Evaluation of the Effects of a Moderate Intensity Static Magnetic Field Application on Human Osteoblast-Like Cells

There is a general interest in the effects of magnetic fields on human tissue, for biomedical imaging, cancer therapy or tissue engineering applications. In particular the orthopaedic field may greatly benefit from magnetic scaffolds, magnetic fixation and magnetic delivery techniques. In this study, MG-63 osteoblast-like cells were analysed in vitro after exposure to a 320mT static magnetic field (SMF), either continuously or applied fo r 1h at any 24-hour interval. Results demonstrate that SMF causes a reduction in cell number after 7 days of exposure, as demonstrated by the MTT assay. This reduction in proliferation is not associated to an increase in Lactate Dehydrogenase production, a marker of cellu lar stress and/or disruption of membrane integrity. Osteocalcin secretion increased at day 3 for the condition 1h/day exposure and this effect was reversed after 7 days. Instead, the continuous application of a SMF resulted in a significantly decreased osteocalcin release at day 7. Cell distribution, morphology and cytoskeleton organization were unaltered, with the typical osteoblastic morphology maintained and normal distribution of cytoplasmic actin fibrils, as demonstrated by phalloidin staining. Gene expression analysis of COL1A1, ALPL and RUNX2 show no alterations respect to control. These results suggest that such a moderate intensity static magnetic field has a detrimental effect on cell proliferation and osteocalcin secretion, while maintaining morphological features and gene expression unaltered. The in vitro effects of magnetic fields depend on cell type, magnetic field intensity and modality of applicat ion. Th is study gives a contribution to understand moderate strength static magnetic field effects on human tissue, with particular importance for the orthopaedic field.


Introduction
There has been an increasing interest in the study of magnetic field employ ment in the clinical setting, either for magnetic resonance imag ing, for magnetically guided delivery of bio molecu les for tissue engineering applications or cancer therapy. In particular in the orthopaedic field, the use of static magnetic fields finds a broad range of applications, fro m the ones mentioned to the possibility of having in situ magnetic fixed stations for prosthesis or scaffold fixat ion. In fact, our laboratory has recently developed a simu lated scaffold fixation technique for osteochondral defect surgery that makes use of magnetic forces attracting the scaffold towards the imp lanted position 1 .
Also the use of superparamagnetic nanoparticles (MNPs) for bio logical and med ical purposes has been increasing, either as contrast agent for diagnostic MRI imaging, fo r its hyperthermia effect, for magnetic drug delivery and for tissue engineering applicat ions [2][3][4][5][6] . We have recently demonstrated that intrinsically magnetic iron-substituted hydroxyapatite nanoparticles significantly increase osteoblast proliferation and activity when exposed to a static magnetic field 7 and the same nanoparticles have been used together with a biodegradable poly(caprolactone) core for the development of a magnetic scaffold for bone tissue engineering 8 . Also, we have introduced magnetite into the structure of a macroporous hydroxyapatite ceramic scaffold, which demonstrated in vitro biocompatibility, either in the presence or absence of a magnetic field, and good in vivo osteointegration 9 . Moreover, we have designed two hydroxyapatite/collagen (70/30 wt %) magnetic scaffolds, magnetized with two different techniques: direct nucleation of bio mimetic phase and MNPs on self-assembling co llagen fibers and scaffold impregnation in a ferro-fluid solution 10,11 . Recently, we have designed magnetic biohybrid scaffo lds made of co llagen and intrinsically magnetic iron-substituted M agnetic Field Application on Human Osteoblast-Like Cells hydroxyapatite for bone regeneration 12 . The possibility of using superparamagnetic phases mostly relies on the application of an external magnetic field able to activate such materials and/or to drive bio mo lecule loaded MPN to desired specific sites. The effects of such magnetic fields on the human tissue need to be well investigated in order to understand the possible range of application of such magnetic devices and its implication for the different biomed ical applications. Cell culture studies directed at exploring both the biocompatibility of magnetic devices and also the effects of magnetic fields in biological tissues are needed. In this context, this study analysed for the first time the effect of a moderate intensity static magnetic field (320 mT) on M G-63 hu man osteoblast cell culture, and intends to give a contribute to deepen the knowledge on possible applications of magnetic devices driven by magnetic forces in the clinical setting.

In Vitro Cell Cul ture
MG-63 hu man osteoblast-like cells (Lonza, Italy) were cultured in DM EM/F12 med iu m (PAA, Austria) containing 10% FBS and Penicillin -Streptomycin (100 U/ ml-100 µg/ ml) and kept at 37℃ in an at mosphere of 5% CO 2 . Cells were detached from cu lture flasks by trypsinization, centrifuged and resuspended. Cell nu mber and viability were assessed with the trypan-blue dye exclusion test. 2.5x104 cells per well were plated into a 24-mu ltiwell and cultured for 1, 3 and 7 days, in the absence (control) or presence of an external magnet, which relied on the applicat ion of a co mmercially available static magnetic field of 320 mT under the mu lt iwell plates (MagnetoFACTOR-24, Chemicell, Germany), which ensures equal application of the magnetic field under each well. A third condition relied on the exposure of cells to the static magnetic field for 1 hour at any 24-hour interval.
All cell handling procedures were performed in a sterile laminar flow hood. Cell cu lture incubation was performed at 37℃ with 5% CO 2 .

MTT Assay
The MTT reagent (3-(4,5-d imethylthiazol-2-yl)-2,5diphenyltetrazoliu m b ro mide) (Invitrogen) was prepared at 5 mg/ ml in 1x PBS. Cells were incubated with the MTT reagent 1:10 for 2 h at 37℃. Mediu m was d iscarded and cells incubated with 1 ml of Dimethyl sulfo xide (Sig ma) for 15 min. In this assay, the metabolically active cells react with the tetrazoliu m salt in the MTT reagent to produce a formazan dye that can be observed at λmax of 570 n m, using a Multiskan FC Microplate Photometer (Thermo Scientific). This absorbance is directly proportional to the nu mber of metabolically active cells. Mean values of absorbance were determined. Three samples were analysed per group per time point.

Lactate Dehydrogenase Assay
Liqui Lactate Dehydrogenase (LDH) assay (Futura System) was performed according to manufacturer's instructions. Briefly, LDH solution was prepared by mixing 4 parts of Buffer solution to 1 part of Substrate solution. 20 µl of cell mediu m was added to 1 ml of LDH solution. Absorbance was read at 0, 1, 2 and 3 min at λmax of 340 n m, using a Multiskan FC M icroplate Photometer (Thermo Scientific). LDH activ ity was determined by calculating the ∆OD/ min. LDH activ ity was expressed as U/l. Three samples were analysed per group per time point.

Human Osteocalcin Elisa
Osteocalcin (OST) secretion was measured by the Human Osteocalcin ELISA kit (Invitrogen), following manufacturer's instructions. Briefly, 25 µl o f cell mediu m was incubated with 100 µl of anti-OST-HRP antibody for 2 hours at room temperature, within the supplied microtiter plate, coated with a second anti-OST antibody. After washing, 100 µl of chro mogenic solution was added to the wells and plate incubated at room temperature in the dark. Reaction was stopped 25 min after addit ion of 100 µl of stop solution. Absorbance was read at λmax of 450 n m, using a Multiskan FC M icroplate Photometer (Thermo Scientific). The amount of osteocalcin was determined by interpolation fro m a standard curve of known concentrations. Osteocalcin concentration was expressed as ng/ml. Three samples were analysed per group per time point.

Phalloi din Immunofluorescence Staining
Cells were washed with 1x PBS for 5 min, fixed with 4% (w/v) paraformaldehyde for 15 min and washed with 1x PBS for 5 min. Permeabilizat ion was performed with 1x PBS with 0.1% (v/v) Triton X-100 for 5 min. FITC-conjugated Phallo idin antibody (Invitrogen) 1:500 in 1x PBS was added for 20 min at roo m temperature in the dark. Cells were washed with 1x PBS for 5 min and incubated with 300 nM DAPI (4'-6-Diamid ino-2-phenylindole) solution (Invitrogen) in 1x PBS for 5 min . Images were acquired by an Inverted Ti-E fluorescence microscope (Nikon). Two samp les per group were analysed.

Total RNA Extraction and Reverse Transcription
At each time point, cells were collected by trypsinization, centrifuged, resuspended in RNAlater solution and kept at -20℃. Total RNA isolation was performed by use of the Tris reagent, followed by the Purelin k RNA Min i kit according to manufacturer's instructions. In particular, we have used a on-column DNAse treat ment by use of the Purelin k DNAse set. Purified total RNA was eluted with THE RNA storage solution and kept at -80℃ until reverse transcription. RNA integrity was analysed by native agarose gel electrophoresis (1.5% agarose in 1x TBE, with 1 µg/ ml of ethid iu m bro mide), run in 1x TBE buffer and quantification performed by the Qubit® 2.0 Fluoro meter (Invitrogen), together with the Qubit® RNA BR assay kit, following manufacturer's instructions. cDNA was obtained by using the High capacity cDNA reverse transcription kit, accord ing to manufacturer's instructions. In particular, we have used 1.5 µg of total RNA in a 20 µl final reaction volu me for each sample, as fo llo ws: 2 µl of 10x RT buffer, 0.8 µl of 25x d NTP mix, 2 µl of 10x RT primers, 1 µl of RNAse inhibitor, 3.2 µl o f Nuclease free water plus 10 µl of temp late. Reaction conditions: 25℃ for 10 min, 37℃ for 120 min, 85℃ for 5 min. cDNA was kept at -20℃ until qPCR. All reagents and instruments were fro m Life Technologies.

Quantitati ve Real-ti me Pol ymerase Chai n Reacti on (qPCR)
Quantificat ion of Collagen, type I, alpha 1 (COL1A1), Alkaline Phosphatase (ALPL), Runt-related transcription factor 2 (RUNX2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression was perfo rmed by use of the StepOne™ Real-Time PCR System (Applied Biosystems). We used the Taqman gene expression mastermix and the following Taq man gene expression assays: GA PDH: Hs99999905_ m1, COL1A 1: Hs00164004 _m1, A LPL: Hs01029144_ m1 and RUNX2: Hs00231692_ m1, all with the Fam™ dye as reporter. 1 µl of cDNA was used in a 10 µl final reaction volu me, as follows: 5 µl of 2x mastermix, 0.5 µl of 20x gene expression assay, 3.5 µl of Nuclease free water p lus 1 µl of template. qPCR reaction was performed on a 48-well plate adhesive sealed, as follows: 40 cycles at 95℃ for 15 sec, 60℃ for 1 min, after an init ial denaturation step of 50℃ fo r 2 min and 95℃ for 10 min. Data was collected using the OneStep Software (Applied Biosystems) and relative quantification was performed using the comparative threshold (Ct) method (ΔΔCt), where relative gene expression level equals 2−ΔΔCt (Livak and Schmittgen, 2001), as described by the manufacturer. In detail, changes in gene expression level of the target genes COL1A 1, A LPL and RUNX2 were calcu lated by normalizat ion to the reference GAPDH and by normalization to the control condition, used as calibrator, within each sample set. Changes in gene expression were reported as fo ld changes of target relative to controls and represent averages fro m trip licate measurements. All reagents and instruments used for gene expression analysis were from Life Technologies.

Statistical Analysis
For MTT, LDH and Osteocalcin experiments, results were expressed as MEAN±SEM plotted on graph (n=3). Analysis of differences between groups was made by one-way ANOVA fo r each time point, fo llo wed by Dunnett's post-hoc test. For gene exp ression experiments, results were expressed as RQ with MAX and MIN values plotted on graph (n=3). All statistical analysis made use of the GraphPad Pris m software (version 5.0), with statistical significance set at p≤0.05.

Results and Discussion
In this study, we analysed in vitro the effect on a MG-63 osteoblast culture of a moderate strength static magnetic field (SM F) of 320 mT, a value that is within the range (1mT to 1T) demonstrated to have particular imp licat ions in a number of biological phenomena 13,14 . We have used two different conditions: continuous exposure and exposure for 1h per day, envisaging a possible clin ical setting application, where the patient would be submitted to therapy for a restricted period of time. Experimental time points were for all experiments 1, 3 and 7 days, except for gene expression analysis where only day 7 was considered.

Application of a Static Magnetic Fiel d Reduces Cell Proliferati on
The MTT assay is a reliable method to quantify cell proliferation at each time point. Results demonstrate that when exposed for either 1h/day or continuously to a SMF, cells continue to grow fro m day 1 to day 7 (Fig. 1A), but presenting a considerably reduced number of cells respect to control, at day 7 (p≤0.01). St ill, this reduction in proliferation observed at day 7 is not associated to an increase in cellular apoptosis, stress or disruption of memb rane integrity, in fact quantification of the cellular stress marker Lactate Dehydrogenase (LDH), released when cell memb rane integrity is disrupted, showed no difference between groups at any of the time points (Fig. 1B). Figure 1. Analysis of cell proliferation and cellular stress. Quantification of cell proliferation was performed by the MTT assay (A) and quantification of cellular stress was performed by the Lactate Dehydrogenase (LDH) assay (B) at 1, 3 and 7 days for control osteoblasts, osteoblasts exposed for 1h/day to a static magnetic field (SMF) or continuously exposed to a SMF. n=3, ** p≤0.01 M agnetic Field Application on Human Osteoblast-Like Cells The observed reduction in proliferat ion is in contrast to a previous study that used murine MC3T3-E1 osteoblasts and showed that a moderate SMF resulted in enhanced proliferation 13 . Still, this was a different cell line and the intensity of the SMF was less than half (150 mT) the one used in this study (320 mT), which could exp lain different results.
Another study instead demonstrated MG-63 osteoblasts increased proliferation when exposed to a 400 mT SMF, yet this study was performed on a poly-L-lactide substrate 15 . Figure 2. Analysis of the osteogenic marker osteocalcin. Quantification of released osteocalcin was performed by ELISA at 1, 3 and 7 days for control osteoblasts, osteoblasts exposed for 1h/day to a static magnetic field (SMF) or continuously exposed to a SMF (n=3). * p≤0.05, *** p≤0.001

Osteocalcin is Reduced after 7 Days of Expos ure
Osteocalcin is secreted by osteoblasts and is the major non collagenous component of the bone extracellular matrix, being used as a reliable marker for bone formation. The quantification of the osteocalcin released by M G-63 osteoblasts to the cell culture was performed by the Human Osteocalcin Elisa Kit. Results demonstrate that application of a SMF for 1h/day increases osteocalcin release at day 3, respect to control (p≤0.05), while this effect is reversed after 7 days (Fig. 2). Instead, the continuous application of a SMF resulted in decreased osteocalcin release, respect to control (p≤0.001). These results might indicate a beneficial effect of the application of a SM F for only 1h/day at the short time, which may result prejudicial if applied continuously and for a more extended period of time. Another study has demonstrated that application of a 150 mT SMF on murine MC3T3-E1 osteoblasts induced increased osteocalcin secretion after 15 days 13 . Overall, such results suggest important differences between cell types, magnetic field intensity and duration and mode of application, so that individual conditions must be optimized for specific clinical application set-ups.

No di fferences in Cellul ar Distri buti on or Morphology
In order to determine effects of the SMF on cellular distribution, mo rphology and cytoskeleton organization, at day 7, we have performed immunofluorescence analysis against phalloidin, a to xin that binds F-actin filaments, and DAPI, a nucleic acid stain co mmonly used as cell nuclei marker. Overall, we have not observed any differences between osteoblasts cultured as control or exposed to the SMF. Cells were nicely distributed throughout the cell culture well (Fig. 3A,B), connections between the cells re mained unaltered and the typical osteoblastic morphology was not lost (Fig. 3C,D). The d istribution of the actin fibrils on the cell cytoplasm was normal (Fig. 4). A previous study instead has shown that MG-63 cells treated with 400 mT SMF had mo re d ifferentiated morphologic features already after 3 days 16 , while a SM F of a mere 6 mT has been shown to influence cell membrane integrity 17 . Our morphological results instead are coherent with the LDH assay results, which measure membrane disruption, in showing an absence of SMF effects on cell membrane integrity.

No Alteration in COL1 A1, ALPL or RUNX2 Gene Expression
Gene exp ression analysis was performed by reverse transcription quantitative real time PCR, at day 7, for groups control, SMF and 1h/day SMF. Collagen type I, alpha 1 (COL1A1), alkaline phosphatase (ALPL) and Runt-related transcription factor 2 (RUNX2) were analysed, using GA PDH as reference. Collagen type I is the main extracellular matrix co mponent of osteoblast cultures, alkaline phosphatase is a marker of osteoblast activity and Run x2 is associated with osteoblastic differentiat ion. No difference was observed in their relative gene expression for any of these proteins. Instead, a previous study demonstrated that a lo w intensity SMF has resulted in decreased ALPL and COL1A 1 gene expression on osteoblast cell cultures for up to 14 days 18 .

Conclusions
This study analysed the effects of a static magnetic field intensity of 320 mT on MG-63 hu man osteoblast-like cells in vitro. Our results suggest a detrimental effect on cell proliferation by the applicat ion of a SMF, either continuously or applied for 1h/day. This reduction was not associated to an increase in cellular apoptosis, stress or disruption of membrane integrity, quantified by the cellular stress marker Lactate Dehydrogenase. Quantification of osteocalcin release, a marker for bone format ion, demonstrated that application of a SM F fo r 1h/day increases osteocalcin release at day 3, while this effect was reversed after 7 days. Instead, the continuous SMF application resulted in decreased osteocalcin release, which might indicate a beneficial effect of the applicat ion of a SMF for only 1h/day at the short time, which may result prejudicial if applied continuously and for a more extended period of time. No differences were seen by immunofluorescence analysis in cellular distrib ution, m orp holo gy or c ytosk elet on organization after 7 days in culture. Quantitative PCR analysis for COL1A 1, A LPL and RUNX2, all markers of osteoblast activity, identified no alterations in gene expression introduced by the SMF to the cell culture. For sure the effects of magnetic fields on in vitro cell culture depend on cell type, magnetic field intensity and duration and mode of application. Th is study gives a contribution to understand moderate strength static magnetic field effects on human tissue.