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Burn wounds pose a serious threat to patients and often require surgical treatment. Skin grafting aims to achieve wound closure but requires a well-vascularized wound bed. The secretome of peripheral blood mononuclear cells (PBMCs) has been shown to improve wound healing and angiogenesis. We hypothesized that topical application of the PBMC secretome would improve the quality of regenerating skin, increase angiogenesis, and reduce scar formation after burn injury and skin grafting in a porcine model. Full-thickness burn injuries were created on the back of female pigs. Necrotic areas were excised and the wounds were covered with split-thickness mesh skin grafts. Wounds were treated repeatedly with either the secretome of cultured PBMCs (Sec(PBMC)), apoptotic PBMCs (Apo-Sec(PBMC)), or controls. The wounds treated with Apo-Sec(PBMC) had an increased epidermal thickness, higher number of rete ridges, and more advanced epidermal differentiation than controls. The samples treated with Apo-Sec(PBMC) had a two-fold increase in CD31+ cells, indicating more angiogenesis. These data suggest that the repeated application of Apo-Sec(PBMC) significantly improves epidermal thickness, angiogenesis, and skin quality in a porcine model of burn injury and skin grafting.

Introduction: Autograft take and rapid wound closure is essential for the survival of severely burned patients. Loss of skin grafts typically occurs during the first few days after coverage, mainly due to shear forces and inadequate contact with the wound bed. Slow-clotting fibrin sealant, applied with a spray-on device, has been shown to improve healing of skin grafts in large wounds. However, its use in burn wounds has not been studied so far., Study Aim: To evaluate the effectiveness of sprayed fibrin sealant in excised and grafted full-thickness burns., Material and Methods: Ten female Yorkshire pigs (30-45 kg) received a full-thickness contact burn of approximately 15% total body surface area. The burns were excised to the level of the muscular fascia after 24 h and covered with meshed skin autograft (mesh ratio 1:3). Wounds were randomized to either fibrin sealant (n=20) or standard skin staples (n=16) for graft fixation. Fibrin sealant was used as a slow-clotting spray (4 IU thrombin/ml). Outcome measurements included clinical scoring at days 2, 5, 9 and 14 postoperatively, planimetric analysis of wound closure, and histological examination of epidermal and dermal thickness 14 days after autografting., Results: In the fibrin sealant group, graft adherence scores were significantly increased (p<0.02) and graft dislocation scores significantly decreased (p<0.01) at days 2 and 5 postoperatively, when compared to controls. Planimetric analysis of remaining open mesh interstices showed acceleration of wound closure in the fibrin sealant group but did not reach statistical significance (day 14 p=0.04 at significance level p<0.025). Wound contraction, occurrence of hematoma, and dermal as well as epidermal thickness were not different between the groups at 14 days postoperatively., Conclusion: The results indicate that the use of slow-clotting fibrin sealant spray for autograft fixation is advantageous over skin staples. Easy handling and reduced graft dislocation at early time points are key qualities of this method., (Copyright © 2011. Published by Elsevier Ltd.)

Objective: To assess the time-dependent treatment effects of extracorporeal shock wave therapy (ESWT) in a standard rodent ischemic epigastric flap model., Background: ESWT has been shown to accelerate tissue repair in acute and chronic wounds and improve graft survival, but the mechanism remains incompletely understood., Methods: Shock waves at 0.1 mJ/mm and 5 impulses/s (total 300 impulses) were applied to the epigastric flap ischemic region at various times pre-, immediately and 24 hours postischemic insult. Flap survival; vascular perfusion; vessel number; von Willebrand factor and smooth muscle actin protein expression as well as in vivo vascular endothelial growth factor receptor 2 expression were evaluated at 1, 3, and 7 days postoperatively in ESWT-treated and untreated controls., Results: Flap perfusion, microvessel number, and survival (through reduced flap contraction and necrosis) were significantly enhanced in the treated groups compared with controls, irrespective of timing of shock wave treatment (preischemia vs. postischemia). Vascular endothelial growth factor receptor 2 expression was dynamically upregulated in response to ESWT., Conclusion: Shock wave preconditioning and treatment postischemic insult improves skin flap survival through neovascularization and early upregulation of angiogenesis-related growth factors.

Liposomal gene transfer effectively enhances dermal and epidermal regeneration in burned rodents. To advance this treatment to clinical studies, we investigated the efficacy of liposomal gene transfer in a clinically relevant porcine wound model. Mimicking the clinical scenario, six female Yorkshire pigs (40-50 kg) received up to 12 burns of 50 cm(2) area that were fully excised and covered with skin autograft meshed at 4:1 ratio 24 h post-burn. Animals received control injections (empty liposomes), liposomes (DMRIE-C) containing 1 mg LacZ-cDNA, or liposomes (DMRIE-C) with 1 mg of platelet-derived growth factor (PDGF)-cDNA, or the naked PDGF gene. Serial biopsies were taken from different wound sites at multiple time points up to 12 days post-wounding. Transfection efficacy and transfection rate of LacZ and localization of beta-gal were determined by immunohistochemical and immunofluorescent techniques. RT-PCR and multiplex protein analysis (ELISA) were used to measure levels of growth factor mRNA transcribed and growth factor protein translated. Wound re-epithelialization and graft adhesion was evaluated using planimetric analysis and clinical scores. We found that peak transfection of liposomal beta-galactosidase occurred on day 2, with a fluorescence increase of 154% to baseline (P<0.001). Transfection intensity dropped to 115% above baseline on day 4 (P<0.001) and 109% on day 7. Immunohistochemistry showed a maximum transfection rate of 34% of cells in wound tissue. Gene transfer of liposomal PDGF-cDNA resulted in increased PDGF-mRNA and protein expression on days 2 and 4, and accelerated wound re-epithlialization as well as graft adhesion on day 9 (P<0.05). In this study, we showed that liposomal cDNA gene transfer is possible in a porcine wound model, and by using PDGF-cDNA we further showed that dermal and epidermal regeneration can be improved. These data indicate that liposomal gene transfer can be a new therapeutic approach to improve wound healing in humans.

Acute burn wounds often require early excision and adequate coverage to prevent further hypothermia, protein and fluid losses, and the risk of infection. Meshed autologous skin grafts are generally regarded as the standard treatment for extensive full-thickness burns. Graft take and rate of wound healing, however, depend on several endogenous factors. This paper describes a standardized reproducible porcine model of burn and skin grafting which can be used to study the effects of topical treatments on graft take and re-epithelialization. Procedures provide a protocol for successful porcine burn wound experiments with special focus on pre-operative care, anesthesia, burn allocation, excision and grafting, postoperative treatment, dressing application, and specimen collection. Selected outcome measurements include percent area of wound closure by planimetry, wound assessment using a clinical assessment scale, and histological scoring. The use of this standardized model provides burn researchers with a valuable tool for the comparison of different topical drug treatments and dressing materials in a setting that closely mimics clinical reality.

The aim of the present study was to experimentally evaluate whether topical fibrin-mediated administration of a vascular endothelial growth factor (VEGF)-A plasmid to the wound bed can protect skin flaps from necrosis. A plasmid expression vector containing the VEGF-A cDNA was constructed. The plasmid was then administered to the wound bed of rat abdominal skin flaps in a fibrin sealant. The percentage of viable, ischemic and necrotic tissue was assessed postoperatively as a baseline and after 3 and 7 days using digital surface area morphometry. Laser Doppler imaging of the flaps and VEGF-A Western blot analysis of flap tissue were performed to assess angiogenesis and VEGF-A tissue levels. Flaps treated with VEGF plasmids in the presence of uptake enhancing Lipofectamine transfection reagent increased flap survival 7 days postoperatively significantly associated with markedly elevated tissue perfusion and enhanced tissue VEGF-A protein expression. Our results indicate that topical fibrin-mediated administration of a VEGF-A plasmid may serve as an alternative to previous strategies in treating ischemic skin flaps. The suggested therapeutic approach is easily applicable and inexpensive in preparation. Thus, this protocol may also enhance wound healing in posttrauma skin lacerations or in skin grafts.

Human fibrin sealant (FS) has been proven effective for skin grafting after severe burn, however no systematic evaluation of application conditions has been performed so far. In order to find the optimal FS amount for fixation of skin grafts to deep defects, we created four full thickness wounds (8 cmx4 cm) on the dorsum of six male pigs. Wounds were covered with unmeshed split thickness skin grafts and fixed either with a thin layer (0.05 ml/cm2) or a thick layer (0.15 ml/cm2) of fibrin sealant (FS) without additional sutures. Sutures served as controls. FS was used as a slow clotting spray (4-5 IUthrombin/ml). Outcome measurements revealed that hematoma formation (day of surgery) was more extensive and occurred more frequently in the suture group as compared to FS 0.05 ml/cm2 (p<0.05). Areas of graft dislocation tended to be larger in the suture group versus the FS 0.05 ml/cm2. The FS 0.05 ml/cm2 graft take on day 5 appeared to be enhanced in comparison to the suture group. Excellent outcome was notable on the final observation day (day 21) in the FS 0.05 ml/cm2 group with a take of 99.7% (IQR 96.1-100%). Corresponding values in the FS 0.15 ml/cm2 group were 96.9% (IQR 92.2-99%) and 95.9% (IQR 93.2-98%) in the suture group. The results indicate, that the usage of a sprayed thin FS layer (0.05 ml/cm2) in a slow clotting rate (4-5 IUthrombin/ml) is an appropriate fixation method in split thickness skin transplantation.