Bioelectromagnetics 29:125^132 (2008)
The Effects of Pulsed Electromagnetic Fields on the Cellular Activity of SaOS-2 Cells Carlos F. Martino,1 Dmitry Belchenko,1 Virginia Ferguson,1* Sheila Nielsen-Preiss,2 and H. Jerry Qi1 1
Department of Mechanical Engineering, University of Colorado at Boulder, Boulder, Colorado 2 Department of Medicine, University of Colorado at Denver and Health Science Center, Aurora, Colorado Although pulsed electromagnetic fields (PEMFs) have been used for treatments of nonunion bone fracture healing for more than three decades, the underlying cellular mechanism of bone formation promoted by PEMFs is still unclear. It has been observed that a series of parameters such as pulse shape and frequency should be carefully controlled to achieve effective treatments. In this article, the effects of PEMFs with repetitive pulse burst waveform on the cellular activity of SaOS-2 osteoblast-like cells were investigated. In particular, cell proliferation and mineralization due to the imposed PEMFs were assessed through direct cell counts, the MTT assay, tissue nonspecific alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining. PEMF stimulation with repetitive pulse burst waveform did not affect metabolic activity and cell number. However, the ALP activity of SaOS-2 cells and mineral nodule formation increased significantly after PEMF stimulation. These observations suggest that repetitive pulse burst PEMF does not affect cellular metabolism; however, it may play a role in the enhancement of SaOS-2 cell mineralization. We are currently investigating cellular responses under different PEMF waveforms and Western blots for protein expression of bone mineralization specific proteins. Bioelectromagnetics 29:125–132, 2008. 2007 Wiley-Liss, Inc. Key words: PEMFs; SaOS-2; mineralization; electromagnetic stimulation
INTRODUCTION While external fields have been used extensively to stimulate bone formation in the treatment of nonunion fractures, factors that trigger biomineralization at the cellular level are still unknown and the coupling mechanism between distinctive external fields and cellular behavior appears to be diverse. External fields may be applied in the form of mechanical stimulus imposed by implanted electrodes, which are allowed to move [Spadaro, 1997], electric stimulus induced by electrodes driven with direct current [Becker et al., 1977; Brighton et al., 1977; Spadaro et al., 1986; Spadaro, 1997], or electromagnetic fields established by Hemholtz coils [Bassett et al., 1978]. These studies were motivated by the discovery of the electromechanical properties of bone by Yasuda, Fukuda, Bassett and others, which led to the hypothesis that external electric currents may stimulate bone growth [Spadaro, 1997]. Spadaro [1997] reviewed work conducted in bone formation induced by three types of electrodes: DC only, DC with motion, and motion only and also considered various combinations of pulsed electromagnetic fields (PEMFs) and motion with the conclusion that a concurrent stimulus may be needed for electrical stimulation. 2007 Wiley-Liss, Inc.
In the past few years, stimulation of cellular behavior by PEMFs has attracted more attention. Electric and electromagnetic fields have been used to stimulate extracellular matrix (ECM) and mineral formation of osteoblast-like cells in vitro [Heermeier et al., 1998; Hartig et al., 2000; Wiesmann et al., 2001; Chang et al., 2004]. Osteoblast-like cells from the calvaria of ICR mice stimulated for 8 h with a repetitive pulse burst consisting of 22 pulses at 15 Hz resulted in an increase in cellular proliferation and a decrease in alkaline phosphatase (ALP) activity [Chang et al., 2004]. Collagen I, osteocalcin, and osteopontin mRNA expression did not change during the 14-day ————— — Grant sponsor: Council on Research and Creative Work at the University of Colorado at Boulder Graduate School. *Correspondence to: Virginia Ferguson, Department of Mechanical Engineering, University of Colorado at Boulder, Boulder, CO 80309. E-mail:
[email protected] Received for review 11 September 2006; Final revision received 30 July 2007 DOI 10.1002/bem.20372 Published online 20 November 2007 in Wiley InterScience (www.interscience.wiley.com).
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experiment. Bovine periosteal cells stimulated using a capacitive-coupled electric field induced by saw tooth pulses at 16 Hz with an external peak voltage of 100 V imposed between the two plates of the capacitor had higher levels of osteonectin, osteocalcin, and sialoprotein production. In contrast, the former study by Chang et al. did not show an increase in proteins that contribute to ECM formation. Similarly, mechanical stimuli have been shown to promote cellular responses in the form of centrifugal force induced RUNX2 expression in osteoblasts [Baumert et al., 2004], mechanical strain of SaOS-2 cells in vitro affects expression of collagen genes types I and III [Liu et al., 2005], and changes in the mechanical environment such as simulated microgravity or substrate deformation have been shown to affect cellular behavior [Granet et al., 2001, 2002]. Electromagnetic stimuli of cell cultures are a promising tool for stimulating cell proliferation and activity and may ultimately be used to speed up growth of engineered tissues for implantation in vitro or encourage healing of tissues in vivo. However, the fundamental science underlying the effects of PEMFs on factors such as cell phenotype, proliferation, stress, and longevity are poorly understood. The overall objective of this research is to investigate the feasibility of PEMFs with repetitive pulse burst waveform to stimulate biomineralization response in SaOS-2 cells. The choice of this waveform stems from the plethora of evidence in orthopedic application of PEMFs in fracture repair. Pilla [2006] recently reviewed therapeutic applications of PEMFs including the repetitive pulse system. In the field of orthopedics PEMFs have been used widely for the treatment of nonunions and congenital pseudarthrosis [Bassett et al., 1974, 1977; Brighton et al., 1975]. SaOS-2 cells were used because they are relatively easy to maintain and are a well-characterized osteosarcoma human cell line. An electric field of about 1–20 mV/cm was induced in the medium by Hemholtz coils; the corresponding magnetic intensity was about 20 G. The effects of PEMFs on SaOS-2 cell activities were assessed through direct cell counting and a cellular proliferation (MTT) assay, ALP activity, mineral nodule staining with Alizarin Red S (ARS) and ARS extraction with cetylpyridinium chloride (CPC). The present study is the first step in an investigation of the effects of PEMFs on the production of proteins in SaOS-2 cell line associated with mineralization (e.g., collagen I, osteonectin, and osteocalcin) and in comparing response to other cell types. MATERIALS AND METHODS SaOS-2 Cell Culture SaOS-2 cells were cultured in RPMI-1640 supplemented with 10% FBS, MEM vitamin solution, sodium Bioelectromagnetics
bicarbonate, sodium pyruvate, and MEM nonessential amino acids (all reagents were provided by GIBCO, Carlsbad, CA), as in Gillette and Nielsen-Preiss [2004]. The cells were cultured in a 75 cm2 flask to expand cell number. After reaching confluence, the cells were seeded in cell culture plates; seeding techniques varied according to the assay used. For the MTT assay, the cells were seeded in 24 well plates at a density of 2.5 104 cells per well. For ALP and ARS staining assays, the cells were seeded in 6 well plates and allowed to grow to confluence. Once the cells reached confluence, the medium was aspirated off and changed to a-MEM containing 10% FBS with and without mineralization-inducing components including ascorbic acid (50 mg/ml) and b-glycerophosphate disodium salt (7.5 mM; Sigma Co., St. Louis, MO). Medium was then changed every 2 days. The cultures were incubated in a 5% CO2 atmosphere at 37 8C. Control and experimental cultures were kept in different incubators with similar coils; the coil of the control group was not energized. Stimulating System The study used two O-shaped air-cored coils to stimulate cells situated horizontally. The coils generating the pulsed magnetic field were 18 cm in diameter, 10 cm height separation, and had 5 turns of 16 AWG magnetic wire. The cells were placed in plates centered vertically between the coils. The waveform of the applied voltage is shown in Figure 1. Figure 1A shows the train of pulses repeated at 15 Hz. Each burst consists of 20–21 pulses of 200 ms wide each as shown in Figure 1B,C. The magnetic field penetrated the cells perpendicular to the plane of the plates; a circumferential electric field on the plane of the cells was induced. A sensing coil directly connected to the oscilloscope was used to determine the induced voltage in air at the center of the coils. The search coil consisted of 12 turns of 30 AWG with an internal diameter of 12.8 mm. The actual induced electric potential in the medium was also measured by using a Pilla-type probe [Pilla et al., 1983] connected directly to the oscilloscope. The measurements were made with the probe submerged in the fluid and the tips aligned with the electric field lines. The electric potential was measured at 2 cm from the center of the Petri dish. Several waveforms were used to test the probe and the resulting induced electric potential 2 cm from the center of the Petri dish for the waveform that was used in this study (Fig. 1) is shown in Figure 2. Computation of the electric field at different points in a 6 well plate can be made from this measurement. From simple electromagnetic theory, it follows that the induced electric field is calculated from Ej ¼ dB/dt r/2, where j is the
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of the coils only. The induced electric field intensity at different radii is tabulated in Table 1. This result can be used to estimate electric field intensities at different points inside the culture plates. Proliferation Assay and Cell Counting The effect of PEMFs stimulation on cellular viability and growth was determined by MTT (Sigma Co.) assay and direct count of cell numbers using Trypan blue exclusion after 1–3 days of stimulation. For MTT assay, 10 wells in 24 well cell culture plates were seeded at a concentration of 2.5 104 cells per well and were incubated in 5% CO2 at 37 8C for 2 days prior to subjecting the experimental group to PEMFs. For cell counting assay, 8.0 104 cells per well in 6 well cultures plates were seeded as above. After PEMFs stimulation cycle of 4 h followed by 16 h of rest, the cells in 6 wellplates were counted twice using Trypan blue. After cell counting, 100 ml of MTT solution was added to each of 10 wells of 24 well plates, and the culture plates were incubated for 4 h. Following incubation, 1 ml of 0.04 N HCl in isopropanol was added to dissolve the crystals. The plates were read after 12 h using a Bio-Tek ELx800 plate reader (BioTek Instruments, Inc., Winooski, VT) at a test wavelength of 570 nm.
Fig. 1. The input waveform to the circuitry that drives the coil is shown above. A: Train of pulses repeated at 15 Hz. B: Each burst consists of 20 ^ 21pulses. C:Each pulse is 200 ms wide.
azimuthal angle of constant electric field lines, r is the distance from the center of the dish, and dB/dt is a parameter independent of the sample. At a radius of 2 cm, the electric field was measured to be 9 mV/cm; this corresponds to a change in magnetic field of 0.9 mT/10 ms. This parameter depends on the geometry
Alkaline Phosphatase (ALP) Assay The ALP assay was conducted on cells with and without mineralization-inducing agents. The level of ALP activity in SaOS-2 cells was evaluated immediately after PEMFs stimulation. The magnetic protocol followed differs from proliferation assay. The rationale for this discrepancy is that changes in cell number are very difficult to observe after short periods of stimulus; however, changes in gene expressions and enzyme levels are readily quantifiable even after minutes of external stimulus [Baumert et al., 2004]. For day 1 of cell culture, the cells were exposed to PEMFs for 4 h and immediately lysed where each cell monolayer was first washed with 1 ml of phosphate buffered saline (PBS), then lysed in 200 ml of RIPA (Upstate, New York, NY) lysis buffer. Cell lysates were then placed on ice for 15 min and then centrifuged for 5 min at 8000g. For subsequent time points (days 2 and 3), PEMF stimulation was turned off for 20 h and turned on again TABLE 1. Electric Field Intensities Are Shown at Different Radii in Culture Plate Radius cm
Fig. 2. The induced electric potential measured in the medium 2 cm from the centerofthe Petri dish.
2 3 4 5
E in V/m
E in mV/cm
0.9 1.35 1.8 2.25
9 13.5 18 22.5 Bioelectromagnetics
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for 4 h after which the above procedure was repeated. For the ALP (Sigma Co.) assay, 100 ml of ALP reagent was added to 10 ml of cell lysate supernatant, incubated in the dark at room temperature for 30 s and read using a plate reader at a test wavelength of 405 nm. Alizarin Red S Staining and CPC Extraction The formation of calcium phosphate by SaOS-2 cells was determined using ARS staining [Stanford et al., 1995]. ARS binds to calcium salts [Puchtler et al., 1969]. Two experimental groups of cells were maintained for the ARS (Sigma Co.) assay. Mineral formation of the cells in culture was assessed using ARS staining as described in Gillette and NielsenPreiss [2004] and Jensh and Brent [1966] at the end of 24 h cycle for each termination point. The cell monolayers were washed with 1 ml of PBS per well, then fixed with 1 ml of 100% methanol for 10 min at 20 8C. Cells were then stained with ARS solution for 10 min and washed three times with sodium acetate buffer solution (pH ¼ 6.3). Mineral nodules were documented by photomicroscopy at random locations within each well. ARS staining was then quantified by incubating the cell culture plates at room temperature with 3 ml of CPC (Fisher Scientific, U.S. Headquarters, Pittsburgh, PA) per well for 1 h with gentle agitation. The dyed solution was then removed and 100 ml aliquots were transferred to a 96 well plate, diluted with 100 ml of dH2O and read at 570 nm.
Statistical Analysis Statistical analysis was performed using the Student’s t-test with a minimal confidence level of 0.05 for statistical significance. Each experiment was performed at least three times with a minimum of 6 samples per termination point, resulting in a total number of 18 samples for each experiment. The data shown constitutes a representative sample of experiments performed. RESULTS Repetitive Pulse PEMFs Do Not Change Cellular Metabolism and Cell Number The optical density of MTT assay and cell counts increased throughout the cell culture period. The average cell number in PEMFs group was consistently 10% higher, although statistically indistinguishable. The optical density of MTT assay for control and PEMF group did not differ after days 1–3 of stimulation (Fig. 3). Figure 4 demonstrates that PEMFs stimulation did not affect cell counts. PEMFs Change ALP Membrane Bound Activities in Medium With and Without Mineralization Agents In the experiments with and without mineralization agents, there were significant increases in ALP
Fig. 3. The effect of repetitive pulse PEMFs on the cellular viability of SaOS-2 cells. Day 0 corresponds to the viability of cells at the start of the experiment. The optical density determined by the MTT assay for both control and PEMFs group did not show any statistical difference fordays1^ 3. Bioelectromagnetics
Cellular Activity of SaOS-2 Cells
Fig. 4. The effect of PEMFs on SaOS-2 cell number. Cell counts for the control and PEMFs stimulated group did not show any statistical difference fordays1^ 3.
membrane bound activity after PEMFs stimulation. For the group with medium consisting of a-MEM supplemented with 10% FBS but without mineralization ingredients, ALP activity increased by 85% after 4 h of stimulation (Fig. 5, day 1). For subsequent time points at days 2 and 3, ALP activities of PEMFs stimulated group increased by 43% and 80% over the control group. For the group with a-MEM supplemented with 10% FBS and mineralization-inducing agents, a similar trend is observed (Fig. 6). The ALP activity increased by over 90% for each day of PEMFs stimulation compared to the control group. For both groups, the ALP activity gradually increased during days 1 and 2 of culture, and decreased slightly during day 3.
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Fig. 6. Changes of ALP activity in lysates for cells with culture media consisting of aMEM and 10% FBS with mineralization ingredients (n ¼ 6).The ALPactivityin PEMFsgroupincreased significantly over control group on day1of PEMF stimulation (P < 0.01). At day 3 ofculture,ALPactivityofthe PEMFsgroupwasabout twice that of the control group (P < 0.001).
PEMFs Increase Mineral Nodules for Cells With Mineralization Agents in Medium Four days after plating, cells reached confluence and PEMFs stimulation was started. SaOS-2 cells underwent biomineralization when cultured under normal conditions and in the presence of PEMFs. To examine the effect of PEMFs on mineral formation of SaOS-2 cells, the mineral nodules were stained with ARS stain. Figure 7 shows the results for ARS stain assay. SaOS-2 cells normally did not exhibit mineral formation until 24–36 h of introducing medium with mineralization ingredients. On day 2, PEMFs-stimulated cell cultures showed mineral nodule formation. For days 3 and 4 of culture, nodule size increased for both the control and experimental groups; however, the PEMFs group exhibited a greater concentration of nodules which were, on average, larger than those nodules in the control group. The PEMF group also exhibited a darker cell associated ARS stain. Biomineralization was not observed for the cell group cultured in the medium without mineralization-inducing agents. ARS was extracted from the monolayer by incubation of the monolayer in 3 ml of CPC for 1 h for quantification. The results confirmed a significant increase in mineralization by day 2 of PEMFs exposure (Fig. 8).
DISCUSSION Fig. 5. Changes of ALP activity in lysates for cells with culture media consisting of aMEM and 10% FBS without mineralization ingredients (n ¼ 6). After PEMFs stimulation, the ALP activities in lysates were always significantly higher than those in control group.The ALP activity in the PEMFs group increased 85%, 43%, and 80% compared to the control group on days 1^ 3 of PEMF (P < 0.01forall days of PEMFs stimulation).
While PEMFs appeared to have little effect on the phenotype and number of SaOS-2 cells, various metrics of cell activity were increased with PEMF stimulation. Our present work demonstrates an increase in ALP activity for cells stimulated by PEMF and cultured in medium with and without mineralization ingredients Bioelectromagnetics
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Fig. 7. Mineral nodule formation in cell culture observed under microscope with10 magnification. Therow onthe topisthe controlgroup and onthe bottomisthe PEMFs group.The cellswere cultured with aMEM supplemented with 10% FBS and mineralization ingredients. [The color figure for this article is available online at www.interscience.wiley.com.]
compared to the control group; in all three groups ALP activity decreases slightly by the third day of culture. However, the pattern of increase and decrease in ALP activity may be explained by the peak of ALP activity for SaOS-2 on day 2 of culture period which occurs coincidently with the initiation of mineralization. While reports on the effects of PEMF stimulation on ALP activity have been contradictory, we found a general increase in ALP activity with PEMF stimulation. When cultured in medium containing mineralization ingredients, SaOS-2 cells stimulated with PEMF formed many, larger mineral nodules by day 2 (Fig. 7)
Fig. 8. Mineralnodulesstainedwith ARS extractedby CPC (n ¼ 6). The optical density of ARS extracted by CPC in the control and PEMFs group increased during the culture period. The optical density increased significantly in the PEMFs group over control during days 2 ^ 4 of culture (P < 0.001foreach day, respectively). Bioelectromagnetics
whilst the control group showed only few, small nodules. The significant ARS staining was also observed for PEMFs group during days 3 and 4 of the culture period and the control group did not match this staining until days 4 and 5. PEMFs thus appear to accelerate the induction of mineral formation in the matrix by SaOS-2 cells. The results presented herein pose many more questions than answers. We look forward to expanding the sophistication and breadth of our future work. To further evaluate the spatial deposition of ECM in control and stimulated cultures, future studies will utilize immunoblots to quantify changes in osteonectin and bone deposition associated protein expression. We also seek to address the nature of the inorganic mineral salts deposited in control and PEMFs-stimulated cultures. High-resolution microscopy techniques will be applied to further study the mineral nodules such as: low vacuum scanning electron microscopy to visualize the location of nodules with respect to specific cells, energy dispersive X-ray analysis (EDAX) to characterize the mineral’s elemental composition, and transmission electron microscopy (TEM) to infer the crystal structure and chemical composition of the mineral particles. A study of mineral produced by SaOS-2 cells in culture demonstrated membrane vesicles containing calcium phosphate, thus revealing the utility of such techniques [Ayers et al., 2006]. Additionally, confocal microscopy can be used to monitor calcium flux in specific cellular regions and organelles during PEMF stimulation by tagging fluorescent proteins to calcium [Palmer and Tsien, 2006].
Cellular Activity of SaOS-2 Cells
The apparent lack of separation of electric and magnetic effects in the current work was unanticipated. Here, an induced electric field of 1 mV/cm was measured near the center of the 10 cm Petri dish; an electric field of 20 mV/cm was measured at the edge. These measurements can be used to estimate the induced electric field at the inner and outer wells of the 12.5 cm by 8.5 cm 6 well plates used in this study. Taking the measurement from the Petri dish, it was estimated that the electric field was 1 mV/cm near the central well of the plate, and more than 20 mV/cm at the four corner wells. If cellular responses are associated with electric fields strength ranging from 1 to 2 mV/cm [Bassett et al., 1978], a spatial dependence of the mineralization response would be expected. Several experiments were conducted to evaluate mineralization processes in this study; however, no difference was observed in mineral deposition for cells seeded in the inner and outer wells, indicating no spatial dependence on the induced electric field intensity. CONCLUSION Cellular activities and biomineralization of SaOS-2 cells under PEMFs with repetitive pulse burst waveform were investigated. Our results demonstrate the application of inductive coupling electromagnetic fields to stimulate SaOS-2 cells. PEMF stimulation with repetitive pulse burst waveform did not affect metabolic activity or cell number. However, the ALP activities of SaOS-2 cells and mineral nodule formation increased significantly after PEMF stimulation. Future studies will focus on the investigation of the effects of waveform to cellular activities, and Western blots for protein expression of bone mineralization specific proteins and mineral content analysis of calcium deposits. We also seek to implement a more sensitive assay to quantify early stages of biomineralization [Gregory et al., 2004]. Overall, the observations presented herein suggest that repetitive pulse burst PEMF may play a significant role in the enhancement of SaOS-2 cell biomineralization. REFERENCES Ayers R, Nielsen-Preiss S, Ferguson V, Gotolli G, Moore JJ, Kleebe H-J. 2006. Osteoblast-like cell mineralization induced by multiphasic calcium phosphate ceramic. Mater Sci Eng C26 6(8):1333–1337. Bassett CA, Pawluk RJ, Pilla AA. 1974. Acceleration of fracture repair by electromagnetic fields. A surgically noninvasive method. Ann N Y Acad Sci 238:242–262. Bassett CA, Pilla AA, Pawluk RJ. 1977. A non-operative salvage of surgically-resistant pseudarthroses and non-unions by
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