Resistance of Bacteria toward 475 nm Blue Light Exposure and the Possible Role of the SOS Response.

The increase in antibiotic resistance represents a major global challenge for our health systems and calls for alternative treatment options, such as antimicrobial light-based therapies. Blue light has shown promising results regarding the inactivation of a variety of microorganisms; however, most often, antimicrobial blue light (aBL) therapy is performed using wavelengths close to the UV range. Here we investigated whether inactivation was possible using blue light with a wavelength of 475 nm. Both Gram-positive and -negative bacterial strains were treated with blue light with fluences of 7.5-45 J/cm2. Interestingly, only some bacterial strains were susceptible to 475 nm blue light, which was associated with the lack of RecA, i.e., a fully functional DNA repair mechanism. We demonstrated that the insertion of the gene recA reduced the susceptibility of otherwise responsive bacterial strains, indicating a protective mechanism conveyed by the bacterial SOS response. However, mitigating this pathway via three known RecA inhibiting molecules (ZnAc, curcumin, and Fe(III)-PcTs) did not result in an increase in bactericidal action. Nonetheless, creating synergistic effects by combining a multitarget therapy, such as aBL, with an RecA targeting treatment could be a promising strategy to overcome the dilemma of antibiotic resistance in the future.

Novel Human Placenta-Based Extract for Vascularization Strategies in Tissue Engineering.

There is critical unmet need for new vascularized tissues to support or replace injured tissues and organs. Various synthetic and natural materials were already established for use of two-dimensional (2D) and three-dimensional (3D) in vitro neovascularization assays, however, they still cannot mimic the complex functions of the sum of the extracellular matrix (ECM) in native intact tissue. Currently, this issue is only addressed by artificial products such as Matrigel™, which comprises a complex mixture of ECM proteins, extracted from animal tumor tissue. Despite its outstanding bioactivity, the isolation from tumor tissue hinders its translation into clinical applications. Since nonhuman ECM proteins may cause immune reactions, as are frequently observed in clinical trials, human ECM proteins represent the best option when aiming for clinical applications. Here, we describe an effective method of isolating a human placenta substrate (hpS) that induces the spontaneous formation of an interconnected network of green fluorescence-labeled human umbilical vein endothelial cells (gfpHUVECs) in vitro. The substrate was biochemically characterized by using a combination of bicinchoninic acid (BCA) assay, DNA, and glycosaminoglycan (GAG) content assays, sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis and Western blot, angiogenesis arrays, chromatographic thrombin detection, high performance liquid chromatography (HPLC)-based amino acid quantification analysis, and assessment of antimicrobial properties. 2D in vitro cell culture experiments have been performed to determine the vasculogenic potential of hpS, which demonstrated that cell networks developed on hpS show a significantly higher degree of complexity (number of tubules/junctions; total/mean tube length) when compared with Matrigel. As 3D cell culture techniques represent a more accurate representation of the in vivo condition, the substrate was 3D solidified using various natural polymers. 3D in vitro vasculogenesis assays have been performed by seeding gfpHUVECs in an hpS-fibrinogen clot. In conclusion, hpS provides a potent human/material-based alternative to xenogenic-material-based biomaterials for vascularization strategies in tissue engineering.

Antimicrobial photodynamic therapy fighting polymicrobial infections - a journey from in vitro to in vivo.

Rapidly evolving multidrug resistance renders conventional antimicrobial strategies increasingly inefficient. This urges the exploration of alternative strategies with a lower potential of resistance development to control microbial infections. A promising option is antimicrobial photodynamic therapy (aPDT), especially in the setting of wound infections. In this study its effectiveness was tested as a treatment option for polymicrobially infected wounds in both in vitro and in vivo models. First, aPDT was applied to wound-relevant Gram-positive and Gram-negative bacteria in planktonic culture as the standard in vitro test system and compared different media to show a possible dependency of the therapy on the surrounding environment. In a second step, aPDT was investigated in an in vitro model mimicking the wound bed conditions using fibrin-coated culture plates. Finally, we tested aPDT in vivo in a polymicrobial infected wound healing model in immunocompromised BALB/c mice. In vitro, it was shown that the bactericidal effectiveness of aPDT was strongly dependent on the surrounding environment of the phototoxic reaction. In vivo, the significant delay in wound healing induced by polymicrobial infection was drastically diminished by a two-times application of aPDT using 100 µM methylene blue (generally regarded as safe for topical application on human skin) and 24 J cm-2 pulsed red LED light. Our experiments suggest that aPDT is capable of significantly improving wound healing also in complicated polymicrobially infected wound situations.

Contamination of wounds with fecal bacteria in immuno-suppressed mice.

Immunocompromised patients are predisposed to chronically infected wounds. Especially ulcers in the dorsal region often experience secondary polymicrobial infections. However, current wound infection models mostly use single-strain bacteria. To mimic clinically occurring infections caused by fecal contamination in immunocompromised/immobile patients, which differ significantly from single-strain infections, the present study aimed at the establishment of a new mouse model using infection by fecal bacteria. Dorsal circular excision wounds in immunosuppressed mice were infected with fecal slurry solution in several dilutions up to 1:8,000. Impact of immunosuppressor, bacterial load and timing on development of wound infections was investigated. Wounds were analyzed by scoring, 3D imaging and swab analyses. Autofluorescence imaging was not successful. Dose-finding of cyclophosphamide-induced immunosuppression was necessary for establishment of bacterial wound infections. Infection with fecal slurry diluted 1:166 to 1:400 induced significantly delayed wound healing (p < 0.05) without systemic reactions. Swab analyses post-infection matched the initial polymicrobial suspension. The customized wound score confirmed significant differences between the groups (p < 0.05). Here we report the establishment of a simple, new mouse model for clinically occurring wound infections by fecal bacteria and the evaluation of appropriate wound analysis methods. In the future, this model will provide a suitable tool for the investigation of complex microbiological interactions and evaluation of new therapeutic approaches.

In vitro effects of 635 nm photobiomodulation under hypoxia/reoxygenation culture conditions.

Photobiomodulation (PBM), especially in the red wavelength range, has been demonstrated to be an effective treatment option for superficial and chronic wounds. However, ischemia and subsequent reperfusion can further challenge wound healing. Therefore, we investigated the effect of pulsed red LED light at 635 nm on cellular function in an in-vitro model of hypoxia/reoxygenation (H/R) challenge. Mouse myoblasts and fibroblasts were incubated in oxygen-deprived starvation medium (hypoxia) for 3 h after which the media was changed to oxygenated, fully supplemented media to simulate reperfusion. Cells were then treated with pulsed red LED light at a wavelength of 635 nm at 40 mW/cm2. Mitochondrial respiratory activity, ATP production and ROS levels were analysed immediately post-illumination. The effects on cellular metabolic activity and proliferation were measured at 6 h and 24 h and apoptosis/necrosis was measured at 24 h post-illumination. Our results show that both cell types reacted differently to H/R challenge and PBM. PBM of H/R-challenged cells enhanced mitochondrial activity and rescued decreased ATP levels, with significant effects in fibroblasts. This was associated with increased cell proliferation rates in both cell types. The increase was again more pronounced in fibroblasts. Our study concluded that PBM with red LED light significantly restored ATP levels during H/R and effectively promoted cell growth under both normoxic and H/R conditions. In clinical applications, PBM has been repeatedly reported to resolve difficult clinical situations in which ischemia/reperfusion injuries are a major issue. Our study confirms the beneficial effects of PBM especially in H/R-challenged cells.