Hyaluronan Films Research Articles

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.

Intraperitoneally administered native and lauroyl-modified hyaluronan films: Pharmacokinetic and metabolism studies

Hyaluronan is being investigated extensively as a biocompatible and biodegradable material for use in biomedical applications. While the derivatization of hyaluronan broadens its potential therapeutic use, the pharmacokinetics and metabolization of the derivatives must be thoroughly investigated. The fate of intraperitoneally-applied native and lauroyl-modified hyaluronan films with varying degrees of substitution was investigated in-vivo employing an exclusive stable isotope-labelling approach and LC-MS analysis. The materials were gradually degraded in peritoneal fluid, lymphatically absorbed, preferentially metabolized in the liver and eliminated without any observable accumulation in the body. Hyaluronan acylation prolongs its presence in the peritoneal cavity depending on the degree of substitution. The safety of acylated hyaluronan derivatives was confirmed via a metabolic study that revealed its degradation into non-toxic metabolites, i.e. native hyaluronan and free fatty acid. Stable isotope-labelling with LC-MS tracking comprises a high-quality procedure for the investigation of the metabolism and biodegradability of hyaluronan-based medical products in-vivo.

A quartz crystal microbalance method to quantify the size of hyaluronan and other glycosaminoglycans on surfaces

Hyaluronan (HA) is a major component of peri- and extra-cellular matrices and plays important roles in many biological processes such as cell adhesion, proliferation and migration. The abundance, size distribution and presentation of HA dictate its biological effects and are also useful indicators of pathologies and disease progression. Methods to assess the molecular mass of free-floating HA and other glycosaminoglycans (GAGs) are well established. In many biological and technological settings, however, GAGs are displayed on surfaces, and methods to obtain the size of surface-attached GAGs are lacking. Here, we present a method to size HA that is end-attached to surfaces. The method is based on the quartz crystal microbalance with dissipation monitoring (QCM-D) and exploits that the softness and thickness of films of grafted HA increase with HA size. These two quantities are sensitively reflected by the ratio of the dissipation shift (ΔD) and the negative frequency shift (− Δf) measured by QCM-D upon the formation of HA films. Using a series of size-defined HA preparations, ranging in size from ~ 2 kDa tetrasaccharides to ~ 1 MDa polysaccharides, we establish a monotonic yet non-linear standard curve of the ΔD/ − Δf ratio as a function of HA size, which reflects the distinct conformations adopted by grafted HA chains depending on their size and surface coverage. We demonstrate that the standard curve can be used to determine the mean size of HA, as well as other GAGs, such as chondroitin sulfate and heparan sulfate, of preparations of previously unknown size in the range from 1 to 500 kDa, with a resolution of better than 10%. For polydisperse samples, our analysis shows that the process of surface-grafting preferentially selects smaller GAG chains, and thus reduces the average size of GAGs that are immobilised on surfaces comparative to the original solution sample. Our results establish a quantitative method to size HA and other GAGs grafted on surfaces, and also highlight the importance of sizing GAGs directly on surfaces. The method should be useful for the development and quality control of GAG-based surface coatings in a wide range of research areas, from molecular interaction analysis to biomaterials coatings.

Insoluble hyaluronan films obtained by heterogeneous crosslinking with iron(III) as resorbable implants

We present water-insoluble hyaluronan films crosslinked by trivalent iron developed as potential resorbable implants. The films were crosslinked by sorption of ferric salt into solid HA films in water/2-propanol bath. These heterogeneously crosslinked films (het-FeHA) remained tough and dimensionally stable when rehydrated in saline. In contrast, films prepared by drying the well-known homogeneous ferric hyaluronate gels (hom-FeHA) softened upon rehydration and expanded rapidly. Differences between hom-FeHA and het-FeHA result from polymer network topology (dominant inter- or intra-molecular crosslink, respectively). Moreover, Mössbauer spectroscopy of het-FeHA revealed diiron complexes, while iron in the hom-FeHA was present exclusively as γ-FeOOH nanoparticles or amorphous FeOOH. The biocompatibility tests of het-FeHA did not show any adverse effect and the sample disintegrated within one day when implanted in mice peritoneum. In conclusion, we developed implantable hyaluronan-based free-standing film with minimal swelling that can be resorbed quickly enough to avoid induction of foreign-body reaction.

Lauroyl hyaluronan films for local drug delivery: Preparation and factors influencing the release of small molecules

Lauroyl derivatives of hyaluronan are safe and biodegradable materials that seem promising for application in medicine. However, their potential in the field of drug delivery was not yet explored. We thus prepared lauroyl hyaluronan films loaded with various drugs and studied the effects of lauroyl hyaluronan properties, drug hydrophobicity and medium composition on the drug release. Since biomolecules will always be present in real clinical applications, media supplemented by albumin were also included. The amphiphilic character of lauroyl hyaluronan enabled convenient loading of the films by both hydrophilic and hydrophobic drugs. Dominant factors influencing drug release were drug hydrophobicity and the presence of albumin. Hydrophilic diclofenac was released rapidly in all cases, while triclosan with medium hydrophobicity exhibited slower release sensitive to other parameters, reaching equilibrium values in the used experimental setup. The release of hydrophobic octenidine into pure buffer was almost negligible, but the addition of albumin did promote its release. The strong effect of albumin highlights the importance of considering biomolecules in the design of release experiments.

Polysaccharide Multilayer Films in Sensors for Detecting Prostate Tumor Cells Based on Hyaluronan-CD44 Interactions.

The increasing need for point-of-care diagnosis has sparked the development of label-free sensing platforms, some of which are based on impedance measurements with biological cells. Here, interdigitated electrodes were functionalized with layer-by-layer (LbL) films of hyaluronan (HA) and chitosan (CHI) to detect prostatic tumor cells (PC3 line). The deposition of LbL films was confirmed with atomic force microscopy and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), which featured the vibrational modes of the HA top layer capable of interacting specifically with glycoprotein CD44 receptors overexpressed in tumor cells. Though the CHI/HA LbL films cannot be considered as a traditional biosensor due to their limited selectivity, it was possible to distinguish prostate tumor cells in the range from 50 to 600 cells/µL in in vitro experiments with impedance spectroscopy. This was achieved by treating the impedance data with information visualization methods, which confirmed the distinguishing ability of the films by observing the absence of false positives in a series of control experiments. The CD44–HA interactions may, therefore, be exploited in clinical analyses and point-of-care diagnostics for cancer, particularly if computational methods are used to process the data.

Non-linear rheological study of hydrogel sliding friction in water and concentrated hyaluronan solution

Studying sliding friction of hydrogels in liquid is important for understanding biological friction. This work applies the non-linear rheology approach to study the friction of elastic hydrogels against adhesive glass in water and in concentrated hyaluronan (HA) solution. Using a rheometer in parallel plate geometry, stress-strain Lissajous curves are obtained from the oscillatory strain sweep test. Non-linearity and energy dissipation are characterized the strain-hardening ratio S, and “energy dissipation occupancy” κ, respectively. For friction in water, a clear transition from a linear viscoelastic to non-linear viscoelastic response occurs at a critical strain amplitude. Whereas, in HA solution, significant friction reduction and non-linear viscoelastic response occurs, related to the rheological behaviors of HA film intercalated at the gel/glass interface.

Hyaluronan biofilms reinforced with partially deacetylated chitin nanowhiskers: Extraction, fabrication, in-vitro and antibacterial properties of advanced nanocomposites

Fabrication of nanocomposite biofilms with enhanced mechanical and antibacterial properties was successfully achieved from hyaluronan (HA) and partially deacetylated chitin nanowhiskers (ChNWs) by a casting-evaporation method. The hydrolysis process of chitin showed an important role in the dimensions, stability, and the crystallinity of extracted ChNWs in a time-dependent manner. The volume fraction of ChNWs nanofiller varying from (0.001 to 0.5) exhibited a great influence on the mechanical properties of the biofilms (young modulus, strength) was enhanced by the high load-bearing capacity of NWs compared with net HA film. The antibacterial activity of the nanocomposite biofilms exhibited significant bactericidal activity against different types of bacteria (−/+ gram). HA/ChNWs Nanocomposite biofilms did not show any toxicity against normal human dermal fibroblasts (NHDF) and human primary osteogenic sarcoma (Saos-2) cell lines. The new biofilms with unique properties like edibleness, environmental friendliness, high mechanical properties, antibacterial performance, and non-cytotoxicity that could be used in skin tissue regenerations, and drug delivery applications.

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.

Boundary lubrication by macromolecular layers and its relevance to synovial joints

We review here two recent works on boundary lubrication by macromolecules with the aim of obtaining an insight into the extremely efficient lubrication of mammalian joints. The first work emulates the structure of the cartilage superficial layer by reconstructing films of hyaluronan (HA) and of HA/aggrecan complexes stabilized by cartilage link protein and studies normal and shear interactions between two atomically smooth mica surfaces coated with such molecules using a surface force balance. These molecules have been conjectured to act as boundary lubricants while sliding past each other at the interface between the articular cartilage surfaces and the synovial fluid. It was discovered that the HA/aggrecan/link protein macromolecular complexes provide better boundary lubrication compared with HA alone, most likely as a result of the higher charge density allowing a more efficient hydration lubrication effect. However, the results suggest that such complexes by themselves cannot be responsible for the very efficient lubrication of synovial joints. On the other hand, in the second work, phosphatidylcholine vesicles were shown to provide extremely efficient lubrication at physiological pressures as long as the robustness of the layer is maintained. Normal and shear interactions between two surfaces bearing different phosphatidylcholine vesicles, having the same head group but varying acyl chain length, were studied systematically. In addition to hydration, robustness of the surface layer plays a major role in the lubrication efficiency of the system. Implications of the results of these studies to the lubrication mechanism of synovial joints are suggested. Copyright © 2014 John Wiley & Sons, Ltd.

Spatially Controlled Photochemical Peptide and Polymer Conjugation on Biosurfaces

An efficient phototriggered Diels-Alder conjugation is utilized to graft in an effective and straightforward approach poly(trifluoro ethyl methacrylate) (Mn = 3700 Da, Đ = 1.27) and a model peptide (GIGKFLHS) onto thin hyaluronan films and cellulose surfaces. The surfaces were functionalized with an o-quinodimethane moiety - capable of releasing a caged diene - via carbodiimide mediated coupling. The o-quinodimethane group is employed as a photoactive linker to tether predefined peptide/polymer strands in a spatially controlled manner onto the biosurface by photoenol ligation. An in-depth characterization employing XPS, ToF-SIMS, SPR, ellipsometry, and AFM was conducted to evidence the effectiveness of the presented approach.

Neocartilage formation from mesenchymal stem cells grown in type II collagen–hyaluronan composite scaffolds

Three-dimensional (3D) collagen type II–hyaluronan (HA) composite scaffolds (CII–HA) which mimics the extracellular environment of natural cartilage were fabricated in this study. Rheological measurements demonstrated that the incorporation of HA increased the compression modulus of the scaffolds. An initial in vitro evaluation showed that scaffolds seeded with porcine chondrocytes formed cartilaginous-like tissue after 8 weeks, and HA functioned to promote the growth of chondrocytes into scaffolds. Placenta-derived multipotent cells (PDMC) and gingival fibroblasts (GF) were seeded on tissue culture polystyrene (TCPS), CII–HA films, and small intestinal submucosa (SIS) sheets for comparing their chondrogenesis differentiation potentials with those of adipose-derived adult stem cells (ADAS) and bone marrow-derived mesenchymal stem cells (BMSC). Among different cells, PDMC showed the greatest chondrogenic differentiation potential on both CII–HA films and SIS sheets upon TGF-β3 induction, followed by GF. This was evidenced by the up-regulation of chondrogenic genes (Sox9, aggrecan, and collagen type II), which was not observed for cells grown on TCPS. This finding suggested the essential role of substrate materials in the chondrogenic differentiation of PDMC and GF. Neocartilage formation was more obvious in both PDMC and GF cells plated on CII–HA composite scaffolds vs. 8-layer SIS at 28 days in vitro. Finally, implantation of PDMC/CII–HA constructs into NOD–SCID mice confirmed the formation of tissue-engineered cartilage in vivo.

Inter-α-inhibitor Impairs TSG-6-induced Hyaluronan Cross-linking

Under inflammatory conditions and in the matrix of the cumulus-oocyte complex, the polysaccharide hyaluronan (HA) becomes decorated covalently with heavy chains (HCs) of the serum glycoprotein inter-α-inhibitor (IαI). This alters the functional properties of the HA as well as its structural role within extracellular matrices. The covalent transfer of HCs from IαI to HA is catalyzed by TSG-6 (tumor necrosis factor-stimulated gene-6), but TSG-6 is also known as a HA cross-linker that induces condensation of the HA matrix. Here, we investigate the interplay of these two distinct functions of TSG-6 by studying the ternary interactions of IαI and TSG-6 with well defined films of end-grafted HA chains. We demonstrate that TSG-6-mediated cross-linking of HA films is impaired in the presence of IαI and that this effect suppresses the TSG-6-mediated enhancement of HA binding to CD44-positive cells. Furthermore, we find that the interaction of TSG-6 and IαI in the presence of HA gives rise to two types of complexes that independently promote the covalent transfer of heavy chains to HA. One type of complex interacts very weakly with HA and is likely to correspond to the previously reported covalent HC·TSG-6 complexes. The other type of complex is novel and binds stably but noncovalently to HA. Prolonged incubation with TSG-6 and IαI leads to HA films that contain, in addition to covalently HA-bound HCs, several tightly but noncovalently bound molecular species. These findings have important implications for understanding how the biological activities of TSG-6 are regulated, such that the presence or absence of IαI will dictate its function.

Treatment and Stability of Sodium Hyaluronate Films in Low Temperature Inductively Coupled Ammonia Plasma

Surface of sodium hyaluronate films was modified in the inductively coupled low temperature ammonia plasma. The amount of bonded nitrogen was measured by the XPS method. The optical emission and temperature of the sample surface were measured during sample processing. Increased processing time and increased discharge power caused a rise of nitrogen concentration on the surface of hyaluronan films, though this effect is limited due to surface saturation and sample destruction at elevated discharge power.

Films of End-Grafted Hyaluronan Are a Prototype of a Brush of a Strongly Charged, Semiflexible Polyelectrolyte with Intrinsic Excluded Volume

Hyaluronan (HA) is a naturally occurring linear, negatively charged polysaccharide that plays a vital role in the organization and function of pericellular coats and extracellular matrices in vertebrates, and that is becoming increasingly popular in biomedical applications. To gain insight into the physical phenomena that govern the mechanical behavior of HA assemblies, we have studied the response of films of end-grafted HA to compression over a large range of ionic strength. Compression forces were measured as a function of the absolute distance between a colloidal probe and the planar surface on which the HA film was constructed, using a combined atomic force microscopy and reflection interference contrast microscopy setup. The HA films were well-defined in the sense that they are made of chains with a narrow size distribution that are grafted at controlled density to a solid support. Detailed comparison of the experimental data with analytical expressions derived from polymer and polyelectrolyte brush theory reveals that films of end-grafted HA behave as strongly charged polyelectrolyte brushes. To quantitatively reproduce the experimental data, intrinsic excluded volume interactions and chain stiffness of the polymer backbone must be taken into account. At low ionic strength, chains become almost fully stretched. In our experimental system, several micrometer thick films are formed that reach a hydration of up to 99.98%, and the brush thickness decreases by more than 5-fold with increasing ionic strength. More generally, the study provides quantitative theoretical predictions for the film thickness and compressive response as a function of HA length, grafting density and ionic strength.

Combining Colloidal Probe Atomic Force and Reflection Interference Contrast Microscopy to Study the Compressive Mechanics of Hyaluronan Brushes

We describe a method that combines colloidal probe atomic force microscopy (AFM) and reflection interference contrast microscopy (RICM) to characterize the mechanical properties of thin and solvated polymer films. When analyzing polymer films, a fundamental problem in colloidal probe AFM experiments is to determine the distance at closest approach between the probe and the substrate on which the film is deposited. By combining AFM and RICM in situ, forces and absolute distances can be measured simultaneously. Using the combined setup, we quantify the compressive mechanics of films of the polysaccharide hyaluronan that is end-grafted to a supported lipid bilayer. The experimental data, and comparison with polymer theory, show that hyaluronan films are well-described as elastic, very soft and highly solvated polymer brushes. The data on these well-defined films should be a useful reference for the investigation of the more complex hyaluronan-rich coats that surround many living cells.

Compressive Mechanics of Hyaluronan Brushes - A Study with a Combined Colloidal Probe AFM/RICM Setup

Around many biological cells, in the transition zone between the plasma membrane and the extracellular matrix, lies the micrometer thick pericellular coat (PCC): hyaluronan (HA), a long and flexible, charged glycosaminoglycan and HA-binding proteins self-organize into a hydrated gel-like coat which serves crucial mechanical functions of the cell. For a thorough investigation of the physical principles underlying the biological functions of these coats and provided the instrumental limitations in studying these highly hydrated systems in vivo, model systems are useful.Atomic force microscopy (AFM) is a widely used analytical approach to determine the behavior of molecules or thin films under mechanical force. Colloidal probe reflection interference contrast microscopy (RICM) is an established microinterferometric technique to determine the thickness of soft hydrated films. In this study we combine colloidal probe AFM and RICM to investigate the mechanical properties of a well-defined model system of the pericellular coat: films of hyaluronan that is grafted to supported lipid bilayers (Richter et.al. 2007, JACS, 129:5306-7). The combination provides interaction forces as a function of the absolute distance between the two approaching surfaces, information that cannot easily be obtained with either technique alone. We quantify the thickness of HA films, and their resistance to compression forces as a function of external salt concentration. From the experimental data, and comparison with scaling and mean-field theories, we conclude that grafted HA films are well-described as a polyelectrolyte brush. Addition of cartilage proteoglycan aggrecan induced a drastic increase in thickness and resistance to compression. The novel combined AFM/RICM setup can serve as a powerful tool to quantify the mechanical properties of soft hydrated biopolymer films with precise control of probe sample separation.

The Inflammation-associated Protein TSG-6 Cross-links Hyaluronan via Hyaluronan-induced TSG-6 Oligomers

Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyaluronan (HA)-binding protein that plays important roles in inflammation and ovulation. TSG-6-mediated cross-linking of HA has been proposed as a functional mechanism (e.g. for regulating leukocyte adhesion), but direct evidence for cross-linking is lacking, and we know very little about its impact on HA ultrastructure. Here we used films of polymeric and oligomeric HA chains, end-grafted to a solid support, and a combination of surface-sensitive biophysical techniques to quantify the binding of TSG-6 into HA films and to correlate binding to morphological changes. We find that full-length TSG-6 binds with pronounced positive cooperativity and demonstrate that it can cross-link HA at physiologically relevant concentrations. Our data indicate that cooperative binding of full-length TSG-6 arises from HA-induced protein oligomerization and that the TSG-6 oligomers act as cross-linkers. In contrast, the HA-binding domain of TSG-6 (the Link module) alone binds without positive cooperativity and weaker than the full-length protein. Both the Link module and full-length TSG-6 condensed and rigidified HA films, and the degree of condensation scaled with the affinity between the TSG-6 constructs and HA. We propose that condensation is the result of protein-mediated HA cross-linking. Our findings firmly establish that TSG-6 is a potent HA cross-linking agent and might hence have important implications for the mechanistic understanding of the biological function of TSG-6 (e.g. in inflammation).

The immobilization of titania nanoparticles on hyaluronan films and their photocatalyticproperties

We have developed a method to bind titania nanoparticles onto hyaluronic films (HA)photoimmobilized on silanized glass. Titania nanoparticles were deposited on the HAfilms from commercially available dispersions by casting and dip-coating methodsat various pH values. XPS was used to monitor the deposition of titania andto estimate the surface coverage of the nanoparticles. The topography of thetitania-modified HA films was investigated by means of AFM. XPS results indicatethat the titania surface coverage depends on the preparation method and thepH of the dispersion. We found that the maximum titania nanoparticle surfacecoverage was obtained by the casting method with the formation of aggregates andmultilayers of particles. The titania surface coverage for the surfaces prepared bythe dip-coating method is pH-dependent. The surfaces prepared at pH 2 show asurface coverage of 65% and a rather uniform distribution of particles. We foundthat titania nanoparticles are anchored in a stable way to the HA substrate in aphosphate buffer solution (PBS) and that the interaction between the HA andthe titania is through the carbonyl group of carboxylates and amidic groups ofthe polymer. AFM images clearly show that titania nanoparticles are uniformlydistributed over the HA films. By measuring the average diameter and the averageheight of the nanoparticles deposited on HA films it appears that the particles arepartially embedded in the polysaccharide films. The results of the study on thephotobleaching of methylene blue indicate that the characteristic photocatalytic activity oftitania is maintained when the nanoparticles are anchored to the HA substrate.

The Influence of Hofmeister Series Ions on Hyaluronan Swelling and Viscosity

The dissolution of hyaluronan in water leads to its degradation, and as a result its molecular weight decreases. The degradation of hyaluronan is mainly influenced by temperature, solution composition, and also its pH. This study describes the influence of Hofmeister series ions on hyaluronan behaviour and hyaluronan film swelling by solutions of these ions. It was found that Hofmeister ions show lyotropic effects influencing the entanglement of hyaluronan coils and their expansion from solid polymer films into swollen gel state. The hydrophobic and hydrophilic interactions in the structure of hyaluronan macromolecules are represented by the mutual diffusion coefficient D(c), the mean mutual diffusion coefficient Ds, the expansion work of coil swelling RAδ,s, the activation enthalpy of diffusion connected with swelling HD,s and kinematic viscosity of hyaluronan-ions solutions ν.