Structural, macro- and microrheological properties of Hyaluronic Acid (HA) cryogels
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Event Abstract Back to Event Supramolecular hyaluronic acid-based hydrogels with dynamic viscoelasticity Adrianne M. Rosales1*, Christopher B. Rodell2*, Minna Chen2*, Jason A. Burdick2* and Kristi S. Anseth1, 3* 1 University of Colorado Boulder, Chemical and Biological Engineering, United States 2 University of Pennsylvania, Bioengineering, United States 3 Howard Hughes Medical Institute, United States Introduction: The extracellular matrix provides many mechanical cues to cells during growth, development, and disease. While hydrogels have emerged as desirable cell culture platforms because they can mimic soft tissues, few hydrogels are capable of providing mechanical cues in a dynamic fashion. Furthermore, almost no hydrogels can stiffen and soften reversibly, which could have important applications in models of diseases such as fibrosis. Here, we present hyaluronic acid (HA)-based hydrogels with photoresponsive guest-host crosslinks (between azobenzene and cyclodextrin), allowing for in situ changes in crosslinking density and therefore modulus in the presence of cells (A). Materials and Methods: HA was functionalized via esterification with 4-phenylazobenzoic acid (AzoHA) or via amidation with β-cyclodextrin (CDHA) (Figure, Part A). The complementary HA polymers were mixed in a 1:1 ratio to form supramolecular gels. The viscoelastic modulus was measured using shear rheology, and light cues were introduced in situ with a mercury lamp containing either a 365 nm or a 400-500 nm filter (10 mW/cm2). For cell studies, HA also contained 2 mM RGD as an adhesion ligand, and NIH 3T3 fibroblasts were either encapsulated in the gels upon mixing or seeded on the gels post-mixing. Results and Discussion: The use of non-covalent photoresponsive crosslinks allows for supramolecular assembly that has been shown to be disrupted upon exposure to light[1]. In our system, azobenzene forms a guest-host complex with β-CD with an affinity of approximately 500 M-1, leading to supramolecular gelation. Upon irradiation with 365 nm light for 10 min, the azobenzene undergoes a trans to cis isomerization, which decreases the binding affinity with β-CD to approximately 200 M-1. The decrease in binding affinity leads to the dissociation of crosslinks and results in an overall decrease in the viscoelastic modulus by up to 60% (B). This transition is reversible upon irradiation with visible light in the 400-500 nm range. Although light is a minimally invasive stimulus to cells[2],[3], the relaxation of the cis to trans transition can be controlled by introducing substituents on the azobenzene, allowing for a half-life of up to 9 hours at 37°C and avoiding the need for continuous irradiation. Encapsulated NIH 3T3 fibroblasts are highly viable (>95%, C) in these gels for up to 7 days, and seeded 3T3 cells can show a spread morphology after 1 day (C). Ongoing experiments are examining the effect of dynamic mechanical cues on cell morphology. Conclusions: A cytocompatible, supramolecular hydrogel with a tunable, reversible modulus has been developed, and ongoing work is probing the effect of dynamic mechanical cues on cell morphology. Due to the importance of mechanics in cellular phenotype, these materials may be broadly applicable to many cell types. HHMI; NSF DMR 1408955; NIH postdoctoral fellowship 5 F32 HL121986-02 (to AMR); Burroughs Wellcome Fund Postdoctoral Enrichment Program award (to AMR)References:[1] Tamesue, S., Harada, A., et. al. Angewandte Chemie, 2010, 49(41), 7461-7464.[2] Kloxin, A.M., Anseth, K.S., et. al. Science 2009, 324(5923), 59-63.[3] Guvendiren, M., Burdick, J.A. Nature Communications 2012, 3:792. Keywords: Hydrogel, mechanical property, stimuli-response, biomacromolecule Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: New Frontier Oral Topic: Nano-structured materials for unique functions Citation: Rosales AM, Rodell CB, Chen M, Burdick JA and Anseth KS (2016). Supramolecular hyaluronic acid-based hydrogels with dynamic viscoelasticity. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.01669 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. * Correspondence: Dr. Adrianne M Rosales, University of Colorado Boulder, Chemical and Biological Engineering, Boulder, CO, United States, adrianne.rosales@colorado.edu Dr. Christopher B Rodell, University of Pennsylvania, Bioengineering, Philadelphia, PA, United States, cbrodell@gmail.com Dr. Minna Chen, University of Pennsylvania, Bioengineering, Philadelphia, PA, United States, minnac@seas.upenn.edu Dr. Jason A Burdick, University of Pennsylvania, Bioengineering, Philadelphia, PA, United States, burdick2@seas.upenn.edu Dr. Kristi S Anseth, University of Colorado Boulder, Chemical and Biological Engineering, Boulder, CO, United States, kristi.anseth@colorado.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Adrianne M Rosales Christopher B Rodell Minna Chen Jason A Burdick Kristi S Anseth Google Adrianne M Rosales Christopher B Rodell Minna Chen Jason A Burdick Kristi S Anseth Google Scholar Adrianne M Rosales Christopher B Rodell Minna Chen Jason A Burdick Kristi S Anseth PubMed Adrianne M Rosales Christopher B Rodell Minna Chen Jason A Burdick Kristi S Anseth Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.
Supramolecular Polymers
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In this study, macroporous, elastic, three-dimensional scaffolds formed of hyaluronic acid mixed with ethylene glycol diglycidyl ether as a chemical cross-linker have been prepared by cryogelation for application in tissue engineering. These cryogels are characterized by large interconnected pores of size ∼50-300 μm and pore wall thickness of ∼5-30 μm as determined from confocal microscopy images. Variation of pH, freezing temperature, and polymerization time allows for control of pore size and shape as well as matrix thickness. These structural properties then determine mechanical strength as well as swelling capacity. Furthermore, increasing hyaluronic acid concentration decreases cryogel pore size, reduces swelling properties, and reinforces mechanical properties. On the other hand, decreasing cross-linker concentration, at a constant hyaluronic acid concentration, increases pore size and swelling capacity but provides less rigidity. Additionally, for the first time, local elastic properties of the polymer matrix and viscous properties of the pores have been characterized using multiple particle tracking microrheology. Local matrix elasticity, relaxation time of hyaluronic acid chains, and the degree of heterogeneity are discussed in detail. These latter properties are crucial for the development of new tissue engineering constructs and will help to understand how local matrix viscoelasticity affects cell cultivation. Finally, elastic moduli obtained in bulk rheology are much higher than corresponding values deduced from microrheology. This discrepancy might be explained by the formation of very highly cross-linked cores in the network where no tracer particle can penetrate.
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Chemical Modifications, Solid Phase, Radio‐Chemical and Enzymatic Transformations of Hyaluronic Acid
This chapter focuses on two subjects related to the field of mechanically stimulated reactions: (1) chemical and photochemical cross-linking of HA in aqueous solutions and (2) advanced methods of solid-phase polysaccharide modification. From these processes, the cross-linked hyaluronic hydrogels acquire a number of valuable properties that significantly extend the range of their medical applications. Radio-chemical transformations of hyaluronan aqueous solutions are studied by the radiation chemistry of biopolymers. Radiolysis of hyaluronan aqueous solutions could be described as destructive oxidative processes in which hydroxyl radical OH* plays the major role. Understanding of the mechanism of radiolysis of hyaluronan is necessary in order to develop a strategy to protect the polysaccharide from the destructive effects of oxidative radicals.
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For the first time, macroporous, elastic, three-dimensional hyaluronic acid cryogels were prepared with genipin as non-cytotoxic crosslinking agent. These cryogels are characterized by a lamellar porous structure with a homogeneous pore size of ~100 µm, shear elasticity of ~2 kPa and a swelling ratio of 2.5 in water. Additionally, multiple particle tracking based microrheology measurements reveal the formation of a heterogeneous network. This novel biomaterial owns great potential as non-cytotoxic alternative for application in drug delivery, as tissue engineering scaffold or wound healing substrate and can help reducing toxicity of artificial skin grafts or tissue equivalents.
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To design models of tissue surfaces, films of soft gels of hyaluronic acid (HA) (a ubiquitous constituent of the extracellular matrix) are covalently grafted to glass substrates functionalized with aminosilane monolayers. Gelation is achieved by coupling of carboxyl groups of HA through carbodiimide. The elasticity of the gel is controlled through the HA concentration. The magnetic bead microrheometry and colloidal bead deformation field mapping techniques are applied to measure the surface viscoelastic moduli and the effective Young moduli of the HA gel as a function of the gel density. For this purpose, magnetic beads and nonmagnetic beads are coupled to the surface of the HA films. Soft homogeneous films exhibiting Young elastic moduli between 3 and 250 Pa were generated. The shear deformation field induced by tangential force pulses applied to the magnetic beads is measured and analyzed in terms of the theory of elastic deformation of half-spaces by local forces. An unconventional vortex-deformation field is observed for gel film thicknesses of several hundred micrometers, which is attributed to the nonlinear elasticity of the gels. We finally show that amoeba-like cells of the slime mold Dictyostelium discoideum spontaneously adhere to the HA film, while fibroblast adhesion can be mediated through coupling fibronectin to the surface.
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Hydrogels are used to create 3D microenvironments with properties that direct cell function. The current study demonstrates the versatility of hyaluronic acid (HA)-based hydrogels with independent control over hydrogel properties such as mechanics, architecture, and the spatial distribution of biological factors. Hydrogels were prepared by reacting furan-modified HA with bis-maleimide-poly(ethylene glycol) in a Diels–Alder click reaction. Biomolecules were photopatterned into the hydrogel by two-photon laser processing, resulting in spatially defined growth factor gradients. The Young's modulus was controlled by either changing the hydrogel concentration or the furan substitution on the HA backbone, thereby decoupling the hydrogel concentration from mechanical properties. Porosity was controlled by cryogelation, and the pore size distribution, by the thaw temperature. The addition of galactose further influenced the porosity, pore size, and Young's modulus of the cryogels. These HA-based hydrogels offer a tunable platform with a diversity of properties for directing cell function, with applications in tissue engineering and regenerative medicine.
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Hyaluronic acid (HA) hydrogels, obtained by cross-linking HA molecules with divinyl sulfone (DVS) based on a simple, reproducible, and safe process that does not employ any organic solvents, were developed. Owing to an innovative preparation method the resulting homogeneous hydrogels do not contain any detectable residual cross-linking agent and are easier to inject through a fine needle. HA hydrogels were characterized in terms of degradation and biological properties, viscoelasticity, injectability, and network structural parameters. They exhibit a rheological behaviour typical of strong gels and show improved viscoelastic properties by increasing HA concentration and decreasing HA/DVS weight ratio. Furthermore, it was demonstrated that processes such as sterilization and extrusion through clinical needles do not imply significant alteration of viscoelastic properties. Both SANS and rheological tests indicated that the cross-links appear to compact the network, resulting in a reduction of the mesh size by increasing the cross-linker amount. In vitro degradation tests of the HA hydrogels demonstrated that these new hydrogels show a good stability against enzymatic degradation, which increases by increasing HA concentration and decreasing HA/DVS weight ratio. Finally, the hydrogels show a good biocompatibility confirmed by in vitro tests.
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Development of injectable high molecular weight hyaluronic acid hydrogels for cartilage regeneration
The study focuses on developing hyaluronic acid (1200 kilo Dalton) hydrogels for cartilage regeneration. In spite of being highly biocompatible; a large amount of water absorption and easily degrading nature restricts the use of hyaluronic acid in the field of tissue regeneration. This can be rectified by crosslinking hyaluronic acid with a crosslinking agent such as divinyl sulfone; which results in a biocompatible hydrogel with superior rheological properties. Different amounts of divinyl sulfone have been used for crosslinking hyaluronic acid to get three types of hydrogels with differing properties. Swelling studies, rheology analysis, enzymatic degradation and scanning electron microscopic analysis were conducted on all the different types of hydrogels prepared. Viscoelastic properties of the hydrogel were analyzed so that a hydrogel with better elastic property and stability is obtained. Scanning electron microscopy was used to study the morphology of the HA hydrogels. The cytotoxicity testing was conducted to prove the non-toxic nature of the hydrogels and cell culture studies using adipose mesenchymal stem cells showed better adhesion and proliferation properties in all the three hydrogels. Thus hyaluronic acid hydrogel makes a promising material for cartilage regeneration.
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