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Bovine lactoferricin (LFcinB) has antimicrobial and immunomodulatory properties; however, the effects on diabetic wound healing remain poorly understood. The wound healing potential of LFcinB was investigated with in vitro, ex vivo, and in vivo models. Cell migration and proliferation were tested on keratinocytes and on porcine ears. A type 1 diabetic mouse model was also used to evaluate wound healing kinetics, bacterial diversity patterns, and the effect of LFcinB on oxidative stress, macrophage phenotype, angiogenesis, and collagen deposition. LFcinB increased keratinocyte migration in vitro (p < 0.05) and ex vivo (p < 0.001) and improved wound healing in diabetic mice (p < 0.05), though not in normoglycemic control mice. In diabetic mouse wounds, LFcinB treatment led to the eradication of Bacillus pumilus, a decrease in Staphylococcus aureus, and an increase in the Staphylococcus xylosus prevalence. LFcinB increased angiogenesis in diabetic mice (p < 0.01), but this was decreased in control mice (p < 0.05). LFcinB improved collagen deposition in both diabetic and control mice (p < 0.05). Both oxidative stress and the M1-to-M2 macrophage ratios were decreased in LFcinB-treated wounds of diabetic animals (p < 0.001 and p < 0.05, respectively) compared with saline, suggesting a downregulation of inflammation in diabetic wounds. In conclusion, LFcinB treatment demonstrated noticeable positive effects on diabetic wound healing., Graphical Abstract, Topical delivery of bovine lactoferricin (LFcinB) has antimicrobial and immunomodulatory actions and improves wound closure and changes the bacterial diversity of diabetic wounds. Hallmarks of diabetic wound healing impairments like inflammation, oxidative stress, disrupted angiogenesis, and collagen deposition improved by LFcinB indicate its therapeutic potential. more...

Antimicrobial peptides can have a dual role with both antimicrobial activity against a broad range of bacteria and immunomodulatory effect, making them attractive as therapeutic treatment of difficult wounds. Nisin A is widely known for its antimicrobial activity, and a preliminary study demonstrated that it increased wound closure, but the mechanism behind its effect is unknown. The aim of this study is to elucidate the wound healing potential of Nisin A and the mechanism behind. First, an epithelial and endothelial cell line, human keratinocyte (HaCaT) and human umbilical vein endothelial cell, were used to demonstrate migration and proliferation effects in vitro. From HaCaT cells and peripheral blood mononuclear cell, changes in cytokine levels were shown by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Second, the ex vivo porcine wound healing model was used to investigate the re-epithelization potential of Nisin A. Finally, the model Galleria mellonella was used to confirm antimicrobial activity and to investigate potential immunomodulatory effects in vivo. Here, we demonstrated that Nisin A affected migration significantly of both human umbilical vein endothelial cell and HaCaT cells (p < 0.05) but not proliferation, potentially by decreasing the levels of proinflammatory cytokines tumor necrosis factor-α, interleukin-6, and interleukin-8 (p < 0.001). Furthermore, Nisin A treatment diminished lipopolysaccharide-induced tumor necrosis factor-α levels from peripheral blood mononuclear cells and monocyte chemoattractant protein-1 from HaCaT cells (p < 0.001). Furthermore, Nisin A did not affect proliferation ex vivo either but increased re-epithelization of the porcine skin. Nisin A improved survival of G. mellonella significantly from Staphylococcus epidermidis (p < 0.001) but not from Escherichia coli, indicating that Nisin A did not help the larvae to survive the infection in a different than direct antimicrobial way. All together this makes Nisin A a potential treatment to use in wound healing, as it increases the mobility of skin cells, dampens the effect of lipopolysaccharide and proinflammatory cytokines, and decreases bacterial growth. more...

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time. more...

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time. more...

Neurotensin, substance P, and insulin have been demonstrated to improve wound healing in vivo. However, the mechanism behind their effect is still not fully understood. This study investigates the effects leading to enhanced scratch closure by these peptides in vitro. The skin keratinocyte cell line, HaCaT, was used to test scratch closure effects of the peptides and alterations of cytokine levels. HUVEC cells were used to test the angiogenic effect of the peptides. Furthermore, clinical isolates of Staphylococcus lugdunensis were used to examine the potential antimicrobial activity of each peptide. Our results demonstrate that neurotensin, substance P, and insulin had significant migratory effects in scratch assays were neurotensin had the lowest effect. Furthermore, we investigated use of the peptides in combination. When substance P was used in combination with neurotensin, the cell migratory capacity was decreased, and the peptides showed a negative correlation (r = -0.298, P < .001). Neurotensin and insulin significantly increased levels of monocyte chemoattractant protein-1 (P < .001) secreted from white blood cells, whereas substance P showed a tendency. Interestingly, neurotensin increased the level of monocyte chemoattractant protein-1 significantly compared to substance P (P < .01). Additionally, the peptides decreased TNFα mRNA levels (P < .001) in HaCaT cells, whereas only neurotensin and insulin decreased IL-8 mRNA (P < .001) but had no significant effect on IL-6 mRNA levels. Surprisingly, substance P increased IL-6 mRNA 9-fold (P < .001). Furthermore, we demonstrate that the peptides increased angiogenesis in the HUVEC cells (P < .001). Finally, S. lugdunensis isolates were not susceptible to the peptides. We demonstrate that the peptides worked differently on HaCaT cells, but substance P acted differently than neurotensin on cytokine levels expression as well as on migration of HaCaT cells. On the contrary, neurotensin and insulin worked similarly. All of these aspects are crucial for proper wound healing, and the results suggest multiple mechanisms for wound-healing properties of these peptides. more...

Staphylococcus epidermidis is a common nosocomial pathogen able to form biofilms in indwelling devices, resulting in chronic infections, which are refractory to antibiotics treatment. Staphylococcal biofilms are also associated with the delayed reepithelization and healing of chronic wounds. The human cathelicidin peptide LL-37 has been proven active against S. epidermidis biofilms in vitro and to promote wound healing. As previous studies have demonstrated that fragments of LL-37 could possess an equal antibacterial activity as the parent peptide, we tested whether shorter (12-mer) synthetic fragments of LL-37 maintained the antibiofilm and/or immune modulating activity, aiming at the identification of essential regions within the LL-37 parent sequence. Three fragments of LL-37 displayed improved activity against S. epidermidis in terms of biofilm inhibition and eradication, a reduced cytotoxicity to human keratinocytes and erythrocytes. In addition, KR-12 and VQ-12V26 enhanced wound healing potential, relative to LL37. FK-12 and KR-12 are truncated version of the cathelicidin, previously reported as valid antimicrobials, whereas VQ-12V26 is a single substituted LL-37 fragment. Remarkably, the single substitution aspartic acid to valine in position 26 caused gain of antimicrobial function in the inactive VQ-12 fragment. The combination of antibiofilm, wound healing potential, and low cytotoxicity makes KR-12 and VQ-12V26 promising therapeutic agents and lead compounds for further improvement and understanding of antibiofilm and wound healing properties. more...