Gel Stiffness Research Articles

Abstract 146: Investigating and Inhibiting Neutrophil Extracellular Trap Formation in the Heart

Neutrophil extracellular traps (NETs) have been observed in multiple diseases of the cardiovascular system, such as myocardial infarction and atherosclerosis. We have recently shown that adenosine (ADO) inhibits NET formation and may be an important endogenous regulator. CD73 is expressed on many cell types and catalyzes the extracellular formation of ADO from AMP. Here, we first attempt to implicate NETs in cardiac injury, and we posit that human NETs negatively impact cardiac function by influencing native leukocyte recruitment and activation. Second, we prototype a CD73-functionalized hydrogel delivery vehicle for adenosine and test its function in vitro. We introduced NETs obtained from stimulated neutrophils into healthy hearts by intramyocardial injection in two-month-old rats. We used echo to assess cardiac function at days 0, 1 and 3 of injection. We measured the abundance of cardiac leukocyte subpopulations by flow cytometry at days 1 and 3 after injection, with immunostaining for CD45 (all leukocytes), myeloperoxidase (MPO, neutrophils), CD68 (macrophages), CD3 (T-cells), B220 (B-cells) and citrullinated histone 3 (citH3, NETs). To deliver adenosine as a NET antagonist, we designed a polyethylene (PEG) hydrogel composed of a 4-armed PEG incorporating a VPM protease-degradable crosslinker for cargo release, and an RGD peptide to enhance gel-tissue attachment. We tested gel polymerization by measuring storage and loss moduli at 4% and 6% PEG content (w/v) and 0, 1 uM and 1mM RGD concentrations. We functionalized the gel with CD73 to test its adenosine production capability. We found a significant decreased in cardiac function assessed by global longitudinal strain at day 1 in the NET group compared to saline. By flow cytometry, we found an increase in neutrophils, macrophages and NET formation at day 1, while B- and T-cells were increased at day 3. We found that increasing the PEG content of our hydrogel, but not RGD, increases gel stiffness. We also found that the CD73 functionalized hydrogel successfully catalyzes the formation of adenosine in vitro. In conclusion, we found that NETs negatively impact cardiac function, and successfully tested an adenosine hydrogel source in vitro. Next, we will study the effect of our gel on NETs in vivo.

Substrate Stiffness-Dependent Carbon Nanotube-Induced Lung Fibrogenesis.

Most living tissues exhibit the specific stiffness, which has been known to have profound influence on cell behaviors, yet how the stiffness affects cellular responses to engineered nanomaterials has not been elucidated. Particularly, discrepancies exist between in vitro and in vivo nanotoxicological studies. Here, we investigated the effects of substrate stiffness on the fibrogenic responses of normal human lung fibroblasts (NHLFs) to multiwalled carbon nanotubes (MWCNTs). NHLFs were grown on polyacrylamide (PAAm) hydrogels with the stiffness comparable to that of human normal and fibrotic lung tissues, and treated with MWCNTs for various time. The fibrogenic responses, including cell proliferation, reactive oxygen species production, and collagen I expression, of NHLFs to MWCNTs were observed to be regulated by substrate stiffness in a time-dependent manner. NHLFs generally were rounded on soft hydrogels and required a long treatment time to exhibit fibrogenic responses, while on stiff hydrogels the cells were well-spread with defined stress fibers and short-time MWCNTs treatment sufficiently induced the fibrogenic responses. Mechanistic studies showed that MWCNTs induced fibrogenesis of NHLFs through promoting expression and phosphorylation of focal adhesion kinase (FAK), while attenuating intracellular tension in the cells on stiff gels could increase MWCNTs uptake and thus elevate the induced fibrogenic responses. Moreover, we proposed a time-stiffness superposition principle to describe the equivalent effects of treatment time and substrate stiffness on nanomaterials-induced fibrogenesis, which suggested that increasing substrate stiffness expedited fibrogenesis and shed light on the rational design of in vitro models for nanotoxicological study.

Physical and mechanical properties of RAFT-stabilised collagen gels for tissue engineering applications

Cell behavior is influenced by the mechanical and structural properties of their substrate environment. Also, materials mechanically resistant to surgical handling and similar to the host site are required in tissue engineering to minimise the chance of an adverse host response. RAFT-stabilisation is a commercially available technique for creating stabilised hydrogels. Properties of RAFT-stabilised collagen (RsC) gels are governed by size, composition and arrangement of fibrils and their interaction with the fluid trapped within the matrix. The stabilisation process, using hydrophilic porous absorbers, produces dense matrices by rapid expulsion of fluid, and the structure obtained has mechanical properties suitable for tissue engineering. However, protocols to define and compare the physical properties and mechanical behavior of RAFT-stabilised collagen gels are not standardised across the field. Here, we investigate the fundamental mechanical and structural properties of RsC gels, and propose a new empirical relationship that correlates the measured stiffness of gels to varying frequency of strain oscillation. The results provide quantitative data characterising this extracellular environment for future tissue engineering studies.

Extracellular and intercellular force distribution in circularly shaped epithelia

Stress homeostasis in multicellular organisms is essential to tissue growth, development, and repair. However, how cellular forces are generated and transmitted within a multicellular organism to maintain stress homeostasis remains poorly understood. Here we employ cellular force microscopies to quantify extracellular traction and intercellular tension of circularly shaped cohesive epithelia seeded on hydrogels of various stiffness. Our experimental measurements show that the traction force is both colony size and gel stiffness dependent. A minimal mechanics model is developed and predicts exponential decay of extracellular traction from the periphery to the center of the epithelial monolayers, which agrees with the experimental data. Our modeling analyses further show that the colony size and gel stiffness dependent traction is originated from the line tension effect and stiffness-dependent cell contractility, respectively. Our study lays down a foundation for future modeling and experimental measurements of cellular force generation, transmission, and distribution in multicellular organisms, which is critical to mechanobiological understanding of normal and disease development.

Dextran modulates physical properties of rennet‐induced milk gels

SummaryPhysical properties of rennet‐induced milk gels as core intermediates of cheese production are mainly affected by milk composition, type and amount of coagulation enzyme and starter culture activity. We investigated model systems of reconstituted milk and dextran, which triggers effects on milk gels similar to exopolysaccharides from lactic acid bacteria. Furthermore, clotting activity (0.02 or 0.04 IMCU mL−1) and milk pH adjusted prior to renneting (6.5–5.7) were studied. A lower pH at renneting resulted in an earlier gelation onset, a higher gelation velocity and gel stiffness. The addition of dextran stabilised the gels especially at higher pH, and microstructural analysis revealed larger, more interconnected protein aggregates. However, at pH 5.7, a reverse effect was observed, indicating a destabilisation of the casein network. The current study indicates that altering milk pH and addition of polysaccharides gives the potential to change textural properties of cheese by affecting rennet‐induced gelation.

Quantifying heart valve interstitial cell contractile state using highly tunable poly(ethylene glycol) hydrogels

Valve interstitial cells (VIC) are the primary cell type residing within heart valve tissues. In many valve pathologies, VICs become activated and will subsequently profoundly remodel the valve tissue extracellular matrix (ECM). A primary indicator of VIC activation is the upregulation of α-smooth muscle actin (αSMA) stress fibers, which in turn increase VIC contractility. Thus, contractile state reflects VIC activation and ECM biosynthesis levels. In general, cell contraction studies have largely utilized two-dimensional substrates, which are a vastly different micro mechanical environment than 3D native leaflet tissue. To address this limitation, hydrogels have been a popular choice for studying cells in a three-dimensional environment due to their tunable properties and optical transparency, which allows for direct cell visualization. In the present study, we extended the use of hydrogels to study the active contractile behavior of VICs. Aortic VICs (AVIC) were encapsulated within poly(ethylene glycol) (PEG) hydrogels and were subjected to flexural-deformation tests to assess the state of AVIC contraction. Using a finite element model of the experimental setup, we determined the effective shear modulus μ of the constructs. An increase in μ resulting from AVIC active contraction was observed. Results further indicated that AVIC contraction had a more pronounced effect on μ in softer gels (72 ± 21% increase in μ within 2.5 kPa gels) and was dependent upon the availability of adhesion sites (0.5–1 mM CRGDS). The transparency of the gel allowed us to image AVICs directly within the hydrogel, where we observed a time-dependent decrease in volume (time constant τ=3.04 min) when the AVICs were induced into a hypertensive state. Our results indicated that AVIC contraction was regulated by both the intrinsic (unseeded) gel stiffness and the CRGDS peptide concentrations. This finding suggests that AVIC contractile state can be profoundly modulated through their local micro environment using modifiable PEG gels in a 3D micromechanical-emulating environment. Moving forward, this approach has the potential to be used towards delineating normal and diseased VIC biomechanical properties using highly tunable PEG biomaterials. Statement of Significance1.Hydrogels are a popular choice for studying cells in a 3D environment due to their tunable properties and optical transparency, which allows for direct cell visualization. In this study, we extended the use of hydrogels to study the active contractile behavior of VICs in 3D.2.Results indicated that AVIC contraction has a more pronounced effect on macroscale properties in softer gels (72 ± 21% increase in shear moduli within 2.5 kPa gels) and is dependent upon the availability of adhesion sites (0.5–1 mM CRGDS).3.Our results indicate that AVIC contraction is regulated by both the intrinsic (unseeded) gel stiffness and the CRGDS peptide concentration. This finding suggests that AVIC contraction can be modulated through their local micro environment.

Peptide gels of fully-defined composition and mechanics for probing cell-cell and cell-matrix interactions in vitro

Current materials used for in vitro 3D cell culture are often limited by their poor similarity to human tissue, batch-to-batch variability and complexity of composition and manufacture. Here, we present a “blank slate” culture environment based on a self-assembling peptide gel free from matrix motifs. The gel can be customised by incorporating matrix components selected to match the target tissue, with independent control of mechanical properties. Therefore the matrix components are restricted to those specifically added, or those synthesised by encapsulated cells. The flexible 3D culture platform provides full control over biochemical and physical properties, allowing the impact of biochemical composition and tissue mechanics to be separately evaluated in vitro. Here, we demonstrate that the peptide gels support the growth of a range of cells including human induced pluripotent stem cells and human cancer cell lines. Furthermore, we present proof-of-concept that the peptide gels can be used to build disease-relevant models. Controlling the peptide gelator concentration allows peptide gel stiffness to be matched to normal breast (<1 kPa) or breast tumour tissue (>1 kPa), with higher stiffness favouring the viability of breast cancer cells over normal breast cells. In parallel, the peptide gels may be modified with matrix components relevant to human breast, such as collagen I and hyaluronan. The choice and concentration of these additions affect the size, shape and organisation of breast epithelial cell structures formed in co-culture with fibroblasts. This system therefore provides a means of unravelling the individual influences of matrix, mechanical properties and cell-cell interactions in cancer and other diseases.

Effects of Freeze-Thaw Cycles on Gel-Forming Ability and Protein Denaturation in Alaska Pollock Frozen Surimi

Frozen surimi may be damaged by freeze-thaw cycles (refreezing) under various conditions. However, few studies have examined the deterioration of the quality of refrozen surimi. The objective of this study was to determine the deterioration mechanism of refrozen surimi. We used Alaska pollock frozen surimi, which has been studied extensively for gel formation. Refreezing decreased the breaking strength and breaking strain of the heated gel. The length of the line in the diagram between breaking strength and gel stiffness (L value, which indicates the level of change in the breaking strength and gel stiffness with setting time) was also decreased. In contrast, the effect of refreezing on the texture of the gel without the setting process was small. The polymerization rate of myosin heavy chain in refrozen surimi during the setting process was slower than that in nonrefrozen surimi. Additionally, the Ca-ATPase activity of surimi was reduced by approximately 30% with each refreezing. These results demonstrate that the reduction in gel-forming ability by refreezing was caused by the decreased polymerization rate of myosin heavy chain because of myosin head denaturation. We also found that quality deterioration including myofibrillar protein denaturation of frozen surimi occurred mostly during first refreezing rather than during second refreezing. Overall, refreezing and/or repeated refreezing of surimi simply decreased the suwari gel-forming ability without changing the characteristics of surimi. The primary cause of the decrease in gel-forming ability induced by refreezing is considered to be the suppression of myosin heavy-chain polymerization during setting.

Comparative study of whey protein isolate gel and polydimethylsiloxane as tribological surfaces to differentiate friction properties of commercial yogurts

To predict in-mouth perception of semi-solid foods like yogurt, their lubricating behavior needs to be understood. In food industrial applications of oral tribology, it is preferable and more efficient to use a material with a hydrophilic surface to mimic the tongue in the in vitro setup. We investigated and compared whey protein isolate (WPI) gel and polydimethylsiloxane (PDMS), based on their physicochemical characteristics and their ability to discriminate the friction behavior of a set of commercial yogurts known to elicit different mouthfeels. Stiffness and wettability of WPI gel were more similar to the human tongue than PDMS. The friction factors of Newtonian fluids measured on WPI gel allowed to construct a master Stribeck curve; this was not the case for PDMS. The yogurts could be clearly discriminated when measured on WPI gel. Their friction factors in the mixed regime correlated to the fat content and to perceived creaminess. Yogurts’ discrimination was not optimal on PDMS. The measurements on WPI gel, done in the speed range 1–0.001 m/s, were compared to a second speed range (0.2–0.002 m/s), believed to be more relevant for in-mouth conditions. Yet, the first speed range gave the best discrimination, also between samples with small differences in fat content (semi vs full-fat). Statistical analyses were performed on the datasets obtained using WPI gel, for a detailed characterization of its discriminative predictive power, demonstrating that the average friction in the mixed regime, and the slope in the mixed regime are the most discriminating features for tribological behavior.

Molecular tailoring to improve polypyrrole hydrogels\u2019 stiffness and electrochemical energy storage capacity

This research looks at ways of tailoring and improving the stiffness of polypyrrole hydrogels for use as flexible supercapacitor electrodes. Molecules providing additional cross-linking between polypyrrole chains are added post-polymerisation but before gelation, and are found to increase gel stiffness by up to 600%, with the degree of change dependent on reactant type and proportion. It was also found that addition of phytic acid led to an increase in pseudocapacitive behaviour of the hydrogel, and thus a maximum specific capacitance of 217.07 F·g−1 could be achieved. This is an increase of 140% compared to pristine polypyrrole hydrogels produced by this method.

Shear induced molecular changes of exopolysaccharides from lactic acid bacteria

Exopolysaccharide (EPS) producing lactic acid bacteria are known for their positive effect on the texture of fermented dairy products, with their high water binding capacity causing changes in rheology and syneresis reduction. During manufacture, product texture is affected by shearing with an unknown impact on the properties of the EPS. By subjecting aqueous solutions of viscosity enhancing EPS from Streptococcus thermophilus DGCC7710 or dextran to defined shear treatments with a Microfluidizer, a cell disruption system or a toothed ring dispersion unit to apply different energy input, we investigated macromolecular properties and the behaviour in solution. Despite the breakage of covalent bonds, spectroscopic NMR analyses revealed that repeating unit structure remained unchanged, whereas changes in macromolecular properties such as intrinsic viscosity, hydrodynamic volume, and molecular mass strongly depended on energy input. The decrease of the dynamic viscosity of the polysaccharide solutions was significantly related to the observed decrease in molecular mass, and the impact of the EPS on technofunctional properties such as contribution to gel stiffness during acid gel formation was also affected. The results therefore indicate that mechanical energy input through shearing should be considered because product texture may be altered as a consequence of a mechanical breakdown of EPS molecules.

Development of a collagen-like peptide polymer via end-to-end disulfide cross-linking and its application as a biomaterial

Collagen is the most abundant protein in the animal kingdom and has a unique triple-helical structure. It not only provides mechanical strength to tissues, but also performs specific biological functions as a multifaceted signaling molecule. Animal-derived collagen is therefore widely used as a biocompatible material in vitro and in vivo. In this study, we developed a novel peptide-based material that mimicked both the polymeric properties and a selected biological function of native collagen. This material was prepared by end-to-end multiple disulfide cross-linking of chemically synthesized triple-helical peptides. The peptide polymer showed a gel-forming property, and receptor-specific cell binding was observed in vitro by incorporating a peptide harboring an integrin α2β1-binding sequence. Furthermore, cell signaling activity and biodegradability were tunable according to the polymer contents. The results demonstrated the potential of this material as a designer collagen. Statement of SignificanceCollagen is a useful biomaterial with the gel-forming property. It also exhibits various biological activities through the interaction of specific amino acid sequences displayed on the triple helix with functional biomacromolecules. Here we report a novel synthetic material, artificial collagen, by end-to-end cross-linking of chemically synthesized collagen-like triple-helical peptides. The material allows independent regulation of polymer properties, i.e. gel stiffness, and sequence-specific bioactivities by altering peptide compositions. This material can also be variously shaped, for example, thin films with high transparency. In addition, it has low inflamatogenic properties and tunable biodegradability in vivo.

Tunable Supramolecular Gel Properties by Varying Thermal History.

The possibility of using differential pre-heating prior to supramolecular gelation to control the balance between hydrogen-bonding and aromatic stacking interactions in supramolecular gels and obtain consequent systematic regulation of structure and properties is demonstrated. Using a model aromatic peptide amphiphile, Fmoc-tyrosyl-leucine (Fmoc-YL) and a combination of fluorescence, infrared, circular dichroism and NMR spectroscopy, it is shown that the balance of these interactions can be adjusted by temporary exposure to elevated temperatures in the range 313-365 K, followed by supramolecular locking in the gel state by cooling to room temperature. Distinct regimes can be identified regarding the balance between H-bonding and aromatic stacking interactions, with a transition point at 333 K. Consequently, gels can be obtained with customizable properties, including supramolecular chirality and gel stiffness. The differential supramolecular structures also result in changes in proteolytic stability, highlighting the possibility of obtaining a range of supramolecular architectures from a single molecular structure by simply controlling the pre-assembly temperature.

Nanocellulose Xerogels With High Porosities and Large Specific Surface Areas.

Xerogels are defined as porous structures that are obtained by evaporative drying of wet gels. One challenge is producing xerogels with high porosity and large specific surface areas, which are structurally comparable to supercritical-dried aerogels. Herein, we report on cellulose xerogels with a truly aerogel-like porous structure. These xerogels have a monolithic form with porosities and specific surface areas in the ranges of 71–76% and 340–411 m2/g, respectively. Our strategy is based on combining three concepts: (1) the use of a very fine type of cellulose nanofibers (CNFs) with a width of ~3 nm as the skeletal component of the xerogel; (2) increasing the stiffness of wet CNF gels by reinforcing the inter-CNF interactions to sustain their dry shrinkage; and (3) solvent-exchange of wet gels with low-polarity solvents, such as hexane and pentane, to reduce the capillary force on drying. The synergistic effects of combining these approaches lead to improvements in the porous structure in the CNF xerogels.

Zwitterionic Copolymer‐Supported Ionogel Electrolytes: Impacts of Varying the Zwitterionic Group and Ionic Liquid Identities

AbstractA series of copolymer scaffolds containing zwitterionic (ZI) functional groups, synthesized in situ in two alkylimidazolium cation‐based ionic liquids, has been created by using two different ZI monomers to yield nonvolatile ionogel electrolytes that exhibit high ionic conductivities and a wide range of elastic moduli. Although all of the examined ionogel formulations consist of 80 mol % ionic liquid, gel properties are observed to depend both on zwitterion content within the copolymer scaffold, as well as the specific ionic liquid/zwitterion pairing. Low zwitterion contents (ca. 1 mol %) generate a notable increase in ionogel room‐temperature ionic conductivity compared to that of their non‐ZI ionogel analogues, whereas higher zwitterion contents (up to 18.5 mol %) yield stiff gels containing a large density of ZI noncovalent cross‐links. This highlights the dual functionality of the ZI groups within zwitterion‐containing copolymer‐supported ionogel electrolytes. All the ionogels synthesized in this study exhibit room‐temperature ionic conductivity values in the range of 3–12 mS cm−1 with compressive elastic modulus values varying between approximately 1–1000 kPa, suggesting their suitability for the design of safer flexible electrochemical devices, including wearable sensors or supercapacitors.

Clustering of Streptococcus thermophilus Strains to Establish a Relation between Exopolysaccharide Characteristics and Gel Properties of Acidified Milk.

In situ produced extracellular polysaccharides (EPS) from lactic acid bacteria are generally known to affect the texture of fermented dairy products; however, the interplay between EPS and product properties is still poorly understood. The aim of this study was to establish a relationship between concentration and properties of EPS, and gel formation of milk analysed by noninvasive Multispeckle Diffusing Wave Spectroscopy. Twenty Streptococcus thermophilus strains were classified with respect to EPS concentration (8–126 mg GE/kg) and ropiness (thread length: 15–80 mm). Five groups identified by cluster analysis demonstrate the high strain-to-strain variability even within one species of lactic acid bacteria. Results from acidification and gelation experiments averaged per cluster indicate that fermentation time and gel stiffness is higher for strains that produce ropy EPS. A further increase in gel stiffness was detected for strains that also produced cell-bound EPS, which underlines the importance of both ropy and cell-bound EPS for improving acid gel properties. The results may be helpful for a proper selection of EPS-producing starter cultures.

Heat-induced gelation of plant globulins

Globulins are one of the four major classes of plant proteins and are the main protein component in legumes such as soy and pea. An important functionality of globulins is that they can form gels when heated in aqueous solution. Thermal gelation of these plant proteins is reviewed and it is concluded that though the general features are similar to that of globular proteins from animal sources, there is a strong variation in specific rheological properties such as the gel stiffness. The variation found for globulins from different sources of the same plants and for different extraction methods is often as important as for globulins from different plants.

Comparative study of the dynamics of laser and acoustically generated bubbles in viscoelastic media.

Experimental observations of the growth and collapse of acoustically and laser-nucleated single bubbles in water and agarose gels of varying stiffness are presented. The maximum radii of generated bubbles decreased as the stiffness of the media increased for both nucleation modalities, but the maximum radii of laser-nucleated bubbles decreased more rapidly than acoustically nucleated bubbles as the gel stiffness increased. For water and low stiffness gels, the collapse times were well predicted by a Rayleigh cavity, but bubbles collapsed faster than predicted in the higher stiffness gels. The growth and collapse phases occurred symmetrically (in time) about the maximum radius in water but not in gels, where the duration of the growth phase decreased more than the collapse phase as gel stiffness increased. Numerical simulations of the bubble dynamics in viscoelastic media showed varying degrees of success in accurately predicting the observations.

Analysis of the Effect of Processing Conditions on Physical Properties of Thermally Set Cellulose Hydrogels.

Cellulose-based hydrogels were prepared by dissolving cellulose in aqueous sodium hydroxide (NaOH)/urea solutions and casting it into complex shapes by the use of sacrificial templates followed by thermal gelation of the solution. Both the gelling temperatures used (40–80 °C), as well as the method of heating by either induction in the form of a water bath and hot press or radiation by microwaves could be shown to have a significant effect on the compressive strength and modulus of the prepared hydrogels. Lower gelling temperatures and shorter heating times were found to result in stronger and stiffer gels. Both the effect of physical cross-linking via the introduction of additional non-dissolving cellulosic material, as well as chemical cross-linking by the introduction of epichlorohydrin (ECH), and a combination of both applied during the gelation process could be shown to affect both the mechanical properties and microstructure of the hydrogels. The added cellulose acts as a physical-cross-linking agent strengthening the hydrogen-bond network as well as a reinforcing phase improving the mechanical properties. However, chemical cross-linking of an unreinforced gel leads to unfavourable bonding and cellulose network formation, resulting in drastically increased pore sizes and reduced mechanical properties. In both cases, chemical cross-linking leads to larger internal pores.

Matrix crosslinking enhances macrophage adhesion, migration, and inflammatory activation.

Macrophages are versatile cells of the innate immune system that can adopt a variety of functional phenotypes depending on signals in their environment. In previous work, we found that culture of macrophages on fibrin, the provisional extracellular matrix protein, inhibits their inflammatory activation when compared to cells cultured on polystyrene surfaces. Here, we sought to investigate the role of matrix stiffness in the regulation of macrophage activity by manipulating the mechanical properties of fibrin. We utilize a photo-initiated crosslinking method to introduce dityrosine crosslinks to a fibrin gel and confirm an increase in gel stiffness through active microrheology. We observe that matrix crosslinking elicits distinct changes in macrophage morphology, integrin expression, migration, and inflammatory activation. Macrophages cultured on a stiffer substrate exhibit greater cell spreading and expression of αM integrin. Furthermore, macrophages cultured on crosslinked fibrin exhibit increased motility. Finally, culture of macrophages on photo-crosslinked fibrin enhances their inflammatory activation compared to unmodified fibrin, suggesting that matrix crosslinking regulates the functional activation of macrophages. These findings provide insight into how the physical properties of the extracellular matrix might control macrophage behavior during inflammation and wound healing.