Acellular Amniotic Emebraneand Small Intestinal Submucosa Promotes Skin Reparationand Vascularization

ObjectiveSkin has an important physical,chemical and biological Protection function,skin trauma caused by local or systemic damage with loss of skin barrier,and as soon as possible to repair the skin defect and restore maximum possible function and appearance has become an important treatment goal.Traditional repair methods are: auto-skin graft,allogeneic graft,heterologous graft and synthetic application of skin products.However,because lack of immune rejection and the corresponding district, The spread of using traditional treatment methods is limited to the application,looking for an ideal skin substitute has been a clinical Medicine problem in urgent need of solution.Tissue engineering technology has been used in natural or synthetic material to inoculation of skin stem cells,gradually degradation in the process of biological scaffold absorption.Hyperplasia of the cells to continue to plant breeding,shaped the formation of a new tissue-engineered skin,reached the repair and reconstruction of the trauma of the purpose of self-organization.So,"Tissue engineering" provide a new and challenging method for the proposed construction of artificial skin substitutes.In skin tissue engineering,biological scaffold material has been a hotspot research,which product for the development of blood vessels has been seen as the bottleneck problem. In recent years the study shows that Small Intestinal Submucosa(SIS) and acellular Human Amniotic Membrane(HAM) are two good biological scaffold materials.In other pre-clinical experiments have shown these two biological scaffold material is a good biocompatibility,to promote wound healing and,more importantly,damage to various tissue regeneration and there is specific remodeling.Research has shown that these two biological scaffold materials for skin wound healing have similar repairing effects,however,another study shows that the regeneration of the skin basement membrane has important significance,while the most important difference lies about the two types of Scaffold material in the fact of there is a natural basement membrane.The basement membrane mainly be composed of by collagen typeⅣ,Ⅶtype collagen and laminin composed of laminin LN,is made up of different protein molecules consisting of a protein family,are extracellular matrix (ECM) in the non-collagenous glycoprotein.Timpl was the first in 1979 found and named ESH from mouse sarcoma.It is widely distributed of the transparent layer in basement membrane,which Close to the bottom of cell-based,Combination of typeⅣcollagen and basement membrane formation of the skeleton,affect cell adhesion, movement,regulation of cell growth and differentiation,basement membrane of amniotic membrane is thick and continuous.LN basement membrane as a membrane protein-specific and major components,with typeⅣcollagen,heparins sulfate binding site,etc..At the same time which also be combined with the epithelial cells, triggering intracellular signal transduction,promoting their adhesion on the collagenⅣ,Thereby promoting cell migration and proliferationOur research will put off cell membrane and acellular small intestinal submucosa covered in SD rats on traumatic skin defects,Observe their effect on wound healing,At the same time,in the level of gene expression to detected vascular endothelial growth factor(VEGF) expression,Observation of their traumatic impact of skin angiogenesis, In order to find a vessel of the skin which has a better biological effect of scaffold materials.Methods1.Acellular small intestinal submucosa and acellular amnion Preparation:Put fresh pig jejunum washed repeatedly with water and remove it's outside layer and muscularis serosa,reversed it after removal of the mucosa.Using NaOH-HCL United Ways deal with cell treatment,PBS rinse repeatedly,then freeze-drying and packaging, sealed and disinfected with peracetic acid,and stored it back-up refrigerator in -70 degrees.Check the health section of the human amniotic membrane,washed blood clean with normal saline and repeated rinsing.Using 0.5%SDS with 37 degrees shocks the human amniotic membrane and placed it overnight,then using 0.25%trypsin digestion it for 2 hours and repeated washed it with PBS,freeze-dried,sealed packaging,with peracetic acid poisoning,under -70 degrees back-up refrigerator storage.2.Scanning transmission electron microscope to observe the production of biological scaffold material and the size of the aperture in surface3.Set up the SD rat model of full-thickness skin defects:use of SD rats 24,on both sides of the rats back,make a full-thickness skin defect with circle 1.8cm in diameter, Wounds were randomly divided into A group,B group and C group.After use of saline flushing,A group with HAM coverage,B group with ASIS coverage,C group were covered up with gauze.4.Using immunohistochemistry to observe the skin of rats in each group in the repair process of the distribution of K19-positive cells and the expression of VEGF.5.Using RT-PCR detection of each experimental group in two weeks time in the expression of VEGFmRNA6.Combined with image acquisition and analysis system,the data with mean±standard((?)±5) deviationindicated that come from the application of statistical software SPSS 11.5,using one-way ANOVA for group comparison,when P＜0.05 for statistical significance,when P＜0.01 to have significant statistical significance.Results1.two scaffold material volume than its surface area is large enough to ensure that a large number of cells can grow rapidly.At 80-90%porosity,suitable pore size and porosity of appropriate inter-connected structure can enhance cell ingrowth from the surrounding tissue of the speed and effectiveness.2.K19-positive cells in the host of two scaffold materials in the more invasive and is conducive to wound healing.3.Two types of scaffold materials to repair skin defects of rats,the vascular endothelial growth factor expression are stronger than the control group,and acellular amniotic membrane group of vascular endothelial growth factor expression is stronger than acellular small intestinal submucosa.ConclusionsThere is an existence acellular basement membrane,increased membrane SD rat skin wound healing in the number of K19-positive cells,enhanced dermal vascular endothelial growth factor secretion and is conducive to the healing of the skin; simultaneously to further explore the issue of blood vessels direction.