| Difficult-to-heal wounds caused by trauma,burns and various diseases often lead to wound infection,which delays wound healing.Local wound infection and growth factor(GFs)decreases are important causes of wound nonunion.Platelet-rich plasma(PRP)is enriched in a variety of GFs,which can effectively promote soft tissue repair.However,the use of PRP alone provides minimal significant improvements due to the short half-life and premature inactivation of GFs.In addition,PRP has limited antibacterial capacity.The purpose of this study was to explore the use of chitosan-silk fibroin moisturizing dressing as an ideal vehicle for PRP,which could improve the stability of PRP,maintaining the activity of GFs and releasing them in a sustained manner.In this study,a solution chitosan fibroin emulsion with added Silver Nanoparticles(AgNPs)was freeze-dried to be the scaffold,and an asymmetric coating was formed on one side.PRP was loaded onto the composite scaffold using a secondary lyophilization technology to prepare the tissue engineering dressings.AgNPs were characterized using a transmission electron microscope.The morphologies of the composite dressing were examined under a scanning electron microscope.The silver content of the dressing was measured by inductively coupled plasma mass spectrometry.The asymmetric wettability of the composite dressing was demonstrated by water contact angle measurement.Relatively high porosity,favourable moisture retention capability and appropriate tensile strength were observed by measuring the physical and mechanical properties.Satisfactory antibacterial properties against various bacteria and microbial isolation performance were observed by the antibacterial effect analysis in vitro.The total protein slow-release property was measured using the BCA assay.Good biocompatibility and lower sensitization were examined both in vitro and in vivo.In addition,the healing effciency of the composite dressing on infected wound were examined in mice infected wound models.Analysis of wound healing rates,bacterial cultures of wound exudate,whole blood cell analysis and histological examination all showed satisfactory results.These results are demonstrated to provide a potential and possible pathway to promote wound tissue repair and regeneration.Part 1 Preparation and characterization of chitosan/silk fibroin AgNPs antibacterial moisturizing dressing loaded with PRPObjective:Investigate the antibacterial and moisturizing dressing of chitosan/fibroin AgNPs loaded with PRP,observe its cross-sectional topological structure,and preliminarily evaluate the possibility of applying the dressing to repair skin wounds.Methods:AgNPs were added to the chitosan-silk fibroin blend emulsion,and freeze-dried to form a loose porous sponge-like dressing scaffold to form an asymmetric wetting coating on the smooth surface of the dressing material.Finally,PRP was loaded onto the composite scaffold using a secondary freeze-drying technique to prepare a tissue engineering dressing(CTS-SF/Ag/SA/PRP).The nano-silver particles were observed under transmission electron microscope.The topography of the cross-section of the composite dressing was observed under a scanning electron microscope.The content of Ag in the composite dressing was detected by ICP-MS.Results:AgNPs with good particle dispersibility and uniform particle size were successfully prepared.Rabbit-derived PRP was successfully prepared.Asymmetric wet-modified chitosan/silk fibroin nanosilver antibacterial moisturizing dressing(CTS-SF/Ag/SA/PRP)loaded with PRP was successfully prepared.The unmodified surface is covered with micropores,and the whole is diffusely reflected,and the modified surface is relatively smooth and flat with slight reflection.The particle size of the prepared AgNPs was uniform under transmission electron microscope.Under the scanning electron microscope,the cross-sectional surface of the CTS-SF/Ag/SA/PRP composite dressing had a perforated pore structure with a pore size of about 30-100?m.The surface of the channel was relatively rough.It can be seen that the AgNPs are attached to the pore walls.The content of Ag in CTS-SF/Ag/SA/PRP composite dressing was 9.0998±0.1337?g/g by ICP-MS,which was equivalent to Ag content in Acosin dressing(9.75999±0.3131?g/g).Conclusion:1)AgNPs with good particle dispersibility and uniform particle size were successfully prepared.2)Rabbit-derived PRP was successfully prepared.3)Asymmetric wet-modified chitosan/silk fibroin nano-silver antibacterial moisturizing dressing(CTS-SF/Ag/SA/PRP)loaded with platelet-rich plasma was successfully prepared.Part 2 Physical and chemical properties and antibacterial activity of chitosan/silk fibroin AgNPs antibacterial moisturizing dressing loaded with PRPObjective:To provide evidence for the use of materials as wound dressings by testing the physicochemical properties,mechanical properties,antibacterial properties and sustained release properties of CTS-SF/Ag/SA/PRP.Methods:The porosity,swelling,moisturizing time,tensile strength and asymmetric modification of CTS-SF/Ag/SA/PRP were measured to determine chitosan loaded with platelet-rich plasma/Antibacterial properties,microbial isolation properties and total protein sustained release propertiesResults:Ink drop test and static water contact angle measurements showed asymmetry in the CTS-SF/Ag/SA/PRP composite dressing.The high porosity and swelling degree of the CTS-SF/Ag/SA/PRP composite dressing ensure that the dressing has a moisturizing time of nearly 20 hours.The tensile strength of the CTS-SF/Ag/SA/PRP composite dressing enables the material to be used for wound care.CTS-SF/Ag/SA/PRP compound dressing has good killing effect on Staphylococcus aureus,Pseudomonas aeruginosa Escherichia coli and Candida albicans,and its modified surface can effectively block the invasion of external bacteria.Has a good microbial barrier effect.The BCA test results show that the CTS-SF/Ag/SA/PRP composite dressing can release the protein contained therein slowly and thus continue to act on the wound surface.Conclusion:1)The asymmetric modification of chitosan/silk fibroin nanosilver antibacterial moisturizing dressing loaded with PRP has no significant effect on the porosity of the material,and can effectively prolong the moisturizing time of the dressing material,and effectively block external microorganisms.The invasion,60.41%±1.20%porosity is conducive to the absorption of wound exudate and material exchange and metabolism of wound tissue,while the moisturizing time of nearly 20 hours also helps protect the wound in a wet state;2)The addition of AgNPs reduces the mechanical strength of the chitosan/silk fibroin AgNPs antibacterial moisturizing dressing loaded with PRP,but at the time the composite dressing still has a tensile strength of nearly 0.40 MPa which can still be used as wound dressing.The loading of PRP and the asymmetric modification of stearic acid have little effect on the tensile strength of the dressing;3)Chitosan/silk fibroin AgNPs antibacterial moisturizing dressing loaded with PRP has relatively satisfactory killing effect on Pseudomonas aeruginosa,Escherichia coli,Staphylococcus aureus and Candida albicans;4)Chitosan/silk fibroin AgNPs antibacterial moisturizing dressing loaded withPRP can slowly release proteins into the wound,and various growth factors contained in the wound healing will continue to act on the wound to promote tissue repair.Part 3 Effect of chitosan/silk fibroin AgNPs antibacterial moisturizing dressing loaded with PRP on proliferation of different cells and its biosafety evaluationObjective:To detect the effect of chitosan/silk fibroin AgNPs antibacterial moisturizing dressing on PRP on cell proliferation,and to test its biosafety,and provide evidence for further in vivo experiments.Methods:The cytotoxicity of extracts from CTS-SF/Ag/SA/PRP composite dressings was evaluated by L929 cells and human dermal fibroblasts.The biosafety of the dressings was evaluated by subcutaneous sensitization in guinea pigs.Results:The human dermal fibroblasts were successfully isolated and cultured to form a swirling or fence-like arrangement.The cell morphology was clear and diverse,and it was fusiform,irregularly star-shaped,polygonal,and the cytoplasm protruded outward.The total value-added curve is of the "S" type.The activity of L929 and human fibroblasts in CTS-SF/Ag/SA/PRP extract was higher than that in the control group.Subcutaneous sensitization experiments in guinea pigs showed that the CTS-SF/Ag/SA/PRP extract did not cause an allergic reaction.Comprehensive cytotoxicity experiments and sensitization experiments can demonstrate that CTS-SF/Ag/SA/PRP has good biosafety.Conclusion:1)Successfully extracting,isolating and cultivating human dermal fibroblasts.2)Chitosan/silk fibroin AgNPs antibacterial moisturizing dressing loaded with PRP can promote the proliferation of human dermal fibroblasts and L929 cells.3)The chitosan/silk fibroin AgNPs antibacterial moisturizing dressing extract loaded with PRP showed no obvious irritation to the subcutaneous skin of guinea pigs,and the biocompatibility was better.Part 4 In vivo evaluation of chitosan/silk fibroin AgNPs antibacterial moisturizing dressing loaded with PRP for infective wound repairObjective:To establish an infective wound model of the back skin of mice,and to analyze the wound healing condition under the treatment of CTS-SF/Ag/SA/PRP composite dressing by in vivo experiment,and provide further evidence for the material as a wound dressing.Methods:A model of infective wound in the back skin of mice was established.The wounds were treated with CTS-SF/Ag/SA/PRP,CTS-SF/Ag/SA,Acosin,Gauze-PRP and Gauze.The wound healing rate,wound exudate bacterial culture,whole blood cell analysis,histological analysis and 12-day postoperative organ silver content were analyzed to comprehensively evaluate the CTS-SF/Ag/SA/PRP composite dressing for infectious wounds.treatment effect.Results:The wound healing rate analysis showed that CTS-SF/Ag/SA/PRP was the best(98.40±0.8%),CTS-SF/Ag/SA was the second(93.44±2.1%),and Acosin was in the middle(84.26±1.9%),Gauze-PRP(76.28±4.4%)and Gauze(74.14±2.1%)were poor and not statistically different.On the third day after surgery,bacterial culture of wound exudate showed no colony formation in the CTS-SF/Ag/SA/PRP,CTS-SF/Ag/SA and Acosin treatment groups,while the culture medium of Gauze-PRP and Gauze group grew colonies.Analysis of white blood cell count,neutrophil percentage and lymphocyte percentage in whole blood of mice showed that CTS-SF/Ag/SA/PRP group effectively killed Pseudomonas aeruginosa in wounds and shortened the infection caused by wounds.The defense/inflammation stage of wound healing and the ability to regulate the body's immune response to some extent.HE staining on the 7th day after surgery showed that CTS-SF/Ag/SA/PRP and CTS-SF/Ag/SA showed less inflammation,no necrosis,no inflammatory exudation,and hyperplastic granulation.There is scattered inflammatory cell infiltration in the tissue,and the epithelial surface of the epithelium has climbed into the epithelium,but the basal layer is still not obvious.HE-stained tissue sections on the 12th day after surgery showed that the wounds in the CTS-SF/Ag/SA/PRP group healed well,covering the thin layer of epithelium,abundant in the dermis,and scattered neutrophils,lymphocytes and plasma cells.The infiltration indicates that the immune response is better.In contrast,the wounds of the CTS-SF/Ag/SA group were not completely covered by the new thin layer of epithelium.It can be seen that the CTS-SF/Ag/SA/PRP group wounds have been basically repaired and rebuilt on the 12th day.The results of Masson staining were basically consistent with the results of HE staining 12 days after surgery.The collagen deposition in the wounds in CTS-SF/Ag/SA/PRP and CTS-SF/Ag/SA groups was more dense and regular,of which CTS-SF/The dermis layer of the Ag/SA/PRP group showed the most regular and dense collagen.On the 12th day after operation,the silver content of the mouse organs detected by ICP-MS showed that the deposition of silver was mainly concentrated in the liver,and the deposition of silver in the CTS-SF/Ag/SA/PRP group was lower than that of CTS-SF/Ag/SA group and the Acosin group.Conclusion:1)Successfully established an animal model of infectious wounds on the back skin of mice;2)CTS-SF/Ag/SA/PRP composite dressing can effectively control the infection degree of infectious wounds,significantly reduce the inflammatory reaction and promote the repair process of wounds,and improve the quality of wound repair. |
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