Synthesis of photo-crosslinkable hyaluronan with tailored degree of substitution suitable for production of water resistant nanofibers.

In this work, hyaluronan (HA) was grafted by a novel and an efficient mixed anhydrides methodology with (hetero)-aryl and aliphatic acrylic moieties suitable for cross-linking. A precise control of stoichiometry was achieved. Derivatives with degree of substitution (DS) below 20% did not show self-crosslinking. Due to mild reaction conditions, a negligible degradation of the polysaccharide was obtained. The influence of the feed components on the reaction efficiency and DS were studied up to 200 g/batch. The structure of the modified HA was characterized by Infrared Spectroscopy, Nuclear Magnetic Resonance, SEC-MALS and chromatographic analyses. Enzymatic degradation of derivatives was performed and isolated dimers demonstrated to be non-cytotoxic. The feasibility of the grafted HA for electrospinning with subsequent photo-crosslinking to avoid nanofibers water dissolution was demonstrated. The biocompatibility of the material, its degradation products, and the formation of honeycomb porous structures also proved the potential of the material for future in vivo applications.

α,ß-Unsaturated aldehyde of hyaluronan--Synthesis, analysis and applications.

Hyaluronic acid (HA) modified with an aldehyde group (HA-CHO or HA-aldehyde) has been extensively used for various biomedical applications. The main advantage of the aldehyde moieties is the ability to react with a wide range of amino compounds under physiological conditions. Reactions of aldehydes with primary amines in water are reversible and equilibrium is thoroughly shifted towards starting aldehyde and amine. This work presents an unique modification of HA: α,ß-unsaturated aldehyde of HA (4,5-anhydro-6(GlcNAc)-oxo HA or ΔHA-CHO), which allows the primary amines to be attached to HA more effectively in comparison to the saturated HA-CHO. Higher hydrolytic stability is caused by the conjugation of imine with an adjacent --C=C-- double bond. Two strategies for the preparation of unsaturated HA-aldehyde were developed and chemical structures were studied in details. Cross-linked materials prepared from this precursor are biocompatible and suitable for applications in drug delivery and regenerative medicine.

Solid-state photocrosslinking of hyaluronan microfibres.

Hyaluronan (HA) was chemically modified to a photocurable derivative by the acylation with a mixed anhydride of trans-cinnamic acid and characterized by UV, IR and 2D NMR spectroscopy. Photocurable HA was processed to a microfibrous structure by wet-spinning technology. Water solubility of otherwise water-soluble HA microfibres was reduced significantly by the solid-state photocrosslinking. We also investigated that the nature of polymer structure had a great impact to a final crosslink ratio. The analysis of the mechanical properties showed higher ultimate tensile strength and increased rigidity of the photocrosslinked fibres in comparison to the untreated ones. Moreover all tested materials are regarded as biocompatible according to the tests of cell viability, phototoxicity and enzymatic degradability, which make them suitable candidates for numerous biomedical applications.

Epilobium angustifolium extract demonstrates multiple effects on dermal fibroblasts in vitro and skin photo-protection in vivo.

Stress-induced fibroblast senescence is thought to contribute to skin aging. Ultraviolet light (UV) radiation is the most potent environmental risk factor in these processes. An Epilobium angustifolium (EA) extract was evaluated for its capacity to reverse the senescent response of normal human dermal fibroblasts (NHDF) in vitro and to exhibit skin photo-protection in vivo. The HPLC-UV-MS analysis of the EA preparation identified three major polyphenol groups: tannins (oenothein B), phenolic acids (gallic and chlorogenic acids) and flavonoids. EA extract increased the cell viability of senescent NHDF induced by serum deprivation. It diminished connective tissue growth factor and fibronectin gene expressions in senescent NHDF. Down-regulation of the UV-induced release of both matrix metalloproteinase-1 and -3 and the tissue inhibitor of matrix metalloproteinases-1 and -2, and also down-regulation of the gene expression of hyaluronidase 2 were observed in repeatedly UV-irradiated NHDF after EA extract treatment. Interestingly, EA extract diminished the down-regulation of sirtuin 1 dampened by UV-irradiation. The application of EA extract using a sub-irritating dose protected skin against UV-induced erythema formation in vivo. In summary, EA extract diminished stress-induced effects on NHDF, particularly on connective tissue growth factor, fibronectin and matrix metalloproteinases. These results collectively suggest that EA extract may possess anti-aging properties and that the EA polyphenols might account for these benefits.

Preparation of hyaluronan polyaldehyde-a precursor of biopolymer conjugates.

Native hyaluronan (HA) has been oxidized to polyaldehyde polymers with a degree of substitution (DS) of up to 50%. Two different procedures enabling the control of the degree of substitution were followed in this study. Selective oxidation of primary hydroxyl groups of N-acetyl-D-glucosamine of hyaluronan was performed either in an aqueous solution containing AcNH-TEMPO/NaBr/NaOCl or in an aprotic solvent containing Dess-Martin periodinane (DMP). It was found that a change of reaction parameters (reaction time and temperature, type of catalyst, oxidant-to-HA ratio, presence of nitrogen, buffer type, and concentration) had an influence on the degree of substitution and molecular weight. The derivatives were characterized by MS, NMR spectroscopy, and SEC-MALLS. Degradation of hyaluronic acid by the oxidant was observed and confirmed by SEC. The effect of oxidized derivatives of hyaluronan on cells was studied by means of NIH 3T3 fibroblast viability, which indicates that prepared hyaluronan polyaldehydes are biocompatible and suitable for medical applications and tissue engineering. The function of polyaldehyde as precursor for other modification was illustrated in the reaction with lysine.

Modulation of keratin 1, 10 and involucrin expression as part of the complex response of the human keratinocyte cell line HaCaT to ultraviolet radiation.

Skin exposure to ultraviolet (UV) light evokes a complex stress response in keratinocytes. Keratin filament organization provides structural stability and mechanical integrity of keratinocytes. Involucrin is a transglutaminase substrate protein contributing to the formation of insoluble cornified envelopes. However, a more complex role for keratins and involucrin has been proposed, including the regulation of cell stress response. The aim was to evaluate modulations of keratin 1, 10 and involucrin expression in HaCaT in the light of the complex response of these cells to UV-B radiation, including effects on c-Jun and matrix metalloproteinase 1 (MMP-1) gene expression and production of interleukin (IL) 6 and 8. A UV-B (300±5 nm) dose of 10 mJ/cm(2) was selected since this dose resulted in a partial decrease in cell viability in contrast to higher UV-B doses, which induced complete cell death 48 h after treatment. The UV-B radiation induced significant expression of keratin 1 and 10 and decreased expression of involucrin. This was accompanied by increased expression of c-Jun and MMP-1 and IL-6 and IL-8 production. The data suggest that the expression of keratin 1, 10 and involucrin is modulated in HaCaT keratinocytes as a part of the complex stress response to UV radiation.