Cross-linked Alginate Hydrogel Research Articles

Self-cross-linkable hydrogels composed of partially oxidized alginate and gelatin for myocardial infarction repair

Calcium cross-linked alginate hydrogel has shown positive results for the treatment of myocardial infarction in both acute and chronic rat models. However, cross-linked alginate hydrogels possess poor mechanical properties, uncontrollable degradation, and poor cell adhesion and infiltration. In this study, covalent cross-linking of partially oxidized alginate and gelatin hydrogel was developed for myocardial infarction treatment, as compared to cross-linked alginate hydrogel and saline. Specifically, the gelation process, mechanical properties, and biodegradation of both cross-linked alginate hydrogel and oxidized alginate and gelatin hydrogel were examined in vitro and in vivo; and a rat myocardial infarction model was developed by injecting hydrogel into hearts, and postinjection, echocardiography was performed at 2, 4, and 6 weeks, respectively. Matrix metalloprotease-2/9 activity was also examined by in situ zymography on frozen slices of the treated hearts. Based on the results, that both hydrogels enhanced scar thickness and attenuated heart remodeling compared with the saline control group and that, compared to cross-linked alginate hydrogel, oxidized alginate and gelatin hydrogel exhibited the increased mechanical strength, enhanced angiogenesis, and stronger cell-recruiting capacity. It has also been illustrated that oxidized alginate and gelatin hydrogel–treated hearts have much weaker cardiac remodeling, enhanced extracellular matrix accumulation, and reduced matrix metalloprotease activity than cross-linked alginate hydrogel–treated ones. All results suggest oxidized alginate and gelatin hydrogel may be more suitable for the myocardial infarction treatment.

Photodegradable Iron(III) Cross-Linked Alginate Gels

Biocompatible photoresponsive materials are of interest for targeted drug delivery, tissue engineering, 2D and 3D protein patterning, and other biomedical applications. We prepared light degradable hydrogels using a natural alginate polysaccharide cross-linked with iron(III) cations. The "hard" iron(III) cations used to cross-link the alginate hydrogel were found to undergo facile photoreduction to "soft" iron(II) cations in the presence of millimolar concentrations of sodium lactate. The "soft" iron(II) cations have a decreased ability to cross-link the alginate which results in dissolution of the hydrogel and the formation of a homogeneous solution. The photodegradation is done using long wave UV or visible light at neutral pH. The very mild conditions required for the photodegradation and the high rate at which it occurs suggest applications for iron(III) cross-linked alginate hydrogels as light-controlled biocompatible scaffolds.

Hyaluronic acid/mildly crosslinked alginate hydrogel as an injectable tissue adhesion barrier

Although hyaluronic acid (HA) has been conventionally utilized as a tissue adhesion barrier material, its rapid clearance in the body still remains as a big challenge in the clinical practice. In this study, we prepared a hydrogel of HA embedded in mildly crosslinked alginate (HA/mcALG hydrogel), which is injectable, easily covers injured tissues, and remains stably at the applied site during wound healing (by muco-adhesive HA embedded in the network structure of the mcALG hydrogel). The HA/mcALG hydrogel was highly effective for the prevention of peritoneal tissue adhesion compared to HA and mcALG hydrogels, and did not lead to any abnormal tissue responses during wound healing. The HA/mcALG hydrogel can be a good candidate as an injectable tissue adhesion barrier for clinical applications.

Electrochemically stimulated release of lysozyme from an alginate matrix cross-linked with iron cations

An electrochemically generated alginate matrix cross-linked with Fe3+ cations was used to entrap lysozyme and then release it upon application of an electrochemical signal. The switchable behavior of the alginate hydrogel was based on the different interaction of Fe3+ and Fe2+ cations with alginate. The oxidized Fe3+ cations strongly interact with alginate resulting in its cross-linking and formation of the hydrogel, while the reduced Fe2+ cations weakly interact with alginate and do not keep it in the hydrogel state. Thus, the electrochemical oxidation of iron cations at +0.8 V (Ag/AgCl) in the presence of alginate and lysozyme resulted in the Fe3+-cross-linked alginate hydrogel thin-film on the electrode surface with the physically entrapped lysozyme. On the other hand, application of reductive potentials (e.g. −1.0 V) converted the iron cations to the Fe2+ state, thus resulting in dissolution of the alginate thin-film and lysozyme release. The bactericidal effect of the electrochemically released lysozyme was tested on the Gram-positive bacterium Micrococcus luteus demonstrating the same activity as the unadulterated lysozyme commercially supplied by Sigma-Aldrich. The present result represents the first step towards drug delivering systems (exemplified by the lysozyme release) based on alginate hydrogels and activated by electrochemical stimuli.

Improving Viability of Stem Cells During Syringe Needle Flow Through the Design of Hydrogel Cell Carriers

Cell transplantation is a promising therapy for a myriad of debilitating diseases; however, current delivery protocols using direct injection result in poor cell viability. We demonstrate that during the actual cell injection process, mechanical membrane disruption results in significant acute loss of viability at clinically relevant injection rates. As a strategy to protect cells from these damaging forces, we hypothesize that cell encapsulation within hydrogels of specific mechanical properties will significantly improve viability. We use a controlled in vitro model of cell injection to demonstrate success of this acute protection strategy for a wide range of cell types including human umbilical vein endothelial cells (HUVEC), human adipose stem cells, rat mesenchymal stem cells, and mouse neural progenitor cells. Specifically, alginate hydrogels with plateau storage moduli (G') ranging from 0.33 to 58.1 Pa were studied. A compliant crosslinked alginate hydrogel (G'=29.6 Pa) yielded the highest HUVEC viability, 88.9% ± 5.0%, while Newtonian solutions (i.e., buffer only) resulted in 58.7% ± 8.1% viability. Either increasing or decreasing the hydrogel storage modulus reduced this protective effect. Further, cells within noncrosslinked alginate solutions had viabilities lower than media alone, demonstrating that the protective effects are specifically a result of mechanical gelation and not the biochemistry of alginate. Experimental and theoretical data suggest that extensional flow at the entrance of the syringe needle is the main cause of acute cell death. These results provide mechanistic insight into the role of mechanical forces during cell delivery and support the use of protective hydrogels in future clinical stem cell injection studies.

Covalent stabilization of alginate hydrogel beads via Staudinger ligation: Assessment of poly(ethylene glycol) and alginate cross‐linkers

Cellular encapsulation within alginate hydrogel capsules has broad applications in tissue engineering. In seeking to improve the inherent instability of ionically cross-linked alginate hydrogels, we previously demonstrated the covalent stabilization of Ba(2+) cross-linked alginate-azide beads via chemoselective Staudinger ligation using a 1-methyl-2-diphenylphosphino-terephthalate (MDT) terminated poly(ethylene glycol) (PEG) linker. In this study, we functionalized variant PEG, linear and branched, and alginate polymers with MDT groups to evaluate the effect of size, structural design, number of functional groups, and charge on the resulting hydrogel bead. All cross-linkers resulted in enhanced covalent stabilization of alginate beads, with significant decreases in swelling and resistance to dissolution via Ba(2+) chelation. The MDT-functionalized alginate resulted in the most stable and homogeneous bead, with the most restrictive permeability even after EDTA exposure. Co-encapsulation of MIN6 cells within the cross-linked alginate hydrogel beads resulted in minimal effects on viability, whereas the degree of proliferation following culture varied with cross-linker type. Altogether, the results illustrate that manipulating the cross-linker structural design permits flexibility in resulting alginate beads characteristics. Covalent stabilization of alginate hydrogel beads with these chemoselective alginate and PEG-based cross-linkers provides a unique platform for cellular encapsulation.

A hydrophobically-modified alginate gel system: utility in the repair of articular cartilage defects

Alginate is a family of natural polysaccharides, widely used in industry and medicine for many applications, with its non toxic nature, gentle sol/gel transition procedure and low cost, alginate inferior biomechanical properties have limited its utility especially in tissue engineering. Additionally, ionically cross-linked alginate hydrogels generally lose most of their initial mechanical and swelling properties within a few hours in physiological solution. In order to overcome these limitations, the referenced alginate was treated by covalent fixation of octadecyl chains onto the polysaccharide backbone by esterification. In semi dilute solution, intermolecular hydrophobic interactions of long alkyl chains result in the formation of physical hydrogels, which can then be reinforced by the addition of calcium chloride. FTIR studies clearly showed the presence of ester bonds at 1612 and 1730cm(-1) indicating that the alkyl groups are incorporated in the backbone of resulting polymer. The endothermic peak and exothermic peak present in the DSC thermogram of Alg-C18 had shifted to lower temperatures comparing to native alginate (from 106 to 83°C and from 250 to 245°C, respectively) due to the esterification reaction that leads to high hydrophobic nature of the modified sample. From rheological experiments, it can be inferred that the combination of both calcium bridges and intermolecular hydrophobic interaction in the treated alginate leads to enhanced gel strength accompanied by more stable structure in physiological solution comparing to native sodium alginate hydrogel. Finally, the modified alginate tended to have no toxic effects on mesenchymal stem cell culture, rather it supported MSC chondrogenic differentiation.

A novel strategy for preparing mechanically robust ionically cross-linked alginate hydrogels

The properties of alginate films modified using two cross-linker ions (Ca2+ and Ba2+), comparing two separate cross-linking techniques (the traditional immersion (IM) method and a new strategy in a pressure-assisted diffusion (PD) method), are evaluated. This was achieved through measuring metal ion content, water uptake and film stability in an ionic solution ([Ca2+] = 2 mM). Characterization of the internal structure and mechanical properties of hydrated films were established by cryogenic scanning electron microscopy and tensile testing, respectively. It was found that gels formed by the PD technique possessed greater stability and did not exhibit any delamination after 21 day immersion as compared to gels formed by the IM technique. The Ba2+ cross-linked gels possessed significantly higher cross-linking density as reflected in lower water content, a more dense internal structure and higher Young's modulus compared to Ca2+ cross-linked gels. For the Ca2+ cross-linked gels, a large improvement in the mechanical properties was observed in gels produced by the PD technique and this was attributed to thicker pore walls observed within the hydrogel structure. In contrast, for the Ba2+ cross-linked gels, the PD technique resulted in gels that had lower tensile strength and strain energy density and this was attributed to phase separation and larger macropores in this gel.

Phosphorylation of Alginate: Synthesis, Characterization, and Evaluation of in Vitro Mineralization Capacity

Phosphorylation of alginate was achieved using a heterogeneous urea/phosphate reaction. The degree and stereoselectivity of phosphorylation as well as the effects on the physical properties of the polysaccharide were investigated by Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopies, inductively coupled plasma optical-emission spectroscopy (ICP-OES), and size exclusion chromatography (SEC). Multidimensional NMR studies of the phosporylated alginate revealed that phosphorylation of the M residues occurred predominantly at the C3 (equatorial) carbon of the polysaccharide ring. In addition, a more comprehensive assignment of the (1)H NMR spectrum of alginate, compared with those previously reported in the literature, is provided here. Hydrogel materials were formed from ionically cross-linked blends of phosphorylated alginate and alginate. These blended hydrogels showed an enhanced resistance to degradation by chelating agents compared with cross-linked alginate hydrogels and a reduction in their mineralization potential.

Poroelasticity of a covalently crosslinked alginate hydrogel under compression

This paper studies the poroelastic behavior of an alginate hydrogel by a combination of theory and experiment. The gel—covalently crosslinked, submerged in water, and fully swollen—is suddenly compressed between two parallel plates. The gap between the plates is held constant subsequently, and the force on the plate relaxes while water in the gel migrates. This experiment is analyzed by using the theory of linear poroelasticity. A comparison of the relaxation curve recorded in the experiment and that derived from the theory determines the elastic constants and the permeability of the gel. The material constants so determined agree well with those determined by using a recently developed indentation method. Furthermore, during relaxation, the concentration of water in the gel is inhomogeneous, resulting in tensile hoop stresses near the edge of the gel, and possibly causing the gel to fracture.

Interferometric characterization of swelling of covalently crosslinked alginate gel and changes associated with polymer impregnation

We report on integration of covalently crosslinked alginate hydrogels at the end of an optical fiber suitable for determination of changes in optical length with resolution of 2 nm, and the characterization of such alginate hydrogels, in particular following impregnation with a polycation. The ionic strength dependence of the changes in the optical length reflect the swelling, and the results showed typical expansion of swelling volume on reducing the ionic strength for an ionic hydrogel. Polycation impregnation of the alginate hydrogels by immersion in aqueous solution containing either poly- l-lysine or chitosan resulted in changes of the optical signal during the impregnation differing between the two polycations. The ionic strength dependence of the hydrogel swelling was also affected. A strategy for modelling of the resulting swelling of the impregnated hydrogel, aiming at a method for nanomechanical characterization of the supported impregnation is suggested.

Shear‐reversibly Crosslinked Alginate Hydrogels for Tissue Engineering

Injectable delivery vehicles in tissue engineering are often required for successful tissue formation in a minimally invasive manner. Shear-reversibly crosslinked hydrogels, which can recover gel structures from shear-induced breakdown, can be useful as an injectable, because gels can flow as a liquid when injected but re-gel once placed in the body. In this study, injectable and shear-reversible alginate hydrogels were prepared by combination crosslinking using cell-crosslinking and ionic crosslinking techniques. The addition of a small quantity of calcium ions decreased the number of cells that were required to form cell-crosslinked hydrogels without changing the shear reversibility of the system. The physical properties and gelation behavior of the gels were dependent on the concentration of both the cells and the calcium ions. We found that gels crosslinked by combination crosslinking methods were effective to engineer cartilage tissues in vivo. Using both ionic and cell-crosslinking methods to control the gelation behavior may allow the design of novel injectable systems that can be used to deliver cells and other therapeutics for minimally invasive therapy, including tissue engineering.

Construction of anticoagulant poly (lactic acid) films via surface covalent graft of heparin-carrying microcapsules

Heparin has been widely investigated in medicine and biomedical applications as an anticoagulant. This paper described a covalent modification of poly (lactic acid) (PLA) films with heparin-carrying microcapsules. The heparin-carrying microcapsules were achieved by layer-by-layer (LbL) self-assembly technology by which poly (allylamine hydrochloride) (PAH) and heparin were alternately coated on the Ca 2+-cross-linked alginate hydrogel. The microcapsules were characterized by scanning electron microscopy (SEM), zeta potential analysis and Fourier transform infrared spectroscopy (FTIR), and then grafted to PLA films by –CONH– linkage between surface PLA molecules with residual primary amino groups on the outer PAH layer of microcapsules, in the presence of catalysts of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). SEM and FTIR revealed the formation of –CONH– linkage. In vitro antithrombogenicity by the method of the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) tests showed that surface-modified PLA films had superior coagulation properties to original PLA films. It is suggested that the present method would provide a potential effective tool for biomaterial modification.

Distinct Intervertebral Disc Cell Populations Adopt Similar Phenotypes in Three-Dimensional Culture

Tissue engineering strategies have the potential to improve upon current techniques for intervertebral disc repair. However, determining a suitable biomaterial scaffold for disc regeneration is difficult due to the complex fibrocartilaginous structure of the tissue. In this study, cells isolated from three distinct regions of the intervertebral disc, the outer and inner annulus fibrosus and nucleus pulposus, were expanded and seeded on resorbable polyester fiber meshes and encapsulated in calcium crosslinked alginate hydrogels, both chosen to approximate the native tissue architecture. Three-dimensional (3D) constructs were cultured for 14 days in vitro and evaluated histologically and quantitatively for gene expression and production of types I and II collagen and proteoglycans. During monolayer expansion, the cell populations maintained their distinct phenotypic morphology and gene expression profiles. However, after 14 days in 3D culture, there were no significant differences in morphology, gene expression, or protein production between all three cell populations grown in either alginate or polyester fiber meshes. The results of this study indicate that the culture environment may have a greater impact on cellular behavior than the intrinsic origin of the cells, and suggest that only a single-cell type may be required for intervertebral disc regenerative therapies.

Maintaining dimensions and mechanical properties of ionically crosslinked alginate hydrogel scaffolds in vitro

Ionically crosslinked alginate hydrogels are attractive scaffolds because of their biocompatibility and mild gelation reaction that allows for gentle cell incorporation. However, the instability of ionically crosslinked hydrogels in an aqueous environment is a challenge that limits their application. This report presents a novel method to control the dimensions and mechanical properties of ionically crosslinked hydrogels via control of the ionic concentration of the medium. Homogeneous calcium-alginate gels were incubated in physiological saline baths adjusted to specific calcium ion concentrations. Swelling and shrinking occurred at low and high ionic concentrations of the medium, respectively, while an "optimal" intermediate calcium ion concentration of the medium was found to maintain original size and shape of the hydrogel. This optimal calcium ion concentration was found to be a function of crosslinking density and polymer concentration of the hydrogel and chemical composition of the alginate. The effects of optimal and high calcium ion concentrations of the medium on swelling behavior, calcium content, dry weight, and mechanical properties of the immersed hydrogels were investigated. It was found that the resulting hydrogel composition and mechanical properties depended on not only the calcium concentration of the medium, but also the crosslinking density and polymer concentration of the gel. In an 8-week experiment, controlled dimensions and mechanical properties of alginate gels in an aqueous environment were demonstrated. This new technique significantly enhances the potential of alginate hydrogels for tissue engineering and other biomedical applications.

Spontaneous Loading of Positively Charged Macromolecules into Alginate-Templated Polyelectrolyte Multilayer Microcapsules

A simple and high-efficiency approach to loading macromolecules into microscale carriers is presented. Calcium-cross-linked alginate hydrogel microspheres were fabricated by an emulsification technique and then used as negatively charged templates to form polyelectrolyte multilayer coatings. A calcium ion chelator, EDTA, was used to free the Ca(2+)-cross-linked alginate hydrogel within {poly(allylamine hydrochloride)/poly (styrene sulfonate)}(4) ({PAH/PSS}(4)) coating, allowing partial release of alginate. The retention of alginate in {PAH/PSS}(4) microcapsule was confirmed by FTIR spectroscopy and confocal microscopy. Real-time confocal microscopy was used to investigate the loading process of positively charged macromolecules (dextran-amino, and peroxidase) into alginate-templated microcapsules, which showed the loading occurred in <2 min for dextran-amino and <10 min for peroxidase, respectively. A high loading efficiency of 25 mug peroxidase in approximately 1.0 x 10(7) microcapsules (2.5 pg POx/capsule) was achieved with a low concentration of peroxidase loading solution (10 mug/mL). This spontaneous loading technique for encapsulating positively charged molecules in alginate-templated polyelectrolyte microcapsules shows strong potential for biosensor and drug delivery applications.

Comparison of bone marrow cell growth on 2D and 3D alginate hydrogels

Calcium cross-linked sodium alginate hydrogels have several advantageous features making them potentially suitable as tissue engineering scaffolds and this material has been previously used in many biomedical applications. 3D cell culture systems are often very different from 2D petri dish type cultures. in this study the effect of alginate hydrogel architecture was investigated by comparing rat bone marrow cell proliferation and differentiation on calcium cross linked sodium alginate discs and 1mm internal diameter tubes. It was found that bone marrow cell proliferation was diminished as the concentration of alginate in the 2D hydrogel substrates increased, yet proliferation was extensive on tubular alginate constructs with high alginate contents. Alginate gel thickness was found to be an important parameter in determining cell behaviour and the different geometries did not generate significant alterations in BMC differentiation profiles.

Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: Part 1. Structure, gelation rate and mechanical properties

Alginate gels have been used in both drug delivery and cell encapsulation applications in the bead form usually produced by dripping alginate solution into a CaCl 2 bath. The major disadvantages to these systems are that the gelation rate is hard to control; the resulting structure is not uniform; and mechanically strong and complex-shaped 3-D structures are difficult to achieve. In this work controlled gelation rate was achieved with CaCO 3–GDL and CaSO 4–CaCO 3–GDL systems, and homogeneous alginate gels were formulated as scaffolds with defined dimensions for tissue engineering applications. Gelation rate increased with increasing total calcium content, increasing proportion of CaSO 4, increasing temperature and decreasing alginate concentration. Mechanical properties of the alginate gels were controlled by the compositional variables. Slower gelation systems generate more uniform and mechanically stronger gels than faster gelation systems. The compressive modulus and strength increased with alginate concentration, total calcium content, molecular weight and guluronic acid (G) content of the alginate. MC3T3-E1 osteoblastic cells were uniformly incorporated in the alginate gels and cultured in vitro . These results demonstrated how alginate gel and gel/cell systems could be formulated with controlled structure, gelation rate, and mechanical properties for tissue engineering and other biomedical applications.

Controlling Material Properties of Ionically Cross-Linked Alginate Hydrogels by Varying Molecular Weight Distribution

ABSTRACTThe mechanical properties of alginate hydrogels are critical to their successful use in tissue engineering. We hypothesized that combining alginates of differing molecular weights would allow one to readily control and decouple the viscosity of the pre-gel solution from the post-gel stiffness. To test this, binary hydrogels were prepared by incorporating low molecular weight (MW) alginate (Mn ~ 3.3 x 104 g/mol) into 1 %(w/w) alginate solutions composed of high MW alginate (Mn ~ 2.2 x 105 g/mol). The addition of low MW alginate had minimal effects on the solution viscosity, allowing high solids loading in the solution while maintaining a workable solution. However, the increased mass loading resulted in much higher stiffness gels following ionic cross-linking with calcium. These data may be interpreted in relation to the microstructure of the solution versus hydrogel.

Controlling Diffusion of Solutes Through Ionically Crosslinked Alginate Hydrogels Designed for Tissue Engineering

AbstractTissue engineering aims at creating new tissues as alternatives to organ transplants. Our approach is to incorporate cells into biodegradable polymer scaffolds designed to temporarily support new tissue formation. An important requirement of scaffolds is homogeneity. Homogeneity ensures structural integrity, uniform distribution of cells, and uniform porosity throughout the scaffold. Controlling pore sizes is necessary to regulate exchange of nutrients and waste products for cells. Pores too large can provide entryway to immune cells that can harm allogenic cells and developing tissue. We have fabricated three-dimensionally defined, homogeneous, ionically crosslinked alginate gels with a controlled slow-gelation system involving CaCO3 and D-glucono-δ-lactone. We varied the structural parameters and alginate types of these gels to control the diffusion of glucose, vitamin B12 and FITC-dextran (molecular weights of 180, 1355 and 9500, respectively) through the gels. Experiments were performed with gel discs placed between side-by-side donor and receptor chambers in a humidified incubator at 37°C. Samples were taken periodically and measured on a UV-Vis spectrophotometer. Generally, diffusion coefficient (D) increased with decreasing solute size. Varying structural parameters of the gels did not have a significant effect on diffusivity of vitamin B12. In contrast, for gels made with a Ca2+ to carboxyl molar ratio of 0.36, D of FITCdextran increased from (2.88 ± 0.52)×10-7 to (4.66 ± 0.48)×10-7 cm2/sec as alginate concentration decreased from 3.2% to 1.5%, respectively. D of FITC-dextran also increased from (3.17 ± 0.30)×10-7 to (4.66 ± 0.48)×10-7 cm2/sec as crosslinking density decreased for 1.5% alginate gels from a Ca2+ to carboxyl molar ratio of 0.72 to 0.36, respectively. D was highest for alginate gels with the highest guluronic acid content. Controlling diffusivity allows alginate gels with specific properties to be fabricated for tissue engineering scaffolding and other biomedical applications.