The authors have declared that no competing interests exist.
Conceived and designed the experiments: PBZ Y. Wei XSC. Performed the experiments: Y. Wang ZXW HTC. Analyzed the data: NPW. Contributed reagents/materials/analysis tools: ZLW. Wrote the paper: PBZ Y. Wang.
Current address: Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, People’s Republic of China
Electrical stimulation (ES) is therapeutic to many bone diseases, from promoting fracture regeneration to orthopedic intervention. The application of ES offers substantial therapeutic potential, while optimal ES parameters and the underlying mechanisms responsible for the positive clinical impact are poorly understood. In this study, we assembled an ES cell culture and monitoring device. Mc-3T3-E1 cells were subjected to different frequency to investigate the effect of osteogenesis. Cell proliferation, DNA synthesis, the mRNA levels of osteosis-related genes, the activity of alkaline phosphatase (ALP), and intracellular concentration of Ca2+ were thoroughly evaluated. We found that 100 Hz could up-regulate the mRNA levels of collagen I, collagen II and Runx2. On the contrary, ES could down-regulate the mRNA levels of osteopontin (OPN). ALP activity assay and Fast Blue RR salt stain showed that 100 Hz could accelerate cells differentiation. Compared to the control group, 100 Hz could promote cell proliferation. Furthermore, 1 Hz to 10 Hz could improve calcium deposition in the intracellular matrix. Overall, these results indicate that 100Hz ES exhibits superior potentialities in osteogenesis, which should be beneficial for the clinical applications of ES for the treatment of bone diseases.
Electrical stimulation (ES) is clinically beneficial in the treatment of fracture, while 5%-10% of fractures show impaired healing and require additional orthopedic intervention. Fracture healing involves a complex multistep process. Serious periosteal and soft tissue damage at the time of fracture can lead to the formation of an atrophic nonunion. In clinic, fracture patients suffering from nonunion are mainly treated with surgery and orthopedic treatment, including bone grafts[
As ES is successful for healing bone fracture, the exact molecular mechanisms for ES of promoting osteogenesis remain relatively unclear. Cells differentiate into osteoblasts including the following principal development periods. First of all, cell periodic cycle and cell proliferation is activated. At the same time, extracellular matrix protein (EMC) is expressed. Secondly, alkaline phosphatase (ALP), a maker of osteoblasts proliferation, is up-regulated. In the third period, maturation of bone-like tissue, and osteopontin (OPN) and osteocalcin (OC) are maximally expressed. At last, the expression of collagenase is up-regulated and apoptotic occurs. Many research work has demonstrated that ES could promote the process of cells differentiated into osteoblasts. BRIGHTON, CT et al. show that newborn rat calvarial bone cells subjected to a matrix of sine wave 60 kHz, 20mV/CM capacitively coupled electrical signals could accelerate cell proliferation when the signal is applied continuously for six hours[
The overall setup for ES is shown in
(A) Platinum electrode chamber design for direct coupling; (B) ES setup including signal function generator for power supply; (C) Oscilloscope for ES verification.
MC-3T3-E1 cells were obtained from the Cell Culture Centre of Institute of Basic Medical Sciences Chinese Academy of Medical Sciences (Shanghai, China). MC 3T3-E1 cells were cultured and expanded in basal medium containing Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 units/ml penicillin (Sigma), and 100 mg/ml streptomycin (Sigma). Cells at passage 2–3 were grown in 24 well cell culture plate (Corning, USA) at initial seeding cell densities of 20,000 cells/well under controlled condition of temperature (37°C), a humidified atmosphere of 95% air and CO2 (5%). Medium was replaced with fresh medium every 2 days [
Cells were subjected to different frequencies for 30 minutes per day continued 1–3 days. Next, 50μL of MTT (Sigma) stock solution in PBS (5 mg/mL) was added into each well with a final concentration of 0.5 mg/mL MTT. The cell plate was then incubated at 37°C for another 4 h. The medium was removed and 500μL of DMSO (Sigma) was added to dissolve the formazan crystals. The optical density (OD) was measured at 492 nm by a plate reader (Tecan M200). Control group was taken as negative control with 100% viability. The cell proliferation (%) of treated group was calculated by [abs] treated group/ [abs] control group×100.
The DNA synthesis was determined by measuring the intensity of fluorescence using PicoGreen dsDNA Kit (Invitrogen). BCA Kit (Pierce) was used to measure the protein concentration of cell lysis in treated group and control group based on a BSA standard curve. The DNA synthesis can be compared by the ratio of relative fluorescence intensive and protein concentration.
The mRNA of cells was extracted using trizol (Invitrogen) and collected using the Qiagen RNEasy Extraction kit (Qiagen). Samples were stored at -80°C until reversed transcription. Total RNA concentration and purity were detected by Nanodrop Assay (Tecan M200). The first strand cDNA was synthesized by reverse transcriptase as described of M-MLV manual (Promega). Gene-specific primers including GAPDH, COL I, COL II, OPN, and Runx2 were designed using the primer design software of beacon 5.0 (
Gene Annotation | Primer Sequence (5’-3’) | Length (bp) | Reference |
---|---|---|---|
20 | NM_007742.3 | ||
21 | |||
24 | NM_007742.3 | ||
21 | |||
21 | NM_007742.3 | ||
24 | |||
21 | NM_007742.3 | ||
21 | |||
21 | NM_007742.3 | ||
21 |
Intracellular Ca2+ concentration was evaluated on day 1 and 3 day after ES using kit (Fluo-4 NW Calcium Assay Kit, Invitrogen). Cell medium was removed, and 100 μL of the dye loading solution was quickly but carefully added to each well following the manual of kit. The plates were incubated at 37°C for 30 minutes, and then at room temperature for an added 30 min. Fluorescence was measured using plate reader with setting the exaction wavelength at 494 nm and emission wavelength at 533 nm.
MC-3T3-E1 cells were cultured in DMEM medium at an initial density of 2×104 cells/well in 24-well culture plates and collected from each well after 3 days ES stimulation as described above. After the different frequency ES stimulation, the cells were assayed for ALP activity with a p-Nitrophenyl Phosphate (pNPP) Liquid Substrate System (Sigma) following the manufacturer’s instructions. First, cells were lysed in a lysis buffer (Sangon) and incubated for 30min at 37°C and centrifuged at 12,000g force for 10min at 4°C. The clear cell lysis was transferred into a new 1.5ml centrifuge tube for the following ALP assay. Add 10μl clear cell lysis into a 96-well transparent plate. Add 200 μl of pNPP solution to each well. Incubate the plate in the dark for approximately 30 minutes at room temperature. After the incubation period, read the plate at 405 nm on a multiwell plate reader (Tecan M200). The enzymatic activity was expressed as mmoles of p-NP/min/mg Protein.
Histochemical analysis of ALP activity was also carried out. Cells were stained using alkaline-dye mixture of 0.01% (W/V) Naphthol AS-MX Phosphate Alkaline Solution and 0.24 mg/ml Fast Blue RR salt at 18–26°C for 30 minutes. After 30minutes, nucleuses were stained using Mayer’s Hematoxylin Solution for 10 minutes. The expression of ALP was observed by light microscopy.
Cell proliferation and DNA content are important indicators in ES frequency studies. Here, we carry out a detailed analysis of cell proliferation and DNA content to determine potential effects of ES frequency. We find that cell proliferation is similar on 1 K and on control, while cell proliferation is decreased on 1, 10, 10 K, and 100 K compared to control at day 1. However, only the cell proliferation of 100Hz is higher than control group at day 1 and day 3. The cell proliferation of 100 Hz is 8.5% more than 10 Hz (P < 0.001), 12.1% more than 1 Hz (P < 0.05), 9.3% more than 10 KHz (P < 0.05), and 7.2% more than 100 KHz (P < 0.05) at day 1. Cell proliferation at day 3 has the similar tendency as day1. Cell proliferation has a maximum increase on 100 Hz compared to control. Cell proliferation is inhibited on 10 KHz, and 100 KHz compared to control at day 3 (
MTT assay to assess proliferation of the MC-3T3-E1 cells after ES. 100 Hz group has the highest proliferation rate at days 1, 3 compared to the other frequency and control group. 1K Hz group showed similar proliferation compared to control group at days 1, 3. 1 Hz, 10 Hz, 10 KHz, and 100 KHz showed lower proliferation than control group at day 1. (P < 0.05, indicated statistically significant difference)
Mc-3T3-E1 cells were subjected to different frequency ES for 3 days. Pico-green DNA assay was used to quantified DNA concentration of cells. 100 Hz group has the higher DNA concentration than other ES groups and control.
Collagen I, Collagen II, OPN, and Runx2 were evaluated as early osteosis-related genes (
MC-3T3-E1 cells were subjected to different frequency ES for 3 days. The mRNA levels of Collagen I, Collagen II, OPN, and RUNX2 were quantified by real-time PCR. The data points represent the mean value ±SD (n = 3), *P<0.05.
A slightly increase in mRNA of collagen II was observed after the stimulation of 10 Hz and 100 Hz, compared to control group. The mRNA level of collagen II was not significantly regulated by the stimulation of 1 Hz, 1 KHz, 10 KHz, 100 KHz, compared to control group (
To study the effect of ES frequency on calcium uptake by Mc-3T3-E1 cells, Fluo-4 NW calcium assay kit was used to measure the intracellular concentration of Ca2+. In this experiment, conditions of ES were fixed at 200mV/CM, 50% duty cycle, and rectangular waveform. The frequency of ES was variable. Mc-3T3-E1 cells were subjected to different frequency (1 Hz, 10Hz, 10 Hz, 1 KHz, 10 KHz, and 100 KHz) for 3 days. The Ca2+ concentration of ES groups are significantly higher than control group. The intracellular Ca2+ concentration of 1 Hz and 10 Hz group are greatly higher than other ES groups and are higher by 14.2% and 15.3% (P<0.05) than control group at 1 day, 19.3% and 21.8% (P<0.05) at 3 days, respectively. These results pointed out that frequency from 1Hz to 10Hz can positively up-regulate calcium uptake by MC-3T3 cells (
MC-3T3-E1 cells subjected to different ES frequency for 1 day and 3 days. Fluo-4 NW was used to assay relative Ca2+ concentration. 1 Hz and 10 Hz groups shown an increasing regulation on intracellular Ca2+ concentration compared to control group. * P < 0.05, indicated statistically significant difference compared to 1Hz and 10Hz.
The ALP activity in MC3T3-E1 cells was examined to evaluate the effect of frequency on cells osteogenic differentiation. After 3 days of ES stimulation, the ALP activity per unit protein in MC3T3-E1 cells was examined. The ALP activity of 10 Hz and 100 Hz group is increased, compared to control group. However, the ALP activity of 10 KHz, 100 KHz group is slightly decrease compared to control group. The ALP activity of 1Hz and 1 KHz does not specially differ to control group (
Cells were exposed to different frequency ES for 30min a day, continued 3 days. Values are the mean ± SD of three independent cultures. *P < 0.05 vs. 1Hz, 10Hz, 100 kHz group.
ALP activity increased in a ES groups reached the highest level at 100 Hz ES for 3 days.(A)1 Hz,(B)10 Hz,(C)100 Hz,(D)1 KHz,(E)10 KHz,(F)100 KHz, bar = 100um.
Previous studies show that ES play a pivotal role in regulating bone-cell’s behavior and development. However, little is known about which ES frequency is actually responsible for the bone-cell's response. This study evaluates the role of ES frequency to determine which frequency most affects osteogenesis differentiation. In our experiments, MC-3T3-E1 cells were subjected to different frequency. After ES induced, we evaluated cell proliferation, DNA content, the mRNA level of osteosis-related genes (COL I, COL II, OPN, and Runx2), markers of osteogenesis differentiation (ALP activity), and intracellular Ca2+ concentration.
As shown in
From our results, frequency around 100 Hz is effective in proliferation of cells. At the same time, EMC (COL I, COL II) is also largely expressed. When frequency ranges from 10 Hz to 100 Hz, the expression of EMC (COL I, COL II) is obviously higher than control group. This promotion is also occurrence in ALP activity of Mc-3T3-E1 cells followed cultures after 3 days stimulation. Histochemical analysis of ALP activity also shows that 100 Hz can obviously enhance ALP activity. The increase of ALP activity means that osteogenesis differentiation of Mc-3T3-E1 cells is started.
Some genes play an important roles in the osteogenesis differentiation. Pluripotent mesenchymal stem cells and osteoprogenitor cells can be regulated to differentiate into osteoblasts by some key cell cytokines and functional proteins, including BMPs family, Runx2, and some ECM. In our test, ES frequency was set at a wide range from 1Hz to 100 KHz. As the results showed in
As the results showed in
Runx2, also called cbfa1 (core-binding factor), is an essential transcription factor for osteoblast differentiation and bone formation, which belongs to the runt-domain gene family. Runx2 expression at appropriate level, times and spaces during fetal development is essential for skeletal formation. When the expression of Runx2 was disrupted in fetal mice, the skeletal systems showed a complete lack of [
Levels of calcium ions plays an important role in the regulation of cell proliferation and differentiation of MC3T3-E1 cells [
The molecular mechanism of ES promoting osteogenic differentiation is unclear. ES and pulsed electromagnetic field (PEMF) can alter the protein’s structure and interactions of proteins and other molecules, such as drugs, ligands and so on. We hypothesize that appropriate ES and EMF frequency can affect the cell signal transduction of osteogenic differentiation because of the structure change of osteogenic related cytokines and receptors [
In conclusion, ES is a flexible, safety, and cheap way to treat bone fracture and nonunions by promoting cell proliferation, DNA synthesis, ALP activity, mRNA level of osteosis-genes, and the concentration of Ca2+. When MC3T3-E1 cells are subjected to ES in different frequency, 100Hz is better than other frequency can significantly promote the up-regulation of osteosis-related genes, activity of ALP, cell proliferation, and DNA synthesis. Our findings also indicate that 1 Hz to 10 Hz can significantly increase the concentration of Ca2+. Conversely, osteogenesis differentiation will be inhibited by using an inappropriate frequency of ES. From the results of our experiments, we find that frequency is the dominant factor affecting the bone-cell’s proliferation, the mRNA level of osteosis-related genes, ALP activity, and the intracellular concentration of Ca2+. This work approves the importance of frequency for ES to promoting osteogenesis differentiation, which is helpful for ES treatments applied to bone fracture and nonunions in clinic.
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