| Objective: Accidents caused by the leakage of radioactive substances and radiation exposed in medical and industrial events have become the unignorable risks of destroying human health.Due to the feature that skin is the largest organ and the protective barrier of human beings to the outside,radiation-induced skin injury is a common complication during radiotherapy and radiation accidents.At present,radiation-induced skin injury has been treated by a series of strategies,such as surgery and topical medication,while the development of tissue-engineered skin substitutes provides a broad sight.Silk fibroin(SF)and chitosan(CS)could be cross-linking through amidation reaction and gradient freeze-drying to form a three-dimensional porous scaffold with excellent biocompatibility which can promote the proliferation and migration of skin cells.Stem cells and cytokines binding to hydrogels,or basing on nano-microspheres and micro-needle injection devices have achieved satisfactory results in clinical trials.The purpose of this study is to investigate the repairing effect and the possible mechanism of silk fibroin/chitosan scaffold combined with adiposederived stem cells on radiation-induced skin injury.Methods: Adipose-derived stem cells(ADSCs)were extracted by enzymolysis method,cell surface markers were detected by flow cytometry,osteogenic and adipogenic differentiation cultures were performed to identify their differentiation ability,in vivo imaging and tissue sections were used to observe the localization and survival situation of ADSCs in vivo after local injection.ADSCs exosomes(ADSCsEXOs)were extracted by ultracentrifugation,the membrane markers of exosomes were detected by Western blot,the structures were observed by transmission electron microscopy,and the size of exosomes was detected by particle size analysis.Mem DyeRed staining was applied to label exosomes.After the composite SF/CS scaffolds were prepared,the growth of ADSCs,human epidermal keratinocytes(Ha Ca T)and human dermal fibroblasts(WS1)cells seeding on the scaffolds was observed by scanning electron microscope.Taking Ha Ca T and WS1 as the research objects.After irradiation,the cells were co-cultured with SF/CS scaffolds,ADSCs,ADSCs-EXOs,and EXOsdepleted conditioned medium.The proliferation ability of skin cells was detected by plate clone,CCK8,Ed U488 cell proliferation assay.The migration ability of cells was detected by scratch assay and Transwell assay.The senescence of cells was detected byβ-galactosidase activity.The apoptosis rate was detected by TUNEL apoptosis assay and flow cytometry.Human vascular endothelial cells EC-304 and human lymphatic endothelial cells HLEC were used as another experimental objects.After irradiation,the cells were co-cultured with scaffolds and ADSCs to detected the fluence in tube formation.As the animal level,40 Gy electron beam was used to construct a rat model of radiation induced skin injury.After ulcer formation,the wounds were treated with SF/CS scaffolds and ADSCs.The wound healing rate of each group was calculated and compared to the control group.HE and MASSON staining of skin slices were observed to evaluated the epidermal thickness,vascularization,and collagen deposition.Results: ADSCs and exosomes were successfully extracted,identification and analysis.Inducing differentiation of ADSCs to osteogenesis and adipocyte were succussed.IVIS showed that the cells could been survived at the injection site for more than 2 weeks.The exosomes extracted by ultracentrifugation were sized comply with the standard,which could incorporate into cells within several hours.ADSCs,Ha Ca T and WS1 cells were well-growth in the scaffolds.In vitro,SF/CS scaffold combined with ADSCs could promote the proliferation and migration of Ha Ca T and WS1 cells after irradiation,inhibit senescence,reduce apoptosis,and enhance the tube formation ability of EC and LEC cells.In vivo,SF/CS scaffold combined with ADSCs could approaching "scarless healing" of radiation-induced skin injury in rats via promoting wound re-epithelialization,increasing angiogenesis,improving collagen remodeling,and stimulating the regeneration of skin appendages.Conclusion: SF/CS scaffold combined with ADSCs can ameliorate the damage effect of ionizing radiation on skin cells,promote skin cell proliferation and migration,inhibit senescence and apoptosis,stimulate angiogenesis,promote wound re-epithelialization,increase collagen deposition and skin appendage regeneration,which has a significant effect on repairing the radiation-induced skin injury. |
