Anandev N. Gurjala

Development of a novel, highly quantitative in vivo model for the study of biofilm-impaired cutaneous wound healing

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 Role of the Epidermis and the Mechanism of Action of Occlusive Dressings in Scarring

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 antiscar therapies.

Age-dependent response of primary human dermal fibroblasts to oxidative stress: cell survival, pro-survival kinases, and entrance into cellular senescence

A central question in cell biology is how cells become senescent. After a finite number of cell divisions, normal cultured human cells enter a state of irreversible growth arrest, termed “replicative senescence.” Alternatively, oxidative stress in the form of hydrogen peroxide (H2O2) can render human dermal fibroblasts (HDFs) nonproliferative and quiescent, a phenomenon known as stress‐induced premature senescence (SIPS). Although critical to the understanding of the pathophysiological basis of many diseases, there is no research to date that has simultaneously examined the interactions between age, oxidative stress, and SIPS. Therefore, the goals of this study were to examine in concert the interactions between these three factors in primary HDFs, and to test our central hypothesis that aging lowers the ability of primary HDFs to respond to oxidative stress. Our data provide, for the first time, evidence that aging dramatically reduces the capacity of primary HDFs to respond to the challenge of hydrogen peroxide. Specifically, aged HDFs showed decreased cell viability, decreased phosphorylation (activation) of pro‐survival kinases (Akt and ERK 1/2), and increased entrance into a senescent state when compared with their younger counterparts. Another important conclusion of this study is that blockade of transforming growth factor‐β1 had a pronounced “rescue effect” in the aged, preventing entrance of HDFs into cellular senescence.

Understanding the host inflammatory response to wound infection : An in vivo study of Klebsiella pneumoniae in a rabbit ear wound model

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.

In vivo evaluation of a novel mesh suture design for abdominal wall closure

Background: The authors present a novel mesh suture design aimed at minimizing the early laparotomy dehiscence that drives ventral hernia formation. The authors hypothesized that modulation of the suture-tissue interface through use of a macroporous structure and increased aspect ratio (width-to-height ratio) would decrease the suture pull-through that leads to laparotomy dehiscence. Methods: Incisional hernias were produced in 30 rats according to an established hernia model. The rat hernias were randomized to repair with either two 5-0 polypropylene sutures or two midweight polypropylene mesh sutures. Standardized photographs were taken before repair and 1 month after repair. Edge-detection software was used to define the border of the hernia defect and calculate the defect area. Histologic analysis was performed on all mesh suture specimens. Results: Seventeen hernias were repaired with mesh sutures and 13 were repaired with conventional sutures. The mean area of the recurrent defects following repair with mesh suture was 177.8 ± 27.1 mm2, compared with 267.3 ± 34.1 mm2 following conventional suture repair. This correlated to a 57.4 percent reduction in defect area after mesh suture repair, compared with a 10.1 percent increase in defect area following conventional suture repair (p < 0.0007). None (zero of 34) of the mesh sutures pulled through the surrounding tissue, whereas 65 percent (17 of 26) of the conventional sutures demonstrated complete pull-through. Excellent fibrocollagenous ingrowth was observed in 13 of 17 mesh suture specimens. Conclusions: Mesh sutures better resisted suture pull-through than conventional polypropylene sutures. The design elements of mesh sutures may prevent early laparotomy dehiscence by more evenly distributing distracting forces at the suture-tissue interface and permitting tissue incorporation of the suture itself.

World's First Baby Born Through Natural Insemination by Father With Total Phalloplasty Reconstruction.

AbstractTechniques for neophallus reconstruction have become increasingly refined, fulfilling more criteria for what is considered to be the ideal penis reconstruction. For both trauma and transgender populations, the radial forearm free flap remains the gold standard, although the pedicled or free anterolateral thigh flap is becoming a favored alternative. Despite the remarkably high rates of sexual activity reported by patients having benefited from these techniques, sexual function remains a significant challenge due to frequent complications including autologous and prosthetic stiffener failure, fistula formation, and inadequate erogenous sensation. Perhaps the ultimate criterion for neophallus reconstruction is one which not only avoids these complications by meeting the immediate goals of a competent neourethra, sensitivity, bulk, and aesthetic form but also successfully combines them into their true overarching function: procreation. In this article, we report the case of a pedicled anterolateral thigh flap neophallus reconstruction which allowed a patient to naturally conceive a child through penetrative intercourse without use of a stiffener, and led to pregnancy and subsequent birth of a baby son. We review the surgical techniques and factors that led to this patients successful progeny.

Evaluating the effects of subclinical, cyclic ischemia-reperfusion injury on wound healing using a novel device in the rabbit ear

ObjectiveThis study aimed to evaluate the effect of cyclic ischemia-reperfusion (IR) injury on wound healing using a novel rabbit ear model. Materials and MethodsA lightweight clamp apparatus was developed for reversible occlusion of the central ear artery. Ventral ear wounds were analyzed postoperatively for epithelialization and granulation as well as gene expression after 3 consecutive days of IR cycling. ResultsBy postoperative day #7, ears showed no gross tissue necrosis, but histologic analysis of wounds confirmed a significant impairment in epithelial and granulation tissue gaps as well as total epithelial and granulation tissue areas (P < 0.001). Quantitative polymerase chain reaction analysis of IR wounds indicated significant up-regulation of heat shock protein-70 and down-regulation of superoxide dismutase 1 relative to sham controls (P < 0.05). ConclusionsA novel rabbit ear model for the induction of subclinical, cyclic IR injury in cutaneous tissue has been developed that will serve as a valuable tool for the testing of new therapeutics.

Quantifying tissue level ischemia: Hypoxia response element-luciferase transfection in a rabbit ear model

Ischemia is a common underlying factor in a number of pathologic conditions ranging from cardiac dysfunction to delayed wound healing. Previous efforts have shown the resulting hypoxia activates the hypoxia inducible factor, a transcription factor with signaling effects through an intranuclear hypoxia response element (HRE). We hypothesized that ischemic conditions should activate these hypoxic signaling pathways in a measurable manner. We tested our hypothesis using variations of an established rabbit ear ischemic wound model and an HRE‐luciferase‐reporter gene construct. This plasmid construct was transfected into the ears of young, female New Zealand White rabbits, harvested at day 7 and processed to yield a reactive solution. Luminometry was used to quantify luciferase expression in each solution as a marker for HRE activation in each wound. Quantitative readings of hypoxic signaling as measured by luminescence yielded profound and statistically significant differences between the various ischemic models. Our results suggest that the biologic systems for hypoxic signaling can be used to detect local ischemia. HRE‐luciferase transfection is an effective tool for quantifying the degree of tissue hypoxia. The caudal ischemic rabbit ear model showed significantly higher levels of hypoxia. Use of a validated model that produces sufficient tissue levels of hypoxia is recommended for meaningful study of ischemic wound healing.