous microcarrier was prepared by complex the SF and GC in the way of application emulsified-chemical crosslinking, treated with polarity solution and freeze-drying. the macroporous microcarrier were observed by Scanning electron microscopy that the surface and internal of macrocarrier was an open pore microstucture.It was open field, and hole distribution. It is suitable for high-density culture hepatocytes. The galactosyl has been successfully compounded into the scaffold material which was confirmed through the1HNMR spectrum, FITR spectroscopy characterization. The scaffold material was introduced to specific ligand of the liver cell surface asialoglycoprotein receptor-galactosyl. Pre-cytocompatibility experiments show that C3A cells can hepatocyte-specific adhesion to material, growth gathered around the microcarrier and a good show of vitality. However, the in vivo environment is more complex with respect to the in vitro environment, silk fibroin-based macroporous microcarrier-attached C3A cells is suitable for survival? Can the model of the animal acute liver failure effectively improve the survival rate, improve liver function, alleviate liver damage? The depth of these problems will undoubtedly contribute to the development of cell transplantation techniques. Based on the results of the work of current research, This experiment will adopt the method of SF/GC macroporous microcarriers-C3A cells suspension culture complex intra-abdominal transplantation, to observe the therapeutic effect of acute liver failure. The purpose of this study is to explore the SF/GC macroporous microcarriers-C3A cell complex in the feasibility of transplanting animal model of acute liver failure in vivo, provide the necessary theoretical and experimental basis for the better in the future clinical application of cell transplantation in the treatment of acute liver failure.ObjectiveLiver cell line C3A cells as transplanted cells, SF/GC macroporous microcarriers as the carrier, cultivate the SF/GC macroporous microcarriers with C3A cells and detect its biological characteristics. Explore the SF/GC macroporous microcarriers adhesion to cultured the C3A cell implanted into a rat model of acute liver failure which was induced by the removal of80%of the liver. Observe the survival rate, the level of liver function and observe histological changes of liver and graft tissue, evaluation of its effect on the treatment of acute liver failure in rats, and to provide the necessary theoretical and experimental basis for future clinical applications.MethodsSF/GC macroporous microcarrier with a porous struscture were prepared in the methods of comprehend application of emulsified-chemical crosslinking, cultivate with C3A cells and detect its biocompatibility and cell function, to determine the best period of the macroporous microcarriers C3A composite grafts to transplant. Remove the80%of the liver of rat to build up the model of acute liver failure. We intraperitoneally transplanted macroporous microcarriers C3A composite grafts (group A) and The C3A cell suspension group (group B) to rats with ALF which were induced by remove the80%of the liver. We compared the survival rate of the three groups during one week after transpiantation. the effects of transpiantation were evaluated by compared the liver function such as ALBã€ASTã€ALT, and the pathologic changes of hepatic tissue.ResultsThere is the highest albumin and glucose consumption level on the third day. The survival rate of group microcarrier was much higher than those of the group during one week after transplantation, Group A was50%, group B was30%, group C was10%, the discrepancy between three groups was significant (P<0.05). During1week after transplantation, the hepatic function of group microcarriers gradually decreased, the7days after transplantation,the hepatic function gradually to near normal levels, there is a statistically significant difference (P<0.05). the hepatic function level of the control group had no significant downward trend, ALT levels reached a peak on the first day after transplantation. Seven days after transplantation, pathological recovery occurs to some extent in the livers of model rats in group microcarriers. Compared with other two groups have improved significantly.ConclusionUse the natural polymeric materials with good biocompatibility include silk fibroin, chitosan and Lactobionate as raw material, galactose was successfully grafted on the chitosan. Then a liver cell-specific SF/GC macroporous microcarrier was prepared by complex the SF and GC in the way of application emulsified-chemical crosslinking, treated with polarity solution and freeze-drying. the macroporous microcarrier were observed by Scanning electron microscopy that the surface and internal of macrocarrier was an open pore microstucture. It was open field, and hole distribution. It is suitable for high-density culture hepatocytes. There is the highest albumin and glucose consumption level on the third day. so the third day may be the optimal time of applying microcarrier culture C3A cells for HCT. Remove the80%of the liver tissue of rat successfully induced to build acute liver failure model. Silk fibroin/galactose-chitosan macroporous microcarriers has good biocompatibility, application of SF/GC microcarrier culture C3A cells intraperitoneal transplantation can improve the hepatic function and increase the survival rates of rat with ALF. |
