Shengmeng Di

Large Gradient High Magnetic Fields Affect Osteoblast Ultrastructure and Function by Disrupting Collagen I or Fibronectin/αβ1 Integrin

The superconducting magnet generates a field and field gradient product that can levitate diamagnetic materials. In this study a specially designed superconducting magnet with a large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels (μ-g, 1-g, and 2-g), was used to simulate a space-like gravity environment. The effects of LG-HMF on the ultrastructure and function of osteoblast-like cells (MG-63 and MC3T3-E1) and the underlying mechanism were investigated by transmission electromicroscopy (TEM), MTT, and cell western (ICW) assays. Under LG-HMF significant morphologic changes in osteoblast-like cells occurred, including expansion of endoplasmic reticulum and mitochondria, an increased number of lysosomes, distorted microvilli, and aggregates of actin filaments. Compared to controls, cell viability and alkaline phosphatase (ALP) secretion were significantly increased, and collagen I (col I), fibronectin (FN), vinculin, integrin α3, αv, and β1 expression were changed under LG-HMF conditions. In conclusion, LG-HMF affects osteoblast ultrastructure, cell viability, and ALP secretion, and the changes caused by LG-HMF may be related to disrupting col I or FN/αβ1 integrin.

Large gradient high magnetic field affects FLG29.1 cells differentiation to form osteoclast-like cells.

Abstract Purpose: We aimed to investigate the effects of different apparent gravities (μ g, 1 g and 2 g) produced by large gradient high magnetic field (LGHMF) on human preosteoclast FLG29.1 cells. Materials and methods: FLG29.1 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium. Cells were exposed to LGHMF for 72 h. On culture day 1, 2, 3, cell proliferation was detected by 3-(4,5)-dimethylthiahi-azo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method. On day 3, cell apoptosis and necrosis were assayed by Hoechst and propidium iodide (PI) staining. After cells were exposed to LGHMF for 72 h with the induction of 12-o-tetradecanoylphorbol 13-acetate (TPA), Tartrate-Resistant Acid Phosphatase (TRAP) positive cells and nitric oxide (NO) release were detected by TRAP staining and Griess method, respectively. Intracellular TRAP activity was measured using nitrophenylphosphate (pNPP) as the substrate. Results: MTT detection revealed that compared to control, FLG 29.1 cell proliferation in the μ g and 2 g groups were promoted. However, there is no obvious difference between the 1 g and control groups. Hoechst-PI staining showed that LGHMF promoted cell apoptosis and necrosis, especially in the 2 g group. Exposure to LGHMF inhibited the NO concentration of supernatant. Both the TRAP activity and the number of TRAP positive cells were higher in cells of μ g group than those in 2 g group. In the 1 g group, they were decreased significantly compared to control. Conclusions: These findings indicate that LGHMF could directly affect human preosteoclast FLG29.1 cells survival and differentiation. High magnetic flux inhibited osteoclasts formation and differentiation while reduced apparent gravity enhanced osteoclastogenesis.

Human mesenchymal stem cells are sensitive to abnormal gravity and exhibit classic apoptotic features

The aim of the present study was to investigate the effects of abnormal gravity on human mesenchymal stem cells (hMSCs). Strong magnetic field and magnetic field gradient generate a magnetic force that can add to or subtract from the gravitational force. In this study, this is defined as a high-magneto-gravitational environment (HMGE). The HMGE provides three apparent gravity levels, i.e. hypogravity (μg), hypergravity (2g) and normal gravity with strong magnetic field (1g) conditions. After hMSCs were subject to HMGE for 12 h, the proliferation, morphology, structure and apoptosis were investigated. Results showed that the proliferation of hMSCs was inhibited under μg condition. The abnormal gravity induced morphologic characteristics of apoptosis cells, such as cell shrinkage, membrane blebbing, nuclear chromatin condensation and margination, decreased cell viability, and increased caspase-3/7 activity. The rate of apoptosis under μg condition is up to 56.95%. The F-actin stress fibers and microtubules were disrupted under abnormal gravity condition. Under μg-condition, the expression of p53 at mRNA and protein levels was up-regulated more than 9- and 6 folds, respectively. The Pifithrin-α, an specific inhibitor of p53, inhibited the apoptosis and prevented the disruption of cytoskeleton induced by abnormal gravity. These results implied that hMSCs were sensitive to abnormal gravity and exhibited classic apoptotic features, which might be associated with p53 signaling.

BONE CELLS UNDER MICROGRAVITY

Weightlessness environment (also microgravity) during the exploration of space is the major condition which must be faced by astronauts. One of the most serious adverse effects on astronauts is the weightlessness-induced bone loss due to the unbalanced bone remodeling. Bone remodeling of human beings has evolved during billions of years to make bone tissue adapt to the gravitational field of Earth (1g) and maintain skeleton structure to meet mechanical loading on Earth. However, under weightlessness environment the skeleton system no longer functions against the pull of gravity, so there is no necessity to keep bone strong enough to support the bodys weight. Therefore, the balance of bone remodeling is disrupted and bone loss occurs, which is extremely deleterious to an astronauts health during long-term spaceflight. Bone remodeling is mainly orchestrated by bone mesenchymal stem cells, osteoblasts, osteocytes, and osteoclasts. Here, we review how these bone cells respond to microgravity environment.

IMPACT OF OSTEOCLAST PRECURSORS SUBJECTED TO RANDOM POSITIONING MACHINE ON OSTEOBLASTS

Osteoblast-osteoclast interaction plays an important role in the bone remodeling. During long duration space flight, astronauts undergo serious bone loss mainly due to the disruption of equivalence between bone formation and bone resorption. Osteoclast precursors often operate under the control of osteoblasts. However, here we show that the osteoclast precursors could in turn influence osteoblasts. RAW264.7 cells, the murine osteoclast precursors, were treated in the simulated weightlessness produced by a Random Positioning Machine (RPM). After 72 h, conditioned mediums (CM) by the RAW264.7 cells from RPM (RCM) or static control (CCM) were collected and were used to culture osteoblastic-like MC3T3-E1 cells. The results showed that the RCM culture inhibited cell viability and slightly altered cell cycle, but the morphology of the MC3T3-E1 cells was not changed by RCM compared to that of CCM. Furthermore, the intracellular ALP level, NO release and expression of osteoblastic marker genes were all down-regulated by RCM culture. These results suggest that osteoclast precursors subjected to RPM exert negative regulation on osteoblasts.

Selection of suitable reference genes from bone cells in large gradient high magnetic field based on GeNorm algorithm

Studies of animals and humans subjected to spaceflight demonstrate that weightlessness negatively affects the mass and mechanical properties of bone tissue. Bone cells could sense and respond to the gravity unloading, and genes sensitive to gravity change were considered to play a critical role in the mechanotransduction of bone cells. To evaluate the fold-change of gene expression, appropriate reference genes should be identified because there is no housekeeping gene having stable expression in all experimental conditions. Consequently, expression stability of ten candidate housekeeping genes were examined in osteoblast-like MC3T3-E1, osteocyte-like MLO-Y4, and preosteoclast-like FLG29.1 cells under different apparent gravities (μg, 1 g, and 2 g) in the high-intensity gradient magnetic field produced by a superconducting magnet. The results showed that the relative expression of these ten candidate housekeeping genes was different in different bone cells; Moreover, the most suitable reference genes of the same cells in altered gravity conditions were also different from that in strong magnetic field. It demonstrated the importance of selecting suitable reference genes in experimental set-ups. Furthermore, it provides an alternative choice to the traditionally accepted housekeeping genes used so far about studies of gravitational biology and magneto biology.