| Objective: To explore the mechanism of adipose-derived stem cells extracellular matrix/stromal vascular fraction-gel(SVF-gel)accelerating wound healing of full-thickness skin defect in thymus-free mice.Methods: 1.Using the technique of Coleman to obtain human adipose tissue specimens from patients undergoing liposuction in the Department of Burns and Plastic Surgery,Affiliated Hospital of Zunyi Medical University.SVF-gel was prepared by pure physical methods of static,centrifugation,repeated injection,flocculation and centrifugation from the above specimens.The obtained SVF-gel was divided into two parts,one part of SVF-gel was digested with collagenase I and cultured adherently after SVF suspension was obtained,and after several culture medium changes and passages,P3 cells were obtained for cytological identification,and the other part of SVF-gel was cultured directly in the DMEM medium containing 2% fetal bovine serum(FBS)at 37℃and 5% CO2 incubator.2.The morphology of adherent cells digested by collagenase was observed under inverted microscope.And the three lines were induced to differentiation,which were identified by alizarin red,alicin blue and oil red O staining respectively.Flow cytometry to detect the expression of cell surface markers.3.SVF-gel was cultured in vitro for 24 hours.Conditioned medium(CM)of SVF-gel was collected by low-speed centrifugation and frozen In-80℃ refrigerator.4.To establish full-thickness skin defect models on both sides of the back and spine of all thymus-free mice,which were randomly divided into three groups: DMEM treatment group,40% CM treatment group and 80% CM.treatment group.A total of 18 mice were treated with 12 wounds in each group,and labeled well.Injection experiments was started immediately after successful modeling.DMEM group was injected withDMEM culture medium containing 2%FBS,40% CM group was injected with CM from40% SVF-gel(diluted with DMEM culture medium containing 2%FBS),and 80% CM group was injected with CM from 80% SVF-gel(diluted with DMEM culture medium containing 2%FBS).Each wound was divided into four quadrants,with a total injection volume of 20 ul per quadrant,and 80 ul/wound was injected into the base and edge of the wound to observe the healing of the wound.On the 3rd,7th and 10 th day after injection,the wound was photographed by digital camera,and the healing rate was calculated by Image J software.5.On the 10 th day after injection,all the mice were sacrificed and wound tissues were cut for histological examination: HE staining was performed to observe the skin regeneration structure of wound tissue;Masson staining was performed to observe the collagen deposition in wound tissue;CD31 immunofluorescence staining was performed to observe the vascular regeneration of wound tissue.6.The frozen CM from SVF-gel were thawed at 37℃.The contents of transforming growth factor-β1(TGF-β1)and vascular endothelial growth factor(VEGF)in CM were determined by enzyme-linked immunosorbent assay(ELISA).7.Statistical analysisThe experimental results were expressed by mean±standard deviation(±s),and were analyzed by statistical software SPSS 19.0.Homogeneity test of variance was carried out on the experimental results.If the variance was homogeneous,one-way ANOVA was used.P value < 0.05 was considered statistical significance.Results: 1.The adherent cells extracted from SVF-gel had much impurity and slow-speed of adherence.Most of the cells were spindle-shaped and polygonal-shaped after 48 hours of culture.Intercellular cross-linkages were observed between the cells.Cell morphology was regular,long spindle-shaped and cross-linkages in the third generation.2.The adherent cells were found to posess the characteristic of osteogenic,chondrogenic and adipogenic differentiation functions after three-line induction and differentiation.The results of flow cytometry showed that CD73,CD90 and CD105 were positive,while CD11 b,CD19,CD34,CD45 and HLADR were negative.It can be seenthat the above adherent cells from SVF-gel were ADSCs.3.The model of full-thickness skin defect of back skin in thymic-free mice was successfully set up.On the 3rd day after operation,all of the wound area(80% CM,40%CM and DMEM group)were decreased.The wound healing rates of the three groups were56.02±1.37%,27.88±2.03% and 23.80±2.21% respectively.On the seventh day after operation,the wound area of three treatment groups was further reduced,and the wound healing rates of the three groups were 75.96±0.14%,66.44±4.24% and 58.24±1.90%respectively.On the 10 th day,80% CM group healed almost completely,with the smallest remaining wound area and the highest wound healing rate.The wound healing rates of the three groups were 96.86±2.74%、86.30±0.94%、87.36±2.32% respectively.4.Histological examination showed that the epidermis and dermis of 80% CM group were thicker,the connection between epidermis which was closer,the regenerated skin appendages were more obvious,and the collagen deposition was more(62.94±1.20%)and the collagen arrangement was more neat.However,in 40% CM group and DMEM group,the connection between epidermis and dermis was looser,with a little space,less regeneration of skin appendages,thinner dermis and less collagen deposition,and their collagen deposition fraction was(56.68±2.3%)and(57.81±2.1%)respectively.The results of CD31 immunofluorescence staining showed that angiogenesis was found in all the treatment groups,and more in 80% CM group.5.The results of ELISA showed that The levels of TGF-β1 and VEGF in the supernatant of control group were 328.00±4.89pg/ml and 3.36±0.16pg/ml respectively,and the levels of ones in the experimental group CM were 409.30±33.45pg/ml and8.05±0.29pg/ml respectively.The above results showed that the expression of TGF-β1 and VEGF in SVF-gel CM was significantly higher than one in the supernatant of DMEM.It is suggested that SVF-gel can paracrine TGF-β1 and VEGF.Conclusion: Human SVF-gel CM could accelerate wound healing in thymus-free mice.The mechanism may be related to the presence of a large number of ADSCs and their paracrining TGF-β1 and VEGF. |
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