| PART 1Isolation,culture of rat adipose-derived stem cells and passage tracing after CM-DiI labeling in vitroObjective: To establish a method for isolation,culture and cell labeling of rat adipose-derived stem cells(ADSCs)and investigate the passage tracing feasibility of CM-DiI labeled ADSCs in vitro.Methods : Adipose tissue was harvested from the inguinal fat pad of Sprague-Dawley rats under sterile conditions.By means of combining zymogen digestion with differential velocity adherence,the primary cells were isolated.They were used on serial subcultivation.The morphology of cells was observed by inverted phase contrast microscope.The molecular phenotype and differentiation potentiality of three passaged ADSCs were identified.Three passaged ADSCs were labeled by CM-DiI and they were used on serial subcultivation.Fluorescence labeling of cells were observed under the fluorescence inverted microscope and laser scanning confocal microscope.The labeling rate was detected by flow cytometry.Results: ADSCs showed long spindle shape and vortex like growth.After passages,the growth and proliferation rate of cells was accelerated.The cells were identified positive for CD29 and CD90 makers,and they showed the lack of CD34,CD45 and CD31 expression.Alizarin red and oil red O staining showed ADSCs could be differentiated to osteoblasts and adipocytes.The red fluorescence of CM-DiI labeling existed in the cell membrane and cytoplasm,which did not exist in the cell nucleus.Afterpassages,the fluorescence was attenuated,and the fluorescence labeling rate of the third,sixth and ninth passage was 97.09%,66.21% and 37.86%,respectively.Conclusion: Rat ADSCs can grow and proliferate rapidly,and they have multiple differentiation potential.CM-DiI can label ADSCs simply and effectively.These can provide the basis for the following experiment in vivo.PART 2 Injectable small intestinal submucosa is co-cultured with adipose-derived mesenchymal stem cells in vitroObjective: To prepare the injectable small intestinal submucosa and investigate it to be injectable biological scaffold material with rat adiposederived stem cells composite culture in vitro.Methods: Primary cultured ADSCs and they were used on serial subcultivation.Three passaged ADSCs were labeled by CM-DiI.The molecular phenotype of ADSCs was identified.Injectable SIS was prepared and co cultured it with ADSCs.Scanning electron microscope was used to observe the injectable materials and cocultured it with ADSCs.The fluorescence microscope was used to observe the co culture of them.CCK-8 kit was used to test the effect of co cultured injectable scaffold material with ADSCs on proliferation at the fist third fifth and seventh day.Live/dead staining kit was used to show the percentage of live cells.Results:ADSCs were isolated successful and it expressed mesenchymal stem cell-related antigen.Scanning electron microscopy showed that injectable SIS had more than the size of the three-dimensional pores and ADSCs were attached wellto the material in the shape of an oval and strip shape.CCK-8 kit showed that the proliferation rate of ADSCs co cultured with material was increased.Live/dead staining kit showed that ADSCs co cultured with material was survived well.Conclusion: Injectable SIS has a good three-dimensional structure,which has good biocompatibility and it can promote the growth and proliferation of ADSCs.So it can be used as a kind of injectable natural biological scaffold material for the adipose tissue engineering.PART 3Injectable small intestinal submucosa and adipose-derived stem cells composite gel for the deep partial thickness burns repairation.Objective: Regenerative medicine provides many treatments for burn wounds.It is a trend to find suitable skin substitutes for the treatment of burn wounds.The purpose of this study is to discuss the feasibility of injectable small intestinal submucosa(SIS)and rat adipose-derived mesenchymal stem cells(ADSCs)composite gel to repair the deep partial thickness burns in rats.Methods: By using the method of coin toss,64 rats were randomly divided into A group(control group),B group(ADSCs group),C group(injectable SIS group),D group(injectable SIS + ADSCs group),16 rats in each group.The burn model was created by contacting the dorsal surface directly with boiled water for 10 seconds.After scalding,the wound edge and the central area wereinjected for treatment.A,B,C and D groups were injected with 1ml PBS solution,1ml containing 2 ×107 ADSCs of PBS solution,1ml 20% injectable SIS,1ml mixture of 2 ×107 ADSCs and 20% injectable SIS,respectively.And then vaseline gauze and sterile gauze were covered the burn wound,breathable tape were wrapped around the trunk of rats.At 3,7,14 and 21 days post injection treatment,gross observation,wound microvascular density assessment,histopathology examination and immunofluorescence were performed.Results: The wound closure percentages on postoperative day 3 in A,B,C and D groups were 7.75±1.30%,13.90 ±1.01%,14.7 ±1.10%,19.14 ±1.56%,respectively.The wound closure percentages on postoperative day 7 in A,B,C and D groups were 21.45 ±1.19%,41.34 ±.80%,40.62±4.93%,58.28 ±2.62%,respectively.The wound closure percentages on postoperative day 14 in A,B,C and D groups were 50.91 ±1.28%,67.15 ±0.86%,69.54 ± 0.81%,85.31±1.05%,respectively.The wound closure percentages on postoperative day 21 in A,B,C and D groups were 62.68±3.14%,82.19±1.69%,88.77±0.73%,100±0%,respectively.The wound closure percentages of D group was significantly higher than that of A,B and C groups(P < 0.01).The microvascular density on postoperative day 3 in A,B,C and D groups were(11.5±4.6,24.6±6.3,20.1±9.7,39.2±7.3)/cm2,respectively.The microvascular density of D group was significantly greater than A,B and C groups(P < 0.05).Compared with A,B and C groups,there were more new blood vessels and less inflammatory infiltration cells in D group.On postoperative day 7,CM-DiI labeled ADSCs colocalized with the vascular endothelial cell marker CD31 could be detected,showing an oval shaped vascular structure.On postoperative day 21,the newly generated epidermis was detected in B,C and D groups.The newly generated epidermis in D group was the most thick,while the A group was no significant new epidermal formation.CM-DiI labeled ADSCs colocalized with the epidermal keratinocyte maker CK10 could be detected,showing the epidermoid structure.Conclusion: Theinjectable SIS and ADSCs composite gel were able to enhance the wound closure percentages,promote angiogenesis,reduce the infiltration of inflammatory cells and improve the re-epithelialization to repair burn wounds. |
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