Changes in human burn fluid biological activity during normal burn healing

The main goal of this work was monitoring the changes occurring in human burn fluid biological activity during normal burn healing. The fluid available in the burn until healing makes a good material for controlling biochemical microenvironment of burn cells. This environment involves factors, such as extracellular matrix proteins and matrix metalloproteinases. In this work our previous studies of the influence of wound and burn fluids on the functional activity of cells were extended to include the effect of burn fluid on fibroblasts and keratinocytes, i. e. human skin cells present in the wound and involved in wound healing. It was shown that human burn fluid biological activity depends on the time that passed after burning, and on the correctness of healing. Migration of human fibroblasts becomes more intensive under the influence of such a fluid independently on the time of fluid sampling. Unlike, keratinocyte migration was inhibited by burn fluid sampled 1-3 days after burning but was enhanced by fluids sampled 6 days following burning. The obtained data are to be necessarily taken into consideration at burn treatment and also at transplantation of cells for healing of wounds of different nature.     

Electron microscopic analysis of the fibroblast differentiation of amebocytes in the common snail, Lymnaea stagnalis L. (Mollusca, Gastropoda) in a lesion focus

The amoebocytes and fibroblasts of intact connective tissues of the common pond snail foot, as well as the amoebocytes of the burn wound were studied by means of electron microscope. The found transitional forms from the amoebocytes to the fibroblasts allowed to conclude that there were cells--precursors of fibroblasts among the amoebocytes migrating in the wound. A tentative scheme of histogenetic series of amoebocytes is given.     

Role of senescent fibroblasts on alkali-induced corneal neovascularization

Cellular senescence acts as a potent regulator of tumor suppression and fibrosis limitation; however, its contribution and crosstalk with neovascularization during normal wound healing has not been examined. Here, we explored the role of senescent fibroblasts on neovascularization with a mouse model of alkali-induced corneal wound healing. Senescent cells accumulated in corneal stroma from day 7 to 27 after alkali burn and peaked on day 14, which was consistent with the development of corneal neovascularization (CNV). In vitro and in vivo assays confirmed that the senescent cells were derived primarily from activated corneal fibroblasts. Furthermore, senescent corneal fibroblasts exhibited enhanced synthesis and secretion of extracellular matrix-degrading enzymes (matrix metalloproteinases 2, 3, and 14 and tissue- and urokinase-type plasminogen activators) and angiogenic factors (vascular endothelial growth factor) and decreased expression of anti-angiogenic factors (pigment epithelium-derived factor and thrombospondins), which supported the proliferation, migration, and promotion of tube formation of vascular endothelial cells. Intrastromal injection of premature senescent fibroblasts induced CNV earlier than that of normal fibroblasts, while matrix metalloproteinase inhibitors blocked the early onset of senescent cell-induced CNV. Therefore, senescent fibroblasts promoted the alkali-induced CNV partially via the enhanced secretion of matrix metalloproteases.     

Pirfenidone attenuates the profibrotic contractile phenotype of differentiated human dermal myofibroblasts

Dysregulated wound healing after burn injury frequently results in debilitating hypertrophic scarring and contractures. Myofibroblasts, the main effector cells for dermal fibrosis, develop from normal fibroblasts via transforming growth factor beta 1 (TGF-β1). During wound healing, myofibroblasts produce extracellular matrix (ECM) proteins, modulate ECM stability, and contract the ECM using alpha smooth muscle actin (α-SMA) in contractile stress fibers. The antifibrotic pirfenidone has previously been shown to inhibit the initial differentiation of fibroblasts into myofibroblasts in vitro and act as a prophylactic measure against hypertrophic scar development in a mouse burn model. To test whether pirfenidone affects differentiated myofibroblasts, we investigated the in vitro effects of pirfenidone treatment after three to five days of stimulation with TGF-β1. In assays for morphology, protein and gene expression, and contractility, pirfenidone treatment produced significant effects. Profibrotic gene expression returned to near-normal levels, further α-SMA protein expression was prevented, and cell contraction within a stressed collagen matrix was reduced. These in vitro results promote pirfenidone as a promising antifibrotic agent to treat existing scars and healing wounds by mitigating the effects of differentiated myofibroblasts.     

Nanostructured lipid carriers containing rapamycin for prevention of corneal fibroblasts proliferation and haze propagation after burn injuries: In vitro and in vivo

Chemical burns are a major cause of corneal haze and blindness. Corticosteroids are commonly used after corneal burns to attenuate the severity of the inflammation-related fibrosis. While research efforts have been aimed toward application of novel therapeutics. In the current study, a novel drug delivery system based nanostructured lipid carriers (NLCs) were designed to treat corneal alkaline burn injury. Rapamycin, a potent inhibitor of mammalian target of rapamycin pathway, was loaded in NLCs (rapa-NLCs), and the NLCs were characterized. Cell viability assay, cellular uptake of NLCs, and in vitro evaluation of the fibrotic/angiogenic genes suppression by rapa-NLCs were carried out on human isolated corneal fibroblasts. Immunohistochemistry (IHC) assays were also performed after treatment of murine model of corneal alkaline burn with rapa-NLCs. According to the results, rapamycin was efficiently loaded in NLCs. NLCs could enhance coumarin-6 fibroblast uptake by 1.5 times. Rapa-NLCs efficiently downregulated platelet-derived growth factor and transforming growth factor beta genes in vitro. Furthermore, proliferation of fibroblasts, a major cause of corneal haze after injury, reduced. IHC staining of treated cornea with alpha-smooth muscle actin and CD34 ⁺ antibodies showed efficient prevention of myofibroblasts differentiation and angiogenesis, respectively. In conclusion, ocular delivery of rapamycin using NLCs after corneal injury may be considered as a promising antifibrotic/angiogenic treatment approach to preserve patient eyesight.     

Acute downregulation of miR-155 leads to a reduced collagen synthesis through attenuating macrophages inflammatory factor secretion by targeting SHIP1

Fibrosis, tightly associated with fibroblasts collagen synthesis, is related closely with inflammatory response. Our previously study found that acute downregulation of miR-155 at wound sites leads to a reduced fibrosis, however its particular mechanism is unclear. Herein, we aimed to explore the mechanism of miR-155 in reducing fibrosis. We first found that down-regulation of miR-155 inhibited macrophages transforming growth factor-β1 (TGF-β1) and IL-1β secretion. Next, we found that co-cultured with macrophages increased the proliferation and collagen synthesis of fibroblasts, and downregulation of miR-155 in macrophages could effectively attenuate the accelerative effects. We further identified SH2 domain containing inositol-5-phosphatase 1 (SHIP1) as a direct target of miR-155 in macrophages, and the expression of SHIP1 was negatively correlated with the level of miR-155. We further confirmed that PI3K/Akt pathway was involved in this process. Last, we found that downregulation of miR-155 leads to a reduced fibrosis in sever burn rat. Taken together, these results indicate that down-regulation of miR-155 leads to a reduced fibroblasts proliferation and collagen synthesis through attenuating macrophages TGF-β1 and IL-1β secretion by targeting SHIP1 via PI3K/Akt pathway, suggesting its potential therapeutic effects on the treatment of skin fibrosis.     

Sensitivity and feasibility of a one-dimensional morphoelastic model for post-burn contraction

We consider a one-dimensional morphoelastic model describing post-burn scar contraction. Contraction can lead to a limited range of motion (contracture). Reported prevalence of burn scar contractures are 58.6% at 3–6 weeks and 20.9% at 12 months post-reconstructive surgery after burns. This model describes the displacement of the dermal layer of the skin and the development of the effective Eulerian strain in the tissue. Besides these components, the model also contains components that play a major role in the skin repair after trauma. These components are signaling molecules, fibroblasts, myofibroblasts, and collagen. We perform a sensitivity analysis for many parameters of the model and use the results for a feasibility study. In this study, we test whether the model is suitable for predicting the extent of contraction in different age groups. To this end, we conduct an extensive literature review to find parameter values. From the sensitivity analysis, we conclude that the most sensitive parameters are the equilibrium collagen concentration in the dermal layer, the apoptosis rate of fibroblasts and myofibroblasts, and the secretion rate of signaling molecules. Further, although we can use the model to simulate significant distinct contraction densities in different age groups, our results differ from what is seen in the clinic. This particularly concerns children and elderly patients. In children we see more intense contractures if the burn injury occurs near a joint, because the growth induces extra forces on the tissue. Elderly patients seem to suffer less from contractures, possibly because of excess skin.

     

Pirfenidone Nanoparticles Improve Corneal Wound Healing and Prevent Scarring Following Alkali Burn

Purpose

To evaluate the effects of pirfenidone nanoparticles on corneal re-epithelialization and scarring, major clinical challenges after alkali burn.

Methods

Effect of pirfenidone on collagen I and α-smooth muscle actin (α-SMA) synthesis by TGFβ induced primary corneal fibroblast cells was evaluated by immunoblotting and immunocytochemistry. Pirfenidone loaded poly (lactide-co-glycolide) (PLGA) nanoparticles were prepared, characterized and their cellular entry was examined in primary corneal fibroblast cells by fluorescence microscopy. Alkali burn was induced in one eye of Sprague Dawley rats followed by daily topical treatment with free pirfenidone, pirfenidone nanoparticles or vehicle. Corneal re-epithelialization was assessed daily by flourescein dye test; absence of stained area indicated complete re-epithelialization and the time for complete re-epithelialization was determined. Corneal haze was assessed daily for 7 days under slit lamp microscope and graded using a standard method. After 7 days, collagen I deposition in the superficial layer of cornea was examined by immunohistochemistry.

Results

Pirfenidone prevented (P<0.05) increase in TGF β induced collagen I and α-SMA synthesis by corneal fibroblasts in a dose dependent manner. Pirfenidone could be loaded successfully within PLGA nanoparticles, which entered the corneal fibroblasts within 5 minutes. Pirfenidone nanoparticles but not free pirfenidone significantly (P<0.05) reduced collagen I level, corneal haze and the time for corneal re-epithelialization following alkali burn.

Conclusion

Pirfenidone decreases collagen synthesis and prevents myofibroblast formation. Pirfenidone nanoparticles improve corneal wound healing and prevent fibrosis. Pirfenidone nanoparticles are of potential value in treating corneal chemical burns and other corneal fibrotic diseases.     

In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors.

Human keratinocytes isolated from a skin biopsy and cultured in vitro on a feeder-layer of irradiated fibroblasts reconstitute a stratified squamous epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3-J2 cells can partially substitute for the intact feeder-layer, we studied the possible involvement of insulin-like growth factors acting in a paracrine fashion. IGFs were measured (after Sephadex G-50 gel-chromatography in acid conditions) in media conditioned by a feeder-layer of lethally irradiated 3T3-J2 fibroblasts on which keratinocytes were grown. Immunoreactive (IR) IGF-I, IGF-II, and IGF binding activity were present in the medium conditioned by the feeder-layer. The medium conditioned by keratinocytes showed nearly undetectable amounts of IR IGF-I and IGF-II, suggesting that keratinocytes are unable to synthesize IGFs peptides. Recombinant IGF-I and IGF-II, and conditioned medium from 3T3-J2 cells, caused a dose-dependent increase of 3H-thymydine incorporation in cultured keratinocytes. The stimulatory effect of IGF and of 3T3-J2 conditioned medium was inhibited by the MoAb Sm 1.2, which recognizes both IGF-I and IGF-II but not insulin, and by the MoAb alpha IR-3, which is a specific antagonist of type-I IGF receptor. Fetal mouse-derived 3T3-J2 cells and adult human skin fibroblasts were equally able to sustain keratinocyte growth and in both cases addition of Sm 1.2 MoAb causes a 50% decrease in the keratinocyte number. When the non-IGF-producing BALB/c 3T3 cells were used as a feeder-layer, the keratinocytes number was similar to that observed with 3T3-J2 and with human fibroblasts plus the Sm 1.2 MoAb. IGF-I and IGF-II restored the BALB/c 3T3 growth promoting activity to the level of 3T3-J2 and of normal human fibroblasts. Our results suggest that fetal mouse 3T3-J2 and human fibroblasts synthesize IGF peptides, while keratinocytes do not. Fibroblast-derived IGFs stimulate keratinocyte growth in a paracrine fashion, suggesting their role in the regulation of keratinocyte proliferation in skin growth and in wound healing.     

Pulmonary changes in a mouse model of combined burn and smoke inhalation-induced injury.

The morbidity and mortality of burn victims increase when burn injury is combined with smoke inhalation. The goal of the present study was to develop a murine model of burn and smoke inhalation injury to more precisely reveal the mechanistic aspects of these pathological changes. The burn injury mouse group received a 40% total body surface area third-degree burn alone, the smoke inhalation injury mouse group received two 30-s exposures of cotton smoke alone, and the combined burn and smoke inhalation injury mouse group received both the burn and the smoke inhalation injury. Animal survival was monitored for 120 h. Survival rates in the burn injury group, the smoke inhalation injury group, and the combined injury group were 70%, 60%, and 30%, respectively. Mice that received combined burn and smoke injury developed greater lung damage as evidenced by histological changes (septal thickening and interstitial edema) and higher lung water content. These mice also displayed more severely impaired pulmonary gas exchange [arterial PO2 (PaO2)/inspired O2 fraction (FiO2)<200]. Lung myeloperoxidase activity was significantly higher in burn and smoke-injured animals compared with the other three experimental groups. Plasma NO2-/NO3-, lung inducible nitric oxide synthase (iNOS) activity, and iNOS mRNA increased with injury; however, the burn and smoke injury group exhibited a higher response. Severity of burn and smoke inhalation injury was associated with more pronounced production of nitric oxide and accumulation of activated leukocytes in lung tissue. The murine model of burn and smoke inhalation injury allows us to better understand pathophysiological mechanisms underlying cardiopulmonary morbidity secondary to burn and smoke inhalation injury.     

Cardiac effects of burn injury complicated by aspiration pneumonia-induced sepsis

Early fluid resuscitation, antimicrobials, early excision, and grafting have improved survival in the early postburn period; however, a significant incidence of pneumonia-related sepsis occurs after burn injury, often progressing to multiple organ failure. Recent studies have suggested that this initial injury (burn injury) primes the subject, producing an exaggerated response to a second insult, such as pneumonia-related sepsis. We developed an experimental animal model that included a third-degree burn over 40% of the total body surface area, followed by sepsis (intratracheal administration of Streptococcus pneumoniae, 4 x 106 colony-forming unit), which was produced either 48 or 72 h after burn injury in adult male rats. Hearts harvested after either burn alone, sepsis alone, or burn plus sepsis were used to assess either contractile function (Langendorff) or cardiomyocyte secretion of tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and IL-10 (ELISA). Experimental groups included the following: 1). sham (sham burn and no sepsis); 2). burn injury alone studied either 24, 48, or 72 h postburn; 3). pneumonia-related sepsis in the absence of burn injury; and 4). pneumonia-induced sepsis studied either 48 or 72 h after an initial burn injury. Burn injury alone (24 h) or sepsis alone produced myocardial contractile defects and increases in pro- and anti-inflammatory cytokine secretion by cardiomyocytes. Sepsis that occurred 48 h postburn exacerbated the cardiac contractile defects seen with either burn alone or sepsis alone. Sepsis that occurred 72 h postburn produced contractile defects resembling those seen in either burn alone or sepsis alone. In conclusion, our data suggest that burn injury primes the subject such that a second insult early in the postburn period produces significantly greater cardiac abnormalities than those seen with either burn alone or sepsis alone.     

Melatonin reduces oxidative damage to skin and normalizes blood coagulation in a rat model of thermal injury

This study was designed to determine the effect of melatonin treatment on the glutathione (GSH) and lipid peroxidation (LPO) levels in the skin as well as prothrombin time (PT) and fibrin degradation products (FDPs) in the blood of rats with thermal injury. Under ether anaesthesia, the shaved dorsum of the rats was exposed to 90 degrees C bath for 10 s to induce burn injury. Rats were decapitated either 3 or 24 hours after burn injury. Melatonin (10 mg/kg) was administered i.p. immediately after burn injury to same animals. In the 24 hour burn group, melatonin injections were repeated for two more occasions 8 and 16 h after burn injury. In the control group the same protocol was applied except that the dorsum was exposed to a 25 degrees C water bath for 10 s. Severe skin scald injury (30% of total body surface area) caused a significant decrease in PT at post burn 3 and 24 hours. FDPs was not increased at post burn 3 hour but was significantly increased at post burn 24 hour. GSH levels were significantly depressed at post burn 3 hour but were not changed at post burn 24 hour. LPO levels were significantly increased both at post burn 3 and 24 hours. Skin protein levels were significantly reduced at post burn 24 hour as evidenced by electrophoresis. Treatment of rats with melatonin normalized PT levels both at post burn 3 and 24 hours. FDP decreased at post burn 24 hour due to melatonin treatment. GSH levels significantly increased as a result of melatonin treatment both at post burn 3 and 24 hours melatonin treatment. LPO levels were not changed by melatonin at post burn 3 hour; however, the melatonin significantly decreased LPO values at post burn 24 hours. In conclusion, exogenously administered melatonin reduced skin oxidant damage and normalized the activated blood coagulation induced by thermal trauma.     

Investigating the Role of P311 in the Hypertrophic Scar

The mechanisms of hypertrophic scar formation are not fully understood. We previously screened the differentially expressed genes of human hypertrophic scar tissue and identified P311 gene as upregulated. As the activities of P311 in human fibroblast function are unknown, we examined the distribution of it and the effects of forced expression or silencing of expression of P311. P311 expression was detected in fibroblast-like cells from the hypertrophic scar of burn injury patients but not in peripheral blood mononuclear cells, bone marrow mesenchymal stem cells, epidermal cells or normal skin dermal cells. Transfection of fibroblasts with P311 gene stimulated the expression of alpha-smooth muscle actin (α-SMA), TGF-β1 and α1(I) collagen (COL1A1), and enhanced the contraction of fibroblast populated collagen lattices (FPCL). In contrast, interference of fibroblast P311 gene expression decreased the TGF-β1 mRNA expression and reduced the contraction of fibroblasts in FPCL. These results suggest that P311 may be involved in the pathogenesis of hypertrophic scar via induction of a myofibroblastic phenotype and of functions such as TGF-β1 expression. P311 could be a novel target for the control of hypertrophic scar development.     

Prognostic significance of erythroblasts in burns

Changes in hematopoiesis that occur in humans after a burn injury may have important effects on morbidity and mortality. In patients with a variety of severe diseases, the presence of erythroblasts in peripheral blood is known to be indicative of a poor prognosis. However, the prognostic significance of erythroblasts in peripheral blood of burn patients has not yet been estimated. This study included 464 consecutive burn patients, of whom 81 did not survive their injuries (17.5 percent). Together with erythroblasts in blood, data on age, sex, total burn surface area, third-degree burn, inhalation trauma, white blood cell count, C-reactive protein, and hemoglobin were studied. The mortality rate of patients with erythroblasts in peripheral blood (n = 53) amounted to 56.6 percent (n = 30; total burn surface area, 39 percent), which is significantly higher (p < 0.001) than the mortality rate of patients without erythroblasts (12.4 percent, n = 51; total burn surface area, 18.69 percent). None of the 10 patients with more than 1000 erythroblasts x 10/liter survived. The detection of erythroblasts in the peripheral blood of burn patients is highly predictive of death, with the odds ratio after adjustment for the other known prognostic factors being 8.3 (95 percent confidence interval, 4.5 to 15.3). Erythroblasts were detected for the first time on average 10 +/- 4 days (median, 6 days) after admission and 13 +/- 6 days (median, 7 days) before death. Detection of erythroblasts in burn patients is of high prognostic power with regard to in-hospital mortality, providing physicians with a strong prognostic method with which to identify seriously threatened patients. It seems attractive to think about an incorporation of erythroblasts into further refinements of burn scores.     

Skeletal muscle apoptosis after burns is associated with activation of proapoptotic signals

Critical illness is associated with muscle wasting and muscle weakness. Using burn injury as a model of local and systemic inflammatory response, we tested the hypothesis that thermal injury causes apoptosis in muscle. After a 40% body surface area burn to rats, abdominal muscles beneath the burn and limb muscles distant from the burn were examined for apoptosis at varying times after burn. Ladder assay, ELISA, and histological methods showed evidence of apoptosis in the abdominal muscles within 4-12 h with peak changes occurring at 3-7 days. Maximal apoptosis was also evident at distant limb muscles at 3-7 days. Investigation of proapoptotic pathways indicated mitochondrial membrane potential to be altered by 1 h after burn. Starting at 15 min after burn, cytochrome c was released from the mitochondria into the cytosol, followed by increased activity of caspase-3, starting at 6 h after burn. These studies suggest that mitochondria and caspase-mediated apoptotic pathways may be an additional mechanism of muscle weight loss in burns and may be potential therapeutic targets for prevention of muscle wasting.     

IFN-gamma production from liver mononuclear cells of mice in burn injury as well as in postburn bacterial infection models and the therapeutic effect of IL-18

Hosts after severe burn injury are known to have a defect in the Th1 immune response and are susceptible to bacterial infections. We herein show that liver NK cells are potent IFN-gamma producers early after burn injury. However, when mice were injected with LPS 24 h after burn injury, IFN-gamma production from liver mononuclear cells (MNC; which we previously showed to be NK cells) was suppressed, and the serum IFN-gamma concentration did not increase, while serum IL-10 conversely increased compared with control mice. Interestingly, a single injection of IL-18 simultaneously with LPS greatly restored the serum IFN-gamma concentration in mice with burn injury and also increased IFN-gamma production from liver MNC. Nevertheless, a single IL-18 injection into mice simultaneously with LPS was no longer effective in the restoration of serum IFN-gamma and IFN-gamma production from the liver MNC at 7 days after burn injury, when mice were considered to be the most immunocompromised. However, IL-18 injections into mice on alternate days beginning 1 day after burn injury strongly up-regulated LPS-induced serum IFN-gamma levels and IFN-gamma production from liver and spleen MNC of mice 7 days after burn injury and down-regulated serum IL-10. Furthermore, similar IL-18 therapy up-regulated serum IFN-gamma levels in mice with experimental bacterial peritonitis 7 days after burn injury and greatly decreased mouse mortality. Thus, IL-18 therapy restores the Th1 response and may decrease the susceptibility to bacterial infection in mice with burn injury.     

The role of nasal carriage in Staphylococcus aureus burn wound colonization

Thermal injury destroys the physical skin barrier that normally prevents invasion of microorganisms. This and concomitant depression of local and systemic host cellular and humoral immune responses are important factors that contribute to colonization and infection of the burn wound. One of the most common burn wound pathogens is Staphylococcus aureus. Staphylococcus aureus is both a human commensal and a frequent cause of infections leading to mild to life-threatening diseases. Despite a variety of infection control measures, for example patient cohorting and contact precaution at burn centres, S. aureus is still frequently encountered in burn wounds. Colonization with S. aureus has been associated with delayed wound healing, increased need for surgical interventions, and prolonged length of stay at burn centres. In this minireview, we focus on S. aureus nasal carriage in relation to S. aureus burn wound colonization and subsequent infection, and its impact on strategies for infection control.     

Enhancement of dendritic cell production by fms-like tyrosine kinase-3 ligand increases the resistance of mice to a burn wound infection

Fms-like tyrosine kinase-3 ligand (Flt3L) is a hemopoietic cytokine that stimulates the production of dendritic cells. This study evaluated the ability of Flt3L-enhanced dendritic cell production to increase the resistance of mice to a burn wound infection with Pseudomonas aeruginosa, a common source of infections in burn patients that have impaired immunity and are susceptible to opportunistic microorganisms. Treatment of mice with Flt3L for 5 days caused a significant increase in dendritic cell numbers in the spleen and significantly increased survival upon a subsequent burn wound infection. Improved survival in Flt3L-treated mice was associated with limited bacterial growth and spread within the burn wounds and a decrease in systemic dissemination of P. aeruginosa. Resistance to burn wound infection could also be conferred to recipient mice by the adoptive transfer of dendritic cells that had been isolated from spleens of Flt3L-treated mice. Adoptive transfer of the same number of splenic dendritic cells from nontreated mice did not confer resistance to burn wound infection. These data indicate that Flt3L can increase the resistance of mice to a P. aeruginosa burn wound infection through both stimulation of dendritic cell production and enhancement of dendritic cell function.