Is It Possible to Monitor the Safest Time to Perform Secondary Surgery on Free Flaps? A Clinical Evaluation of the Tewameter®.

Background and Objectives: Postoperative monitoring, following free flap surgery, plays a crucial role in ensuring the survival of the flap. However, in microsurgery, not only the immediate postoperative monitoring period but also the choice of the right time for secondary surgeries is crucial for the free flap survival. There is no clear consensus concerning the right choice of timing for secondary surgery. Our aim was to evaluate transepidermal water loss (TEWL), with the objective evaluation tool Tewameter® in free flap surgery to monitor flap autonomization. Materials and Methods: Transepidermal water loss was assessed in 20 patients with microsurgically transplanted free anterior lateral thigh (ALTP) flaps. The transplantation of the ALTP-flap and the postoperative care were administered in accordance with the standard of care of the department. Measures were taken on the free flap and normal skin at follow-ups of 1, 3, and 6 months after initial free flap transplantation. Results: Transepidermal water loss gradually increased to the values found in normal skin, after 6 months. The differences between the two areas demonstrated the smallest variance after 6 months, specifically in the ALTP-flap region. The largest disparities were observed between month 1 and month 6, followed by month 3 and month 6, and month 1 and month 3. Conclusions: Free flap autonomization and physiology are complex processes. TEWL might be a valuable parameter to monitor flap autonomization. Our results indicate that TEWL in the free flap is nearly "normal" after six months. For a clear consensus of when to perform individual secondary surgery, further studies are needed.

High-Intensity Blue Light (450-460 nm) Phototherapy for Pseudomonas aeruginosa-Infected Wounds.

Background: Nosocomial wound infection with Pseudomonas aeruginosa (PA) is a serious complication often responsible for the septic mortality of burn patients. Objective: High-intensity antimicrobial blue light (aBL) treatment may represent an alternative therapy for PA infections and will be investigated in this study. Methods: Antibacterial effects of a light-emitting diode array (450-460 nm; 300 mW/cm2; 15/30 min; 270/540 J/cm2) against PA were determined by suspension assay, biofilm assay, and a human skin wound model and compared with 15-min topically applied 3% citric acid (CA) and wound irrigation solution (Prontosan®; PRT). Results: aBL reduced the bacterial number [2.51-3.56 log10 colony-forming unit (CFU)/mL], whereas PRT or CA treatment achieved a 4.64 or 6.60 log10 CFU/mL reduction in suspension assays. aBL reduced biofilm formation by 60-66%. PRT or CA treatment showed reductions by 25% or 13%. Here, aBL reduced bacterial number in biofilms (1.30-1.64 log10 CFU), but to a lower extend than PRT (2.41 log10 CFU) or CA (2.48 log10 CFU). In the wound skin model, aBL (2.21-2.33 log10 CFU) showed a bacterial reduction of the same magnitude as PRT (2.26 log10 CFU) and CA (2.30 log10 CFU). Conclusions: aBL showed a significant antibacterial efficacy against PA and biofilm formation in a short time. However, a clinical application of aBL in wound therapy requires effective active skin cooling and eye protection, which in turn may limit clinical implementation.

High-Intensity Blue Light (450-460 nm) Phototherapy for Pseudomonas aeruginosa-Infected Wounds.

Background: Nosocomial wound infection with Pseudomonas aeruginosa (PA) is a serious complication often responsible for septic mortality of burn patients. High-intensity antimicrobial blue light (aBL) treatment may represent an alternative therapy for PA infections. Methods: Antibacterial effects of an light-emitting diode (LED) array (450-460 nm; 300 mW/cm2; 15/30 min; 270/540J/cm2) against PA were determined by suspension assay, biofilm assay, and a human skin wound model and compared with 15-min topically applied 3% citric acid (CA) and wound irrigation solution (Prontosan®; PRT). Results: The aBL reduced the bacterial number (2.51-3.56 log10 CFU/mL), whereas PRT or CA treatment achieved a 4.64 or 6.60 log10 CFU/mL reduction in suspension assays. The aBL reduced biofilm formation by 60%-66%. PRT or CA treatment showed reductions by 25% or 13%. In this study, aBL reduced bacterial number in biofilms (1.30-1.64 log10 CFU), but to a lower extent than PRT (2.41 log10 CFU) or CA (2.48 log10 CFU). In the wound skin model, aBL (2.21-2.33 log10 CFU) showed a bacterial reduction of the same magnitude as PRT (2.26 log10 CFU) and CA (2.30 log10 CFU). Conclusions: The aBL showed a significant antibacterial efficacy against PA and biofilm formation in a short time. However, a clinical application of aBL in wound therapy requires effective active skin cooling and eye protection, which in turn may limit clinical implementation.

Experimental evidence for Parthanatos-like mode of cell death of heat-damaged human skin fibroblasts in a cell culture-based in vitro burn model.

The cellular mechanisms of burn conversion of heat damaged tissue are center of many studies. Even if the molecular mechanisms of heat-induced cell death are controversially discussed in the current literature, it is widely accepted that caspase-mediated apoptosis plays a central role. In the current study we wanted to develop further information on the nature of the mechanism of heat-induced cell death of fibroblasts in vitro. We found that heating of human fibroblast cultures (a 10 s rise from 37 °C to 67 °C followed by a 13 s cool down to 37 °C) resulted in the death of about 50% of the cells. However, the increase in cell death started with a delay, about one hour after exposure to heat, and reached the maximum after about five hours. The lack of clear evidence for an active involvement of effector caspase in the observed cell death mechanism and the lack of observation of the occurrence of hypodiploid nuclei contradict heat-induced cell death by caspase-mediated apoptosis. Moreover, a dominant heat-induced increase in PARP1 protein expression, which correlated with a time-delayed ATP synthesis inhibition, appearance of double-strand breaks and secondary necrosis, indicate a different type of cell death than apoptosis. Indeed, increased translocation of Apoptosis Inducing Factor (AIF) and Macrophage Migration Inhibitory Factor (MIF) into cell nuclei, which correlates with the mentioned enhanced PARP1 protein expression, indicate PARP1-induced, AIF-mediated and MIF-activated cell death. With regard to the molecular actors involved, the cellular processes and temporal sequences, the mode of cell death observed in our model is very similar to the cell death mechanism via Parthanatos described in the literature.

Which Moisturizer to Use in Scar Therapy after Burn Injuries? A Subjective and Objective Skin and Scar Evaluation after Topical Treatment with Dexpanthenol, Aloe Vera, and Plant Oil.

Background and Objectives: Good scar management in burn care is essential. Nevertheless, there are no consistent recommendations regarding moisturizers for scar management. Our aim was to investigate and compare the effects of commonly used products on normal skin and burn scars. Materials and Methods: A total of 30 skin-healthy (control group) and 12 patients with burn scars were included in this study. For an intraindividual comparison, each participant received creams consisting of dexpanthenol (P), aloe vera (A), and a natural plant oil (O) with instructions to apply them daily to a previously defined area for at least 28 days. Objective scar evaluation was performed with Visioscan®; Tewameter®; Cutometer®, and the Oxygen To See® device. Subjective evaluation was performed with an "application" questionnaire, the Patient and Observer Scar Assessment Scale (POSAS), and with the "best of three" questionnaire. Results: After (A) a high trend of amelioration of +30%, TEWL was detected on the scar area. Blood flow increased slightly on healthy skin areas after (A) application to +104%. The application of (A) on healthy skin demonstrated a positive effect on the parameters of scaliness (+22%, p < 0.001), softness (+14%, p = 0.046), roughness R1 (+16%, p < 0.001) and R2 (+17%, p = 0.000), volume (+22%, p < 0.001), and surface area (+7%, p < 0.001) within the control group. After (P), a significant improvement of the baseline firmness parameter of +14.7% was detected (p = 0.007). (P) also showed a beneficial effect on the parameters of R1 (+7%, p = 0.003), R2 (+6%, p = 0.001), and volume (+17%, p = 0.001). (O) lead to a statistically significant improvement of volume (+15%, p = 0.009). Overall, most study participants stated (A) to be the "best of three". Conclusions: (A) performed statistically best, and is a well-tolerated moisturizing product. However, further quantitative studies are needed to provide statistically significant clarification for uniform recommendations for scar therapy.

Phototherapy of Pseudomonas aeruginosa-Infected Wounds: Preclinical Evaluation of Antimicrobial Blue Light (450-460 nm) Using In Vitro Assays and a Human Wound Skin Model.

Objective: To determine effective treatment strategies against bacterial infections of burn wounds with Pseudomonas aeruginosa, we tested different treatment regimens with antibacterial blue light (BL). Background: Infections of burn wounds are serious complications and require effective and pathogen-specific therapy. Hereby, infections caused by P. aeruginosa pose a particular challenge in clinical practice due to its resistance to many antibiotics and topical antiseptics. Methods: LED-based light sources (450-460 nm) with different intensities and treatment times were used. Antibacterial effects against P. aeruginosa were determined by colony-forming unit (CFU) assays, human skin wound models, and fluorescence imaging. Results: In suspension assays, BL (2 h, 40 mW/cm2, 288 J/cm2) reduced bacterial number (>5 log10 CFU/mL). Applying 144 J/cm2, using 40 mW/cm2 for 1 h was more effective (>4 log10 CFU) than using 20 mW/cm2 for 2 h (>1.5 log10 CFU). BL with low irradiance (24 h, 3.5 mW/cm2, 300 J/cm2) only revealed bacterial reduction in thin bacteria-containing medium layers. In infected in vitro skin wounds only BL irradiation (2 h, 40 mW/cm2, 288 J/cm2) exerted a significant antimicrobial efficacy (2.94 log10 CFU/mL). Conclusions: BL treatment may be an effective therapy for P. aeruginosa-infected wounds to avoid radical surgical debridement. However, a significant antibacterial efficacy can only be achieved with higher irradiances and longer treatment times (min. 40 mW/cm2; >1 h), which cannot be easily integrated into regular clinical treatment protocols, for example, during a dressing change. Further studies are necessary to establish BL therapy for infected burns among tissue compatibility and interactions with previous therapeutic agents.

Characterization of Blue Light Treatment for Infected Wounds: Antibacterial Efficacy of 420, 455, and 480 nm Light-Emitting Diode Arrays Against Common Skin Pathogens Versus Blue Light-Induced Skin Cell Toxicity.

Objective: To determine effective treatment strategies against bacterial infections of chronic wounds, we tested different blue light (BL)-emitting light-emitting diode arrays (420, 455, and 480 nm) against wound pathogens and investigated in parallel BL-induced toxic effects on human dermal fibroblasts. Background: Wound infection is a major factor for delayed healing. Infections with Pseudomonas aeruginosa and Staphylococcus aureus are clinically relevant caused by their ability of biofilm formation and their quickly growing antibiotics resistance. BL has demonstrated antimicrobial properties against various microbes. Methods: Determination of antibacterial and cell toxic effects by colony-forming units (CFUs)/biofilm/cell viability assays, and live cell imaging. Results: A single BL irradiation (180 J/cm2), of P. aeruginosa at both 420 and 455 nm resulted in a bacterial reduction (>5 log10 CFU), whereas 480 nm revealed subantimicrobial effects (2 log10). All tested wavelengths of BL also revealed bacteria reducing effects on Staphylococcus epidermidis and Escherichia coli (maximum 1-2 log10 CFU) but not on S. aureus. Dealing with biofilms, all wavelengths using 180 J/cm2 were able to reduce significantly the number of P. aeruginosa, E. coli, and S. epidermidis. Here, BL420nm achieved reductions up to 99%, whereas BL455nm and BL480nm were less effective (60-83%). Biofilm-growing S. aureus was more BL sensitive than in the planktonic phase showing a reduction by 63-75%. A significant number of cell toxic events (>40%) could be found after applying doses (>30 J/cm2) of BL420nm. BL455nm showed only slight cell toxicity (180 J/cm2), whereas BL480nm was nontoxic at any dose. Conclusions: BL treatment can be effective against bacterial infections of chronic wounds. Nevertheless, using longer wavelengths >455 nm should be preferred to avoid possible toxic effects on skin and skin cells. To establish BL therapy for infected chronic wounds, further studies concerning biofilm formation and tissue compatibility are necessary.

The effect of the macrophage migration inhibitory factor (MIF) on excisional wound healing in vivo.

Background: The macrophage migration inhibitory factor (MIF) has been determined as a cytokine exerting a multitude of effects in inflammation and angiogenesis. Earlier studies have indicated that MIF may also be involved in wound healing and flap surgery. Methods: We investigated the effect of MIF in an excisional wound model in wildtype, Mif-/- and recombinant MIF treated mice. Wound closure rates as well as the macrophage marker Mac-3, the pro-inflammatory cytokine tumor necrosis factor α (TNFα) and the pro-angiogenic factor von Willebrand factor (vWF) were measured. Finally, we used a flap model in Mif-/- and WT mice with an established perfusion gradient to identify MIF's contribution in flap perfusion. Results: In the excision wound model, we found reduced wound healing after MIF injection, whereas Mif deletion improved wound healing. Furthermore, a reduced expression of Mac-3, TNFα and vWF in Mif-/- mice was seen when compared to WT mice. In the flap model, Mif-/- knockout mice showed mitigated flap perfusion with lower hemoglobin content and oxygen saturation as measured by O2C measurements when compared to WT mice. Conclusions: Our data suggest an inhibiting effect of MIF in wound healing with increased inflammation and perfusion. In flaps, by contrast, MIF may contribute to flap vascularization.

Vitamin D deficiency may stimulate fibroblasts in Dupuytren's disease via mitochondrial increased reactive oxygen species through upregulating transforming growth factor-ß1.

Dupuytren's disease, a benign fibroproliferative disorder of the palmar fascia, represents an ideal model to study tissue fibrosis. Transforming growth factor-ß1 (TGF-ß1) and its downstream Smad signaling system is well established as a keyplayer during fibrogenesis. Vitamin D has been extensively studied as an anti-fibrotic agent in malignant chronic diseases. A number of studies have shown that myofibroblasts are main target cells of 1,25(OH)2D3 inhibitory action. The myofibroblast in the palmar aponeurosis of patients in different stages of Dupuytren's disease was found by electron microscopy to contain a large number of mitochondria. Mitochondria play a critical role in cell metabolism being the major source of reactive oxygen species (ROS) in cells. TGF-ß1 has been shown to increase mitochondrial ROS production in different cell types, which mediate fibrosis related gene expression and myofibroblast differentiation. TGF-ß1 increases mitochondrial ROS production in patients with Dupuytren's contracture potentially in consequence of Vitamin D deficiency, leading to myofibroblast differentiation. Thus, targeting this basic pathomechanism seems suitable to establish new treatment strategies.

Fatal Necrotizing Fasciitis following Episiotomy.

Introduction. Necrotizing fasciitis is an uncommon condition in general practice but one that provokes serious morbidity. It is characterized by widespread fascial necrosis with relative sparing of skin and underlying muscle. Herein, we report a fatal case of necrotizing fasciitis in a young healthy woman after episiotomy. Case Report. A 17-year-old primigravida underwent a vaginal delivery with mediolateral episiotomy. Necrotizing fasciitis was diagnosed on the 5th postpartum day, when the patient was referred to our tertiary care medical center. Surgical debridement was initiated together with antibiotics and followed by hyperbaric oxygen therapy. The patient died due to septic shock after 16 hours from the referral. Conclusion. Delay of diagnosis and consequently the surgical debridement were most likely the reasons for maternal death. In puerperal period, a physician must consider necrotizing fasciitis as a possible diagnosis in any local sings of infection especially when accompanied by fever and/or tenderness. Early diagnosis is the key for low mortality and morbidity.

Adipose-derived stem cells and keratinocytes in a chronic wound cell culture model: the role of hydroxyectoine.

Chronic wounds represent a major socio-economic problem in developed countries today. Wound healing is a complex biological process. It requires a well-orchestrated interaction of mediators, resident cells and infiltrating cells. In this context, mesenchymal stem cells and keratinocytes play a crucial role in tissue regeneration. In chronic wounds these processes are disturbed and cell viability is reduced. Hydroxyectoine (HyEc) is a membrane protecting osmolyte with protein and macromolecule stabilising properties. Adipose-derived stem cells (ASC) and keratinocytes were cultured with chronic wound fluid (CWF) and treated with HyEc. Proliferation was investigated using MTT test and migration was examined with transwell-migration assay and scratch assay. Gene expression changes of basic fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF), matrix metalloproteinases-2 (MMP-2) and MMP-9 were analysed by quantitative real-time polymerase chain reaction (qRT-PCR). CWF significantly inhibited proliferation and migration of keratinocytes. Addition of HyEc did not affect these results. Proliferation capacity of ASC was not influenced by CWF whereas migration was significantly enhanced. HyEc significantly reduced ASC migration. Expression of b-FGF, VEGF, MMP-2 and MMP-9 in ASC, and b-FGF, VEGF and MMP-9 in keratinocytes was strongly induced by chronic wound fluid. HyEc enhanced CWF induced gene expression of VEGF in ASC and MMP-9 in keratinocytes. CWF negatively impaired keratinocyte function, which was not influenced by HyEc. ASC migration was stimulated by CWF, whereas HyEc significantly inhibited migration of ASC. CWF induced gene expression of VEGF in ASC and MMP-9 in keratinocytes was enhanced by HyEc, which might partly be explained by an RNA stabilising effect of HyEc.