| Rationale and objectives：Cell transplantation therapy can significantly improve the quality of woundhealing but not dependent on the skin graft surgical.Basic and clinical research ofcell transplantation therapy related is a hotspot and has a good application prospect,There are many kinds of seed cells of cell transplantation therapy. With thedevelopment of material science in recent years, choose a kind of biologicalengineering materials used in refractory wound healing is very important Andchitosan as a kind of excellent biocompatibility of biodegradable material, and it hasanti-inflammatory, antioxidant, hemostatic, promote wound healing, analgesia,anti-infection and so on many kinds of good for the body to repair the biologicalcharacteristics, is a very suitable for used in painless wound repair of the biologicalengineering materials. Chitosan hydrogel has been widely applied in the fields ofbiological medicine, clinical trials have made some achievements, the mechanism isnot clear, but its serious limits its further clinical application.In this paper, throughexperimental synthesis of chitosan hydrogel, joint between fat used in painlessmesenchymal stem cells for the treatment of wounds, the study of chitosan hydrogeljoint ADSCs of painless wound and to explore the efficacy of some of its biologicalmechanisms, painless sex for clinical treatment provides a new method of thewound.Objectives：To establish a highly efficient separation of ADSCs cultivation system, observethe biological characteristics of ADSCs, and through appropriate training, we canobtain the large-scale expansion of ADSCs in order to meet the required for celltransplantation therapy. We exploratory research the production the method ofchitosan hydrogel, and systematic analysis of its membranous, cytotoxicity, and surface structure. We study the chitosan hydrogel joint ADSCs promote refractorywound healing, and discussing the biological mechanisms of promote woundhealing.Methods：1. ADSCs conform to the morphological features of mesenchymal stem cells.Theoriginal generation of ADSCs cultivation cell collection rate is50%at3days,7dayscollection rate is90%.Take the third generation of ADSCs, using adipogenic,osteogenic, chondrogenic liquid cell culture and flow cytometry analysisrespectively, transplanted skin wounds, according to the different groups ofbiological fluids coated chitosan membrane covering the wound dressing on thewound.2. Chitosan, gelatin, acetic acid, glycerin dissolve in normal saline,37℃waterunder shock dissolved, made from chitosan hydrogel after filtering impurities, in thefilm is56℃under the condition of dry and incubation after48h to form chitosanmembrane, to observe the physical properties of the membrane body directly, andscanning electron microscopy test of its surface structure.Preparation of theimmersion of the material at the same time, with the concentration50%,100%respectively of chitosan membrane immersion test ADSCs toxicity.3.1. Preparation of mouse full-thickness skin model: To establish an ordinarymouse skin defect model with C57mice, the ADSCs suspension were transplantedon the skin wounds, and then coated with chitosan in wound fluid biologicalmembranes covering the wound dressing. ADSCs and CSH application on wounds:ADSCs transplantation and coating covering the wound CSH groups, respectively,depending on the wound. The suspension ADSCs transplanted skin wounds, andthen coated in a wound CSH cover the wound. Group A: saline group; Group B:CSH; Group C: ADSCs; D: CSH+ADSCs. Or without postoperative woundinfections every day, secretions, healing area, healing quality and so on. Measuringwound healing area: surgery, close observation of the wound, the postoperative0d, 3d,7d,10d,14d,18d,21d size of the wound tracings in a sterile transparent film,according to a formula to calculate the rate of wound contraction, recording thetreatment group and a control group healing time. without postoperative woundinfections every day, secretions, healing area, healing quality and so on. Measuringwound healing area: After inoculation, the situation closely observed wounds,postoperative0,3,7,10,14,18,21days will be the size of the wound tracings in asterile transparent film, according to a formula to calculate the rate of woundcontraction, record treatment group and control group healing time. Postoperative14d under sterile conditions were cut with a scalpel wound tissue for histologicalobservation, frozen sections of wound tissue epidermis and dermis and thepercentage of GFP-positive cells, in order to observe tissue wound healing cellsources the dynamic changes VEGF expression.Results：1. ADSCs conform to the morphological features of mesenchymal stem cells.Theoriginal generation of ADSCs cultivation cell collection rate is50%at3days,7dayscollection rate is90%.Flow Cytometry results showed that ADSCsectomesenchymal marker CD10, CD13, CD166and adhesion factor CD29andCD54is highly expressed;Low expression of CD34hematopoietic cell surfacemarkers and vascular endothelial antigen CD45.Conclusion: ADSCs isolation and culture system established in this study can get alot of ADSCs,to provide a favorable environment to ensure cell transplantion. 2. CSH is transparent, half liquid gel.CSD volume shrinkage, color is a bit deeper,have certain flexibility and water absorption. Scanning electron microscopy resultsshowed that after50000times magnification can be clearly showed that chitosanmembrane membrane surface like granular, fissure with sizes.Cell toxicity experiments show that the chitosan membrane has good biocompatibility andbiodegradability, degradation products generally have no obvious toxic effect.3. CSH applied on the wound: the CSH can be applied to the wound after naturalfilm, transplanted to the wound after wound moist no abnormal secretions, twoweeks after the film body gradually disappeared, the wound no swelling, infectionand other inflammatory reaction.Adipose-derived mesenchymal stem cells in thewound:1x107ml of ADSCs0.1ml of cell suspension was applied to the wound andmulti-point injection after local skin wound edge, no oozing wounds, infections andother reactions, the application of wound healing ADSCs group rate of transplantedcells after10days was significantly higher (P <0.05).The wound was observed: allanimals infected and death did not occur during the experiment. healing time:healing rates between the four groups in the10days before no difference (P>0.05),the tenth day: A group, group B, group C healing rate was significantly greater thanthe saline group D group (P <0.05). Light microscopy: HE staining was found ingroup A, group B, group C speed wound healing and inflammatory cell infiltrationcondition D group than the control group. Wound protein expression: Western blotanalysis of four experimental groups wound tissue VEGF protein expressionincreased, with VEGF protein expression ADSCs+CSH group is the highest.Conclusion:The application of ADSCs joint CSD can significantly improve the quality ofrefractory wound healing, and the healing rate of wound healing factor proteinexpression are better than the control group. |
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