Hyaluronan Backbone Research Articles

Hyaluronan: Sources, Structure, Features and Applications.

Hyaluronan (HA) is a non-sulfated glycosaminoglycan that is present in a variety of body tissues and organs. Hyaluronan has a wide range of biological activities that are frequently influenced by molar mass; however, they also depend greatly on the source, purity, and kind of impurities in hyaluronan. High-molar-mass HA has anti-inflammatory, immunosuppressive, and antiangiogenic properties, while low-molar-mass HA has opposite properties. A number of chemical modifications have been performed to enhance the stability of HA and its applications in medical practice. Hyaluronan is widely applied in medicine, such as viscosupplementation, ophthalmology, otolaryngology, wound healing, cosmetics, and drug delivery. In this review, we summarized several medical applications of polymers based on the hyaluronan backbone.

Astrocyte 3D culture and bioprinting using peptide functionalized hyaluronan hydrogels

ABSTRACT Astrocytes play an important role in the central nervous system, contributing to the development of and maintenance of synapses, recycling of neurotransmitters, and the integrity and function of the blood–brain barrier. Astrocytes are also linked to the pathophysiology of various neurodegenerative diseases. Astrocyte function and organization are tightly regulated by interactions mediated by the extracellular matrix (ECM). Engineered hydrogels can mimic key aspects of the ECM and can allow for systematic studies of ECM-related factors that govern astrocyte behaviour. In this study, we explore the interactions between neuroblastoma (SH-SY5Y) and glioblastoma (U87) cell lines and human fetal primary astrocytes (FPA) with a modular hyaluronan-based hydrogel system. Morphological analysis reveals that FPA have a higher degree of interactions with the hyaluronan-based gels compared to the cell lines. This interaction is enhanced by conjugation of cell-adhesion peptides (cRGD and IKVAV) to the hyaluronan backbone. These effects are retained and pronounced in 3D bioprinted structures. Bioprinted FPA using cRGD functionalized hyaluronan show extensive and defined protrusions and multiple connections between neighboring cells. Possibilities to tailor and optimize astrocyte-compatible ECM-mimicking hydrogels that can be processed by means of additive biofabrication can facilitate the development of advanced tissue and disease models of the central nervous system.

Regulation of perineuronal net components in the synaptic bouton vicinity on lumbar α-motoneurons in the rat after spinalization and locomotor training: New insights from spatio-temporal changes in gene, protein expression and WFA labeling

Chondroitin sulfate proteoglycans (CSPGs) consist of core proteins and glycosaminoglycan side chains. Tenascins, and hyaluronan and proteoglycan link protein 1 (HAPLN), link CSPGs with a hyaluronan backbone to constitute perineuronal nets (PNNs), which ensheath preferentially highly active neurons to maintain architecture and stabilize synapses, but restrict repair plasticity. Spinal cord injury increases CSPG core protein levels in the lesion proximity, limiting permissiveness of the extracellular milieu for fiber regrowth, however regulation of PNNs structure in the vicinity of distant α-motoneurons (MNs) in the course of degeneration and reorganization of their inputs requires research. Here, we examined early and late changes in CSPGs, HAPLN1, tenascin-R, and glial activation along the spinal cord in male rats with complete spinal cord transection (Th10), and their impact on PNNs ensheathing lumbar MNs innervating ankle extensor and flexor muscles, which are in different loading states in paraplegic rats. We show that (1) distance from the lesion site and time after injury (2–5 weeks) differentiate degree of changes in transcription rates (measured with RT-qPCR) of PNNs proteins with increased CSPGs and decreased HAPLN1 transcripts, suggesting long-term PNN destabilization in majority of spinal segments, (2) in lumbar segments PNN composition is not MN-class (extensor vs flexor) specific, both showing early decrease and late upregulation of Wisteria floribunda agglutinin (WFA) labeling in vicinity of synaptic boutons on MNs, (3) long-term locomotor training tends to reduce WFA(+) PNNs, but not their protein components (immunofluorescence measurements) around MNs. Our results suggest that training-induced regulation may target glycan structures of CSPGs.

Biomimetic cartilage-lubricating polymers regenerate cartilage in rats with early osteoarthritis.

The early stages of progressive degeneration of cartilage in articular joints are a hallmark of osteoarthritis. Healthy cartilage is lubricated by brush-like cartilage-binding nanofibres with a hyaluronan backbone and two key side chains (lubricin and lipid). Here, we show that hyaluronan backbones grafted with lubricin-like sulfonate-rich polymers or with lipid-like phosphocholine-rich polymers together enhance cartilage regeneration in a rat model of early osteoarthritis. These biomimetic brush-like nanofibres show a high affinity for cartilage proteins, form a lubrication layer on the cartilage surface and efficiently lubricate damaged human cartilage, lowering its friction coefficient to the low levels typical of native cartilage. Intra-articular injection of the two types of nanofibre into rats with surgically induced osteoarthritic joints led to cartilage regeneration and to the abrogation of osteoarthritis within 8 weeks. Biocompatible injectable lubricants that facilitate cartilage regeneration may offer a translational strategy for the treatment of early osteoarthritis.

The SAgA of Antigen-Specific Immunotherapy for Type 1 Diabetes.

Islet antigen-specific strategies are the holy grail of immunotherapy for type 1 diabetes (T1D) (1), as they selectively target the autoimmune responses involved in β-cell destruction (2). The idea is to induce a response opposite to that of a conventional vaccine. The administration of antigen(s) in the absence of inflammation (e.g., adjuvants) should potentiate the outcomes of physiological immune homeostasis, i.e., anergy, regulatory polarization, and, to a lesser extent, deletion. Such antigens can be delivered as proteins, peptides, bacteria engineered to secrete these products, DNA plasmids, or nanoparticles coated with antigens (either alone or preloaded on MHC molecules). Peptides are easy to synthesize by amino acid chemistry, but they are variably water-soluble and short-lived in vivo (in the order of minutes), which makes it difficult to achieve the steady concentrations more suitable for tolerance induction. Nonetheless, peptide-based immunotherapies have proven safe and, in some cases, documented encouraging immune and clinical outcomes (3). In this issue of Diabetes , Firdessa-Fite et al. (4) give peptides a boost by mounting them on a hyaluronan backbone to obtain a soluble antigen array (SAgA). First, this backbone accounts for their superior solubility. The linker used was either stable (“click” chemistry, cSAgA) or hydrolysable (hSAgA), to facilitate peptide release upon uptake by antigen-presenting cells (APCs). Second, SAgAs deliver multiple peptide copies to individual APCs, thus providing a multivalent (high avidity) engagement of T cells that is more efficient for signal transduction. This may be …

The Labeling, Visualization, and Quantification of Hyaluronan Distribution in Tumor-Bearing Mouse Using PET and MR Imaging.

Hyaluronan (HA) based biomaterials are widely used as tissue scaffolds, drug formulations, as well as targeting ligands and imaging probes for diagnosis and drug delivery. However, because of the presence of abundant endogenous HA presented in various tissues in vivo, the pharmacokinetic behavior and biodistribution patterns of exogenously administered HAs have not been well characterized. The HA backbone was modified with Diethylenetriamine (DTPA) to enable the chelation of gadolinium (Gd) and aluminum (Al) ions. Series of PET and MR imaging were taken after the injection of HA-DTPA-Gd and HA-DTPA-Al18F while using18F-FDG and Magnevist(DTPA-Gd) as controls. The Tomographic images were analyzed and quantified to reveal the distribution and locations of HA in tumor-bearing mice. The labeled HAs had good stability in plasma. They retained binding affinity towards CD44s on tumor cell surface. The injected HAs distributed widely in various organs, but were found to be cleared quickly except inside tumor tissues where the signals were higher and persisted longer. Medical imaging tools, including MR and PET, can be highly valuable for examining biomaterial distribution non-invasively. The HA tumor accumulation properties may be explored for the development of active targeting drug carriers and molecular probes.

Stability Evaluation and Degradation Studies of DAC® Hyaluronic-Polylactide Based Hydrogel by DOSY NMR Spectroscopy.

The stability and the degradation of polymers in physiological conditions are very important issues in biomedical applications. The copolymer of hyaluronic acid and poly-D,L-lactic acid (made available in a product called DAC®) produces a hydrogel which retains the hydrophobic character of the poly-D,L-lactide sidechains and the hydrophilic character of a hyaluronic acid backbone. This hydrogel is a suitable device for the coating of orthopedic implants with structured surfaces. In fact, this gel creates a temporary barrier to bacterial adhesion by inhibiting colonization, thus preventing the formation of the biofilm and the onset of an infection. Reabsorbed in about 72 h after the implant, this hydrogel does not hinder bone growth processes. In the need to assess stability and degradation of both the hyaluronan backbone and of the polylactic chains along time and temperature, we identified NMR spectroscopy as a privileged technique for the characterization of the released species, and we applied diffusion-ordered NMR spectroscopy (DOSY-NMR) for the investigation of molecular weight dispersion. Our diffusion studies of DAC® in physiological conditions provided a full understanding of the product degradation by overcoming the limitations observed in applying classical chromatography approaches by gel permeation UV.

Dynamic peptide-folding mediated biofunctionalization and modulation of hydrogels for 4D bioprinting

Hydrogels are used in a wide range of biomedical applications, including three-dimensional (3D) cell culture, cell therapy and bioprinting. To enable processing using advanced additive fabrication techniques and to mimic the dynamic nature of the extracellular matrix (ECM), the properties of the hydrogels must be possible to tailor and change over time with high precision. The design of hydrogels that are both structurally and functionally dynamic, while providing necessary mechanical support is challenging using conventional synthesis techniques. Here, we show a modular and 3D printable hydrogel system that combines a robust but tunable covalent bioorthogonal cross-linking strategy with specific peptide-folding mediated interactions for dynamic modulation of cross-linking and functionalization. The hyaluronan-based hydrogels were covalently cross-linked by strain-promoted alkyne-azide cycloaddition using multi-arm poly(ethylene glycol). In addition, a de novo designed helix-loop-helix peptide was conjugated to the hyaluronan backbone to enable specific peptide-folding modulation of cross-linking density and kinetics, and hydrogel functionality. An array of complementary peptides with different functionalities was developed and used as a toolbox for supramolecular tuning of cell-hydrogel interactions and for controlling enzyme-mediated biomineralization processes. The modular peptide system enabled dynamic modifications of the properties of 3D printed structures, demonstrating a novel route for design of more sophisticated bioinks for four-dimensional bioprinting.

Mechanical Properties and Cell Compatibility of Agarose Hydrogels Containing Proteoglycan Mimetic Graft Copolymers.

Proteoglycans have vital biochemical and biomechanical functions. Their proteolytic degradation results in loss of these functions. We have previously reported nonprotein proteoglycan-mimetic graft copolymers that stabilize and deliver growth factors and are not subject to proteases. Here we expand our investigation of these proteoglycan mimics by also investigating their effects on hydrogel mechanical properties. Four polysaccharide side chains, chondroitin sulfate, heparin, dextran, and dextran sulfate, are each grafted to a hyaluronan backbone. The polysaccharides and graft copolymers are added to agarose hydrogels. Cyclic compression and stress relaxation tests reveal how the addition of the polysaccharides and graft copolymers influence hydrogel modulus. Cells encapsulated in agarose hydrogels containing chondroitin sulfate and the chondroitin sulfate graft copolymer have decreased cell viability and metabolic activity compared to cells in unmodified agarose hydrogels. These multifunctional additives can be used to improve both the biochemistry and biomechanics of materials, warranting further optimization to overcome the potentially negative effects these may have on cell viability and activity.

Pulmonary Administration of Soluble Antigen Arrays Is Superior to Antigen in Treatment of Experimental Autoimmune Encephalomyelitis

Antigen-specific immunotherapy has been used to hyposensitize patients to allergens and offers an enticing approach for attenuating autoimmune diseases. Applying antigen-specific immunotherapy to mucosal surfaces such as the lungs may engage unique immune response pathways to improve efficacy. Pulmonary delivery of soluble antigen arrays (SAgAs) was explored in mice with experimental autoimmune encephalomyelitis (EAE), a multiple sclerosis model. SAgAs were designed to impede immune response to autoantigen epitopes and are composed of a hyaluronan backbone with peptides PLP139-151 (proteolipid protein) and LABL, a disease-causing proteolipid peptide epitope and an intracellular cell-adhesion molecule-1 ligand, respectively. Pulmonary instillation of SAgAs decreased disease score, improved weight gain, and decreased incidence of disease in EAE mice compared to pulmonary delivery of hyaluronic acid polymer, LABL, or PLP. Interestingly, treating with PLP alone also showed some improvement. Splenocytes from SAgA-treated animals showed increased interferon-gamma levels, and interleukin-6 (IL-6) and IL-17 were elevated in SAgA-treated animals compared to PLP treatments. IL-10, IL-2, and tumor necrosis factor-alpha levels showed no significant difference, yet trends across all cytokines suggested SAgAs induced a very different immune response compared to treatment with PLP alone. This work suggests that codelivery of peptide components is essential when treating EAE via pulmonary instillation, and the immune response may have shifted toward immune tolerance.

Fabrication of cell-compatible hyaluronan hydrogels with a wide range of biophysical properties through high tyramine functionalization.

Enzymatically mediated crosslinked hyaluronan-tyramine hydrogels (HA-Tyr) are promising matrices for tissue engineering and regenerative medicine. However, due to relatively low tyramine modifications of the hyaluronan backbone achieved, HA-Tyr matrices have a weak and narrow range of mechanical properties. The iterative use of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as a coupling agent increased the yields of tyramine functionalization, which was reflected by a two-fold increase in Young's modulus of HA-Tyr hydrogels. The accurate control over hydrogel degradation was also facilitated. Viable encapsulation of human mesenchymal stem cells, with 85-98% over 6 days, was achieved in all hydrogels and distinct cellular spreading was observed in the absence of additional binding cues. The biophysical properties of the tunable HA-Tyr hydrogels are improved to study a wide range of cellular behaviors.

Hyaluronan-based multifunctional hydrogels formed via enzyme-mediated crosslinking

Event Abstract Back to Event Hyaluronan-based multifunctional hydrogels formed via enzyme-mediated crosslinking Daniela S. Ferreira1, 2, Dominic W. Collis1, 2 and Helena S. Azevedo1, 2 1 Queen Mary, University of London, School of Engineering and Materials Science, United Kingdom 2 Queen Mary University of London, Institute of Bioengineering, United Kingdom Introduction: Hydrogels have generated great interest to develop 3D artificial matrices that recapitulate features of the natural extracellular matrix (ECM) for applications in tissue regeneration or cell expansion[1],[2]. Covalent crosslinking is a common method to obtain stable hydrogels with precise control over crosslinking density. However, many of the crosslinking reagents used are toxic, precluding their use for cell encapsulation applications. Enzyme-mediated crosslinking reactions are finding increasing applications for developing hydrogels due to their mild conditions and enzyme specificity[3]. FXIIIa is a transglutaminase that catalyses the reaction between an ε-amine of lysine (K) onto a γ-carboxamide residue of glutamine (Q), resulting in the covalent bond formation of an ε-(γ-glutamyl)lysine isopeptide which is highly resistant to proteolytic degradation[4]. This crosslinking mechanism naturally occurs in vivo during the processes of wound healing. Hyaluronan (HA), a linear polysaccharide present in the ECM of many tissues, has been chemically modified to develop hydrogels[5]-[7]. In this work, HA was functionalized with two different transglutaminase peptide substrates to obtain HA hydrogels by enzyme-mediated crosslinking. Materials and Methods: Peptides were synthesized manually using standard Fmoc based solid phase chemistry and purified, as described elsewhere[8]. Hyaluronan (60 kDa) was functionalized with acrylate groups using a two-step synthesis (Fig 1-A, as described elsewhere[9]. Peptides were coupled to acrylated HA via Michael-type addition by reacting 1.2-fold molar excess of peptide over acrylate groups in 0.3 M triethanolamine (pH 8.0) overnight at 37°C. Results and Discussion: Peptides were designed as factor XIIIa substrates, containing either a lysine donor peptide CH3CONH-GFKGG-ERCG-CONH2 (TG-K, Mw = 950.08 g/mol) or a glutamine acceptor peptide - CH3CONH-GNQEQVSPL-ERCG-CONH2 (TG-Q, Mw = 1457.58 g/mol). In addition, a glutamine acceptor containing a cell adhesion ligand RGDS, which is recognized by several integrin receptors, was also synthesized, CH3CONH--GNQEQVSPLRGDSPG-CONH2 (TG-Q-RGDSPG, Mw = 1581.67 g/mol). To render hydrogels susceptible to degradation by proteolytic enzymes, like natural fibrin, a lysine donor peptide was synthesized bearing a substrate for matrix metalloproteases (MMPs) Ac-GFKGGGPQGIWGQ-ERCG-CONH2 (TG-MMP-K, Mw = 1581.67 g/mol). Acrylated HA was obtained by a two-step reaction and the degree of acrylation was found to be 16%, as determined by NMR (Fig. 1-B). Transglutaminase peptide substrates were grafted onto acrylate groups of HA backbone via Michael-type addition as confirmed by NMR analysis (Fig. 2). Conclusion: HA macromers functionalized with transglutaminase substrate peptides were successfully obtained. These HA-peptide macromers will be used as precursors to develop hydrogels that can be formed in situ (e.g. wound site), where FXIIIa is produced. The incorporation of integrin-specific adhesive ligands and cell-sensitive proteolytic domains in these hydrogels will aid in guiding and enhancing local tissue regeneration. The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007‐2013) under REA grant agreement n° 608765.

The neuronal extracellular matrix restricts distribution and internalization of aggregated Tau-protein

Alzheimer’s disease (AD) is a chronic degenerative disorder characterized by fibrillary aggregates of Aß and Tau-protein. Formation and progression of these pathological hallmarks throughout the brain follow a specific spatio-temporal pattern which provides the basis for neuropathological staging. Previously, we could demonstrate that cortical and subcortical neurons are less frequently affected by neurofibrillary degeneration if they are enwrapped by a specialized form of the hyaluronan-based extracellular matrix (ECM), the so called ‘perineuronal net’ (PN). PNs are composed of large aggregating chondroitin sulfate proteoglycans connected to a hyaluronan backbone, stabilized by link proteins and cross-linked via tenascin-R. Recently, PN-associated neurons were shown to be better protected against iron-induced neurodegeneration compared to neurons without PN, indicating a neuroprotective function. Here, we investigated the role of PNs in distribution and internalization of exogenous Tau-protein by using organotypic slice cultures of wildtype mice as well as mice lacking the ECM-components aggrecan, HAPLN1 or tenascin-R. We could demonstrate that PNs restrict both distribution and internalization of Tau. Accordingly, PN-ensheathed neurons were less frequently affected by Tau-internalization, than neurons without PN.Finally, the PNs as well as their three investigated components were shown to modulate the processes of distribution as well as internalization of Tau.

Synthesis and characterization of proteoglycan-mimetic graft copolymers with tunable glycosaminoglycan density.

Proteoglycans (PGs) are important glycosylated proteins found on the cell surface and in the extracellular matrix. They are made up of a core protein with glycosaminoglycan (GAG) side chains. Variations in composition and number of GAG side chains lead to a vast array of PG sizes and functions. Here we present a method for the synthesis of proteoglycan-mimetic graft copolymers (or neoproteoglycans) with tunable GAG side-chain composition. This is done using three different GAGs: hyaluronan, chondroitin sulfate, and heparin. Hyaluronan is functionalized with a hydrazide-presenting linker. Either chondroitin sulfate or heparin is grafted by the reducing end on to the hyaluronan backbone through reductive amination. PG mimics with heparin or chondroitin sulfate side chains and four different ratios of GAG side chain result in graft copolymers with a wide range of sizes. The chemistry is confirmed through attentuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and (1)H NMR. Effective hydrodynamic diameter and zeta potential are determined using dynamic light scattering and electrophoretic mobility measurements. Graft copolymers were tested for their ability to bind and deliver basic fibroblast growth factor (FGF-2) to mesenchymal stem cells (MSCs). The chondroitin sulfate-containing graft copolymers successfully deliver FGF-2 to cells from surfaces. The lowest graft density of heparin-containing PG mimic also performs well with respect to growth factor delivery from a surface. This new method for preparation of GAG-based graft copolymers enables a wide range of graft density, and can be used to explore applications of PG mimics as new biomaterials with tunable biochemical and biomechanical functions.

Modification of Hyaluronan by Heavy Chains of Inter-α-Inhibitor in Idiopathic Pulmonary Arterial Hypertension

We previously reported an altered hyaluronan (HA) metabolism in idiopathic pulmonary arterial hypertension (IPAH) lung tissue and cultured smooth muscle cells. Hyaluronan was present in the smooth muscle cell layer surrounding the pulmonary vasculature and in plexigenic lesions. Additionally, cultured pulmonary artery smooth muscle cells produced spontaneous HA "cable" structures, without additional stimuli, that were leukocyte-adhesive. We now present evidence that the HA that accumulates in IPAH plexigenic lesions is a pathological form of HA in which heavy chains (HCs) from the serum-derived proteoglycan inter-α-inhibitor are covalently attached to the HA backbone to form a pathological HC-HA complex. CD45-positive leukocytes were identified within these HC-HA matrices. Elevated mRNA levels of the enzyme that transfers HCs to HA, known as tumor necrosis factor-stimulated gene 6, were detected in IPAH lung tissue.

Aggrecan, link protein and tenascin-R are essential components of the perineuronal net to protect neurons against iron-induced oxidative stress

In Alzheimer's disease (AD), different types of neurons and different brain areas show differential patterns of vulnerability towards neurofibrillary degeneration, which provides the basis for a highly predictive profile of disease progression throughout the brain that now is widely accepted for neuropathological staging. In previous studies we could demonstrate that in AD cortical and subcortical neurons are constantly less frequently affected by neurofibrillary degeneration if they are enwrapped by a specialized form of the hyaluronan-based extracellular matrix (ECM), the so called ‘perineuronal net' (PN). PNs are basically composed of large aggregating chondroitin sulphate proteoglycans connected to a hyaluronan backbone, stabilized by link proteins and cross-linked via tenascin-R (TN-R). Under experimental conditions in mice, PN-ensheathed neurons are better protected against iron-induced neurodegeneration than neurons without PN. Still, it remains unclear whether these neuroprotective effects are directly mediated by the PNs or are associated with some other mechanism in these neurons unrelated to PNs. To identify molecular components that essentially mediate the neuroprotective aspect on PN-ensheathed neurons, we comparatively analysed neuronal degeneration induced by a single injection of FeCl3 on four different mice knockout strains, each being deficient for a different component of PNs. Aggrecan, link protein and TN-R were identified to be essential for the neuroprotective properties of PN, whereas the contribution of brevican was negligible. Our findings indicate that the protection of PN-ensheathed neurons is directly mediated by the net structure and that both the high negative charge and the correct interaction of net components are essential for their neuroprotective function.

Thermoreversible Hyaluronan Hydrogels Support Mesenchymal Stem Cells Discogenic Differentiation in Vitro and Ex Vivo

Introduction Hyaluronan is a proteoglycan widely present in the nucleus pulposus (NP) of the intervertebral disk (IVD). Recently, thermoreversible hyaluronan (HA) based hydrogels, which are injectable, minimally invasive, allow for encapsulation and maintenance of viable cells and promote re-differentiation of NP cells, have been developed.1 While it is known that hyaluronan is recognized by mesenchymal stem cells (MSCs)2 and can induce anabolic pathways, growth factors may further support MSC differentiation in HA-based hydrogels. Therefore, the objectives of the present work were: (i) to assess whether the addition of growth factors3 could support MSC differentiation towards the disk cell phenotype in HA based hydrogels and (ii) to determine if MSC preconditioning (culture under “chondrogenic” conditions) would improve the survival rate and differentiation potential of MSC supplemented to an IVD cultured ex vivo. Materials and Methods Hydrogel Thermoreversible HA-based hydrogels were prepared by grafting a thermoreversible segment poly (N-isopropylacrylamide) (pNIPAM) on a functionalized HA backbone, as previously described.1 In vitro Human bone marrow-derived MSCs (P2-P4) were cultured for 7 days in HA-pNIPAM hydrogel under hypoxic conditions (5% O2) in chondrogenic medium, with or without recombinant human growth and differentiation factor-5 (rhGDF-5) supplementation. Cell viability, DNA, and glycosaminoglycans (GAG) content were quantified by Live-dead, Picogreen, and 1,9 dimethylmethylene blue assays, respectively. Gene expression analysis was performed to assess the disc-like differentiation of MSCs. The expression of collagen type I and II (COL1 and COL2) aggrecan (ACAN), cytokeratin-19, sox-9, cluster of differentiation 24 (CD24), hyaluronan synthase I and II (HAS1 and HAS2), forkhead box protein F1 (FOXF1) and carbonic anhydrase 12 (CA12) was analyzed with 18s as endogenous control. Ex Vivo Bovine caudal IVDs were excised and nucleotomy performed through the endplate (Fig. 1A,B). MSCs were either suspended in HA-pNIPAM and directly supplied to the IVDs or precultured (under hypoxic conditions and with supplementation of GDF-5) for 1 week in HA-pNIPAM and then supplied to the IVDs. After 1 week of culture, gene expression analyses were performed using the same markers as for the in vitro study. Statistics After confirming the normality of the data distribution, one-way analysis of variance with Bonferroni posthoc was performed ( p < 0.05). Results MSC viability was maintained>90% over 1 week of culture in the HA-pNIPAM hydrogel both in vitro and ex vivo. DNA and GAG did not show significant variation over the 7 days of in vitro culture. Gene expression revealed 10–100-fold upregulation of the transcription factor SOX9 and 100–1000, fold upregulation of COL2, while COL1 was only slightly enhanced. The expression of the potential NP cell marker KRT19 was also increased. With the addition of rhGDF-5, an improved COL2/COL1 expression ratio was found. No significant differences were found for the other genes. Ex vivo cultures of nucleotomized discs supplemented with MSCs in HA-pNIPAM confirmed the positive effect of the hydrogel and the IVD environment on MSC differentiation towards the disc-like phenotype (Fig. 1C), with upregulation of collagen type II ( p < 0.05), CD24, and FOX-F1. No significant differences were found for the other genes. Preculture of MSCs before the supplementation in the IVD did not enhance MSC differentiation. Figure 1 Bovine IVD after nucleotomy: (A) top view and (B) view of the nucleotomized area. (C) Gene expression of human MSCs after 1 week of culture in bovine IVDs normalized to the mRNA levels at day 0 of culture: human MSCs were either suspended in HA-pNIPAM and directly supplied to bovine IVDs (white bars) or precultured for 1 week in HA-pNIPAM hydrogel and then supplied to bovine IVDs (grey bars);* p < 0.05, outliers. Conclusion The thermoreversible hyaluronan-based hydrogel was able to induce human MSC differentiation toward a “disc-like” phenotype without the addition of growth factors, although GDF-5 supplementation resulted in an improved gene expression profile. Preculture of MSCs before the supplementation in the IVD is not required with the thermoreversible hyaluronan-based hydrogel. Therefore, this type of hydrogel could be an appropriate carrier for MSCs supplementation to compromised discs. Investigations are ongoing to evaluate its biomechanical behavior under loading. Acknowledgements This study was partially supported by a NASS Research Grant. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Peroglio M, et al. European Spine Journal (Epub ahead of print) Zhu H et al. Stem cells 2006; 24:928–935 Gantenbein-Ritter B, et al. European Spine Journal 2011;20(6):962–971

Multiple Membrane Tethers Probed by Atomic Force Microscopy

Using the atomic force microscope to locally probe the cell membrane, we observed the formation of multiple tethers (thin nanotubes, each requiring a similar pulling force) as reproducible features within force profiles recorded on individual cells. Forces obtained with Chinese hamster ovary cells, a malignant human brain tumor cell line, and human endothelial cells (EA hy926) were found to be 28 ± 10 pN, 29 ± 9 pN, and 29 ± 10 pN, respectively, independent of the nature of attachment to the cantilever. The rather large variation of the tether pulling forces measured at several locations on individual cells points to the existence of heterogeneity in the membrane properties of a morphologically homogeneous cell. Measurement of the summary lengths of the simultaneously extracted tethers provides a measure of the size of the available membrane reservoir through which co-existing tethers are associated. As expected, partial disruption of the actin cytoskeleton and removal of the hyaluronan backbone of the glycocalyx were observed to result in a marked decrease (30–50%) in the magnitude and a significant sharpening of the force distribution indicating reduced heterogeneity of membrane properties. Taken together, our results demonstrate the ability of the plasma membrane to locally produce multiple interdependent tethers—a process that could play an important role in the mechanical association of cells with their environment.

Comparison of the antioxidant properties of HYAFF ®-11p75, AQUACEL ® and hyaluronan towards reactive oxygen species in vitro

In chronic wounds, a number of host factors are released which perpetuate the inflammatory process, including polymorphonuclear leukocyte (PMN)-derived reactive oxygen species (ROS), such as superoxide radical (O 2 − ) and hydroxyl radical ( OH) species. The glycosaminoglycan, hyaluronan, has been shown to act as an antioxidant towards ROS, although the potential for biomaterials, such as HYAFF ®-11p75 (the 75% benzyl ester of hyaluronan) and AQUACEL ® (carboxymethylcellulose), to act in this manner has yet to be elucidated. This study compared the antioxidant properties of high and low molecular weight hyaluronan (HMWT HA and LMWT HA), HYAFF ®-11p75, AQUACEL ® and an AQUACEL ®/hyaluronan composite (AQUACEL ®/HA) against O 2 − and OH. The antioxidant capacities of each material were assessed by their ability to inhibit cytochrome C reduction by O 2 − fluxes, generated via the oxidation of hypoxanthine by xanthine oxidase, and their inhibition of 2-deoxy- d-ribose degradation by OH fluxes, generated by the reaction of hydrogen peroxide (H 2O 2) and iron (Fe 2+). All materials studied possessed dose dependent antioxidant properties towards O 2 − , with HYAFF ®-11p75 having the greatest antioxidant potential towards these species, followed by AQUACEL ®, HMWT HA, AQUACEL ®/HA and LMWT HA. Only HMWT HA exhibited dose dependent antioxidant properties towards OH at the fluxes examined. Gas chromatography/mass spectrometry analysis implied that ester bonds between the hyaluronan backbone and benzyl groups of HYAFF ®-11p75 are highly susceptible to O 2 − hydrolysis, with the de-esterified benzyl alcohol being rapidly degraded in the presence of OH. This data supports the hypothesis that HYAFF ®-11p75 has greater antioxidant capacity towards O 2 − , due to the esterified benzyl groups providing alternative sites for O 2 − attack other than the hyaluronan backbone of HYAFF ®-11p75 itself and explains the inability of HYAFF ®-11p75 to scavenge OH, due to benzyl alcohol degradation by OH. The antioxidant activities of these biomaterials, particularly HYAFF ®-11p75 and AQUACEL ®, towards O 2 − could be beneficial, as the scavenging of PMN-derived O 2 − may remove initial sources of O 2 − and further prevent the secondary formation of OH. These ROS are thought to be a primary causal factor for the extensive degradation and metabolic alterations observed in chronic wounds.

The enhanced stability of the cross-linked hylan structure to hydroxyl (OH) radicals compared with the uncross-linked hyaluronan

A comparison has been made of the relative stabilities of hyaluronan and hylan to degradation by OH radicals produced by γ-irradiation of aqueous solutions in N 2O, when G (yield per 100 eV) for OH radicals is 5.6 and H atoms 0.6. Using low angle light scattering and viscometric methods, the change in molecular weight of the polysaccharides was measured with increasing dose. From the yield/dose curves (expressed as breaks per molecule), the initial G value for hyaluronan degradation is ∼ 4. A further slow post-irradiation decrease in molecular weight is observed, which can be brought to completion by incubating the solutions for 1 h at 60°C. Thereafter, the G value for degradation is ∼ 6. A similar post-irradiation degradation was found for hylan. A technique using tetranitromethane (TNM) has been used to distinguish between two types of radicals formed on the hyaluronan backbone. Radicals of the 1-hydroxy-2-alkoxy type (C-2, C-4, C-2 and C3 of the glucuronic acid) would induce strand breakage by alkoxy elimination. For the equivalent alkoxy radical at C6 of the acetamido monosaccharide, ring opening would occur with formation of a hemi-acetal, leading also to strand breakage. The C-2 and C-3 radicals would eliminate water rather than produce breaks by β-alkoxy elimination. Thus three out of the initially formed radicals would produce breaks by β-alkoxy formation. These can be stabilised with TNM and distinguished. It is concluded that these are the radicals involved in the post-irradiation thermal degradation process. Comparison of hylan and hyaluronan is, therefore, most valid when this post-irradiation process has been completed. Therefore, all G values for degradation were measured after incubation for 1 h at 60°. This investigation establishes the greater stability of hylan ( G degradation = 2) compared to hylan ( G degradation = 6). Therefore, in an environment such as supplementation of an inflammed joint where OH radicals are released, hylan is able to retain its integrity as a viscoelastic macromolecule three times better than hyaluronan. Its potential as a viscosupplementation material, or as an inflammatory drug release matrix inserted within the joint is, therefore, greater than non-cross-linked hyaluronan.