Porcine SIS Bilayer Bionic Wound Dressing Composed Of SIS Membrane With SIS Cryogel

Background and ObjectivesPain,physiological dysfunction and physical appearance changes caused by chronic non-healing wound and/or tissue defect seriously affects the patient’s quality of life and movement,and even endangers life.The application of autologous and allogeneic grafts for covering wound is limited by the risk of infection,ethics,and infectious diseases,while the tissue engineering dressings with good performance are more potential.Recent studies focus on animal-derived heterogeneous biomaterials.The biomaterials which is easy to be prepared have the advantages of abundant source,good biocompatibility,safety and reliability,and is a good scaffold for tissue regeneration without ethical problem.Small intestinal submucosa(SIS)derived from pig is non-immunogenic,biocompatible,degradable and packed with growth factors,which is an ideal biomaterial.However,SIS still has some disadvantages:first,SIS is a thin film lacking of enough three-dimensional structure,which limits its applications;second,the SIS is compact,lacking of pores,which is not conducive to the migration of cells and the exchange of nutrients.In the early study,our research group found a new approach for SIS crosslinking and innovatively prepare d SIS cryogel.The new 3D scaffold biomaterials prepared in this way has the property of elastic deformation and shape memory.Basis on SIS cryogel,we created SIS-based double-layered bionic full-thickness skin dressing composed of acellular SIS membrane and SIS cryogel.Its feasibility as a scaffold for cell transplantation and a dressing for wound is evaluated by biological and physical tests and provide theoretical basis for the repair of wound and tissue reconstruction.The complex structure was observed by scanning electron microscope.The mechanical properties,cytotoxicity and the ability to promote wound healing were tested.Cell culture in vitro confirmed that the lower layer of SIS gel could absorb the cells,and the cells could adhere to the scaffolds.The bilayer materials own good biological properties,physical properties,strong compression resistance,low porosity,strong tensile capacity and resistance to bacteria.It could be a support for cell transplantation,also be used alone for its ability of capturing nucleated cells to form tissue engineering scaffolds,which has a wide application prospect in tissue reconstruction and wound repair.MethodsThe study can be divided into 5 parts.Part 1:preparation of SIS membrane and SIS powderpreparation of acellular SIS membrane by mechanical scraping and degreasing,trypsin digestion,detergent cleaning and other sequential steps.The SIS membrane crushed in low temperature,was digested by pepsin,then centrifuged,salting out,dialysis and freeze-dried to make SIS powder.Part 2:the preparation of SIS cryogel and bionic full skin scaffoldThe SIS powder was freeze cross-linked with SIS membrane to form a double layer structure.Observation of microstructure and pore size of bilayered SIS by S canning electron microscopy.Study on mechanical properties and water vapor transmission rate(WVTR)of bilayer SIS.Part 3:The resistance of SIS bilayer bionic skin scaffold to bacterial invasion.The bacteria were seeded on the surface of SIS bilayer bionic skin scaffold compared control materials,then the amount of bacteria penetrated through various materials was measured by plate count and the resistance to bacterial invasion was evaluated.Part 4:Cell culture on SIS bilayer bionic skin scaffold in vitro.Isolation and culture of bone marrow mesenchymal stem cells derived from GFP transgenic mice.Observation by fluorescence microscopy and confocal laser scanning microscopy.Cytotoxicity was detected by CCK-8 kit compared with commercial collagen sponge.Part 5:SIS bilayer bionic full-thickness skin scaffold for wound healing.After the preparation of full-thickness skin defect in rat model,the effectsof SIS bilayered bionic full-thickness skin scaffold on wound healing was evaluated,comparing with the blank control,the commercial gelatin sponge,SIS membrane and SIS cryogel.The expression of PCNA,E-cadherin and CD31 was measured by q RT-PCR,and the effect of the bilayered bionic material on proliferation of cell in wound margin and wound vascularization.Results:1.Acellular SIS membrane and SIS powderwas prepared successfully.The surface of SIS membrane showed a reticular structure without cell underscanning electron microscopy.2.SIS cryogel and SIS bilayer bionic full-thickness skin scaffold composed of SIS cryogel and SIS acellular membrane were prepared successfully.Observation under scanning electron microscopy showed that the surface covered by acelluler SIS membrane is closely connected with SIS cryogel below to form a bilayered composite with dense surface layer and porous lower layer.The mechanical and water vapor permeation tests show that the material has excellent mechanical properties:Young’s modulus 10.95MPa,tensile strength 1.26MPa,elongation at break 35.41%,water vapor permea bility656g/m~2/day.3.Bacterial penetration experiments showed that SIS bilayer bionic full-thickness skin scaffold and SIS membrane can prevent bacteria significantly,comparing with vaseline gauze,commercial gelatin sponge and SIS cryogel.(p<0.05,n=3).4.The bone marrow mesenchymal stem cells(MSCs)isolated from GFP transgenic mice were cultured in vitro,and then the cells were loaded with SIS cryogel.The results of fluorescence microscopy and laser scanning confocal microscope(LSCM)showed that t he SIS bilayer material had cell adhesion,the cells attached to the surface of the scaffold and the pore,they were flat,spindle like,and linear.CCK-8 kit test results showed that SIS bilayer group was higher than commercial gelatin sponge.5.The mice full-thickness skin defect wound healing process.Three days after injury,SIS bilayer was healing faster than gelatin sponge group and blank control group,the healing rate was 63.22%,21.32%,12.18%,respectively.Seven days of wound healing after injury,90.63%,67.27%,47.96%,respectively,the difference was statistically significant(P<0.01,n=3).The wound healing rate of SIS bilayer group was significantly higher than that of gelatin sponge group and blank control group at 3 and 7 days after inj ury;Quantitative Real-time PCR detection,SIS bilayer group cell proliferation marker PCNA expression level is the highest,SIS film and gelatin expression was lower,the difference was statistically significant(P<0.05,n=3);at the same time to det ect the E-cadherin expression in the SIS group,it was the lowest group,about 35.32,significantly lower than the maximum value of 72.09 that in Gelatin sponge group,the difference was statistically significant(P<0.05,n=3);CD31 was found on the 7 day bilayer group(4.67)was significantly higher than that of SIS membrane group(2.05),SIS cryogel group(2.39)and gelatin gelatin sponge group(1.21)(P<0.05,n=3).Conclusion1.Successfully developed the SIS bilayer bionic skin scaffold.The utility model relates to a three-dimensional tissue engineering scaffold which is composed of a SIS cryogel with elastic deformation and shape memory function and an acellular SIS membrane.The SIS membrane is the surface of the bilayer,which is tightly co mbined with SIS cryogel,the two are closely connected,the surface layer is dense,the lower layer is porous.The mechanical and water vapor permeation tests showed that the material has excellent mechanical properties and appropriate water vapor permeabi lity.And it has good antibacterial effect.2.SIS bilayer bionic skin dressing can promote the proliferation of cells.The porous structure of SIS gel can promote cell migration and nutrient exchange.Crosslinking agent EDC does not increase the cytotoxicity of SIS materials.Cell attached and grew on the scaffold surface and pore.3.SIS bilayer bionic skin dressing can promote wound healing.The upper dense SIS membrane has the function of preventing the invasion of bacteria and strengthen mechanical properties and keep the appropriate moisture.The porous structure of SIS gel is beneficial to the proliferation and repair of wound.In addition,SIS material has good physical and chemical properties and biological characteristics,can effectively protect t he wound surface and establish a good local micro-environment,promote wound repair.