Karthik Mukundakrishnan

Escherichia coli Biofilms Formed under Low-Shear Modeled Microgravity in a Ground-Based System

Bacterial biofilms cause chronic diseases that are difficult to control. Since biofilm formation in space is well documented and planktonic cells become more resistant and virulent under modeled microgravity, it is important to determine the effect of this gravity condition on biofilms. Inclusion of glass microcarrier beads of appropriate dimensions and density with medium and inoculum, in vessels specially designed to permit ground-based investigations into aspects of low-shear modeled microgravity (LSMMG), facilitated these studies. Mathematical modeling of microcarrier behavior based on experimental conditions demonstrated that they satisfied the criteria for LSMMG conditions. Experimental observations confirmed that the microcarrier trajectory in the LSMMG vessel concurred with the predicted model. At 24 h, the LSMMG Escherichia coli biofilms were thicker than their normal-gravity counterparts and exhibited increased resistance to the general stressors salt and ethanol and to two antibiotics (penicillin and chloramphenicol). Biofilms of a mutant of E. coli, deficient in sigma(s), were impaired in developing LSMMG-conferred resistance to the general stressors but not to the antibiotics, indicating two separate pathways of LSMMG-conferred resistance.

Sedimentation of an ellipsoid inside an infinitely long tube at low and intermediate Reynolds numbers

The motion of a heavy rigid ellipsoidal particle settling in an infinitely long circular tube filled with an incompressible Newtonian fluid has been studied numerically for three categories of problems, namely, when both fluid and particle inertia are negligible, when fluid inertia is negligible but particle inertia is present, and when both fluid and particle inertia are present. The governing equations for both the fluid and the solid particle have been solved using an arbitrary Lagrangian-Eulerian based finite-element method. Under Stokes flow conditions, an ellipsoid without inertia is observed to follow a perfectly periodic orbit in which the particle rotates and moves from side to side in the tube as it settles. The amplitude and the period of this oscillatory motion depend on the initial orientation and the aspect ratio of the ellipsoid. An ellipsoid with inertia is found to follow initially a similar oscillatory motion with increasing amplitude. Its orientation tends towards a flatter configuration, and the rate of change of its orientation is found to be a function of the particle Stokes number which characterizes the particle inertia. The ellipsoid eventually collides with the tube wall, and settles into a different periodic orbit. For cases with non-zero Reynolds numbers, an ellipsoid is seen to attain a steady-state configuration wherein it falls vertically. The location and configuration of this steady equilibrium varies with the Reynolds number.

Bone cell survival in microgravity: evidence that modeled microgravity increases osteoblast sensitivity to apoptogens.

Abstract: Studies were performed to evaluate the effects of modeled microgravity on the induction of osteoblast apoptosis. MC3T3‐E1 osteoblast‐like cells were cultured in alginate carriers in the NASA‐approved high aspect ratio vessel (HARV). This system subjects the cells to a time‐averaged gravitational field (vector‐averaged gravity) to simulate low gravity conditions. Cells were cultured in the HARV for five days, and then examined for apoptosis. In simulated microgravity, the cells remained vital, although analysis of expressed genes indicated that there was loss of the mature osteoblast phenotype. Additionally, we noted that there was a loss of the mitochondrial membrane potential, a low level of the antiapoptotic protein Bcl‐2, as well as Akt protein, and the redox status of the cells was disturbed. All of these parameters indicated that vector‐averaged gravity disrupts mitochondrial function, thereby sensitizing osteoblasts to apoptosis. We then used a challenge assay to evaluate the apoptotic sensitivity of the cells subjected to vector‐averaged gravity. When challenged with staurosporine, cells subjected to vector‐averaged gravity evidenced elevated levels of cell death relative to control cell populations. Another objective of the study was to improve upon conventional carriers by using alginate encapsulation to support cells in the HARV. We have demonstrated that the alginate carrier system affords a more robust system than surface‐seeded carriers. This new system has the advantage of shielding cells from mechanical damage and fluid shear stresses on cells in the HARV, permitting carefully controlled studies of the effects of vector‐averaged gravity.

The effect of simulated microgravity on osteoblasts is independent of the induction of apoptosis

Bone loss during spaceflight has been attributed, in part, to a reduction in osteoblast number, altered gene expression, and an increase in cell death. To test the hypothesis that microgravity induces osteoblast apoptosis and suppresses the mature phenotype, we created a novel system to simulate spaceflight microgravity combining control and experimental cells within the same in vitro environment. Cells were encapsulated into two types of alginate carriers: non‐rotationally stabilized (simulated microgravity) and rotationally stabilized (normal gravity). Using these specialized carriers, we were able to culture MC3T3‐E1 osteoblast‐like cells for 1–14 days in simulated microgravity and normal gravity in the same rotating wall vessel (RWV). The viability of cells was not affected by simulated microgravity, nor was the reductive reserve. To determine if simulated microgravity sensitized the osteoblasts to apoptogens, cells were challenged with staurosporine or sodium nitroprusside and the cell death was measured. Simulated microgravity did not alter the sensitivity of C3H10T‐1/2 stem cells, MC3T3‐E1 osteoblast‐like cells, or MLO‐A5 osteocyte‐like cells to the action of these agents. RT‐PCR analysis indicated that MC3T3‐E1 osteoblasts maintained expression of RUNX2, osteocalcin, and collagen type I, but alkaline phosphatase expression was decreased in cells subjected to simulated microgravity for 5 days. We conclude that osteoblast apoptosis is not induced by vector‐averaged gravity, thus suggesting that microgravity does not directly induce osteoblast death. J. Cell. Biochem. 102: 483–495, 2007.

Bubble Motion in a Blood Vessel: Shear Stress Induced Endothelial Cell Injury

Mechanisms governing endothelial cell (EC) injury during arterial gas embolism have been investigated. Such mechanisms involve multiple scales. We have numerically investigated the macroscale flow dynamics due to the motion of a nearly occluding finite-sized air bubble in blood vessels of various sizes. Non-Newtonian behavior due to both the shear-thinning rheology of the blood and the Fahraeus-Lindqvist effect has been considered. The occluding bubble dynamics lends itself for an axisymmetric treatment. The numerical solutions have revealed several hydrodynamic features in the vicinity of the bubble. Large temporal and spatial shear stress gradients occur on the EC surface. The stress variations manifest in the form of a traveling wave. The gradients are accompanied by rapid sign changes. These features are ascribable to the development of a region of recirculation (vortex ring) in the proximity of the bubble. The shear stress gradients together with sign reversals may partially act as potential causes in the disruption of endothelial cell membrane integrity and functionality.

Numerical modeling of oxygen distributions in cortical and cancellous bone: oxygen availability governs osteonal and trabecular dimensions

Whereas recent work has demonstrated the role of oxygen tension in the regulation of skeletal cell function and viability, the microenvironmental oxemic status of bone cells remains unknown. In this study, we have employed the Krogh cylinder model of oxygen diffusion to predict the oxygen distribution profiles in cortical and cancellous bone. Under the assumption of saturation-type Michaelis-Menten kinetics, our numerical modeling has indicated that, under steady-state conditions, there would be oxygen gradients across mature osteons and trabeculae. In Haversian bone, the calculated oxygen tension decrement ranges from 15 to 60%. For trabecular bone, a much shallower gradient is predicted. We note that, in Haversian bone, the gradient is largely dependent on osteocyte oxygen utilization and tissue oxygen diffusivity; in trabecular bone, the gradient is dependent on oxygen utilization by cells lining the bone surface. The Krogh model also predicts dramatic differences in oxygen availability during bone development. Thus, during osteon formation, the modeling equations predict a steep oxygen gradient at the initial stage of development, with the gradient becoming lesser as osteonal layers are added. In contrast, during trabeculum formation, the oxygen gradient is steepest when the diameter of the trabeculum is maximal. Based on these results, it is concluded that significant oxygen gradients exist within cortical and cancellous bone and that the oxygen tension may regulate the physical dimensions of both osteons and bone trabeculae.

Effect of a soluble surfactant on a finite-sized bubble motion in a blood vessel

We present detailed results for the motion of a finite sized gas bubble in a blood vessel. The bubble (dispersed phase) size is taken to be such as to nearly occlude the vessel. The bulk medium is treated as a shear thinning Casson fluid and contains a soluble surfactant that adsorbs and desorbs from the interface. Three different vessel sizes, corresponding to a small artery, a large arteriole, and a small arteriole, in normal humans, are considered. The hematocrit (volume fraction of RBCs) has been taken to be 0.45. For arteriolar flow, where relevant, the Fahraeus-Lindqvist effect is taken into account. Bubble motion cause temporal and spatial gradients of shear stress at the cell surface lining the vessel wall as the bubble approaches the cell, moves over it and passes it by. Rapid reversals occur in the sign of the shear stress imparted to the cell surface during this motion. Shear stress gradients together with sign reversals are associated with a recirculation vortex at the rear of the moving bubble. The presence of the surfactant reduces the level of the shear stress gradients imparted to the cell surface as compared to an equivalent surfactant-free system. Our numerical results for bubble shapes and wall shear stresses may help explain phenomena observed in experimental studies related to gas embolism, a significant problem in cardiac surgery and decompression sickness.

Bubble Motion through a Generalized Power‐Law Fluid Flowing in a Vertical Tube

Intravascular gas embolism may occur with decompression in space flight, as well as during cardiac and vascular surgery. Intravascular bubbles may be deposited into any end organ, such as the heart or the brain. Surface interactions between the bubble and the endothelial cells lining the vasculature result in serious impairment of blood flow and can lead to heart attack, stroke, or even death. To develop effective therapeutic strategies, there is a need for understanding the dynamics of bubble motion through blood and its interaction with the vessel wall through which it moves. Toward this goal, we numerically investigate the axisymmetric motion of a bubble moving through a vertical circular tube in a shear‐thinning generalized power‐law fluid, using a front‐tracking method. The formulation is characterized by the inlet Reynolds number, capillary number, Weber number, and Froude number. The flow dynamics and the associated wall shear stresses are documented for a combination of two different inlet flow conditions (inlet Reynolds numbers) and three different effective bubble radii (ratio of the undeformed bubble radii to the tube radii). The results of the non‐Newtonian model are then compared with that of the model assuming a Newtonian blood viscosity. Specifically, for an almost occluding bubble (effective bubble radius = 0.9), the wall shear stress and the bubble residence time are compared for both Newtonian and non‐Newtonian cases. Results show that at low shear rates, for a given pressure gradient the residence time for a non‐Newtonian flow is higher than that for a Newtonian flow.

Modeling of Phosphate Ion Transfer to the Surface of Osteoblasts under Normal Gravity and Simulated Microgravity Conditions

Abstract: We have modeled the transport and accumulation of phosphate ions at the remodeling site of a trabecular bone consisting of osteoclasts and osteoblasts situated adjacent to each other in straining flows. Two such flows are considered; one corresponds to shear levels representative of trabecular bone conditions at normal gravity, the other corresponds to shear level that is representative of microgravity conditions. The latter is evaluated indirectly using a simulated microgravity environment prevailing in a rotating wall vessel bioreactor (RWV) designed by NASA. By solving the hydrodynamic equations governing the particle motion in a RWV using a direct numerical simulation (DNS) technique, the shear stress values on the surface of the microcarriers are found. In our present species transfer model, osteoclasts release phosphate ions (Pi) among other ions at bone resorption sites. Some of the ions so released are absorbed by the osteoblast, some accumulate at the osteoblast surface, and the remainder are advected away. The consumption of Pi by osteoblasts is assumed to follow Michaelis‐Menten (MM) kinetics aided by a NaPi cotransporter system. MM kinetics views the NaPi cotransporter as a system for transporting extracellular Pi into the osteoblast. Our results show, for the conditions investigated here, the net accumulation of phosphate ions at the osteoblast surface under simulated microgravity conditions is higher by as much as a factor of three. Such increased accumulation may lead to enhanced apoptosis and may help explain the increased bone loss observed under microgravity conditions.

Computational simulation of hematocrit effects on arterial gas embolism dynamics.

BACKGROUND Recent computational investigations have shed light into the various hydrodynamic mechanisms at play during arterial gas embolism that may result in endothelial cell (EC) injury. Other recent studies have suggested that variations in hematocrit level may play an important role in determining the severity of neurological complications due to decompression sickness associated with gas embolism. METHODS To develop a comprehensive picture, we computationally modeled the effect of hematocrit variations on the motion of a nearly occluding gas bubble in arterial blood vessels of various sizes. The computational methodology is based on an axisymmetric finite difference immersed boundary numerical method to precisely track the blood-bubble dynamics of the interface. Hematocrit variations are taken to be in the range of 0.2-0.6. The chosen blood vessel sizes correspond to small arteries and small and large arterioles in normal humans. RESULTS Relevant hydrodynamic interactions between the gas bubble and EC-lined vessel lumen have been characterized and quantified as a function of hematocrit levels. In particular, the variations in shear stress, spatial and temporal shear stress gradients, and the gap between bubble and vascular endothelium surfaces that contribute to EC injury have been computed. DISCUSSION The results suggest that in small arteries, the deleterious hydrodynamic effects of the gas embolism on an EC-lined cell wall are significantly amplified as the hematocrit levels increase. However, such pronounced variations with hematocrit levels are not observed in the arterioles.