| Skin is the first protective barrier of human body.Full-thickness skin damage caused by trauma,tumor removal,burns and so on is difficult to restore the body’s function through normal repair mechanism,which brings serious physical and mental health problems to people and causes serious economic losses to society.Stem cell therapy is a new treatment in the field of trauma repair in recent years.Studies have shown that mesenchymal stem cells can be directly transferred or embedded into the scaffold and delivered to the wound site,secreting cytokines to alleviate inflammation and stimulate angiogenesis leading to faster wound closure.However,the clinical application of mesenchymal stem cells may be limited by the potential risks of tumorigenesis,foreign body formation and low survival rate of implanted cells.Therefore,it is necessary to develop acellular therapy with mesenchymal stem cells(MSCs).Exosomes(Exo)of MSCs are the key active vesicles responsible for paracellular secretion,carrying a variety of active components from cells,including cytokines,growth factors,m RNA,lipids and proteins,which mediate various processes of wound healing.Exosomes are abundant,easy to handle and store,without immunogenicity,and have good application prospects in the field of tissue regeneration.However,it is difficult to maintain a high concentration of exosomes in the process of local administration,and multiple administrations are required,which brings certain difficulties to the treatment.Therefore,there is an urgent to design a carrier to improve the difficulty of exosomes delivery,maintain the effective release of exosomes and improve their efficacy.In this study,exosome-based chitosan thermosensitive gel(Exo@CS-Gel)was constructed to improve the stability of exosomes,help long-term release of exosomes,and provide temporary extracellular matrix for cell infiltration and adhesion,accelerate wound re-epithelialization,and provide new ideas for the construction a new exosomethermosensitive gel for wound repair.The specific research work includes the following parts:1.Preparation and characteristics of exosome-thermosensitive gel(Exo@CS-Gel)According to the requirements of the subject and the characteristics of thermosensitive gel,the prescription of chitosan thermosensitive gel was optimized by single factor analysis and orthogonal test,and CS-Gel was successfully prepared.The optimized formulation was CS 2%,β-glycerophosphate disodium 40%,and sodium bicarbonate 0.4 mol/L.It showed a colorless transparent sol state at 4℃,and became a solid gel after gelation at 37℃.The initial gel temperature of the prepared CS-Gel was34.1±1.3℃,and the viscosity of the initial solution was 3222.3±438.1 m Pa·s,which was moderate and easy to be coated on the wound.In addition,CS-Gel showed irregular pore structure with a pore diameter of about 50 μm and a certain pore wall thickness,which provided a possibility for Exo loading.Exo showed uniform spherical shape with particle size of about 30-100 nm under cryo-scope.The rheological results showed that CS-Gel had a sol-gel transition at34.5℃.In vitro test,the retention rate of Exo in Exo@CS-Gel for three days was92.45±0.76 %,with good retention ability.In addition,in vitro release test showed that Exo released slowly in the gel to ensure that Exo sustained efficacy.2.Study on the in vitro wound repair promotion effect of Exo@CS-GelAt present,Exo@CS-Gel has been successfully prepared,and rat fibroblasts are used as cell models to investigate its ability to repair trauma.The results of in vitro cell proliferation experiment showed that the proliferation rate of rat fibroblasts treated with Exo@CS-Gel reached 387.7±26.6% five days later,which was about 1.2 times that of Exo group.In addition,Exo@CS-Gel showed strong scratch repair effect in vitro scratch test,and its relative wound healing efficiency was 52.9±3.5%,1.3 times that of Exo group.In conclusion,Exo@CS-Gel has the ability to promote proliferation and migration of rat fibroblasts.3.Transport mechanism of Exo in cellsExo enters cells mainly through micropinocytosis and the clathrin dependent endocytosis pathway.Subsequently,Exo was located on the endoplasmic reticulum and Golgi apparatus,indicating the Exo could delivery growth factors horizontally in cells by avoiding lysosome pathway,which was conducive to regulating the physiological functions of recipient cells.4.In vivo wound repair of Exo@CS-GelIn order to evaluate the ability of Exo@CS-Gel to repair rats after trauma,a fullthickness skin damage model was established.In vivo imaging results showed that Exo@CS-Gel could remain on rat epidermis for more than three days.The pharmacodynamic evaluation results showed that the healing rate of the Exo@CS-Gel group was 95.6±1.3% after two weeks,indicating faster wound healing.H&E staining showed that Exo@CS-Gel group showed the best re-epithelialization and regeneration of skin appendages such as hair follicles.Masson trichrome staining showed that Exo@CS-Gel could promote wound repair though collagen fiber regeneration.The preparation process of Exo@CS-Gel is simple,which is conducive to process amplification.The sol state at low temperature was conducive to wound administration,and the gel state at body surface temperature are conducive to drug retention and controllable release,reduce the number of administrations,improve patient compliance,and have high transformation value in clinic.In addition,exosome-based acellular therapy has many advantages,such as low immunogenicity,rapid wound healing,wound scar-free repair,and has great potential in wound healing and tissue regeneration.Local administration of Exo@CS-Gel at the wound site improves the targeting of exosomes and enhances the stability and activity of exosomes,which is conducive to the efficacy of exosomes and is a promising local delivery system. |
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