| Background:Diabetic chronic refractory ulcer refers was caused by skin and soft tissue blood supply disorders and neuropathy.In the case of skin damage can easily caused the wound to be delayed for more than 3 months.Skin,as the body’s first physical barrier and the most vulnerable organ,its damage repair process was important.For the treatment of diabetic chronic refractory ulcers,there is a lack of targeted and effective methods,whatever china or other places.Stem cells are a new hope for the treatment of it.Umbilical cord mesenchymal stem cells were widely used in the treatment of diabetic chronic refractory ulcers because they had the functions such as remodeling blood vessels,enhancing blood flow of lower limbs,repairing peripheral neuropathy of diabetes,and regulating immunity.However,the clinical risks,inefficiency,and ethical issues of stem cell transplantation were needed to be solved urgently.At the same time,it is currently considered that umbilical cord mesenchymal stem cells mainly perform damage repair effects by paracrine.And,the exosomes secreted by umbilical cord mesenchymal stem cells have the same effect as MSCs.A number of studies have shown that non-cell therapy based on stem cell exosomes is safer and more convenient than cell therapy.However,there are problems such as insufficient targeting and limited duration of exosome treatment.Combined with our previous studies,hydrogel-loaded exosomes for treatment is a viable idea.Thus,we combined the exosomes derived from umbilical cord mesenchymal stem cells with Pluronic F 127,then we preliminary verified the foundation of it.All this works will lay the foundation of our follow-up studies.METHODS:Human umbilical cord mesenchymal stem cells supernatants were collected after 96 hours of starvation culture.Exosomes were extracted by precipitation method.Concentration and purity identifications were determined by BCA protein quantification,western-blot,scanning electron microscopy,and nanosight.PKH67 invasion assay will determine the invasive effect of exosomes.The scratched test of human umbilical vein endothelial cells will determine the effect of the exosome-gel complex on migration and proliferation.RESULTS:Western-blot showed exosomes had a high expression of CD63,CD81,and low expression of GAPDH;scanning electron microscopy showed it had typical exosomal structures;BCA-kit shown its concentration was 1617.2 ug/ml;nanosight detected a concentration of 4.19×1011/mL,where the 30 nm-150 nm range accounted for 44.4%.The thermo-responsive property measurement showed that with the increase of the concentration of Pluronic F 127,the initial gelation temperature of the exosome hydrogel complex and the gel time at 37℃decreased gradually.The PKH67 invasion assay showed that the number of exosomes entering the cell in the exosome-gel complex was much greater than that of the exosome alone.The scratch test showed that exosomes promoted the endothelial cell migration strongest,the supernatant poorer,and the hydrogel was roughly equivalent to the control group.Conclusion:We identified the purity and concentration of exosomes in first part,indicating that exosomes was feasible and the exosomes were available for the construction of composite hydrogels.In the second part,PKH67 invasion test showed that the composite hydrogel worked better than other group.Scratch test means exosome is the major component of the paracrine pathway of mesenchymal stem cells that promotes endothelial cell migration.ALL of this showed that the newly constructed exosome composite hydrogel has a good effect,but still needed further animal experiments and clinical trials to verify. |
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