| Section 1 IntroductionWith the coming of the aging society,the implantation rate of cardiac implantable electronic device(CIED)is increasing recently.At least 1.7 million patients receive CIED every year,which greatly improves the patient's quality of life.However,in recent years,the infection rate of CIED has increased,which has become an urgent problem to be solved in clinical work.About 60-90% of CIED infections are caused by Staphylococcus epidermidis and Staphylococcus aureus.In recent years,it has been found that the proportion of CIED infection caused by a variety of coagulase negative staphylococci is gradually increasing.Despite many precautions are taken against CIED infection,it is regrettable that the incidence rate,hospitalization rate and mortality rate are increasing in those year.At present,the measures to prevent CIED infection mainly include skin preparation,washing with antibacterial agents,perioperative use of antibiotics,Aigis Rx antibacterial envelope and other emerging materials.Antibacterial hydrogel is mainly divided into two categories.The first is hydrogel directly loaded with antimicrobial agents,such as nano metal,antibiotics,antimicrobial peptides,etc.Silver nanoparticles,the most widely used in clinical applications,have been widely used in wound dressings.The second type is the macromolecule material of the hydrogel itself,which has antibacterial properties,such as terpenes and chitosan polymers.HA is a natural carbohydrate polymer.Because of its inherent biocompatibility and biodegradability,it has been widely used in the construction of hydrogel materials.Firstly,HA has good biosafety and degradability.Secondly,HA an absorb several times of its own volume of water swelling,and can combine with cell membrane receptor CD44,providing conditions for wound / incision healing.Finally,the HA backbone contains active groups such as carboxyl and amino groups,which facilitate chemical and / or physical crosslinking to form hydrogels.As is known to all,physically cross-linked HA hydrogel carriers are injectable and exhibit good re-modelling capabilities through surrounding tissues,but usually exhibit poor mechanical properties and rapid degradation in vivo.On the contrary,chemically cross-linked HA hydrogel usually has mechanical stability and stability,but it has poor plasticity after gelation.CHX is considered to be a promising antibacterial drug.Its excellent and broad-spectrum antibacterial properties are mainly attributed to the presence of a large number of imino groups in its structure.It can attach to anions on the cell wall surface of various bacteria,or destroy the chemical bonds of the bacterial wall,which blocks the process of protein transcription and translation in cells and eventually leads to the death of bacteria.At the same time,CHX has the characteristics of stable structure,convenient production and low irritation,so CHX has been widely used in the field of biomedicine.Therefore,we intend to build a hybrid crosslinking strategy of antibacterial hydrogel,combined with chemical and physical crosslinking methods,Which eventually used to prevent CIED bag infection.Section 2 Construction of an injectable and antibacterial hyaluronic acid hydrogel based on hybrid crosslinking strategyCHA hydrogels were obtained through chemical crosslinking and partial improvement.BDDE was used as a crosslinking agent,followed by freeze-drying,grinding,and the crosslinking density was calculated by NMR.The CHA hydrogel was successfully synthesized.CHA hydrogel was implanted into the SD rat pouch to verify the degradability and degradation time of the hydrogel was about 15 days.The degradation time could well cover the infection period of CIED pocket,and it was not easy to cause chronic reaction.The preparation process of CHA/CHX hydrogel is convenient and can be prepared only by mixing CHA hydrogel and CHX in equal proportion.The rheological properties of CHA/CHX hydrogel were characterized by dynamic oscillation,frequency dependence,dynamic time scanning and thixotropic properties.It can be found that both G 'and G' of CHA/CHX hydrogel exhibit similar nonlinear rheological behavior,and their value increases with the increase of scanning frequency,indicating that the microstructure of CHA/CHX hydrogel may be similar.Moreover,the storage modulus of all hydrogels is far higher than the loss modulus in the whole frequency range,indicating that the gel state of CHA/CHX hydrogel can be maintained.Then the shear modulus of CHA hydrogel was 165 Pa at the constant time frequency(1 Hz)and the strain amplitude(1%)time sweep test.But the shear modulus of CHA-0.1X hydrogel and CHA-0.3X was 311 Pa and 330 Pa respectively.These results indicate that CHX can be re-physically cross-linked by electrostatic interactions with pre-crosslinked HA,thus significantly improving the mechanical properties of CHA hydrogels.Then,at a constant frequency(1 Hz),the transient viscosity modulus of CHA/CHX hydrogel was obtained by dynamic time sweep rheology test in the range of 0%-60% strain amplitude.The shear modulus of CHA/CHX hydrogel decreases,and the viscosity modulus decreases with the increase of strain amplitude.CHA/CHX hydrogel can be injected by 26 G injector(needle diameter 0.46 mm),which proves that CHA/CHX hydrogel has good shear thinning properties.The CHA-0.5X hydrogel was used to further explore the thixotropic behavior of the CHA/CHX hydrogel at the constant range of frequency(1 Hz)and the dynamic strain rheometer at the entire range or 0.1% and 1000%alternating strain amplitudes.It was observed that the hydrogel was completely collapsed under the strain amplitude of 1000%.At 1% and 1000% strain amplitude alternation,CHA/CHX hydrogel can collapse and restore cycle.The results showed that CHA/CHX hydrogel had good shear thinning and self-healing behavior.The microstructure of CHA/CHX hydrogel was characterized by scanning electron microscopy.The CHA/CHX hydrogel had a lamellar superimposed lamellar structure,and the surface was covered by microfilaments through local amplification,indicating that the carboxyl group of CHA was physically crosslinked with imino of CHX successfully.The release behavior of CHA/CHX hydrogel was characterized by ultraviolet spectrophotometer.The cumulative release amount of CHX was 72.3%and 49.4% in the initial 24 hours or without hyaluronidase,and the cumulative release rate was 77.3% and 65.8% within 120 hours.The release of CHX was well released,and the release of CHX was hyaluronidase responsive.The microstructure of CHA/CHX hydrogel was characterized by scanning electron microscopy.The CHA/CHX hydrogel had a lamellar superimposed lamellar structure.The surface was covered by microfilaments by local amplification,indicating that the carboxyl group of CHA was physically crosslinked with CHX imino group.The release behavior of CHA/CHX hydrogel was characterized by ultraviolet spectrophotometer.The cumulative release amount of CHX was 72.3% and 49.4% in the initial 24 hours or without hyaluronidase,and the cumulative release rate was77.3% and 65.8% within 120 hours.The release of CHX was well released,and the release of CHX was hyaluronidase responsive.Section 3 Biocompatibility evaluation of CHA/CHX hydrogelThrough the co-culture of CHA and CHA/CHX hydrogel and L929 cells,the proliferation toxicity of cells was detected by CCK-8.The results showed that the survival rate of L929 cells in CHA/CHX hydrogel group was more than 90%.By live/dead cells staining,the number of L929 cells in CHA/CHX hydrogel group did not increase or decrease,and the morphology did not show obvious abnormalities,indicating that CHA/CHX hydrogel had good cytocompatibility.By co-incubation of CHA/CHX hydrogel and human red blood cell suspension for 1 hours,the hemolysis rate of CHA/CHX hydrogel was lower than 5% of the international standard of biomaterial industry.Therefore,CHA/CHX hydrogel has good blood compatibility.After injection of CHA/CHX hydrogel into subcutaneous tissue of Kunming mice,there was no obvious infiltration of inflammatory cells observed in CHA/CHX hydrogel injection area after 14 days.It was proved that CHA/CHX hydrogel had biocompatibility.Section 4 In vitro antibacterial evaluation of CHA/CHX hydrogelIt was expressed by the size of inhibition zone of Gram-positive bacteria and Gram-negative bacteria.Staphylococcus aureus was selected to represent gram positive bacteria and Escherichia coli to represent gram negative bacteria.PBS,CHA,CHA-0.1X,CHA-0.3X and CHA-0.5X hydrogels were added to the diameter of Staphylococcus aureus or Escherichia coli solid medium respectively.Adding PBS and CHA hydrogel did not produce bacteriostatic circle against Staphylococcus aureus and Escherichia coli.But CHA-0.1X,CHA-0.3X and CHA-0.5X hydrogels showed excellent antibacterial effect.And with the increase of CHX concentration,the inhibition zone of CHA/CHX hydrogel was larger.The inhibition zone of CHA-0.1X,CHA-0.3X and CHA-0.5X hydrogels on Staphylococcus aureus were14.4 mm,16.4 mm and 16.7 mm respectively.The inhibition zone of CHA-0.1X,CHA-0.3X and CHA-0.5X hydrogel on Escherichia coli was 14.3 mm,15.8 mm and16.4 mm respectively.It was proved that CHX released from CHA/CHX hydrogel had excellent bacteriostatic effect on Gram positive bacteria and Gram negative bacteria,and the bacteriostatic effect was positively correlated with the concentration of CHX.By transferring CHA,CHA-0.1X,CHA-0.3X and CHA-0.5X hydrogels onto the surface of aseptic slide,PBS was used as negative control group,and Staphylococcus aureus and Escherichia coli were transferred to PBS,CHA,CHA-0.1X,CHA-0.3X and CHA-0.5X hydrogel surfaces respectively.The surface of CHA hydrogel has no bactericidal ability.The concentration of Staphylococcus aureus on CHA-0.1X,CHA-0.3X and CHA-0.5X hydrogel decreased by 5.4 times,6.5 times and 6.5 times respectively,while the concentration of Escherichia coli decreased 4.1 times,4.2 times and 6.1 times respectively,indicating that the surface of CHA/CHX hydrogel could not only kill gram positive bacteria but also kill gram negative bacteria,and the surface antibacterial properties of CHA/CHX hydrogels.It is mainly related to the concentration of CHX in CHA/CHX hydrogel,and CHA-0.5X hydrogel has the best antibacterial activity.The effect of CHA/CHX hydrogel on the membrane structure of Staphylococcus aureus and Escherichia coli was observed by scanning electron microscope.Many studies had confirmed that CHX mainly destroyed the integrity of bacterial membrane.Staphylococcus aureus and Escherichia coli were dripped on the surface of CHA-0.5X hydrogel and agar medium for 4 hours in the incubator at37? and the structure of Staphylococcus aureus and Escherichia coli was observed after treatment.On the surface of CHA-0.5X hydrogel,Staphylococcus aureus and Escherichia coli were damaged in cell membrane integrity,the contents were outflow,and the morphology of bacteria changed greatly.On the surface of agar gel,Staphylococcus aureus and Escherichia coli were retained,indicating that CHA/CHX hydrogel destroyed the structure of Staphylococcus aureus and Escherichia coli causing bacterial death.The antibacterial activity of CHA/CHX hydrogel was evaluated by live/dead staining.Staphylococcus aureus and Escherichia coli were transferred to agar medium or CHA-0.5x hydrogel surface,incubated for incubate in 37? bacterial incubator for 1 hour,and then stained by SYPO9 and propidium iodide,followed by confocal microscopy.Under the confocal fluorescence microscope,it could be observed that the surface of agar medium was almost green fluorescence,and only a small amount of red fluorescence showed that Staphylococcus aureus and Escherichia coli were alive.However,the CHA/CHX hydrogel group showed almost red fluorescence and no obvious green fluorescence,indicating that the surface was all dead Staphylococcus aureus and Escherichia coli,indicating that CHA/CHX hydrogel has excellent antibacterial properties.Section 5 Evaluation of CHA/CHX hydrogel in preventing CIED pocket infectionBy constructing the CIED pocket infection model,two mutually isolated pocket were made on the back of each experimental rabbit,and all of them were implanted with pacemakers and bacterial suspension.When only the pacemaker was implanted,the pocket was dry and free of purulent secretions.When the pacemaker and bacterial suspension were implanted in the bag,the pacemaker was filled with purulent secretions.At the same time,the pacemaker surface was covered with a layer of viscous membrane,which proved that the CIED pocket infection model was successfully constructed.CHA-0.5X hydrogels can be conveniently applied through sterile syringes.First of all,the appropriate size of pacemaker bag was made and implanted.Then,the CHA/CHX hydrogel was directly injected into the CIED pocket by 26 G syringe,and it was evenly molded around the pacemaker by kneading the bag.Finally,the surgical site was closed by suture.From the whole CHA/CHX hydrogel operation process,it could be found that it was easily to be applied to the CIED pocket by means of injectors and capsular methods,and did not need to enlarge the volume of the capsule or extend the length of the incision.Compared with the CHA/CHX hydrogel,the CHA/CHX hydrogel group had no excessive tension after suturing the wound,and did not increase the risk of surgical incision dehiscence.It proved that CHA/CHX hydrogel had good application convenience and plasticity.The effect of CHA-0.5X on the prevention of pacemaker pocket infection was observed by re incision subcutaneous pocket on the 7th day after implantation.Whether pacemaker and bacterial suspension were implanted or pacemaker,bacterial suspension and CHA hydrogel were implanted,a large number of purulent secretions could be seen around the pocket and pacemaker.There were sticky membranous substances on the pacemaker surface.However,after pacemaker implantation,bacterial suspension and CHA-0.5X hydrogel implantation,there was almost no purulent secretion around the pacemaker.The results show that CHX could also be released from CHA/CHX hydrogel in vivo to kill Staphylococcus aureus and prevented CIED pocket infection.By collecting the surrounding tissues of pacemakers for HE staining,we can evaluate the effectiveness of CHA/CHX hydrogel in preventing CIED pocket infection from the organizational level.Whether pacemaker and bacterial suspension,pacemaker,bacterial suspension and CHA hydrogel group,the fascia layer of pleura muscle layer adhered to a large number of exudates,and there were a large number of inflammatory cell infiltration,indicating a large number of bacterial infiltration.There was no obvious exudation on the surface of the fascia layer of pacemaker,bacterial suspension and CHA-0.5X hydrogel group,and no bacterial invasion was observed.The CHA/CHX hydrogel was proved to have good antibacterial properties in vivo at the tissue and cell level.By collecting the surrounding tissues of pacemakers,the bacterial counts in capsular tissue can be quantitatively evaluated for the antibacterial efficacy of CHA/CHX hydrogel.The weight of tissue around each bag was weighed,and the bacterial suspension was obtained by homogenization method.Then the bacterial amount of each group was obtained by gradient dilution and coating method.Finally,the logarithm was taken as the ordinate by the ratio of bacterial amount to weight.Whether the pacemaker and bacterial suspension,or pacemaker,bacterial suspension and CHA hydrogel group,there were a large number of Staphylococcus aureus in the capsular tissue,and the logarithm of bacterial weight to weight ratio in bacterial group and CHA hydrogel group was 8.13 and 8.79,while only a litter Staphylococcus aureus could be cultured in pacemaker,bacterial suspension and CHA-0.5X hydrogel group.The logarithm of bacterial weight to weight ratio in CHA/CHX hydrogel group is 1.98,the Staphylococcus aureus colony of CHA-0.5X hydrogel group was far lower than that of pacemaker and bacterial suspension group.It could quantitatively prove that CHA/CHX hydrogel could kill Staphylococcus aureus and prevent CIED pocket infection.Through the collection of bacteria,CHA hydrogel and CHA/CHX hydrogel pacemakers in live / dead bacteria staining analysis,it can reflect the killing effect of CHA/CHX hydrogel on Staphylococcus aureus.Chose SYTO 9 and propyl iodide organism used for live/dead cells,respectively under the confocal fluorescence microscope,both pacemaker and bacterial suspension,pacemaker,bacterial suspension and CHA hydrogel group were almost green fluorescent,and only a small amount of red fluorescence showed that there were a large number of viable Staphylococcus aureus on the surface of the pacemaker,but CHA/CHX hydrogel group.Almost red fluorescence,indicating a large number of Staphylococcus aureus,proved that CHA/CHX hydrogel can kill Staphylococcus aureus in vivo and can effectively prevent CIED pocket infection.Section 6 SummaryIn this paper,we successfully constructed a hybrid antibacterial HA hydrogel,which combines chemical and physical crosslinking strategy.It has good biocompatibility and can effectively prevent CIED pocket infection.It provides a theoretical basis for the later clinical transformation and application of antibacterial hydrogels. |
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