Biomimetic carbohydrate substrates of tunable properties using immobilized dextran hydrogels

Glycidylmethacrylate-modified dextran macromers (Dex-GMA) of different degrees of substitution (DS) were synthesized. The elastic modulus of the hydrogels produced using one-component and two-component macromer systems was measured using rheometry. When one macromer of DS 1/10 was used, a hydrogel modulus in the range of 0.2 Pa to 42 kPa was obtained by varying the Dex-GMA concentration from 80 to 200 mg/mL. When a mixture of two macromers of different DS (1/10 and 1/23) was used, a more uniform variation of modulus in the range of 0.4 Pa to 42 kPa was achieved by controlling the ratio of the two macromers. When dextran hydrogels were functionalized with fibronectin and immobilized onto glass substrates, the attachment, spreading, and growth of human aortic smooth muscle cells were modulated by the elastic properties of the dextran matrix. The dextran hydrogel system with tunable mechanical and biochemical properties appears promising for applications in cell culture and tissue engineering.     

Hydrolytically degradable hyaluronic acid hydrogels with controlled temporal structures

Polysaccharides are being processed into biomaterials for numerous biological applications due to their native source in numerous tissues and biological functions. For instance, hyaluronic acid (HA) is found abundantly in the body, interacts with cells through surface receptors, and can regulate cellular behavior (e.g., proliferation, migration). HA was previously modified with reactive groups to form hydrogels that are degraded by hyaluronidases, either added exogenously or produced by cells. However, these hydrogels may be inhibitory and their applications are limited if the appropriate enzymes are not present. Here, for the first time, we synthesized HA macromers and hydrogels that are both hydrolytically (via ester group hydrolysis) and enzymatically degradable. The hydrogel degradation and growth factor release was tailored through the hydrogel cross-linking density (i.e., macromer concentration) and copolymerization with purely enzymatically degradable macromers. When mesenchymal stem cells (MSCs) were encapsulated in the hydrogels, cellular organization and tissue distribution was influenced by the copolymer concentration. Importantly, the distribution of released extracellular matrix molecules (e.g., chondroitin sulfate) was improved with increasing amounts of the hydrolytically degradable component. Overall, this new macromer allows for enhanced control over the structural evolution of the HA hydrogels toward applications as biomaterials.     

Synthesis and degradation test of hyaluronic acid hydrogels

Hyaluronic acid (HA) hydrogels prepared with three different crosslinking reagents were assessed by in vitro and in vivo degradation tests for various tissue engineering applications. Adipic acid dihydrazide grafted HA (HA-ADH) was synthesized and used for the preparation of methacrylated HA (HA-MA) with methacrylic anhydride and thiolated HA (HA-SH) with Traut's reagent (imminothiolane). (1)H NMR analysis showed that the degrees of HA-ADH, HA-MA, and HA-SH modification were 69, 29, and 56 mol%, respectively. HA-ADH hydrogel was prepared by the crosslinking with bis(sulfosuccinimidyl) suberate (BS(3)), HA-MA hydrogel with dithiothreitol (DTT) by Michael addition, and HA-SH hydrogel with sodium tetrathionate by disulfide bond formation. According to in vitro degradation tests, HA-SH hydrogel was degraded very fast, compared to HA-ADH and HA-MA hydrogels. HA-ADH hydrogel was degraded slightly faster than HA-MA hydrogel. Based on these results, HA-MA hydrogels and HA-SH hydrogels were implanted in the back of SD rats and their degradation was assessed according to the pre-determined time schedule. As expected from the in vitro degradation test results, HA-SH hydrogel was in vivo degraded completely only in 2 weeks, whereas HA-MA hydrogels were degraded only partially even in 29 days. The degradation rate of HA hydrogels were thought to be controlled by changing the crosslinking reagents and the functional group of HA derivatives. In addition, the state of HA hydrogel was another factor in controlling the degradation rate. Dried HA hydrogel at 37 degrees C for a day resulted in relatively slow degradation compared to the bulk HA hydrogel. There was no adverse effect during the in vivo tests.     

Diels-Alder Click cross-linked hyaluronic acid hydrogels for tissue engineering

Hyaluronic acid (HA) is a naturally occurring polymer that holds considerable promise for tissue engineering applications. Current cross-linking chemistries often require a coupling agent, catalyst, or photoinitiator, which may be cytotoxic, or involve a multistep synthesis of functionalized-HA, increasing the complexity of the system. With the goal of designing a simpler one-step, aqueous-based cross-linking system, we synthesized HA hydrogels via Diels-Alder "click" chemistry. Furan-modified HA derivatives were synthesized and cross-linked via dimaleimide poly(ethylene glycol). By controlling the furan to maleimide molar ratio, both the mechanical and degradation properties of the resulting Diels-Alder cross-linked hydrogels can be tuned. Rheological and degradation studies demonstrate that the Diels-Alder click reaction is a suitable cross-linking method for HA. These HA cross-linked hydrogels were shown to be cytocompatible and may represent a promising material for soft tissue engineering.     

Superporous IPN hydrogels having enhanced mechanical properties

The objective of this study was to improve the mechanical properties of superporous hydrogels (SPHs), which were used to develop gastric retention devices for long-term oral drug delivery. The main approach used in this study was to form an interpenetrating polymer network by incorporating a second polymer network inside an SPH structure. Polyacrylonitrile was used as the second network inside an SPH. Mechanical properties including compression strength and elasticity were significantly improved, up to 50 times as compared with the control SPHs. The enhanced mechanical properties were a result of the scaffold-like fiber network structures formed inside the cell walls of SPHs. The fast swelling property of SPHs was not affected by the incorporation of the second polymer network because the interconnected pore structures were maintained. Gastric retention devices based on superporous IPN hydrogels (SPIHs) with the improved mechanical properties are expected to withstand compression pressure and mechanical frictions in the stomach better than the control SPHs.     

Surface rheological properties of hyaluronic acid solutions

Using an oscillating ring surface rheometer, surface shear rheological studies of hyaluronic acid solutions at physiological pH have demonstrated the elastico-viscous nature of the surface films. The properties of these surface films change with time and are shown to be related to bulk concentration, ionic strength and pH. This ageing behaviour can be explained on the basis of molecular conformational changes and molecular segmental kinetics. The results are discussed in relation to the postulated function of hyaluronic acid in synovial fluid.     

Synthetic biomimetic hydrogels incorporated with ephrin-A1 for therapeutic angiogenesis

Eph receptors and ephrin ligands are essential for vascular development and angiogenic remodeling. In this work, we developed biomimetic poly(ethylene glycol)-diacrylate hydrogels incorporated with ephrin-A1 and examined their angiogenic properties. Ephrin-A1 was covalently immobilized on the surface of hydrogels by chemical modification and photopolymerization. Ephrin-A1 immobilized on hydrogels was found to retain its capacity to stimulate endothelial cell adhesion in a dose-dependent manner as similar findings were observed on polystyrene culture wells pre-adsorbed with ephrin-A1. Cell adhesion stimulated by ephrin-A1 was abolished by treatment with soluble RGDS and anti-alpha(v)beta3 integrin but not anti-alpha(v)beta5 integrin antibodies, suggesting that ephrin-A1 activates cell adhesion through alpha(v)beta3 integrins. Also, surface immobilized ephrin-A1 was found to induce endothelial tubule formation with luminal diameters ranging 5-30 microm on hydrogels. The results of these studies demonstrate that pro-angiogenic properties of ephrin-A1 are preserved in hydrogels and suggest potential applications of this hydrogel system in regenerative medicine and tissue engineering.     

Reductive alkylation of hyaluronic acid for the synthesis of biocompatible hydrogels by click chemistry

Hyaluronan (HA) based hydrogels have been synthesized combining chemical modification of the polysaccharide by partial oxidation, reductive amination and 'click chemistry'. HA was oxidized by 4-acetamido-TEMPO-mediated reaction, using sodium hypochlorite as primary oxidant and NaBr in buffered pH, so that the produced aldehyde moieties (hemiacetals) were trapped in situ by adding primary amines containing azide or alkyne-terminal groups. The structure of the reaction products, oxidized-HA and primary amines bonded to HA, was elucidated using 2D NMR spectroscopy. SEC-MALLS analysis of the modified substrates showed a negligible degradation of the polysaccharide using this procedure. Furthermore, azido- and alkynyl derivatives underwent cross-linking by click chemistry into hydrogels, which were characterized by NMR, FT-IR, swelling degree and mechanical properties. Possible application of the material as scaffold for tissue engineering was tested by seeding and proliferation of chondrocytes for up to 15 days.     

Repeated rapid shear-responsiveness of peptide hydrogels with tunable shear modulus

A pair of mutually attractive but self-repulsive decapeptides, with alternating charged/neutral amino acid sequence patterns, was found to co-assemble into a viscoelastic material upon mixing at a low total peptide concentration of 0.25 wt %. Circular dichroism spectroscopy of individual decapeptide solutions revealed their random coil conformation. Transmission electron microscopy images showed the nanofibrillar network structure of the hydrogel. Dynamic rheological characterization revealed its high elasticity and shear-thinning nature. Furthermore, the co-assembled hydrogel was capable of rapid recoveries from repeated shear-induced breakdowns, a property desirable for designing injectable biomaterials for controlled drug delivery and tissue engineering applications. A systematic variation of the neutral amino acids in the sequence revealed some of the design principles for this class of biomaterials. First, viscoelastic properties of the hydrogels can be tuned through adjusting the hydrophobicity of the neutral amino acids. Second, the beta-sheet propensity of the neutral amino acid residue in the peptides is critical for hydrogelation.     

Facile synthesis and characterization of disulfide-cross-linked hyaluronic acid hydrogels for protein delivery and cell encapsulation

Injectable hyaluronic acid (HA) hydrogels cross-linked via disulfide bond are synthesized using a thiol-disulfide exchange reaction. The production of small-molecule reaction product, pyridine-2-thione, allows the hydrogel formation process to be monitored quantitatively in real-time by UV spectroscopy. Rheological tests show that the hydrogels formed within minutes at 37 °C. Mechanical properties and equilibrium swelling degree of the hydrogels can be controlled by varying the ratio of HA pyridyl disulfide and macro-cross-linker PEG-dithiol. Degradation of the hydrogels was achieved both enzymatically and chemically by disulfide reduction with distinctly different kinetics and profiles. In the presence of hyaluronidase, hydrogel mass loss over time was linear and the degradation was faster at higher enzyme concentrations, suggesting surface-limited degradation. The kinetics of hydrogel erosion by glutathione was not linear, nor did the erosion rate correlate linearly with glutathione concentration, suggesting a bulk erosion mechanism. A cysteine-containing chemokine, stromal cell-derived factor 1α, was successfully encapsulated in the hydrogel and released in vitro without chemical alteration. Several different cell types, including fibroblasts, endothelial cells, and mesenchymal stem cells, were successfully encapsulated in the hydrogels with high cell viability during and after the encapsulation process. Substantial cell viability in the hydrogels was maintained up to 7 days in culture despite the lack of adhesion between the HA matrix and the cells. The facile synthesis of disulfide-cross-linked, dual-responsive degradable HA hydrogels may enable further development of bioactive matrices potentially suitable for tissue engineering and drug delivery applications.     

Synthesis and characterization of an injectable hydrogel with tunable mechanical properties for soft tissue repair

Injectable polymers are attractive materials for the fixation or augmentation of soft tissues. Thermosensitive hydrogels, especially poly(N-isopropylacryamide), have been investigated for these applications to exploit the lower critical solution temperature (LCST) which falls between room and body temperatures. One limitation to the material is the ability to withstand loading. In this work, we evaluated an injectable material system, poly(N-isopropylacryamide)-co-poly(ethyleneglycol) dimethacrylate, with the addition of trimethacryloxypropyltrimethoxysilane (MPS). Our goal was to investigate the potential to tune the mechanical behavior of the injectable hydrogel. Addition of MPS to the hydrogel increased the compressive modulus but did not affect the LCST of the hydrogel. An increase in ion concentration of the immersion media resulted in less solution uptake by the hydrogels, regardless of MPS presence in the system. The challenge of this material system is to balance the network-forming and modulus-enhancing MPS while maintaining an injectable hydrogel for potential soft tissue repair.     

Isolation and properties of hyaluronic acid from bovine heart valves

1. A soluble extract of bovine heart valves was obtained after the tissue had been pulverized at liquid-nitrogen temperatures in a mill. 2. Hyaluronic acid was isolated from the crude extract by sedimentation equilibrium in a caesium chloride density gradient (Franek & Dunstone, 1966). 3. Analysis of the product indicated that it contained 15% of protein and the molar ratio of glucuronic acid to glucosamine was 1.27. 4. Its physicochemical properties, as determined by lightscattering, viscosity and sedimentation studies, suggested that its molecular size and configuration were similar to those of hyaluronic acid isolated from ox synovial fluid (Preston, Davies & Ogston, 1965).     

Microstructure,mechanical, and biomimetic properties of fish scales from Pagrus major

The fish scale of Pagrus major has an orthogonal plywood structure of stratified lamellae, 1-2 microm in thickness, consisting of closely packed 70- to 80-nm-diameter collagen fibers. X-ray diffraction, energy-dispersive X-ray analysis, and infrared spectroscopy indicate that the mineral phase in the scale is calcium-deficient hydroxyapatite containing a small amount of sodium and magnesium ions, as well as carbonate anions in phosphate sites of the apatite lattice. The tensile strength of the scale is high (approximately 90 MPa) because of the hierarchically ordered structure of mineralized collagen fibers. Mechanical failure occurs by sliding of the lamellae and associated pulling out and fracture of the collagen fibers. In contrast, demineralized scales have significantly lower tensile strength (36 MPa), indicating that interactions between the apatite crystals and collagen fibers are of fundamental importance in determining the mechanical properties. Thermal treatment of fish scales to remove the organic components produces remarkable inorganic replicas of the native orthogonal plywood structure of the fibrillary plate. The biomimetic replica produced by heating to 873 K consists of stratified porous lamellae of c-axis-aligned apatite crystals that are long, narrow plates, 0.5-0.6 microm in length and 0.1-0.2 microm in width. The textured inorganic material remains intact when heated to 1473 K, although the size of the constituent crystals increases as a result of thermal sintering.     

Engineering strategies to recapitulate epithelial morphogenesis within synthetic three-dimensional extracellular matrix with tunable mechanical properties

The mechanical properties (e.g. stiffness) of the extracellular matrix (ECM) influence cell fate and tissue morphogenesis and contribute to disease progression. Nevertheless, our understanding of the mechanisms by which ECM rigidity modulates cell behavior and fate remains rudimentary. To address this issue, a number of two and three-dimensional (3D) hydrogel systems have been used to explore the effects of the mechanical properties of the ECM on cell behavior. Unfortunately, many of these systems have limited application because fiber architecture, adhesiveness and/or pore size often change in parallel when gel elasticity is varied. Here we describe the use of ECM-adsorbed, synthetic, self-assembling peptide (SAP) gels that are able to recapitulate normal epithelial acini morphogenesis and gene expression in a 3D context. By exploiting the range of viscoelasticity attainable with these SAP gels, and their ability to recreate native-like ECM fibril topology with minimal variability in ligand density and pore size, we were able to reconstitute normal and tumor-like phenotypes and gene expression patterns in nonmalignant mammary epithelial cells. Accordingly, this SAP hydrogel system presents the first tunable system capable of independently assessing the interplay between ECM stiffness and multi-cellular epithelial phenotype in a 3D context.