| [Background]Chronic diabetic wounds are one of the most severe complications caused by diabetes.They primarily manifest in the foot,making it a difficult clinical challenge.As the number of diabetic patients worldwide continues to rise（537 million by 2021）,so do the incidences of chronic and refractory wounds.In China alone,8.1%of diabetic patients experienced new wounds within a year,and the recurrence rate was as high as 31.6%.Patients with amputations caused by diabetic foot ulcers have a lower 5-year survival rate than most cancer patients.In addition,their treatment cost exceeds that of many common cancers,thus,posing a grave threat to human life and health.Angiogenesis is involved in the entire wound healing process and is a crucial factor for chronic wound healing in diabetic patients.Its function is mainly performed by vascular endothelial cells.The metabolic abnormalities caused by the diabetic environment result in the excessive production of reactive oxygen species（ROS）,which triggers several cellular mechanisms,including the activation of protein kinase C,NF-k B mediated vascular inflammation,etc.,resulting in structural and functional lesions of the vascular system,which in turn affects vascular permeability,neovascularization,and cell proliferation.This is the most common cause of endothelial damage and vasculopathy in diabetic wounds.Thus,to promote wound healing and improve the vascular health of diabetic wounds,it is crucial to reduce the excessive production of ROS.Due to their robust capacity for self-renewal and multiple differentiation,mesenchymal stem cells are widely employed in tissue repair and regeneration.Adipose tissue-derived mesenchymal stem cells（ADSCs）can be easily obtained by minimally invasive techniques;additionally,they are capable of multi-directional differentiation and demonstrate self-renewal and accelerated wound healing abilities.However,the cell survival rate is usually low after ADSC injection,and the therapeutic activity remains poor.In addition,it may induce an immune and inflammatory response after cell transplantation,thereby exacerbating the clinical application of ADSCs.As an essential mode of intercellular communication,adipose-derived mesenchymal stem cell exosomes can promote angiogenesis,regulate oxidative stress and inflammatory response,and be the primary contributor to the role of ADSCs.However,it is rapid clearance and short half-life in vivo limit its use in wound treatment.Hydrogels have excellent hydrophilicity,biocompatibility,and a three-dimensional porous structure similar to the extracellular matrix,which aids in optimizing the biological and molecular events associated with wound healing.Moreover,it can load drugs or bioactive substances as intelligent delivery systems for wounds,thereby reducing the immediate clearance issues caused by direct injection of exosomes.Metormin is a well-known drug for the treatment of diabetes.By activating the AMP-activated protein kinase pathway of vascular endothelial cells,NF-κB signaling can be inhibited,thus inhibiting the expression of proinflammatory factors and adhesion molecules,and exerting vascular protection.Herein,we selected adipose-derived mesenchymal stem cell exosomes and metformin as model drugs and bioactive substances and loaded them into polyethylene glycol（PEG）conductive hydrogels to study whether the dual-loaded intelligent hydrogels can produce a synergistic effect on the diabetic chronic wounds.[Aim]The current study aims the following,based on diabetic wound model and ADSC-exos:1.To explore the role of ADSC-exos in diabetic chronic wound healing.2.To construct the PEG/Ag-series hydrogel and evaluate its physicochemical structure and multifunctional properties.3.To evaluate the repair effect of the PEG/Ag-series hydrogel on diabetic chronic wounds.4.To explore the effect of the PEG/Ag-series hydrogel on vascular endothelial cells.5.To explore related mechanisms.[Methods]1.The therapeutic effect of ADSC-exos on diabetic chronic wounds was detected.Human ADSCs were isolated,cultured,and identified using surface marker fluorescence staining,flow cytometry,and adipogenic and osteogenic ability detection.Additionally,differential centrifugation was used to isolate and collect ADSC-exos,which was then identified by electron microscopy,flow nanotechnology,and Western blotting.A full-thickness injury model of circular skin with a 1 cm-diameter was created on the back of diabetic mice.Correspondingly,the wound healing rate,oxidative stress（malondialdehyde,total antioxidant capacity,and superoxide dismutase）,and angiogenesis（vascular endothelial growth factor and vascular cell adhesion molecule）were measured in each group.Using reverse transcription polymerase chain reaction（RT-PCR）,CCK8,scratch,and tube formation experiments,the effect of ADSC-exos on the angiogenesis of vascular endothelial cells in a high glucose environment was analyzed.Moreover,flow cytometry,mitochondrial membrane potential staining,and RT-PCR,were used to evaluate the effects of exosomes on reactive oxygen species,mitochondrial protection,and anti-inflammatory effects of Human Umbilical Vein Endothelial Cells（HUVECs）under high glucose stimulation.In addition,Sirt3 si RNA was transfected into vascular endothelial cells to investigate the role of Sirt3 in treating diabetic chronic wounds with ADSC-exos.2.Construction of PEG/Ag-series hydrogel.Carbon nanotubes（CNTs）（4 mg/m L）were dispersed after the 4-arm PEG sulfhydryl group was dissolved in 100μg/ml exosomes.Subsequently,4 mg/m L of metformin hydrochloride（MET）was dissolved in the above-mentioned mixture.Concurrently,Ag NO3 was dissolved in phosphate-buffered saline（PBS）（p H=7.4）to yield the Ag NO3 mother liquor with a concentration of 3mg/m L.Finally,500μL of 4-Arm-PEG-Thiol/exosome/CNTs/MET solution and 500μL of the Ag NO3 stock solution were mixed to prepare an injectable,adhesive,self-healing exosome hydrogel.The morphology and structure of the PEG/Ag-series hydrogels were observed by scanning electron microscopy,while,their capability to adhere to the host tissue was evaluated using fresh pig skin.The self-healing properties of hydrogels were evaluated using a rheometer（TA instrument）.In addition,its injectability,swelling,biodegradability,conductivity,and blood compatibility were tested.3.The biocompatibility of PEG/Ag-series hydrogels was detected.The high glucose control group consisted of HUVECs cultured in a high glucose environment,while the high glucose experimental group consisted of vascular endothelial cells co-cultured with various hydrogels in a high glucose environment.Correspondingly,the HUVECs were detected for live/dead cells.Cell proliferation,migration,and tube formation capabilities were determined using Ki67 fluorescence staining,Transwell,and tube formation.In addition,Western blotting was used to detect vascular function-related factors;mitochondrial Fission inhibitor Mdivi-1 and mitochondrial Fission activator Fccp were used as controls.Furthermore,flow cytometry and fluorescence staining were used to detect ROS expression in cells and mitochondria.To detect mitochondrial morphology,F-actin expression,and Dynamin-related protein 1（DRP1）expression,fluorescence staining was utilized.Western blotting was used to detect the expression of proteins associated with mitochondrial fission and fusion.Finally,vascular function-related tests were conducted.4.PEG/Ag-series hydrogel for the treatment of diabetic chronic wounds.A full-thickness skin injury model was developed for diabetic mice,and each group’s wound healing images were captured.The wound samples were stained with hematoxylin and eosin stain and Masson,and Ki67 and a SMA/CD31 double fluorescence staining was used to detect tissue proliferation and angiogenesis.In addition,each group’s expression of inflammatory factors was determined using immunohistochemistry.Moreover,the vascular and mitochondrial morphology of wounds in each group wounds were observed using electron microscopy.Furthermore,ROS fluorescence staining was used to detect the ROS expression level of in each group’s wounds.[Results]1.The ADSCs isolated and cultured in this study demonstrated a typical long spindle shape that could undergo osteogenic and adipogenic differentiation.The detection of surface markers MET the identification criteria for stem cells.The membrane structure of the extracted ADSC-exos was a typical double-layer cup shape.In addition,the in vitro studies demonstrated that ADSC-exos could reduce wound oxidative stress and inflammation-related indicators and promote wound angiogenesis and closure.Moreover,the ability of HUVECs to proliferate,migrate,and form tubes increased in a high glucose environment,whereas ROS production decreased,mitochondrial membrane potential increased,inflammatory-related molecules decreased,and Sirt3/SOD2 expression increased.Vascular protection and angiogenesis were weakened after using si Sirt3.2.The constructed PEG/Ag-series hydrogel exhibited a uniform macroporous structure while demonstrating conductivity,injectability,adhesion,and self-healing abilities.PBS with a p H of 7.4 was used to simulate the physiological environment and evaluate the swelling properties of PEG/Ag hydrogels.The results demonstrated that each group of hydrogels had excellent swelling abilities,and their in vitro degradation times ranged from 12 to 14 days.In addition,the hemolysis rate of hydrogels in each group was less than 5%,indicating good blood compatibility.3.Staining for live/dead cells confirmed the biocompatibility of each group of hydrogels.The PEG/Ag/CNT-M+E group contained the greatest number of Ki67-positive cells,and migration and tube formation functions were significantly enhanced.Compared to other groups,the vascular barrier function and detection of vascular inflammation proteins were also significantly improved.In addition,the expression of both total ROS and mitochondrial ROS decreased.Mitochondrial staining revealed that mitochondria in the high glucose group became smaller and punctate and shrunk considerably in length.In contrast,the majority of mitochondria in the hydrogel-treated group displayed a long filamentous morphology.Moreover,the expression of mitochondrial fission-related molecules DRP1,FIS,and MFF decreased,whereas the expression of mitochondrial fusion-related molecules MFN1 and MFN2 increased.Staining for DRP1 further confirmed that DRP1 lesions were significantly reduced.Concurrently,the treatment group maintained the filamentous F-actin structure.In addition,the treatment groups and fission inhibitors were able to induce endothelial cell migration and angiogenesis,improve vascular integrity and barrier function,and reduce vascular inflammation,offset by the mitochondrial fission activator Fccp.4.The double-loaded hydrogel group had the quickest healing rate compared to other groups of hydrogels.The wound length was the shortest,the epithelial regeneration was complete,the collagen was neatly arranged,and the healing quality was the highest compared to the other groups.Electron microscopy results demonstrated a decrease in diabetic wound vascular edema,lumen stenosis,basement membrane thickening,and the number of tight junctions.These pathological morphologies were partially reversed by the double-loaded hydrogel.Furthermore,as revealed from the results of mitochondrial morphology,the outer membrane of the mitochondria in the wounds of diabetic mice was found to be damaged;the overall mitochondrial structure further disappeared and fragmented into numerous circular fragments of varying sizes.In this regard,the intelligent double-loaded hydrogel group was able to restore the intracellular mitochondrial morphology partially.At the same time,the double-loaded hydrogel had the most Ki67-positive cells,the largest vascular diameter,and the highest vascular density.Moreover,the vascular integrity and barrier function were better than the other groups,the vascular inflammation was lower than the other groups,and the wound inflammation was lower than the other groups.Thus,the expression of ROS in skin tissue was significantly improved.[Conclusion]1.By regulating the Sirt3/SOD2 pathway,ADSC-exos could improve oxidative stress and inflammatory microenvironment,as well as vascular endothelial cell dysfunction caused by hyperglycemia,promote angiogenesis,and enhance diabetic chronic wound healing.2.The PEG/Ag/CNT-M+E hydrogel demonstrated self-healing,antibacterial,and conductive properties and could release exosomes and metformin continuously.In addition,it accelerated the process of wound epithelization by promoting angiogenesis and cell proliferation in vivo and in vitro and exhibited a profound anti-inflammatory effect.Further studies demonstrated that the intelligent dual-loaded hydrogel could reduce the production of ROS in a high glucose environment and protect F-actin homeostasis by interfering with mitochondrial fission,thereby promoting vascular endothelial cell migration and neovascularization.Thus,it demonstrated an excellent therapeutic effect and provided the theoretical foundation for treating chronic diabetic wounds. |
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