| BackgroundThe treatment of acute and chronic liver failure is one of the major clinical studies needed to be solved, about 300 thousands patients dying from this disease every year. Although medical treatment has gained great progress, there are still only 30% of patients suffering from this disease can survive. Liver transplantation is the most effective treatment of liver failure, but its development has been greatly restricted because of the lack of donor liver.The liver tissue engineering brought new hope for the terminal stage liver disease. Tissue engineering is a new cross-disciplinary which was developed from the late 20th century. In recent years, the researchers have got great progress in various tissues and organs regeneration in vitro accompanying with development of life science, materials science and engineering science. Some of which has been achieved commercialization such as cartilage and skin. Liver tissue engineering is a new discipline which combined biology with engineering principles. The core is to establish a three-dimensional compomers of cells and biological materials, which was a living tissue with vitality used for rebuilding the organization with lesion morphology,structure and function,to achieve a permanent replacement. The research topics include the selection of seed cells,,extracellular matrix material and the construction of tissue and organ regeneration. Hepatocytes are anchorage-dependent cells,they need an insoluble extracellular matrix,in order to survive, reorganization, proliferation and exert function, the biocompatibility and cell adhesion capacity of scaffold materials is very important. In recent years, people come to realize the matrix material plays an important role in tissue engineering, the matrix material is not only a signaling molecule or cell carrier, but also holder for the growth of new organizations. Therefore, such as searching an appropriate matrix material,studying its interaction relationship with the tissues and cells, exploring the signaling molecules, assembly model about cells and carrier, all are important research contents in tissue engineering.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 providing a greater space to grow and attachment area., The microcarriers are favorable for nutrients into and metabolite eduction from cell, improveing the metabolism of density and cell cultures. With the research of tissue engineering scaffolds, the researchers found that polymer materials are 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. In the liver tissue engineering scaffolds, Galactosyl-modified in a variety of natural and artificial polymers, going through the liver cell surface glycoprotein sialic acid receptor and high concentration of polymer chains on the galactose-based molecular recognition between the unique features, can improve liver cell-specific function. Galactosyl are surface asialoglycoprotein receptor of the liver cell, 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 enhance liver cells in the scaffold of extracellular matrix on the adhesion and proliferation. Our group have been successfully prepared a SF/GC macroporous microcarrier with hepatocyte-specific affinity,which can be prepared by using the silk fibroin,chitosan as raw material, through the conduct of chitosan-modified galactose glycosylation, and then complex the SF and GC in the way of application emulsified-chemical crosslinking, then treated with polarity solution and freeze-drying. Currently most of the liver tissue engineering using composite materials as scaffolds, and generally get a uniform cross-linking strength by chemical crosslinking methods, but the choice of crosslinking agent is critical. Glutaraldehyde is the most common amino acid cross-linking agent, but there is still toxic even the residual glutaraldehyde concentration was as low as 3.0 mg/L. The Water-soluble 1-ethyl -3--(3 - dimethyl aminopropyl) - carbodiimide (C8H17N3·HCl, EDC) and N-hydroxysuccinimide (C4H5NO3, NHS) are new crosslinker with non-toxic and good biocompatibility, which can trigger cross-linking of collagen to formed amide bond. In the cross-linking process the EDC/NHS does not enter to the end profile but convering to a water-soluble urea derivatives, so it is little toxicity. As a crosslinking agent to prepare scaffolds for tissue engineering it is expected to enhance the biocompatibility and optimize liver cell function.ObjectiveTo prepare a high-performance SF/GC macroporous microcarrier with hepatocyte-specific characteristics, using the silk fibroin, chitosan, lactose acid as raw material,completely innoxious EDC/NHS as crosslinking agent.And to study the biocompatibility of the neotype macroporous microcarrier we use the MTT testing and cell function testing.MethodsAt first, the initial dose of the EDC/NHS was initially definited through consulting the literature, meanwhile it is divided into high, medium and low groups by preliminary experiment. Then the silk fibroin solution and galactosyl chitosan solution were prepared. The silk fibroin aqueous solution and GC were divided into three groups after mixing according to a certain percentage, then the high, medium and low three groups of cross-linking agent were added to the three group of mix solution, At last three groups of liver cell-specific SF/GC macroporous microcarrier with a porous struscture were prepared in the methods of comprehend application of emulsified--chemical crosslinking, treated with polarity solution and freeze-drying. And the water absorption and density of the SF/GC macroporous microcarrier were determinated preliminary; The morphology of macro- porous microcarrier was observed under the inverted microscope and scanning ele- ctron microscopy.Then the SF/GC macroporous microcarriers of EDC/NHS as the cross-linking agent were prepared as the experimental group while the SF/GC macroporous microcarriers of glutaraldehyde as the cross-linking agent were prepared as the control group.The biological toxicity of the two group macroporous microcarriers were texted by the MTT texting. And then the cell function was compared after the two group of macroporous microcarriers were co-cultured with immortalized liver cell C3A. the data were analyzed by SPSS13.0 and P<0.05 is significant.ResultsThis research use the natural polymeric materials with good bio- compatibility includeing silk fibroin and chitosan as raw material, galactose was successfully grafted on the chitosan at the activation of EDC and NHS. 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.By comparing the macroporous microcarrier prepared by different doses of crosslinker we found when the dose of Crosslinking agent were EDC:0.6g, NHS:0.14g that the diameter of microcarriers ranged from 200 to 400μm, pore diameter ranged from 40 to 80μm and density ranged from 1.03 to 1.08g/ml,which was most suitable for liver cell growth. the macroporous microcarrier were observed uneder Scan- ning electron microscopy(SEM)that the surface of macrocarrier was an open pore microstucture. The pore sizes ranged from 40 to 80μm.It was open field, hornshaped, and hole distribution. the internal is also porous structure which are suitable for high- density culture hepatocytes. Compared to the MTT texting of the experimental group SF/GC macroporous microcarriers wit the control group we found that the cell activity of experimental group macroporous microcarrier was significantly higher than the control group (P<0.05). By comparing the cell function of experimental and control groups found that the glucose consumption of the experimental group were significantly higher than the control group(P<0.05), while the secretion of albumin was not significantly different (P> 0.05). The ALT and AST leakage of the experimental group were lower than the control group (P<0.05).Conclusion(1) A hepatocyte-specific affinity of the SF/GC macroporous microcarrier can be prepared by use the silk fibroin, chitosan as raw material, completely innoxious EDC/NHS as crosslinker,through the conduct of chitosan-modified galactose glycosylation, and then complex the SF and GC in the way of application emulsified-chemical crosslinking, treated with polarity solution and freeze-drying. (2) when the dose of Crosslinking agent were EDC:0.6g, NHS:0.14g that the diameter of microcarriers ranged from 200 to 400μm, pore diameter ranged from 40 to 80μm and density ranged from 1.03 to 1.08g/ml,which was most suitable for liver cell growth. the macroporous microcarrier were observed uneder Scan- ning electron microscopy(SEM)that the surface of macrocarrier was an open pore microstucture. The pore sizes ranged from 40 to 80μm.lt was open field, hornshaped, and hole distribution. the internal is also porous structure which are suitable for high- density culture hepatocytes. (3) The SF/GC macroporous microcarriers with EDC/NHS as the crosslinking agent are more adventage in biological safety,Metabolism and detoxification function of the co-culture cell than the The SF/GC macroporous microcarriers with glutaraldehyde as the crosslinking agent. while the synthesis function of the co-culture cell is not significantly different. |
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