Research Of Application Mechanisms Of Negative Pressure Wound Therapy In Infected Soft-tissue Wounds

BackgroundWound infection is a long challenged problem in the realm of orthopedics. Previous study indicate that infection is a complicated procedure of the interaction between bacteria and wound local microenvironment. The pathogenicity of bacteria depends more on characteristic bacteriology and expression of diverse virulence factors, not only on the bioburden. Furthemore, The pathogenic bacteria can coordinate the patterns of diverse virulence factors expression in response to environmental changes. Negative pressure wound therapy (NPWT) is an effective physical method to treat infected soft-tissue wounds, which has shown significant clinical benefits in infected wound healing. Differentiated with the traditional therapies for wound care which lead to the decrease of bacteria bioburden, the mechanisms of infection control of NPWT is unclear.ObjectiveFirstly, we intend to investigate the impact of NPWT on bacterial behavior, including colony morphology, spatial distribution, and bacterial bioburden. Secondly, we intend to evaluate the virulence of Staphylococcus aureus, including activation of accessory gene regulator, gene expression and production of virulence factors. Thirdly, we intend to test the impact of NPWT on local expression of proinflammatory cytokines, and the amount of neutrophils in different layer of wound.Methods1. Full-thickness soft-tissue wounds were created on the back of rabbits, and were inoculated with bioluminescent Staphylococcus aureus. The wounds were treated with sterile gauze dressings and NPWT. SEM and TEM were performed to determine the characteristic bacteriology. LSCM was performed to obtain bioluminescent images which were used to observe spatial distribution of the GFP-labeled S. aureuswithin the tissue and quantify the bacterial bioburden.2．Wound samples were harvested with the same methods. The gene expression of S.aureus virulence factors was analyzed through real-time PCR, the amount ofvirulence factors in wound beds was investigated by western blot.3. The proinflammatory cytokines gene expression of IL-1β, IL-8and TNF-α wasevaluated through real-time PCR. Wound biopsies were examined histologically forneutrophils quantification in different layers of tissue.Results1. NPWT resulted in sparse amounts of scattered bacteria on the wound surface or assparsely spaced single colonies within the tissue，and the less bacterial invasiondepth.2. Wound bioburden on day8in the NPWT and gauze groups was34.6±5.5%and141.9±15.4%of the baseline values, respectively (P＜0.01). But according to thecalculation of relative percent, the bacterial load only reduced an order of magnitude.3. NPWT depressed the the agrA expression of accessory gene regulator.4. The transcription and amount of virulence factors including EAP, SpA and α-toxinwere significantly decreased in NPWT group.5. NPWT treated wounds which exhibited earlier and greater peaking of IL-1β andIL-8expression and a decrease in TNF-α expression.6. Histologic examination revealed significantly increased neutrophil countpresenced in the shallow layer in wound biopsies of NPWT treatment at the day2.Conclusion1．NPWT can impact on the infection behavior of S. aureus, including colonymorphology and spatial distribution both on the surface and within wound tissue, andreduces bacterial invasion depth.2．To a certain extent that NPWT can reduced the bacterial load within wound, but itis not the main mechanism of infection control.3．The changes of bacterial colony morphology treated with NPWT depressed the activation of accessory gene regulator,which is quorum-sensing system.4． Inhibition of accessory gene regulator by negative pressure reduced virulencefactor expression, and adequate drainage of synergy to further decreased the amountof virulence factors in wound.5． NPWT enhances local inflammatory responses in acute infected soft-tissuewound to prevent the infection spread.