Effect of crosslinker length on the elastic and compression modulus of poly(acrylamide) nanocomposite hydrogels

Polymer hydrogelshave shown to exhibit improved properties upon the addition of nanoparticles; however, the mechanical underpinnings behind these enhancements have not been fully elucidated. Moreover, fewer studies have focused on developing an understanding of how polymer parameters affect the nanoparticle-mediated enhancements. In this study, we investigated the elastic properties of silica nanoparticle-reinforced poly(acrylamide) hydrogels synthesized using crosslinkers of various lengths. Crosslinker length positively affected the mechanical properties of hydrogels that were synthesized with or without nanoparticles. However the degree of nanoparticle enhancement was negatively correlated to crosslinker length. Our findings enable the understanding of the respective roles of nanoparticle and polymer properties on nanoparticle-mediated enhancement of hydrogels and thereby the development of next-generation nanocomposite materials

A Novel 2.5D Culture Platform to Investigate the Role of Stiffness Gradients on Adhesion-Independent Cell Migration

Current studies investigating the role of biophysical cues on cell migration focus on the use of culture platforms with static material parameters. However, migrating cells in vivo often encounter spatial variations in extracellular matrix stiffness. To better understand the effects of stiffness gradients on cell migration, we developed a 2.5D cell culture platform where cells are sandwiched between stiff tissue culture plastic and soft alginate hydrogel. Under these conditions, we observed migration of cells from the underlying stiff substrate into the alginate matrix. Observation of migration into alginate in the presence of integrin inhibition as well as qualitative microscopic analyses suggested an adhesion-independent cell migration mode. Observed migration was dependent on alginate matrix stiffness and the RhoA-ROCK-myosin-II pathway; inhibitors specifically targeting ROCK and myosin-II arrested cell migration. Collectively, these results demonstrate the utility of the 2.5D culture platform to advance our understanding of the effects of stiffness gradients and mechanotransductive signaling on adhesion-independent cell migration

Function, Structure, and Stability of Enzymes Confined in Agarose Gels

Research over the past few decades has attempted to answer how proteins behave in molecularly confined or crowded environments when compared to dilute buffer solutions. This information is vital to understanding in vivo protein behavior, as the average spacing between macromolecules in the cell cytosol is much smaller than the size of the macromolecules themselves. In our study, we attempt to address this question using three structurally and functionally different model enzymes encapsulated in agarose gels of different porosities. Our studies reveal that under standard buffer conditions, the initial reaction rates of the agarose-encapsulated enzymes are lower than that of the solution phase enzymes. However, the encapsulated enzymes retain a higher percentage of their activity in the presence of denaturants. Moreover, the concentration of agarose used for encapsulation had a significant effect on the enzyme functional stability; enzymes encapsulated in higher percentages of agarose were more stable than the enzymes encapsulated in lower percentages of agarose. Similar results were observed through structural measurements of enzyme denaturation using an 8-anilinonaphthalene-1-sulfonic acid fluorescence assay. Our work demonstrates the utility of hydrogels to study protein behavior in highly confined environments similar to those present in vivo; furthermore, the enhanced stability of gel encapsulated enzymes may find use in the delivery of therapeutic proteins, as well as the design of novel strategies for biohybrid medical devices

Influence of Macromolecular Crowding and Confinement on Enzyme Activity and Structure under Native and Denaturing Conditions

It is now well appreciated that both crowding and confinement influence enzyme structure and function due to excluded volume effects; however, the relative efficacies of these environments on protein fates remain unclear due to a lack of direct comparison studies. In this study, we explore the use of the biopolymer alginate to develop an in vitro platform to investigate the effects of both crowding and confinement on the behavior of two model enzymes - horseradish peroxidase and β-galactosidase. Alginate, in its solution phase, can be used as a crowding agent and, in its gel phase by crosslinking using divalent cations, to encapsulate and confine proteins, thereby allowing us to use the same system to directly compare the effects of crowding and confinement. Different degrees of crowding and confinement were achieved by varying the alginate concentration, and these studies demonstrated a clear dependence of enzyme activity on the degree of crowding and confinement. Moreover, our data also suggested that protein confinement in crosslinked alginate gels led to higher enhancements in enzyme activity under denaturing conditions relative to non-crosslinked crowded environments. Results from the kinetic analyses were corroborated using structural measurements of protein denaturation using the 8-anilinonaphthalene-1-sulfonic acid fluorescence assay

Advances in homology directed genetic engineering of human pluripotent and adult stem cells

The ability to introduce precise genomic modifications in human cells has profound implications for both basic and applied research in stem cells, ranging from identification of genes regulating stem cell self-renewal and multilineage differentiation to therapeutic gene correction and creation of in vitro models of human diseases. However, the overall efficiency of this process is challenged by several factors including inefficient gene delivery into stem cells and low rates of homology directed site-specific targeting. Recent studies report the development of novel techniques to improve gene targeting efficiencies in human stem cells; these methods include molecular engineering of viral vectors o efficiently deliver episomal genetic sequences that can participate in homology directed targeting, as well as the design of synthetic proteins that can introduce double-stranded breaks in DNA to initiate such recombination events. This review focuses on the potential of these new technologies to precisely alter the human stem cell genome and also highlights the possibilities offered by the combination of these complementary strategies

Soft microenvironments promote the early neurogenic differentiation but not self-renewal of human pluripotent stem cells

Human pluripotent stem cells (hPSCs) are of great interest in biology and medicine due to their ability to self-renew and differentiate into any adult or fetal cell type. Important efforts have identified biochemical factors, signaling pathways, and transcriptional networks that regulate hPSC biology. However, recent work investigating the effect of biophysical cues on mammalian cells and adult stem cells suggests that the mechanical properties of the microenvironment, such as stiffness, may also regulate hPSC behavior. While several studies have explored this mechanoregulation in mouse embryonic stem cells (mESCs), it has been challenging to extrapolate these findings and thereby explore their biomedical implications in hPSCs. For example, it remains unclear whether hPSCs can be driven down a given tissue lineage by providing tissue-mimetic stiffness cues. Here we address this open question by investigating the regulation of hPSC neurogenesis by microenvironmental stiffness. We find that increasing extracellular matrix (ECM) stiffness in vitro increases hPSC cell and colony spread area but does not alter self-renewal, in contrast to past studies with mESCs. However, softer ECMs with stiffnesses similar to that of neural tissue promote the generation of early neural ectoderm. This mechanosensitive increase in neural ectoderm requires only a short 5-day soft stiffness “pulse,” which translates into downstream increases in both total neurons as well as therapeutically relevant dopaminergic neurons. These findings further highlight important differences between mESCs and hPSCs and have implications for both the design of future biomaterials as well as our understanding of early embryonic development

Role of Nanoparticle–Polymer Interactions on the Development of Double-Network Hydrogel Nanocomposites with High Mechanical Strength

Extensive experimental and theoretical research over the past several decades has pursued strategies to develop hydrogels with high mechanical strength. Our study investigated the effect of combining two approaches, addition of nanoparticles and crosslinking two different polymers (to create double-network hydrogels), on the mechanical properties of hydrogels. Our experimental analyses revealed that these orthogonal approaches may be combined to synthesize hydrogel composites with enhanced mechanical properties. However, the enhancement in double network hydrogel elastic modulus due to incorporation of nanoparticles is limited by the ability of the nanoparticles to strongly interact with the polymers in the network. Moreover, double-network hydrogel nanocomposites prepared using lower monomer concentrations showed higher enhancements in elastic moduli compared to those prepared using high monomer concentrations, thus indicating that the concentration of hydrogel monomers used for the preparation of the nanocomposites had a significant effect on the extent of nanoparticle-mediated enhancements. Collectively, these results demonstrate that the hypotheses previously developed to understand the role of nanoparticles on the mechanical properties of hydrogel nanocomposites may be extended to double-network hydrogel systems and guide the development of next-generation hydrogels with extraordinary mechanical properties through a combination of different approaches

Exploring the Role of Nanoparticles in Enhancing Mechanical Properties of Hydrogel Nanocomposites

Over the past few decades, research studies have established that the mechanical properties of hydrogels can be largely impacted by the addition of nanoparticles. However, the exact mechanisms behind such enhancements are not yet fully understood. To further explore the role of nanoparticles on the enhanced mechanical properties of hydrogel nanocomposites, we used chemically crosslinked polyacrylamide hydrogels incorporating silica nanoparticles as the model system. Rheological measurements indicate that nanoparticle-mediated increases in hydrogel elastic modulus can exceed the maximum modulus that can be obtained through purely chemical crosslinking. Moreover, the data reveal that nanoparticle, monomer, and chemical crosslinker concentrations can all play an important role on the nanoparticle mediated-enhancements in mechanical properties. These results also demonstrate a strong role for pseudo crosslinking facilitated by polymer–particle interactions on the observed enhancements in elastic moduli. Taken together, our work delves into the role of nanoparticles on enhancing hydrogel properties, which is vital to the development of hydrogel nanocomposites with a wide range of specific mechanical properties

Directed Evolution of Adeno-associated Virus for Enhanced Gene Delivery and Gene Targeting in Human Pluripotent Stem Cells

Efficient approaches for the precise genetic engineering of human pluripotent stem cells (hPSCs) can enhance both basic and applied stem cell research. Adenoassociated virus (AAV) vectors are of particular interest for their capacity to mediate efficient gene delivery to and gene targeting in various cells. However, natural AAV serotypes offer only modest transduction of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), which limits their utility for efficiently manipulating the hPSC genome. Directed evolution is a powerful means to generate viral vectors with novel capabilities, and we have applied this approach to create a novel AAV variant with high gene delivery efficiencies (~50%) to hPSCs, which are importantly accompanied by a considerable increase in gene-targeting frequencies, up to 0.12%. While this level is likely sufficient for numerous applications, we also show that the gene-targeting efficiency mediated by an evolved AAV variant can be further enhanced (\u3e1%) in the presence of targeted double stranded breaks (DSBs) generated by the co-delivery of artificial zinc finger nucleases (ZFNs). Thus, this study demonstrates that under appropriate selective pressures, AAV vectors can be created to mediate efficient gene targeting in hPSCs, alone or in the presence of ZFNmediated double-stranded DNA breaks