Repeatability of Noninvasive Break-Up Time Measurements using Keratograph Oculus 3.

AIM: The primary aim of this study is to evaluate the repeatability of noninvasive break-up time (NIBUT) measurement by keratograph when it is determined from one, two or three partial measurements, and to recommend a suitable methodology for practice. Another goal is to verify that repeated measurements do not affect the measured value. MATERIAL AND METHODS: Thirty-eight healthy volunteers (30 women and 8 men) aged between 19 and 50 years old were included in the study, in which only one eye of each volunteer was measured. The study was designed as a prospective one. Each subject adapted to the local conditions of the laboratory for 15 minutes and subsequently underwent two series of NIBUT measurements (test, retest) on an OCULUS 3 Keratograph. The minimum time interval between the two series was 10 minutes, in which each series contained three partial measurements approximately 3 three measurements in the given series. Repeatability was assessed by a Bland-Altman analysis and expressed as a repeatability coefficient. In every case, only the time of the first break-up of the tear film was monitored. RESULTS: The statistical analysis did not show statistically significant differences both between partial measurements of NIBUT in the individual series (p = 0.92, p = 0.81) and when comparing all six measurements (p = 0.95). The mean values of the partial measurements ranged from 13.6 s to 14.4 s. The repeatability coefficients were found to be 15.0 s, 12.1 s and 10.0 s for methodologies A, B and C, respectively. A supplementary analysis for 12 eyes with low NIBUT (< 10 s) showed statistically significantly better repeatability in this group, with coefficients of 7.0 s (methodology A), 6.0 s (B) and 4.6 s (C). CONCLUSION: Determination of NIBUT from three consecutive measurements (with a sufficient interval of ideally a few minutes) significantly improves repeatability. Such repeated NIBUT measurements do not have a significant effect on the measured value. The mentioned methodology for measuring NIBUT on a keratograph can be recommended for use in practice.

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.

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.

Oxidized polysaccharides for anticancer-drug delivery: What is the role of structure?

Study provides an in-depth analysis of the structure-function relationship of polysaccharide anticancer drug carriers and points out benefits and potential drawbacks of differences in polysaccharide glycosidic bonding, branching and drug binding mode of the carriers. Cellulose, dextrin, dextran and hyaluronic acid have been regioselectively oxidized to respective dicarboxylated derivatives, allowing them to directly conjugate cisplatin, while preserving their major structural features intact. The structure of source polysaccharide has crucial impact on conjugation effectiveness, carrier capacity, drug release rates, in vitro cytotoxicity and cellular uptake. For example, while branched structure of dextrin-based carrier partially counter the undesirable initial burst release, it also attenuates the cellular uptake and the cytotoxicity of carried drug. Linear polysaccharides containing ß-(1→4) glycosidic bonds and oxidized at C2 and C3 (cellulose and hyaluronate) have the best overall combination of structural features for improved drug delivery applications including potentiation of the cisplatin efficacy towards malignances.

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.

Number of flankers influences foveal crowding and contour interaction differently.

Nearby flanking objects degrade visual resolution. If the flankers are similar to the acuity target, this influence is called crowding (CW), whereas if the flanking stimuli are simple bars then the phenomenon is known as contour interaction (CI). The aim of this study was to compare the influence of the number and position of flankers on foveal CW and CI to investigate possible differences in mechanism of these two effects. Five normal observers viewed single, foveally presented Sloan letters surrounded by 1, 2 or 4 flankers (either a Sloan letter or one-stroke-width bars), presented at several edge-to-edge separations. Single flankers were presented in the right, left, top or bottom position, 2 flankers were placed equally to the right and left or top and bottom of the central target, and 4 flankers were equally spaced in all four directions. Percent correct letter identification was determined for each type, number, position and separation of flankers and confusion matrices were constructed for separations equal to 20% and 100% letter width. Increasing the number of flankers caused an increase in the magnitude of both phenomena. CW showed a greater magnitude than CI for higher numbers of flankers. Analysis of confusion matrices suggests that in addition to the edge-to-edge interaction that appears to mediate CI, letter substitution and feature pooling contribute significantly to CW when higher numbers of flankers are presented. Foveal CW is more strongly influenced by an increase in the number of flankers than CI, which can be explained by the presence of additional interaction effects.

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.

The Effect of Plasma Pretreatment and Cross-Linking Degree on the Physical and Antimicrobial Properties of Nisin-Coated PVA Films.

Stable antimicrobial nisin layers were prepared on the carrying medium-polyvinyl alcohol (PVA) films, crosslinked by glutaric acid. Surface plasma dielectric coplanar surface barrier discharge (DCSBD) modification of polyvinyl alcohol was used to improve the hydrophilic properties and to provide better adhesion of biologically active peptide-nisin to the polymer. The surface modification of films was studied in correlation to their cross-linking degree. Nisin was attached directly from the salt solution of the commercial product. In order to achieve a stable layer, the initial nisin concentration and the following release were investigated using chromatographic methods. The uniformity and stability of the layers was evaluated by means of zeta potential measurements, and for the surface changes of hydrophilic character, the water contact angle measurements were provided. The nisin long-term stability on the PVA films was confirmed by tricine polyacrylamide gel electrophoresis (SDS-PAGE) and by antimicrobial assay. It was found that PVA can serve as a suitable carrying medium for nisin with tunable properties by plasma treatment and crosslinking degree.

Contour interaction under photopic and scotopic conditions.

In the present study, we asked whether contour interaction undergoes significant changes for different luminance levels in the central and peripheral visual field. This study included nine normal observers at two laboratories (five at Palacky University Olomouc, Czech Republic and four at the University of Houston, USA). Observers viewed a randomly selected Sloan letter surrounded by four equally spaced bars for several separations measured edge-to-edge in min arc. Stimuli were viewed foveally under photopic and mesopic luminances and between 5° and 12° peripherally for four different background luminances of the display monitors, corresponding to photopic, mesopic, scotopic, and dim scotopic levels. The extent of the contour interaction in the fovea is approximately 20 times smaller than in the periphery. Whereas the magnitude of foveal contour interaction markedly decreases with decreasing luminance, no consistent luminance-induced change occurs in peripheral contour interaction. The extent of contour interaction does not scale with the size of the target letter, either in the fovea or peripherally. The results support a neural origin of contour interaction consistent with the properties of center-surround antagonism.

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.

Explicit treatment of active-site waters enhances quantum mechanical/implicit solvent scoring: Inhibition of CDK2 by new pyrazolo[1,5-a]pyrimidines.

We present comprehensive testing of solvent representation in quantum mechanics (QM)-based scoring of protein-ligand affinities. To this aim, we prepared 21 new inhibitors of cyclin-dependent kinase 2 (CDK2) with the pyrazolo[1,5-a]pyrimidine core, whose activities spanned three orders of magnitude. The crystal structure of a potent inhibitor bound to the active CDK2/cyclin A complex revealed that the biphenyl substituent at position 5 of the pyrazolo[1,5-a]pyrimidine scaffold was located in a previously unexplored pocket and that six water molecules resided in the active site. Using molecular dynamics, protein-ligand interactions and active-site water H-bond networks as well as thermodynamics were probed. Thereafter, all the inhibitors were scored by the QM approach utilizing the COSMO implicit solvent model. Such a standard treatment failed to produce a correlation with the experiment (R2 = 0.49). However, the addition of the active-site waters resulted in significant improvement (R2 = 0.68). The activities of the compounds could thus be interpreted by taking into account their specific noncovalent interactions with CDK2 and the active-site waters. In summary, using a combination of several experimental and theoretical approaches we demonstrate that the inclusion of explicit solvent effects enhance QM/COSMO scoring to produce a reliable structure-activity relationship with physical insights. More generally, this approach is envisioned to contribute to increased accuracy of the computational design of novel inhibitors.