Changes in interstitial fluid flow, mass transport and the bone cell response in microgravity and normogravity

Abstract

In recent years, our scientific interest in spaceflight has grown exponentially and resulted in a thriving area of research, with hundreds of astronauts spending months of their time in space. A recent shift toward pursuing territories farther afield, aiming at near-Earth asteroids, the Moon, and Mars combined with the anticipated availability of commercial flights to space in the near future, warrants continued understanding of the human physiological processes and response mechanisms when in this extreme environment. Acute skeletal loss, more severe than any bone loss seen on Earth, has significant implications for deep space exploration, and it remains elusive as to why there is such a magnitude of difference between bone loss on Earth and loss in microgravity. The removal of gravity eliminates a critical primary mechano-stimulus, and when combined with exposure to both galactic and solar cosmic radiation, healthy human tissue function can be negatively affected. An additional effect found in microgravity, and one with limited insight, involves changes in dynamic fluid flow. Fluids provide the most fundamental way to transport chemical and biochemical elements within our bodies and apply an essential mechano-stimulus to cells. Furthermore, the cell cytoplasm is not a simple liquid, and fluid transport phenomena together with viscoelastic deformation of the cytoskeleton play key roles in cell function. In microgravity, flow behavior changes drastically, and the impact on cells within the porous system of bone and the influence of an expanding level of adiposity are not well understood. This review explores the role of interstitial fluid motion and solute transport in porous bone under two different conditions: normogravity and microgravity.

Fig. 1

A multilayered porous system in bone allows for dynamic fluid flow (green arrows) at the nano, micro- and macroscale. a A microCT image of healthy murine trabecular bone. The bone structure consists of micro- and macropores. b A schematic demonstrating osteocytes within the LCS. c Representative photomicrographs of a longitudinal section prepared from the distal femur of a healthy rat. The pores are inhabited by bone marrow containing blood vessels and multiple cell groups. The pores provide an environment for fluid movement (red arrows) and the generation of fluid-induced cellular mechanostimulation. d Representative images of osteocytes within the LCS. The osteocyte nucleus (blue) and cell processes (green) are observed within the canaliculi and allow communication between cells that are located at distant sites. Images were taken from cryo-sections prepared from healthy rat bone. Not presented are the vascular porosities within the Volkmann and Haversian canals, which provide an additional pore structure, as well as the collagen-hydroxyapatite porosities, which comprise the smallest pore size in bone

Fig. 2

Scanning electron microscopy (SEM) images of cultured cells in vitro. a Human osteocytes are stellate in shape and are found within the lacunae-canalicular system. The cell body varies in size between 5 and 20 µm in diameter and sits within the lacunae. Each cell body contains 40–60 cell processes, which are approximately 23–50 µm in length. Cell processes occupy the canaliculi system and establish a communication network. The cell-to-cell distance is approximately 20–30 µm. b Image demonstrating rounded M0 murine macrophages, approximately 10-20 µm in size. When activated toward a pro- (M1) or anti-inflammatory (M2) phenotype, cytoplasmic extensions appear, and cell size increases with elongation of the cell body associated with the M2 phenotype (inset b). c Human mesenchymal stem cells (MSCs) are a heterogeneous cell population that are typically large, flat and spindle shaped. MSCs range between 15 and 40 µm in size

Fig. 3

Gravity and external mechanical stimuli influence the growth, development, and maintenance of healthy tissues. a Cells sense their mechanical environment (e.g., tensile stretch, compressive strains, and shear stimuli) via mechanisms involving cilia, adherens junctions, ion channels, and focal adhesion. b The controlled fluid environment provided by 2D microfluidic devices is a commonly used method to examine the cell response under different flows. c Representative micrographs of IDG-SW3 murine late osteoblasts/preosteocytes taken using confocal microscopy showing cell nuclei (DAPI (blue)) and F-actin cytoskeletal filaments [phalloidin (red)]. Cells were cultured within a microfluidic device. A flow rate of 0.15 mL·s⁻¹ was applied, and the mechanosensitive actin filaments responded by realigning their structure, becoming more parallel in orientation (white arrows). Images show the fluid-induced response after 24 h of culture. d Representative micrographs of macrophages within microfluidic devices (24 h) and examined using fluorescence microscopy (×20 mag.). Images show red cytoplasmic actin filaments (phalloidin) and blue nuclei (DAPI). a Static-flow conditions and following the application of continuous fluid flow delivered at b 0.1 dyn per cm², c 1.1 dyn per cm² and d 10.7 dyn per cm² (physiological) fluid shear to cells. The cells were observed to respond differently to changes in fluid shear. M0 (nonactivated) macrophages are characterized by their small size (~10 µm) and abundant number, and this phenotype is indicated in the unstimulated static control group. Following the application of an extremely low fluid shear (0.1 dyn per cm²), the cells were fewer in number and slightly larger (~15–20 µm), displaying a more M1-like, proinflammatory, osteodestructive phenotype. Notably, there were fewer nuclei, suggesting cell death. Remarkably, when exposed to higher shear rates, the cells become much larger (~80–100 µm) and are round or spindle shaped, suggesting an osteoprotective M2 phenotype

Fig. 4

A schematic showing the mechanical loads borne by bone in normogravity. με: microstrain, BMD: bone mineral density, ALP: alkaline phosphatase, OCN: osteocalcin. Several studies have suggested that the rate (determined by the frequency and amplitude) rather than the magnitude alone of the applied loading stimulus correlates with bone formation.^, This implies that bone formation is enhanced by dynamic loading, and therefore, both the magnitude (or amplitude) and the frequency of loading are important parameters. It has been shown that low magnitude [<10 με (<1 g; 1 g = 9.8 m·s⁻²)] and high frequency (10–100 Hz) loading stimulate bone growth, inhibiting disuse osteoporosis.^, Peak dynamic strain magnitudes within the physiological range of 1 500–3 000 microstrain (μɛ) are reported to result in bone modeling and an increase in mass. Strain within the disuse range of 100–300 μɛ activates osteoclastic activity and bone resorption. Strain levels above 3 000 μɛ are considered overuse, and those above 5 000 are considered pathological overload

Fig. 5

The effect of microgravity on fluid flow within bone and the subsequent cell response reported in the literature. Bone tissue is a complex mechanical environment that provides a specialized habitat for numerous mechanosensitive cell types

Fig. 6

Images of healthy and osteoporotic bone in a rodent model developed in normogravity. A greater level of adiposity is observed within the osteoporotic architecture. a MicroCT images showing that the trabecular architecture is slowly resorbed, leading to alterations in pore number, size and shape. b Histological images demonstrate narrowing of the trabeculae (T) and the generation of an increased level of lipids in osteoporotic bone compared with healthy tissue. Sudan Black B stained phospholipids (gray) and intracellular lipids (black). c Perilipins are found exclusively on adipocytes, and using immunohistochemistry (IHC), a greater amount of positive staining is observed within the marrow of osteoporotic bone. Positive lipid staining is indicated by red arrows

Fig. 7

a A schematic demonstrating alterations in the stresses derived by fluid flow in healthy and osteoporotic bone. As fluid flows within the porous bone network, it imparts pressure, shear stress and tensile strain on cells within the local environment. These fluid-induced stresses are influenced by the degree of pore curvature, surface topography, stiffness and level of adiposity. The increased marrow adipocyte content that occurs in microgravity may lower the fluid-induced microstrain levels to the cells within the bone marrow, and this effect may be further amplified in microgravity as fluid flow is estimated to be reduced by up to 99.97%. It is conceivable that a reduced interstitial fluid flow combined with increased adiposity contributes to the accelerated bone loss observed in microgravity. b Using CFD modeling, disparate levels of fluid velocity were measured within the structure of healthy versus osteoporotic rat bone. Notably, the contribution of adiposity was not modeled. While these models identify changes in fluid flow within trabecular bone under these two conditions, more complex analyses are essential to determine the role of increasing adiposity and its role, if any, in shielding cells against fluid shear stress and accelerating bone loss

Fig. 8

Based on the current literature, it is reasonable to speculate that extremely low fluid shear stresses induced via microgravity, alterations in osteoporotic bone architecture, and increased adiposity will together guide cells toward a phenotype that results in osteodestruction. Hypothesized mechanisms of bone loss due to extremely reduced fluid shear stress: osteocytes release more RANKL than OPG, thereby activating osteoclastic activity and a proinflammatory macrophage phenotype. The reduced OPG and increased proinflammatory cytokine release (e.g., IL-1β, IL-6, and TNFα) and ROS suppress the osteogenic differentiation of MSCs as well as the proliferation and deposition of new bone by osteoblasts. Increased myostatin and decreased irisin expression further activate osteoclastic activity and inhibit osteoblastic activity. MSCs preferentially differentiate into adipocytes (via increased leptin and decreased adiponectin, for example) while secreting proinflammatory cytokines that contribute to maintaining the M1 macrophage phenotype. It is conceivable that the concerted response of osteocytes, osteoclasts, macrophages, adipocytes, myocytes, and MSCs, among other key cells not shown (e.g., fibroblasts and endothelial cells), will serve to keep cells within a cycle of osteodestruction. The role of mechanoresponsive HSCs remains unknown. Experimentally unconfirmed associations are labeled in green