Initial Exploration On The Autophagic Inductive Activity Of Degradation Products Of ECM And Recycling Of Deep Second-degree Burn Skin

Background To Repair of deep degree burns, especially in functional position, is always a challenge in the burn Medical. Clinical indicates that lacking of autoskin of large area deep degree burn patients causes difficulty of closed wound, but large sheet of split-thickness autoskin or cultured epidermal sheets grafting with the acellular dermal matrix could be a choice for the management of deep degree burn wound, which not only solves the wound closed difficult problem, but also reduces scar formation and contracture obviously. But the allogenetic scaffold material biological activity is limited and costs high, which were difficult to meet the need of large area shin transplant for burn patients. In recent studies, recovery of deep burn with the preservation of denatured dermis together with split-thickness autoskin could restore the function and appearance of the burn wound, which demonstrated that the denatured dermis will recover to normal or close to normal structure and morphology. The research of the reuse of denatured dermis discarded after escharectomy becomes the hotspots.In recent years, autophagy is the hottest research fields of life science, autophagy is a common life characteristic of the eukaryotic cells, which plays an important role in maintaining cell self-steady state, promoting cell survival and widely involving in various physiological and pathological process. Research showed that lysosomal enzymes can be transferred to the inside of the cytoplasm and even secreted to the outside of the cell to degrade ECM, at the same time, the mechanism of cell autophagy was connected with releasing of lysosomal enzyme, we speculate that there is a correlation between the degradation products of ECM and cell autophagy and had cell autophagic inductive activity.Therefore, this study is to explore whether the degradation products of the ECM of deep second-degree burns skin (DBS) has autophagic inductive activity or not, and to explore the preparation methods, tissue compatibility and the feasibility of deep burn wounds be repaired as a dermal substitute of deep second-degree burns dermal matrix (DBDM).Abstract:Objective (1) To observe expression of autophagy-related gene Beclin Ion deep second-degree burn skin (DBS) in rats, and investigate the relation of autophagy and scald tissues.(2) To explore the autophagic inductive activity of DBDM and the feasibility of DBS as dermal substitute for constructing model of complex skin transplanting in repairing wounds. Methods (1)16Wistar rats were randomly divided into2groups:the normal group (control group) and the scald group (experimental group),8rats in each group. Second-degree scald models were established in experimental group, and scald tissues were obtained1,3and7days after scald. The rat skin in each group was evaluated by histologically examination. Expression of Beclin1in each group was detected by the immunohistochemiscal technique and conducted by image processing techniques. Statistical analyses were performed.(2) Implanting experiment:20Wistar rats were randomly divided into the deep second-degree burn dermal matrix (DBDM) implanting group (experimental group) and acellular dermal matrix (ADM) implanting group (control group),10rats in each group. After a second-degree scald, the skin of two rats in experimental group was treated with enzyme digestion to remove cells to prepare DBDM. ADM was prepared by normal skin in control group. Implanting tissues were obtained3days and1,2and3weeks after operation. Cell growth in dermal scaffolds of each group was evaluated by histologically examination. Expression of Beclin1in times of each group was detected by the immunohistochemiscal technique and conducted by image processing techniques. Statistical analyses were performed. Transplanting experiment:20Wistar rats were randomly divided into the deep second-degree burn dermal matrix (DBDM) transplanting group (experimental group) and acellular dermal matrix (ADM) transplanting group (control group),10rats in each group. After a second-degree scald, the skin of two rats in experimental group was treated with enzyme digestion to remove cells to prepare DBDM. The back skin of newborn rats was integrally taken and the subcutaneous and dermal tissues were removed. The thin skin overlapped with mesh DBDM were transplanted into the back skin wound of full-thickness in size of2cm×2cm or so. After14days, the situation of composite skin growth was observed. ADM was prepared by normal skin in control group, and other experiment steps were the same as the experimental group. Results (1) Expression of Beclin1was positive in experimental group, but negative in control group(P<0.05).(2) The freshly prepared DBDM was yellow-white, less soft in texture of flexibility and extensibility compared with ADM. Collagen fibers of DBDM were thickened unevenly with hyaline changing and arranging in disorder. No cellular, blood vessels, hair follicles and other components were observed in DBDM under light microscope. Implanting experiment:The incision in each group healed well without local swelling and inflammatory response after grafting. The grafts have compact contacts with the wounds. Fibroblasts, neutrophils and lymphocytes migrated into the grafts from3post grafting day.1week after implanting, fibroblasts and micro vessel developed evidently.2and3weeks after implanting, there were many micro vessels in implanting tissues with a large range. Expression of Beclin1reached the highest value in experimental group. There was significant difference between experimental groups and control group (P<0.05). Transplanting experiment:Fourteen days after the composite skin transplantation experiments, the survival rate of rats and transplants in the two groups had significant difference (P<0.05). The bilayer composite skin and subcutaneous tissue connected closely. The deep DBDM or ADM was pink. The skin after operation had no significant swelling or inflammation. Infiltration of inflammatory cells and ingrowth of fibroblast and capillary appeared more earlier in DBDM group compared with those in ADM group. Inflammatory cells were mainly neutrophils and macrophages. New collagen deposition was rapid and collagen fibers arranged in a regular and dense state. Conclusion (1) Expression of Beclin1in the experimental group was apparently higher than that in the control group, and autophagy was speculated to participate repair process of second-degree scald.(2) Sources of DBDM are wide and preparation method is simple. Biocompatibility of transplants is good, burn toxin-like effect is weak and enhance autophagic inductive activity, inducing cell autophagy to remove damaged organelles, keeping cells from further injury and apoptosis, creating conditions for transfering to repair period, have the potential of return to normal structure. DBDM can act as dermal substitute of autologous split thickness skin. The experiment may provide new ideas for clinical transformation and utilization of DBS.