Uday Chippada

Neural lineage differentiation of embryonic stem cells within alginate microbeads

Cell replacement therapies, using renewable stem cell sources, hold tremendous potential to treat a wide range of degenerative diseases. Although many studies have established techniques to successfully differentiate stem cells into different mature cell lineages using growth factors or extracellular matrix protein supplementation in both two and three-dimensional configurations, they are often limited by lack of control and low yields of differentiated cells. Previously, we developed a scalable murine embryonic stem cell differentiation environment which maintained cell viability and supported ES cell differentiation to hepatocyte lineage cells. Differentiated hepatocyte function was contingent upon aggregate formation within the alginate microbeads. The present studies were designed to determine the feasibility of adapting the alginate encapsulation technique to neural lineage differentiation. The results of our studies indicate that by incorporating the soluble inducer, retinoic acid (RA), into the permeable microcapsule system, cell aggregation was decreased and neural lineage differentiation enhanced. In addition, we demonstrated that even in the absence of RA, differentiation could be directed away from the hepatocyte and toward the neural lineage by physical cell-cell aggregation blocking. In conjunction with the mechanical and physical characterization of the alginate crosslinking network, we determined that 2.2% alginate microencapsulation can be optimally adapted to ES neural differentiation. This study offers insights into targeting cellular differentiation toward both endodermal and ectodermal cell lineages, and could potentially be adaptable to differentiation of other stem cell types given the correct inducible factors and material properties.

Effect of the Structural Water on the Mechanical Properties of Collagen-like Microfibrils: A Molecular Dynamics Study

The objective of this paper is to investigate the role played by the structural water on the intermolecular sliding between collagen-like 1QSU peptides in a microfibril under deformation. Three modes of deformation are used to generate intermolecular sliding: forced axial stretching (case I) or sliding (case II) of a central peptide monomer (while other surrounding monomers are fixed); and cantilever bending (case III) under a terminal lateral load. The force–displacement curve of each deformation mode is derived using a module called Steered Molecular Dynamics (SMD) in a molecular dynamics package NAMD under the CHARMM22 force field. Each calculation is carried out twice, one in the presence of structural water, one without. It is found that the structural water is a weak “lubricant” in forced axial stretching (case I), but it functions as a “glue” in forced axial sliding (case II) and cantilever bending (case III). A change in the pulling speed does not significantly alter the force–displacement behavior in axial stretching (case I) and sliding (case II), but it does in cantilever bending (case III). The additional resistance contributed by the structural water is attributed to the additional energy cost in breaking the water-mediated hydrogen bonds (water bridges).

Simultaneous determination of Young's modulus, shear modulus, and Poisson's ratio of soft hydrogels

Besides biological and chemical cues, cellular behavior has been found to be affected by mechanical cues such as traction forces, surface topology, and in particular the mechanical properties of the substrate. The present study focuses on completely characterizing the bulk linear mechanical properties of such soft substrates, a good example of which are hydrogels. The complete characterization involves the measurement of Youngs modulus, shear modulus, and Poissons ratio of these hydrogels, which is achieved by manipulating nonspherical magnetic microneedles embedded inside them. Translating and rotating these microneedles under the influence of a known force or torque, respectively, allows us to determine the local mechanical properties of the hydrogels. Two specific hydrogels, namely bis-cross-linked polyacrylamide gels and DNA cross-linked polyacrylamide gels were used, and their properties were measured as a function of gel concentration. The bis-cross-linked gels were found to have a Poissons ratio that varied between 0.38 and 0.49, while for the DNA-cross-linked gels, Poissons ratio varied between 0.36 and 0.49. The local shear moduli, measured on the 10 μm scale, of these gels were in good agreement with the global shear modulus obtained from a rheology study. Also the local Youngs modulus of the hydrogels was compared with the global modulus obtained using bead experiments, and it was observed that the inhomogeneities in the hydrogel increases with increasing cross-linker concentration. This study helps us fully characterize the properties of the substrate, which helps us to better understand the behavior of cells on these substrates.

A Nonintrusive Method of Measuring the Local Mechanical Properties of Soft Hydrogels Using Magnetic Microneedles

Soft hydrogels serving as substrates for cell attachment are used to culture many types of cells. The mechanical properties of these gels influence cell morphology, growth, and differentiation. For studies of cell growth on inhomogeneous gels, techniques by which the mechanical properties of the substrate can be measured within the proximity of a given cell are of interest. We describe an apparatus that allows the determination of local gel elasticity by measuring the response of embedded micron-sized magnetic needles to applied magnetic fields. This microscope-based four-magnet apparatus can apply both force and torque on the microneedles. The force and the torque are manipulated by changing the values of the magnetic field at the four poles of the magnet using a feedback circuit driven by LABVIEW. Using Hall probes, we have mapped out the magnetic field and field gradients produced by each pole when all the other poles are held at zero magnetic field. We have verified that superposition of these field maps allows one to obtain field maps for the case when the poles are held at arbitrary field values. This allows one to apply known fields and field gradients to a given microneedle. An imaging system is employed to measure the displacement and rotation of the needles. Polyacrylamide hydrogels of known elasticity were used to determine the relationship between the field gradient at the location of the needles and the force acting on the needles. This relationship allows the force on the microneedle to be determined from a known field gradient. This together with a measurement of the displacement of the needle in a given gel allows one to determine the stiffness (Fdelta) of the gel and the elastic modulus, provided Poisons ratio is known. Using this method, the stiffness and the modulus of elasticity of type-I collagen gels were found to be 2.64+/-0.05 nNmicrom and 284.6+/-5.9 Pa, respectively. This apparatus is presently being employed to track the mechanical stiffness of the DNA-cross-linked hydrogels, developed by our group, whose mechanical properties can be varied on demand by adding or removing cross-linker strands. Thus a system that can be utilized to track the local properties of soft media as a function of time with minimum mechanical disturbance in the presence of cells is presented.

Complete mechanical characterization of soft media using nonspherical rods

Hydrogels have been used as substrates for studying the cellular processes by many researchers. The stiffness of such gels was also characterized previously. However, in most of the cases, these soft Poissons ratio was assumed incompressible and Poissons ratio is assumed to be one-half. This may not be true in many cases, and might alter the calculation of the stiffness of the gels. In this study, we present equations for the complete characterization of soft media, i.e., calculation of Youngs modulus, shear modulus, and Poissons ratio. The method involves the individual measurement of either the displacement or rotation of cylindrical rods embedded in the soft media, under the application of an external force or torque. Equations involving shear modulus and Poissons ratio for rotation of the rod and Youngs modulus and Poissons ratio for the displacement of the rod are independently derived. In addition, the displacement and rotation of the rods embedded in an elastic medium, under the application of either a force or a torque, respectively, were also calculated using finite element analysis. These values compared well with the displacements and rotations obtained using closed form equations.

Mechanical Properties of DNA-Crosslinked Polyacrylamide Hydrogels with Increasing Crosslinker Density

Abstract DNA-cross-linked polyacrylamide hydrogels (DNA gels) are dynamic mechanical substrates. The addition of DNA oligomers can either increase or decrease the crosslinker density to modulate mechanical properties. These DNA-responsive gels show promise as substrates for cell culture and tissue-engineering applications, since the gels allow time-dependent mechanical modulation. Previously, we reported that fibroblasts plated on DNA gels responded to modulation in elasticity via an increase or decrease in crosslinker density. To better characterize fibroblast mechanical signals, changes in stress and elastic modulus of DNA gels were measured over time as crosslinker density altered. In a previous study, we observed that as crosslinker density decreased, stress was generated, and elasticity changed over time; however, we had not evaluated stress and elastic modulus measurements of DNA gels as crosslinker density increased. Here, we completed this set of fibroblast studies by reporting stress and elastic modulus measurements over time as the crosslinker density increased. We found that the stress generated and the elastic modulus alterations were correlated. Hence, it seemed impossible to separate the effect of stress from the effect of modulus changes for fibroblasts plated on DNA gels. Yet, previous results and controls revealed that stress contributed to fibroblast behavior.

Fibroblast Behavior on Tunable Gels With Decreasing Elasticity

Cells sense and react to various extracellular matrix (ECM) cues including chemical and physical cues. Previous studies in our laboratory and others have used static substrates, where the elastic properties remain unchanged throughout the culture period, to examine the effects of mechanical stiffness on neuron and fibroblast behavior [1–4]. However, in vivo, the ECM is dynamic and alters due to pathological, developmental, and external factors [5]. To study the effects of dynamic ECM changes on cell behavior, we developed a DNA-crosslinked, polyacrylamide gel (DNA gel) that allows us to study how dynamic changes in ECM stiffness affect cell behavior [6, 7].Copyright

Alteration of Fibroblast Cell Behavior due to Contraction of Substrate

Many researchers have utilized hydrogels as substrates for cell attachment. The stiffness of these substrates has been found to influence the cellular behavior such as morphology, proliferation, growth and differentiation. Lo et al. deformed polyacrylamide substrates with a blunted microneedle and observed the movement of NIH 3T3 fibroblasts. In both pulling and pushing, the cells reversed their direction and moved away from the needle. This shows that cellular behavior is also affected by stretching the underlying substrates. In a previous study, Lin et al. have demonstrated the ability to contract DNA-crosslinked polyacrylamide hydrogels (‘DNA gels’ in short) by addition of crosslinks. Jiang et al. have utilized these DNA gels as substrates to observe the cellular responses of L929 and GFP fibroblasts to both static and dynamic substrate compliances.Copyright

Simultaneous Determination of E, G and ν of Soft Hydrogels Using Theory of Elasticity

Hydrogels have been used as substrates by many researchers in the study of cellular processes. The mechanical properties of these gels play a significant role in the growth of the cells. Significant research using several methods like compression, indentation, atomic force microscopy and manipulation of beads has been performed in the past to characterize the stiffness of these substrates. However, most of the methods employed assume the gel to be incompressible, with a Poisson’s ratio of 0.5. However, Poisson’s ratio can differ from 0.5. Hence, a more complete characterization of the elastic properties of hydrogels requires that one experimentally obtain the value of at least two of the three quantities: Poisson’s ratio, shear modulus, and elastic modulus.Copyright

The Effect of Dynamic Alterations in Stiffness of the Substrate on Cell Growth

Cells reside in a dynamic environment composed of extracellular matrix (ECM) and other cells, and take a variety of cues, of which mechanical stresses and strains are an important subset. ECM undergoes constant synthesis and degradation, and its mechanical stiffness can also be altered, with ageing, upon external assault or via pathological processes. Particularly in load barring tissues, the mechanical properties of the ECM can vary, by exposure to changing load conditions through, for example, collagen realignment. Tissue-implant interfaces also present medically important dynamic mechanical environment. Furthermore, recent studies revealed that the ranges of mechanical stiffness of ECM or substrates can alter specific cellular properties in distinct ways. From an engineering viewpoint, it is thus beneficial to be able to modify the physical properties of the biomaterials for the implants, providing optimal conditions for a specific desired outcome at different points during time progression. All of these reasons make it desirable to have a dynamic culture system with controlled property changes.Copyright