The hyaluronan metabolism in the UV-irradiated human epidermis and the relevance of in vitro epidermal models.

Exposure to the sun affects the skin and may eventually result in UV-induced skin damage. It is generally known that hyaluronan (HA) is one of the main structural and functional components of the skin. However, UV-related changes in the HA metabolism in the skin have not yet been elucidated. Using qRT-PCR, confocal microscopy and LC-MS/MS we compared the naturally sun-exposed (SE), sun-protected, experimentally repeatedly UVA + UVB-exposed and acutely (once) UVA + UVB irradiated skin of Caucasian women. The epidermis was harvested by means of suction blistering 24 h after the acute irradiation. In addition, the epidermis was compared with a UV-irradiated in vitro reconstituted 3D epidermis (EpiDerm) and an in vitro 2D culture of normal human keratinocytes (NHEK). The amount of HA was found to be statistically significantly enhanced in the acutely irradiated epidermis. The acute UV evinced the upregulation of HA synthases (HAS2 and HAS3), hyaluronidases (HYAL2 and HYAL3), Cluster of differentiation 44 (CD44), and Cell Migration Inducing Proteins (CEMIP and CEMIP2), while only certain changes were recapitulated in the 3D epidermis. For the first time, we demonstrated the enhanced gene and protein expression of CEMIP and CEMIP2 following UV irradiation in the human epidermis. The data suggest that the HA metabolism is affected by UV in the irradiated epidermis and that the response can be modulated by the underlying dermis.

An in vitro model that mimics the foreign body response in the peritoneum: Study of the bioadhesive properties of HA-based materials.

A cascade of reactions known as the foreign body response (FBR) follows the implantation of biomaterials leading to the formation of a fibrotic capsule around the implant and subsequent health complications. The severity of the FBR is driven mostly by the physicochemical characteristics of implanted material, the method and place of implantation, and the degree of immune system activation. Here we present an in vitro model for assessing new materials with respect to their potential to induce a FBR in the peritoneum. The model is based on evaluating protein sorption and cell adhesion on the implanted material. We tested our model on the free-standing films prepared from hyaluronan derivatives with different hydrophobicity, swelling ratio, and rate of solubilization. The proteomic analysis of films incubated in the mouse peritoneum showed that the presence of fibrinogen was driving the cell adhesion. Neither the film surface hydrophobicity/hydrophilicity nor the quantity of adsorbed proteins were decisive for the induction of the long-term cell adhesion leading to the FBR, while the dissolution rate of the material proved to be a crucial factor. Our model thus helps determine the probability of a FBR to materials implanted in the peritoneum while limiting the need for in vivo animal testing.

Retinoic Acid Grafted to Hyaluronic Acid Activates Retinoid Gene Expression and Removes Cholesterol from Cellular Membranes.

All-trans-retinoic acid (atRA) is a potent ligand that regulates gene expression and is used to treat several skin disorders. Hyaluronic acid (HA) was previously conjugated with atRA (HA-atRA) to obtain a novel amphiphilic compound. HA-atRA forms micelles that incorporate hydrophobic molecules and facilitate their transport through the skin. The aim of this study was to determine the influence of HA-atRA on gene expression in skin cells and to compare it with that of unbound atRA. Gene expression was investigated using microarrays and a luciferase system with a canonical atRA promoter. HA-atRA upregulated gene expression similarly to atRA. However, HA-atRA activated the expression of cholesterol metabolism genes, unlike atRA. Further investigation using HPLC and filipin III staining suggested that the treated cells induced cholesterol synthesis to replenish the cholesterol removed from the cells by HA-atRA. HA modified with oleate (HA-C18:1) removed cholesterol from the cells similarly to HA-atRA, suggesting that the cholesterol removal stemmed from the amphiphilic nature of the two derivatives. HA-atRA induces retinoid signaling. Thus, HA-atRA could be used to treat skin diseases, such as acne and psoriasis, where the combined action of atRA signaling and anti-inflammatory cholesterol removal may be potentially beneficial.

Silver distribution in chronic wounds and the healing dynamics of chronic wounds treated with dressings containing silver and octenidine.

Although silver is an efficient antimicrobial and is a widely used antiseptic in wound healing, previous studies have reported the cytotoxic in vitro effects of silver dressings. Moreover, few studies have addressed the distribution of silver in chronic wounds. The study compares the healing of chronic wounds treated with a standard-of-care silver dressing (Ag-CMC) and a dressing containing antiseptic octenidine (OCT-HA). Biopsies were taken from two wound areas before the commencement of treatment (baseline), after 2 weeks and after 6 weeks (the end of the study). We analyzed the histopathologic wound-healing score, silver distribution, and expression of selected genes. The wound-healing score improved significantly in the wounded area treated with OCT-HA after 2 weeks compared to the baseline and the Ag-CMC. The Ag-CMC wound areas improved after 6 weeks compared to the baseline. Moreover, collagen maturation and decreases in the granulocyte and macrophage counts were faster in the OCT-HA parts. Treatment with OCT-HA resulted in less wound slough. The silver, visualized via autometallography, penetrated approximately 2 mm into the wound tissue and associated around capillaries and ECM fibers, and was detected in phagocytes. The metallothionein gene expression was elevated in the Ag-CMC wound parts. This exploratory study determined the penetration of silver into human chronic wounds and changes in the distribution thereof during treatment. We observed that silver directly affects the cells in the wound and elevates the metallothionein gene expression. Octenidine and hyaluronan dressings provide a suitable alternative to silver and carboxymethyl cellulose dressings without supplying silver to the wound.

In vitro investigation of hyaluronan-based polymeric micelles for drug delivery into the skin: The internalization pathway.

In our previous research, we concluded that polymeric micelles based on hyaluronic acid are able to penetrate into the deeper layers of skin tissue. The aim of this work was to characterize the mechanisms involved in the uptake by skin cells, which is important for understanding the influence of the carrier composition on the drug penetration. To reach this goal, we used micelles encapsulating curcumin made of oleyl-hyaluronan (HAC18:1) and hexyl-hyaluronan (HAC6) covalently linked with fluorescent Nile Blue. This labeling enabled us to track the micelle-forming derivative and also micelle payload into the keratinocytes and fibroblasts by fluorescent microscopy and flow cytometry. The regulation of both the passive and active cellular uptake was used to determine the mechanism of micelle internalization. Furthermore, the changes of membrane fluidity were measured for these derivatives by FRAP. Using these methods we concluded that carriers entered the cells using both active and passive transport. Passive transport was facilitated by the affinity of the carrier to the cell membrane, especially in the case of HAC18:1 carrier, which changed significantly the membrane fluidity. The active transport was dependent on cell type, but mainly driven by the clathrin-mediated endocytosis and macropinocytosis. Surprisingly, the main HA receptor, CD44, was not involved in the uptake. We can conclude that these carrier systems could be used for the local transport of active substances or hydrophobic drugs into the skin cells using the advantage of passive transport of oleyl-HA derivative.

Optimization of cell growth on palmitoyl-hyaluronan knitted scaffolds developed for tissue engineering applications.

Polysaccharides meet several criteria for a suitable biomaterial for tissue engineering, which include biocompatibility and ability to support the delivery and growth of cells. Nevertheless, most of these polysaccharides, for example dextran, alginate, and glycosaminoglycans, are highly soluble in aqueous solutions. Hyaluronic acid hydrophobized by palmitic acid and processed to the form of wet-spun fibers and the warp-knitted textile scaffold is water non-soluble, but biodegradable material, which could be used for the tissue engineering purpose. However, its surface quality does not allow cell attachment. To enhance the biocompatibility the surface of palmitoyl-hyaluronan was roughened by freeze drying and treated by different cell adhesive proteins (fibronectin, fibrinogen, laminin, methacrylated gelatin and collagen IV). Except for collagen IV, these proteins covered the fibers uniformly for an extended period of time and supported the adhesion and cultivation of dermal fibroblasts and mesenchymal stem cells. Interestingly, adipose stem cells cultivated on the fibronectin-modified scaffold secreted increasing amount of HGF, SDF-1, and VEGF, three key growth factors involved in cardiac regeneration. These results suggested that palmitoyl-hyaluronan scaffold may be a promising material for various applications in tissue regeneration, including cardiac tissue repair. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1488-1499, 2018.

Segmentation of Total Cell Area in Brightfield Microscopy Images.

Segmentation is one of the most important steps in microscopy image analysis. Unfortunately, most of the methods use fluorescence images for this task, which is not suitable for analysis that requires a knowledge of area occupied by cells and an experimental design that does not allow necessary labeling. In this protocol, we present a simple method, based on edge detection and morphological operations, that separates total area occupied by cells from the background using only brightfield channel image. The resulting segmented picture can be further used as a mask for fluorescence quantification and other analyses. The whole procedure is carried out in open source software Fiji.

A novel photopolymerizable derivative of hyaluronan for designed hydrogel formation.

A new photopolymerizable derivative of hyaluronan (methacrylhydrazide-HA, MAHA) was prepared by carbodiimide chemistry. The reaction conditions were optimized for molecular weight (Mw), reaction time and amount of reagents with a degree of methacrylation (DM) ranging from 2% to 58%. Methacrylhydrazide-HA was hydrolytically stable (PBS, 7days, 37°C) in contrast to commonly used methacrylester analoque (23% hydrolyzed). MAHA readily photopolymerized into densely crosslinked hydrogels under physiological conditions. The varied DM, Mw, irradiation time (texp) and macromer concentration in photocrosslinking afforded hydrogels with different physical (swelling ratio, degradation rate) and mechanical properties (stiffness, toughness). Three-dimensional fabrication and surface patterning of MAHA hydrogels were demonstrated by photolithography and light mediated micromolding. A live-dead assay with skin fibroblasts showed convenient biocompatibility of MAHA (16%, 116kDa) for potential scaffolding applications in tissue engineering and regenerative medicine.

Hyaluronan polymeric micelles for topical drug delivery.

Nanosized materials offer promising strategy for topical drug delivery due to their enhancing effect on drug percutaneous transport across the stratum corneum barrier. In this work, polymeric micelles made from hydrophobized hyaluronic acid (HA) were probed for skin delivery. Compared to non-polymeric micelle solutions containing similar drug amount, in vitro skin penetration analysis indicated 3 times larger deposition of drug in the epidermis and 6 times larger drug deposition in the dermis after 5h of topical treatment in Franz diffusion cells. The drug deposition was further increased with prolonged time of topical treatment. Laser confocal microscopy revealed the accumulation of both, the HA forming the vehicle and the payload, in the epidermis and dermis. Although fluorescent labeling of the HA would suggest co-transport of the HA and the drug, loading FRET pair dyes in the micellar core clearly demonstrated gradual micelle disruption with increasing skin depth. Transcellular penetration was the predominant pathway for the loaded drug. The HA polymeric micelles also demonstrated increased bioactivity of loaded compound in vitro and in vivo. In addition, the loaded micelles were found to be stable in cream formulations and thus they have great potential for topical applications for cosmetic and pharmaceutical purposes.

Fabrication of biodegradable textile scaffold based on hydrophobized hyaluronic acid.

In this work, we report on the preparation of a novel biodegradable textile scaffold made of palmitoyl-hyaluronan (palHA). Monofilament fibres of palHA with a diameter of 120µm were prepared by wet spinning. The wet-spun fibres were subsequently processed into a warp-knitted textile. To find a compromise between swelling in water and degradability of the final textile scaffold, a series of palHA derivatives with different degrees of substitution of the palmitoyl chain was synthesized. Freeze-drying not only provided shape fixation, but also speeded up scaffold degradation in vitro. Fibronectin, fibrinogen, laminin and collagen IV were physically adsorbed on the textile surface to enhance cell adhesion on the material. The highest amount of adsorbed cell-adhesive proteins was achieved with fibronectin (89%), followed by fibrinogen (81%). Finally, textiles modified with fibronectin or fibrinogen both supported the adhesion and proliferation of normal human fibroblasts in vitro, proving to be a useful cellular scaffold for tissue engineering.