| As the first defensive line of human body,skin is vulnerable to various damages.In specific,skin in oral and maxillofacial region,one of the most prominent part of the body,is more liable to suffer injuries.Restoring the aesthetic appearance and biological function of damaged skin is of great importance for maintaining oral and maxillofacial homeostasis.Currently,a variety of strategies have been explored,among which MSCs-based therapy shows a great potential.Due to their capacity of self-renewal,multi-directional differentiation and immunoregulation,MSCs have been applied in treatment for several diseases and tissue injuries.It has been reported that exogenous MSCs have therapeutic effects on cutaneous wound healing,while the underlying mechanism is still elusive.Within recent years,the paracrine function of MSCs has been widely concerned,through which MSCs secrete multiple soluble factors and EVs to regulate the function of other cells.Exosomes are most attractive EVs,which can be secreted by almost all living cells.With bi-layer membranes and diverse cargoes,exosomes play a critical role in mediating cell-cell communications.To explore whether exosomes contribute to the therapeutic effects of exogenous MSCs on cutaneous wound healing,we firstly verified the therapeutic effects of exogenous BMMSCs in mice skin wound model,and found that exogenous BMMSCs promoted the vascularization level of damaged skin.Furthermore,we extracted BMMSCs-derived exosomes,and verified their therapeutic effects in mice skin wound model.Meanwhile,we examined the effects of BMMSCs-derived exosomes on endothelial cells in vitro.In our previous work,we found that exogenous MSCs undergo extensive apoptosis within a short time after transplantation.The apoptotic MSCs contribute to tissue repair by enhancing the immunoregulation capacity.Apoptotic cells also release a large amount of AB s,which are a kind of EVs.However,the biological function of ABs is still unclear due to the lack of studies.To explore whether ABs contribute to the therapeutic effects of exogenous MSCs,we extracted BMMSCs-derived ABs and verified their therapeutic effects in mice skin wound model.Meanwhile,we demonstrated that macrophages are the target cells of BMMSCs-derived AB s,and examined the effects of BMMSCs-derived ABs on macrophages both in vivo and in vitro.Based on the background above,we designed and carried out the following experiments.Ⅰ.Studies on the therapeutic effects of BMMSCs in mice skin wound model1.Isolation,culture and characterization of BMMSCsBMMSCs were extracted from the bone marrow of 8-week-old female C57BL/6J mice,and cells of the second passage was used for characterization.(1)The morphology of primary cells and cells of the second passage was observed by using an inverted optical microscope.(2)The MSCs related surface makers(CD11b,CD29,CD45,Sca-1,CD34,CD90,CD 105,CD 146)were examined by flow cytometric analysis.(3)The colony-forming capacity was detected by colony-forming unit assays.(4)Cells were cultured in osteogenic and adipogenic induction medium respectively.Then ALP staining,Alizarin red staining and Oil Red O staining were carried out to detect the multi-directional differentiation capacity.2.Establishment and treatment of mice skin wound modelEight-week-old female C57BL/6J mice were used to establish full-thickness wound model on the dorsal skin.BMMSCs were embedded in commercial hydrogel PF-127 gel and locally applied on wound area.The treatment was carried out three times on day 0,3 and 7 respectively.Photos of the wound area were taken on day 0,3,7,10 and 14.The skin tissues were harvest on day 14.Mice treated by PBS or PF-127 gel were set as two control groups.(1)Wound healing rate and body weight were calculated.(2)H&E staining and Masson staining were carried out on paraffin sections to observe the healing of the skin.(3)Immunofluorescent staining was carried out on frozen sections to examine the expression of KRT14 and CD31 in healed skin.Ⅱ.Studies on the therapeutic effects of BMMSCs-derived exosomes in mice skin wound model1.Isolation and characterization of BMMSCs-derived exosomesBMMSCs-derived exosomes were extracted by ultracentrifugation and underwent characterization.(1)The morphology of exosomes was observed by using a transmission electron microscopy.(2)The size distribution of exosomes was measured by dynamic light scattering analysis.(3)The expression of CD81,CD63,and TSG101 were detected by Western blot.2.Establishment and treatment of mice skin wound modelEight-week-old female C57BL/6J mice were used to establish full-thickness wound model on the dorsal skin.BMMSCs-derived exosomes were embedded in commercial hydrogel PF-127 gel and locally applied on wound area.The treatment was carried out three times on day 0,3 and 7 respectively.Photos of the wound area were taken on day 0,3,7,10 and 14.The skin tissues were harvest on day 14.Mice treated by PBS or PF-127 gel were set as two control groups.(1)Wound healing rate and body weight were calculated.(2)H&E staining and Masson staining were carried out on paraffin sections to observe the healing of the skin.(3)Immunofluorescent staining was carried out on frozen sections to examine the expression of KRT14 and CD31 in healed skin.Ⅲ.Effects of BMMSCs-derived exosomes on endothelial cells1.In vitro phagocytic test of exosomesBMMSCs-derived exosomes were labelled by PKH26.Then,HUVECs were cultured and treated by PKH26-labelled exosomes with different time(0,3,6,12,24,48 h)or different concentrations(0,5,10,25,50,100 μg/ml).Photos were taken by using a confocal microscope,and relative fluorescence intensity was calculated.The location of exosomes was determined by layer scanning under high magnification.2.Detection of the function of endothelial cellsHUVECs were cultured and treated with BMMSCs-derived exosomes.(1)The expression of Ki67 was examined by immunofluorescent staining to detect the proliferation capacity of HUVECs.(2)Scratch assay was carried out to detect the migration capacity of HUVECs.(3)Tube formation assay was carried out to detect the tube formation capacity of HUVECs.(4)Western blot was carried out to detect the protein level of ANGII and VEGF in HUVECs.Ⅳ.Studies on the therapeutic effects of BMMSCs-derived ABs in mice skin wound model1.Detection of the apoptosis of exogenous BMMSCs in vivoBMMSCs were labelled by PKH26 and applied in treatment for mice skin wound model according to the methods in part Ⅰ.Skin samples were harvested 24 h after transplantation and performed TUNEL staining.2.Isolation and characterization of BMMSCs-derived ABsBMMSCs-derived ABs were extracted by STS induction and gradient centrifugation,and underwent characterization.(1)The morphology of AB s was observed by using a scanning electron microscopy.(2)The size distribution of AB s was measured by dynamic light scattering analysis.(3)The expression of Clq and Annexin V were detected by immunofluorescent staining.(4)The expression of Caspase-3 and cleaved Caspase-3 were detected by Western blot.3.Establishment and treatment of mice skin wound modelEight-week-old female C57BL/6J mice were used to establish full-thickness wound model on the dorsal skin.BMMSCs-derived AB s were embedded in commercial hydrogel PF-127 gel and locally applied on wound area.The treatment was carried out three times on day 0,3 and 7 respectively.Photos of the wound area were taken on day 0,3,7,10 and 14.The skin tissues were harvest on day 14.Mice treated by PBS or PF-127 gel were set as two control groups.(1)Wound healing rate and body weight were calculated.(2)H&E staining and Masson staining were carried out on paraffin sections to observe the healing of the skin.(3)Immunofluorescent staining was carried out on frozen sections to examine the expression of KRT14 and CD206 in healed skin.(4)BMMSCs derived ABs were labelled by PKH26 and applied in treatment for mice skin wound model according to the methods above.Skin samples were harvested 6 h after transplantation and performed immunofluorescent staining to observe the internalization of ABs in vivo.Ⅴ.Effects of BMMSCs-derived ABs on macrophages1.In vitro phagocytic test of ABsBMMSCs-derived ABs were labelled by PKH26.Then,BMDMs were cultured and treated by PKH26-labelled ABs with different time(0,3,6,12,24,48 h)or different concentrations(0,5,10,25,50,100 μg/ml).Photos were taken by using a confocal microscope,and relative fluorescence intensity was calculated.The location of ABs was determined by layer scanning under high magnification.2.Detection of the function of macrophagesBMDMs were cultured and treated by BMMSCs-derived ABs with different concentrations(0,5,10,20,30 μg/ml).LPS was used to mimic inflammatory microenvironment in vitro.(1)The expression of iNOS and CD206 was detected by immunofluorescent staining.(2)The content of IL-6,TNF-α,IL-10 and TGF-β in supernatant were detected by ELISA.(3)The expression of Il-10,Arg-1 and Tgf-β were detected by RT-qPCR.(4)The expression of ARG-1 and TGF-β were detected by Western blot.3.Detection of the function of fibroblastsFibroblasts were cultured and treated by BMMSCs-derived ABs with different concentrations(0,5,10,20,30 μg/ml).Meanwhile,supernatant of BMDMs in above experiments were collected and used to treat fibroblasts.(1)The expression of Ki67 was examined by immunofluorescent staining to detect the proliferation capacity of fibroblasts.(2)Cell migration assay was carried out to detect the migration capacity of fibroblasts.The results are as follows.Ⅰ.Studies on the therapeutic effects of BMMSCs in mice skin wound model1.Isolation,culture and characterization of BMMSCsThe BMMSCs we cultured showed a typical fibroblast-like morphology,and highly expressed CD29,CD90,CD 105,CD 146 and Sca-1,while CD11b,CD34 and CD45 were not detected.Meanwhile,we observed the capacity of colony-forming and multi-directional differentiation.Therefore,the BMMSCs we used in this study were consistent with ISCT standard and can be used in subsequent experiments.2.Establishment and treatment of mice skin wound modelThe results of in vivo treatment showed that the wound healing rate of Gel+MSCs group was significantly higher than that of PBS group and Gel group.The results of H&E staining and Masson staining showed better cutaneous structure in Gel+MSCs group than the other two groups.The results of immunofluorescent staining showed higher expression of KRT14 and CD31 in Gel+MSCs group than the other two groups.There was no significant difference in body weight among the three groups.Ⅱ.Studies on the therapeutic effects of BMMSCs-derived exosomes in mice skin wound model1.Isolation and characterization of BMMSCs-derived exosomesThe exosomes we extracted were bilayer vesicles with an average diameter of 100 nm,and highly expressed CD81,CD63 and TSG101.Therefore,the exosomes we used in this study were consistent with literatures and can be used in subsequent experiments.2.Establishment and treatment of mice skin wound modelThe results of in vivo treatment showed that the wound healing rate of Gel+Exos group was significantly higher than that of PBS group and Gel group.The results of H&E staining and Masson staining showed better cutaneous structure in Gel+Exos group than the other two groups.The results of immunofluorescent staining showed higher expression of KRT14 and CD31 in Gel+Exos group than the other two groups.There was no significant difference in body weight among the three groups.Ⅲ.Effects of BMMSCs-derived exosomes on endothelial cells1.In vitro phagocytic test of exosomesBMMSCs derived exosomes were engulfed by HUVECs in a time-and concentration-dependent way.Exosomes were located within endothelial cells rather than attached to the cell surface2.Detection of the function of endothelial cellsAfter treatment with BMMSCs-derived exosomes,Ki67 positive cells increased significantly in HUVECs.In the scratch assay,the wound healing rate increased significantly when treated with BMMSCs-derived exosomes.In the tube formation assay,HUVECs formed more complete and clear tube structure when treated with BMMSCs-derived exosomes.Western blot showed the increased protein level of ANGII and VEGF in HUVECs.Ⅳ.Studies on the therapeutic effects of BMMSCs-derived ABs in mice skin wound models1.Detection of the apoptosis of exogenous BMMSCsExtensive apoptosis of BMMSCs were observed 24 h after transplantation.2.Isolation and characterization of BMMSCs-derived ABsThe ABs we extracted were vesicles with an average diameter of 1μm and highly expressed Clq,AnnexinV and cleaved Caspase-3.Therefore,the ABs we used in this study were consistent with literatures and can be used in subsequent experiments.3.Establishment and treatment of mice skin wound modelsThe results of in vivo treatment showed that the wound healing rate of Gel+ABs group was significantly higher than that of PBS group and Gel group.The results of H&E staining and Masson staining showed better cutaneous structure in Gel+ABs group than the other two groups.The results of immunofluorescent staining showed higher expression of KRT14 and CD206 in Gel+ABs group than the other two groups.There was no significant difference in body weight among the three groups.The results of in vivo phagocytosis showed that the PKH26-labelled ABs were engulfed by F4/80 positive macrophages 6 h after transplantation.Ⅴ.Effects of BMMSCs-derived ABs on macrophages1.In vitro phagocytic test of ABsBMMSCs-derived ABs were engulfed by BMDMs in a time-and concentration-dependent way.ABs were located within BMDMs rather than attached to the cell surface2.Detection of the function of macrophagesThe expression level of iNOS in BMDMs increased when stimulated by LPS and was reduced by BMMSCs-derived ABs.The secretion of both IL-6 and TNF-α of BMDMs were decreased by BMMSCs-derived ABs.The expression level of CD206 in BMDMs decreased when stimulated by LPS and was rescued by BMMSCs-derived ABs in a concentration-dependent manner.The secretion of both IL-10 and TGF-β of BMDMs were increased by BMMSCs-derived ABs.The expression level of genes like Il-10,Arg-1 and Tgf-β,and proteins like ARG-1,TGF-β and p-STAT3 in BMDMs were increased by BMMSCs-derived AB s treatment.3.Detection of the function of fibroblastsThe Ki67 positive cell numbers and wound healing rate in fibroblasts were not changed by treating with BMMSCs-derived ABs.Nevertheless,the conditioned medium from BMDMs treated with BMMSCs-derived ABs enhanced both the two parameters of fibroblasts.Based on the results,we draw conclusions as follows.1.Exogenous BMMSCs promoted murine cutaneous wound healing by improving the vascularization level of damaged skin.2.Exosomes mediated the therapeutic effects of exogenous BMMSCs on cutaneous wound healing by enhancing the function of endothelial cells and improving the vascularization level of damaged skin.3.Transplanted BMMSCs promoted cutaneous wound healing partially via releasing ABs during apoptosis.The ABs were largely engulfed by macrophages and thus triggered the polarization of macrophages towards M2 phenotype,which further enhanced the function of fibroblasts,collectively contributing to cutaneous wound healing.Collectively,we investigated the therapeutic effects of BMMSCs derived EVs on cutaneous wound healing,including exosomes secreted by living cells and ABs released during apoptosis.In this way,we have further revealed the therapeutic mechanism of exogenous MSCs,broadened the knowledge about the biological functions of EVs,and provided insights into establishment of new strategies. |
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