| For the diabetic patients,impaired of the vessels and nerves,high blood glucose burden,and the high expression of the inflammation factors are often lead to the chronic delayed or even non-healing of the skin wound,which could lead the gangrene and amputation.Diabetic chronic wound not only not only leads to high economic burden but also seriously impairs the quality of life in DM patients.Many studies have shown that stem cell derived exosomes can promote the healing of the skin wound effectively,which provide a new way to achieve the cell-free therapy.With many favorable biological properties,gingival mesenchymal stem cells(GMSCs)have been proved that it can promote the healing of the skin defects in the in vivo studies.Because accumulated evidences supported that the tissue repair function the stem cells are largely relies on the paracrine effect.While as the most important contents of the cell paracrine effects,exosome play a critical role in the transmitting messages and in the cell-to-cell communication.Therefore we hypothesized that GMSCs derived exosomes may also play an important role in tissue repair during wound healing.However,the GMSCs derived exosomes have not yet been isolated and characterized.In the currently study,we will isolate and identify the human GMSCs and GMSCs derived exosomes,then we will evaluate the effects of GMSCs derived exosomes on the biological characteristics of human skin fibroblasts(HSFs).Currently,the application methods of the exosomes in the wound healing researches are mainly by subcutaneous injection to the site around the wound or to the intravenous administration to the body.However,the injection method may produce secondary injury to the skin,and may also cause loss and waste.Because of the neuropathy and peripheral vascular disease in diabetic patients,even a small wound is likely to lead to non-healing ulcers and increase the risk of bacterial infections,which may put patients at risk of additional severe complications,especially in the extremities.Because of the benefit effects of the exosomes on the tissue wound healing and regeneration,the researchers has been exploring the new methods to apply exosomes.Different from injection,combination of the exosomes and biomaterials are one of the major topics,but the related studies are limited.In the previous studies,our team has prepared a chitosan/silk porous hydrogel sponge by freeze-dry method.Based on this hydrogel sponge wound dressing,after loading with nano-particles of silver,we have successfully promote the healing of the infectious skin wound in a mouse model.In the current study,we will prepare the chitosan/silk hydrogel sponge based on our previous experience.After loading the GMSCs derived exosomes on the hydrogel sponge,we will applied them on the diabetic rat skin defect model to evaluate the effects GMSCs derived exosomes on the diabetic wound healing and explore the feasibility of combination of exosomes and porous hydrogel sponge applied on the wound healing.Our study will provide new evidences for the GMSCs derived exosomes on the wound healing.By exploring the feasibility of combination of exosomes and porous hydrogel sponge applied on the wound healing,our study lay a foundation of clinical application of exosomes and provide new ideas for the treatment of diabetic chronic wound,which is of potential value.Fart 1:Isolation and identification of human gingival mesenchymal stem cells,gingival mesenchymal stem cells derived exosomes and human skin fibroblastsAims:To isolate and identify human GMSCs from human gingiva tissue;to isolate GMSCs derived exosomes from the culture supernatant;to isolate human skin fibroblasts from foreskin tissue.Methods:GMSCs were isolated by enzyme digestion methods,and characterized by growth characteristic,surface antigen analysis and in vitro multipotent differentiation.The culture supernatant were collected to isolate the GMSCs derived exosomes.The HSFs were isolated and identified from foreskin tissue.Results:1.We have successfully derived GMSCs and the cells exhibited spindle-like morphology and had "S" shape of growth curve in vitro.GMSCs positively expressed CD44,CD73,CD90,CD105,CD29 and were negative for CD31,CD34 and CD45.The results of differentiation experiments demonstrated GMSCs could differentiate into osteoblasts,chondrocytes and adipocytes.2.GMSC-derived exosomes were successfully isolated and were observed as spherical structures.The exosomes have a single peak(?80 nm)diameter and the mean diameter is 127±55.9 nm.The results of Western blot analysis showed that GMSC-derived exosomes expressed the exosomal markers CD9,CD81,Tsg 101.3.The HSFs were polygonal and spindle-shaped with distinct cell borders,which were growth quickly and had"S" shape of growth curve.HSFs were positively express vimentin and negatively express CK 19.Conclusions:In this part we have successfully isolated GMSCs,GMSCs derived exosomes and HSFs,which lay a foundation for the follow up study.Part 2:Isolation and identification of human gingival mesenchymal stem cells,gingival mesenchymal stem cells derived exosomes and human skin fibroblastsAims:After treatment of HSFs by different concentrations of GMSCs exosomes,to explore the biological effects on the HSFs such as proliferation,migration,apoptosis under normal glucose and high glucose condition.Methods:After treated the HSFs by the concentration of 25?g/ml and 50?g/ml exosomes,we evaluated the cell proliferation,division,migration and chemotaxi seffects of the exosomes on the HSFs in the normal glucose condition.Afterward,we use same concentration of exosomes to treat the HSFs under high glucose concentrations(35mM),to evaluate the effect of cell proliferation,migration,apoptosis,and the reactive oxygen species(ROS)accumulation.Results:1.Under the normal culture condition,25?g/ml?50?g/ml GMSCs derived exosomes could effectively promote the HSFs proliferation,division and migration.The role of 50?g/ml exosomes was stronger than 25?g/ml.2.The high glucose condition(35mM)inhibited the HSFs proliferation,division and migration,promote the HSFs apoptosis and accumulation of ROS.While the addition of exosomes could effectively attenuate the inhibition effects and promote HSFs proliferation and migration,inhibit the cell apoptosis and ROS accumulation.Conclusions:1.The GMSCs derived exosomes could promote the HSFs proliferation,division and migration effectively in a dose dependent manner.2.The GMSCs derived exosomes could also promote the HSFs proliferation,division and migration,inhibit the high glucose-induced cell apoptosis and ROS accumulation effectively in a dose dependent manner.Part 3:Preparation and characterization of chitosan/silk hydrogel spongeAims:To preparation and characterization of chitosan/silk hydrogel sponge by freeze-dry method based on our previously study and to explore the feasibility as exosomes scaffolds.Methods:According to our previously experience and methods,the hydrogel was prepared using the freeze-drying method.The microstructure of the hydrogel sponge was evaluated by scanning electron microscopy(SEM).The degree of swelling rate(SR)and the moisture retention capacity was evaluated.The functional groups of the hydrogel were detected by Fourier transform infrared(FTIR)spectroscopy.After loading the exosomes to the hydrogel,the presence of the exosomes on the hydrogel particles and the release of the protein from the hydrogel was detected.Results:The chitosan/silk hydrogel sponge we prepared is thin and porous with good swelling property:at 12 hours,in the deionized water the SR is 2110.00%,in the PBS is 1750.73%,in the SBF is1556.86%,in the FBS is 1440.92%.The hydrogel is also of good moisture retention capacity.The results of FITR shows related characteristic bands.The SEM images show that the surface of the hydrogel sponge has many pores ranging in size from 50 to 150 ?m;while in the cross-section,larger pores are observed as ranging in size from 200 to 500 ?m.In addition,the pore wall surface appears smooth and thick.According to the LSCM images,compared to the control chitosan/silk hydrogel,we detected more green fluorescence in the hydrogel after loading the DiO-labeled exosomes,which confirmed the presence of the exosome particles.In the SEM images,the surface morphology and structure of the hydrogel sponge after adding the exosomes was not noticeably different.In the low-resolution images,compared with the control hydrogel(without exosomes),the hydrogel adding exosomes give the impression of coarseness and grittiness.In the high-resolution images,exosome particles can be observed on the hydrogel sponge.After loading the exosomes to hydrogel,obvious burst release of the protein is observed and it reaches 91.05%after third day.Conclusions:1.We have successfully prepared chitosan/silk sponge,which are of uniform micro pores,with good swelling moisture retention capacity.2.The exosomes can be loaded on the chitosan/silk hydrogel sponge,and the spherical exosomes can be detected under SEM without affect the morphology of the sponge.Part 4:Evaluation of wound healing in a diabetic rat skin defect model using GMSCs exosomes combined with chitosan/silk hydrogel spongeAims:To develop a diabetic rats model and make full thick skin defects,then evaluate the wound healing effects of the GMSCs derived exosomes combined with chitosan/silk hydrogel sponge.Methods:The diabetic rats model were established by injection of STZ after fed with high-sugar and high-lipid fodder.After full-thickness wounds were created on the upper back of the diabetic rats,the rats were divided to three groups:Control group:the wound was covered by gauze;Hydrogel group:the wound was covered by a hydrogel;Hydrogel-Exosomes group:the wound was covered by a hydrogel after loading GMSCs derived exosomes.The healing rates,lengths of the neo-epithelium,collagen content in the wound area,micro vessel density(MVD)and nerve density were compared between the groups.Results:1.We have successfully established a diabetic rats model:blood glucose levels of all the rats were higher than the 11 mmol/L and this condition was stable for the following 5 weeks.In addition,the typical symptoms of body weight-loss,polyphagia,polydipsia,and polyuria after the injection of STZ were observed in the rats.Compared with normal rats,the H&E staining of the pancreatic tissue revealed that islet ?-cells were damaged in the diabetic rats.2.The Hydrogel-Exosomes groups are of highest wound healing rate,re-epithelialization degree,content of collagen,MVD and nerve fiber density,which is of statistical significance compared with the other two groups.Besides,wound healing rate,re-epithelialization degree,content of collagen,MVD in the Hydrogel group are higher than the Control group,which are of also statistical significance.Conclusions:GMSCs can promote the diabetic wound healing effectively.The method using porous hydrogel sponge loading exosomes is a noninvasive and effective method to applying exosomes on the wound healing.The chitosan/silk hydrogel sponge are also of benefit effects on the wound healing. |
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