A Hypomagnetic Field Aggravates Bone Loss Induced by Hindlimb Unloading in Rat Femurs
et al. (2014) A Hypomagnetic Field Aggravates Bone Loss Induced by Hindlimb Unloading in Rat Femurs. PLoS
ONE 9(8): e105604. doi:10.1371/journal.pone.0105604
A Hypomagnetic Field Aggravates Bone Loss Induced by Hindlimb Unloading in Rat Femurs
Bin Jia 0
Li Xie 0
Qi Zheng 0
Peng-fei Yang 0
Wei-ju Zhang 0
Chong Ding 0
Ai-rong Qian 0
Peng Shang 0
Luc Malaval, Universite Jean Monnet, France
0 Key Laboratory for Space Bioscience and Biotechnology, Institute of Special Environmental Biophysics, School of Life Sciences, Northwestern Polytechnical University , Xi'an , China
A hypomagnetic field is an extremely weak magnetic field-it is considerably weaker than the geomagnetic field. In deepspace exploration missions, such as those involving extended stays on the moon and interplanetary travel, astronauts will experience abnormal space environments involving hypomagnetic fields and microgravity. It is known that microgravity in space causes bone loss, which results in decreased bone mineral density. However, it is unclear whether hypomagnetic fields affect the skeletal system. In the present study, we aimed to investigate the complex effects of a hypomagnetic field and microgravity on bone loss. To study the effects of hypomagnetic fields on the femoral characteristics of rats in simulated weightlessness, we established a rat model of hindlimb unloading that was exposed to a hypomagnetic field. We used a geomagnetic field-shielding chamber to generate a hypomagnetic field of ,300 nT. The results show that hypomagnetic fields can exacerbate bone mineral density loss and alter femoral biomechanical characteristics in hindlimbunloaded rats. The underlying mechanism might involve changes in biological rhythms and the concentrations of trace elements due to the hypomagnetic field, which would result in the generation of oxidative stress responses in the rat. Excessive levels of reactive oxygen species would stimulate osteoblasts to secrete receptor activator of nuclear factor-kB ligand and promote the maturation and activation of osteoclasts and thus eventually cause bone resorption.
Funding: This work was supported by the National Basic Research Program of China (2011CB710903). The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
The geomagnetic field (GMF, ,50 mT) is a natural component
of the environment, and plays an important role in the growth and
evolution of living organisms. In addition, the GMF is essential
for life and protects the Earth against high-energy particles from
cosmic and solar winds. Moreover, in deep-space exploration
missions, biological objects transported on long-term
interplanetary missions or on the surface of the moon or Mars would
experience extremely weak magnetic fieldsi.e., hypomagnetic
fields (HMFs)because the galactic magnetic field is 0.12 nT,
whereas the lunar magnetic field is approximately 1000 times
weaker than the Earths GMF. However, the effects of an
HMF on the functions of biological organisms are still
insufficiently understood despite active study.
A few studies have clearly confirmed that HMFs greatly affect
the functional state and even the morphology of organisms[1,68].
An experiment in which rabbits passed through embryogenesis
and grew to an age of 1 month in an HMF environment indicated
the development of degenerative disturbances in the liver,
myocardium, and gastrointestinal tract; structural and energy
metabolism perturbations; marked inhibition of enzymatic
systems; and a significantly higher mortality rate compared with the
controls. In a shielded 5-nT environment, the incidence of
somatic defects increased in the developing larvae of Japanese
newts (Cynops pyrrhogaster); in particular, bi-headedness, intestinal
protrusion, spinal curvature, malformed eyes, and retarded or
blocked development were observed, especially after 20 days of
shielding. A brief 2-hour exposure to an HMF (,200 nT) is
sufficient to interfere with the development of Xenopus embryos in
the cleavage stages. Long-term GMF deprivation results in
animals exhibiting various behavioral and mood disorders such as
inactivation, depression, mania, and anxiety, suggesting that their
central nervous systems have been affected. Adult male rats
exposed to an HMF for 3 months exhibited significantly decreased
work capacity, endurance, and behavioral activity as well as
significant increases in heart rate and conditioned reflex
development time. After they were housed in an ambient 20-nT field
for 7 months, the concentrations of certain elements in the hair of
laboratory rats, especially iron, manganese, copper, and
chromium, were significantly altered. In addition, BALB/c mice
housed in a GMF-shielding room (,300 nT) exhibited altered
blood leukocyte and platelet counts at different times during the
28-day breeding period.
Bone loss in space is one of the most important problems
endangering the health of astronauts. Previous studies suggest that
the health of the skeletal system is dependent on Earths gravity.
Under microgravity conditions in space, the loss of bone calcium
in the skeletal system, especially from the weight-bearing bones, is
unavoidable; furthermore, continuous bone loss in space may
cause fractures and renal calculus in astronauts, which may affect
both their health and the mission[16,17].
HMFs are extremely weak static magnetic fields, and are far
weaker than the GMF. However, their effects on the skeletal
system are unclear and have not been reported. Nevertheless,
several studies demonstrate that a static magnetic field that is
stronger than the GMF can affect bone remodeling and the
activities of bone-related cells. An average flux of 290 mT in a
static magnetic field accelerates the osteogenic differentiation and
mineralization of dental pulp cells. Exposure of an
intramedullary implant to a static magnetic field (2226 mT) radiologically
improves bone healing in the first 2 weeks, and the difference in
the configuration of the magnetic poles also affects bone
quality. In rabbit tibiae, the use of sand-blasted large-grid
acid-etched titanium implants with a neodymium magnet
(15.34 mT) can trigger quicker early peri-implant bone formation
as compared with implants without a magnet. A small disc
magnet with a maximum magnetic flux density of 180 mT that
was implanted in the right side of the spinous process of the third
lumbar vertebra reportedly increased the bone mineral density
(BMD) of osteoporotic lumbar vertebrae in ovariectomized
As mentioned above, many previous studies indicate that not
only gravity but also static magnetic fields can affect the skeletal
system, and may thus play pivotal roles in maintaining skeletal
system health. In space environments, microgravity is one of the
complex factors, along with HMF and radiation that should be
carefully considered with regard to its effects on astronauts. For
astronauts who have acclimatized to the GMF, an HMF is a novel
environmental factor. It remains unclear whether the absence of
the GMF or HMF plus microgravity condition influences the
skeletal system health. In an effort to provide scientific support to
the health care of astronauts on deep-space missions, we
investigated the effects of an HMF and the complex effects of an
HMF plus hindlimb unloading (HLU) on bone loss in rats as well
as the role and mechanism of HMF on bone loss in microgravity.
Materials and Methods
To obtain a stable experimental HMF environment, a
1.861.661.5-m GMF-shielding chamber (NORINDAR
International, Shijiazhuang, Hebei, China) was constructed (Figure 1A).
The shielding chamber consisted of an aluminum alloy frame,
several layers of highly permeable permalloy, silicon steel sheets,
and pure iron sheets. The chamber can generate a hypomagnetic
environment with an average magnetic field intensity of ,300 nT.
The mean magnetic field intensity in Xian, Shaanxi, China, is
approximately 50 mT. A mandatory ventilation system was
installed in the shielding chamber, with a ventilation frequency
of 15 times per hour. Incandescent lights were used for
illumination inside the shielding chamber (20 lm). The
illumination switch was synchronized with a 12-h light/12-h dark cycle.
As a control, we constructed a 1.561.461.5-m wooden
experimental box that has no shielding effect against the GMF
but has the same illumination and ventilation conditions as the
GMF shielding chamber.
Experimental animals and tail-suspension animal model
A total of 60 male adult Sprague-Dawley rats weighing
260610 g were provided by The Lab Animal Center of the
Fourth Military Medical University, Xian, Shaanxi, China. The
experiment was performed after the rats were allowed to acclimate
for 3 days. During the experiment, each cage contained 1 HLU rat
or 3 other rats, and feed and water were controlled. The entire
experiment was performed in accordance with the
recommendations of the local animal ethics association as well as the principles
of laboratory animal welfare (Regulations for the Administration
of Affairs Concerning Laboratory Animals in Shaanxi Province,
China). The protocol was approved by the Lab Animal Ethics and
Welfare Committee, Northwestern Polytechnical University (The
Form of Animal Experimental Ethical Inspection, No. 1017).
Blood sample collection and execution were performed under
sodium pentobarbital anesthesia, and all efforts were made to
NASAs rat model involving tail suspension, HLU, and 230u
downward head tilting was used  with some minor
improvements to the fixation of the rat tails (Figure 1C).
Briefly, the strip-type medical adhesive tape pasted on both sides of
the rats tail was fixed by 2 flexible Band-Aid brand adhesive
bandages (Johnson & Son, Racine, WI, USA), forming a ring
around the tail. The part of the Band-Aid that touched the skin of
the rat tails was the nonsticky layer (Figure 1B). This improved
fixation method can effectively avoid lesions on the rats tails
caused by improper tape fixation. Because the Band-Aid is made
of elastic materials, the frequency of replacing the adhesive tape
can be reduced during the 28-day experiment. Tail-suspended rats
had access to food and water ad libitum. During the experiment, a
few rats had learned to climb to the top of the cage using the cord
used for tail suspension or by leaning their hind limbs against the
cage wall, which led to model failure; such rats, accounting for
17.2% of animals that underwent tail suspension, were excluded
from the analysis.
Experimental animals were randomly divided into 5 groups: (1)
the baseline group (BL), in which rats were executed to get basal
data on day 0 of the experiment. (2) the control group, in which
rats were kept inside the wooden experimental box with the
normal GMF environment; (3) the HLU group, in which rats
received tail suspension, HLU, and 230u downward head tilting,
and were kept inside the wooden experimental box; (4) the HMF
group, in which rats were kept inside the GMF-shielding chamber;
and (5) the HMF+HLU group, in which HLU rats were kept
inside the GMF-shielding chamber.
The experiment lasted for 4 weeks. On day 0, 28 of the
experiment, the rats of BL group and other groups were sent for a
BMD scan and serum sample collection from the inferior vena
cava under anesthesia respectively. The rats were subsequently
killed by cervical vertebra luxation under anesthesia, and their
bilateral femurs were separated from the soft tissues. For
biomechanical analysis, the left femur was immediately bisected
and fixed with 4% paraformaldehyde. Decalcification treatment
was also performed before the femur was sent for
immunohistochemical analysis. The right femur was used to obtain micro-CT
Dual-energy x-ray absorptiometry (DEXA) (Lunar Prodigy; GE
Medical Systems, Madison, WI, USA) in the small-animal mode
was used to measure the BMD of femurs in vivo. On day 0, 28 of
the experiment, BL group and all experimental animals were
anesthetized with an intraperitoneal injection of 3% pentobarbital
sodium (1 mL/kg) and placed in the prone position with the lower
limbs naturally extended on the absorptiometry machine for BMD
measurement. The scan results were analyzed with enCORE
software version 10.50 (GE Medical Systems) for assessing the
BMD and bone mineral content (BMC) of bilateral femurs.
Micro-CT femur 3D reconstruction and analysis
An eXplore Locus SP micro-CT (GE Medical Systems) was
used for femur 3D reconstruction and analysis. The scan
parameters were as follows: scan resolution, 14 mm; rotation
angle, 360u; rotation angle increment, 0.4u; voltage, 80 kV;
current, 80 mA; exposure time, 3000 ms. After the scan was
completed, MicroViewTM (version 2.1.2) was used for 3D
reconstruction. Analysis was performed with MicroViewTM
Advance Bone Analysis(ABA)-specific bone analysis software. A
2.019-mm-thick trabecular bone chip under the epiphyseal plate
in the lower end of the femur was selected as the region of interest
(ROI) for 3D reconstruction and measurements. The following
parameters were measured: BMC, BMD, tissue mineral content
(TMC), tissue mineral density (TMD), structure model index
(SMI), bone volume fraction (BVF, i.e., the ratio of bone
volume to tissue volume [BV/TV]), bone surface to bone volume
(BS/BV), trabecular thickness (Tb.Th), trabecular separation
(Tb.Sp), degree of anisotropy, Euler number, and connectivity
density (1/mm3). A 1.512-mm-thick cortical bone chip in the
middle of the femur was selected as the ROI for 3D reconstruction
and measurements of BMC, BMD, mean thickness, inner
perimeter, outer perimeter, marrow area, and cortical area.
On day 28 of the experiment, the rats were anesthetized and
killed by cervical dislocation, and the soft tissues of both hind limbs
and the bilateral femurs were removed. The mechanical properties
of the femurs were tested using the conventional 3-point bending
test. A universal testing machine (Instron, Canton, MA, USA) was
used to support the platform. The span between 2 load-supporting
points was set at 20 mm. Femurs from each experimental group
were placed on the supporting platform in the same orientation,
and load was added evenly at the midpoint of the femur at 2 mm/
min until the femur fractured. A load-deformation curve was
simultaneously obtained during loading, through which the
mechanical properties of the femur were determined, including
ultimate force, toughness factor, and elastic modulus.
The abdominal wall was cut open under anesthesia to expose
the abdominal aortic vein. A syringe was then used to directly
collect blood from the abdominal aortic vein. After the blood
sample was collected, the animal was killed by cervical dislocation.
The collected blood sample was stored at 4uC for 1 h and then
centrifuged at 1500 rpm for 10 min. Serum was collected by
suction and stored in a fridge at 270uC. The serum
concentrations of bone alkaline phosphatase (bALP), deoxypyridinoline
(DPD), and glucocorticoids (GCs) were determined using an
enzyme-linked immunoassay detection kit (Beijing Chenglin
Biotechnology Co. Ltd.(Beijing, China); primary antibodies were
obtained from Abcam(Cambridge, UK)).
Trace elements in serum were detected by an atomic absorption
spectrophotometer (ZEEnit700P; Analytik Jena AG, Jena,
Germany). Serum manganese was detected using the graphite furnace
atomic absorption spectroscopy method, whereas other elements
including iron, copper, zinc, calcium, and magnesium, were
detected using the flame method.
After biomechanical tests, fresh femurs were split at the center
line and fixed in 4% paraformaldehyde for 24 hours. The
paraformaldehyde solution was then discarded, and decalcification
solution (20% EDTA) was added and subsequently changed every
4 days. Decalcification was performed continuously for 28 days.
The decalcified femurs were dehydrated and embedded in paraffin
to prepare paraffin sections. After dewaxing, the
streptavidinbiotin complex method was used to detect the expression of
receptor activator of nuclear factor-kB ligand (RANKL) in bone
tissue, by using its antibody (anti-RANKL; Abcam). A
semiquantitative analysis was conducted for determining the percentage of
RANKL-positive cells and the intensity of positivity, which was
classified into 4 degrees (0, negative; 1, slightly positive; 2,
distinctly positive; and 3, strongly positive). Moreover, the intensity
of RANKL positivity was quantified as the H-score, where
H-score = g(i + 1) 6 Pi, in which i is the degree of positivity as
classified above (from 03) and Pi is the percentage of the positive
cells which fluctuates from 0% to 100%. Five vision fields (6 400)
were randomly selected on each section, and RANKL-positive
cells were counted in accordance with the above method.
All statistical analyses were performed by using the GraphPad
Prism for Windows statistical software (version 5, GraphPad
Software, Inc., La Jolla, CA, USA). The differences between the
BL group and the other experimental groups were revealed using
an ordinary one-way ANOVA. Two-way analysis of variance was
applied to test the variation trends and differences between the
experimental groups. The results are expressed as mean 6
standard deviation. For all statistical tests, a P value of ,0.05 was
considered to be statistically significant.
Effects of HMF and HMF+HLU on the body weight of rats
The differences in body weights between the experimental
groups except the BL group are shown in Figure 2. During the
experimental period, the body weight in all the rats increased. The
body weights of the rats in the HLU and HMF+HLU groups were
significantly decreased compared with those of the rats in the
control group from days 14 to 28 (P,0.05 in the HLU group at 14
days, P,0.01 in the HLU and HMF+HLU group at 21 and 28
days.). At day 28, compared with the HMF group, the body
weights of the rats in the HLU and HMF+HLU groups were
significantly decreased (P,0.01).
Effects of HMF and HMF+HLU on the mechanical
properties of the rat femur
The mechanical properties of the femur in the 3-point bending
test are shown in Figure 3. The ultimate load (Fmax) in the
control, HLU, HMF and HMF+HLU groups were significantly
increased, compared with BL group. The HLU and HMF+HLU
groups had significantly lower Fmax than the control and HMF
groups (P,0.01); however, according to two-way ANOVA, the
interaction between the two factors was no statistically significant.
Compared with BL group, the toughness factor of all experiment
groups were decreased obviously. For the toughness factor, there
was a significant effect for HLU (P,0.05) and HMF (P,0.05).
Likewise, the interaction HLU*HMF was significant (P,0.05).
The post-hoc test revealed that the toughness factor, in the HMF+
HLU group, was significantly greater than that of the HMF group,
but significantly lower than that of the HLU group (P,0.05).
Moreover, the elastic modulus of the HMF+HLU group was
significantly greater than that of the HMF group (P,0.05), but
significantly lower than that of the HLU group (P,0.05). The
elastic modulus of the BL group was similar to the control and
HMF group was lower than that of the HLU and HMF+HLU
Effects of HMF and HMF+HLU on femoral BMD and BMC
DEXA analysis through two-way ANOVA showed that there
was a significant effect for HLU (P,0.01) and HMF (P,0.01) in
the femoral BMD and the interaction HLU*HMF was significant
(P,0.05) in the femoral BMD. For the femoral BMC, the
interaction HLU*HMF was no significant. Further analysis, the
femoral BMD and BMC of the HLU and HMF+HLU groups
were significantly less than those of the control and HMF groups
(all P,0.01). The femoral BMD was lower in the HMF+HLU
group than in the HLU group (P,0.01), whereas the BMC of the
femurs was similar between groups. The differences in femoral
BMD and BMC were not significant between the HMF and
control groups (Figure 4). BMD and BMC of all experimental
groups were higher than the BL groups except the BMD of the
Effects of HMF and HMF+HLU on femoral trabecular and
The three-dimensional (3D) reconstruction of the
2.019-mmthick trabecular bone ROI at the epiphyseal end of the femur
(Figure 5A) by MicroViewTM version 2.1.2 revealed significant
differences in the ROI of the femurs of HLU rats in a normal
GMF environment compared with that in the control group, with
both the thickness and number of bone trabeculae significantly
reduced. The femoral ROI of the HMF rats showed no significant
differences compared with that of the control group, although the
femoral ROI of HMF+HLU rats showed significant differences
compared with that of the control, HMF, and HLU groups;
moreover, the HMF+HLU group showed more significant bone
trabecular changes than the HLU group. Compared with the BL
group, only the BVF in the control and HMF groups, the BMC in
the HMF group were no significant differences (Table 1).
Two-way ANOVA revealed a significant effect for HLU
(P,0.01) and HMF (P,0.01) in the femoral BMD, BMC,
TMC, BVF, EUN and COD. Likewise, the interaction
HLU*HMF was significant (P,0.01). The 3D BMD and BMC
values were significantly lower in the HMF+HLU group than in
the control, HLU, and HMF groups (P,0.01); however, no
significant differences in these values were observed between the
HMF and control groups. The 3D TMC was significantly lower in
the HMF+HLU group than in the control, HLU, and HMF
groups (P,0.01); however, no significant difference was observed
in these values between the HMF and control groups. There were
no significant differences between groups with respect to TMD
The SMI of the HMF+HLU group differed significantly from
that of the control and HMF groups (P,0.01); however, there was
Figure 2. Changes in bodyweight from the original weight of rats in different groups. Rats in all 4 groups were weighed every 7d. C: Rats
were raised in a wooden box with a normal GMF for 28 days; HLU: Rats were suspended, unloaded with 230u downward head tilting, and raised in a
wooden box; HMF: Rats were raised normally in a GMF-shielded room; HMF+HLU: HLU rats were raised in a GMF-shielded room. **P,0.01 vs C, *P,
0.05 vs. C, ##P,0.01 vs. HMF.
also a significant difference in this value between the HMF and
control groups (P,0.01). The BVF of the HMF+HLU group was
significantly different from that of the control and HMF groups
(P,0.01). The absolute value of the Euler number and
connectivity density were significantly lower in the HMF+HLU
group than in the control, HLU, and HMF groups (P,0.01);
however, no significant differences were observed in these values
between the HMF and control groups (Table 1).
With regard to the degree of anisotropy, the a1/a3 plane value
of the HMF+HLU and HLU groups was significantly lower than
that of the control group and BL respectively (P,0.05, P,0.01),
and the a1/a2 plane value of the HLU group was significantly
lower than that of the control group and BL (P,0.01). Moreover,
the degrees of anisotropy of the a1/a2 and a2/a3 planes of the
HMF+HLU group were not significantly different from those of
the control group and BL, and were significantly lower than those
of the HLU group (P,0.01) and BL (P,0.05) (Figure 5B).
The axial values of BV/TV at the x, y, and z axes as well as the
mean values exhibited consistent changes. Two-way ANOVA
revealed a significant effect for HLU (P,0.01) and HMF (P,
0.01) in BV/TV, Tb.N and Tb.Sp. The interaction HLU*HMF
was significant (P,0.01). The BV/TV of the HMF+HLU group
differed significantly from those of the HLU and control groups
(P,0.01). The axial values of BS/BV at the x, y, and z axes as well
as the mean values were significantly higher in the HMF+HLU
and HLU groups than in the control group (P,0.01); however, no
significant differences in these values were observed between the
HMF+HLU and HLU groups. The Tb.Th of the HMF+HLU
and HLU groups was significantly lower than that of the control
group (P,0.01); however, no significant differences in this value
were observed between the HMF+HLU and HLU groups. The
trabecular number of the HMF+HLU and HLU groups were
significantly lower than that of the control group (P,0.01);
however, the trabecular number of the HMF+HLU group was
significantly higher than that of the HLU group (P,0.01).
Furthermore, the Tb.Sp of the HMF+HLU and HLU groups
was significantly higher than that of the control group (P,0.01);
however, the Tb.Sp was significantly higher in the HMF+HLU
group than in the HLU group (P,0.01). Compared with BL,
significant differences in BS/BV, Tb.Th, Tb.N and Tb.Sp of other
groups were observed (P,0.01 or P,0.05); however, no
significant differences in BV/TV of control and HMF groups
were observed (Table 2).
The 3D reconstruction of the 1.5012-mm-thick cortical bone
ROI at the middle of the femur yielded the following findings
(Table 3): compared with the control group, the BMC, mean
thickness, and cortical area in the HLU and HMF+HLU groups
were significantly reduced; the HMF group showed no significant
differences compared with the control group; and the HMF+HLU
group showed significant differences compared with the HLU
groups in terms of the BMC and cortical area. Compared with the
BL group, the BMC, mean thickness, inner Perimeter and cortical
area in the HLU group,the BMD, BMC and mean thickness in
HMF+HLU group, the mean thickness in HMF group were no
significant changes. Certainly,the work preceding the
abovementioned analysis revealed that, only in BMC (P,0.01) and
Cortical Area (P,0.05), the interaction HLU*HMF was
Effects of HMF and HMF+HLU on RANKL expression in
The expression of RANKL could be clearly observed in the
femoral trabecular bone in the HLU and HMF+HLU groups,
whereas no obvious expression was observed in the control or
HMF group (Figure 6A). A semiquantitative analysis yielded a
positive result in the HLU and HMF+HLU groups, with H-scores
that were much higher than 100. The H-scores in the control and
HMF groups were less than 100a negative finding (Figure 6B).
Effects of HMF and HMF+HLU on serum bALP, DPD and
The serum bALP, DPD, and GC concentrations in each group
exhibited consistent changes (Figure 7). Two-way ANOVA
revealed a significant effect for HLU (P,0.01) and HMF (P,
0.01) in bALP, DPD, and GC concentrations. The interaction
HLU*HMF was significant (P,0.01). These concentrations were
significantly greater in the HLU, HMF, and HMF+HLU groups
than in the control group (P,0.01). Furthermore, the HMF and
HMF+HLU groups also exhibited significant differences in these
values compared with the HLU group; in particular, bALP
concentrations were significantly higher in the HMF and HMF+
HLU groups than in the HLU group (P,0.05). Moreover, DPD
and GC concentrations differed significantly between the HMF
and HLU groups (P,0.05); furthermore, the HMF+HLU groups
exhibited an increasing trend in these values compared with the
HLU group, although the difference was not significant.
Compared with BL group, the bALP, PDP and GC concentrations in
control group were significantly decreased, and other groups were
no significant changes.
Effects of HMF and HMF+HLU on serum trace element
The changes in the concentrations of trace elements in each
group are shown in Figure 8. Two-way ANOVA showed that the
interaction between the two factors (HLU*HMF) was statistically
significant only in the serum iron concentrations.
The serum iron concentrations in the HMF and HMF+HLU
groups were significantly greater than those in the BL, control and
HLU groups (P,0.01); furthermore, the serum iron concentration
was significantly higher in the HMF+HLU group than in the
HMF group (P,0.01). However, the serum calcium
concentrations were significantly lower in the HLU, HMF, and HMF+HLU
Figure 4. Changes in femoral BMD and BMC measured by DEXA. BL: The baseline group. Rats were executed to get basal data on day 0 of
the experiment. C: Rats were raised in a wooden box with a normal GMF for 28 days; HLU: Rats were suspended, unloaded with 230u downward
head tilting, and raised in a wooden box; HMF: Rats were raised normally in a GMF-shielded room; HMF+HLU: HLU rats were raised in a GMF-shielded
room. BMD: bone mineral density; BMC: bone mineral content; **P,0.01.
groups than in the BL and control group (P,0.05, P,0.01, and
The serum copper concentrations tended to be greater in the
HLU, HMF, and HMF+HLU groups than in the control group,
although a significant difference was observed only between the
HMF+HLU and control groups (P,0.05). The serum magnesium
concentrations of the HMF and HMF+HLU groups were
significantly lower than those of the BL, control and HLU groups
(P,0.01). A high serum manganese concentration was noted in
the control group but not in the other groups, which was lower
level in the BL group, and was not detected in the HLU, HMF
and HMF+HLU groups. All experimental groups had significantly
lower serum zinc concentrations compared with the BL and
control group (P,0.05, P,0.01, and P,0.01, respectively); the
serum zinc concentrations were lower in the HMF and HMF+
HLU groups and were significantly different between the HMF+
HLU and HLU groups (P,0.05).
This land-based animal study used tail-suspended HLU rats to
simulate weightlessness. Experimental animals were kept in an
illumination- and ventilation-controlled GMF-shielding chamber
to simulate the hypomagnetic and microgravity environment in
deep space. Using tail-suspended HLU rats or mice as an animal
model of simulated weightlessness to study bone loss in space is
widely approved. In the present study, rats were exposed to an
HMF and simulated microgravity. The biomechanical properties,
2D BMD and BMC, were assessed, and a quantitative analysis of
femoral trabecular and cortical bone using micro-CT was
performed, to determine the effects of an HMF with and without
simulated microgravity on the skeletal system. In addition, serum
biochemical analysis and femur immunohistochemistry were
performed to determine the possible mechanisms through which
an HMF and simulated weightlessness influence the skeletal
Body weight has a close relationship with bone. Because the
Sprague-Dawley rats we used were still in their growing period
during the 28-day experiment, the weights of the rats in each
group significantly increased. Nevertheless, the weight gain in the
2 tail-suspension groups was significantly less than that in the other
2 groups, possibly because unloading induces resistance to
insulinlike growth factor-I and transforming growth factor-b2.
There were no significant differences in weight gain between the
HLU and HMF+HLU groups, implying that the HMF
environment had no effect on body weight.
BMD and BMC rise in direct proportion to the rats growth
period. However, this increase was notably suppressed in the HLU
and HLU+HMF groups, the latter of which shows particularly
obvious effect. DEXA revealed that BMD and BMC were
significantly lower in the HLU and HMF+HLU groups than in
the control group; BMD was lower in the HMF+HLU group than
in the HLU group. This indicates that HMF further promotes the
reduction of femur BMD in HLU rats, whereas HMF alone does
not cause a BMD reduction.
The biomechanical tests revealed that the ultimate loads of the
HLU and HMF+HLU groups were significantly lower than that of
the control group, whereas no significant differences were
observed in these values between the HLU and HMF+HLU
groups or the HMF and control groups. This indicates that the
hypomagnetic environment in this study did not significantly
influence the ultimate load of the femur in the 3-point bending
test. Therefore, HLU is the primary reason for the decreased
ultimate load. With regard to the toughness factor and elastic
modulus, compared with the control and HMF groups, the femurs
of the HMF+HLU and HLU groups exhibited higher toughness
and lower elasticity, which led to reduced load tolerance capacity,
thereby increasing the chance of bone fracture. Compared with
the HLU group, the HMF+HLU groups exhibited decreased
femur toughness and elasticity. Although BMD was significantly
reduced in both groups, for growing rats, the mechanical
properties differed, indicating that the HMF environment might
have influenced the reconstruction characteristics of bone minerals
BMD, bone mineral density (mg/cm3), refers to the total BMD of the ROI; BMC, bone mineral content (mg), refers to the total BMC of the ROI, including the ossature and
soft tissue; TMC, tissue mineral content (mg), refers to the mineral content of the ossature of the ROI; TMD, tissue mineral density (mg), refers to the mineral density of
the ossature of the ROI; SMI, structure model index, is a method for determining the plate- or rod-like geometry of trabecular structures; BVF, bone volume fraction,
refers to the ratio of bone volume to tissue volume; EUN,Euler number indicates the decrease in osteoporosis; COD, connectivity density shows the number of
connections in the trabecular networks, which is decreased in osteoporosis. **P,0.01vs. C, ##P,0.01vs.HLU, ggP,0.01vs.BL, gP,0.05vs.BL.
inside the femur. This is the first study demonstrating that HMFs
might affect the bone reconstruction process in vivo. The finding
that a static magnetic field can affect bone reconstruction both in
vivo and in vitro corroborates the importance of magnetic fields in
bone reconstruction from another perspective.
To confirm the effect of HMF on bone reconstruction,
microCT was used to further analyze changes in cancellous bone
trabeculae. In the selected ROI of 3D cancellous bone, each group
exhibited consistent trends regarding BMD, BMC, and TMC;
these parameters were significantly lower in the HLU and HMF+
HLU groups than in the BL, control and HMF groups, and the
parameters were even lower in the HMF+HLU group than in the
HLU group. However, no significant changes in TMD was
observed between the experimental groups, but was higher than in
the BL group. TMD is defined as the BMD of the area considered
to be skeletal, according to the threshold binarization within an
ROI, following exclusion of the nonskeletal parts. The lack of a
significant difference in TMD indicates that bone reconstruction
BV/TV, bone volume to tissue volume (%); BS/BV, bone surface to bone volume (1/mm); Tb.Th, trabecular thickness (mm); Tb.N, trabecular number (1/mm); Tb.Sp,
trabecular separation (mm); The x, y, and z axes express the quantities of the 3D structure; Avg, average quantities of the vector sum of the x, y, and z axes.*P,0.05
vdso.i:C1,0.*1*3P7,10/j.o0u1rnvas.l.pCo,n#eP.0,1005.06504v.st.0H02LU, ##P,0.01 vs. HLU, ggP,0.01 vs. BL, gP,0.05 vs. BL.
BMD, bone mineral density (mg/cm3), refers to the total BMD of the ROI; BMC, bone mineral content (mg), refers to the total BMC of the ROI, including the ossature and
soft tissue; Mean Thickness, average thickness of cortical bone; Inner Perimeter, the inside perimeter of the cortical bone; Outer Perimeter, the outside perimeter of the
cortical bone; Marrow Area, the area of the cortical bone inside; Cortical Area, the area of the cortical bone.*P,0.05 vs. C, **P,0.01vs. C, #P,0.05vs.HLU, ##P,0.01
vs.HLU, ggP,0.01vs.HL, gP,0.05vs.HL.
did not influence the physical appearance of bone tissue. BMD,
BMC and TMC in HLU and HMF+HLU groups were lower than
in the BL group, which declare that bone formation was inhibited,
especially in HMF+HLU. HLU inhibits the process of bone
formation in which HMF increases the inhibiting level. The extent
of the decreases in the 3D BMD and BMC of cancellous bone
within the ROI was greater than that in the 3D data obtained by
DEXA, indicating that bone mineral loss in the HLU and HMF+
HLU groups mainly involved cancellous bone. Bone loss was
simultaneous with bone remodeling.
The shape of bone trabeculae can be described by the SMI.
In osteoporosis, bone trabeculae are converted from a plate shape
to a rod shape, and the SMI is increased. The HLU and HMF+
HLU groups had almost rod-shaped bone trabeculae, and both
exhibited osteoporosis. The control and HMF groups had smaller
SMI values and exhibited plate-shaped trabeculae. But, their
significant differences were still existing, which implied that HMF
affected the bone remodeling. However, the SMI is insufficient to
describe the degree of osteoporosis. The BVF (or BV/TV), Euler
number, and connectivity density (1/mm3) of bone trabeculae can
be used to further determine the degree of osteoporosis, .
For growing rats, BVF in control and HMF groups were no
obvious changes and the HMF environment alone does not cause
the changes of BVF. But in the HLU and HMF+HLU groups, all
of these parameters were significantly lower than in the BL,
control and HMF groups and in the HMF+HLU group the values
were the lowest. Hence, the microscopic 3D structure of femurs in
the present study further confirms that HMF aggravates bone loss
in HLU rats and that the HMF environment alone does not cause
bone loss in rats.
The degree of anisotropy of bone trabeculae indicated certain
changes in the directionality and symmetry of bone trabecular
structure among the experimental groups; in particular, the
changes in major/minor axis ratios of a1/a2 and a2/a3 in the oval
planes were significantly different between the HMF+HLU and
HLU groups. This indicates that the HMF environment influences
the directionality and symmetry of the bone trabecular structure
during reconstruction, which may affect the biomechanical
properties of the femur, thus resulting in the observed decreases
in femur toughness and elasticity in the HMF+HLU group.
Data regarding the x, y, and z axial directions as well as their
mean values in cancellous bone ROI were obtained using
MicroViewTM. The BV/TV value of normal growth rats was
basically unchanged and the single factor of HMF environment
could not affect it, while HLU and HMF+HLU caused a
significant reduction of BV/TV. And simultaneously, a significant
difference was observed in this value between the HMF+HLU and
HLU groups. Moreover, BS/BV and Tb.Th exhibited consistent
changes in all 3 axial directions, and no significant differences were
observed in these values between the HMF+HLU and HLU
groups. The inconsistencies with respect to BV/TV and BS/BV
indicate that although osteoporosis was observed in the HMF+
HLU and HLU groups, their bone trabeculae had different
appearances. Even though the HMF+HLU and HLU groups
showed no significant changes in Tb.Th, the former exhibited
significantly decreased trabecular number and significantly
increased Tb.Sp. These findings suggest increased bone
resorption. Hence, we concluded that osteoporosis was more severe in
the HMF+HLU group than in the HLU group.
In the case of bone loss, the loss of cortical bone always occurs
after that of cancellous bone. In this study, BMC and mean
cortical bone thickness was apparently decreased in both the HLU
and HMF+HLU groups, showing that the HMF environment
affected not only cancellous bone but also cortical bone; this
decrease was particularly greater in the HMF+HLU group.
With regard to immunohistochemistry, because tissue section
selection was inconsistent in each group, normalization was
difficult. However, apparent RANKL expression was observed on
the femoral bone trabecular surface in both the HMF+HLU and
HLU groups. High RANKL expression is the major cause of bone
loss. Osteoblasts on the bone trabecular surface form cell-cell
contacts with preosteoclasts via RANKL expression on the cell
surface to promote osteoclast formation. Mature osteoclasts
erode the bone trabecular surface and cause bone loss.
The serum concentrations of bALP, DPD and GC in the BL
group were in higher level, which may be related to vigorous
metabolism of young rats. As the growth of the age, theirs
concentrations were significantly lower. But in the three challenge
groups, theirs concentrations were maintained in higher level. The
bALP concentration is a common index used to evaluate bone
formation and turnover; bALP is secreted by osteoblasts and
reflects osteoblast activity. It hydrolyzes phosphates during
osteogenesis to provide the phosphoric acid required for
hydroxyapatite deposition, while simultaneously hydrolyzing
pyrophosphates and preventing their inhibition of bone mineral formation,
thus promoting bone formation. The ALP expression of
unloaded skeleton is considered to be significantly reduced based
on the finding of decreased proliferation ability of osteoblasts in
tail-suspended rats. The early serum ALP level of unloaded
rats also shows a decreasing trend. However, after 2 weeks of
unloading, the serum ALP level returns to normal. In this
study, the unloading of rats lasted for 28 days, and the serum ALP
level increased significantly. This phenomenon may be related to
feedback regulation caused by the early decrease in the serum ALP
level. DPD is only present in type I collagenous fiber of bone and is
released into the blood as the degradation product of type I
collagenous fibers, when osteoclasts are active. DPD is unaffected
by diet and can be used as a specific index to reflect bone
resorption. Changes in DPD reflect the degree of bone resorption
during the bone turnover process[39,40]. In our study, serum
bALP and DPD concentrations increased significantly to different
extents in each experimental group, indicating active bone tissue
metabolism in each group and significantly elevated bone
formation and resorption.
Interestingly, the serum GC content was significantly increased
in each experimental group. An excessive level of GC is the most
common cause of osteoporosis. GCs can directly act on osteoblasts
and osteocytes to induce apoptosis, thus reducing bone
formation[41,42]. Increases in GCs are usually caused by stress
responses to changes in the surrounding environment. Excessive
GC levels further promote the production of reactive oxygen
species (ROS)[43,44]. Under normal physiological conditions,
ROS produced by osteoclasts stimulate and promote bone tissue
resorption[45,46]. Oxidative stress increases the sensitivity of
osteocytes to GCs. GCs can directly act on osteoblasts, and reduce
the number of osteoblasts via both oxidative stress-dependent and
-independent pathways. When ROS production exceeds the
capacity of the natural antioxidant defense mechanisms of the
body, related oxidative stress responses cause major bone loss and
weaken bones, and may even manifest certain characteristics of
osteoporosis. This is because ROS such as H2O2 and
superoxide anion can stimulate bone marrow mesenchymal cells
and osteoblasts to express RANKL, consequently inducing
osteoclast differentiation and maturation as well as promoting
Maintaining a normal biological rhythm is important to avoid
excessive oxidative stress responses. Changes in GMF activity
must reach a certain magnitude in order to affect the amplitude of
melatonin production. If the fluctuation is sufficiently large (i.e., .
80 nT/3 h), it significantly affects melatonin concentration in
saliva. Because life on Earth has adapted to the existing GMF,
the much weaker HMFs (i.e., ,300 nT in the present study) are
novel environments for organisms. In environments in which there
are no circadian rhythm changes due to the GMF but only a
constant extremely weak magnetic field, the biological rhythms of
animals change[56,57]. In a direct response to this change, the
24-hour circadian melatonin secretion rhythm is disturbed, and
melatonin itself can weaken the effect of GCs, thus decreasing
the bodys antioxidant capacity. In addition, to adapt to HMF
environments, endogenous GC secretion by the adrenal glands is
triggered via the pituitary-adrenal axis, to increase blood GC
Figure 8. Effects of HMF and HMF+HLU on the concentrations of serum trace elements. BL: The baseline group. Rats were executed to get
basal data on day 0 of the experiment. C: Rats were raised in a wooden box with a normal GMF for 28 days; HLU: Rats were suspended, unloaded with
230u downward head tilting, and raised in a wooden box; HMF: Rats were raised normally in a GMF-shielded room; HMF+HLU: HLU rats were raised
in a GMF-shielded room. Serum concentrations (mg L21) of (A) iron, (B) calcium, (C) copper, (D) magnesium, (E) manganese, and (F) zinc. *P,0.05,
concentrations, thus resulting in more intense oxidative stress
Long-term GMF deprivation alters the concentrations of trace
elements in rats. A perturbed balance of trace elements in the
body affects metabolic processes. Trace elements are closely
associated with the development of osteoporosis; sufficient
zinc, copper, magnesium, calcium, and manganese uptake is
critical for maintaining a healthy skeleton. In the present study, the
serum iron concentrations of rats in the HMF and HMF+HLU
groups were significantly higher (approximately double) than those
of the BL, control and HLU groups, indicating that HMF
environments cause iron accumulation in the body. Serum
calcium, magnesium, and zinc concentrations in each
experimental group were decreased to different degrees. Magnesium
specifically decreased in the 2 groups of rats subjected to the
HMF environment, indicating that the pituitary adrenal axis
affects the blood magnesium concentration. Although we used
graphite furnace atomic absorption spectroscopy, which is high
sensitivity for detecting trace elements, serum manganese was not
detected in any experimental group.
Iron is a trace element that has important functions in vivo.
In the skeletal system, both excess and insufficient iron can reduce
bone mass. In vitro, iron can even inhibit the growth of
hydroxyapatite crystals. Iron ions promote RANKL-induced
osteoclast differentiation, accompanied by increased ROS levels
and oxidative stress responses. Excess ROS production confirms
that iron ions promote osteoblast differentiation and bone
resorption[68,69]. A study on the relationships between the serum
iron levels, oxidative stress, and bone resorption of astronauts
living for a long duration on the International Space Station
revealed that variations in serum iron concentration are positively
correlated with an oxidative stress marker,
8-hydroxy-29-deoxyguanosine, suggesting that increased iron storage during
spaceflight might be a risk factor that causes oxidative damage and bone
Zinc is an important enzyme reaction catalytic factor and
structural cofactor for many enzymes and some other proteins.
Although Zn2+ has no redox activity under physiological
conditions, numerous studies demonstrate that zinc deficiency
intensifies the oxidative stress responses, resulting in oxidative
damage to DNA, proteins, and lipids. Thus, zinc has an indirect
antioxidant effect. Moreover, zinc deficiency causes DNA
damage by increasing oxidative stress and blocking DNA
repair. Thus, zinc plays an important role in maintaining
DNA integrity. Zinc deficiency in growing rats reduces bone mass
accumulation, which might be an important pathogenic
mechanism of osteoporosis. Insulin treatment has considerable
efficacy against diabetic osteoporosis. However, the efficacy of
this treatment is greatly reduced in diabetic osteoporosis combined
with zinc deficiency.
Manganese deficiency can cause disorders of serum
boneregulating hormones and bone metabolic enzymes. Moreover,
manganese supplementation can effectively inhibit bone loss in
Decreases in serum calcium concentration can directly cause
parathyroid hormone (PTH) secretion, thus triggering bone
calcium release. One of the underlying mechanisms for this action
involves the effect of PTH on osteocytes; PTH promotes
cytoplasmic processes to release alkaline phosphatase and
proteolytic enzymes into the surrounding environment, thus accelerating
dissolution of the walls of bone canaliculi. Another mechanism
involves the increase in the stimulation of osteoblasts to express
RANKL, thus inducing the differentiation and maturation of
preosteoclasts, which then increase the number of osteoclasts and
accelerate bone dissolution and resorption. Therefore, continuous
low serum calcium levels increase the risk of osteoporosis.
Both high and low magnesium levels adversely affect the
skeleton; therefore, strictly controlling magnesium balance is vital
to bone health. The most direct effect of magnesium
deficiency on the skeletal system is the change in the
hydroxyapatite crystal structure of bone tissue through its effect on osteocytes,
consequently weakening the load-bearing capacity of bones.
Magnesium deficiency is related to decreased PTH and vitamin D
levels. Moreover, magnesium supplementation can correct
osteoporosis combined with PTH and vitamin D deficiency.
Magnesium deficiency is related to low-grade inflammation, and
the inflammatory cytokines produced by inflammatory responses
stimulate bone reconstruction and decrease bone mass. In
addition, magnesium deficiency causes endothelial dysfunction,
and the resultant vascular disease is also a risk factor for
In this study, femoral BMD decreased significantly in the HLU
group, demonstrating that HLU is a reliable model for studying
weightlessness-induced bone loss in rats. The additional decrease
in femoral BMD in the HMF+HLU group indicates that HMF
environments further promote bone loss in HLU rats. However, in
the HMF group, which was conventionally housed in a
hypomagnetic environment, although serum GC, DPD, and
bALP concentrations increased and trace elements were adversely
altered, the femurs did not exhibit bone loss. One of the reasons
for this is that the skeletal systems of HMF rats were continuously
stimulated by gravity, which is the most important environmental
factor that maintains bone health. Another possible reason is
that even though the HMF environment caused oxidative stress
responses in HMF rats, these responses did not exceed the rats
antioxidant capacity. Moreover, in HLU rats, bone loss directly
caused by hindlimb unloading, the 230u downward head-tilting
position also caused a stress responses in rats simultaneously,
which, combined with the effect of the HMF environment,
increased the oxidative stress responses in the body, resulting in
increased bone loss. This interesting phenomenon requires more
direct evidence for confirmation in future studies.
In conclusion, HMFs can promote additional bone loss in rats in
simulated weightlessness. The underlying mechanism might
involve changes in biological rhythms by the HMF, which may
induce oxidative stress responses; in turn, excessive levels of ROS
stimulate osteoblasts to secret RANKL and promote the
maturation and activation of osteoclasts, eventually causing bone
resorption. In addition, changes in the concentrations of trace
elements in the rats bodies caused by HMFs might also be one of
the important reasons for the oxidative stress responses.
I gratefully acknowledge the support of the Key Laboratory for Space
Bioscience and Biotechnology, and would like to thank NORINDAR
International for shielding chamber construction and other colleagues who
have contributed to this paper.
Conceived and designed the experiments: JB PS. Performed the
experiments: JB LX QZ WJZ PFY CD. Analyzed the data: JB ARQ.
Contributed reagents/materials/analysis tools: LX QZ ARQ. Wrote the
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