Controlled Structuring of Hyaluronan Films by Phase Separation and Inversion.

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.

Synthesis and Exfoliation of Calcium Organophosphonates for Tailoring Rheological Properties of Sodium Alginate Solutions: A Path toward Polysaccharide-Based Bioink.

Layered nanoparticles with surface charge are explored as rheological modifiers for extrudable materials, utilizing their ability to induce electrostatic repulsion and create a house-of-cards structure. These nanoparticles provide mechanical support to the polymer matrix, resulting in increased viscosity and storage modulus. Moreover, their advantageous aspect ratio allows for shear-induced orientation and decreased viscosity during flow. In this work, we present a synthesis and liquid-based exfoliation procedure of phenylphosphonate-phosphate particles with enhanced ability to be intercalated by hydrophilic polymers. These layered nanoparticles are then tested as rheological modifiers of sodium alginate. The effective rheological modification is proved as the viscosity increases from 101 up to 103 Pa·s in steady state. Also, shear-thinning behavior is observed. The resulting nanocomposite hydrogels show potential as an extrudable bioink for 3D printing in tissue engineering and other biomedical applications, with good shape fidelity, nontoxicity, and satisfactory cell viability confirmed through encapsulation and printing of mouse fibroblasts.

Interactions between Sodium Hyaluronate and ß-Cyclodextrin as Seen by Transport Properties.

Knowledge of mass transport parameters, diffusion, and viscosity of hyaluronic acid (HA) in the presence of cyclodextrins is of considerable importance for areas such as food packaging and drug delivery, among others. Despite a number of studies investigating the functionalization of HA or the corresponding sodium salt by cyclodextrins, only a few studies have reported the effect of cyclodextrins on the mass transport of HA in the presence of these oligosaccharides. Here, we report the tracer binary and ternary interdiffusion coefficients of sodium hyaluronate (NaHy) in water and aqueous ß-cyclodextrin solutions. The diffusion behavior of sodium hyaluronate was dependent on the reduced viscosity of NaHy, which, in turn, presented a concave dependence on concentration, with a minimum at approximately 2.5 g dm-3. The significant decrease in the limiting diffusion coefficient of NaHy (at most 45%) at NaHy concentrations below 1 g dm-3 in the presence of ß-cyclodextrin, taking water as the reference, allowed us to conclude that NaHy strongly interacted with the cyclodextrin.

Formulation of Magneto-Responsive Hydrogels from Dually Cross-Linked Polysaccharides: Synthesis, Tuning and Evaluation of Rheological Properties.

Smart hydrogels based on natural polymers present an opportunity to fabricate responsive scaffolds that provide an immediate and reversible reaction to a given stimulus. Modulation of mechanical characteristics is especially interesting in myocyte cultivation, and can be achieved by magnetically controlled stiffening. Here, hyaluronan hydrogels with carbonyl iron particles as a magnetic filler are prepared in a low-toxicity process. Desired mechanical behaviour is achieved using a combination of two cross-linking routes-dynamic Schiff base linkages and ionic cross-linking. We found that gelation time is greatly affected by polymer chain conformation. This factor can surpass the influence of the number of reactive sites, shortening gelation from 5 h to 20 min. Ionic cross-linking efficiency increased with the number of carboxyl groups and led to the storage modulus reaching 103 Pa compared to 101 Pa-102 Pa for gels cross-linked with only Schiff bases. Furthermore, the ability of magnetic particles to induce significant stiffening of the hydrogel through the magnetorheological effect is confirmed, as a 103-times higher storage modulus is achieved in an external magnetic field of 842 kA·m-1. Finally, cytotoxicity testing confirms the ability to produce hydrogels that provide over 75% relative cell viability. Therefore, dual cross-linked hyaluronan-based magneto-responsive hydrogels present a potential material for on-demand mechanically tunable scaffolds usable in myocyte cultivation.

New approach to prepare cytocompatible 3D scaffolds via the combination of sodium hyaluronate and colloidal particles of conductive polymers.

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.

Hierarchically Structured Surfaces Prepared by Phase Separation: Tissue Mimicking Culture Substrate.

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.

Cross-Linked Gelatine by Modified Dextran as a Potential Bioink Prepared by a Simple and Non-Toxic Process.

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.

Surface Modification of Metallic Inserts for Enhancing Adhesion at the Metal-Polymer Interface.

A combination of mechanical and chemical treatments was utilized to modify the surface textures of copper and duralumin inserts in order to enhance the adhesion at the metal-polymer interface and provide an adhesive joint with a high loadbearing capacity. Pretreatment of the surfaces with sandblasting was followed by etching with various chemical mixtures. The resulting surface textures were evaluated with a scanning electron microscope (SEM) and an optical confocal microscope. Surface geometry parameters (Sa, Sz, and Sdr) were measured and their relationships to the adhesion joint strength were studied. It was found that the virgin and purely mechanically treated inserts resulted in joints with poor loadbearing capacity, while a hundredfold (duralumin) and ninetyfold (copper) increase in the force to break was observed for some combinations of mechanical and chemical treatments. It was determined that the critical factor is overcoming a certain surface roughness threshold with the mechanical pretreatment to maximize the potential of the mechanical/chemical approach for the particular combination of material and etchant.

Hierarchically Structured Polystyrene-Based Surfaces Amplifying Fluorescence Signals: Cytocompatibility with Human Induced Pluripotent Stem Cell.

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.

Effect of Hofmeister Ions on Transport Properties of Aqueous Solutions of Sodium Hyaluronate.

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.

Factors determining self-assembly of hyaluronan.

The conditions determining network-forming and aggregation properties of hyaluronan on the mica surface were studied. The hyaluronan was deposited on the surface from aqueous and saline solutions and attached by a bivalent cation. The morphology of the immobilized assemblies was characterized by atomic force microscopy. The experimental results show that the morphology and size of the aggregates as well as the density of the interconnecting fibrillar network, both made of hyaluronan, at the liquid-solid phase interface are determined not only by its molecular weight or concentration in solution, but also by the dissolution conditions and storage time. These findings extend the current state of knowledge about the conformational variability of this biologically important polymer. Understanding the conformational variability is of great importance, as it governs the physiological functions of hyaluronan, as well as its processability and formulations. That in turn determines its usability in different pharmacological and biomaterial applications.

Electrospinning of Hyaluronan Using Polymer Coelectrospinning and Intermediate Solvent.

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 behaviour of hyaluronan solutions in the presence of Hofmeister ions: A light scattering, viscometry and surface tension study.

Dynamic light scattering (DLS), viscosity and surface tension (SFT) measurements were used to characterize influence of salts containing ions of Hofmeister series (Na2SO4, (NH4)2SO4, NaSCN, NH4SCN and NaCl) on the behaviour of hyaluronan in diluted solutions at a temperature range of 15-45 °C. The results of the study showed that chaotropic and kosmotropic ions notably influenced the folding and unfolding of hyaluronan coils due to interactions between a respective ion and hydrophilic or hydrophobic patches present in the backbone of the polymer chains. This was mainly proved by viscosity and light scattering measurements. The temperature dependence of the hydrodynamic diameter of the hyaluronan coil determined by DLS demonstrated that combinations of chaotropic and kosmotropic ions in one salt (NaCl, NaSCN and (HN4)2SO4) can stabilize the size of the coil in a wide range of temperatures. Tensiometry measurements indicated that certain types of ions present in the solution caused an unfolding of the hyaluronan coils, leading to a decrease of SFT.

Preparation of Textured Surfaces on Aluminum-Alloy Substrates.

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.

Hyaluronan hydrogels modified by glycinated Kraft lignin: Morphology, swelling, viscoelastic properties and biocompatibility.

Effects of the addition of water soluble glycinated Kraft lignin (WS/KL) on the mechanical stability and biocompatibility of hyaluronan (NaHy) hydrogels were evaluated in this work. Water soluble lignin was obtained by the modification of Kraft lignin via a Mannich reaction. It was found that WS/KL is highly compatible with hyaluronan due to its improved water solubility, which favours its use in designing new advanced composite hydrogels. The effects of the concentration of WS/KL on morphological, swelling and creep/recovery behaviours of hyaluronan hydrogels were investigated. It was detected that the creep resistance and creep recovery of NaHy hydrogels was improved by the incorporation of up to 3% (w/w) of WS/KL. In contrast, the swelling capacity of hydrogels was decreased. The cytotoxicity tests proved that glycinated KL lignin limits the viability of cells only slightly, and the final hyaluronan/lignin hydrogels were non-toxic materials.

Variations of Polymer Porous Surface Structures via the Time-Sequenced Dosing of Mixed Solvents.

A new approach to polystyrene surface treatment via the time-sequenced dispensing of good and poor solvent mixtures on the rotating surface of treated substrate is presented in this study. It is demonstrated that the variation of the sequencing together with other variables (e.g., temperature and solvent concentration) affects the size and depth of pores evolving on the polystyrene surface. A model of the surface pore creation, associated with the viscoelastic phase separation, surface tension, and secondary flows caused by temperature variations and the rapid evaporation of the good solvent is proposed. Experimental results of profilometric, goniometric, and optical measurements show that this approach enables the simple and quick preparation of surfaces with various numbers, diameters, and depths of individual pores, which ultimately affects not only the wetting characteristics of the surfaces but also the fate of cells cultivated there. The presented method allows the easy preparation of a large number of structured substrates for effective cell cultivation and proliferation.

Characterization at 25 °C of sodium hyaluronate in aqueous solutions obtained by transport techniques.

Mutual diffusion coefficients, D, were determined for aqueous solutions of sodium hyaluronate (NaHy) at 25 °C and concentrations ranging from 0.00 to 1.00 g·dm(-3) using the Taylor dispersion technique. From these experimental data, it was possible to estimate some parameters, such as the hydrodynamic radius Rh, and the diffusion coefficient at infinitesimal concentration, D0, of hyaluronate ion, permitting us to have a better understanding of the structure of these systems of sodium hyaluronate in aqueous solutions. The additional viscosity measurements were done and Huggins constant, kH, and limiting viscosity number, [η], were computed for interaction NaHy/water and NaHy/NaHy determination.

Viscoelastic and mechanical properties of hyaluronan films and hydrogels modified by carbodiimide.

This study investigated an effect of different ways of the preparation of insoluble hyaluronan material on its mechanical and viscoelastic properties. Hyaluronan (NaHy) of molecular weight Mw=500,000 g mol(-1) was modified with N-(3-dimethylaminopropyl-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), to be able absorb liquid without changing its mechanical properties. The modified, water insoluble NaHy materials were prepared in different geometry; as modified films and modified cylinders with exact dimensions. The occurrence of modification was confirmed by FT-IR (Fourier transform infrared spectroscopy) and (1)H NMR (proton nuclear magnetic resonance) spectroscopy and swelling test. The determined mechanical and viscoelastic properties of unmodified and modified hyaluronan revealed the high dependency of elasticity changes depending on the gel processing method. Moreover, NaHy gels in the cylindrical form with the sponge-like structure predominant them as a convenient geometry for application in a humid environment.

The influence of quarternary salt on hyaluronan conformation and particle size in solution.

Interaction of hyaluronan (NaHy) with the quaternary salt, benzalkonium chloride (BAC), was studied. Based on the DLS experiments, viscometry and surface tension measurements executed on hyaluronan samples with two molecular weights of Mw=1.8 MDa and Mw=0.35 MDa, the hypothesis was proposed suggesting that at certain BAC concentrations, hyaluronan can form aggregates, which lead to increase of the polymer coil size measured as z-average diameter. Moreover, it was confirmed that within the whole range of BAC concentrations, repeated variations in size and conformations of polymer coils occur, being connected with the critical micellar concentration of BAC and with hydrophobic interactions of non-polar segments of BAC with hyaluronan hydrophobic domains. Tensiometry, DLS and viscometry data support the assumption that variations of thermodynamical "favourability" of BAC-BAC or NaHy-BAC interactions take place in hyaluronan solutions, based on the BAC concentration.

The measurement of polymer swelling processes by an interferometric method and evaluation of diffusion coefficients.

The amorphous polymer film swelling in a liquid solvent below the glass transition temperature was characterized by a few kinetic parameters (especially the mutual diffusion coefficient of swelling and its mean value) obtained by interference of monochromatic light in the wedge arrangement. This interferometric method allows one to determine the concentration field in the swollen surface layer and consequently to compute the concentration-dependent diffusion coefficient. A software system developed at the Department of Physics and Material Engineering at TBU in Zlin has been used for the evaluation of the main kinetic parameters of the swelling process. The software can be used for the on-line analyses of interferograms during the swelling process. The main application outputs are the computation of the concentration profile, the concentration gradient, the mutual diffusion coefficient of the swelling by the solvent and its mean value.