Thomas Edison State College Physics in the News Article Paper The forum topic this week will allow you to select a topic in the news related to physics, a

Thomas Edison State College Physics in the News Article Paper The forum topic this week will allow you to select a topic in the news related to physics, a discovery in physics, or an application of physics. The requirements are:1. Summarize the article, indicating the laws or theories of physics that are relevant to the article. 2. The article is posted below. 3. APA format 3-4 pages Zhang et al. Stem Cell Research & Therapy
https://doi.org/10.1186/s13287-019-1497-1
(2019) 10:407
RESEARCH
Open Access
Electromagnetic field treatment increases
purinergic receptor P2X7 expression and
activates its downstream Akt/GSK3?/?catenin axis in mesenchymal stem cells
under osteogenic induction
Yingchi Zhang1†, Wenkai Li1†, Chaoxu Liu1, Jiyuan Yan1, Xuefeng Yuan2, Wei Wang1, Huaixi Wang3, Hua Wu1* and
Yong Yang1*
Abstract
Background: Imbalance in bone formation and resorption is a crucial component of the pathological process
leading to osteoporosis. Electromagnetic fields (EMFs) have been reported to be beneficial to osteogenesis,
although the exact mechanism has not been fully clarified. Purinergic receptor P2X7 is expressed in osteoblasts and
is reported to participate in the regulation of bone metabolism.
Objectives: To elucidate the link between EMFs and P2X7 expression and investigate its potential as a novel
therapeutic target in osteoporosis.
Method: We investigated the effect of EMFs on P2X7 expression and downstream signaling in human bone
marrow mesenchymal stem cells (h-MSCs). We also established an ovariectomized (OVX) osteoporosis rat model to
evaluate the therapeutic efficacy of combining EMFs with P2X7 agonists.
Results: EMF treatment increased P2X7 expression in h-MSCs under conditions of osteogenic induction but not under
regular culture conditions. P2X7 or PI3K/Akt inhibition partially inhibited the pro-osteogenic effect of EMF and lowered
the EMF-stimulated activity of the Akt/GSK3?/?-catenin axis. No additive effect of this suppression was observed
following simultaneous inhibition of P2X7 and PI3K/Akt. EMF treatment in the presence of a P2X7 agonist had a
greater effect in increasing osteogenic marker expression than that of EMF treatment alone. In the OVX osteoporosis
model, the therapeutic efficacy of combining EMFs with P2X7 agonists was superior to that of EMF treatment alone.
Conclusions: EMF treatment increases P2X7 expression by h-MSCs during osteogenic differentiation, leading to
activation of the Akt/GSK3?/?-catenin axis, which promotes the osteogenesis. Our findings also indicate that combined
EMF and P2X7 agonist treatment may be an effective novel strategy for osteoporosis therapy.
Keywords: Electromagnetic fields (EMFs), Purinergic receptor P2X7, Human bone marrow mesenchymal stem cells (hMSCs), Osteogenic differentiation, Akt/GSK3?/?-catenin signaling pathway
* Correspondence: wuhua360@aliyun.com; yangyong0127@hotmail.com
†
Yingchi Zhang and Wenkai Li contributed equally to this work and should
be considered co-first authors.
1
Department of Orthopedics, Tongji Hospital, Tongji Medical College,
Huazhong University of Science and Technology, 1095 Jiefang Avenue,
Wuhan 430030, China
Full list of author information is available at the end of the article
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Zhang et al. Stem Cell Research & Therapy
(2019) 10:407
Introduction
Osteoporosis, which literally means porous bone, is a
disease in which the density and quality of bone are reduced. Currently, there are millions of osteoporosis patients worldwide, most of which are postmenopausal
women. It is characterized by reduced bone mass and
micro-architectural degradation of bone tissue, resulting
in increased bone fragility and higher fracture risk [1].
Common treatments for osteoporosis include dietary
and lifestyle changes as well as pharmacologic therapies,
such as teriparatide, denosumab, and bisphosphonates
[2, 3]; however, these interventions are limited by multiple side-effects, high cost, and low patient compliance.
Although the specific pathogenesis of osteoporosis is unclear, there is increasing evidence that dysplasia of bone
marrow stromal cells (BMSCs) is major cause of structural abnormalities in osteoporosis bones [4–6]. BMSCs
are self-renewable, multipotent stem cells that can be
differentiated into different lineages of chondrocytes, osteoblasts, adipocytes, and other mesenchymal tissues,
after culturing with appropriate hormonal inducers or
growth factors under appropriate conditions [7, 8]. Uncoupling between osteoblast and osteoclast activity and/
or loss of the balance between osteogenic differentiation
and adipogenic differentiation of BMSCs lead to
osteoporosis.
As a clinically safe, effective, and noninvasive treatment, electromagnetic field (EMF) therapies have been
well received during recent decades. In the field of orthopedics, EMF therapy is commonly used to treat bone
fractures [9] and musculoskeletal disorders, including
osteoarthritis and rheumatoid arthritis [10]. Based on
pre-clinical studies and prospective clinical trials, the
Food and Drug Administration, USA, approved pulsed
EMF therapy as a safe and effective method for treating
delayed union or nonunion fractures [11, 12]. In the last
few years, EMFs have been widely reported to positively
affect the balance of osteoblast and adipocyte differentiation of mesenchymal stem cells [13–15] and the balance between bone formation and bone resorption [16],
which are critical components of the development of
osteoporosis. These reports indicated that EMFs can be
used to improve the state of osteoporosis. Previous studies confirmed that EMF treatment directly induced [17]
or accelerated [18] the osteogenic differentiation of
BMSCs. However, the mechanism by which EMFs induce or accelerate osteogenic differentiation of MSC remains to be fully elucidated.
Extracellular nucleotides, such as ATP and UTP, as
soluble factors released into cellular matrix in response
to mechanical stimuli, signal in the autocrine or paracrine manner through specifically binding cell surface P2
receptors [19–21] There is increasing evidence that
extracellular nucleotides play an important role in bone
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metabolism [21–23]. Depending on molecular structure
and activation of signal pathway, P2 purinergic receptors
fall into two categories in mammalian cells, including
seven P2X subtypes and eight P2Y subtypes [24].
In particular, the purinergic receptor P2X7, a ligandgated ion channel, is closely involved in bone remodeling
and mechanical transduction. P2X7 knockout resulted in
an osteopenic phenotype, in which the formation of
periosteal bone is reduced in long bones while there is
no significant difference in length of bone or trabecular
bone resorption [25]. Further in vivo loading experiments showed that appositional growth of the long bone
is reduced and skeletal responses to mechanical loading
are weakened in these mice [26]. An in vitro study has
since indicated that shockwaves enhance osteogenic differentiation of human mesenchymal stem cells through
ATP release and P2X7 activation [27]. P2X7-induced
membrane blebbing promotes osteogenesis and
mineralization of postmenopausal bone marrow-derived
mesenchymal stem cells [28]. However, a link between
EMF exposure and P2X7 expression in osteogenic differentiation of MSCs has not yet been reported.
In this study, we found that P2X7 expression was increased after EMF exposure. Furthermore, the increased
P2X7 expression contributed to MSC osteogenic differentiation via the Akt/GSK3?/?-catenin axis. We also
found that utilization of P2X7 agonists improved the
therapeutic effects of EMF in osteoporosis.
Materials and methods
EMF device
The EMF generation device which comprises a waveform generator, amplifier, oscilloscope, and Helmholtz
coils was designed and manufactured by the Naval University of Engineering of China (Wuhan, China). Signals
produced by the waveform generator were amplified and
then output to the Helmholtz coils. The Helmholtz coils
(diameter 30 cm, 15 cm apart) which were wound with
coated copper wire (diameter 0.8 mm) generated the
EMF which is used in the following experiment. For the
in vitro experiments, the Helmholtz coils were vertically
placed in a CO2 incubator (Thermo Scientific, Wilmington, DE, USA) (Additional file 1: Figure S1A). For the
in vivo experiments, the Helmholtz coils were vertically
placed in a ventilated environment at 26 °C and the
Plexiglas cages (length 35 cm, width 30 cm, height 45
cm) were placed between the coils, in which rats were
housed individually with free access to clean tap water
and standard rodent chow (Animal Center of Tongji
Medical College, Wuhan, China) (Additional file 1: Figure S1B). EMF generated by the device were at
frequency range of 0–100 Hz and an intensity range of
0–5.0 mT. A sinusoidal EMF was used as it showed a
satisfactory effect in our previous studies [29, 30]. The
Zhang et al. Stem Cell Research & Therapy
(2019) 10:407
intensity of the EMF was measured using a gauss meter
(GM55A; TinDun Industry, Shanghai, China). The uniformity of the EMF was approximately 90% in the 7 cm
of the spherical region (from the coil center to the
origin).
Human MSC culture and stimulation
Human bone marrow MSCs (h-MSC) (Cell Bank of the
Chinese Academy of Sciences, Shanghai, China) were
identified by detecting mesenchymal stem cell surface
markers through flow cytometry (Additional file 1: Figure S2A) and evaluating the potential for differentiation
towards adipocytes, osteoblasts, and chondrocytes (Additional file 1: Figure S2A). The h-MSCs were cultured in
Gibco Dulbecco’s modified Eagle medium/Ham’s F-12
(DMEM/F12) supplemented with 10% FBS and 100 U/
mL penicillin–streptomycin (Gibco, USA) under condition of 5% CO2, 37 °C, and 100% humidity. Upon the
cells were approximately 90% confluence, 0.25% trypsin
(Gibco, USA) were used to detach the cells, which then
passaged at a ratio of 1:2 or 1:3. Passages between 3 and
5 were used in the following experiment. For the EMF
treatment, MSCs were exposed for 8 h per day. To inhibit P2X7 or PI3K/Akt activity, cells were treated with
5 ?M P2X7 blocker A740003 (Sigma–Aldrich, St. Louis,
MO, USA) or 10 ?M PI3K/Akt inhibitor LY294002
(Sigma–Aldrich).
RNA extraction and qRT-PCR analysis
Total RNA was extracted from human mesenchymal
stem cells (h-MSCs) using TRIzol reagent (Invitrogen,
Carlsbad, CA, USA). The concentration and purity of
RNA samples were assessed by spectrophotometric analysis. Three micrograms of RNA was used to reverse
transcription by using an EasyScript First-Strand cDNA
Synthesis Super Mix kit (TransGen Biotech, Beijing,
China). The relative mRNA expression of human P2X7,
RUNX2, ALP, OPN, and GAPDH (Invitrogen) was evaluated by quantitative real-time PCR (qRT-PCR) by using
the BioRad myiQ2 Sequence Detection System (BioRad,
Hercules, CA, USA) and TransStart Eco Green qPCR
Super Mix (TransGen Biotech, Beijing, China). Primers
were purchased from Invitrogen, and the sequences were
listed in Additional file 1: Table S1. Cycle conditions
followed primers’ introduction. Relative gene expressions
were normalized with GAPDH and analyzed by the
2???Ct method.
Western blot analysis
The h-MSCs were cultured in 6-well plates (5 × 105
cells/well) in expansion medium for 24 h. After incubation and stimulation, cells were washed using icecold PBS (Boster Biol Tech, Wuhan, China) and centrifuged at 1000 rpm for 10 min. Total proteins were
Page 3 of 13
isolated by lysing cells in radioimmunoprecipitation
assay lysis buffer containing 1% phosphatase inhibitors
(Boster Biol Tech) for 30 min on ice. Lysates were centrifuged at 13,000 rpm for 15 min at 4 °C, then the harvested supernatants were stored at ? 20 °C prior to
analysis. Nuclear and Cytoplasmic Protein Extraction
Kit (Beyotime, Shanghai, China) was used to extract
cytoplasmic and nuclear proteins according to the
manufacturer’s protocol. BCA protein assay kit (Applygen, Beijing, China) was used to assess protein concentration. Proteins in equivalent amount from each
sample were separated by 10% SDS-PAGE (BioRad)
electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore,
Billerica, MA, USA). 5% BSA (Boster Biol Tech) in 1×
TBST (0.1% Tween-20) was used to block the membranes for 1 h at room temperature. After blocking,
membranes were incubated with primary antibodies
(Cell Signaling Technologies Inc., Milan, Italy) against
P2X7, RUNX2, OPN, Akt, p-Akt, GSK3?, p-GSK3?, Ecadherin, Fibronectin, Vimentin, Snail, ALDH1A1, ?catenin, lamin B1 (all 1:1000), and GAPDH (1:400) at
4 °C overnight. The next day, membranes were washed
three times with TBST on ice for 10 min and then incubated with HRP-conjugated goat anti-mouse or goat
anti-rabbit IgG secondary detection antibodies (Boster
Biol Tech) (1:5000) at room temperature for 1 h.
Membranes were washed three times in TBST for 10
min each. Enhanced chemiluminescence (ECL) reagent
was used to detect immunostained protein bands
through autoradiography. Image-Lab software (BioRad,
Hercules, CA, USA) was used to analyze the band intensities. Relative expression was normalized using
GAPDH or lamin B1 as a loading control, and data
were presented as a percentage of the expression of
the reference genes.
Alizarin red S staining and Oil red O staining
To induce osteogenic differentiation, h-MSCs were
seeded on plastic dishes (diameter 35 mm) with a density
of 2 × 104 cells/cm2 and cultured for 7 to 21 days in human osteogenic induction mediums (Cyagen). The formulation of the human osteogenic induction medium is
175 mL h-MSC osteogenic differentiation basal medium
with 20 mL fetal bovine serum, 2 mL penicillin–streptomycin, 2 mL glutamine, 400 ?L ascorbate, 2 mL ?glycerophosphate, and 20 ?L dexamethasone.
For Alizarin red S staining, h-MSCs were washed with
PBS and fixed with 4% paraformaldehyde at room
temperature for 30 min. Then the fixed cells were
washed with PBS and stained with 1 mL Alizarin red S
working solution for 3–5 min. After two to three times
rinse with PBS, then the cells were photographed.
Zhang et al. Stem Cell Research & Therapy
(2019) 10:407
Page 4 of 13
Ovariectomy models
Results
Sprague Dawley rats were purchased from the Experimental Animal Center of Huazhong University of Science and Technology (Wuhan, China) Twenty-four
female Sprague Dawley rats were randomly divided into
four groups: sham-operated controls, OVX controls,
OVX with EMF stimulation, and OVX with both EMF
stimulation and BzATP administration. Rats underwent
the sham operation or ovariectomy at age of 8 weeks
(weighing 200–220 g) and recovered for 2 weeks. All experimental procedures were performed on the animals
with the official approval of the Ethics and Animal Research Committee of Huazhong University of Science
and Technology. From 10 weeks of age, the shamoperated controls and OVX controls received daily intraperitoneal injections of 0.9% NaCl. The OVX+EMF and
OVX+EMF+BzATP groups were exposed to EMFs (15
Hz/1 mT) for 8 h per day (from 9:00 AM to 5:00 PM).
The OVX+EMF+BzATP group administrated daily of
BzATP (5 mg/kg/day) by intraperitoneal injection. All
rats were euthanized after 12-week treatment. The right
femur and tibia of these rats were excised and fixed with
4% paraformaldehyde for further analysis.
Various frequencies of EMFs increased P2X7 expression in
h-MSCs under osteogenic differentiation
Micro-CT scanning and analysis
Scanco viva CT 40 instrument (Scanco, Brüttisellen,
Switzerland) was used to scan the right femur and tibia
excised from all groups of rats. The micro-structure of
above bones was analyzed as reported in our previous
study [30]. Briefly, contiguous cross-sectional images
(10.5 ?m) were obtained at 70 kV and 113 mA. The constant threshold for trabecular and cortical bone was set
at 180 and 220, respectively, in order to distinguish the
bone from the bone marrow. For trabecular bones, the
region of interest (ROI) began at 10 slices (105 ?m)
below the lowest point of the growth plate and extended
downward for 200 slices (2100 ?m). For the cortical
bones, the ROI was selected from the whole femoral.
The trabecular morphometry parameters were analyzed
including the relative bone volume (bone volume/total
volume, BV/TV).
Statistical analysis
All data were presented as mean values ± standard deviation (SD). Differences among each time points or
among each treatment groups were determined by oneway analysis of variance (ANOVA). Following the oneway ANOVA, significances between every pair of time
points or treatment groups were determined by Tukey’s
post hoc analysis. Statistical analysis was performed
using the software of Statistical Package for Social Sciences (SPSS 15.0 for Windows; SPSS, Chicago, IL). P < 0.05 was accepted as significant difference. h-MSCs were exposed to various frequencies of EMFs (7.5, 15, 30, 50, and 75 Hz/1 mT) in the presence of osteogenic induction medium for 7, 14, and 21 days. P2X7 expression was detected at the mRNA and protein levels by qRT-PCR and Western blot analysis, respectively. We found that 1 mT EMFs at all these frequencies (7.5, 15, 30, 50, and 75 Hz) increased P2X7 expression to varying degrees at both the mRNA and protein levels from 7 to 21 days. Among the frequencies, 15 Hz exhibited the optimal ability to increase P2X7 expression (Fig. 1a, b) and was therefore selected for use in further research. The EMF-induced P2X7 expression in h-MSCs was detected during osteogenic differentiation but not in regular culture To examine the conditions and timing of EMFstimulated P2X7 expression, we cultured h-MSCs with basic medium or osteogenic medium for 0, 1, 3, 7, 14, and 21 days prior to analysis of P2X7 expression. The qRT-PCR analysis showed that EMF did not affect hMSC expression of P2X7 mRNA under basic medium culture conditions. In contrast, under osteogenic culture conditions, h-MSC expression of P2X7 mRNA was slightly increased (approximately 20%) on day 3 and was increased by more than twofold on days 7 to 21. This effect was significantly enhanced by exposure to EMFs. hMSC cultured in osteogenic medium combined with EMF exposure exhibited a marked increase (80%) in P2X7 mRNA expression on day 3, with further increases (four- to fivefold) in P2X7 expression detected on days 7 to 21 (Fig. 2a). In h-MSCs cultured in basic medium, Western blot analysis showed no significant differences in P2X7 protein expression at the various time points, with or without EMF exposure (Fig. 2b). In h-MSCs cultured in osteogenic medium without EMF exposure, P2X7 protein expression increased by 30% on days 7 to 21, but was increased by 100 to 200% on days 3 to 21 in the presence of EMFs (Fig. 2c). These results suggested that EMFs significantly promote P2X7 expression in hMSCs during osteogenic differentiation. P2X7 expression was linked to EMF-enhanced osteogenic differentiation in h-MSCs Our previous study showed that 15 Hz/1 mT EMF exposure significantly promoted osteogenic differentiation of rat MSCs [31]. In this study, we verified the proosteogenic effect of EMF exposure on h-MSCs cultured in osteogenic medium. Furthermore, we used A740003, a specific P2X7 antagonist, to investigate whether this effect was related to P2X7 expression. The qRT-PCR Zhang et al. Stem Cell Research & Therapy (2019) 10:407 Page 5 of 13 Fig. 1 Effects of exposure to various frequencies of EMF on P2X7 expression in osteogenic differentiated h-MSCs. a qRT-PCR and b Western blot analysis of P2X7 expression in h-MSCs exposed to various frequencies of EMF (7.5, 15, 30, 50, and 75 Hz of 1 mT) for 7, 14, and 21 days. The mRNA and protein expression of P2X... Purchase answer to see full attachment