The Preparation Of Small Intestinal Submucosa Extracellular Matrix Hydrogel And Its Effect On The Liver Tissue Damage Repair

Background and aims:Small intestinal submucosa(SIS) of extracellular matrix(ECM) is remove intestinal serosa layer, muscular layer and mucosa, and a series of processing and after disinfection treatment, obtain a excluding cells of extracellular matrix in [1]. SIS is an ideal tissue engineering biomaterial, in the aspect of the body tissue repair has the following three aspects: biological advantages: 1. Has the histocompatibility, SIS mainly consists of Ⅰ, Ⅲtype collagen, and contains rich protein fiber connection and growth factor, even after a series of processing the SIS and the growth factor. Such as, vascularβfibroblast growth factor-2, the transfer of growth factor- endothelial growth factor, platelet-derived growth factor, etc., these substances are the normal components of extracellular matrix. Type of Ⅰcollagen protein and fiber connections are available in the cell membrane, the specific binding of pathway to promote cell adhesion and activate cells, and other various growth factors can stimulate cell proliferation. SIS, therefore, not only can offer the better for the host cell adhesion and migration of the natural environment, can also quickly with the host organization, promote angiogenesis and tissue function recovery. 2. the immunogenicity and safety, through mechanical method and chemical method combined processing of the SIS does not contain cells, its carrying pathogens probability is very low, and the weak immunogenicity, even also does not produce rejection after xenotransplantation. SIS after disinfection treatment, of various components are not lost or inactivation, ultrastructure and mechanical properties have no obvious change. Therefore, after disinfection treatment SIS has higher security. At the same time Sarikaya A etc. [2] points out that the SIS leaching liquid of E.coli and staphylococcus aureus has inhibition, can inhibit the growth of bacteria. 3. With biomechanical performance, small intestinal submucosa is a kind of nonlinear material, the vertical axis direction, whether digging wall or the tensile strength of tendon and ligament between 1/7 and a quarter of the organization. SIS as tissue engineering materials in the body, with the appropriate mechanical properties, can withstand the pressure from the surrounding tissue and cells to provide enough space for regeneration. Because of its good biological advantage, SIS materials and tissue engineering has been widely used in modern biological medicine, such as tendon reconstruction and lower urinary tract reconstruction, repair the dura mater, vascular remodeling, and repair parts or full-thickness skin damage, etc. [3]. But now research on SIS in liver tissue damage repair is less, but it has great research value and application potential. Therefore, this study used the pig intestine homemade small intestinal mucosa under matrix, by applying the pig small intestinal mucosa under matrix(small intestinal submucosa, or SIS) hydrogel, injection to induced by carbon tetrachloride(CCL4) injury of liver damage, as to research in the SIS effect on chemical liver injury repair. Through the study on the feasibility of injectable SIS treating chemical liver injury, for the clinical for the repair and treatment of acute and chronic liver injury provide basis and new method.Methods:1. Take fresh pig small intestine tissue, using SDS cells and enzyme digestion method, preparation of small intestinal extracellular matrix hydrogel; Cell-free effect matrix material with H&E staining method; And test the temperature of the water gel materials produced by the sensitivity; Determined by MTT method to detect water gel material biocompatibility of human normal liver cells.2. Cultivate people LO2 normal liver cells, were divided into normal control group, carbon tetrachloride CCL4 injury group, hydrogel CCL4 damaged cells, using flow cytometry to test each cell apoptosis, Ed U cell proliferation staining method to detect each cell proliferation, cell culture supernatant ELISA method to detect the groups in b EGF, VEGF and NGF plays, IL- 4, the change of IL- 6, IL- 8 content, to detect homemade small intestine hydrogel matrix of CCL4 damage of liver cell repair function.3. Use by peritoneal injection of carbon tetrachloride method for 3 months in a row, C57/BL chemical liver injury in mice model was established. Under ultrasound guidance, with 31 g needle homemade small intestinal mucosal damage liver subcapsular matrix hydrogel injection to. Collect the serum and liver tissue of mice after processing, SIS hydrogel on laboratory mice serum albumin(propagated), alanine aminotransferase(ALT), aspartate transaminase(AST), and the influence of liver tissue pathological changes.Results:Through SDS after treatment and enzyme digestion method, H&E detection can completely get rid of the pig small intestinal mucosa tissue outside the cell, thus successful preparation of the pig intestine extracellular matrix hydrogel material, and has obvious temperature sensitivity and biocompatibility. Flow cytometry detection found that small intestine extracellular matrix hydrogel materials can reduce the damage of liver cell apoptosis induced by CCL4; And Ed U detection method found small intestine extracellular matrix hydrogel materials can promote the injury of liver cell proliferation induced by CCL4; ELISA to detect small intestine extracellular matrix hydrogel materials can promote the liver cell culture environment b EGF, VEGF and NGF plays cytokine levels, and reduce proinflammatory factor the production of IL-6, IL-8, anti-inflammatory factor and increase production of IL-4; Using CCL4 to injury C57/BL mice liver model was established, successful under ultrasound guidance will be homemade small intestinal mucosal damage liver subcapsular matrix hydrogel injection to, after tests found water gel treatment group of a marked increase in the mice serum propagated, and the content of ALT and AST, ALT decreased significantly, while the H&E test also found that the pig intestine extracellular matrix hydrogel injection help damage morphology of liver tissue recovery.Conclusion:This study into pig small intestine extracellular matrix water gel preparation, testing found that it has win min and biocompatibility, and can by improving the related content of cytokine secretion, promote the injury of liver cell proliferation and inhibit apoptosis, thus reduce liver cell damage, and can be injected to the liver injury of mice, helped by its liver repair and liver functional recovery. Through this study will help to explore the new methods for clinical liver damage treatment and for tissue engineering of biological materials research and development and clinical use provides the basis.