Experiment Study Of Non-cultured Autologous Epidermal Cells Suspension Combined With Fish Source Collagen Membrane To Repair Large Area Deep Burn Wounds

ObjectiveThis topic explores a kind of quick separation of epidermis and the dermis to prepare the cell suspension with high cell number and cell activity.And it also proposed for making collagen membrane to cover the deep burn wounds or to be dermal substitute for transplantation,and to provide a new method to choose from to repair large area deep burn wounds.Mehtods1.Exploration of the best digestion condition and preparation of the epidermal cells suspension(1)Separate epidermis and dermis by enzyme digestion with one step,prepare epidermal cells suspension,measure cell number and cell activity by FCM(Flow Cytometry),identify the best digestion condition.(2)Validate the degree of separation of epidermis and dermis and the remaining of the basal layer cells by immunohistochemical P63 staining.2.Preparation of the collagen membrane and test of it’s physical and biological characteristics(1)Dissolve fish scale by acid,extract collagen,prepare the collagen membrane by the vacuum freeze-drying.(2)Observe the structure characteristics of the collagen membrane by HE staining,Masson staining and electron microscope scanning.(3)Test the density of the collagen membrane by the method of GB/T4472-2011.(4)Test the cell toxicity of the collagen membrane by the method of CCK-8.3.Experiments of epidermal cells suspension transplantation of the nude mouse(1)Prepare human epidermal cells suspension,make nude mouse as experimental object to prepare deep burn wound,make the collagen membrane as covering to transplant human epidermal cells suspension.(2)Observe the wound healing,calculate the wound healing rate.(3)Take specimens after transplantation,observe the condition of cell infiltration by HE staining,observe the condition of the collagen arrangement by Masson staining,identify whether human source cells involved in the epithelium by immunohistochemical HLA-ClassⅠstaining.Results1.The cell number and cell activity were affected by the digestive enzyme concentration and the digestion time.When digested for 20 min with 0.25%DispaseⅡ-0.25% Trypsin,the cell number and cell activity were the highest.With digestive enzyme concentration increased,the cell number and cell activity were increased after the first with the same digestion time.Along with the prolonged digestion,the cell number and the cell activity were increased after the first with the same digestion concentration.With immunohistochemical P63 staining,the basal layer was positive before separation.After separation,the basal layer of the epidermis was positive and no dermis was seen,and the basal layer of the dermis was negative and no epidermis was seen.2.Surface of the collagen membrane was smooth with good flexibility.The thickness is about 0.3mm,and the density was(42.76±0.13)kg/m3.The collagen was arranged orderly by by HE and Masson staining,and formed the size of mesh structure.Electron microscopy scanning shows the collagen fibers were arranged mesh,and the pore size was 100 um ~ 200 um.Cell toxicity experiment shows that The Ralative Cell Viability increased with the extension of time,and there is no significant statistical difference at the same time between the two groups(P > 0.05).3.Two weeks after transplantation,each group did not the collagen membrane residues,and skin island formed in part of the wounds.To 4 weeks after transplantation,most wound of the composite transplantation group has healed completely,and the wound healing rate was more than 90%.Part of the wound has healed in the epidermal cells suspension transplantation group,and the wound healing rate was less than the composite transplantation group.While there was only a modest and edge epithelium with remnant wounds in the collagen membrane transplantation group and the blank control group.Immunohistochemical HLA-ClassⅠstaining of the basal layer in the composite transplantation group was positive.The human source cells involved in the epithelium.Inflammatory cell infiltration and new blood vessels formation were ween with HE staining,while there were less inflammatory cells or new blood vessels in the other groups.Masson staining shows that the collagen was arranged orderly in all of the groups.Conclusions1.When using 0.25%DispaseⅡ-0.25% Trypsin to digest thigh split-thickness skin(the thickness is 0.3 ~ 0.4mm,and the size is 1cm × 1cm)for 20 min under the condition of 25℃,the cell number and the cell activity were the highest in the unit volume of the prepared cell suspension.2.Surface of the collagen membrane prepared by fish scale is smooth.It’s thickness is about 0.3mm.The collagen fibers are arranged mesh and the pore size is 100 um ~ 200 um.The collagen membrane is non-toxic and can be used as a covering of autologous epidermal cells suspension transplantation.3.Composite transplantation of collagen membrane and epidermal cells suspension is a good method to repairing the deep burn wound,the mechanism of which may be the skin island formation.It is expected to become one of the alternative methods to repairing large area deep burn wounds.