Integrating Negative Pressure And Exogenous Electric Fields Via A Flexible Porous Conductive Dressing To Accelerate Wound Healing

Background and purposeWound healing results from overlapping and interacting biological processes,including hemostasis,inflammation,proliferation,and remodeling.Any disorder in these processes will lead to chronic or even nonhealing wounds.Currently,a large number of modern biomaterials and medical technologies have been designed for wound treatments.However,these therapies are limited because they have a single specific function,and comprehensive strategies to improve wound healing are laking.Therefore,it is highly imperative to develop an ideal treatment with multifunctionality and multitargeted applications to accelerate wound healing.Among the many treatments available,negative pressure wound therapy(NPWT)is believed to be an effective method because it supports the adequate drainage of pus and necrotic tissue,mitigates inflammation,and enhances perfusion by continuous negative pressure suction.However,wound healing is an extremely complex process,and wound re-epithelialization is the key to wound healing.Chronic wound healing or delayed wound healing is mainly due to abnormal re-epithelialization.Re-epithelialization of wounds involves several keratinocyte functions:proliferation,migration,and differentiation.Keratinocyte migration is the rate-limiting step and key link of re-epithelialization.However,negative pressure wound therapy on promoting keratinocyte migration is very limited.Many recent studies on keratinocyte migration focused on cell behavior control demonstrate that wound electric fields(EFs)are an overriding signal that directs the cell migration.When a wound occurs,lateral wound EFs are formed from the surrounding intact tissue toward the center of the wound.In turn,abnormal wound EFs or their tendency to collapse are the crucial causes of difficulty in the repair of problematic wounds.Thus,the synergistic combination of NPWT and exogenous directed wound EFs is extremely likely to successfully accelerate wound healing.Moreover,considering that the four wound healing phases overlap and interact,these two methods should exert simultaneous implementation.NPWT serves as an exudate drainage and inflammation control system,while exogenous wound EFs enhance or replenish the electrical signals to facilitate re-epithelialization.Construction of an artificial wound microenvironment that supports drainage,controls inflammatory,and facilitates proliferation and re-epithelialization within one system is a promising and novel strategy.However,this combination is difficult to achieve due to limitation of implementation path of exogenous wound EFs.Conventional metal electrodes cause unsatisfactory contact in terms of a mechanical mismatch with human tissues.Moreover,the wound exudate is extremely likely to corrode the electrode,leading to unstable conduction and toxic degradation products.Aim:The purpose of this study was to construct a highly conductive,flexible foam wound dressing with the properties of porous structure,bending-compression durability,and long-term stability under wet conditions to integrate NPWT and exogenous wound EFs.This "all-in-one" treatment system could optimize the artificial wound microenvironment at multiple stages of the wound healing process.NPWT reduced inflammation during the inflammatory phase,while exogenous wound EFs stimulated cell proliferation and angiogenesis,and electrical signals could direct the keratinocyte migration to achieve re-epithelialization during the proliferation phase.Contents and methods:1.Construction of a flexible porous conductive foam wound dressingThe conductive foam dressing(TPFS-AgNWs-PU)was constructed via a solution-based method.The AgNWs used as conductive components,while PU sponge acted as a skeleton to support 3D conductive networks.2.Characterization of TPFS-AgNWs-PU foam wound dressing.The micronetwork structure of TPFS-AgNWs-PU foam was assess by SEM.The electrical conductivity of the TPFS-AgNWs-PU foam was regulated by the number of spray-coating cycles.The wettability of the foam was evaluated by measuring the water contact angles on the surface by a sessile drop technique.EDS elemental mapping used to analysis the distribution of TPFS-coated AgNWs on the surface of the PU foam.3.Biocompatibility of AgNWs-PU foam wound dressingTo achieve applying in vivo,the biocompatibility was assessed by live/dead cell staining and CCK-8 assay.To study the stability of the TPFS-AgNWs-PU foam dressing,the release behavior of the AgNWs in PBS was determined by ICP-MS.4.Application of exogenous EFs via TPFS-AgNWs-PU foamWith TPFS-AgNWs-PU as the conductive section and ordinary PU foam dressing as nonconductor,annular exogenous EFs whose negative pole was in the wound center and positive pole was in the wound margin could be successfully applied on wounds.The center of the conductive wound dressing in the experimental group(TPFS-AgNWs-PU/EF)was connected to the negative electrode of the external power,and the outer ring of the conductive dressing was connected to the positive electrode,which formed directional EFs from the margin to the center of the wound.Exogenous EFs(70%duty cycle,0.1 Hz)with an applied intensity of+100 mV/mm were created in the direction of endogenous EFs of the wound.A negative-pressure drainage at 75 mm Hg was implemented simultaneously.The real-time EFs values across the wound were detected.5.Detection of therapeutic effect on wound healing in a pig modelThe therapeutic effect of integrating negative pressure and exogenous electric fields via TPFS-AgNWs-PU foam dressing was detected by full-thickness skin defects in Bama pigs.The immunohistochemical assay and western blot analysis were used to study the novel "all-in-one" device presented intrinsic multifunctionality,including the drainage of pus and necrotic tissue,mitigation of inflammation,promotion of cell proliferation,direction of keratinocyte migration,and induction of angiogenesis.Result:1.After modification,the resistance of the TPFS-AgNWs-PU foam gradually decreased from approximately 1156? to approximately 24.5?,and an ultralow resistance of 3.8? was obtained under 83.3%compression deformation.2.The live/dead cell staining and CCK-8 assay results illustrated that TPFS-AgNWs-PU foam displayed good biocompatibility(RGR>80%),indicating the acceptability of TPFS-AgNWs-PU foam for application in covering wounds.3.The total EF strength was approximately 200 mV/mm after exogenous EFs at a strength of 100 mV/mm superimposing on the endogenous wound EFs,which proved that the exogenous EFs had been successfully implemented.4.On the 7th day after wounding,TPFS-AgNWs-PU/EF group had the lowest residual wound area percentage(30.30±1.75%),followed by the TPFS-AgNWs-PU foam group(61.94±3.86%),while the blank group had the largest residual wound area percentage(72.54±3.89%).On the 14th day,the percentage of the residual wound area in the AgNWs-PU/EF foam group was only 3.07±1.23%,that in the AgNWs-PU group was 18.35±3.83%,and that in the blank group was 25.82±3.52%.5.On the 14th day after injury,the length in the TPFS-AgNWs-PU/EF foam group was 10.23±1.01 mm,that in the TPFS-AgNWs-PU group(NPWT without exogenous EFs)was 7.29±0.34 mm,and that in the blank group was 6.68±0.41 mm.6.On the 7th day,there were 27±4 inflammatory cells in the AgNWs-PU/EF group,61±6 inflammatory cells in the AgNWs-PU group,and 152±8 inflammatory cells in blank group.7.TPFS-AgNWs-PU/EF group showed the highest positive staining result on the 7th day,which was 1.23 times that in the TPFS-AgNWs-PU group and 2.03 times that in the blank group.Positive staining for Ki67 on the 14th day was 142%higher in the TPFS-AgNWs-PU/EF group than in the blank group,while that of the TPFS-AgNWs-PU group was 85%higher than that of the blank group.8.On the 7th day of wound formation,the expression level of E-cadherin in the TPFS-AgNWs-PU/EF group was 62.99%lower than that in the TPFS-AgNWs-PU group,and 39.88%lower than that in the blank group.On the 14th day,the expression level of E-cadherin in TPFS-AgNWs-PU/EF group was 65.50%lower than that in TPFS-AgNWs-PU group,and 52.32%lower than that in blank group.9.The result of western blot analysis on the 7th day showed that the expression of PI3K and Akt remained stable,while phosphorylated PI3K and phosphorylated Akt were upregulated in the tissues subjected to TPFS-AgNWs-PU/EF group.10.Compared to the blank control group,the AgNWs-PU/EF group exhibited 1.67 times vascular density on the 7th day and 1.37 times vascular density on the 14th days,while that of the AgNWs-PU group was 4.06 times on the 7th day and 3.21 times on the 14th day compared to vascular density in the blank control group on the corresponding days.Conclusion:This study focused on constructing a 3D foam dressing with the ability to facilitate both conduction and drainage by combining AgNWs with an ultra-high length-diameter ratio with PU foam so that negative pressure and exogenous wound EFs simultaneously promoted wound healing.This combined therapeutic system showed enhanced effects on necrotic tissue drainage,cell proliferation and migration,and angiogenesis by optimizing the wound microenvironment,thus facilitating wound healing at multiple stages.Supported by additional in vivo experiments in Bama pigs,the results of our study not only demonstrated the innovation of existing wound treatment methods but also provide a basis for follow-up clinical research.