Low-Dose Blue Light (420 nm) Reduces Metabolic Activity and Inhibits Proliferation of Human Dermal Fibroblasts.

Hypertrophic scarring in burn wounds is caused by overactive fibroblasts and myofibroblasts. Blue light reveals wavelength- and dose-dependent antibacterial and antiproliferative effects and may serve as a therapeutic option against wound infection and fibrotic conditions. Therefore, we evaluated in this study the effects of single and multiple irradiations with blue light at 420 nm (BL420) on the intracellular ATP concentration, and on the viability and proliferation of the human skin fibroblast (HDFs). In addition, possible BL420-induced effects on the catalase expression and differentiation were assessed by immunocytochemical staining and western blot analyses. Furthermore, we used RNA-seq analyses to identify BL420-affected genes. We found that BL420 induced toxicity in HDFs (up to 83%; 180 J/cm2). A low dose of 20 J/cm2 reduced the ATP concentration by ~50%. Multiple irradiations (4 × 20 J/cm2) inhibited proliferation without visible toxicity and reduced catalase protein expression by ~37% without affecting differentiation. The expression of about 300 genes was significantly altered. Many downregulated genes have functions in cell division/mitosis. BL420 can strongly influence the fibroblast physiology and has potential in wound therapy. However, it is important to consider the possible toxic and antiproliferative effects, which could potentially lead to impaired wound healing and reduced scar breaking strength.

Silk proteins in reconstructive surgery: Do they possess an inherent antibacterial activity? A systematic review.

The field of reconstructive surgery encompasses a wide range of surgical procedures and regenerative approaches to treat various tissue types. Every surgical procedure is associated with the risk of surgical site infections, which are not only a financial burden but also increase patient morbidity. The surgical armamentarium in this area are biomaterials, particularly natural, biodegradable, biocompatible polymers, including the silk proteins fibroin (SF) and sericin (SS). Silk is known to be derived from silkworms and is mainly composed of 60-80% fibroin, which provides the structural form, and 15-35% sericin, which acts as a glue-like substance for the SF threads. Silk proteins possess most of the desired properties for biomedical applications, including biocompatibility, biodegradability, minimal immunogenicity, and tunable biomechanical behaviour. In an effort to alleviate or even prevent infections associated with the use of biomaterials in surgery, antibacterial/antimicrobial properties have been investigated in numerous studies. In this systematic review, the following question was addressed: Do silk proteins, SF and SS, possess an intrinsic antibacterial property and how could these materials be tailored to achieve such a property?

Soluble CD83 improves and accelerates wound healing by the induction of pro-resolving macrophages.

To facilitate the recovery process of chronic and hard-to-heal wounds novel pro-resolving treatment options are urgently needed. We investigated the pro-regenerative properties of soluble CD83 (sCD83) on cutaneous wound healing, where sCD83 accelerated wound healing not only after systemic but also after topical application, which is of high therapeutic interest. Cytokine profile analyses revealed an initial upregulation of inflammatory mediators such as TNFα and IL-1ß, followed by a switch towards pro-resolving factors, including YM-1 and IL-10, both expressed by tissue repair macrophages. These cells are known to mediate resolution of inflammation and stimulate wound healing processes by secretion of growth factors such as epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF), which promote vascularization as well as fibroblast and keratinocyte differentiation. In conclusion, we have found strong wound healing capacities of sCD83 beyond the previously described role in transplantation and autoimmunity. This makes sCD83 a promising candidate for the treatment of chronic- and hard-to-heal wounds.

Antibacterial properties of functionalized silk fibroin and sericin membranes for wound healing applications in oral and maxillofacial surgery.

Oral wounds are among the most troublesome injuries which easily affect the patients' quality of life. To date, the development of functional antibacterial dressings for oral wound healing remains a challenge. In this regard, we investigated antibacterial silk protein-based membranes for the application as wound dressings in oral and maxillofacial surgery. The present study includes five variants of casted membranes, i.e., i) membranes-silver nanoparticles (CM-Ag), ii) membranes-gentamicin (CM-G), iii) membranes-control (without functionalization) (CM-C), iv) membranes-silk sericin control (CM-SSC), and v) membranes-silk fibroin/silk sericin (CM-SF/SS), and three variants of nonwovens, i.e., i) silver nanoparticles (NW-Ag), ii) gentamicin (NW-G), iii) control (without functionalization) (NW-C). The surface structure of the samples was visualized with scanning electron microscopy. In addition, antibacterial testing was accomplished using agar diffusion assay, colony forming unit (CFU) analysis, and qrt-PCR. Following antibacterial assays, biocompatibility was evaluated by cell proliferation assay (XTT), cytotoxicity assay (LDH), and live-dead assay on L929 mouse fibroblasts. Findings indicated significantly lower bacterial colony growth and DNA counts for CM-Ag with a reduction of bacterial counts by 3log levels (99.9% reduction) in CFU and qrt-PCR assay compared to untreated control membranes (CM-C and CM-SSC) and membranes functionalized with gentamicin (CM-G and NW-G) (p < 0.001). Similarly, NW-G yielded significantly lower DNA and colony growth counts compared to NW-Ag and NW-C (p < 0.001). In conclusion, CM-Ag represented 1log level better antibacterial activity compared to NW-G, whereas NW-G showed better cytocompatibility for L929 cells. As data suggest, these two membranes have the potential of application in the field of bacteria-free oral wound healing. However, provided that loading strategy and cytocompatibility are adjusted according to the antibacterial agents' characteristic and fabrication technique of the membranes.

Tissue Adhesives in Reconstructive and Aesthetic Surgery-Application of Silk Fibroin-Based Biomaterials.

Tissue adhesives have been successfully used in various kind of surgeries such as oral and maxillofacial surgery for some time. They serve as a substitute for suturing of tissues and shorten treatment time. Besides synthetic-based adhesives, a number of biological-based formulations are finding their way into research and clinical application. In natural adhesives, proteins play a crucial role, mediating adhesion and cohesion at the same time. Silk fibroin, as a natural biomaterial, represents an interesting alternative to conventional medical adhesives. Here, the most commonly used bioadhesives as well as the potential of silk fibroin as natural adhesives will be discussed.

Characterization of Blue Light Treatment for Infected Wounds: Antibacterial Efficacy of 420, 455, and 480 nm Light-Emitting Diode Arrays Against Common Skin Pathogens Versus Blue Light-Induced Skin Cell Toxicity.

Objective: To determine effective treatment strategies against bacterial infections of chronic wounds, we tested different blue light (BL)-emitting light-emitting diode arrays (420, 455, and 480 nm) against wound pathogens and investigated in parallel BL-induced toxic effects on human dermal fibroblasts. Background: Wound infection is a major factor for delayed healing. Infections with Pseudomonas aeruginosa and Staphylococcus aureus are clinically relevant caused by their ability of biofilm formation and their quickly growing antibiotics resistance. BL has demonstrated antimicrobial properties against various microbes. Methods: Determination of antibacterial and cell toxic effects by colony-forming units (CFUs)/biofilm/cell viability assays, and live cell imaging. Results: A single BL irradiation (180 J/cm2), of P. aeruginosa at both 420 and 455 nm resulted in a bacterial reduction (>5 log10 CFU), whereas 480 nm revealed subantimicrobial effects (2 log10). All tested wavelengths of BL also revealed bacteria reducing effects on Staphylococcus epidermidis and Escherichia coli (maximum 1-2 log10 CFU) but not on S. aureus. Dealing with biofilms, all wavelengths using 180 J/cm2 were able to reduce significantly the number of P. aeruginosa, E. coli, and S. epidermidis. Here, BL420nm achieved reductions up to 99%, whereas BL455nm and BL480nm were less effective (60-83%). Biofilm-growing S. aureus was more BL sensitive than in the planktonic phase showing a reduction by 63-75%. A significant number of cell toxic events (>40%) could be found after applying doses (>30 J/cm2) of BL420nm. BL455nm showed only slight cell toxicity (180 J/cm2), whereas BL480nm was nontoxic at any dose. Conclusions: BL treatment can be effective against bacterial infections of chronic wounds. Nevertheless, using longer wavelengths >455 nm should be preferred to avoid possible toxic effects on skin and skin cells. To establish BL therapy for infected chronic wounds, further studies concerning biofilm formation and tissue compatibility are necessary.

Plasma medicine and wound management: Evaluation of the antibacterial efficacy of a medically certified cold atmospheric argon plasma jet.

OBJECTIVES: A major problem for wound healing is contamination with bacteria, often resulting in biofilm formation and wound infection, which, in turn, needs immediate intervention such as surgical debridement and through irrigation. A topical treatment with cold atmospheric pressure plasma (CAP) for wound disinfection may present an alternative and less painful approach. METHODS: This study investigated the antibacterial effects of a cold atmospheric pressure argon plasma jet (kINPen® MED) as a CAP source, using the three-dimensional Staphylococcus aureus immunocompetent biofilm system hpBIOM in addition to a standard planktonic test. Furthermore, skin cell compatibility was evaluated using a keratinocyte (HaCat) model. RESULTS: CAP treatment (0-240 s) followed by incubation (15, 120 min) within the CAP-treated media showed slight bactericidal efficacy under planktonic conditions but no effect on biofilms. However, indirect CAP treatment of keratinocytes performed under the same conditions resulted in a significant decrease in metabolic activity. Short CAP treatment and exposure time (30 s; 15 min) induced a slight increase in the metabolic activity; however, longer treatments and/or exposure times led to pronounced reductions up to 100%. These effects could partially be reversed by addition of catalase, indicating a dominant role of CAP-generated hydrogen peroxide. CONCLUSIONS: These results indicate that plasma treatment does not lead to the desired disinfection or significant reduction in the bacterial burden of Staphylococcus aureus in a wet milieu or in biofilms. Thus, treatment with CAP could not be recommended as a single anti-bacterial therapy for wounds but could be used to support standard treatments.

The impact of non-toxic blue light (453 nm) on cellular antioxidative capacity, TGF-ß1 signaling, and myofibrogenesis of human skin fibroblasts.

Studies have demonstrated that blue light induces biological effects, such as cell death, and inhibition of proliferation and differentiation. Since blue light at longer wavelength (>440 nm) exerts less injurious effects on cells than at shorter wavelengths, (400-440 nm), we have investigated the impact of non-toxic (LED) blue light at 453 nm wavelength on human skin fibroblasts (hsFBs). We found that besides its decreasing effects on the proliferation rate, repeated blue light irradiations (80 J/cm2) also significantly reduced TGF-ß1-induced myofibrogenesis as shown by diminished α-SMA and EDA-FN expression accompanied by reduced protein expression and phosphorylation of ERK 1/2, SMAD 2/3, and p38-key players of TGF-ß1-induced myofibrogenesis. In parallel, catalase protein expression, intracellular FAD concentrations as well as NADP+/NADPH ratio were reduced, whereas intracellular reactive oxygen species (ROS) were increased. We postulate that as a molecular mechanism downregulation of catalase and photoreduction of FAD induce intracellular oxidative stress which, in turn, affects the signaling factors of myofibrogenesis leading to a lower rate of α-SMA and EDA-FN expression and, therefore, myofibroblast formation. In conclusion, blue light even at longer wavelengths shows antifibrotic activity and may represent a suitable and safe approach in the treatment of fibrotic skin diseases including hypertrophic scarring and scleroderma.

Temporal phenotyping of circulating microparticles after trauma: a prospective cohort study.

BACKGROUND: After severe polytrauma the dynamic process of coagulation may deteriorate towards a trauma-induced coagulopathy (TIC) promoting a dramatic increase in morbidity and mortality. Recent evidence suggests that microparticles (MPs) play a pivotal role at the interface between cellular and plasmatic coagulation systems. However, the impact of MPs on functional coagulation has not been clarified yet in the setting of traumatic injuries. We assessed the temporal patterns of circulating MP concentrations including their cellular origin in the context of clinical presentation and global coagulation assays. METHODS: Blood samples from 22 consecutive polytrauma patients (ISS ≥16) from 2015 were collected at hospital admission, after 24 and 72 h and compared to those from healthy individuals and minor injured patients with isolated extremity fractures. Flow cytometry (BD Accuri C6; Heidelberg/Germany) was used to determine MP concentrations and cellular origin using cell-specific markers (platelet derived (PDMP): CD42b+, CD61+, CD62p+; endothelial cell derived (EDMP): CD144+, CD62e+, CD144+/62e+). Results were correlated with clinical data and results from viscoelastic testing (ROTEM). RESULTS: Twenty two polytrauma patients (17 males, agemedian 60 yrs) with a median ISS 26.5 (IQR 14.5) were assessed. PDMP and EDMP concentrations increased significantly in polytrauma patients as compared to healthy individuals and minor injured patients. MP concentrations correlated with injury severity (CD144+: ρsp = 0.79, p < 0.001; CD42b+: ρsp = 0.61, p < 0.001). EDMP displayed a negative correlation with aPTT (CD144/62e+, ρsp = - 0.55, p < 0.05), INR (CD144/62e+, ρsp = - 0.61, p < 0.05) and ROTEM-INTEM CT (CD144/62e+, ρsp = - 0.68, p < 0.05) reflecting increased dynamics of clot formation and an overall procoagulative effect. Additionally, EDMP showed a negative association with FIBTEM values (10 min amplitude, maximum clot firmness) indicating a fibrinolytic potential. DISCUSSION: In a small cohort, analysing most severly injured patients, the association of increased MP levels and altered coagulation parameters could be demonstrated. However, these findings are based on correlation analysis, which do not enable causel evidence. Therefore, further in-vitro studies are needed analysing the underlying pathomechanisms. CONCLUSION: In conclusion, this study could demonstrate that PDMP and EDMP levels increase significantly following polytrauma correlating with injury severity. Although severe coagulopathy was not observed, EDMP levels were associated with improved coagulation parameters suggesting their essential role for regulating blood coagulation after trauma.

Prehospital volume resuscitation--Did evidence defeat the crystalloid dogma? An analysis of the TraumaRegister DGU® 2002-2012.

BACKGROUND: Various studies have shown the deleterious effect of high volume resuscitation following severe trauma promoting coagulopathy by haemodilution, acidosis and hypothermia. As the optimal resuscitation strategy during prehospital trauma care is still discussed, we raised the question if the amount and kind of fluids administered changed over the recent years. Further, if less volume was administered, fewer patients should have arrived in coagulopathic depletion in the Emergency Department resulting in less blood product transfusions. METHODS: A data analysis of the 100 489 patients entered into the TraumaRegister DGU® (TR-DGU) between 2002 and 2012 was performed of which a total of 23512 patients (23.3%) matched the inclusion criteria. Volume and type of fluids administered as well as outcome parameter were analysed. RESULTS: Between 2002 and 2012, the amount of volume administered during prehospital trauma care decreased from 1790 ml in 2002 to 1039 ml in 2012. At the same time higher haemoglobin mean values, higher Quick's mean values and reduced mean aPTT can be observed. Simultaneously, more patients received catecholamines (2002: 9.2 to 2012: 13.0%). Interestingly, the amount of volume administered decreased steadily regardless of the presence of shock. Fewer patients were in the need of blood products and the number of massive transfusions (≥10 pRBC) more than halved. DISCUSSION: The changes in volume therapy might have reduced haemodilution potentially resulting in an increase of the Hb value. During the period observed transfusion strategies have become more restrictiveand ratio based; the percentage of patients receiving MT halved as blood products may imply negative secondary effects. Furthermore, preventing administration of high blood product ratios result in less impairment of coagulation factors and inhibitors and an therfore improved coagulation. CONCLUSION: The volume administered in severely injured patients decreased considerably during the last decade possibly supporting beneficial effects such as minimizing the risk of coagulopathy and avoiding potential harmful effects caused by blood product transfusions. Despite outstanding questions in trauma resuscitation, principle evidence merges quickly into clinical practice and algorithms.

Diversity in clinical management and protocols for the treatment of major bleeding trauma patients across European level I Trauma Centres.

BACKGROUND: Uncontrolled haemorrhage is still the leading cause of preventable death after trauma and the primary focus of any treatment strategy should be related to early detection and control of blood loss including haemostasis. METHODS: For assessing management practices across six European level I trauma centres with academic interest and research in the field of coagulopathy an online survey was conducted addressing local management practice for bleeding trauma patients including algorithms for detection, management and monitoring coagulation disorders and immediate interventions. Each centre provided their locally applied massive transfusion protocol. RESULTS: All participating trauma centres have developed and implemented a local algorithm and protocol for the bleeding trauma patient. These are uniformly activated by clinical triggers and deactivated once the bleeding has stopped according to clinical assessment in combination with laboratory signs of achieved haemostasis. The severity of coagulopathy and shock is mostly assessed via standard coagulation tests and partially used extended viscoelastic tests. All centres have implemented the immediate use of tranexamic acid. Initial resuscitation is started either pre-hospital or after hospital admission by using transfusion packages with pre-fixed universal blood product combinations and ratios following the concept of "damage control resuscitation" at which applied ratios substantially vary. Two centres initially start with transfusion packages but with viscoelastic tests running in parallel to quickly allow a shift towards a viscoelastic test-guided therapy. CONCLUSION: Diversity in the management of bleeding trauma patients such as pre-hospital blood administration and routinely performed viscoelastic tests exists even among level I trauma centres. The paucity of consensus among these centres highlights the need for further primary research followed by clinical trials to improve the evidence for sophisticated guidelines and strategies.

Standardized bending and breaking test for the normal and osteoporotic metaphyseal tibias of the rat: effect of estradiol, testosterone, and raloxifene.

UNLABELLED: The fracture of bone plays a key role in osteoporosis. BMD measurement, however, is only an indirect parameter of this phenomenon. We therefore developed a highly sensitive three-point bending test for the metaphyseal tibias in rats to evaluate stiffness and strength. This was validated in a right-left comparison and a bioassay with soy-free food, estradiol, raloxifene, and testosterone in orchidectomized rats. INTRODUCTION: Osteoporosis becomes manifest predominantly in the metaphyseal rat tibia. The anti-osteoporotic character of substances should, therefore, be tested (mechanically) in this bone area. MATERIALS AND METHODS: We evaluated a new three-point bending test for the metaphyseal tibia in rats in a right-left trial. In an animal experiment, we studied the change of bone quality under estradiol (E)-, raloxifene (R)-, and testosterone (T)-supplemented food and compared it with trabecular BMD (qCT). RESULTS: In the right-left comparison, the mean difference between the metaphyseal loads of both tibias in 37 rats was 8.43% for the maximum load (Fmax) and 6.46% for the failure load (fL). These results show the high reproducibility of the test, because they are close to the usual intraindividual difference of the two extremities. In a second experiment, four groups of 11 3-month-old male orchidectomized rats were fed with soy-free food only (C) or with the additives E, T, or R for 12 weeks. E and R were similar for Fmax and fL. There were significant differences in the stiffness (E = 406.92 N/mm versus R = 332.08 N/mm), the yield load (yL; E = 99.17 N versus R = 83.33 N), and the ratio between yL and Fmax (E = 86.33% versus R = 76.37%). T was similar to the controls concerning F(max), fL, and stiffness. There were significant differences in yL (T = 49.00N versus C = 39.5N) and the ratio between yL and Fmax (T = 64.28% versus C = 51.28%). CONCLUSIONS: Estradiol is superior to raloxifene concerning stiffness and yield load, and both are superior to testosterone. We conclude that the described three-point bending test for the metaphyseal tibia is a highly sensitive method to study hormones and substances with regard to their osteoprotective character. The precision and the low SD of the presented results are superior to the data from qCT and the calculated index of stiffness (SSI).

Stem cell-coated titanium implants for the partial joint resurfacing of the knee.

The goal of the present study was to evaluate the partial surface replacement of the knee with stem cell-coated titanium implants and to provide a basis for a successful treatment of large osteochondral defects. Mesenchymal stem cells (MSCs) were isolated from bone marrow aspirates of adult sheep. Round titanium implants with a diameter of 2 x 7.3 mm were seeded with autologous MSC and inserted into an osteochondral defect in the medial femoral condyle. As controls, defects received either an uncoated implant or were left untreated. Nine animals with 18 defects were sacrificed after 6 months. Histological evaluation was performed by intravital polychrome fluorescent labelling, intravital perfusion with Indian ink, microradiographs and differential staining with toluidine blue. The quality of regenerated cartilage was assessed by in situ hybridization of collagen type II and immunohistochemistry of collagen types I and II. In 50% of the cases, defects treated with MSC-coated implants showed a complete regeneration of the subchondral bone layer. In these cases collagen type II and only traces of collagen type I were detected. A high level of collagen type II mRNA expression compared to articular cartilage indicates regenerating hyaline-like cartilage. A total of 50% of MSC-coated and uncoated implants failed to osseointegrate and formation of fibrocartilage was observed. Untreated defects as well as defects treated with uncoated implants demonstrated incomplete healing of subchondral bone and formation of fibrous cartilage. A modified histological score according to Wakitani significantly demonstrated better results for cell-coated implants (8.8+/-6.4) than for uncoated implants (5.5+/-3.9) and for untreated defects (2.8+/-2.5). Our results demonstrate that, in a significant number of cases, a partial joint resurfacing of the knee with stem cell-coated titanium implants occur. A slow bone and cartilage regeneration and an incomplete healing in half of the MSC-coated implants are limitations of the presented method. To improve our approach and optimize the experimental parameters, further investigations are needed prior to clinical application.