Hydrogels Of Different Stiffness Research Articles

Human induced mesenchymal stem cells display increased sensitivity to matrix stiffness

The clinical translation of mesenchymal stem cells (MSCs) is limited by population heterogeneity and inconsistent responses to engineered signals. Specifically, the extent in which MSCs respond to mechanical cues varies significantly across MSC lines. Although induced pluripotent stem cells (iPSCs) have recently emerged as a novel cell source for creating highly homogeneous MSC (iMSC) lines, cellular mechanosensing of iMSCs on engineered materials with defined mechanics is not well understood. Here, we tested the mechanosensing properties of three human iMSC lines derived from iPSCs generated using a fully automated platform. Stiffness-driven changes in morphology were comparable between MSCs and iMSCs cultured atop hydrogels of different stiffness. However, contrary to tissue derived MSCs, no significant changes in iMSC morphology were observed between iMSC lines atop different stiffness hydrogels, demonstrating a consistent response to mechanical signals. Further, stiffness-driven changes in mechanosensitive biomarkers were more pronounced in iMSCs than MSCs, which shows that iMSCs are more adaptive and responsive to mechanical cues than MSCs. This study reports that iMSCs are a promising stem cell source for basic and applied research due to their homogeneity and high sensitivity to engineered mechanical signals.

TAZ/WWTR1 mediates liver mesothelial-mesenchymal transition induced by stiff extracellular environment, TGF-β1, and lysophosphatidic acid.

Mesothelial cells cover the surface of the internal organs and the walls of body cavities, facilitating the movement between organs by secretion of a lubricating fluid. Upon injury, mesothelial cells undergo a mesothelial-mesenchymal transition (MMT) and give rise to myofibroblasts during organ fibrosis, including in the liver. Although transforming growth factor-β1 (TGF-β1) was shown to induce MMT, molecular and cellular mechanisms underlying MMT remain to be clarified. In the present study, we examined how the extracellular environment, soluble factors, and cell density control the phenotype of liver mesothelial cells by culturing them at different cell densities or on hydrogels of different stiffness. We found that TGF-β1 does not fully induce MMT in mesothelial cells cultured at high cell density or in the absence of fetal bovine serum. Extracellular lysophosphatidic acid (LPA) synergistically induced MMT in the presence of TGF-β1 in mesothelial cells. LPA induced nuclear localization of WW domain-containing transcription regulator1 (WWTR1/TAZ) and knockdown of Taz, which suppressed LPA-induced MMT. Mesothelial cells cultured on stiff hydrogels upregulated nuclear localization of TAZ and myofibroblastic differentiation. Knockdown of Taz suppressed MMT of mesothelial cells cultured on stiff hydrogels, but inhibition of TGF-β1 signaling failed to suppress MMT. Our data indicate that TAZ mediates MMT induced by TGF-β1, LPA, and a stiff matrix. The microenvironment of a stiff extracellular matrix is a strong inducer of MMT.

In vitro and in vivo methods to study bacterial colonization of hydrogel dermal fillers.

Preclinical in vitro and in vivo methods to study bacterial interactions with dermal fillers and infection pathogenesis are lacking. In this work, first in vitro methods to assess protein biofouling and effective pore size of commercial dermal fillers, including degradable hyaluronic acid (HA)-based fillers and other semi-degradable or permanent fillers (non-HA), were developed. The results were then related to Staphylococcus aureus (S. aureus) adhesion rates in vitro. HA fillers had less protein sorption than non-HA fillers and overall had smaller effective pore sizes. The properties correlated with levels of bacterial adhesion, where the control glass surface had the most rapid increase in bacterial cell adhesion, with a slope of 0.29 cm-2 min-1 , three unique non-HA fillers had intermediate adhesion with slopes of 0.11 and 0.06 cm-2 min-1 , and three unique HA fillers had the least adhesion with slopes of 0.02, 0.02, and 0.01 cm-2 min-1 . S. aureus had greater motility on the HA fillers than on non-HA fillers. Next, a mouse model for dermal filler biofilm and infection was developed. Mice were inoculated with a controlled amount of bioluminescent bacteria (Xen36 S. aureus) and polyacrylamide hydrogels of different stiffness were injected. In vivo bioluminescence was monitored longitudinally for 35 days to ensure that lasting colonization was established. The inoculum was optimized to achieve adequate bioluminescent signal, and bacterial bioburden over time and inter-animal variability in bioburden were determined. These in vitro and in vivo approaches can be used for future studies of antimicrobial interventions for dermal fillers.

Destructive fibrotic teamwork: how both microenvironment stiffness and profibrotic interleukin 13 impair alveolar macrophage phenotype and function.

The pulmonary fibrotic microenvironment is characterized by increased stiffness of lung tissue and enhanced secretion of profibrotic soluble cues contributing to a feedback loop that leads to dysregulated wound healing and lung failure. Pinpointing the individual and tandem effects of profibrotic stimuli in impairing immune cell response remains difficult and is needed for improved therapeutic strategies. We utilized a statistical design of experiment (DOE) to investigate how microenvironment stiffness and interleukin 13 (IL13), a profibrotic soluble factor linked with disease severity, contribute to the impaired macrophage response commonly observed in pulmonary fibrosis. We used engineered bioinspired hydrogels of different stiffness, ranging from healthy to fibrotic lung tissue, and cultured murine alveolar macrophages (MH-S cells) with or without IL13 to quantify cell response and analyze independent and synergistic effects. We found that, while both stiffness and IL13 independently influence macrophage morphology, phenotype, phagocytosis and efferocytosis, these factors work synergistically to exacerbate impaired macrophage phenotype and efferocytosis. These unique findings provide insights into how macrophages in fibrotic conditions are not as effective in clearing debris, contributing to fibrosis initiation/progression, and more broadly inform how underlying drivers of fibrosis modulate immune cell response to facilitate therapeutic strategies.

Correlation of cellular traction forces and dissociation kinetics of adhesive protein zyxin revealed by multi-parametric live cell microscopy.

Cells exert traction forces on the extracellular matrix to which they are adhered through the formation of focal adhesions. Spatial-temporal regulation of traction forces is crucial in cell adhesion, migration, cellular division, and remodeling of the extracellular matrix. By cultivating cells on polyacrylamide hydrogels of different stiffness we were able to investigate the effects of substrate stiffness on the generation of cellular traction forces by Traction Force Microscopy (TFM), and characterize the molecular dynamics of the focal adhesion protein zyxin by Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery After Photobleaching (FRAP). As the rigidity of the substrate increases, we observed an increment of both, cellular traction generation and zyxin residence time at the focal adhesions, while its diffusion would not be altered. Moreover, we found a positive correlation between the traction forces exerted by cells and the residence time of zyxin at the substrate elasticities studied. We found that this correlation persists at the subcellular level, even if there is no variation in substrate stiffness, revealing that focal adhesions that exert greater traction present longer residence time for zyxin, i.e., zyxin protein has less probability to dissociate from the focal adhesion.

Regulation of SMC traction forces in human aortic thoracic aneurysms

Smooth muscle cells (SMCs) usually express a contractile phenotype in the healthy aorta. However, aortic SMCs have the ability to undergo profound changes in phenotype in response to changes in their extracellular environment, as occurs in ascending thoracic aortic aneurysms (ATAA). Accordingly, there is a pressing need to quantify the mechanobiological effects of these changes at single cell level. To address this need, we applied Traction Force Microscopy (TFM) on 759 cells coming from three primary healthy (AoPrim) human SMC lineages and three primary aneurysmal (AnevPrim) human SMC lineages, from age and gender matched donors. We measured the basal traction forces applied by each of these cells onto compliant hydrogels of different stiffness (4, 8, 12, 25 kPa). Although the range of force generation by SMCs suggested some heterogeneity, we observed that: 1. the traction forces were significantly larger on substrates of larger stiffness; 2. traction forces in AnevPrim were significantly higher than in AoPrim cells. We modelled computationally the dynamic force generation process in SMCs using the motor-clutch model and found that it accounts well for the stiffness-dependent traction forces. The existence of larger traction forces in the AnevPrim SMCs were related to the larger size of cells in these lineages. We conclude that phenotype changes occurring in ATAA, which were previously known to reduce the expression of elongated and contractile SMCs (rendering SMCs less responsive to vasoactive agents), tend also to induce stronger SMCs. Future work aims at understanding the causes of this alteration process in aortic aneurysms.

Micropatterned soft hydrogels to study the interplay of receptors and forces in T cell activation

The analysis of T cell responses to mechanical properties of antigen presenting cells (APC) is experimentally challenging at T cell-APC interfaces. Soft hydrogels with adjustable mechanical properties and biofunctionalization are useful reductionist models to address this problem. Here, we report a methodology to fabricate micropatterned soft hydrogels with defined stiffness to form spatially confined T cell/hydrogel contact interfaces at micrometer scale. Using automatized microcontact printing we prepared arrays of anti-CD3 microdots on poly(acrylamide) hydrogels with Young's Modulus in the range of 2 to 50 kPa. We optimized the printing process to obtain anti-CD3 microdots with constant area (50 µm2, corresponding to 8 µm diameter) and comparable anti-CD3 density on hydrogels of different stiffness. The anti-CD3 arrays were recognized by T cells and restricted cell attachment to the printed areas. To test functionality of the hydrogel-T cell contact, we analyzed several key events downstream of T cell receptor (TCR) activation. Anti-CD3 arrays on hydrogels activated calcium influx, induced rearrangement of the actin cytoskeleton, and led to Zeta-chain-associated protein kinase 70 (ZAP70) phosphorylation. Interestingly, upon increase in the stiffness, ZAP70 phosphorylation was enhanced, whereas the rearrangements of F-actin (F-actin clearance) and phosphorylated ZAP70 (ZAP70/pY centralization) were unaffected. Our results show that micropatterned hydrogels allow tuning of stiffness and receptor presentation to analyze TCR mediated T cell activation as function of mechanical, biochemical, and geometrical parameters.

Impulsive stimulated Brillouin microscopy for non-contact, fast mechanical investigations of hydrogels.

The mechanical properties of tissues and cells are increasingly recognized as an important feature for the understanding of pathological processes and as a diagnostic tool in biomedicine. Impulsive stimulated Brillouin scattering (ISBS) is promising to overcome shortcomings of other measurement methods such as invasiveness, low spatial resolution and long acquisition time. In this paper, we present for the first time ISBS measurements of hydrogels, which are model materials for biological samples. We demonstrate ISBS measurements discriminating hydrogels of different stiffness. ISBS measurements with lateral resolution close to cellular level are presented. These results underline that ISBS microscopy has a high potential for biomedical applications.

Polyamidoamine dendrimer-PEG hydrogel and its mechanical property on differentiation of mesenchymal stem cells.

Biocompatible hydrogel systems with tunable mechanical properties have been reported to influence the behavior and differentiation of mesenchymal stem cells (MSCs). To develop a functionalized hydrogel system with well-defined chemical structures and tunable mechanical property for regulation of stem cell differentiation. An in situ-forming hydrogel system is developed by crosslinking vinyl sulfone functionalized polyamidoamine (PAMAM) dendrimer and multi-armed thiolated polyethylene glycol (PEG) through a thiol-ene Michael addition in aqueous conditions. The viability and differentiation of MSCs in hydrogels of different stiffness are conducted for 21 days under corresponding induction media. MSCs are viable in synthesized hydrogels after 48 hours of culture. By varying the concentrations of PAMAM dendrimer and PEG, hydrogels of different gelation time and stiffness are achieved. The MSC differentiation indicates that more osteogenic differentiation is observed in hard gel (5,663 Pa) and more adipogenic differentiation is observed in soft gel (77 Pa) in addition to the differentiation caused by each individual induction media during the process of culture. A biocompatible in situ-forming hydrogel system is successfully synthesized. This hydrogel system has shown influences on differentiation of MSCs and may potentially be important in developing therapeutic strategies in medical applications.

Modulation of Huh7.5 spheroid formation and functionality using modified PEG-based hydrogels of different stiffness.

Physical cues, such as cell microenvironment stiffness, are known to be important factors in modulating cellular behaviors such as differentiation, viability, and proliferation. Apart from being able to trigger these effects, mechanical stiffness tuning is a very convenient approach that could be implemented readily into smart scaffold designs. In this study, fibrinogen-modified poly(ethylene glycol)-diacrylate (PEG-DA) based hydrogels with tunable mechanical properties were synthesized and applied to control the spheroid formation and liver-like function of encapsulated Huh7.5 cells in an engineered, three-dimensional liver tissue model. By controlling hydrogel stiffness (0.1–6 kPa) as a cue for mechanotransduction representing different stiffness of a normal liver and a diseased cirrhotic liver, spheroids ranging from 50 to 200 μm were formed over a three week time-span. Hydrogels with better compliance (i.e. lower stiffness) promoted formation of larger spheroids. The highest rates of cell proliferation, albumin secretion, and CYP450 expression were all observed for spheroids in less stiff hydrogels like a normal liver in a healthy state. We also identified that the hydrogel modification by incorporation of PEGylated-fibrinogen within the hydrogel matrix enhanced cell survival and functionality possibly owing to more binding of autocrine fibronectin. Taken together, our findings establish guidelines to control the formation of Huh7.5 cell spheroids in modified PEGDA based hydrogels. These spheroids may serve as models for applications such as screening of pharmacological drug candidates.

Modulation of chondrocyte functions and stiffness-dependent cartilage repair using an injectable enzymatically crosslinked hydrogel with tunable mechanical properties

We developed an injectable hydrogel system to evaluate the effect of hydrogel stiffness on chondrocyte cellular functions in a three-dimensional (3D) environment and its subsequent influence on ectopic cartilage formation and early-stage osteochondral defect repair in a rabbit model. The hydrogels, composed of gelatin-hydroxyphenylpropionic acid (Gtn-HPA) conjugate, were formed using oxidative coupling of HPA moieties catalyzed by hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). The storage modulus (G′) of the hydrogels, which was tunable by changing the H2O2 and Gtn-HPA concentrations, ranged from 570 Pa to 2750 Pa. It was found that the cellular functions of chondrocytes encapsulated in hydrogels, including cell proliferation, biosynthesis of collagen and sulfated glycosaminoglycans (sGAG), as well as gene expression of type I (Col-I) and type II collagen (Col-II), were strongly affected by the stiffness of the hydrogels. Of note, chondrocytes cultured within the Gtn-HPA hydrogel of medium stiffness (G′ = 1000 Pa) produced highest level of sGAG production, as well as highest ratio of Col-II to Col-I gene expression among the Gtn-HPA hydrogels of different stiffness. Consistent with the results from in vitro and in vivo ectopic cartilage formation, osteochondral defect repair in a rabbit model showed stiffness-dependent tissue repair, with defects implanted with chondrocytes in hydrogels of medium stiffness having markedly more hyaline cartilage formation, smoother surface and better integration with adjacent cartilage, compared to defects treated with hydrogels of low or high stiffness. These results suggest that the tunable stiffness of Gtn-HPA hydrogels modulates chondrocyte cellular functions, and has a dramatic impact on cartilage tissue histogenesis and repair.

Deformation Gradients Imprint the Direction and Speed of En Masse Fibroblast Migration for Fast Healing

En masse cell migration is more relevant than single cell migration in physiological processes of tissue formation, such as embryogenesis, morphogenesis and wound healing. In these situations, cells are influenced by the proximity of other cells including interactions facilitated by substrate mechanics. Here we found that when fibroblasts migrated en masse over a hydrogel, they established a well-defined deformation field by traction forces and migrated along a trajectory defined by field gradients. The mechanics of the hydrogel determined the magnitude of the gradient. For materials stiff enough to withstand deformation related to cellular traction forces, such patterns did not form. Furthermore, migration patterns functioned poorly on very soft matrices where only a minimal traction gradient could be established. The largest degree of alignment and migration velocity occurred on the gels with the largest gradients. Granulation tissue formation in punch wounds of juvenile pigs was correlated strongly with the modulus of the implanted gel in agreement with in vitro en masse cell migration studies. These findings provide basic insight into the biomechanical influences on fibroblast movement in early wounds and relevant design criteria for development of tissue-engineered constructs that aim to stimulate en masse cell recruitment for rapid wound healing.

Bioactive polyacrylamide hydrogels with gradients in mechanical stiffness

We propose a novel, single step method for the production of polyacrylamide hydrogels with a gradient in mechanical properties. In contrast to already existing techniques such as UV photo-polymerization with photomasks (limited penetration depth) or microfluidic gradient mixers (complex microfluidic chip), this technique is not suffering such limitations. Young's modulus of the hydrogels was varied by changing the total monomer concentration of the hydrogel precursor solution. Using programmable syringe pumps, the total monomer concentration in the solution fed to the hydrogel mold was varied from 16 wt% down to 5 wt% over the feeding time to obtain a gradient in compliance ranging from 150 kPa down to 20 kPa over a length of 10 mm down to 2.5 mm. Polymerization was achieved with the dual initiation system composed of ammonium persulfate and N,N,N',N'-tetramethylethylenediamine, which were both fed through separate capillaries to avoid premature polymerization. Functionalized with the model ligand collagen I, the substrates were bioactive and supported the attachment of human foreskin fibroblasts (around 30% of the cells seeded attached after 1 h). A kinetic morphology study on homogeneous hydrogels of different stiffness's indicated that fibroblasts tend to spread to their final size within 2 h on stiff substrates, while the spreading time was much longer (ca. 4-5 h) on soft substrates. These trends were confirmed on hydrogels with compliance gradients, showing well spread fibroblasts on the stiff end of the hydrogel after 2 h, while the cells on the soft end still had small area and rounded morphology.

Identification of signalling pathways triggered by changes in the mechanical environment in rat chondrocytes

The aim of this work was to determine the pathways implicated in the mechanosensing of chondrocytes. Rat chondrocytes were cultured in collagen hydrogels of different stiffness (2-20 Pa) in normoxia and hypoxia, in monolayer and embedded inside hydrogels. First, chondrocyte were cultured on hydrogels in the presence of antibodies to block integrins. Second, custom RT-PCR array plates and western blot were used to detect changes in expression of genes implicated in downstream signalling pathways. The results allowed us to demonstrate the mechanosensing of chondrocytes for changes in stiffness in the range of Pascals. We also identified Non-Muscle Myosin II (NMMII) and integrins α1, β1 and β3 as participants in the mechanosensing, since their blockade inhibits the sensing of the stiffness, and they are up-regulated in the process. RT-PCR arrays and western blot detected up-regulation of Paxillin, RhoA, Fos, Jun and Sox9. We detected no expression of Src in the monolayer cultures, but we found a role for this protein in 3D. The expression of HIF-1α was not modified under normoxia but was found to participate under hypoxia. Focal Adhesion Kinase (FAK), showed a direct relationship with the expression of Aggrecan in hypoxia and an inverse one in normoxia. Finally, immunofluorescence analysis located the expression of factors AP-1, Sox-9 and HIF-1α inside the cell nuclei and RhoA, Src, Paxillin and FAK close to the cytoplasmic membrane. We determined here some of the genes that are up-regulated during the process of chondrocyte mechanosensing.