| BackgroundThe treatment of diabetic wounds has been a major clinical challenge,with limited effectiveness in traditional treatment methods.In recent years,stem cell therapy has gained attention as a novel potential treatment.Stem cells from different sources,such as bone marrow stem cells and human umbilical cord mesenchymal stem cells,enhance and optimize cell function by regulating TGF-β and extracellular matrix signaling pathways like Col-I,Col-III,promoting rapid wound healing.Current research indicates some shortcomings in clinical stem cell applications,such as strong immunogenicity leading to immune rejection and potential cancer risks due to their strong proliferative capacity.However,recent studies have revealed that stem cells exert their effects through extracellular vesicles,known as exosomes,which play a crucial role in intercellular communication.Exosomes,containing bioactive molecules like growth factors,miRNA,and proteins,can regulate cell signaling pathways and promote tissue repair.In comparison,exosomes exhibit high activity,stability,low immunogenicity,and are less likely to induce abnormalities or tumors.Therefore,stem cell-derived exosomes are considered a more potent and ideal therapeutic agent.Effective treatment in the field of stem cells relies not only on stem cells and exosomes themselves but also requires suitable carriers to facilitate the release and stability of stem cell-derived exosomes.Dynamic chemical c ROSslinking hydrogels,recognized as intelligent drug release platforms,have garnered widespread attention in the biomedical field.Particularly,gallic acid-modified chitosan(CS-GA)and aldehyde-modified hyaluronic acid(HA-ALD)as novel hydrogel carriers exhibit excellent biocompatibility and tunable drug release characteristics,making them ideal carriers for UCMSC-Exo.This dynamic chemical c ROSslinking hydrogel effectively encapsulates UCMSC-Exo,allowing stable storage and controlled release of bioactive molecules.Using this hydrogel as a carrier enhances the therapeutic effects of UCMSC-Exo and provides sustained release of bioactive factors during wound treatment.In this study,extracellular vesicles derived from hUCMSCs were loaded into dynamic chemical c ROSslinking hydrogels composed of CS-GA and HA-ALD,resulting in a novel multifunctional hydrogel dressing capable of sustained release of exosomes(referred to as Exo@CSGA-HA).Exo@CSGA-HA hydrogel possesses various outstanding characteristics,including good mechanical properties,tissue adhesion,spectrum antibacterial activity,and antioxidant ability.Through validation in a diabetic mouse model,Exo@CSGA-HA hydrogel significantly promoted wound healing.This was achieved by accelerating epidermal regeneration and dermal structure reconstruction through the promotion of extracellular matrix Col-I,Col-III generation,and alteration of TGF-β signaling.This multifunctional hydrogel dressing,Exo@CSGA-HA,provides a new option for the treatment of diabetic wounds,expanding the possibilities of stem cell-derived exosome applications in tissue engineering.Further exploration of wound healing mechanisms includes high-throughput sequencing analysis of miRNA enriched in UCMSC-Exo.Mi R-21-5p closely associated with wound healing was identified and investigated through virus transfection to form miR-21-5p mimics/miR-21-5p inhibitors,exploring the impact of different levels of miR-21-5p on TGF-β/SMAD.This research opens new avenues for the application of stem cell-derived exosomes in the biomedical field and provides new hope for the treatment of diabetic woundsResearch Objectives:1.Extract and validate hUCMSCs and their exosomes.Utilize NTA and high-throughput analysis to compare the different sizes and contents of exosome molecules at different generations,providing an innovative supplement for experimental exosome identification and extraction.2.Innovatively apply a newly synthesized polymer material,Exo@CSGA-HA,for treating diabetic wounds.Explore the impact of umbilical cord mesenchymal stem cell exosomes on diabetic wound healing and HSF cells.3.Investigate the mechanism by transfecting miRNA miR-21-5p fragments through gene transfection technology,preparing exosomes with different miR-21-5p expression levels,and elucidating the role of the miR-21-5p/TGF-β/SMAD/ HSF/ECM signaling pathway in UCMSC-Exo repairing diabetic wounds.Methods1.Cultivate hUCMSCs in DMEM without fetal bovine serum for 48 hours,Collect the supernatant,and store it at-80°C.After centrifugation to remove cell debris,perform ultracentrifugation to extract exosomes.Separate UCMSC-Exo in PBS and identify them through transmission electron mic ROScopy(TEM),Western Blot,and nanoparticle tracking analysis(NTA).Explore the biological properties of stem cell exosomes to provide a scientific theoretical basis for understanding exosome composition and their role in wound healing.2.Load exosomes from human UCMSC-Exo into dynamic chemical c ROSslinking hydrogels composed of CS-GA and HA-ALD to prepare a multifunctional hydrogel dressing capable of sustained exosome release Exo@CSGA-HA.Test the properties of the novel polymer material Exo@CSGA-HA,including preparation and characterization,Fourier-transform infrared spect ROScopy characterization,hydrogel dynamic rheological and adhesive properties,reparability testing,mic ROScopic morphology analysis,exosome release and uptake experiments,and antimicrobial testing.3.Conduct cell experiments to study the effective promotion of tissue repair and accelerated healing of diabetic wounds by the novel composite material Exo@CSGA-HA.Verify the promotion effects of umbilical cord mesenchymal stem cell exosomes and Exo@CSGA-HA on HSF cells through cell experiments,including testing the biocompatibility of Exo@CSGA-HA,its ability to clear intracellular ROS,and its impact on cell proliferation and migration.4.Further construct a diabetic mouse model using streptozotocin(STZ)treatment on male BALB/c mice weighing 18 ~ 22 g to obtain diabetic mice.Create full-thickness skin wounds on the backs of diabetic mice,and categorize them into control group,Exo group,CSGA-HA gel group,and Exo@CSGA-HA hydrogel group(12 mice per group).Observe and record wound microimages using a digital camera,measure wound area with Image J software,and calculate wound healing rates.Evaluate tissue epithelialization,Collagen deposition,angiogenesis,inflammation,and other indicators through HE staining,Masson staining,Sirius Red staining,immunohistochemistry,and immunofluorescence experiments.5.Explore the detection of miRNA associated with promoting wound healing in UCMSC-Exo and investigate the mechanism of miR-21-5p mimics/miR-21-5p inhibitor on the TGF-β/SMAD pathway.This includes isolating and extracting UCMSC-Exo,high-throughput sequencing of miRNA in UCMSC-Exo,predicting wound healing-related pathways,selecting target miRNA,and validating the biological functions of these miRNA on the TGF-β signaling pathway.Verify the effects of miR-21-5p overexpression on Col-III,TGF-β,and SMAD at the cellular level through qRT-PCR and Western Blot.Results1.The UCMSC-Exo was successfully extracted from the cell culture supernatant of hUCMSCs using ultra-high-speed centrifugation.The UCMSC-Exo are lipid membrane-enclosed small vesicles,observed as cup-shaped structures under electron mic ROScopy,with a diameter ranging from approximately 30-200 nm.They contain cytoplasmic and membrane components derived from the source cells and are specific subcellular structures rather than cell debris.The UCMSC-Exo were characterized for their abundant content of m RNA,micro RNA,and protein components.Transmission electron mic ROScopy revealed the cup-shaped morphology of UCMSC-Exo with double membranes and approximate circular shape.The diameter ranged from 30-200 nm.Surface markers CD9,CD63,STG101 of UCMSC-Exo were detected by Western Blot.The concentration and particle size of UCMSC-Exo were determined using nanoparticle tracking analysis based on the Brownian motion trajectory.The diameter of UCMSC-Exo was approximately 30-200 nm,and NTA detected that the majority of particles in the tested samples were around 120 nm.These results confirmed the extracted material as UCMSC-Exo.2.Preparation and characterization of the novel Exo@CSGA-HA hydrogel.The UCMSC-Exo derived from human umbilical cord mesenchymal stem cells were loaded into a dynamically chemically c ROSslinked hydrogel composed of chitosan modified with Cs-GA and HA-ALD,resulting in a multifunctional hydrogel dressing capable of sustained release of the exosomes.The Exo@CSGA-HA hydrogel demonstrated excellent properties,including good mechanical performance,injectability,self-healing ability,strong adhesion,broad-spectrum antibacterial activity,and the capability to release exosomes.This provides a new option for the treatment of refractory wounds by delivering exosomes with polymeric materials.It also expands the potential applications of stem cell exosomes in tissue engineering.3.Cell experiments were conducted to evaluate the effects.The results showed that the degradation products of Exo@CSGA-HA hydrogel cultured with HSF cells did not exhibit toxicity but promoted cell proliferation and improved cell survival,as demonstrated by live/dead cell assays.The positive effects of Exo@CSGA-HA on cell proliferation and migration were verified by CCK8 assays and scratch assays.Additionally,H2O2-induced injury experiments confirmed the anti-ROS properties of Exo@CSGA-HA hydrogel,reducing cellular damage caused by H2O2.The antioxidant mechanism of CSGA was explored using EPR,and the results indicated a gradual weakening of hydroxyl radical signals detected by EPR with increasing reaction time of gallic acid,suggesting the ability of CSGA to scavenge free radicals.4.The diabetic mouse model was successfully established.Animal experiments showed that Exo@CSGA-HA smart hydrogel promoted the repair of diabetic wounds at 0,1,7,and 14 days after full-thickness skin injury.HE staining revealed that the epidermis and dermis in the Exo@CSGA-HA group approached normal tissue,with a good continuous epidermis and increased sweat gland and hair follicle regeneration compared to other groups.Masson staining,PLRS staining,and immunohistochemistry showed that the Collagen fibers in the Exo@CSGA-HA group were thicker,more densely arranged,and more widely distributed,with significantly increased blood vessels compared to other groups.The thickness and Collagen content of granulation tissue were higher,and the Collagen protein markers Col-I,Col-III,the blood vessel marker CD31,and TGF-β,closely related to Collagen and blood vessel formation,were also significantly higher.In addition,immunofluorescence detection of inflammation-related factors showed that IL-β,EZH2,and IL-6 in the Exo@CSGA-HA group were superior to other control groups,possibly benefiting from the shorter transition time from the inflammation stage to the regeneration stage,resulting in earlier blood vessel formation and Collagen synthesis,thus favoring diabetic wound healing.5.Detection of miRNA expression profiles in UCMSC-Exo revealed 740 detectable miRNA.Early and late generation miRNA expression profiles showed some differences,with 316 common genes,and hsa-miR-148a-3p,hsa-miR-122-5p,hsa-miR-21-5p,hsa-miR-143-3p,hsa-miR-151a-3p,hsa-miR-192-5p,hsa-miR-100-5p,hsa-let-7f-5p,hsa-miR-26a-5p,and hsa-miR-423-5p being the predominant expressed miRNA.Comparative analysis with human whole-genome reads showed upregulation of chr2,chr8,chr16,and chr17,while chr2 and chr10 showed downregulation.Scatter plots indicated that early-generation UCMSC-Exo miRNA did not cover late-generation miRNA.Among the differentially expressed miRNA,260 were upregulated,216 were downregulated,and 270 showed no differential expression.Functional enrichment analysis and functional abundance histograms suggested significant enrichment of genes related to apoptosis,cell differentiation,cell division,cell transformation,and cell migration.Target Scan identified miR-21-5p as closely related to the TGF-β/SMAD pathway involved in skin tissue remodeling.Thus,miR-21-5p was chosen for further study.Viral transfection successfully formed miR-21-5p mimics/miR-21-5p inhibitor,producing exosomes with different miR-21-5p levels.qRT-PCR validated the differential expression of miR-21-5p in miR-21-5p mimics and inhibitor UCMSC-Exo.Mi R-21-5p mimics had a positive impact on HSF cell proliferation and migration compared to inhibitor UCMSC-Exo.ELISA results indicated upregulated expression of Col-III and TGF-β1 and downregulated expression of SMAD7 in the miR-21-5p overexpression group compared to the negative control and inhibition groups,consistent with Western Blot results.ConclusionIn this study,we synthesized a novel multifunctional hydrogel dressing,Exo@CSGA-HA,capable of loading and releasing exosomes.Results demonstrated that Exo@CSGA-HA hydrogel possessed suitable properties,including adhesiveness,injectability,broad-spectrum antimicrobial activity,and antioxidative characteristics.Moreover,it exhibited the ability to sustainably release exosomes.Through both cell and animal experiments,the newly developed hydrogel showed good cell compatibility,promoting the proliferation and migration of HSF cells.The smart hydrogel could reduce intracellular ROS levels,mitigating the adverse effects of oxidative stress on cell migration.Exo@CSGA-HA facilitated diabetic wound healing by promoting Collagen deposition,cell proliferation,neovascularization,and accelerated re-epithelialization.This study suggests that Exo@CSGA-HA smart hydrogel provides a novel strategy to enhance cell functionality and facilitate the repair of diabetic wounds.Further experiments,including high-throughput sequencing of UCMSC-Exo miRNA and prediction of UCMSC-Exo-related pathways in wound healing,identified miR-21-5p as associated with wound repair and closely linked to the TGF-β/SMAD pathway.By successfully transfecting and preparing different levels of miR-21-5p mimics and miR-21-5p inhibitor,the positive impact of miR-21-5p on HSF cell proliferation and migration was validated at the cellular level through qRT-PCR,Western Blot,and Elisa methods.Additionally,the expression levels of TGF-β/SMAD/Col-III were influenced,further affecting wound healing. |
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