A growing body of evidence suggests that in addition to hypoxia, ischemia-reperfusion injury, and intrinsic host factors, bacterial biofilms represent a fourth major pillar in chronic wound pathogenesis. Given that most studies to date rely on in vitro or observational clinical data, our aim was to develop a novel, quantitative animal model enabling further investigation of the biofilm hypothesis in vivo. Dermal punch wounds were created in New Zealand rabbit ears, and used as uninfected controls, or inoculated with green fluorescent protein-labeled Staphylococcus aureus to form wounds with bacteria predominantly in the planktonic or biofilm phase. Epifluorescence and scanning electron microscopy revealed that S. aureus rapidly forms mature biofilm in wounds within 24 hours of inoculation, with persistence of biofilm viability over time seen through serial bacterial count measurement and laser scanning confocal imaging at different time points postwounding and inoculation. Inflammatory markers confirmed that the biofilm phenotype creates a characteristic, sustained, low-grade inflammatory response, and that over time biofilm impairs epithelial migration and granulation tissue in-growth, as shown histologically. We have established and validated a highly quantitative, reproducible in vivo biofilm model, while providing evidence that the biofilm phenotype specifically contributes to profound cutaneous wound healing impairment. Our model highlights the importance of bacterial biofilms in chronic wound pathogenesis, providing an in vivo platform for further inquiry into the basic biology of bacterial biofilm-host interaction and high-throughput testing of antibiofilm therapeutics.

The problem of cutaneous scarring has conventionally been approached as a pathology of the dermis. Multiple lines of evidence from the clinic, in vitro experiments, and in vivo animal and human studies, however, increasingly suggest that the epidermis plays a major role in the control of underlying dermal scar. Building on the demonstrated efficacy of silicone gel occlusion, in this paper we review the evidence for epidermal regulation of scar, and propose the novel hypothesis that dermal fibrosis is exquisitely linked to the inflammatory state of the epidermis, which in turn is linked to hydration state as a function of epidermal barrier function. In the spectrum of factors contributing to dermal scar, the epidermis and its downstream effectors offer promising new targets for the development of anti-scar therapies.

Wound infection development is critically dependent on the complex interactions between bacteria and host. Klebsiella pneumoniae has become an increasingly common wound pathogen, but its natural history within wounds has never been studied. Using a validated, in vivo rabbit ear model, wounds were inoculated with K. pneumoniae at different concentrations (10²-10⁷ colony-forming units) with measurement of viable and nonviable bacterial counts, histological wound-healing parameters, and host inflammatory gene expression at multiple time points postinoculation (48, 96, and 240 hours). Bacteria and wound morphologies were evaluated with scanning electron microscopy. Comparable experiments were performed in ischemic ears to model immune response impairment. All wounds, despite different inoculants, equilibrated to similar bacterial concentrations by 96 hours. With a 10⁶ colony-forming units inoculant, wounds at 240 hours showed decreased bacterial counts (p < 0.01), with a corresponding improvement in healing (p < 0.01) and a decrease in inflammatory response (p < 0.05). In contrast, ischemic wounds revealed impaired inflammatory gene expression (p < 0.05) resulting in higher steady-state bacterial concentrations (p < 0.01), impaired healing (p < 0.05), and biofilm formation on scanning electron microscopy. We conclude that a normal inflammatory response can effectively stabilize and overcome a K. pneumoniae wound infection. An impaired host cannot control this bacterial burden, preventing adequate healing while allowing bacteria to establish a chronic presence. Our novel study quantitatively validates the host immune response as integral to wound infection dynamics.

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