Mutual influence of selenium nanoparticles and FGF2-STAB® on biocompatible properties of collagen/chitosan 3D scaffolds : in vitro and ex ovo evaluation

In a biological system, nanoparticles (NPs) may interact with biomolecules. Specifically, the adsorption of proteins on the nanoparticle surface may influence both the nanoparticles' and proteins' overall bio-reactivity. Nevertheless, our knowledge of the biocompatibility and risk of exposure to nanomaterials is limited. Here, in vitro and ex ovo biocompatibility of naturally based crosslinked freeze-dried 3D porous collagen/chitosan scaffolds, modified with thermostable fibroblast growth factor 2 (FGF2-STAB®), to enhance healing and selenium nanoparticles (SeNPs) to provide antibacterial activity, were evaluated. Biocompatibility and cytotoxicity were tested in vitro using normal human dermal fibroblasts (NHDF) with scaffolds and SeNPs and FGF2-STAB® solutions. Metabolic activity assays indicated an antagonistic effect of SeNPs and FGF2-STAB® at high concentrations of SeNPs. The half-maximal inhibitory concentration (IC50) of SeNPs for NHDF was 18.9 µg/ml and IC80 was 5.6 µg/ml. The angiogenic properties of the scaffolds were monitored ex ovo using a chick chorioallantoic membrane (CAM) assay and the cytotoxicity of SeNPs over IC80 value was confirmed. Furthermore, the positive effect of FGF2-STAB® at very low concentrations (0.01 µg/ml) on NHDF metabolic activity was observed. Based on detailed in vitro testing, the optimal concentrations of additives in the scaffolds were determined, specifically 1 µg/ml of FGF2-STAB® and 1 µg/ml of SeNPs. The scaffolds were further subjected to antimicrobial tests, where an increase in selenium concentration in the collagen/chitosan scaffolds increased the antibacterial activity. This work highlights the antimicrobial ability and biocompatibility of newly developed crosslinked collagen/chitosan scaffolds involving FGF2-STAB® and SeNPs. Moreover, we suggest that these sponges could be used as scaffolds for growing cells in systems with low mechanical loading in tissue engineering, especially in dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration. Due to their antimicrobial properties, these scaffolds are also highly promising for tissue replacement requiring the prevention of infection

Healing and angiogenic properties of collagen/chitosan scaffolds enriched with hyperstable FGF2-STAB® protein: in vitro, ex ovo and in vivo comprehensive evaluation

Wound healing is a process regulated by a complex interaction of multiple growth factors including fibroblast growth factor 2 (FGF2). Although FGF2 appears in several tissue engineered studies, its applications are limited due to its low stability both in vitro and in vivo. Here, this shortcoming is overcome by a unique nine-point mutant of the low molecular weight isoform FGF2 retaining full biological activity even after twenty days at 37 °C. Crosslinked freeze-dried 3D porous collagen/chitosan scaffolds enriched with this hyper stable recombinant human protein named FGF2-STAB® were tested for in vitro biocompatibility and cytotoxicity using murine 3T3-A31 fibroblasts, for angiogenic potential using an ex ovo chick chorioallantoic membrane assay and for wound healing in vivo with 3-month old white New Zealand rabbits. Metabolic activity assays indicated the positive effect of FGF2-STAB® already at very low concentrations (0.01 µg/mL). The angiogenic properties examined ex ovo showed enhanced vascularization of the tested scaffolds. Histological evaluation and gene expression analysis by RT-qPCR proved newly formed granulation tissue at the place of a previous skin defect without significant inflammation infiltration in vivo. This work highlights the safety and biocompatibility of newly developed crosslinked collagen/chitosan scaffolds involving FGF2-STAB® protein. Moreover, these sponges could be used as scaffolds for growing cells for dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration

Adenylate Cyclase Toxin Promotes Internalisation of Integrins and Raft Components and Decreases Macrophage Adhesion Capacity

Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT) that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin) family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis

Folia2.qxp

Abstract. Heavy proteinuria may be caused by either increased glomerulal basement membrane permeability or membrane or podocyte structural damage, and also by impairment of secretion-reabsorption tubular processes. The precise composition of modified or degraded urine proteins in proteinuria is not known. However, a possible toxic effect of proteins on tubular cells and disease progression is assumed. In this study, 15 patients with nephrotic proteinuria and other diagnoses (systemic lupus erythematodes with renal involvement (lupus nephritis) and AAV) were analysed by the 2D electrophoresis method. We have studied sample stability during storage, the albumin separation effect on sample analyses using ammonium sulphate, and the effect of proteases on the protein spectrum. In the first step, the proteins were divided by the isoelectric focusing method using polyacrylamide strips (pH 3-10 linear). The second step involved twodimensional SDS electrophoresis performed in 12% polyacrylamide gel, which separated proteins according to their molecular weight. The proteins were visualized by the silver method. The gels were evaluated by Phoretix 2D expression software 2005. We found out that samples are stable for more than 6 months provided that they are frozen to -80ºC. The separation of albumin caused higher lucidity of the urinary proteomes. Without adding protease inhibitors we could detect proteolysis with increased quantity of proteins manifested in the area of about 10 kDa and decreased quantity of proteins detectable in the area with molecular weights about 50 kDa

Thermogelling water solutions of multifunctional macromonomers based on PLGA-PEG-PLGA triblock copolymers

Biodegradable thermosensitive triblock copolymers based on poly(ethylene glycol) and poly[(lactic acid)-co-(glycolic acid)](PLGA-PEG-PLGA) with PLGA/PEG weight ratio in the range of 1.5–3.0 and LA/GA molar ratio equal to 2.4 or 3.0 were prepared via ring opening polymerization (ROP). Prepared copolymers were subsequently modified in “one pot” by itaconic anhydride (ITA) in order to functionalize both ends with carboxylic acid groups and reactive double bonds. Chemical structure was characterized by means of gel permeation chromatography and nuclear magnetic resonance. Aqueous solutions of both modified and unmodified copolymers are able to form free flowing sol at room temperature and clear gel at temperature around 37°C. Therefore, sol-gel transitions of PLGA-PEG-PLGA and ITA/PLGA-PEG-PLGA/ITA copolymers in aqueous solutions were investigated using test tube inverting method. No mater if copolymer is modified or not, it was found that the critical gel temperature (CGT) increased when the PLGA/PEG weight ratio dropped from 2.5 to 2.0 and that the critical gel concentration (CGC) grew up with decreasing molar ratio of LA/GA from 3.0 up to 2.4. However, in all cases the ITA functionalization improved sol-gel characteristics of original PLGA-PEG-PLGA copolymer by approaching gel phase to body temperature. As a result, aqueous solution of ITA/PLGA-PEG-PLGA/ITA having LA/GA = 3 and PLGA/PEG = 2 with concentration higher than 6 wt% might be suitable material for biomedical applications as injectable temporary implants

Synergistic effect of bovine platelet lysate and various polysaccharides on the biological properties of collagen-based scaffolds for tissue engineering : scaffold preparation, chemo-physical characterization, in vitro and ex ovo evaluation

Crosslinked 3D porous collagen-polysaccharide scaffolds, prepared by freeze-drying, were modified with bovine platelet lysate (BPL) and evaluated in terms of chemical, physical and biological properties. Natural antibacterial polysaccharides like chitosan, chitin/chitosan-glucan complex and calcium salt of oxidized cellulose (CaOC) incorporated in collagen scaffolds affected not only chemo-physical properties of the composite scaffolds but also improved their biological properties, especially when BPL was presented. Lipophilic BPL formed microspheres in porous scaffolds while reduced by half their swelling ratio. The resistance of collagen sponges to hydrolytic degradation in water depended strongly on chemical crosslinking varying from 60 min to more than one year. According to in-vitro tests, chemically crosslinked scaffolds exhibited a good cellular response, cell–matrix interactions, and biocompatibility of the material. The combination of collagen with natural polysaccharides confirmed a significant positive synergistic effect on cultivation of cells as determined by MTS assay and PicoGreen method, as well as on angiogenesis evaluated by ex ovo Chick Chorioallantoic Membrane (CAM) assay. Contrary, modification only by BLP of pure collagen scaffolds exhibited decreased biocompatibility in comparison to unmodified pure collagen scaffold. We propose that the newly developed crosslinked collagen sponges involving bioactive additives could be used as scaffold for growing cells in systems with low mechanical loading in tissue engineering, especially in dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration