This work explores application of phase separation phenomena for structuring of films made from hyaluronan. A time-sequenced dispensing of different solution mixtures was applied under rotation of hyaluronan-covered substrates to generate surface textures. This method is applicable in direct surface modification or cover layer deposition. Changes in the surface topography were characterized by atomic force microscopy, optical microscopy, and contact and non-contact profilometry. The mechanical properties of the surface-modified self-supporting films were compared using a universal testing machine. Experimental results show that diverse hyaluronan-based surface reliefs and self-supporting films with improved mechanical properties can be prepared using a newly designed multi-step phase separation process without the need for sacrificial removable templates or additives.

Tracer diffusion coefficients obtained from the Taylor dispersion technique at 25.0 °C were measured to study the influence of sodium, ammonium and magnesium salts at 0.01 and 0.1 mol dm-3 on the transport behavior of sodium hyaluronate (NaHy, 0.1%). The selection of these salts was based on their position in Hofmeister series, which describe the specific influence of different ions (cations and anions) on some physicochemical properties of a system that can be interpreted as a salting-in or salting-out effect. In our case, in general, an increase in the ionic strength (i.e., concentrations at 0.01 mol dm-3) led to a significant decrease in the limiting diffusion coefficient of the NaHy 0.1%, indicating, in those circumstances, the presence of salting-in effects. However, the opposite effect (salting-out) was verified with the increase in concentration of some salts, mainly for NH4SCN at 0.1 mol dm-3. In this particular salt, the cation is weakly hydrated and, consequently, its presence does not favor interactions between NaHy and water molecules, promoting, in those circumstances, less resistance to the movement of NaHy and thus to the increase of its diffusion (19%). These data, complemented by viscosity measurements, permit us to have a better understanding about the effect of these salts on the transport behaviour of NaHy.

The ways of producing porous-like textured surfaces with chemical etching on aluminum-alloy substrates were studied. The most appropriate etchants, their combination, temperature, and etching time period were explored. The influence of a specifically textured surface on adhesive joints' strength or superhydrophobic properties was evaluated. The samples were examined with scanning electron microscopy, profilometry, atomic force microscopy, goniometry, and tensile testing. It was found that, with the multistep etching process, the substrate can be effectively modified and textured to the same morphology, regardless of the initial surface roughness. By selecting proper etchants and their sequence one can prepare new types of highly adhesive or even superhydrophobic surfaces.

Bio-inspired conductive scaffolds composed of sodium hyaluronate containing a colloidal dispersion of water-miscible polyaniline or polypyrrole particles (concentrations of 0.108, 0.054 and 0.036% w/w) were manufactured. For this purpose, either crosslinking with N-(3-dimethylaminopropyl-N-ethylcarbodiimide hydrochloride and N-hydroxysuccinimid or a freeze-thawing process in the presence of poly(vinylalcohol) was used. The scaffolds comprised interconnected pores with prevailing porosity values of ~ 30% and pore sizes enabling the accommodation of cells. A swelling capacity of 92-97% without any sign of disintegration was typical for all samples. The elasticity modulus depended on the composition of the scaffolds, with the highest value of ~ 50 kPa obtained for the sample containing the highest content of polypyrrole particles. The scaffolds did not possess cytotoxicity and allowed cell adhesion and growth on the surface. Using the in vivo-mimicking conditions in a bioreactor, cells were also able to grow into the structure of the scaffolds. The technique of scaffold preparation used here thus overcomes the limitations of conductive polymers (e.g. poor solubility in an aqueous environment, and limited miscibility with other hydrophilic polymer matrices) and moreover leads to the preparation of cytocompatible scaffolds with potentially cell-instructive properties, which may be of advantage in the healing of damaged electro-sensitive tissues.

Essential features of well-designed materials intended for 3D bioprinting via microextrusion are the appropriate rheological behavior and cell-friendly environment. Despite the rapid development, few materials are utilizable as bioinks. The aim of our work was to design a novel cytocompatible material facilitating extrusion-based 3D printing while maintaining a relatively simple and straightforward preparation process without the need for harsh chemicals or radiation. Specifically, hydrogels were prepared from gelatines coming from three sources-bovine, rabbit, and chicken-cross-linked by dextran polyaldehyde. The influence of dextran concentration on the properties of hydrogels was studied. Rheological measurements not only confirmed the strong shear-thinning behavior of prepared inks but were also used for capturing cross-linking reaction kinetics and demonstrated quick achievement of gelation point (in most cases < 3 min). Their viscoelastic properties allowed satisfactory extrusion, forming a self-supported multi-layered uniformly porous structure. All gelatin-based hydrogels were non-cytototoxic. Homogeneous cells distribution within the printed scaffold was confirmed by fluorescence confocal microscopy. In addition, no disruption of cells structure was observed. The results demonstrate the great potential of the presented hydrogels for applications related to 3D bioprinting.

An innovative multi-step phase separation process was used to prepare tissue culture for the polystyrene-based, hierarchically structured substrates, which mimicked in vivo microenvironment and architecture. Macro- (pore area from 3000 to 18,000 µm2; roughness (Ra) 7.2 ± 0.1 µm) and meso- (pore area from 50 to 300 µm2; Ra 1.1 ± 0.1 µm) structured substrates covered with micro-pores (area around 3 µm2) were prepared and characterised. Both types of substrate were suitable for human-induced pluripotent stem cell (hiPSC) cultivation and were found to be beneficial for the induction of cardiomyogenesis in hiPSC. This was confirmed both by the number of promoted proliferated cells and the expressions of specific markers (Nkx2.5, MYH6, MYL2, and MYL7). Moreover, the substrates amplified the fluorescence signal when Ca2+ flow was monitored. This property, together with cytocompatibility, make this material especially suitable for in vitro studies of cell/material interactions within tissue-mimicking environments.

In the current study, we present methods of sodium hyaluronate, also denoted as hyaluronan (HA), nanofiber fabrication using a direct-current (DC) electric field. HA was spun in combination with poly(vinyl alcohol) (PVA) and polyethylene oxide (PEO) and as a pure polymer. Nonaggressive solvents were used due to the possible use of the fibers in life sciences. The influences of polymer concentration, average molecular weight (Mw), viscosity, and solution surface tension were analyzed. HA and PVA were fluorescent-labeled in order to examine the electrospun structures using fluorescence confocal microscopy. In this study, two intermediate solvent mixtures that facilitate HA electrospinning were found. In the case of polymer co-electrospinning, the effect of the surfactant content on the HA/PVA electrospinning process, and the effect of HA Mw on HA/PEO nanofiber morphology, were examined, respectively.

The pseudo 3D hierarchical structure mimicking in vivo microenvironment was prepared by phase separation on tissue culture plastic. For surface treatment, time-sequenced dosing of the solvent mixture with various concentrations of polymer component was used. The experiments showed that hierarchically structured surfaces with macro, meso and micro pores can be prepared with multi-step phase separation processes. Changes in polystyrene surface topography were characterized by atomic force microscopy, scanning electron microscopy and contact profilometry. The cell proliferation and changes in cell morphology were tested on the prepared structured surfaces. Four types of cell lines were used for the determination of impact of the 3D architecture on the cell behavior, namely the mouse embryonic fibroblast, human lung carcinoma, primary human keratinocyte and mouse embryonic stem cells. The increase of proliferation of embryonic stem cells and mouse fibroblasts was the most remarkable. Moreover, the embryonic stem cells express different morphology when cultured on the structured surface. The acquired findings expand the current state of knowledge in the field of cell behavior on structured surfaces and bring new technological procedures leading to their preparation without the use of problematic temporary templates or additives.