Acute and chronic wound fluids inversely influence adipose-derived stem cell function: molecular insights into impaired wound healing.

Wound healing is a complex biological process that requires a well-orchestrated interaction of mediators as well as resident and infiltrating cells. In this context, mesenchymal stem cells play a crucial role as they are attracted to the wound site and influence tissue regeneration by various mechanisms. In chronic wounds, these processes are disturbed. In a comparative approach, adipose-derived stem cells (ASC) were treated with acute and chronic wound fluids (AWF and CWF, respectively). Proliferation and migration were investigated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and transwell migration assay. Gene expression changes were analysed using quantitative real time-polymerase chain reaction. AWF had a significantly stronger chemotactic impact on ASC than CWF (77·5% versus 59·8% migrated cells). While proliferation was stimulated by AWF up to 136·3%, CWF had a negative effect on proliferation over time (80·3%). Expression of b-FGF, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 was strongly induced by CWF compared with a mild induction by AWF. These results give an insight into impaired ASC function in chronic wounds. The detected effect of CWF on proliferation and migration of ASC might be one reason for an insufficient healing process in chronic wounds.

Acute and chronic wound fluids influence keratinocyte function differently.

Wound healing requires a proper functioning of keratinocytes that migrate, proliferate and lead to a competent wound closure. Impaired wound healing might be due to a disturbed keratinocyte function caused by the wound environment. Basically, chronic wound fluid (CWF) differs from acute wound fluid (AWF). The aim of this study was to analyse the effects of AWF and CWF on keratinocyte function. We therefore investigated keratinocyte migration and proliferation under the influence of AWF and CWF using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test and scratch assay. We further measured the gene expression by qRT-PCR regarding growth factors and matrixmetalloproteinases (MMPs) involved in regeneration processes. AWF had a positive impact on keratinocyte proliferation over time, whereas CWF had an anti-proliferative effect. Keratinocyte migration was significantly impaired by CWF in contrast to an undisturbed wound closure under the influence of AWF. MMP-9 expression was strongly upregulated by CWF compared with AWF. Keratinocyte function was significantly impaired by CWF. An excessive induction of MMP-9 by CWF might lead to a permanent degradation of extracellular matrix and thereby prevent wounds from healing.

Staphylococcus aureus bacteremia after thermal injury: the clinical impact of methicillin resistance.

OBJECTIVE: To evaluate the impact of methicillin resistance in Staphylococcus aureus bacteremia (SAB) on mortality and length of stay in burn patients. DESIGN: Retrospective cohort study. SETTING: A 750-bed tertiary care university hospital in Cologne, Germany. PATIENTS: Patients registered in the database of the burn intensive care unit (BICU) between 1989 and 2009 with complete data sets (n=1688). RESULTS: Over the 21-year study period, 74 patients with SAB were identified; 33 patients had methicillin-resistant S. aureus (MRSA) and 41 methicillin-susceptible S. aureus (MSSA). Comparing the MRSA with the MSSA population the following parameters were significantly different in the univariate analysis: BMI (27.2 kg/m(2) vs. 23.6 kg/m(2); P=0.05), extent of deep partial thickness burns (17.8% vs. 9.0% of total body surface area; P=0.007), antibiotic requirement on admission (45.5% vs. 22.0%; P=0.046), median length of hospitalization prior SAB (24 days vs. 7 days; P<0.001), packed red blood cells administration (47.6 units vs. 26.1 units; P=0.003), intubation requirement (100% vs. 80.5%; P=0.007), intubation period (43.5 days vs. 26.8 days; P=0.008), catecholamine requirement (90.9% vs. 61.0%; P=0.004), sepsis (60.6% vs. 34.1%; P=0.035) and organ failures (81.8% vs. 39.0%; P<0.001). Regarding outcome parameters, methicillin resistance was not significantly related with mortality (adjusted OR 1.55, 95% CI 0.56-4.28; P=0.40) and length of BICU stay after SAB (Kaplan-Meier analysis log-rank test P=0.32; Cox's proportional hazards regression HR 1.22, 95% CI 0.65-2.27, P=0.535) in the univariate and multivariate analyses. CONCLUSION: Our data suggest that methicillin resistance is not associated with significant increases in mortality and length of BICU stay among burn patients with SAB.

Vascular endothelial growth factor (VEGF165) plus basic fibroblast growth factor (bFGF) producing cells induce a mature and stable vascular network--a future therapy for ischemically challenged tissue.

BACKGROUND: Vascular endothelial growth factor (VEGF)(165) induces formation of immature blood vessels with increased permeability. In this study, we used a cell-based gene-transfer model of fibroblasts to investigate the effects of a combined in vivo treatment consisting of the VEGF165 and basic fibroblast growth factor (bFGF) proteins on ischemic and non-ischemic tissues. MATERIALS AND METHODS: After controlled in vitro adenoviral transfection we transplanted fibroblasts into either healthy tissue, or into an ischemic skin flap model at different tissue locations and at different time points. Subsequent protein expression and angiogenic effects were measured using ELISA, PCR, immunohistology, planimetry, and microangiography. RESULTS: Transfected fibroblasts temporarily produced VEGF(165) and bFGF. After transdermal implantation we found an up-regulation of genes encoding for both factors in tissue samples. The combined transplantation of VEGF(165) and bFGF modified cells increased the number of sm-actin+/CD31+ blood vessels and reduced necrosis by 25%. The number of functional blood vessels increased over a period of 168 d even in healthy tissue. CONCLUSIONS: We achieved stable vessel growth in healthy tissue by inducing a temporary overexpression of VEGF(165) and bFGF and improved the survival of ischemic tissue. One possible mechanism for the latter observation is the stabilization of VEGF(165)-induced hyperpermeable vessels by a bFGF-mediated pericytial recruitment of smooth muscle cells.

The choice of anesthesia influences oxidative energy metabolism and tissue survival in critically ischemic murine skin.

In surgical animal studies anesthesia is used regularly. Several reports in the literature demonstrate respiratory and cardiovascular side effects of anesthesiologic agents. The aim of this study was to compare two frequently used anesthesia cocktails (ketamine/xylazine [KX] versus medetomidine/climazolam/fentanyl [MCF]) in skin flap mouse models. Systemic blood values, local metabolic parameters, and surgical outcome should be analyzed in critical ischemic skin flap models. Systemic hypoxia was found in the animals undergoing KX anesthesia compared with normoxia in the MCF group (sO(2): 89.2% +/- 2.4% versus 98.5% +/- 1.2%, P < 0.01). Analysis of tissue metabolism revealed impaired anaerobic oxygen metabolism and increased cellular damage in critical ischemic flap tissue under KX anesthesia (lactate/pyruvate ratio: KX 349.86 +/- 282.38 versus MCF 64.53 +/- 18.63; P < 0.01 and glycerol: KX 333.50 +/- 83.91 micromol/L versus MCF 195.83 +/- 29.49 micromol/L; P < 0.01). After 6 d, different rates of flap tissue necrosis could be detected (MCF 57% +/- 6% versus KX 68% +/- 6%, P < 0.01). In summary we want to point out that the type of anesthesia, the animal model and the goal of the study have to be well correlated. Comparing the effects of KX and MCF anesthesia in mice on surgical outcome was a novel aspect of our study.

Venous thromboembolism after severe trauma: incidence, risk factors and outcome.

BACKGROUND: Venous thromboembolic events (VTEs) are common life-threatening complications after trauma, but epidemiology and reported risk factors still vary. The purpose of this investigation was to determine the incidence of VTEs among hospitalised trauma patients, to identify potential risk factors and to assess whether their presence was associated with: (a) the magnitude and pattern of injury, (b) therapeutic interventions and (c) outcome, all by using a large population-based registry. PATIENTS AND METHODS: Patient data from the Trauma Registry of the German Society for Trauma Surgery (TR-DGU) including datasets from more than 35,000 trauma patients were screened for all clinically relevant VTEs, i.e. deep vein thrombosis (DVT) and pulmonary embolism (PE). A total of 7937 patients were identified for further investigation and multivariate logistic regression analyses were performed to assess potential risk factors for VTEs and to evaluate the effect of VTEs on outcome. RESULTS: One hundred forty-six of 7937 patients developed clinically relevant VTEs during post-traumatic hospitalisation corresponding to an overall incidence rate of 1.8%. Two-thirds (97/146) of all VTEs occurred during the first 3 weeks after admission. At the time point of the event 118/146 (80.8%) patients were under either mechanical or chemical prophylaxis. Multivariate analysis with VTE as dependent variable identified injury severity score, the number of operative procedures, pelvic injury (abbreviated injury scale > or = 2) and concomitant diseases (i.e. diabetes, renal failure, malignancies and congenital or acquired coagulation disorders) as independent risk factors. The presence of VTEs was associated with higher frequencies of sepsis (25% vs. 9.1%), single (63.6% vs. 41.3%) and multiple organ failure (49% vs. 25%) and prolonged in-hospital length of stay (52+/-34 days vs. 29+/-30 days; all p<0.001). The mortality in the VTE group totaled 13.7% vs. 7.4% in the non-VTE group (p=0.004). The presence of PE was associated with a mortality rate of 25.7%. The adjusted odds ratio of post-traumatic VTEs for hospital mortality was 2.08 (CI95 1.15-3.78; p=0.016). CONCLUSION: The occurrence of clinically apparent VTEs during post-traumatic hospitalisation is low but associated with increased morbidity and mortality. Conclusions about the effectiveness of different thromboprophylactic measures could not be drawn, since detailed information was not recorded. However, 80.8% of VTE patients had received thromboprophylaxis at the time point of the event.

Severe burn injuries: acute and long-term treatment.

BACKGROUND: The physician that initially sees a patient with an extensive and deep dermal burn injury must be able to provide initial acute treatment and to make a well-founded decision whether to have the patient transported to a burn care center (BCC). Physicians from a variety of specialities will be involved in the management of long-term sequelae. METHODS: This article provides an overview of the treatment of severe burns and their commonest complications. Special attention is paid to initial emergency treatment (first aid) and to late complications, because physicians from multiple specialties are often involved in these phases of treatment. The data and guidelines that are summarized here were obtained through a selective Medline search and supplemented by the authors' experience in their own burn care center. RESULTS: Analgesia, careful fluid balance, and early intubation are important elements of the initial emergency treatment. Long-term complications of burns, such as disfiguring scars on exposed areas of skin and functionally significant contractures, often require surgical treatment. Early measures for scar care may improve the outcome. CONCLUSIONS: The effective treatment of severe burns is interdisciplinary, involving general practitioners and emergency physicians as well as plastic surgeons and physicians of other specialties. Knowledge of the basic principles of treatment enables physicians to care for patients with burns appropriately both in the acute setting and in the long term.

Association of preexisting medical conditions with in-hospital mortality in multiple-trauma patients.

BACKGROUND: Mortality after trauma has been shown to be influenced by host factors, such as age and preexisting medical conditions (PMCs). The independent predictive value of specific PMCs for in-hospital mortality after adjustment for injury severity, injury pattern, age, and presence of other PMCs has not been fully elucidated. STUDY DESIGN: Records of 11,142 trauma patients (18 years of age or older, Injury Severity Score > or = 16, years 2002 to 2007) documented in the Trauma Registry of the German Society for Trauma Surgery were analyzed to assess the association of PMCs with in-hospital mortality. Multiple logistic regression models were used for this analysis. RESULTS: PMCs were affirmed for 3,836 of the 11,142 patients studied (34.4%). An independent statistical association with increased in-hospital mortality was found for 6 of 14 analyzed PMCs after adjustment for age and the Revised Injury Severity Classification score, respectively, ie, heart disease, obesity, hepatitis/liver cirrhosis, malignancies, coagulation disorder, and peripheral arterial occlusive disease stage IV. The association with mortality varied with different injury patterns. CONCLUSION: Specific PMCs were associated with increased mortality after trauma independent from injury severity and age. Knowledge of the identified relevant PMCs could help the medical team to be able to assess the mortality risk profile of trauma patients in a more detailed and quantifiable way.

Timing and targeting of cell-based VEGF165 gene expression in ischemic tissue.

BACKGROUND: Therapeutic angiogenesis has become a key technology in experimental and clinical medicine. Only few data are available on the effects of timing and targeting of therapeutic proteins after cell-based gene transfer. This work investigates such effects after temporary expression of vascular endothelial growth factor 165 (VEGF(165)), the most commonly used angiogenic protein for therapeutic purposes. METHODS: We established a cell-based gene-transfer model using fibroblasts to temporarily produce VEGF(165). Cells were implanted into 40 rats. Protein expression and angiogenic effects were measured by PCR, immunohistology, and microangiography. To determine an improvement for survival of ischemically challenged tissue, cells were implanted in an ischemic flap model at different locations and time points. RESULTS: After implantation of modified cells, a temporary increase was found in the target tissue for VEGF(165), endothelial cell counts, and capillary network formations. Four wk later, histological alterations in the target tissue area were not different from controls. Implantation of modified cells into flap plus wound margin 1 wk before surgery showed significant improvement of tissue survival demonstrated by planimetric measurements and blood vessels counting in the target tissue. CONCLUSION: In our model, temporary expression of VEGF(165) induces therapeutically relevant angiogenesis and improves blood supply only if applied 1 wk before ischemia. It is essential to include the surrounding area for induction of angiogenesis in this model. In contrast, the angiogenic effects are not effective in the target area and its surrounding tissue, if therapeutic gene expression is started during onset of ischemia or 2 wk before ischemia in this model.

Hemoglobin vesicles reduce hypoxia-related inflammation in critically ischemic hamster flap tissue.

OBJECTIVES: The aim of this study was to investigate the effect of a highly viscous, left-shifted hemoglobin vesicle solution (HbV) on the hypoxia-related inflammation and the microcirculation in critically ischemic peripheral tissue. DESIGN: Randomized prospective study. SETTING: University laboratory. SUBJECTS: Twenty-four male golden Syrian hamsters. INTERVENTIONS: Island flaps were dissected from the back skin of anesthetized hamsters for assessment with intravital microscopy. The flap included a critically ischemic, hypoxic area that was perfused via a collateralized vasculature. One hour after completion of the preparation, the animals received an injection of 25% of total blood volume of 0.9% NaCl or NaCl suspended with HbVs at a concentration of 5 g/dL (HbV5) or 10 g/dL (HbV10). MEASUREMENTS AND MAIN RESULTS: Plasma viscosity was increased from 1.32 cP to 1.61 cP and 2.14 cP after the administration of HbV5 and HbV10, respectively (both p < .01). Both HbV solutions raised partial oxygen tension (Clark-type microprobes) in the ischemic tissue from approximately 10 torr to 17 torr (p < .01), which was paralleled by an increase in capillary perfusion by > 200% (p < .01). The 50% increase in macromolecular capillary leakage found over time in the control animals was completely abolished by the HbV solutions (p < .01), which was accompanied by a > 50% (p < .01) reduction in cells immunohistochemically stained for tumor necrosis factor-alpha and interleukin-6 and in leukocyte counts, whereas no such changes were observed in the anatomically perfused, normoxic tissue. CONCLUSIONS: Our study suggests that in critically ischemic, hypoxic peripheral tissue, hypoxia-related inflammation may be reduced by a top-load infusion of HbV solutions. We attributed this effect to a restoration of tissue oxygenation and an increase in plasma viscosity, both of which may have resulted in attenuation of secondary microcirculatory impairments.