Saumendra K Bajpai

In this work, an attempt has been made to analyze the influence of pathological changes in eye globe dimensions towards the mechanical responses of optic nerve head tissues during eye adductions. For this study, a 3D baseline model geometry of posterior ocular tissues has been constructed. The eye globe diameters of the model are modified to mimic the changes in ocular globe morphology in control, glaucoma, myopia and glaucoma with myopia conditions. Adductions are simulated for each modified model as the rotation of the globe from 1ºto 10ºin steps of 1º. von Mises strain in lamina cribrosa (LC) and posterior displacement of LC are estimated. Results show that strains developed in LC and its posterior displacement are higher in diseased eyes compared to healthy eyes. It appears that eyes with higher axial length and globe anisotropy are more susceptible to optic nerve head damage. This study might be extended to assess the progression of glaucomatous optic neuropathy.

Tumor-associated collagen signature-3 (TACS-3) is a prognostic indicator for breast cancer survival. It is characterized by highly organized, parallel bundles of collagen fibers oriented perpendicular to the tumor boundary, serving as directional, confining channels for cancer cell invasion. Here we design a TACS-3-mimetic anisotropic, confined collagen I matrix and examine the relation between anisotropy of matrix, directed cellular migration, and anisotropy of cell membrane-the first direct contact between TACS-3 and cell-using Michigan Cancer Foundation-7 (MCF-7) cells as cancer-model. Using unidirectional freezing, we generated ∼50 μm-wide channels filled with collagen I. Optical tweezer (OT) microrheology shows that anisotropic confinement increases collagen viscoelasticity by two orders of magnitude, and the elastic modulus is significantly greater along the direction of anisotropic confinement compared to that along the orthogonal direction, thus establishing matrix anisotropy. Furthermore, MCF-7 cells embedded in anisotropic collagen I, exhibit directionality in cellular morphology and migration. Finally, using customized OT to trap polystyrene probes bound to cell-membrane (and not to ECM) of either free cells or cells under anisotropic confinement, we quantified the effect of matrix anisotropy on membrane viscoelasticity, both in-plane and out-of-plane, vis-à-vis the membrane. Both bulk and viscous modulus of cell-membrane of MCF-7 cells exhibit significant anisotropy under anisotropic confinement. Moreover, the cell membrane of MCF-7 cells under anisotropic confinement is significantly softer (both in-plane and out-of-plane moduli) despite their local environment being five times stiffer than free cells. In order to test if the coupling between anisotropy of extracellular matrix and anisotropy of cell-membrane is regulated by cell-cytoskeleton, actin cytoskeleton was depolymerized for both free and confined cells. Results show that cell membrane viscoelasticity of confined MCF-7 cells is unaffected by actin de-polymerization, in contrast to free cells. Together, these findings suggest that anisotropy of ECM induces directed migration and correlates with anisotropy of cell-membrane viscoelasticity of the MCF-7 cells in an actin-independent manner.

Normal thermal fluctuations of the cell membrane have been studied extensively using high resolution microscopy and focused light, particularly at the peripheral regions of a cell. We use a single probe particle attached non-specifically to the cell-membrane to determine that the power spectral density is proportional to (frequency)-5/3 in the range of 5 Hz to 1 kHz. We also use a new technique to simultaneously ascertain the slope fluctuations of the membrane by relying upon the determination of pitch motion of the birefringent probe particle trapped in linearly polarized optical tweezers. In the process, we also develop the technique to identify pitch rotation to a high resolution using optical tweezers. We find that the power spectrum of slope fluctuations is proportional to (frequency)-1, which we also explain theoretically. We find that we can extract parameters like bending rigidity directly from the coefficient of the power spectrum particularly at high frequencies, instead of being convoluted with other parameters, thereby improving the accuracy of estimation. We anticipate this technique for determination of the pitch angle in spherical particles to high resolution as a starting point for many interesting studies using the optical tweezers. This journal is

The cell membrane has fluctuations due to thermal and athermal sources. That causes the membrane to flicker. Conventionally, only the normal (perpendicular to the membrane) fluctuations are studied and then used to ascertain the membrane properties like the bending rigidity. It is here that we introduce a different concept, namely the slope fluctuations of the cell membrane which can be modelled as a gradient of the normal fluctuations. This can be studied using a new technique where a birefringent particle placed on the membrane turns in the out of plane sense, called the pitch sense. We introduce the pitch detection technique in optical tweezers relying upon asymmetric scattering from a birefringent particle under crossed polarizers. We then go on to use this pitch detection technique to ascertain the power spectral density of membrane slope fluctuations and find it to be (frequency)-1 while the normal fluctuations yields (frequency)-5/3. We also explore a different regime where the cell is applied with the drug Latrunculin-B which inhibits actin polymerization and find the effect on membrane fluctuations. We find that even as the normal fluctuations now become (frequency)-4/3, the slope fluctuations spectrum still remains (frequency)-1, with exactly the same coefficient as the case when the drug was not applied. Thus, this presents a convenient opportunity to study the membrane parameters like bending rigidity as a function of time after applying the drug. This would be the first time the membrane bending rigidity could be studied as a function of time upon the application of Lat-B without reverting to AFM.

We study the normal fluctuations of an MCF-7 cell membrane and calibrate the optical trap to detect pitch motion to get information about the rocking motion of a birefringent particle. We could show both theoretically and experimentally that the Z power spectrum has a power-law behavior of (frequency)-5/3, and We find that the power spectrum of slope fluctuations is proportional to (frequency)-1. We could extract parameters like bending rigidity directly from the power spectrum fitting parameters in 5 Hz to 1 kHz range. Our method was powerful enough to identify pitch rotation for a spherical birefringent particle to a high resolution using optical tweezers.

Microstructurally-aligned Silk fibroin sponges have emerged as a viable candidate for replicating the anisotropic fibrous microarchitecture that is encountered in several functional tissues in vivo. But for the material to reach its full potential in terms of its application in biomimetic soft tissue constructs or scaffolds, a complete understanding and quantification of its nonlinear viscoelastic response in the finite strain regime is needed. To address this gap from the mechanical modeling perspective, an anisotropic, nonlinear, viscoelastic model is developed within a thermodynamic framework in this study, to capture the macroscopic response of these sponges in a phenomenological manner. The rate-type constitutive equations that are developed in the process are subsequently applied for the case of uniaxial compression, and satisfactorily corroborated against the results from finite strain viscoelastic characterization experiments done on hydrated Silk fibroin sponges under uniaxial compression for different constituent material concentrations of the sponges ranging from 1 w/v % - 4 w/v %.

Development of clinically amenable bio-implants with silk-fibroin (SF) necessitates characterization of cellular-traction generated between cells and the substrate. However, studies on the biomechanical response of cells on SF substrates are limited. In this study, we prepared SF hydrogels of varying compliance (SF30 and SF50) and varying surface-ligands (derivatized with poly-L-lysine (PLL) or Arg-Gly-Asp (RGD) peptide). Subsequently, NIH-3T3 fibroblast cells were grown on these substrates, and the morphological changes was examined. It was observed that the increase in SF stiffness from 0.7 kPa to 3.1 kPa decreased nucleus-to-cytoplasm area-ratio and increased asymmetricity along the major-axis of cells. Moreover, while functionalization of SF with RGD induced increase in cell-area and circularity, functionalization with PLL did not cause any change. Next, using traction-force-microscopy (TFM), we quantified 2D cell-traction for NIH-3T3 cells cultured on SF hydrogels. Cells plated on SF50 hydrogel exhibited significantly high traction stress as compared to SF30; change of functionalization did not show significant change. Also, protrusion traction stress was found to be greater than nuclear traction stress. Put together, our results suggest that surface-stiffness of SF-hydrogel, rather than nature of surface-ligand, regulates both cellular morphology and cellular traction stresses.

A colloidal particle placed inside the cell cytoplasm is enmeshed within a network of cytoskeletal fibres immersed in the cytosolic fluid. The translational mode is believed to yield different rheological parameters than the rotational mode, given that these modes stretch the fibers differently. We compare the parameters for Michigan Cancer Foundation-7 (MCF-7) cells in this manuscript and find that the results are well comparable to each other. At low values of 0 Hz viscosity, the rotational and translational viscoelasticity matches well. However, discrepancies appear at higher values which may indicate that the cytoskeletal modes involved in rotation and translation of the particle are getting invoked. We also show that the 0 Hz viscosity increases as the cell ages under the conditions of constant room temperature of 25°C on the sample chamber.

Silk fibroin (SF) is a model candidate for use in tissue engineering and regenerative medicine owing to its bio-compatible mechanochemical properties. Despite numerous advances made in the fabrication of various biomimetic substrates using SF, relatively few clinical applications have been designed, primarily due to the lack of complete understanding of its constitutive properties. Here we fabricate microstructurally aligned SF sponge using the unidirectional freezing technique wherein a novel solvent-processing technique involving Acetic acid is employed, which obviates the post-treatment of the sponges to induce their water-stability. Subsequently, we quantify the anisotropic, viscoelastic response of the bulk SF sponge samples by performing a series of mechanical tests under uniaxial compression over a wide range of strain rates. Results for these uniaxial compression tests in the finite strain regime through ramp strain and ramp-relaxation loading histories applied over two orders of strain rate magnitude show that microstructural anisotropy is directly manifested in the bulk viscoelastic solid-like response. Furthermore, the experiments reveal a high degree of volume compressibility of the sponges during deformation, and also evince for their remarkable strain recovery capacity under large compressive strains during strain recovery tests. Finally, in order to predict the bulk viscoelastic material properties of the fabricated and pre-characterized SF sponges, a finite strain kinematics-based, nonlinear, continuum model developed within a thermodynamically-consistent framework in a parallel investigation, was successfully employed to capture the viscoelastic solid-like, transversely isotropic, and compressible response of the sponges macroscopically.

Quantitative assessment of cell adhesion during pathological development can potentially lead to a better elucidation of the mechanisms behind disease progression and improve prognostic accuracy. Concurrent developments in the fields of biosensors and multimodal imaging techniques, and improved understanding of the biophysical principles driving a disease, have contributed to the development of techniques for quantification of cell adhesion strength. Together, these measurements have underscored the importance of a tightly regulated cell adhesion phenotype, exhibited by tissues under pathological progression. Here we discuss some of the techniques that evaluate cell-to-cell and cell-to-substrate adhesion strength across multiple scales of length and time, taking cancer metastasis as our model system.