Medical & Biological Engineering & Computing

, Volume 48, Issue 8, pp 793–798

Static magnetic field exposure promotes differentiation of osteoblastic cells grown on the surface of a poly-l-lactide substrate

Authors

  • Sheng-Wei Feng
    • School of DentistryTaipei Medical University
  • Yi-June Lo
    • Dental Department of Wan-Fang HospitalTaipei Medical University
  • Wei-Jen Chang
    • School of DentistryTaipei Medical University
  • Che-Tong Lin
    • School of DentistryTaipei Medical University
  • Sheng-Yang Lee
    • School of DentistryTaipei Medical University
  • Yoshimitsu Abiko
    • Department of Biochemistry, School of Dentistry at MatsudoNihon University
    • Graduate Institute of Biomedical Materials and EngineeringTaipei Medical University
    • Research Center for Biomedical Implants and Microsurgery DevicesTaipei Medical University
    • Research Center for Biomedical DevicesTaipei Medical University
Original Article

DOI: 10.1007/s11517-010-0639-5

Cite this article as:
Feng, S., Lo, Y., Chang, W. et al. Med Biol Eng Comput (2010) 48: 793. doi:10.1007/s11517-010-0639-5

Abstract

This study investigated the effects of static magnetic fields on the differentiation of MG63 cells cultured on the surface of poly-l-lactide (PLLA) substrates. The cells were continuously exposed to a 4,000 Gauss-static magnetic field (SMF) for 5 days. The proliferation effects of the SMF were measured by MTT assay. Morphologic changes and extracellular matrix release were observed by scanning electron microscopy. The effects of the SMF on alkaline phosphatase activity levels were compared between exposed and unexposed cells. The SMF-exposed cells exhibited decreased MTT values after 1 and 3 days of culture. In addition, SMF exposure promoted the expression of extracellular matrix in MG63 cells on the PLLA substrate. After 1 day, the alkaline phosphatase-specific activity of SMF-exposed MG63 cells was significantly increased (P < 0.05) with a ratio of 1.5-fold. These results show that MG63 cells, seeded on a PLLA disc and treated with SMF, had a more differentiated phenotype.

Keywords

Static magnetic fieldPLLAMG63Differentiation

1 Introduction

Biodegradable polymer scaffolds are widely used in the reconstruction of bony defects because of their good biocompatibility, biodegradability, and low toxicity after degradation in vivo [26, 31]. Poly-l-lactide (PLLA) has become the most popular bioresorbable material for fixation or scaffolding because of its controlled physical and chemical surface properties [15]. In the clinical application, PLLA scaffold were applied in the dental therapy for guided tissue regeneration/guided bone regeneration as a barrier. Gottlow et al. [10] proposed PLLA membrane could allow gingival connective tissue integration while excluding epithelium with eventual connective tissue integration with PDL tissue [10]. Furthermore, Cortellini et al. demonstrated PLLA membrane could have 4.6 ± 1.2 mm clinical attachment gain in the clinical study [6]. Because of the above evidences, PLLA membrane application was getting more and more in the modern dentistry. However, low proliferation rate and poor cell attachment were found when cells were cultured on the surface of PLLA [4, 12]. In order to improve cell growth on the PLLA surface, many strategies for surface and structure modification of PLLA scaffolds have been introduced [16, 25].

Several studies have demonstrated that scaffold design, growth factor, dynamic culture system, and physical simulation could promote proliferation and differentiation of cells in culture for tissue engineering [3, 21, 22, 26, 27]. Mechanical loads induced by extracellular fluid flow, low-intensity ultrasound, hydrostatic pressure, and electromagnetic fields (EMF) produced physiological effects on bone cells [19, 28]. In addition, pulse electromagnetic fields (PEMF) have been used extensively in the clinical treatment of non-united bone fractures for more than two decades [9, 18]. In order to enhance osteoblast proliferation and differentiation for tissue engineering, PEMF bioreactors were developed in several studies [8, 26]. These studies used the apparatus to treat osteoblast cells seeded on porous polymer. Their findings show that the electromagnetic bioreactor regulated cell behavior and suggested their potential role in bone tissue engineering.

The permanent magnet is a treatment variant also used in clinical practice [7, 20]. Recently, cell culture studies have demonstrated that, like PEMFs, static magnetic fields (SMF) can induce osteoblastic cell differentiation in the early maturation stage [5, 11, 23, 30]. During SMF stimulation, the cellular membrane is assumed to be the target. It has been reported that phospholipids can be oriented by SMF, resulting in overdeformation of the cellular membrane. This can modify the biological properties of imbedded receptors in the membrane, and thereby alter the proliferation kinetics of the cells [15]. Accordingly, Kim et al. used SMF as a proliferation stimulator for osteoblastic cells seeded on a titanium disc. Their results indicated that SMF had a significant effect on cell attachment and proliferation on the titanium surface [11].

The use of a permanent magnet provides an important advantage for bone cell stimulation in that it does not require a power device and thereby reduces the unexpected thermal effects on treated cells. However, the effect of SMF on cell behavior on the PLLA surface remains unclear. In this study, we used cellular models to examine the effect of SMF on the growth of osteoblast-like cells on a PLLA membrane.

2 Materials and methods

2.1 PLLA sample preparation

In order to prepare PLLA substrates, PLLA chips with molecular weight of 140 kDa (lot# 020301; BioTech One Ltd., Taipei, Taiwan) were vacuum-dried at 70°C overnight to reduce the moisture content. A horizontal injection molding machine was used in this study (Fig. 1a, b). Disc shape specimens (14 mm diameter, 1 mm thickness) were manufactured at an injection temperature of 185°C (Fig. 1c) in a clean room (class 10,000). The temperature settings of the feed, compression, and metering sections were 170, 175, and 180°C, respectively.
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Fig. 1

Setup for PLLA disc fabrication. a Schematic of the molding machine used in this study, b Injection molds for fabrication of PLLA discs, c Geometry of injected PLLA disc

2.2 Surface roughness measurement

Surface roughness of SMF-exposed and sham-exposed cell-free PLLA discs were measured using scanning topography measurement (STM; Talyscan 150, Taylor Hobson Ltd., Leicester, GB) with a diamond-stylus inductive gage (2 μm tip radius, 90o tip angle) for contact measurement. Nine areas (0.5 × 0.5 mm2) on the PLLA surface were randomly moved on the stage to scan the complete measurement areas. Each profile was recorded at a resolution of 1 μm. The roughness (Sz) was defined as the length of vertical movement of the gage.

2.3 Contact angle measurement

Contact angle measurements were used to test the differences in surface property between SMF-exposed and sham-exposed cell-free PLLA discs. The static water contact angles were measured with water using a goniometer (KYOWR, CA-VP 150, Japan) at room temperature. The images of the droplet contacting the PLLA disc were recorded using a digital camera connected to the eyepiece of a microscope. The contact angle (θ) was calculated through measurement of the height and width of drops placed on the PLLA surfaces using image-processing software (Image Pro Plus, Media Cybernetics, Inc., MD, USA). For each data, three drops of water on the sample surfaces were measured. Data are presented as mean ± standard deviation (SD) of four samples.

2.4 Cell culture

In order to test the effects of SMF on cell behavior on the PLLA surface, MG-63 osteoblast-like cells (ATCC CRL-1427) were cultured on PLLA discs that were placed in 24-well polystyrene tissue-culture plates at a density of 2.5 × 104 cells per ml. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; HyClone, Logan, Utah, USA) supplemented with l-glutamine (4 mM), 10% fetal bovine serum (FBS), and 1% penicillin–streptomycin. Cell cultures were incubated in 5% CO2 at 37°C and 100% humidity. The SMF-exposed cells were placed on the surfaces of a neodymium (Nd2Fe14B) magnet (Fig. 2). The average magnetic flux density on the surface was 0.4 T (range, 0.38–0.43 T). In the sham-exposed group, unmagnified neodymium disks were used. Before the cells were plated onto the magnet, both the exposed and control cells were incubated in an unexposed environment for 24 h.
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Fig. 2

Illustration of the cell culture system with SMF stimulation

In order to test the growth differences between SMF-exposed and sham-exposed cells, cell viability was detected every 2 days for 5 days. At each observation time, the cells were incubated with a tetrazolium salt, MTT (MTT kit; Roche Applied Science, Mannheim, Germany). Four hours after the addition of the colorometric substrate, the viable cells have converted the MTT salt to a water-insoluble formazan dye, which can be quantitated after solubilization with 500 μl DMSO for 5 min using a microplate reader (Model 2020; Anthos Labtec Instruments, Wals, Austria) at 570/690 nm. The absorbance directly correlates with the cell number.

2.5 Detection of alkaline phosphatase activity

The alkaline phosphatase activity of MG-63 cells cultured on PLLA with and without SMF exposure was determined as the rate of conversion of p-nitrophenyl phosphate to p-nitrophenol at a pH of 10.2. Briefly, at each observation time, the media was aspirated, the cells were washed three times with PBS, and 300 μl 0.05% (V/V) Triton X-100 (Sigma-Aldrich Co, St Louis, MO) was added per dish. After cell disruption by three freeze/thaw cycles, 50 μl cell lysate from each dish was transferred to a 96-well microtiter plate. Absorbance was measured at 405 nm. Total cell lysate also was used for protein determination. Bicinchoninic acid (BCA Protein Assay Kit; Pierce, Rockford, IL) was added to the cell lysate and incubated for 10 min. Absorbance was read spectrophotometrically at a wavelength of 590 nm. Specific enzyme activity was calculated using these two parameters.

2.6 Scanning electron microscopy

For scanning electron microscopy (SEM), the culture media was removed and the samples were washed three times with PBS. Then, the MG63 cells seeded on the PLLA disc were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde for 20 min, and fixed with 1% osmium tetroxide in 0.1 mol/l PBS for 30 min. After initial fixation, the samples were washed and treated with 1% osmium tetroxide for 1 h for post fixation. The samples were washed with PBS and dehydrated in an ethanol series of the concentrations of 70, 80, 90, 95, and 100% in a critical point dryer (HCP-2; Hitachi Ltd, Tokyo, Japan). A thin layer of gold was coated onto the cell-cultured side of the PLLA substrate with a sputtering apparatus (IB-2; Hitachi, Ltd). The morphologic features of the cells then were examined using a Hitachi S-2400 electron microscope (Hitachi, Ltd).

2.7 Statistical analysis

For the MTT and ALPase assays, data are presented as mean ± standard deviation (SD) of four samples. The differences between two groups were compared using Student’s t-test. Probability values of less than 0.05 were considered significant.

3 Results

The roughness (Sz) measurements for SMF-exposed and sham-exposed cell-free PLLA discs were 2.6 ± 0.5 and 2.3 ± 0.9 μm, respectively. No significant difference was found between the roughness values for the two cell-free disc surfaces. The measured contact angle value of the SMF-exposed cell-free PLLA discs was 65.4° ± 2.6°. No significant difference was found when compared this data to the measured value of SMF-unexposed cell-free PLLA discs (64.1° ± 4.6°).

MG-63 cells were cultured on injection-molded PLLA substrates for 5 days. Significant differences in MTT counts were found after 1 day of culture. The cells, cultured on the PLLA substrates with SMF exposure, had a significantly decreased proliferation rate relative to the unexposed cells, with a maximum 1.2-fold difference at the first 24 h (Fig. 3) (P < 0.05).
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Fig. 3

Cell proliferation assay for growth of MG63 cells on the PLLA substrate. Cells were seeded at 2.5 × 104 cells/ml. Cellular exposure to SMF resulted in significant inhibition of proliferation rates (n = 4 cultures, * P < 0.05)

The cells had more mature morphologic features after SMF exposure (Fig. 4). Initially, cells cultured on PLLA surface were well spread and appeared to form a relatively thin, continuous monolayer (Fig. 4a). After SMF exposure for 1 day, the MG63 cells demonstrated more abundant extracellular matrix (Fig. 2c) compared to the unexposed cells (Fig. 2b).
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Fig. 4

Scanning electron microscopy images show untreated MG63 cells and cells treated with SMF stimulation at 24 h. a Cells cultured on TCP substrate without SMF exposure. b MG63 cells cultured on PLLA substrate without SMF exposure. c Abundant extracellular matrix (black arrows) accumulation was seen around the cells (on PLLA substrate) after 24 h of SMF exposure. (original magnification, ×500)

ALPase activity was actively expressed immediately after MG63 cells were plated onto the dishes and was gradually down-regulated during subsequent culture periods. After day 1, cells cultured on PLLA with SMF demonstrated a significant increase in ALPase specific activity of 1.5-fold compared to the control group (P < 0.01) (Fig. 5).
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Fig. 5

After 1 day of SMF exposure: a the alkaline phosphatase activity of the SMF-exposed cells on PLLA substrate was significantly greater than that of the sham-exposed cells (n = 4 cultures; * P < 0.05, ** P < 0.01)

4 Discussion

The surface roughness of biomaterials has been shown to strongly affect cell behaviors [29, 32]. In this study, measurement of geometric roughness at a resolution of 1 μm was used to control for this variable. Our results showed that SMF exposure had no obvious effect on substrate topography characteristics (Fig. 4). The surface property of biomaterials is another factor that affects cell proliferation and differentiation [2, 27]. In this study, measurement of the contact angle of the injection-molded PLLA revealed no differences between the SMF-exposed and sham-exposed cell-free PLLA samples, suggesting the changes in cell behavior of MG63 cells on the PLLA substrates in this study (Figs. 3, 4, 5) were decoupled from the surface characteristics of the PLLA.

Previous studies found that osteoblast-like cells expressed greater ALP and matrix after SMF exposure [2, 11, 13, 14, 29, 32]. Alkaline phosphatase plays an important role in matrix mineralization. Thus, the activity of ALPase is an early marker of osteoblast phenotypic differentiation. In the present study, the enzyme activity of SMF-exposed cells at 24 h was significantly greater than that of unexposed cells (Fig. 5). Furthermore, our SEM investigation indicated that the release of extracellular matrix, known to be enriched with alkaline phosphatase, was associated with the enzyme-activity changes (Fig. 4c). Aaron and Ciombor [1] also reported a significant increase in extracellular matrix synthesis when osteoblast-like cells were subjected to electromagnetic stimulation [1]. Since extracellular matrix is a promoter of mineral deposition, its release indicates the SMF-exposed cells shown in Fig. 4c progressed into the matrix development/maturation stage.

In order to improve the growth rates of cells seeded on PLLA substrate, Fassina et al. [8] developed a novel bioreactor that integrates PEMF to regulate osteoblast proliferation on polyurethane porous scaffold. Their results demonstrated that electromagnetic stimulation increased osteoblast cell proliferation approximately two times, and enhanced the gene expression specific for differentiation [8]. However, Tsai et al. [26] showed that the PEMF bioreactor inhibited cell proliferation but enhanced ALP activity during the culture period [26]. A previous study by Stein and Lian showed that there is a relationship between proliferation and differentiation during the osteoblast developmental sequence. They found that when the gene associated with extracellular matrix maturation is activated, the proliferation of the cells is down-regulated [24]. Similarly, Tsai et al. showed that the cell number reduction effects of PEMF were due to the differentiation of cells into more mature stages [8, 17].

The main mechanism responsible for PEMF-stimulated bone formation is electrical field in tissue. However, the major effects of SMF on osteoblast differentiation were inducing membrane reorientation and distortion, which alter the membrane properties [11]. Although the physical response of bone cells exposed to SMF is different than when exposed to PEMF, our results are consistent with the findings of Tsai et al. [26]. As shown in Fig. 3, we also found that the proliferation rate of MG63 cells was inhibited by SMF exposure. These results confirmed previous findings that both the continuous SMF- and PEMF-stimulated osteoblasts expressed more ALP activity than control cultures [5, 11, 17].

5 Conclusion

The results of this study suggest that the SMF method may provide an effective physical-simulated resource for cell culture on PLLA substrate. To our knowledge, this is the first study to report this finding.

Acknowledgements

This study was supported, by grants from Wan-Fang Hospital, Taipei Medical University, Taipei, Taiwan (98TMU-WFH-08), and, in part, by Association for Dental Sciences, ROC, Taiwan.

Copyright information

© International Federation for Medical and Biological Engineering 2010