Cornelia Wiegand

Comparative in vitro study on cytotoxicity, antimicrobial activity, and binding capacity for pathophysiological factors in chronic wounds of alginate and silver-containing alginate

Chronic wounds contain elevated levels of proteases, proinflammatory cytokines, and free radicals. The presence of bacteria further exaggerates the tissue‐damaging processes. For successful treatment, the wound dressing needs to manage wound exudates, create a moist environment, inhibit infection, bind pathophysiological factors that are detrimental to wound healing, and provide thermal isolation. Furthermore, it has to relieve pain, be easy to use, show no allergic potency, and not release toxic residues. The present study suggests a comprehensive in vitro approach to enable the assessment of wound dressings to support optimal conditions for wound healing. Three alginate‐based wound dressings: alginate alone, alginate containing ionic silver, and alginate with nanocrystalline silver, were tested for biocompatibility, antimicrobial activity, and influence on chronic wound parameters such as elastase, matrix metalloproteases‐2, tumor necrosis factor‐α, interleukin‐8, and free radical formation. Alginate was found to bind considerable amounts of elastase, reduce the concentration of proinflammatory cytokines and inhibit the formation of free radicals. Furthermore, alginate showed antibacterial activity and high biocompatibility. Incorporation of silver into alginate fibers increased antimicrobial activity and improved the binding affinity for elastase, matrix metalloproteases‐2, and the proinflammatory cytokines tested. Addition of silver also enhanced the antioxidant capacity. However, a distinct negative effect of silver‐containing alginates on human HaCaT keratinocytes was noted in vitro.

Active wound dressings based on bacterial nanocellulose as drug delivery system for octenidine

Although bacterial nanocellulose (BNC) may serve as an ideal wound dressing, it exhibits no antibacterial properties by itself. Therefore, in the present study BNC was functionalized with the antiseptic drug octenidine. Drug loading and release, mechanical characteristics, biocompatibility, and antimicrobial efficacy were investigated. Octenidine release was based on diffusion and swelling according to the Ritger-Peppas equation and characterized by a time dependent biphasic release profile, with a rapid release in the first 8h, followed by a slower release rate up to 96 h. The comparison between lab-scale and up-scale BNC identified thickness, water content, and the surface area to volume ratio as parameters which have an impact on the control of the release characteristics. Compression and tensile strength remained unchanged upon incorporation of octenidine in BNC. In biological assays, drug-loaded BNC demonstrated high biocompatibility in human keratinocytes and antimicrobial activity against Staphylococcus aureus. In a long-term storage test, the octenidine loaded in BNC was found to be stable, releasable, and biologically active over a period of 6 months without changes. In conclusion, octenidine loaded BNC presents a ready-to-use wound dressing for the treatment of infected wounds that can be stored over 6 months without losing its antibacterial activity.

Protease and pro-inflammatory cytokine concentrations are elevated in chronic compared to acute wounds and can be modulated by collagen type I in vitro

Physiological wound repair is a highly regulated, complex process, which leads to formation of new tissue after injury. However, the healing process is not perfect and healing impairments can occur. Delayed healing and formation of chronic wounds has been linked to the excessive production of proteolytic enzymes leading to reduced amounts of growth factors and successive destruction of the extracellular matrix. It has been implied that there is an alteration in the normal control mechanisms regulating the levels of these enzymes. The study presented provides data on the concentration of proteases and cytokines in wound fluid from chronic when compared with acute wounds. Levels of proteases such as PMN elastase, matrix metalloproteinases-2 (MMP-2), and MMP-13 were found to be profoundly elevated in chronic when compared with acute wound fluids. In addition, concentrations of IL-1β, IL-6, and IL-8 were shown to be significantly higher in chronic than in acute wounds. Furthermore, the ability of a wound dressing, consisting of bovine collagen type I, to bind pro-inflammatory cytokines was investigated. Collagen type I was able to bind significant amounts of the pro-inflammatory cytokines in vitro. Thus, it should be able to establish a more physiological wound milieu in vivo and promote healing.

HaCaT keratinocytes in co-culture with Staphylococcus aureus can be protected from bacterial damage by polihexanide

Wound healing is compromised by critical colonization and infection with bacteria. Hence, antimicrobial agents are used clinically to decrease the bacterial load and promote wound healing. Polihexanide (PHMB) has been found to be effective against a broad spectrum of micro‐organisms and is increasingly utilized in rinsing solutions or in combination with wound dressings because of its good biocompatibility. In the present study, a co‐culture of human keratinocytes and Staphylococcus aureus was established to serve as an in vitro model for infected wounds. Incubation of keratinocytes with increasing concentrations of S. aureus led to a dose‐dependent decline of cell viability and proliferation. Lactate dehydrogenase release and interleukin‐8 liberation were found to be elevated under these conditions. Polihexanide dose‐dependently was able to protect keratinocytes from bacterial damage and re‐establish normal human cell proliferation in vitro. Furthermore, a dressing consisting of biocellulose derived from Acetobacter xylinum with the addition of polihexanide was adept to safeguard keratinocytes against S. aureus. In conclusion, the co‐culture system presented embodies a valuable tool as a model system for infected cells in a non‐healing wound. Furthermore, the results obtained support the favorable function of polihexanide in the treatment of infected chronic wounds.

Effect of the sterilization method on the performance of collagen type I on chronic wound parameters in vitro

In the treatment of chronic wounds, it is necessary to establish a physiological wound milieu to improve healing. Application of collagen as wound dressing has been described as beneficial as it possesses the ability to reduce elevated levels of proteases, cytokines, and free radicals. Consequently, a wide range of wound dressings based on collagen have been developed. Native collagen is susceptible to alterations because of influences during the production process; to minimize effects on the molecule itself collagen wound dressings are usually aseptically produced. Common sterilization methods (autoclaving, irradiation, and ethylene oxide (EtO) treatment) can induce changes in the protein chemistry and physical properties, potentially affecting the absorption rate, mechanical strength, or performance. In this study, we have evaluated the influence of gamma- and beta-irradiation as well as EtO sterilization on the binding capacity of collagen type I for selected proteases and cytokines associated with nonhealing wounds. Although a pronounced effect on the physical properties of the collagen was found, there was no significant loss in the binding affinity for polymorphonuclear elastase, matrix metalloproteinase-2, and interleukin-1beta, or in the antioxidant capacity.

Antimicrobial porous hybrids consisting of bacterial nanocellulose and silver nanoparticles

The increasing resistance of pathogens and bacteria is a serious problem in the medical treatment of wounds and injuries. Therefore, new therapeutic agents are not solely based on antibiotics, but also on the use of antimicrobial metal nanoparticles. In this paper we present an innovative method to prepare porous hybrids consisting of bacterial nanocellulose (BNC) and silver nanoparticles (AgNPs). The stepwise modification is based on fairly simple chemical reactions already described for two-dimensional cellulose films. We transferred this method to the three-dimensional, porous network of BNC leading to an antimicrobial activation of its surface. Compared to former approaches, the ultrafine network structure of BNC is less damaged by using mild chemicals. The amount and distribution of the AgNPs on the modified BNC was investigated using scanning electron microscopy. The AgNPs are firmly immobilized on the top and bottom surface of the BNC by chemical interactions. Their size and quantity increase with an increasing concentration of AgNO3 and extended reaction time in the AgNO3 solution. A strong antimicrobial activity of the BNC-AgNP hybrids against Escherichia coli was detected. Furthermore, agar diffusion tests confirmed that this activity is restricted to the modified dressing itself, avoiding a release of NPs into the wound. Therefore, the produced hybrids could be potentially suited as novel antimicrobial wound dressings.

Molecular-Weight-Dependent Toxic Effects of Chitosans on the Human Keratinocyte Cell Line HaCaT

The cationic polysaccharide chitosan possesses bioactive properties such as antimicrobial activity, antitumor effects, hemostatic assets and positive effects on wound healing. The influence of polycations like chitosan on human cells has been reported to depend on their molecular weight. However, the mechanism of cytotoxicity caused by polycations is not yet fully understood. In the study presented, the influence of two chitosans with a similar degree of deacetylation but different molecular weight, chitosan 1130 (120 kDa) and chitosan oligosaccharide (5 kDa), on the human keratinocyte cell line HaCaT was analyzed. The results obtained indicate that chitosans exhibit a molecular-weight-dependent negative effect on HaCaT cell viability and proliferation in vitro. The chitosans tested also stimulated the release of inflammatory cytokines by HaCaT cells depending on incubation time and concentration. Chitosan 1130 and chitosan oligosaccharide induced apoptotic cell death which was mediated by activation of the effector caspases 3/7. At least for chitosan 1130, the involvement of both, extrinsic and intrinsic signal pathways, was shown by activation of caspases 8 and 9.

Poly(ethyleneimines) in dermal applications: biocompatibility and antimicrobial effects.

Cationic polyamines, such as poly(ethyleneimines) (PEIs), may recommend themselves for antimicrobial applications as they can interact with microbial membranes resulting in their disruption. The purpose of the study was the assessment of biocompatibility and antibacterial activity of PEIs with different architectures (branched (b) and linear (l)) and molar masses (0.8-750 kDa). lPEI and bPEI exhibited a strong antibacterial activity against Staphylococcus aureus and Escherichia coli with a more pronounced effect on the Gram-positive bacteria. lPEIs further demonstrated a higher antibacterial efficacy compared to bPEIs but no significant differences between 5 and 25 kDa were observed. In accordance, antibacterial activity of bPEI did not specifically depend on molar mass. Only slightly lower minimal inhibitory concentrations (MIC) were observed at 5 kDa (S. aureus) and 25 kDa (E. coli) in the tests. As PEIs are compelling candidates for use in antimicrobial treatment, two basic aspects have to be investigated: treatment effectiveness and safety. PEIs clearly induced molecular weight dependent cytotoxic effects in vitro. PEIs with low molecular weight (0.8 and 5 kDa) exhibited higher biocompatibility. Nonetheless, the results confirmed a low genotoxic potential of lPEI and bPEIs. In conclusion, 2.5 kDa-lPEI and 0.8 kDa-bPEI can be recommended for use as antimicrobial polymers in dermal applications due to their high biocompatibility with concomitant antibacterial efficacy.

Antibacterial properties of cyclodextrin–antiseptics-complexes determined by microplate laser nephelometry and ATP bioluminescence assay

Antimicrobial effects of substances can be determined with different methods that measure distinct parameters. Thus, a comparison of the results obtained can be difficult. In this study, two in vitro methods were employed to determine concentration and time dependent effects of cyclodextrin (CD)-complexes with the antiseptics chlorhexidine diacetate (CHX), iodine (IOD) and polihexanide (PHMB) on Candida albicans and Malassezia pachydermatis. Using both, microplate laser nephelometry and the ATP bioluminescence assay, it could be shown that CD-antiseptics-complexes tested exhibited significant antifungal effects with the exception of γ-CD-CHX in the case of C. albicans. Microplate laser nephelometry (MLN) is an optical method and enables a quantitative determination of particle concentrations in solution. By means of this method, microbial growth under influence of potential antimicrobial substances can be monitored over a prolonged time period. In addition, the antimicrobial activity was analyzed by measurement of the microbial adenosine triphosphate (ATP) content with a bioluminescent assay. The luminescent signal is directly proportional to the amount of ATP, and thus, a linear function of the number of living microbial cells present. Both methods were compared according to the half maximal inhibitory concentration (IC(50)) calculated and the statistical evaluation of Pearsons correlation coefficient (r). In summary, it could be demonstrated that both methods yield similar results although they differ in the parameter.

Identification of yeast isolated from dermatological patients by MALDI‐TOF mass spectrometry

Species identification of yeasts is based on biochemical (e.g. API ID 32 C®, bioMérieux) and molecular biological approaches. As an alternative to DNA‐dependent methods, mass spectral analysis based identification of micro‐organisms has become increasingly recognized. In a number of studies, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) has been applied for the rapid classification and identification of micro‐organisms. In this study, the applicability of MALDI‐TOF MS for identifying yeasts isolated from dermatological patients was analysed and compared with the results from the API ID 32 C® system. Furthermore, sequencing the internal transcribed spacer (ITS) regions of the ribosomal DNA was employed as reference method. Candida (C.) albicans was isolated in 41.9% of all cases, C. parapsilosis in 20.3%, C. glabrata in 10.8%, and C. krusei in 6, 8.1%. Rarely isolated yeasts were Candida colliculosa, famata, guilliermondii, lusitaniae, and tropicalis as well as Geotrichum candidum, Rhodotorula mucilaginosa and Trichosporon mucoides. The MALDI TOF results were equal to the results gained by ITS sequence analysis in 94%, whereas API ID 32 C® provided the correct diagnosis in 84.3% (of all cases). This lower identification rate is mostly referable to frequent misidentifications of C. krusei as C. inconspicua/norvegensis,Candida tropicalis, or Geotrichum capitatum. In contrast, all C. krusei strains were correctly identified by MALDI TOF MS. In conclusion, species identification by MALDI‐TOF MS was proven to be consistent with ITS sequence analysis; the technique has a resolving power comparatively as high as ITS sequence analysis.