| Objective:The aim of this study was to develop a novel hydrogel(named Sericin/i-PRF hydrogel)and to study and evaluate the effect of Sericin/i-PRF hydrogel on diabetic wound healing by material characterization and full-thickness skin excision model of diabetic nude mice.Methods:Sericin/i-PRF hydrogel was prepared by one-step activation method:the Sericin aqueous solution was mixed with liquid i-PRF at the volume ratio of 1:1 into a homogeneous solution,and an appropriate amount of 25%(w/v)glutaraldehyde was added to the mixed solution,which was placed at room temperature,and the Sericin/iPRF hydrogel was prepared with the activation of the solution from liquid to solid.The structure,gelation time,swelling ratio,rheological rate,compressibility,Fourier transform infrared spectroscopy,in vitro growth factor release,biocompatibility and bioactivity of the Sericin/i-PRF hydrogel were characterized.A type Ⅱ diabetes model was established in nude mice.A round full-layer skin wound with a diameter of 6mm was made on the back of the successfully modeled nude mice,and a silicone ring(with an inner diameter of 8mm)was sutured to the skin with a 5-0 suture to prevent skin shrinkage.The experimental animals were divided into 4 groups:normal saline(NS)group,Sericin hydrogel group,i-PRF group and Sericin/i-PRF hydrogel group.The wound size of nude mice in each group was evaluated regularly after corresponding treatment,and photographs were taken on the scheduled date on the 7th and 14th day,the nude mice were sacrificed and the skin tissues were taken for histomorphological and immunological analysis.The effects of Sericin/i-PRF hydrogel on wound repair and regeneration of diabetes mellitus were studied from the aspects of wound closure,angiogenesis,collagen synthesis and inflammation.Results:(1)Formation and morphology of the Sericin/i-PRF hydrogelAfter adding glutaraldehyde to the mixture of Sericin aqueous solution and liquid iPRF and standing at 25℃ for about 4 minutes,the mixture gradually changed from liquid to solid gel and Sericin/i-PRF hydrogel was prepared.(2)Microstructure observationScanning electron microscope observation showed that the fibrin bundles of i-PRF were interwoven and connected with each other,showing a flake-like complex network structure,and both Sericin hydrogel and Sericin/i-PRF hydrogel had porous 3D structure.The fibrin network from i-PRF can be observed on the pore wall of Sericin/i-PRF hydrogel,which proves the integration of i-PRF and Sericin hydrogel.(3)Gelation timeThe mixture of Sericin aqueous solution and i-PRF was liquid at first.With the activation of fibrin and the cross-linking of sericin,Sericin hydrogel and i-PRF fused to form an interconnected colloidal network after adding glutaraldehyde.When the temperature increased from 4℃ to 37℃,the gelation time of Sericin/i-PRF hydrogel decreased from about 8 minutes to 3 minutes.Its injectability within a few minutes is enough to adapt to a variety of irregular wounds,so that it can play a comprehensive role.(4)Swelling rateThe swelling rate of Sericin/i-PRF hydrogel increased rapidly over time,reaching half of the maximum value within about 4h,and then began to rise slowly.Under alkaline and neutral conditions,the maximum swelling rate of Sericin/i-PRF hydrogel is about 2,which is slightly higher than that under acidic conditions.The hydrogel can swell rapidly in PBS,indicating that it has good water absorption performance.(5)Rheology and compression propertiesThe storage modulus(G’)of Sericin/i-PRF hydrogel is significantly higher than that of the loss modulus(G"),indicating that Sericin/i-PRF hydrogel is a superior elastic material.In compression experiments,Sericin/i-PRF hydrogel can be compressed to 40%without breaking.(6)Fourier transform infrared spectroscopyThe characteristic peaks of Sericin and i-PRF appear in the infrared spectrum of Sericin/i-PRF hydrogels.Among them,962 and 839cm-1 are the oscillations outside the C-H bond plane,which are the new absorption peaks,indicating the integration of sericin and i-PRF.(7)In vitro growth factor release testThe growth factor release curve of Sericin/i-PRF hydrogel was slower and more uniform than that of i-PRF,and the release duration was longer.(8)Biocompatibility and bioactivity detection.Toxicity tests showed that all hydrogels had no cytotoxicity.Cell proliferation experiments showed that Sericin/i-PRF hydrogels showed more effective proliferation promotion behavior than other groups after the third day.Biodegradation experiments in vivo showed that the residual weight of Sericin hydrogels and Sericin/i-PRF hydrogels could still reach nearly 50%at 96h,and this stability supported their clinical application.(9)Full thickness skin excision model in nude mice with diabetesThe wound healing of Sericin/i-PRF hydrogel group was significantly better than that of normal saline(NS)group,Sericin hydrogel group and i-PRF group.The wound area measurement results showed that the wound closure was faster in the Sericin/i-PRF hydrogel group.The histomorphological results showed that the epithelialization was complete,the new epidermis was dense and continuous,and the collagen was arranged in order in the Sericin/i-PRF hydrogel group.Immunological analysis showed that the inflammatory reaction in the Sericin/i-PRF hydrogel group was mild and the angiogenesis was significant.Conclusion:Sericin/i-PRF hydrogel was successfully prepared in this study.The characterization results show that the hydrogel has the advantages of porous 3D structure,good biocompatibility,excellent mechanical properties and continuous release of bioactive molecules.In animal experiments,Sericin/i-PRF hydrogel may accelerate wound healing in diabetic nude mice by promoting angiogenesis,appropriately increasing collagen deposition density and reducing inflammation.This study proves the great potential of Sericin/i-PRF hydrogel in promoting diabetic wound healing and lays a foundation for further study of the mechanism of Sericin/i-PRF hydrogel in the future. |
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