| As the largest organ in humans,skin not only maintains our metabolism by preventing evaporation but also plays an important role in the protection against UV irradiation,chemical substances,foreign organisms and other harmful environmental factors.However,trauma and burns often lead to tissue necrosis and disrupt the defence functions of skin.Wound infection is the most common complication of skin damage and is a major cause of delayed healing.Severe wound infection is associated with the occurrence of bacteremia and sepsis and may even lead to the development of systemic inflammatory response syndrome(SIRS)and multiple organ dysfunction syndrome(MODS),which severely threaten the lives of the patients.Topical drugs are widely used for the treatment of burn wounds.However,when these drugs are used to treat large-area skin wounds,large doses and high administration frequencies are typically required,which often cause varying degrees of side effects.Therefore,improving wound dressings is of great importance for the effective treatment of infected wounds.An ideal wound dressing should retain moisture,maintain electrolyte balance and haemostasis,provide analgesic and antibacterial properties,and promote wound healing.The two sides of a dressing should be equipped with different characteristics.The inside surface(i.e.,the side next to the skin)should have good hydrophilicity,enable the dressing to attach well to the wound,and play a superior role in regenerating skin tissue and fighting infection.The outside of the dressing(the side exposed to the air)should exhibit hydrophobicity,which can enable the dressing to act as a barrier against outside risk factors.Although some wound dressings havebeen commercially available,their treatment outcomes are far from satisfactory.As a type of artificial antibacterial material,silver nanoparticles(AgNP)have a small particle size,large surface area,and broad-spectrum bactericidal effects without causing resistant bacteria.Over the last few decades,the preparation of antibacterial dressings by combining AgNP with polymers has become an important area of research.Chitosan has been considered as one of the promising candidate materials in the application of wound treatment,due to its cell adhesive ability,oxygen permeability,biocompatibility and other important properties.However,the structural characteristics of chitosan made the dressing brittle,and the particles were not strongly adsorbed,which caused them to be easily lost.To resolve the above limitations,we plan to regulate the release of AgNP and endow the dressing with good flexibility by adding silk fibroin fiber.A sponge scaffold containing AgNP was produced in this study by blending and emulsifying chitosan and silk fibroin,adding a specific concentration of a AgNP solution and then freeze drying the final solution.Chitosan and silk fibroin exhibit good tissue repair function.Finally,an antibacterial dressing with an asymmetric coating was prepared by grafting stearic acid(SA)onto the smooth surface.We obtained an asymmetric wettable chitosan-silk fibroin composite dressing with fixed AgNP(CTS-SF Ag/SA).This study disusussed the characterization,physicochemical property,cytocompatibility of dressing and the its effectiveness on infected wounds,as well as evaluate its clinical feasibility and usefulness in scientific research.Part1 Preparation and Characterization of An Asymmetric Wettable Chitosan-Silk fibroin Composite Dressing with Fixed Silver NanoparticlesObjective:Investigate the preparation of an asymmetric wettable chitosan-silk fibroin gel sponge with fixed silver nanoparticles,characterize it and evaluate the possibility of the sponge as a candidate of wound dressing used in the treatment of skin damage.Methods:A gel sponge was produced in this study by blending and emulsifying chitosan and silk fibroin and freeze drying.Then,silver nanoparticles prepared by liquid phase reduction method was loaded in the pore of sponge.Finally,an asymmetric wettable chitosan-silk fibroin gel sponge with fixed AgNP was prepared after modified by stearic acid.Observe the morphology of AgNP and determine its size distribution.Observe the surface morphology of gel sponge and test its silver contentResults:An asymmetric wettable chitosan-silk fibroin gel sponge was successfully prepared.The TEM observation showed the size of AgNP was respectively uniform and the average value of the main peak of the particle size distribution was 49nm.The asymmetric wettable chitosan-silk fibroin gel sponge has regular shape and form uniform surface.The SEM observation of unmodified surface showed the inter-connective porous structures was obvious,the wall of the pore was thin and smooth with AgNP in it.And with an increasing amount of AgNP,the surface pores of the sponges were smaller and uniformer.The SEM observation of modified surface showed the surface was smooth and the pore structures was disappeared.ICP-MS detection result showed that AgNP was successfully loaded in the sponge:CTS-SF/SA(0?g/g),CTS-SFAg0.5/SA(9.7712±0.2266?g/g),CTS-SFAg1.0/SA(22.3911±0.1807?g/g),CTS-SFAg2.0/SA(47.9614±0.1687?/g).Conclusions:(1)AgNP with uniform particle size was successfully prepared and loaded in the pores of gel sponge.(2)An asymmetric wettable chitosan-silk fibroin gel sponge was successfully prepared.Part2 Physicochemical Property and Antibacterial performance of an Asymmetric Wettable Chitosan-Silk fibroin Composite Dressing with Fixed Silver NanoparticlesObjective:Test the physicochemical properties and antibacterial performance of an asymmetric wettable chitosan-silk fibroin gel sponge with fixed silver nanoparticles in order to evaluate the possibility of the sponge as a candidate of wound dressing.Methods:Test the porosity,swelling degree,moisture retention,tensile strength and asymmetric wettability of the gel sponge and evaluate its antibacterial performance,the ability of control and release AgNP,sustained antibacterial property and microbe barrier property.Results:The prepared gel sponge has a high porosity.The modification of single surface made the porosity of sponge decrease somewhat but enlarge 4-7h in moisturizing time.Although the tensile strength of sponge decreased with an increase in the AgNP content,it is suitable for wound care applications.The asymmetric wettable chitosan-silk fibroin gel sponge has a significant effect on P.aeruginosa,S.aureus,M.albicans and E.coli.Compared with the gauze sample loaded with the same AgNP dose,the asymmetric wettable chitosan-silk fibroin gel sponge with fixed silver nanoparticles can effectively control the release of AgNP.ICP-MS test showed that the silver content of the experiment samples loaded with the same AgNP dose markedly increased as the time increased.Meanwhile,no significant difference was observed in the samples loaded with different AgNP dose at the same time.The sponge still has significant antibacterial property after immerged in normal saline for 72h.Also,the gel sponge with single surface modification showed good microbe barrier properties.Conclusions:(1)The asymmetric wettable chitosan-silk fibroin gel sponge with fixed silver nanoparticles has a high porosity which can benefit the transmission of oxygen and nutrients into cells in the wound.(2)Asymmetric wettable modification the porosity of sponge decreased somewhat but enlarge 4-7h in moisturizing time,which can promote the wound healing in a moist environment.(3)Tensile strength of sponge decreased with an increase in the AgNP content,however,it is suitable for wound care applications.(4)The asymmetric wettable chitosan-silk fibroin gel sponge has a significant effect on many pathogenic bacteria and has an excellent ability of control and release AgNP,sustained antibacterial property and microbe barrier property to maintain a sterile condition for wound healing and resist against invasion of microorganism.Part3 The effect of asymmetric wettable chitosan-silk fibroin gel sponge with fixed silver nanoparticlesoncell proliferation and its biosecurity testObjective:Investigate the effect of asymmetric wettable chitosan-silk fibroin gel sponge with fixed silver nanoparticles on cell proliferation and evaluate its biosecurity in order to provide theoretical basis for the further experiment in vivo.Methods:Evaluate the cytotoxicity of asymmetric wettable chitosan-silk fibroin gel sponge with fixed silver nanoparticles by L929 cells,human dermal fibroblasts and human umbilical cord mesenchymal stem cells.And further assess the biosecurity by guinea pig subcutaneous sensitization testResults:Human dermal fibroblasts were successfully extracted and shows spindle or spindly olygon,spiral growth.Its growth curve of the tumor liked "S" with positive expression of Vimentin in immunofluorescence assay.Human umbilical cord mesenchymal stem cells were successfully extracted and the shape of the cells were fibroblast-like,spiral growth,growth curve of the tumor liked "S".Molecular phenotyping showed the cells strongly positively expressed CD105(99.42%),CD44(99.69%),CD73(99.99%)and negatively expressed CD31(0.31%),CD45(0.70%)and CD34(1.06%).The extract liquid of sample of each groupall play a positive role in the proliferation of L929 cells,human dermal fibroblasts and human umbilical cord mesenchymal stem cells,however no difference in the viability of cells was observed between the CTS-SF Ag0.5/SA dressing extracts and the controls.The results of the subcutaneous sensitization test showed mild allergic reaction in CTS-SF Ag1.0/SA and CTS-SF Ag2.0/SA dressing groups,but no allergic reaction was found in CTS-SF/SAand CTS-SFAg0.5/SA groups.Conclusions:(1)Human dermal fibroblasts and human umbilical cord mesenchymal stem cells were successfully extracted.(2)CTS-SF Ag0.5/SA gel spongewas selected as the optimum wound dressing for the treatment of infected wounds.Part4 The effect of asymmetric wettable chitosan-silk fibroin gel sponge with fixed silver nanoparticles on the regeneration and repair of infected woundObjective:By the establishment of infected wound animal model and further evaluate the efficiency of the asymmetric wettable chitosan-silk fibroin gel sponge dressing in the treatment of infected wound in vivo experiment in order to provide more comprehensive experiment results and evidence.Methods:Establish the infected wound animal model and use CTS-SF Ag0.5/SA,CTS-SF/SA,Gauze-Ag and Gauze dressings in the treatment of infected wound.Comprehensively assess the efficiency of CTS-SF Ag0.5/SA gel sponge as wound dressing in the treatment of infected wound by wound healing ratio analysis,exudation cultivation,blood samples analysis,histological analysis and determine the silver content of organs by ICP-MS.Results:On the 14th postoperative day,the wound healing ratio of the CTS-SF Ag0.5/SA group reached 99.38± 1.5%,while the healing ratios in the CTS-SF/SA dressing,Gauze-Ag and Gauze groups were 89.22± 1.3%,69.26±3.7%and 70.23± 1.3%,respectively.Exudation cultivation on the 2nd postoperative day showed there was no colony of strains in the CTS-SF Ag0.5/SA and Gauze-Ag groups after secretion culture,but colony of strains was observed in CTS-SF/SAand Gauze groups.Blood sample analysis showed the the WBC count and NEUT%had a significant increasing trend on the 4th day and sustained growth on the 8th day,while the LYMP%sustained decreased.The WBC count and NEUT%of Gauze-Ag groups had not changed significantly on the 4th day,but surge significantly on the 8th day.However,the changes of WBC count,NEUT%and LYMP%in CTS-SF Ag0.5/SA groups were not obviously.The HE stained tissues on the 8th showed abundant fibroblasts but fewer fibrocyte cells and neovascularization accompanied by considerable amounts of inflammatory cell influx in the Gauze and Gauze-Ag groups.However,considerable capillary vessels and abundant fibrocytes were observed in the CTS-SF Ag0.5/SA and CTS-SF/SA dressing groups,and the former exhibited very few inflammatory cells in the CTS-SF Ag0.5/SA groups.The HE stained tissues and Masson stained tissues both demonstrated that CTS-SF Ag0.5/SA and CTS-SF/SA dressing groups was completely repaired,with keratinization and a dermis that was rich with blood vessels,and the CTS-SF Ag0.5/SA group had a significant collagen expression,and the tissue of the dermis was closer that of normal skin,with cells in the dermis layers arranged regularly.The Gauze and Gauze-Ag groups were still in the process of wound healing,and the Gauze-Ag group was actively repaired in the dermis layer,accompanied by an inflammatory reaction and erosion in parts of the epidermis.ICP-MS test shows silver mainly deposited in the liver of mice,and the amount of silver in the liver of mice treated with the CTS-SF Ag0.5/SA dressing was 1/15 of that in mice treated with the Gauze-Ag dressing.Conclusions:(1)The infected wound animal model was successfully established.(2)The asymmetric wettable chitosan-silk fibroin gel sponge dressing demonstrated good biosecurity and it can effectively control wound infection,reduce inflammation and plays an important role in the wound repair. |
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