Intraluminal release of citrullinated histone 3 from various cellular origins coincides with microvascular thrombosis in burn wounds.

Loss of perfusion in the burn wound might cause wound deepening and impaired healing. We previously showed persistent microvascular thrombosis coinciding with intraluminal neutrophils extracellular traps in human burned skin. This study investigates the presence of intraluminal citrullinated histone 3 (H3cit) from different cellular origins (neutrophils, monocytes, and lymphocytes) in relation to microvascular thrombosis of burn wounds. Eschar was obtained from burn patients (n = 18) 6-40 days postburn with a mean total burned body surface area of 23%. Microvascular presence of tissue factor (TF), factor XII (FXII) and thrombi was assessed by immunohistochemistry. Intramicrovascular cell death was analyzed via immunofluorescent microscopy, combining antibodies for neutrophils (MPO), monocytes (CD14), and lymphocytes (CD45) with endothelial cell markers CD31 and H3cit. Significantly increased microvascular expression of TF, FXII, and thrombi (CD31+) was found in all eschar samples compared with control uninjured skin. Release of H3cit from different cellular origins was observed in the lumen of the dermal microvasculature in the eschar tissue 7-40 days postburn, with release from neutrophilic origin being 2.7 times more abundant. Intraluminal presence of extracellular H3cit colocalizing with either MPO, CD14, or CD45 is correlated to increased microvascular thrombosis in eschar of burn patients.

Efficacy of Alkaline Phosphatase in Critically Ill Patients with COVID-19: A Multicentre Investigator-Initiated Double-Blind Randomised Placebo-Controlled Trial.

BACKGROUND: Efforts to identify therapies to treat hospitalised patients with COVID-19 are being continued. Alkaline phosphatase (AP) dephosphorylates pro-inflammatory adenosine triphosphate (ATP) into anti-inflammatory adenosine. METHODS: In a randomised controlled trial, we investigated the safety and efficacy of AP in patients with SARS-CoV-2 infection admitted to the ICU. AP or a placebo was administered for four days following admission to the ICU. The primary outcome was the duration of mechanical ventilation. Mortality in 28 days, acute kidney injury, need for reintubation, safety, and inflammatory markers relevant to the described high cytokine release associated with SARS-CoV-2 infection were the secondary outcomes. RESULTS: Between December 2020 and March 2022, 97 patients (of the intended 132) were included, of which 51 were randomised to AP. The trial was terminated prematurely based on meeting the threshold for futility. Compared to the placebo, AP did not affect the duration of mechanical ventilation (9.0 days vs. 9.3 days, p = 1.0). No safety issues were observed. After 28 days, mortality was 9 (18%) in the AP group versus 6 (13%) in the placebo group (p = 0.531). Additionally, no statistically significant differences between the AP and the placebo were observed for the other secondary outcomes. CONCLUSIONS: Alkaline phosphatase (AP) therapy in COVID-19 ICU patients showed no significant benefits in this trial.

Comparing the antibacterial and healing properties of medical-grade honey and silver-based wound care products in burns.

Burns are a major global healthcare concern, often complicated by the presence of bacteria such as Pseudomonas aeruginosa in the wounds. Silver-based dressings are commonly used in the treatment of burns but can cause skin irritation and delay healing time. Medical-grade honey (MGH) provides an interesting alternative. This study investigated the antimicrobial effects and possible cytotoxicity of L-Mesitran Soft (MGH-gel) and its individual components, Medihoney (Manuka), Flammazine (silver sulphadiazine), and silver nitrate (AgNO3) in an ex vivo human burn wound model. Bacterial survival and wound healing parameters, including re-epithelialization and keratinocyte proliferation were assessed. L-Mesitran, Flammazine, and AgNO3 reduced P. aeruginosa numbers below detection levels. L-Mesitran Soft exhibited a significantly stronger antimicrobial effect compared to Medihoney. The individual components of L-Mesitran contributed significantly to its antibacterial efficacy, thus suggesting synergistic activities. Moreover, L-Mesitran, Flammazine, and AgNO3 slightly inhibited re-epithelialization while Medihoney treatment resulted in a complete lack of re-epithelialization and keratinocyte proliferation. Furthermore, clinical cases illustrated the effectiveness of MGH therapy in infected burns. Overall, L-Mesitran Soft had similar effects as silver-based products on bacterial load and epidermal regeneration, but outperformed Medihoney. Therefore, supplemented MGH could be used as an effective alternative to silver-based dressings for P. aeruginosa-infected burns.

Kinetics of Inflammatory Mediators in the Immune Response to Burn Injury: Systematic Review and Meta-Analysis of Animal Studies.

Burns are often accompanied by a dysfunctional immune response, which can lead to systemic inflammation, shock, and excessive scarring. The objective of this study was to provide insight into inflammatory pathways associated with burn-related complications. Because detailed information on the various inflammatory mediators is scattered over individual studies, we systematically reviewed animal experimental data for all reported inflammatory mediators. Meta-analyses of 352 studies revealed a strong increase in cytokines, chemokines, and growth factors, particularly 19 mediators in blood and 12 in burn tissue. Temporal kinetics showed long-lasting surges of proinflammatory cytokines in blood and burn tissue. Significant time-dependent effects were seen for IL-1ß, IL-6, TGF-ß1, and CCL2. The response of anti-inflammatory mediators was limited. Burn technique had a profound impact on systemic response levels. Large burn size and scalds further increased systemic, but not local inflammation. Animal characteristics greatly affected inflammation, for example, IL-1ß, IL-6, and TNF-α levels were highest in young, male rats. Time-dependent effects and dissimilarities in response demonstrate the importance of appropriate study design. Collectively, this review presents a general overview of the burn-induced immune response exposing inflammatory pathways that could be targeted through immunotherapy for burn patients and provides guidance for experimental set-ups to advance burn research.

Monocytes and T cells incorporated in full skin equivalents to study innate or adaptive immune reactions after burn injury.

Introduction: Thermal injury often leads to prolonged and excessive inflammation, which hinders the recovery of patients. There is a notable absence of suitable animal-free models for investigating the inflammatory processes following burn injuries, thereby impeding the development of more effective therapies to improve burn wound healing in patients. Methods: In this study, we established a human full skin equivalent (FSE) burn wound model and incorporated human peripheral blood-derived monocytes and T cells. Results: Upon infiltration into the FSEs, the monocytes differentiated into macrophages within a span of 7 days. Burn-injured FSEs exhibited macrophages with increased expression of HLA-DR+ and elevated production of IL-8 (CXCL8), in comparison to uninjured FSEs. Among the T cells that actively migrated into the FSEs, the majority were CD4+ and CD25+. These T cells demonstrated augmented expression of markers associated with regulatory T cell, Th1, or Th17 activity, which coincided with significant heightened cytokine production, including IFN-γ, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IP-10 (CXCL10), and TGF-ß1. Burn injury did not impact the studied effector T cell subsets or cytokine levels. Discussion: Collectively, this study represents a significant advancement in the development of an immunocompetent human skin model, specifically tailored for investigating burn-induced innate or adaptive immune reactions at the site of burn injury.

Short- and Long-term Outcomes of an Acellular Dermal Substitute versus Standard of Care in Burns and Reconstructions: A Phase I/II Intrapatient Randomized Controlled Trial.

OBJECTIVE: Dermal substitutes promote dermal regeneration and improve scar quality, but knowledge gaps remain regarding their efficacy and indications for use. The authors investigated the safety and short- and long-term efficacy of an acellular dermal substitute in patients with full-thickness wounds. METHODS: This intrapatient randomized controlled, open-label, phase I (safety) and phase II (efficacy) study compared treatment with Novomaix (Matricel GmbH), a dermal collagen/elastin-based scaffold, with split-thickness skin graft (STSG) only. The primary safety outcome was graft take at 5 to 7 days postsurgery. Postsurgical scar quality was assessed by measuring elasticity, color, and scores on the Patient and Observer Scar Assessment Scale at 3 months, 12 months, and 6 years. RESULTS: Twenty-five patients were included, of which 24 received treatment allocation. Graft take and wound healing were statistically significantly lower/delayed in the dermal matrix group compared with STSG alone (P < .004). Serious adverse events were delayed epithelialization in four dermal matrix and three STSG study areas. At 12 months postsurgery, skin extension (P = .034) and elasticity (P = .036) were better for the dermal matrix group compared with the group receiving STSG alone. Other scar quality parameters at 12 months and 6 years did not differ between treatment arms. CONCLUSIONS: The dermal substitute was a safe treatment modality for full-thickness wounds. Compared with STSG alone, time to wound healing was slightly increased. Nevertheless, scar quality at 12 months seemed somewhat improved in the wounds treated with the dermal substitute, indicative of enhanced scar maturation. In the long term, final scar quality was similar for both treatment modalities.

Full Skin Equivalent Models for Simulation of Burn Wound Healing, Exploring Skin Regeneration and Cytokine Response.

Healing of burn injury is a complex process that often leads to the development of functional and aesthetic complications. To study skin regeneration in more detail, organotypic skin models, such as full skin equivalents (FSEs) generated from dermal matrices, can be used. Here, FSEs were generated using de-epidermalized dermis (DED) and collagen matrices MatriDerm® and Mucomaix®. Our aim was to validate the MatriDerm- and Mucomaix-based FSEs for the use as in vitro models of wound healing. Therefore, we first characterized the FSEs in terms of skin development and cell proliferation. Proper dermal and epidermal morphogenesis was established in all FSEs and was comparable to ex vivo human skin models. Extension of culture time improved the organization of the epidermal layers and the basement membrane in MatriDerm-based FSE but resulted in rapid degradation of the Mucomaix-based FSE. After applying a standardized burn injury to the models, re-epithelization occurred in the DED- and MatriDerm-based FSEs at 2 weeks after injury, similar to ex vivo human skin. High levels of pro-inflammatory cytokines were present in the culture media of all models, but no significant differences were observed between models. We anticipate that these animal-free in vitro models can facilitate research on skin regeneration and can be used to test therapeutic interventions in a preclinical setting to improve wound healing.

Burn-injured skin is marked by a prolonged local acute inflammatory response of innate immune cells and pro-inflammatory cytokines.

The systemic and local immune response in burn patients is often extreme and derailed. As excessive inflammation can damage healthy tissues and slow down the healing process, modulation of inflammatory responses could limit complications and improve recovery. Due to its complexity, more detailed information on the immune effects of thermal injury is needed to improve patient outcomes. We therefore characterized and quantified subsets of immune cells and mediators present in human burn wound tissue (eschar), sampled at various time points. This study shows that after burn injury, the number of immune cells were persistently increased, unlike the normal wound healing process. There was an immediate, strong increase in neutrophils and a moderate increase in monocytes/macrophages and lymphocytes, especially in the second and third week post burn. The percentage of classical (CD14highCD16-) monocytes/macrophages demonstrated a steady decrease over time, whereas the proportion of intermediate (CD14highCD16+) monocytes/macrophages slowly increased. The absolute numbers of T cells, NK cells and B cells increased up to week 3, while the fraction of γδ T cells was increased only in week 1. Secretome profiling revealed high levels of chemokines and an overall pro-inflammatory cytokine milieu in burn tissue. The local burn immune response shows similarities to the systemic immune reaction, but differs in neutrophil maturity and lymphocyte composition. Altogether, the neutrophil surges, high levels of pro-inflammatory cytokines and limited immunosuppression might be key factors that prolong the inflammation phase and delay the wound healing process in burns.

Burn-Induced Local and Systemic Immune Response: Systematic Review and Meta-Analysis of Animal Studies.

Because burn injuries are often followed by a derailed immune response and excessive inflammation, a thorough understanding of the occurring reactions is key to preventing secondary complications. This systematic review, which includes 247 animal studies, shows the postburn response of 14 different immune cell types involved in immediate and long-term effects in both wound tissue and circulation. Peripheral blood neutrophil and monocyte numbers increased directly after burns, whereas thrombocyte numbers increased near the end of the first week. However, lymphocyte numbers were decreased for at least 2 weeks. In burn wound tissue, neutrophil and macrophage numbers accumulated during the first 3 weeks. Burns also altered cellular functions because we found an increased migratory potential of leukocytes, impaired antibacterial activity of neutrophils, and enhanced inflammatory mediator production by macrophages. Neutrophil surges were positively associated with burn size and were highest in rats. Altogether, this comprehensive overview of the temporal immune cell dynamics shows that unlike normal wound healing, burn injury induces a long-lasting inflammatory response. It provides a fundamental research basis to improve experimental set-ups, burn care, and outcomes.

Antibacterial and safety tests of a flexible cold atmospheric plasma device for the stimulation of wound healing.

Cold atmospheric plasma (CAP) devices generate an ionized gas with highly reactive species and electric fields at ambient air pressure and temperature. A flexible dielectric barrier discharge (DBD) was developed as an alternative antimicrobial treatment for chronic wounds. Treatment of Staphylococcus aureus in collagen-elastin matrices with CAP for 2 min resulted in a 4 log reduction. CAP treatment was less effective on S. aureus on dermal samples. CAP did not affect cellular activity or DNA integrity of human dermal samples when used for up to 2 min. Repeated daily CAP treatments for 2 min lowered cellular activity of dermal samples to 80% after 2 to 4 days, but this was not significant. Repeated treatment of ex vivo human burn wound models with CAP for 2 min did not affect re-epithelialization. Intact skin of 25 healthy volunteers was treated with CAP for 3× 20" to determine safety. Although participants reported moderate pain scores (numerical rating scale 3.3), all volunteers considered the procedure to be acceptable. Severe adverse events did not occur. CAP treatment resulted in a temporarily increased local skin temperature (≈3.4°C) and increased erythema. Lowering the plasma power resulted in a significantly lower erythema increase. Good log reduction (2.9) of bacterial load was reached in 14/15 volunteers artificially contaminated with Pseudomonas aeruginosa. This study demonstrated the in vitro and in vivo safety and efficacy in bacterial reduction of a flexible cold plasma device. Trial registration number NCT03007264, January 2, 2017 KEY POINTS: • CAP strongly reduced bacterial numbers both in vitro and in vivo. • Re-epithelialization of burn wound models was not affected by repeated CAP. • CAP treatment of intact skin was well tolerated in volunteers.

C1 Inhibitor Administration Reduces Local Inflammation and Capillary Leakage, Without Affecting Long-term Wound Healing Parameters, in a Pig Burn Wound Model.

BACKGROUND: Burns induce a boost in local and systemic complement levels as well as immune cell infiltration in the burn wound, which may negatively affect wound healing. OBJECTIVE: In this study, the effects of long-term treatment with complement inhibitor C1 esterase inhibitor (C1inh) on post-burn inflammation and wound healing parameters were analyzed in time up to 60 days post-burn. METHODS: Burned pigs were treated either with or without C1inh up to 15 days post-burn. Burn wound biopsies and blood were collected at different time points up to 60 days post-burn. Thereafter, complement in blood as well as complement and immune cells in the wound, capillary leakage, necrosis, reepithelialization and wound contraction were quantified. RESULTS: No significant differences in complement C3 blood levels were observed at any time point between C1inh-treated and control pigs. In the wound, complement C4 levels were significantly lower in the C1inh group than in controls at day 3-6 and 21-30 post-burn. Similarly, C3 levels, neutrophil and macrophage infiltration in the wound were, although not statistically significant, reduced in C1inh-treated pigs at day 9-14 post-burn. No differences in lymphocyte infiltration in the wound were found between C1inh and control pigs. C1inh-treated pigs also showed reduced capillary leakage. Despite these effects, no significant differences in the long-term wound healing parameters necrosis, reepithelialization and wound contraction were observed between C1inh and control pigs. CONCLUSION: In pigs, 15 days of C1inh treatment after burn, leads to a reduction in local inflammation and capillary leakage in the burn wound without affecting long-term wound healing parameters.

Safety and bactericidal efficacy of cold atmospheric plasma generated by a flexible surface Dielectric Barrier Discharge device against Pseudomonas aeruginosa in vitro and in vivo.

BACKGROUND: Cold atmospheric plasma (CAP), which is ionized gas produced at atmospheric pressure, could be a novel and potent antimicrobial therapy for the treatment of infected wounds. Previously we have shown that CAP generated with a flexible surface Dielectric Barrier Discharge (sDBD) is highly effective against bacteria in vitro and in ex vivo burn wound models. In the current paper, we determined the in vitro and in vivo safety and efficacy of CAP generated by this sDBD device. METHODS: The effect of CAP on DNA mutations of V79 fibroblasts was measured using a hypoxanthine-guanine-phosphoribosyltransferase (HPRT) assay. Furthermore, effects on cell proliferation, apoptosis and DNA damage in ex vivo burn wound models (BWMs) were assessed using immunohistochemistry. Next, 105 colony forming units (CFU) P. aeruginosa strain PAO1 were exposed to CAP in a 3D collagen-elastin matrix environment to determine the number of surviving bacteria in vitro. Finally, rat excision wounds were inoculated with 107 CFU PAO1 for 24 h. The wounds received a single CAP treatment, repeated treatments on 4 consecutive days with CAP, 100 µL of 1% (wt/wt) silver sulfadiazine or no treatment. Wound swabs and punch biopsies were taken to determine the number of surviving bacteria. RESULTS: Exposure of V79 fibroblasts to CAP did not increase the numbers of mutated colonies. Additionally, the number of proliferative, apoptotic and DNA damaged cells in the BWMs was comparable to that of the unexposed control. Exposure of PAO1 to CAP for 2 min resulted in the complete elimination of bacteria in vitro. Contrarily, CAP treatment for 6 min of rat wounds colonized with PAO1 did not effectively reduce the in vivo bacterial count. CONCLUSIONS: CAP treatment was safe but showed limited efficacy against PAO1 in our rat wound infection model.

Persistent Systemic Inflammation in Patients With Severe Burn Injury Is Accompanied by Influx of Immature Neutrophils and Shifts in T Cell Subsets and Cytokine Profiles.

Severe burn injury causes local and systemic immune responses that can persist up to months, and can lead to systemic inflammatory response syndrome, organ damage and long-term sequalae such as hypertrophic scarring. To prevent these pathological conditions, a better understanding of the underlying mechanisms is essential. In this longitudinal study, we analyzed the temporal peripheral blood immune profile of 20 burn wound patients admitted to the intensive care by flow cytometry and secretome profiling, and compared this to data from 20 healthy subjects. The patient cohort showed signs of systemic inflammation and persistently high levels of pro-inflammatory soluble mediators, such as IL-6, IL-8, MCP-1, MIP-1ß, and MIP-3α, were measured. Using both unsupervised and supervised flow cytometry techniques, we observed a continuous release of neutrophils and monocytes into the blood for at least 39 days. Increased numbers of immature neutrophils were present in peripheral blood in the first three weeks after injury (0.1-2.8 × 106/ml after burn vs. 5 × 103/ml in healthy controls). Total lymphocyte numbers did not increase, but numbers of effector T cells as well as regulatory T cells were increased from the second week onward. Within the CD4+ T cell population, elevated numbers of CCR4+CCR6- and CCR4+CCR6+ cells were found. Altogether, these data reveal that severe burn injury induced a persistent innate inflammatory response, including a release of immature neutrophils, and shifts in the T cell composition toward an overall more pro-inflammatory phenotype, thereby continuing systemic inflammation and increasing the risk of secondary complications.

Potential factors contributing to the poor antimicrobial efficacy of SAAP-148 in a rat wound infection model.

BACKGROUND: We investigated the efficacy of a synthetic antimicrobial peptide SAAP-148, which was shown to be effective against Methicillin-resistant Staphylococcus aureus (MRSA) on tape-stripped mice skin. Unexpectedly, SAAP-148 was not effective against MRSA in our pilot study using rats with excision wounds. Therefore, we investigated factors that might have contributed to the poor efficacy of SAAP-148. Subsequently, we optimised the protocol and assessed the efficacy of SAAP-148 in an adapted rat study. METHODS: We incubated 100 µL of SAAP-148 with 1 cm2 of a wound dressing for 1 h and determined the unabsorbed volume of peptide solution. Furthermore, 105 colony forming units (CFU)/mL MRSA were exposed to increasing dosages of SAAP-148 in 50% (v/v) human plasma, eschar- or skin extract or PBS. After 30 min incubation, the number of viable bacteria was determined. Next, ex vivo skin models were inoculated with MRSA for 1 h and exposed to SAAP-148. Finally, excision wounds on the back of rats were inoculated with 107 CFU MRSA overnight and treated with SAAP-148 for 4 h or 24 h. Subsequently, the number of viable bacteria was determined. RESULTS: Contrary to Cuticell, Parafilm and Tegaderm film, < 20% of peptide solution was recovered after incubation with gauze, Mepilex border and Opsite Post-op. Furthermore, in plasma, eschar- or skin extract > 20-fold higher dosages of SAAP-148 were required to achieve a 2-log reduction (LR) of MRSA versus SAAP-148 in PBS. Exposure of ex vivo models to SAAP-148 for 24 h resulted in a 4-fold lower LR than a 1 h or 4 h exposure period. Additionally, SAAP-148 caused a 1.3-fold lower mean LR at a load of 107 CFU compared to 105 CFU MRSA. Moreover, exposure of ex vivo excision wound models to SAAP-148 resulted in a 1.5-fold lower LR than for tape-stripped skin. Finally, SAAP-148 failed to reduce the bacterial counts in an adapted rat study. CONCLUSIONS: Several factors, such as absorption of SAAP-148 by wound dressings, components within wound exudates, re-colonisation during the exposure of SAAP-148, and a high bacterial load may contribute to the poor antimicrobial effect of SAAP-148 against MRSA in the rat model.

Allogeneic platelet-rich plasma (PRP) is superior to platelets or plasma alone in stimulating fibroblast proliferation and migration, angiogenesis, and chemotaxis as relevant processes for wound healing.

BACKGROUND: Platelet-rich plasma (PRP) is frequently used in the treatment of acute and chronic wounds. One of the major problems concerning the use of PRP is the absence of a well-characterized and standardized product, which leads to a high variety in study outcomes. Therefore, more studies on the composition and standardization of PRP in wound healing are needed. STUDY DESIGN AND METHODS: Platelet concentrates derived from healthy blood donors were made in plasma (PC-plasma) or platelet additive solution (PC-PAS). The effects of PC-plasma, PC-PAS, and plasma were then tested on proliferation, differentiation, and migration of fibroblasts, as well as sprouting of endothelial cells in fibrin gels and chemotaxis of white blood cells (WBCs). RESULTS: PC-plasma stimulates the migration and proliferation of human dermal fibroblasts more than plasma or platelets alone. Furthermore, platelet factors decrease the expression of α-smooth muscle actin in dermal fibroblast cultures. PC-plasma also stimulates sprouting of endothelial cells. Finally, PC-plasma also acts as a strong chemoattractant for WBCs. CONCLUSIONS: Allogeneic PC-plasma has beneficial effects on various aspects of wound healing in vitro and is superior to plasma or platelets alone. PC-plasma is an attractive candidate for further in vivo evaluation.

Growth Factor Quantification of Platelet-Rich Plasma in Burn Patients Compared to Matched Healthy Volunteers.

Platelet rich plasma (PRP) is blood plasma with a platelet concentration above baseline. When activated, PRP releases growth factors involved in all stages of wound healing, potentially boosting the healing process. To expand our knowledge of the effectiveness of PRP, it is crucial to know the content and composition of PRP products. In this study, growth factor quantification measurements of PRP from burn patients and gender- and age-matched controls were performed. The PRP of burn patients showed levels of growth factors comparable to those of the PRP of healthy volunteers. Considerable intra-individual variation in growth factor content was found. However, a correlation was found between the platelet count of the PRP and most of the growth factors measured.

Early intervention by Captopril does not improve wound healing of partial thickness burn wounds in a rat model.

The Renin Angiotensin System is involved in fibrotic pathologies in various organs such as heart, kidney and liver. Inhibition of this system by angiotensin converting enzyme antagonists, such as Captopril, has been shown beneficial effects on these pathologies. Captopril reduced the inflammatory reaction but also directly influenced the fibrotic process. Prolonged and excessive inflammatory response is a major cause of hypertrophic scar formation in burns. We therefore evaluated the effect of Captopril on the healing of partial thickness burn wounds in a rat model. Partial thickness contact burns were inflicted on the dorsum of the rats. The rats received either systemic or local treatment with Captopril. The inflammatory reaction and wound healing (scar) parameters were investigated and compared to control animals. In this study we could not detect positive effects of either administration route with Captopril on the inflammatory reaction, nor on wound healing parameters. The local treatment showed reduced wound closure in comparison to the systemic treatment and the control group. Early Captopril treatment of burn wounds did not show the beneficial effects that were reported for fibrotic disorders in other tissues. To influence the fibrotic response Captopril treatment at a later time point, e.g. during the remodeling phase, might still have beneficial effects.

In vivo polarization-sensitive optical coherence tomography of human burn scars: birefringence quantification and correspondence with histologically determined collagen density.

Obtaining adequate information on scar characteristics is important for monitoring their evolution and the effectiveness of clinical treatment. The aberrant type of collagen in scars may give rise to specific birefringent properties, which can be determined using polarization-sensitive optical coherence tomography (PS-OCT). The aim of this pilot study was to evaluate a method to quantify the birefringence of the scanned volume and correlate it with the collagen density as measured from histological slides. Five human burn scars were measured in vivo using a handheld probe and custom-made PS-OCT system. The local retardation caused by the tissue birefringence was extracted using the Jones formalism. To compare the samples, histograms of birefringence values of each volume were produced. After imaging, punch biopsies were harvested from the scar area of interest and sent in for histological evaluation using Herovici polychrome staining. Two-dimensional en face maps showed higher birefringence in scars compared to healthy skin. The Pearson's correlation coefficient for the collagen density as measured by histology versus the measured birefringence was calculated at r=0.80 (p=0.105). In conclusion, the custom-made PS-OCT system was capable of in vivo imaging and quantifying the birefringence of human burn scars, and a nonsignificant correlation between PS-OCT birefringence and histological collagen density was found.

Differential effects of Losartan and Atorvastatin in partial and full thickness burn wounds.

Healing of burn wounds is often associated with scar formation due to excessive inflammation and delayed wound closure. To date, no effective treatment is available to prevent the fibrotic process. The Renin Angiotensin System (RAS) was shown to be involved in fibrosis in various organs. Statins (e.g. Atorvastatin), Angiotensin receptor antagonists (e.g. Losartan) and the combination of these drugs are able to reduce the local RAS activation, and reduced fibrosis in other organs. We investigated whether inhibition of the RAS could improve healing of burn wounds by treatment with Atorvastatin, Losartan or the combination of both drugs. Therefore, full and partial thickness burn wounds were inflicted on both flanks of Yorkshire pigs. Oral administration of Atorvastatin, Losartan or the combination was started at post-burn day 1 and continued for 28 days. Full thickness wounds were excised and transplanted with an autologous meshed split-thickness skin graft at post-burn day 14. Partial thickness wounds received conservative treatment. Atorvastatin treatment resulted in enhanced graft take and wound closure of the full thickness wounds, faster resolution of neutrophils compared to all treatments and reduced alpha-smooth muscle actin positive cells compared to control treatment. Treatment with Losartan and to a lesser extent the combination therapy resulted in diminished graft take, increased wound contraction and poorer scar outcome. In contrast, Losartan treatment in partial thickness wounds decreased the alpha-smooth muscle actin+ fibroblasts and contraction. In conclusion, we showed differential effects of Losartan and Atorvastatin in full and partial thickness wounds. The extensive graft loss seen in Losartan treated wounds is most likely responsible for the poor clinical outcome of these full thickness burn wounds. Therefore, Losartan treatment should not be started before transplantation in order to prevent graft loss. Atorvastatin seems to accelerate the healing process in full thickness wounds possibly by dampening the pro-inflammatory response.

The Local and Systemic Inflammatory Response in a Pig Burn Wound Model With a Pivotal Role for Complement.

In patients with burns, a massive inflammatory response is induced which negatively affects the healing process of the burn wound and additionally exerts systemic effects. An important factor herein is the complement system. Here we analyzed the effects of burns on complement and inflammatory cells both locally and systemically after burn in time in a pig burn wound model. In burned pigs, burn wound biopsies and blood were collected up to 60 days after burn. Complement in blood as well as complement and inflammatory cells in the burn wound and several organs were determined. In the blood, C3 was significantly increased after 9 to 60 days, whereas C4 after 21 to 30 days after burn. In the burn wound, C3 levels were significantly increased after 9 days and C4 after 3 days, whereafter both declined after 21 and 9 days, respectively. Neutrophils, macrophages, and lymphocytes were significantly increased in the burn wound after 3 days, all declined after 21 days after burn. In the heart, at 60 days after burn, an increase of neutrophils and macrophages was observed, mainly in the right atrium. In contrast to the heart, the inflammatory cell infiltrates in the lungs, liver, and kidney of burned pigs were lower than in control pigs. In pigs, following burn there is a prolonged increase in complement levels both in the burn wound and the blood and increased inflammatory cell infiltrate in the burn wound and the heart. However, complement levels in the burn wound and in the blood seem not to be correlated in time.