Elastic Hydrogel Research Articles

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell–substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young's modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces.

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell–substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young's modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Biomimetic, ultrathin and elastic hydrogels regulate human neutrophil extravasation across endothelial-pericyte bilayers.

The vascular basement membrane—a thin, elastic layer of extracellular matrix separating and encasing vascular cells—provides biological and mechanical cues to endothelial cells, pericytes, and migrating leukocytes. In contrast, experimental scaffolds typically used to replicate basement membranes are stiff and bio-inert. Here, we present thin, porated polyethylene glycol hydrogels to replicate human vascular basement membranes. Like commercial transwells, our hydrogels are approximately 10μm thick, but like basement membranes, the hydrogels presented here are elastic (E: 50-80kPa) and contain a dense network of small pores. Moreover, the inclusion of bioactive domains introduces receptor-mediated biochemical signaling. We compare elastic hydrogels to common culture substrates (E: >2GPa) for human endothelial cell and pericyte monolayers and bilayers to replicate postcapillary venules in vitro. Our data demonstrate that substrate elasticity facilitates differences in vascular phenotype, supporting expression of vascular markers that are increasingly replicative of venules. Endothelial cells differentially express vascular markers, like EphB4, and leukocyte adhesion molecules, such as ICAM-1, with decreased mechanical stiffness. With porated PEG hydrogels we demonstrate the ability to evaluate and observe leukocyte recruitment across endothelial cell and pericyte monolayers and bilayers, reporting that basement membrane scaffolds can significantly alter the rate of vascular migration in experimental systems. Overall, this study demonstrates the creation and utility of a new and accessible method to recapture the mechanical and biological complexity of human basement membranes in vitro.

Elastic soft hydrogel supercapacitor for energy storage

The elastic soft (PVA/PPy)(−)//CNTs(+) supercapacitor is first designed who exhibits fascinating mechanical properties and realizes a high working voltage.

General Chemical Characteristics of Pectins and Arabinogalactans from Callus Culture and Native Plant of Tanacetum vulgare and Gelation Properties of the Pectins

Pectin and arabinogalactan (AG) were isolated from Tanacetum vulgare callus and native tissues. The main chain of the callus pectin macromolecule consisted of a linear galacturonan that made up over half of the whole carbohydrate chain and a branched region that was RG-I. Callus pectin was characterized by a higher molecular mass and lower degree of methyl esterification than pectin from the native plant. Callus AG differed from AG of the native plant by a higher galactose content. The strength and elasticity of hydrogels of callus culture pectins were greater than those of native plant pectins.

Microenvironment complexity and matrix stiffness regulate breast cancer cell activity in a 3D in vitro model.

Three-dimensional (3D) cell cultures represent fundamental tools for the comprehension of cellular phenomena both in normal and in pathological conditions. In particular, mechanical and chemical stimuli play a relevant role on cell fate, cancer onset and malignant evolution. Here, we use mechanically-tuned alginate hydrogels to study the role of substrate elasticity on breast adenocarcinoma cell activity. The hydrogel elastic modulus (E) was measured via atomic force microscopy (AFM) and a remarkable range (150–4000 kPa) was obtained. A breast cancer cell line, MCF-7, was seeded within the 3D gels, on standard Petri and alginate-coated dishes (2D controls). Cells showed dramatic morphological differences when cultured in 3D versus 2D, exhibiting a flat shape in both 2D conditions, while maintaining a circular, spheroid-organized (cluster) conformation within the gels, similar to those in vivo. Moreover, we observed a strict correlation between cell viability and substrate elasticity; in particular, the number of MCF-7 cells decreased constantly with increasing hydrogel elasticity. Remarkably, the highest cellular proliferation rate, associated with the formation of cell clusters, occurred at two weeks only in the softest hydrogels (E = 150–200 kPa), highlighting the need to adopt more realistic and a priori defined models for in vitro cancer studies.

Xanthan and κ-carrageenan based alkaline hydrogels as electrolytes for Al/air batteries

Xanthan and κ-carrageenan were used to prepare alkaline hydrogels to be used as electrolytes in aluminium air primary batteries. Two pasty gels were obtained starting from xanthan and KOH solutions (1M and 8M), while only the 8M KOH solution permitted the formation of a stable, elastic and gumminess hydrogel with κ-carrageenan. Discharge tests, performed on three Al/air cells assembled with Al anodes, electrolyte gels and Pt based cathodes, evidenced that all hydrogels exhibited appreciable properties of Al ion conductivities, according to the following performance order: xanthan with KOH 1M<xanthan with KOH 8M<κ-carrageenan KOH 8M.Characterization measurements (XRD, Ionic conductivity by EIS, SEM-EDS) were effected on hydrogels and galvanic cells to explain the behaviour differences detected between the hydrogels.

Fabrication of transparent quaternized PVA/silver nanocomposite hydrogel and its evaluation as an antimicrobial patch for wound care systems.

Grafting of quaternary nitrogen atoms into the backbone of polymer is an efficient way of developing new generation antimicrobial polymeric wound dressing. In this study, an elastic, non-adhesive and antimicrobial transparent hydrogel based dressing has been designed, which might be helpful for routine observation of wound area without removing the dressing material along with maintaining a sterile environment for a longer period of time. Green synthesized silver nanoparticles have been loaded into the quaternized PVA hydrogel matrix to improve its antimicrobial property. Silver nanoparticles loaded quaternized PVA hydrogel showed enhanced mechanical and swelling properties compared to native quaternized PVA hydrogel. Release kinetics evaluated by atomic absorption spectroscopy revealed that the release mechanism of silver nanoparticles from the hydrogel follows Fickian diffusion. Antimicrobial efficacy of the hydrogels was evaluated by disk diffusion test on Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli. After 96 h of release in phosphate buffer, the growth inhibition zone created by silver nanoparticless loaded quaternized PVA hydrogel is comparable to that created by ampicillin. These observations assert that the silver nanoparticles loaded quaternized PVA hydrogel acts as a reservoir of silver nanoparticles, which helps in maintaining a sterile environment for longer time duration by releasing Ag nanocrystallite in sustained manner.

Potential of Agarose/Silk Fibroin Blended Hydrogel for in Vitro Cartilage Tissue Engineering

An osteoarthritis pandemic has accelerated exploration of various biomaterials for cartilage reconstruction with a special emphasis on silk fibroin from mulberry (Bombyx mori) and non-mulberry (Antheraea assamensis) silk worms. Retention of positive attributes of the agarose standard and nullification of its negatives are central to the current agarose/silk fibroin hydrogel design. In this study, hydrogels of mulberry and non-mulberry silk fibroin blended with agarose were fabricated and evaluated in vitro for two weeks for cartilaginous tissue formation. The fabricated hydrogels were physicochemically characterized and analyzed for cell viability, proliferation, and extra cellular matrix deposition. The amalgamation of silk fibroin with agarose impacted the pore size, as illustrated by field emission scanning electron microscopy studies, swelling behavior, and in vitro degradation of the hydrogels. Fourier transform infrared spectroscopy results indicated the blend formation and confirmed the presence of both components in the fabricated hydrogels. Rheological studies demonstrated enhanced elasticity of blended hydrogels with G' > G″. Biochemical analysis revealed significantly higher levels of sulfated glycosaminoglycans (sGAGs) and collagen (p ≤ 0.01) in blended hydrogels. More specifically, the non-mulberry silk fibroin blend showed sGAG and collagen content (∼1.5-fold) higher than that of the mulberry blend (p ≤ 0.05). Histological and immunohistochemical analyses further validated the enhanced deposition of sGAG and collagen, indicating maintenance of chondrogenic phenotype within constructs after two weeks of culture. Real-time PCR analysis further confirmed up-regulation of cartilage-specific aggrecan, sox-9 (∼1.5-fold) and collagen type II (∼2-fold) marker genes (p ≤ 0.01) in blended hydrogels. The hydrogels demonstrated immunocompatibility, which was evidenced by minimal in vitro secretion of tumor necrosis factor-α (TNF-α) by murine macrophages. Taken together, the results suggest promising attributes of blended hydrogels and particularly the non-mulberry silk fibroin/agarose blends as alternative biomaterial for cartilage tissue engineering.

Full-color mechanical sensor based on elastic nanocomposite hydrogels encapsulated three-dimensional colloidal arrays

A label free and easy-to-prepare optical sensor with high mechanical strength for the fast and visible response to tension and pH variables was reported. An elastic nanocomposite hydrogel was successfully fabricated by in situ free-radical co-polymerization of N,N-dimethylacrylamide (DMAA), acrylic acid (AA) and aluminum oxide nanoparticles (Al2O3 NPs). DMAA and AA could form effective cross-linkage with Al2O3 NPs via hydrogen binding, electrostatic interaction and bidentate bridging. By encapsulating polymethyl methacrylate (PMMA) colloidal arrays into the hydrogels, the optical nanocomposite hydrogels (ONHs) can be easily obtained. This three-dimensionally ordered array exhibits a stop band, and the optical hydrogels can tune the bright color from blue to cyan, green, yellow green, yellow, orange and then finally red by mechanical stimulation of uniaxial tension within 50% strain following Bragg’s law. The mechanical and optical properties can be adjusted via changing the fraction of AA and polymerization time. Meanwhile, the mechanical and optical responses were reversible for more than 10 times without deterioration. Besides, this material has rapid response to the changing of pH and the full-color change was observed. Owing to the high biocompatibility of nanocomposite hydrogel (NC gel) system and the excellent mechanical strength and water-stability of these ONHs, this optical sensor has high potential to be applied in biomedical field as tension or pH sensor for the intraocular pressure measurement and tonometry and blood gas analysis.

Elastic hydrogel as a sensor for detection of mechanical stress generated by single cells grown in three-dimensional environment

Cell volume growth occurs in all living tissues. The growth exerts mechanical stresses on surrounding tissues that may alter tissue microenvironment, and have significant implications in health and diseases. However, the level of growth stress generated by single cells in three-dimensional (3D) environment remains to be determined. To this end, we developed a growth force microscopy technique to determine 3D distribution of the stress. The technique was based on encapsulation of cells in elastic hydrogels, and involved 3D particle tracking and mechanical analysis of gel deformation. Data from the study demonstrated that the growth stress was dynamic, and the stress distribution at the gel-cell interface was correlated inversely to the mean surface curvature or the distance to the geometric center of the cell. The stress averaged over the cell surface increased with increasing gel stiffness, suggesting that cells could alter growth stress in response to stiffness change in microenvironment. These findings suggested that the elastic hydrogel-based microscopy technique had a potential to provide new insights into mechanisms of mechanical interactions between cell and its microenvironment.

Biomimetic and photo crosslinked hyaluronic acid/pluronic F127 hydrogels with enhanced mechanical and elastic properties to be applied in tissue engineering

Biosynthetic hydrogels have proved to be a credible solution in developing cost effective scaffolds with superlative mechanical properties for biomedical applications. Elastic hydrogels have emerged with advanced application possibilities for cartilage tissue regeneration and cell implantation. However, a hydrogel scaffold that mimics the properties of biological tissues in terms of elasticity, provision of favorable environment for cell growth and biocompatibility are rarely reported. In this research, we developed photocrosslinked hyaluronic acid/pluronic F127 (HA/PF) porous hydrogels with exceptional mechanical and water sorption properties. In order to retain the micellar phase of PF in the hydrogels, we restrained their concentrations to 8 wt% in the hydrogel matrices. Further optimization such as duration of photocrosslinking resulted in hydrogel scaffolds with remarkable mechanical properties. Topical and cross-sectional scanning electron microscopy images depicted the dense interconnected porous networks within the hydrogel matrices. Rheology studies confirmed the importance of PF concentrations in obtaining the hydrogels with enhanced toughness and mechanical properties. The results from the microscopic rheology studies were further testified by applying macroscopic mechanical distortions over the hydrogels. Hydrogels with higher PF concentrations restrained the degradations and displayed enhanced mechanical properties. By retaining the micellar structures in the HA/PF hydrogel scaffolds, the polypropylene blocks in PF were able to reversibly fold and unfold to favor the energy dissipation during the mechanical deformation and aid in improving the mechanical properties of the hydrogels. The overall properties of the HA-PF hydrogels show optimum feasibility for hard tissue engineering application.

Synthesis and characterization of a new photo-crosslinkable glycol chitosan thermogel for biomedical applications

The major limitations of typical thermogelling polymers for practical applications are low gel stability and weak mechanical properties under physiological conditions. In this study, we have synthesized a new polysaccharide-based thermogelling polymer that can be photo-crosslinked by UV irradiation to form a mechanically resilient and elastic hydrogel. Methacrylated hexanoyl glycol chitosan (M-HGC), was synthesized by a series of chemical modifications, N-hexanoylation and N-methacrylation, of glycol chitosan (GC). Various M-HGC polymers with different methacryl group contents were synthesized and their thermogelling and photo-crosslinkable properties were evaluated. The M-HGCs demonstrated a thermo-reversible sol–gel transition behavior in aqueous solutions. The thermally-induced hydrogels could be chemically crosslinked by UV-triggered photo-crosslinking. From the cytotoxicity studies using MTT and the live/dead assay, the M-HGC hydrogels showed non-cytotoxicity. These photo-crosslinkable thermogelling M-HGC polymers may hold great promises for various biomedical applications, such as an injectable delivery system and 3D cell culture.

Complex Mechanics of Collagen Matrices and their Impact on Remote Intercellular Communication

The ability of cells to sense remotely is critical for a large array of collagen-dependent processes including collective cell migration and cancer cell invasion. However, the impact of the physical properties of collagen, particularly inelasticity, on intercellular communication is not defined. We developed a molecular dynamics model of a fibrous collagen network for examining how inelasticity arising from fiber slippage affects mechanical interactions of adherent cells. Inelastic remodeling of the fibrous pericellular matrix enabled propagation of cell-induced matrix deformations over longer distances (>5 fold) than in linear elastic, or strain-stiffening matrices, which do not exhibit inelastic behavior. Cells generated >10 fold larger forces at distant sites (>5 cell diameters) in matrices that undergo inelastic remodeling compared with linear or nonlinear elastic matrices. The size of the cell-induced matrix deformation fields and the amplitude of forces, which were derived from measurements of force autocorrelation function at remote sites, were proportional to the inelasticity of the fibrous network. This model indicates that inelasticity in the fibrous collagen network enables long-range strain propagation while network stiffening accounts for the transmission of larger forces predicted from linear or nonlinear elastic hydrogels. We conclude that the unusual trait of inelasticity in non-cross-linked collagen arising from fiber slippage, enables long-range mechanosensing that is not observed in synthetic, linear elastic hydrogels or in cross-linked matrices exhibiting nonlinear elasticity.

Photocrosslinking of Silk Fibroin Using Riboflavin for Ocular Prostheses.

A novel method to photocrosslink silk fibroin protein is reported, using riboflavin (vitamin B2) as a photoinitiator and the mechanism of crosslinking is determined. Exposure of riboflavin-doped liquid silk solution to light results in the formation of a transparent, elastic hydrogel. Several applications for this new material are investigated including corneal reshaping to restore visual acuity and photolithography.

Gelation of Soy Milk with Hagfish Exudate Creates a Flocculated and Fibrous Emulsion- and Particle Gel.

Hagfish slime is an ultra dilute, elastic and cohesive hydrogel that deploys within milliseconds in cold seawater from a glandularly secreted exudate. The slime is made of long keratin-like fibers and mucin-like glycoproteins that span a network which entraps water and acts as a defense mechanism against predators. Unlike other hydrogels, the slime only confines water physically and is very susceptible to mechanical stress, which makes it unsuitable for many processing operations and potential applications. Despite its huge potential, little work has been done to improve and functionalize the properties of this hydrogel. To address this shortcoming, hagfish exudate was mixed with a soy protein isolate suspension (4% w/v) and with a soy emulsion (commercial soy milk) to form a more stable structure and combine the functionalities of a suspension and emulsion with those of the hydrogel. Hagfish exudate interacted strongly with the soy systems, showing a markedly increased viscoelasticity and water retention. Hagfish mucin was found to induce a depletion and bridging mechanism, which caused the emulsion and suspension to flocculate, making “soy slime”, a cohesive and cold-set emulsion- and particle gel. The flocculation network increases viscoelasticity and substantially contributes to liquid retention by entrapping liquid in the additional confinements between aggregated particles and protein fibers. Because the mucin-induced flocculation resembles the salt- or acid-induced flocculation in tofu curd production, the soy slime was cooked for comparison. The cooked soy slime was similar to conventional cooked tofu, but possessed a long-range cohesiveness from the fibers. The fibrous, cold-set, and curd-like structure of the soy slime represents a novel way for a cold coagulation and fiber incorporation into a suspension or emulsion. This mechanism could be used to efficiently gel functionalized emulsions or produce novel tofu-like structured food products.

Uncoupling shear and uniaxial elastic moduli of semiflexible biopolymer networks: compression-softening and stretch-stiffening.

Gels formed by semiflexible filaments such as most biopolymers exhibit non-linear behavior in their response to shear deformation, e.g., with a pronounced strain stiffening and negative normal stress. These negative normal stresses suggest that networks would collapse axially when subject to shear stress. This coupling of axial and shear deformations can have particularly important consequences for extracellular matrices and collagenous tissues. Although measurements of uniaxial moduli have been made on biopolymer gels, these have not directly been related to the shear response. Here, we report measurements and simulations of axial and shear stresses exerted by a range of hydrogels subjected to simultaneous uniaxial and shear strains. These studies show that, in contrast to volume-conserving linearly elastic hydrogels, the Young’s moduli of networks formed by the biopolymers are not proportional to their shear moduli and both shear and uniaxial moduli are strongly affected by even modest degrees of uniaxial strain.

Distinct impacts of substrate elasticity and ligand affinity on traction force evolution.

Cell adhesion is regulated by the mechanical characteristics of the cell environment. The influences of different parameters of the adhesive substrates are convoluted in the cell response leading to questions on the underlying mechanisms, like biochemical signaling on the level of adhesion molecules, or viscoelastic properties of substrates and cell. By a time-resolved analysis of traction force generation during early cell adhesion, we wanted to elucidate the contributions of substrate mechanics to the adhesion process, in particular the impact of substrate elasticity and the molecular friction of adhesion ligands on the substrate surface. Both parameters were independently adjusted by (i) an elastic polyacrylamide hydrogel of variable crosslinking degree and (ii) a thin polymer coating of the hydrogel surface controlling the affinity (and the correlated substrate-ligand friction) of the adhesion ligand fibronectin. Our analysis showed two sequential regimes of considerable force generation, whose occurrence was found to be independent of substrate properties. The first regime is characterized by spreading of the cell and a succeeding force increase. After spreading cells enter the second regime with saturated forces. Substrate elasticity and viscosity, namely hydrogel elasticity and ligand affinity, were both found to affect the kinetics and absolute levels of traction force quantities. A faster increase and a higher saturation level of traction forces were observed for a higher substrate stiffness and a higher ligand affinity. The results complement recent modeling approaches on the evolution of forces in cell spreading and contribute to a better understanding of the dynamics of cell adhesion on viscoelastic substrates.

2G45 ハイドロゲルを基板とする伸縮性電極シートの開発と生体応用

Hydrogel-based, molecular permeable electronic devices are considered to be promising for electrical stimulation and recording of living tissues, either in vivo or in vitro. This study reports the fabrication of the hydrogel-based devices that remain highly electrically conductive under substantial stretch and bending. Using a simple technique involving a combination of chemical polymerization and electropolymerization of poly (3,4-ethylenedioxythiophene) (PEDOT), a tight bonding of a conductive composite of PEDOT and polyurethane (PU) to an elastic double-network hydrogel is achieved to make fully organic PEDOT/PU-hydrogel hybrids. Their response to repeated mechanical stretching, hydration-drying cycles, and autoclaving is assessed, demonstrating excellent stability, without any mechanical or electrical damage. The adhesion, proliferation, and differentiation of neural and muscle cells cultured on these hybrids are demonstrated, advancing the field of tissue engineering with integrated electronics.

Contractile dynamics change before morphological cues during fluorescence [corrected] illumination.

Illumination can have adverse effects on live cells. However, many experiments, e.g. traction force microscopy, rely on fluorescence microscopy. Current methods to assess undesired photo-induced cell changes rely on qualitative observation of changes in cell morphology. Here we utilize a quantitative technique to identify the effect of light on cell contractility prior to morphological changes. Fibroblasts were cultured on soft elastic hydrogels embedded with fluorescent beads. The adherent cells generated contractile forces that deform the substrate. Beads were used as fiducial markers to quantify the substrate deformation over time, which serves as a measure of cell force dynamics. We find that cells exposed to moderate fluorescence illumination (λ = 540–585 nm, I = 12.5 W/m2, duration = 60 s) exhibit rapid force relaxation. Strikingly, cells exhibit force relaxation after only 2 s of exposure, suggesting that photo-induced relaxation occurs nearly immediately. Evidence of photo-induced morphological changes were not observed for 15–30 min after illumination. Force relaxation and morphological changes were found to depend on wavelength and intensity of excitation light. This study demonstrates that changes in cell contractility reveal evidence of a photo-induced cell response long before any morphological cues.