Preparation Of Biomimetic Hydrogel Dressing Loaded With Freeze-dried PRP And Its Effect On Diabetic Wound Repair

Chronic wounds are a serious medical problem.The development of a multifunctional dressing that promotes high-quality wound healing is of great significance for the management and treatment of chronic wounds.In nature,the porosity of the extracellular matrix generated during wound healing in African echinoderms may play an important role in the mechanism of scarless healing.The three-period minimal surface(TPMS)is an ideal porous structure with high specific surface area,high porosity,high connectivity and excellent mechanical properties.Inspired by this,we developed a porous biomimetic hydrogel loaded with freeze-dried PRP based on the idea of bionics.The hydrogel was loaded with platelets that can preserve vitality for a long time.The Gel MA/HAMA double network hydrogel was used as the basic material to imitate the extracellular matrix,and the digital light processing(DLP)technology was used to print the porous structure of TPMS structure.Subsequently,it was characterized and analyzed to understand the crosslinking rules and physicochemical characteristics of the double-network hydrogel,as well as the key points and structural characteristics of TPMS printing,and to explore whether it has the potential to promote wound healing in vivo and in vitro experiments to understand its mechanism.It is hoped that this study can provide new ideas for the development of bionic dressings for wound healing.Part 1 Preparation and characterization of dual-network hydrogels for TPMSstructures via 3D PrintingObjective: To characterize Gel MA/HAMA dual-network hydrogels with different additive ratios and prepare bioprinting inks with optimal mechanical properties and biocompatibility.To design TPMS structures and explore the feasibility of 3D printing hydrogel dressings.Methods: 1.Gel MA with a concentration of 15% and HAMA with concentrations of1%,2%,and 3% were prepared with different additive ratios to form hydrogel solutions.Fourier-transform infrared spectroscopy,scanning electron microscopy,mapping analysis,mechanical property analysis,swelling performance analysis,porosity analysis,contact angle analysis,and biocompatibility analysis were performed to select the most suitable dual-network hydrogel ratio based on comprehensive physical properties and biocompatibility.2.Twelve TPMS structures were designed and printed.The mechanical properties and porosity of TPMS structure hydrogels were analyzed.HDF-a cells were threedimensionally cultured on TPMS hydrogel scaffolds,and cell viability was tested using confocal microscopy and CCK-8 assays.Results: 1.Gel MA/HAMA dual-network hydrogels with interpenetrating networks were formed through cross-linking of acrylamide bonds,which could be initiated,propagated,and terminated by the carbon-carbon double bond in the Gel MA/HAMA network.Within a certain range,as the additive ratio and HAMA concentration increased,the cross-linking degree of the dual-network hydrogel increased,resulting in an increase in mechanical properties and a decrease in porosity,but the biocompatibility did not change significantly at different concentrations.2.Twelve TPMS structures,including Diamond(30%,35%,40%),I-WP(35%,40%,45%),Gyroid(40%,45%,50%),and Primitive(45%,50%,55%),were designed based on the structure and appropriate porosity parameters.The TPMS hydrogel structures were printed,and the printed TPMS hydrogel scaffolds showed good formability.The mechanical properties and porosity of different structures were different.HDF-a cells grew preferentially on the sides and pores of the scaffold,and according to the CCK-8 assay,the viability of the cells was higher than that of the control group,with statistically significant differences.Conclusion: Gel MA/HAMA can undergo further cross-linking to enhance mechanical properties.TPMS structures can be used for hydrogel 3D printing and promote cell growth.Part 2.Preparation and evaluation of biomimetic hydrogel dressings loaded withfreeze-dried PRPObjectives: To investigate the preparation method of the bio-ink loaded with freezedried PRP,to prepare biomimetic hydrogels loaded with freeze-dried PRP,and to characterize and evaluate the hydrogels in vitro.Methods: 1.freeze-dried PRP(FDP)was prepared using the trehalose method.The main growth factors,PDGF-BB,TGF-β,and VEGF,were detected using the Elisa method.FDP/PRP was characterized by SEM,and CCK-8 proliferation experiments were performed on HDF-a,HACAT,and HUVEC cells.Scratch experiments were performed on HDF-a cells,and tube formation experiments were performed on HUVEC cells.2.The biomimetic hydrogel loaded with freeze-dried PRP was prepared,and protein release kinetics,mechanical properties,SEM characterization,pore size measurement,swelling and water retention performance evaluation were conducted.The hydrogel extract was used for cell proliferation,scratch and tube formation experiments.Results: 1.The appearance of FDP prepared by the trehalose method was uniform and met the standards of freeze-dried products.The results of PDGF-BB,TGF-β,and VEGF growth factor detection showed that the retention rate of growth factors exceeded(60.31±8.14)% after storage at room temperature for 30 days,indicating the potential to maintain platelet vitality.SEM characterization analysis showed slight wrinkling of the FDP membrane,but the integrity of the cell membrane was still good.FDP promoted cell proliferation in HDF-a,HACAT,and HUVEC cells,promoted HDF-a migration,and promoted HUVEC tube formation ability.2.The biomimetic hydrogel loaded with freezedried PRP had good shape recovery after freeze-drying and rehydration,and the TPMS structure was conducive to the release of proteins from the dressing.The results of mechanical property testing and SEM characterization showed that loading FDP changed the cross-linking degree of the hydrogel.The hydrogel extract promoted cell proliferation,scratch and tube formation ability in HDF-a,HACAT,and HUVEC cells.Conclusion: The biomimetic hydrogel dressing loaded with freeze-dried PRP can maintain the biological activity of PRP and promote cell proliferation,angiogenesis,and migration,with potential for clinical translation.Part 3.Study on the repairing effect of biomimetic structural hydrogel loaded withfreeze-dried PRP on diabetic wounds in ratsObjective: To investigate the in vivo effect of biomimetic hydrogel loaded with freezedried PRP on diabetic wound healing.Methods: In accordance with ISO 10993-10:2010 "Biological Evaluation of Medical Devices",the hydrogel was evaluated for biological safety.A diabetic wound model was induced in SD rats using STZ,and the rats were divided into control group,hydrogel group,TPMS group,and FDP/TPMS group to treat the wound model.On the 4th,8th,and 12 th day,photographs were taken and skin tissues of the rat wound were collected.Measurements of wound size were taken,and skin sections were prepared for staining with HE,immunohistochemistry and Masson.Results: The biomimetic hydrogel loaded with freeze-dried PRP had good in vivo biological safety,could promote wound closure,significantly increase the wound healing rate,promote re-epithelialization and angiogenesis.This effect may be related to the release of bioactive substances from FDP and the formation of a "fusion interface" between TPMS and the wound.Conclusion: The biomimetic hydrogel loaded with freeze-dried PRP can promote diabetic wound healing and has great clinical translational potential.