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Mg ion implantation on SLA-treated titanium surface and its effects on the behavior of mesenchymal stem cell
Materials Science and Engineering C 33 (2013) 1554–1560
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Mg ion implantation on SLA-treated titanium surface and its effects on the behavior of mesenchymal stem cell Beom-Su Kim a, b, Jin Seong Kim a, b, Young Min Park c, Bo-Young Choi d, Jun Lee a, b,⁎ a
Wonkwang Bone Regeneration Research Institute, Wonkwang University, Iksan 570-749, Republic of Korea Bonecell Biotech Inc., 77, Dunsan-ro, Seo-gu, Daejeon 302-830, Republic of Korea DIO Corporation, 66, Centum seo-ro, Haeundae-gu, Busan, Republic of Korea d Department of Oral and maxillofacial Surgery, Wonkwang University Daejeon Dental Hospital, Daejeon 302-830, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 23 August 2012 Received in revised form 16 November 2012 Accepted 17 December 2012 Available online 23 December 2012 Keywords: Magnesium Titanium implant Surface modification Mesenchymal stem cells Vacuum arc source ion implantation
a b s t r a c t Magnesium (Mg) is one of the most important ions associated with bone osseointegration. The aim of this study was to evaluate the cellular effects of Mg implantation in titanium (Ti) surfaces treated with sand blast using large grit and acid etching (SLA). Mg ions were implanted into the surface via vacuum arc source ion implantation. The surface morphology, chemical properties, and the amount of Mg ion release were evaluated by scanning electron microscopy (SEM), Auger electron spectroscopy (AES), Rutherford backscattering spectroscopy (RBS), and inductively coupled plasma-optical emission spectrometer (ICP-OES). Human mesenchymal stem cells (hMSCs) were used to evaluate cellular parameters such as proliferation, cytotoxicity, and adhesion morphology by MTS assay, live/dead assay, and SEM. Furthermore, osteoblast differentiation was determined on the basis of alkaline phosphatase (ALP) activity and the degree of calcium accumulation. In the Mg ion-implanted disk, 2.3 × 10 16 ions/cm 2 was retained. However, after Mg ion implantation, the surface morphology did not change. Implanted Mg ions were rapidly released during the first 7 days in vitro. The MTS assay, live/dead assay, and SEM demonstrated increased cell attachment and growth on the Mg ion-implanted surface. In particular, Mg ion implantation increased the initial cell adhesion, and in an osteoblast differentiation assay, ALP activity and calcium accumulation. These findings suggest that Mg ion implantation using the plasma source ion implantation (PSII) technique may be useful for SLA-treated Ti dental implants to improve their osseointegration capacity. © 2012 Elsevier B.V. All rights reserved.
1. Introduction A dental implant is an artificial tooth root device that is used to support restorations for holding a tooth or bridge in place. Titanium (Ti) has been widely used as a dental implant material because of its excellent mechanical strength, chemical stability, and biocompatibility [1]. For successful clinical performance, osseointegration is an important factor closely related to implant surface characteristics. A biomechanical interlocking between the implant surface and the surrounding bone and biochemical bonding has been proposed as an acceptable osseointegration mechanism. Various techniques have been used to modify the surface of dental implants. Grit blasting, etching, or hybrid techniques such as sand blast with large grit and
Abbreviations: PSII, plasma source ion implantation; SLA, sand blast with large grit and acid etching; SLA Ti, titanium implant treated with sand blast with large grit and acid etching; Mg-SLA Ti, Mg ion implanted titanium implant treated with sand blast with large grit and acid etching. ⁎ Corresponding author at: Wonkwang Bone Regeneration Research Institute, Wonkwang University, Iksan 570-749, Republic of Korea. Tel.: +82 42 341 2800; fax: +82 42 341 2809. E-mail address: [email protected] (J. Lee). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.12.061
acid etching (SLA) have been used for biomechanical interlocking [2,3] and these modifications allow the bone to grow into and maintain the implant surface. The SLA technique combines the advantages of sandblasting and acid-etching techniques to impart macroroughness and micro pits to the implant surface. In addition, several chemical modifications, e.g., sodium hydroxide and heat treatment, anodic oxidation, fluoride treatment, and ion implantation, have been used to add biochemical bonding properties [4–8] and to modify the chemical composition modification of the surface to permit enhanced interactions with osteoblast cells. Furthermore, several studies have attempted to enhance the topographic changes of the implant surface. Among these chemical modifications, ion implantation is a material modification process by which ions are injected into the surface region of a substrate without altering the surface topography. Beam-line ion implantation (BLII) is an ion implantation method. In this process, high-energy ions are produced in an accelerator and directed as a beam onto the surface of the implant surface for ion deposition [9]. However, this technique has some disadvantages. Although this technique is useful for disk, plate, and cylindrical implants, line-of-sight limitations make it difficult to apply it to clinical implants because of shadowed areas such as valleys or flank areas of a thread.
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To overcome the line-of-sight limitations of the BLII technique, plasma source ion implantation (PSII) was developed [9]. Using the PSII technique, ions can be implanted into a surface. In PSII ion implantation, a metal plasma source is used, and an ionized metal ion plasma is generated by a filtered vacuum arc source and implanted into the surface of the implant. The PSII technique can be controlled by the size of the sheath to produce an even ion-implanted layer [10,11] and is frequently used to implant chemical ions into Ti implant surfaces. The tissue response, including cellular attachment, osteoblast activity, and proliferation, may be affected by the chemical properties of the Ti surface. Several studies have reported that variations in the calcium [12], sulfur [13], and magnesium (Mg) content of a surface [14] influences the bone tissue reaction. Mg ion plays a particularly critical role in bone remodeling [15], and Mg ion implantation in a Ti surface positively influenced the initial cellular response and enhanced the bone response [14,15]. Mesenchymal stem cells (MSCs) are important for bone remodeling because they have self-renewal capacity and multi-lineage potential [16]. MSCs can be differentiated into osteoblasts, which promote bone growth. Some studies have reported that Mg ions affect the adherence of MSCs [17,18]. According to Park et al. [19], Mg ions affect the cellular response of osteoblasts. Additionally, Arlin et al. [20] previously reported a good clinical outcome for SLA and titanium plasma-sprayed implants. Although these studies have shown that Mg ions enhance the cellular response of osteoblasts and bone regeneration in vivo, the properties of SLA-treated Ti implants implanted with Mg ions via the PSII technique and the biocompatibility of these implants have not yet been evaluated for MSCs. Therefore, in this study, we investigated the implantation of Mg ions into the surface of SLA-treated Ti implant disks using the PSII technique. Physical and chemical properties were evaluated and biocompatibility factors, including cellular attachment, proliferation, cytotoxicity, and osteoblast differentiation, were evaluated using human bone marrow mesenchymal stem cells (hMSCs) in vitro.
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Fig. 1. Schematic representation of plasma source ion implantation (PSII). A metal plasma source was used, and the ionized metal ion plasma was generated by a filtered vacuum arc source and implanted into the surface of the specimen.
(RBS) analysis, helium ions were used as an ion beam source and accelerated using a Pelltro apparatus (6SDH2, NEC) at a voltage of 2 MeV. The charge of the helium ions was 20 μC, the incident angle was 0°, and the scattered angle was 10°. The measured spectrum was fitted to a theoretical spectrum and a 16-layer sample was measured and quantified. 2.3. Mg ion release
2. Materials and methods 2.1. Preparation of specimens In this study, SLA-treated pure Ti (ASTM grade 4) disks were used as substrate. The specimens had a diameter of 12 mm and a thickness of 1 mm. PSII processing was performed in collaboration with DIO Implant Corporation (Busan, Korea). Fig. 1 shows a schematic representation of the PSII process. When Mg ion plasma was released from an arc spot in a negatively charged surface and then injected into 90 electromagnetic filters, ionized Mg plasma was generated. The ionized Mg plasma accelerated within the electric field between the substrate and sheath and then reached the surface of the substrate. The electric energy of the implantation field was 15 keV. All specimens were sterilized with 25 kGy γ-radiation from the 60Co source at the UTR GAMMA-Pi facility (Greenpia, Yeoju, Korea). 2.2. Surface characterization The surface morphology of the specimens was observed by scanning electron microscopy (SEM; JSM-5800, JEOL, Tokyo, Japan). To analyze the chemistry of the implanted layer, Auger electron spectroscopy (AES; PHI650; Physical Electronics, Chanhassen, MN, USA) was used. For electron excitation in the AES analysis, a primary electron beam (3 keV and 2.6 μA) with a diameter of 40 μm was used. The samples were sputtered by two symmetrically inclined 1 keV argon ion beams at an ion incidence angle of 47° with respect to the surface normal during depth profiling. Using the relative sensitivity factor provided by the instrument producer, the atomic concentrations were calculated. For Rutherford backscattering spectroscopy
The 20 samples were immersed in 10 mL of standard simulated body fluid (SBF) buffer prepared as reported previously [21] and then incubated at 37 °C under gentle shaking. For the analyses of Mg ions, small aliquots were removed during the 21-day experiment, and they were replaced with fresh solution. Additionally, specimens that were not implanted with Mg were used as blanks. The concentration of Mg ions released was determined by inductively coupled plasma-optical emission spectrometer (ICP-OES; Optima™ 8300, PerkinElmer, MA, USA). 2.4. hMSC culture hMSCs were used to evaluate the biocompatibility of the Mg ion-implanted SLA Ti specimen. hMSCs were obtained from Dr. HK You (Wonkwang University, Dental Hospital, Iksan, Korea) and maintained in α-MEM (Gibco-BRL, Gaithersburg, MD) containing 10% fetal bovine serum (FBS; Gibco-BRL) at 37 °C under a 5% CO2 atmosphere. In this study, experiments were performed using hMSCs from passages 4 to 6. To induce osteoblast differentiation, the cells were cultured in the presence of an osteoblast differentiation stimulator (OS; 10 mM β-glycerophosphate, 50 μg/mL ascorbic acid, and 100 nM dexamethasone). The medium and OS were replaced every 2 days. 2.5. Proliferation assay for hMSCs cultured on implant specimen The proliferation of hMSCs cultured on the specimens was measured using CellTiter96® Aqueous One solution (Promega, Madison, WI). hMSCs (1 × 10 4 cells) were seeded on the surface of specimens.
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Fig. 2. Surface morphological image of sandblasting and acid etching (SLA)-treated titanium (Ti) specimen (SLA Ti) and SLA Ti specimen implanted with Mg ions (Mg-SLA Ti) obtained using scanning electron microscopy (SEM).
After 1 h to allow initial adhesion, the specimens were rinsed with PBS to remove the non-adherent cells and placed in a new culture dish. At predetermined time intervals (1, 5, 10, and 15 days), 25 μL of MTS reagent was added per 100 μL of medium and incubated. After incubation for 4 h, the absorbance of the supernatant was measured at 490 nm on an enzyme-linked immunosorbent assay (ELISA) reader (SpectraMAX M3, Molecular Devices, Sunnyvale, CA).
2.8. Measurement of alkaline phosphatase activity
2.6. Viability and cytotoxicity staining
2.9. Measurement of calcium accumulation
The Live/Dead® Viability/Cytotoxicity Kit (Molecular Probe, Eugene, USA) was used to measure the viability and cytotoxicity of the cells according to the manufacturer's protocol. Briefly, 1 × 10 4 hMSCs were seeded on implant specimen and cultured for 5 days. Then, the specimens were incubated for 30 min in PBS containing the Live/Dead® Viability/Cytotoxicity reagent and observed under a fluorescence microscope (DM IL LED Fluo, Leica, Mannheim, Germany). To quantify initial cell adhesion, fluorescently stained cells were counted using an image analyzer software (ImagePro plus 7.0; Media Cybermethics, Silver Spring, MD, USA). The number of counted cells was then normalized against the number of cells counted in the SLA Ti group and presented as a fold change.
Calcium accumulation was determined using AR-S (Sigma) staining. hMSCs were cultured with continuous treatment with OS. After 3 weeks, the samples were fixed with 70% ice-cold ethanol for 1 h at 4 °C, and the ethanol was removed. Calcium accumulation was measured by staining with 40 mM AR-S solution for 10 min at room temperature. The stained portions were photographed with a digital camera (Nikon D80, Nikon, Tokyo, Japan). The deposited stain was leached using 10% cetylpyridinium chloride (CPC) dissolved in 10 mM sodium phosphate buffer and read at 562 nm using an ELISA reader.
2.7. Cell morphology Cellular attachment was evaluated by SEM. To observe the morphology of adherent cells, after 1 h and 72 h of culture, specimens harboring cultured hMSCs were washed with phosphate-buffered saline (PBS) and fixed with 2.5% glutaraldehyde at 4 °C for 2 h. For post-fixation, the samples were treated with 0.1% osmium tetroxide and dehydrated in a series of ethanol washes (25%, 50%, 75%, 95%, and 100%). The dehydrated specimens were sputter-coated with platinum and viewed using SEM.
Alkaline phosphatase (ALP) activity was assessed after 7 days of treatment with OS using p-nitrophenylphosphate (p-NPP) as the substrate according to a previously described method [22]. ALP activity was normalized against the total protein content measured using a BCA protein assay reagent kit (Pierce, Rockford, IL). The ALP activity was measured as nmol/30 min per mg of protein.
2.10. Statistical analysis All experiments were performed in triplicate. The values are expressed as mean ± standard deviation (SD). Statistical analysis was performed using Student's t-test and one-way analysis of variance. P b 0.05 was considered statistically significant. 3. Results 3.1. Surface characterization SEM images of a control specimen surface and a Mg ion-implanted specimen surface are shown in Fig. 2. The SLA Ti surface demonstrated an irregular and rough morphology, and numerous small holes
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Fig. 4. Proliferation as determined by the MTS assay (A). Fluorescence staining image (B) and relative number of adhering cells (C) after 1 h of culture of human mesenchymal stem cells (hMSCs). Proliferation and initial adhesion were higher in cells cultured on Mg-ion-implanted SLA Ti than in those cultured on SLA-Ti. Values are expressed as the mean ± SD. Significance was defined as *P b 0.05 when compared with the SLA Ti specimen. The size bar represents 500 μm.
implantation, and its Ra value did not change after Mg ion implantation (data not shown). RBS analysis was performed to measure the retained dose of implanted Mg ions in the implanted layer (Fig. 3B). Because the Ti and Mg ion peaks overlapped, a pure carbon wafer was used as substrate. Mg ions were implanted with a dose of 2.3 × 10 16 ions/cm 2 in a layer with a thickness of 150 Å. In addition, the concentrations of released Mg ions in the SBF solutions used in the immersion test in vitro are shown in Fig. 3C. During the first 7 days, Mg ions were rapidly released from the Mg-SLA Ti. Subsequently, the ions were slowly released from the specimens.
Fig. 3. Auger electron spectroscopy depth profiling (A), Rutherford backscattering spectroscopy (RBS) analysis (B) and pattern of Mg ion release (C) from the Mg-ion-implanted surface of a sandblasting and acid etching (SLA)-treated titanium (Ti) specimen.
were observed under high magnification (×4000). Furthermore, small micro pits and sharp edges were clearly observed. These morphological characteristics did not change after Mg ion implantation. To determine the elemental content of the outermost surface of the Mg-SLA Ti specimens, AES analysis was used. The AES depth profiles are shown in Fig. 3A. The concentration of Mg, oxygen, and Ti gradually varied from the outermost surface to the bulk without forming a distinct interface. The concentration of Mg ions was approximately 9.8%. The presence of oxygen in the implanted layer, which may have been derived from residual gas, indicated that magnesium oxide formed during ion implantation. The roughness of SLA Ti and Mg-SLA Ti specimens was also measured. A SLA Ti specimen with an average roughness (Ra) of 2.0 ± 0.5 μm was used for Mg ion
3.2. Effect of Mg ion implantation on cell proliferation To evaluate the effect on cell proliferation and toxicity of Mg ion implantation into SLA-treated Ti specimens, hMSCs were seeded and cultured on the specimens. As shown in Fig. 4A, the proliferation of cells on SLA Ti and Mg-SLA Ti gradually increased with cultivation time. At day 1, proliferation of cells cultured on SLA Ti (0.37 ± 0.03) was lower than that of cells cultured on Mg-SLA Ti (0.62 ± 0.06) and this difference was continuously observed until 15 days. To evaluate the effect of Mg ion implantation on initial cell adhesion, adherent cells were stained 1 h after seeding. The number of adherent cells on Mg-SLA Ti was 2.15-fold higher than that on SLA Ti (Fig. 4B and C). These results suggest that Mg ion implantation affects the initial cell adhesion. To evaluate the potential cytotoxic effects of Mg ion implantation, hMSCs were cultured on the Mg-SLA Ti substrate, and live/dead staining was performed after 1 day and 5 days. Although a few dead or unhealthy cells were observed, the fluorescent images showed
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Fig. 5. Live/dead fluorescence imaging of human mesenchymal stem cells (hMSCs) cultured on sandblasting and acid etching (SLA)-treated titanium (SLA Ti) and Mg ion-implanted SLA Ti (Mg-SLA Ti) specimens in normal growth media. The viability/cytotoxicity assay was performed after 1 day and 5 days of culture. Live cells were stained green by calcein acetoxymethyl (calcein AM) and dead cells were stained red by ethidium homodimer-1 (EthD-1). The size bar represents 500 μm.
that nearly all cells were viable and that they maintained a fibroblastic morphology during cultivation (Fig. 5). 3.3. Effect of Mg ion implantation on cell morphology To observe the cell adhesion morphology, SEM analysis was performed after 1 h and 72 h to evaluate initial and growth adhesion behavior, respectively. Fig. 6 demonstrates the interaction between the cells and the Ti surface as observed by SEM. After 1 h of
incubation, almost all cells maintained a spherical morphology, and only a few cells presented a slight extracellular membrane bridge for attachment onto the surface of SLA Ti. However, numerous cells presented a wide cellular membrane bridge and flattened morphology on Mg-SLA Ti. These initial cell adhesion results were consistent with the fluorescent image analysis at 1 h (Fig. 4B) and 1 day (Fig. 5). Furthermore, after 72 h of cultivation, these cells were remarkably elongated and formed a fibroblast-like morphology on both SLA Ti and Mg-SLA Ti.
Fig. 6. Cell adhesion and growth morphology determined by scanning electron microscopy (SEM) of human mesenchymal stem cells (hMSCs) cultured on sandblasting and acid etching (SLA)-treated titanium (SLA Ti) and Mg ion-implanted SLA Ti (Mg-SLA Ti) specimens. hMSCs were seeded and cultured, and the initial cell adhesion and growth morphology were observed at 1 h and 72 h.
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These results agree with those obtained for ALP activity shown in Fig. 7.
4. Discussion
Fig. 7. Alkaline phosphatase (ALP) activity of human mesenchymal stem cells (hMSCs) cultured on sandblasting and acid etching (SLA)-treated titanium (SLA Ti) and Mg ion-implanted SLA Ti (Mg-SLA Ti) specimens. Cells were cultured in media in the presence and absence of an osteoblast differentiation stimulator (OS) for 7 days, and ALP activity was measured. In the presence of OS, the ALP activity was increased by Mg ion implantation. Values are expressed as the mean ± SD. Significance was defined as **P b 0.01 when compared with the SLA Ti specimen.
3.4. Effect of Mg ion implantation on osteoblast differentiation To determine the effect of Mg ion implantation on osteoblast differentiation of cultured hMSCs, OS-containing medium was used as described in the Materials and methods. When the cells on SLA Ti were treated with OS medium, ALP activity increased from 0.23 ± 0.07 nmol/30 min per mg of protein to 1.28 ± 0.07 nmol/30 min per mg of protein. When the cells were cultured on Mg-SLA Ti specimens with OS medium, ALP activity increased from 0.33 ± 0.08 nmol/ 30 min per mg of protein to 2.33 ± 0.11 nmol/30 min per mg of protein. Although, ALP activity increased on both specimen types, ALP activity of cells cultured on Mg-SLA Ti was approximately 1.8-folds higher than that of cells cultured on SLA Ti (Fig. 7). In addition, calcium accumulation was assessed to evaluate osteoblast differentiation after culture for 21 days with OS medium, and AR-S staining was performed (Fig. 8). When treated with OS medium, cells cultured on SLA Ti and Mg-SLA Ti demonstrated calcium accumulation. However, calcium accumulation on Mg-SLA Ti was significantly higher than that on SLA Ti (approximately 1.5-fold higher).
To enhance osseointegration, the topography, free energy, crystal structure, and chemical composition of the surface of dental implants have been modified. Various studies have attempted ion implantation in dental implants using nitrogen, oxygen, calcium, and Mg [8,12,23]. To modify the surface roughness, resorbable blast media (RBM) such as hydroxyapatite and calcium phosphate have been widely used [24]. The SLA technique has also been used to modify the surface roughness [25]. Although Cho et al. [10] recently reported that Mg ions implanted in RBM-treated Ti surfaces using the PSII technique provided enhanced osseointegration capacity, no reports have addressed the effect of Mg ion implantation on SLA-treated Ti. Therefore, in this study, Mg ions were implanted in the surface of SLA-treated commercial pure Ti using the PSII technique. A previous study reported an enhanced cell response after Mg ion implantation and suggested that the response would improve with increasing concentrations of Mg ions [26]. However, the strongest osseointegration was observed in a range of 8.36% to 9.33% for the Mg ion content, which suggested that an optimal surface concentration exists [15]. Cho et al. recommended implantation with a concentration of less than 10% [10]. Therefore, in this study, we used a Mg ion concentration of less than 10%, and Mg ion-implanted specimens were prepared at a final Mg concentration of 2.3 × 10 16 ions/cm2 using the vacuum arc method for the PSII technique. Several methods can be used to produce plasma from a metal, e.g., vaporization, radio-frequency discharge, microwave, sputtering, and vacuum arc, but the vacuum arc technique is simpler than the other methods. After Mg ion implantation, the surface roughness was maintained and the Ra was unchanged compared with SLA Ti; these results were consistent with a previous report [27]. To determine the effect of Mg ion on hMSCs, the cells were observed to proliferate over time. In bone healing and implant osseointegration, MSCs are the key repair cells, and their cellular response is important because successful osseointegration of implants depends on the attachment of MSCs onto the implant surface [28]. Therefore, hMSCs were used in this study to evaluate biocompatibility. In this study, we showed that Mg ion implantation enhanced MSC attachment and proliferation. The results for the initial cell number after 1 h of incubation suggests that the observed differences resulted from the initial cell adhesion to the surface. Our results show that the number of hMSCs that initially attached was higher on Mg-SLA Ti than on SLA Ti, which indicates that Mg ion implantation enhanced the initial cell attachment onto the surface
Fig. 8. Calcium accumulation determined by alizarin red staining of human mesenchymal stem cells (hMSCs) cultured on sandblasting and acid etching (SLA)-treated titanium (SLA Ti) and Mg ion-implanted SLA Ti (Mg-SLA Ti) specimens. Cells were cultured in media in the presence and absence of an osteoblast differentiation stimulator (OS) for 3 weeks, and the stained portions were photographed using a digital camera (A). The stained portion was solubilized using a cetylpyridinium chloride (CPC) solution, and the optical density was then measured (B). In the presence of OS, calcium accumulation was increased by Mg ion implantation. Values are expressed as the mean ± SD. Significance was defined as *P b 0.05 when compared with the SLA Ti specimen.
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of Mg-SLA Ti. These findings may explain the relationship between in vivo osseointegration and MSCs in terms of Mg ion implantation. Several types of ions have been used for surface modification of dental implants [8,12,23]. However, negative physiological and cellular effects have been reported to result from the release of metal ions [29]. In contrast, Klein et al. demonstrated that a low concentration of released metal ions can lead to enhanced cellular adhesion [30]. These reports therefore suggest that the metal ion release pattern is important for successful dental implantation. In this study, the pattern of Mg ion release from Mg-SLA Ti was analyzed in vitro. Although Mg ions were rapidly released within the first 7 days, the released Mg ions were not cytotoxic to hMSCs. Furthermore, our SEM analysis showed the morphology of the interaction between the cells and the Ti surface. After 1 h of incubation, almost all cells maintained a spherical morphology on the surface of SLA Ti. In contrast, numerous cells presented a wide extracellular membrane bridge and flattened morphology on Mg-SLA Ti. Furthermore, spreading cells showed fibroblast-like morphology on the surface of SLA Ti and Mg-SLA Ti. These morphological changes are typical phenomena during the cell adhesion process and they only reflect the differences in the timing of cell adhesion. Therefore, these results indicated that implanted Mg ions facilitate the fast initial cell adhesion of MSCs. Several studies have shown that Mg ions can enhance cell attachment and provide a beneficial effect during the initial stage of the bone response [19,31] and play an essential role in the binding interactions between the integrin family of cell surface receptors and their ligand proteins [31]. Sul et al. [15] reported that Mg ions promote integrin–ligand interactions to provide an improved bone response. However, in the present study of the interaction between hMSCs and Mg ions implanted in a Ti surface, the Mg ions may have played a role in the binding interaction between MSCs and the titanium surface. To evaluate the effect of Mg ion implantation on osteoblast differentiation, ALP activity and calcium accumulation were measured. ALP activity is one of the most commonly used early markers of osteogenesis, and calcium accumulation is used as a late marker [32,33]. In the present study, ALP activity was higher on Mg-SLA Ti than on SLA Ti when the cells were cultured in OS-containing medium. These results suggest that Mg ions increase ALP activity. However, ALP activity was not significantly affected when the cells were cultured in the absence of OS. Therefore, we consider that Mg ion implantation does not directly affect osteoblast differentiation. Similar results for osteoblast differentiation were obtained when AR-S staining was used as a late marker. Intracellular communication is an important factor for cellular differentiation, including osteoblast differentiation [34]. In our results, Mg ion implantation improved the initial cell attachment onto the surface of specimens. Therefore, the observed enhancement of differentiation may have been the result of earlier confluence of cells cultured on Mg-SLA Ti when compared with cells cultured on SLA Ti, which could have resulted in earlier differentiation. 5. Conclusion This study examined the cellular response to SLA-treated Ti implanted with Mg ions using the PSII method. The biocompatibility was evaluated using hMSC cells. Distinct cytotoxicity was not observed, and the initial cellular attachment was improved, with a resulting enhancement of osteoblast differentiation. These findings suggest that
Mg ion implantation into SLA Ti dental implants using the PSII technique may be a useful modification to improve osseointegration. However, further studies are necessary to determine the bone response in an in vivo model.
Acknowledgments This work was supported by a research grant from Wonkwang University in 2012 and Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (2012-040526).
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Mg ion implantation on SLA-treated titanium surface and its effects on the behavior of mesenchymal stem cell Beom-Su Kim a, b, Jin Seong Kim a, b, Young Min Park c, Bo-Young Choi d, Jun Lee a, b,⁎ a
Wonkwang Bone Regeneration Research Institute, Wonkwang University, Iksan 570-749, Republic of Korea Bonecell Biotech Inc., 77, Dunsan-ro, Seo-gu, Daejeon 302-830, Republic of Korea DIO Corporation, 66, Centum seo-ro, Haeundae-gu, Busan, Republic of Korea d Department of Oral and maxillofacial Surgery, Wonkwang University Daejeon Dental Hospital, Daejeon 302-830, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 23 August 2012 Received in revised form 16 November 2012 Accepted 17 December 2012 Available online 23 December 2012 Keywords: Magnesium Titanium implant Surface modification Mesenchymal stem cells Vacuum arc source ion implantation
a b s t r a c t Magnesium (Mg) is one of the most important ions associated with bone osseointegration. The aim of this study was to evaluate the cellular effects of Mg implantation in titanium (Ti) surfaces treated with sand blast using large grit and acid etching (SLA). Mg ions were implanted into the surface via vacuum arc source ion implantation. The surface morphology, chemical properties, and the amount of Mg ion release were evaluated by scanning electron microscopy (SEM), Auger electron spectroscopy (AES), Rutherford backscattering spectroscopy (RBS), and inductively coupled plasma-optical emission spectrometer (ICP-OES). Human mesenchymal stem cells (hMSCs) were used to evaluate cellular parameters such as proliferation, cytotoxicity, and adhesion morphology by MTS assay, live/dead assay, and SEM. Furthermore, osteoblast differentiation was determined on the basis of alkaline phosphatase (ALP) activity and the degree of calcium accumulation. In the Mg ion-implanted disk, 2.3 × 10 16 ions/cm 2 was retained. However, after Mg ion implantation, the surface morphology did not change. Implanted Mg ions were rapidly released during the first 7 days in vitro. The MTS assay, live/dead assay, and SEM demonstrated increased cell attachment and growth on the Mg ion-implanted surface. In particular, Mg ion implantation increased the initial cell adhesion, and in an osteoblast differentiation assay, ALP activity and calcium accumulation. These findings suggest that Mg ion implantation using the plasma source ion implantation (PSII) technique may be useful for SLA-treated Ti dental implants to improve their osseointegration capacity. © 2012 Elsevier B.V. All rights reserved.
1. Introduction A dental implant is an artificial tooth root device that is used to support restorations for holding a tooth or bridge in place. Titanium (Ti) has been widely used as a dental implant material because of its excellent mechanical strength, chemical stability, and biocompatibility [1]. For successful clinical performance, osseointegration is an important factor closely related to implant surface characteristics. A biomechanical interlocking between the implant surface and the surrounding bone and biochemical bonding has been proposed as an acceptable osseointegration mechanism. Various techniques have been used to modify the surface of dental implants. Grit blasting, etching, or hybrid techniques such as sand blast with large grit and
Abbreviations: PSII, plasma source ion implantation; SLA, sand blast with large grit and acid etching; SLA Ti, titanium implant treated with sand blast with large grit and acid etching; Mg-SLA Ti, Mg ion implanted titanium implant treated with sand blast with large grit and acid etching. ⁎ Corresponding author at: Wonkwang Bone Regeneration Research Institute, Wonkwang University, Iksan 570-749, Republic of Korea. Tel.: +82 42 341 2800; fax: +82 42 341 2809. E-mail address: [email protected] (J. Lee). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.12.061
acid etching (SLA) have been used for biomechanical interlocking [2,3] and these modifications allow the bone to grow into and maintain the implant surface. The SLA technique combines the advantages of sandblasting and acid-etching techniques to impart macroroughness and micro pits to the implant surface. In addition, several chemical modifications, e.g., sodium hydroxide and heat treatment, anodic oxidation, fluoride treatment, and ion implantation, have been used to add biochemical bonding properties [4–8] and to modify the chemical composition modification of the surface to permit enhanced interactions with osteoblast cells. Furthermore, several studies have attempted to enhance the topographic changes of the implant surface. Among these chemical modifications, ion implantation is a material modification process by which ions are injected into the surface region of a substrate without altering the surface topography. Beam-line ion implantation (BLII) is an ion implantation method. In this process, high-energy ions are produced in an accelerator and directed as a beam onto the surface of the implant surface for ion deposition [9]. However, this technique has some disadvantages. Although this technique is useful for disk, plate, and cylindrical implants, line-of-sight limitations make it difficult to apply it to clinical implants because of shadowed areas such as valleys or flank areas of a thread.
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To overcome the line-of-sight limitations of the BLII technique, plasma source ion implantation (PSII) was developed [9]. Using the PSII technique, ions can be implanted into a surface. In PSII ion implantation, a metal plasma source is used, and an ionized metal ion plasma is generated by a filtered vacuum arc source and implanted into the surface of the implant. The PSII technique can be controlled by the size of the sheath to produce an even ion-implanted layer [10,11] and is frequently used to implant chemical ions into Ti implant surfaces. The tissue response, including cellular attachment, osteoblast activity, and proliferation, may be affected by the chemical properties of the Ti surface. Several studies have reported that variations in the calcium [12], sulfur [13], and magnesium (Mg) content of a surface [14] influences the bone tissue reaction. Mg ion plays a particularly critical role in bone remodeling [15], and Mg ion implantation in a Ti surface positively influenced the initial cellular response and enhanced the bone response [14,15]. Mesenchymal stem cells (MSCs) are important for bone remodeling because they have self-renewal capacity and multi-lineage potential [16]. MSCs can be differentiated into osteoblasts, which promote bone growth. Some studies have reported that Mg ions affect the adherence of MSCs [17,18]. According to Park et al. [19], Mg ions affect the cellular response of osteoblasts. Additionally, Arlin et al. [20] previously reported a good clinical outcome for SLA and titanium plasma-sprayed implants. Although these studies have shown that Mg ions enhance the cellular response of osteoblasts and bone regeneration in vivo, the properties of SLA-treated Ti implants implanted with Mg ions via the PSII technique and the biocompatibility of these implants have not yet been evaluated for MSCs. Therefore, in this study, we investigated the implantation of Mg ions into the surface of SLA-treated Ti implant disks using the PSII technique. Physical and chemical properties were evaluated and biocompatibility factors, including cellular attachment, proliferation, cytotoxicity, and osteoblast differentiation, were evaluated using human bone marrow mesenchymal stem cells (hMSCs) in vitro.
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Fig. 1. Schematic representation of plasma source ion implantation (PSII). A metal plasma source was used, and the ionized metal ion plasma was generated by a filtered vacuum arc source and implanted into the surface of the specimen.
(RBS) analysis, helium ions were used as an ion beam source and accelerated using a Pelltro apparatus (6SDH2, NEC) at a voltage of 2 MeV. The charge of the helium ions was 20 μC, the incident angle was 0°, and the scattered angle was 10°. The measured spectrum was fitted to a theoretical spectrum and a 16-layer sample was measured and quantified. 2.3. Mg ion release
2. Materials and methods 2.1. Preparation of specimens In this study, SLA-treated pure Ti (ASTM grade 4) disks were used as substrate. The specimens had a diameter of 12 mm and a thickness of 1 mm. PSII processing was performed in collaboration with DIO Implant Corporation (Busan, Korea). Fig. 1 shows a schematic representation of the PSII process. When Mg ion plasma was released from an arc spot in a negatively charged surface and then injected into 90 electromagnetic filters, ionized Mg plasma was generated. The ionized Mg plasma accelerated within the electric field between the substrate and sheath and then reached the surface of the substrate. The electric energy of the implantation field was 15 keV. All specimens were sterilized with 25 kGy γ-radiation from the 60Co source at the UTR GAMMA-Pi facility (Greenpia, Yeoju, Korea). 2.2. Surface characterization The surface morphology of the specimens was observed by scanning electron microscopy (SEM; JSM-5800, JEOL, Tokyo, Japan). To analyze the chemistry of the implanted layer, Auger electron spectroscopy (AES; PHI650; Physical Electronics, Chanhassen, MN, USA) was used. For electron excitation in the AES analysis, a primary electron beam (3 keV and 2.6 μA) with a diameter of 40 μm was used. The samples were sputtered by two symmetrically inclined 1 keV argon ion beams at an ion incidence angle of 47° with respect to the surface normal during depth profiling. Using the relative sensitivity factor provided by the instrument producer, the atomic concentrations were calculated. For Rutherford backscattering spectroscopy
The 20 samples were immersed in 10 mL of standard simulated body fluid (SBF) buffer prepared as reported previously [21] and then incubated at 37 °C under gentle shaking. For the analyses of Mg ions, small aliquots were removed during the 21-day experiment, and they were replaced with fresh solution. Additionally, specimens that were not implanted with Mg were used as blanks. The concentration of Mg ions released was determined by inductively coupled plasma-optical emission spectrometer (ICP-OES; Optima™ 8300, PerkinElmer, MA, USA). 2.4. hMSC culture hMSCs were used to evaluate the biocompatibility of the Mg ion-implanted SLA Ti specimen. hMSCs were obtained from Dr. HK You (Wonkwang University, Dental Hospital, Iksan, Korea) and maintained in α-MEM (Gibco-BRL, Gaithersburg, MD) containing 10% fetal bovine serum (FBS; Gibco-BRL) at 37 °C under a 5% CO2 atmosphere. In this study, experiments were performed using hMSCs from passages 4 to 6. To induce osteoblast differentiation, the cells were cultured in the presence of an osteoblast differentiation stimulator (OS; 10 mM β-glycerophosphate, 50 μg/mL ascorbic acid, and 100 nM dexamethasone). The medium and OS were replaced every 2 days. 2.5. Proliferation assay for hMSCs cultured on implant specimen The proliferation of hMSCs cultured on the specimens was measured using CellTiter96® Aqueous One solution (Promega, Madison, WI). hMSCs (1 × 10 4 cells) were seeded on the surface of specimens.
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Fig. 2. Surface morphological image of sandblasting and acid etching (SLA)-treated titanium (Ti) specimen (SLA Ti) and SLA Ti specimen implanted with Mg ions (Mg-SLA Ti) obtained using scanning electron microscopy (SEM).
After 1 h to allow initial adhesion, the specimens were rinsed with PBS to remove the non-adherent cells and placed in a new culture dish. At predetermined time intervals (1, 5, 10, and 15 days), 25 μL of MTS reagent was added per 100 μL of medium and incubated. After incubation for 4 h, the absorbance of the supernatant was measured at 490 nm on an enzyme-linked immunosorbent assay (ELISA) reader (SpectraMAX M3, Molecular Devices, Sunnyvale, CA).
2.8. Measurement of alkaline phosphatase activity
2.6. Viability and cytotoxicity staining
2.9. Measurement of calcium accumulation
The Live/Dead® Viability/Cytotoxicity Kit (Molecular Probe, Eugene, USA) was used to measure the viability and cytotoxicity of the cells according to the manufacturer's protocol. Briefly, 1 × 10 4 hMSCs were seeded on implant specimen and cultured for 5 days. Then, the specimens were incubated for 30 min in PBS containing the Live/Dead® Viability/Cytotoxicity reagent and observed under a fluorescence microscope (DM IL LED Fluo, Leica, Mannheim, Germany). To quantify initial cell adhesion, fluorescently stained cells were counted using an image analyzer software (ImagePro plus 7.0; Media Cybermethics, Silver Spring, MD, USA). The number of counted cells was then normalized against the number of cells counted in the SLA Ti group and presented as a fold change.
Calcium accumulation was determined using AR-S (Sigma) staining. hMSCs were cultured with continuous treatment with OS. After 3 weeks, the samples were fixed with 70% ice-cold ethanol for 1 h at 4 °C, and the ethanol was removed. Calcium accumulation was measured by staining with 40 mM AR-S solution for 10 min at room temperature. The stained portions were photographed with a digital camera (Nikon D80, Nikon, Tokyo, Japan). The deposited stain was leached using 10% cetylpyridinium chloride (CPC) dissolved in 10 mM sodium phosphate buffer and read at 562 nm using an ELISA reader.
2.7. Cell morphology Cellular attachment was evaluated by SEM. To observe the morphology of adherent cells, after 1 h and 72 h of culture, specimens harboring cultured hMSCs were washed with phosphate-buffered saline (PBS) and fixed with 2.5% glutaraldehyde at 4 °C for 2 h. For post-fixation, the samples were treated with 0.1% osmium tetroxide and dehydrated in a series of ethanol washes (25%, 50%, 75%, 95%, and 100%). The dehydrated specimens were sputter-coated with platinum and viewed using SEM.
Alkaline phosphatase (ALP) activity was assessed after 7 days of treatment with OS using p-nitrophenylphosphate (p-NPP) as the substrate according to a previously described method [22]. ALP activity was normalized against the total protein content measured using a BCA protein assay reagent kit (Pierce, Rockford, IL). The ALP activity was measured as nmol/30 min per mg of protein.
2.10. Statistical analysis All experiments were performed in triplicate. The values are expressed as mean ± standard deviation (SD). Statistical analysis was performed using Student's t-test and one-way analysis of variance. P b 0.05 was considered statistically significant. 3. Results 3.1. Surface characterization SEM images of a control specimen surface and a Mg ion-implanted specimen surface are shown in Fig. 2. The SLA Ti surface demonstrated an irregular and rough morphology, and numerous small holes
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Fig. 4. Proliferation as determined by the MTS assay (A). Fluorescence staining image (B) and relative number of adhering cells (C) after 1 h of culture of human mesenchymal stem cells (hMSCs). Proliferation and initial adhesion were higher in cells cultured on Mg-ion-implanted SLA Ti than in those cultured on SLA-Ti. Values are expressed as the mean ± SD. Significance was defined as *P b 0.05 when compared with the SLA Ti specimen. The size bar represents 500 μm.
implantation, and its Ra value did not change after Mg ion implantation (data not shown). RBS analysis was performed to measure the retained dose of implanted Mg ions in the implanted layer (Fig. 3B). Because the Ti and Mg ion peaks overlapped, a pure carbon wafer was used as substrate. Mg ions were implanted with a dose of 2.3 × 10 16 ions/cm 2 in a layer with a thickness of 150 Å. In addition, the concentrations of released Mg ions in the SBF solutions used in the immersion test in vitro are shown in Fig. 3C. During the first 7 days, Mg ions were rapidly released from the Mg-SLA Ti. Subsequently, the ions were slowly released from the specimens.
Fig. 3. Auger electron spectroscopy depth profiling (A), Rutherford backscattering spectroscopy (RBS) analysis (B) and pattern of Mg ion release (C) from the Mg-ion-implanted surface of a sandblasting and acid etching (SLA)-treated titanium (Ti) specimen.
were observed under high magnification (×4000). Furthermore, small micro pits and sharp edges were clearly observed. These morphological characteristics did not change after Mg ion implantation. To determine the elemental content of the outermost surface of the Mg-SLA Ti specimens, AES analysis was used. The AES depth profiles are shown in Fig. 3A. The concentration of Mg, oxygen, and Ti gradually varied from the outermost surface to the bulk without forming a distinct interface. The concentration of Mg ions was approximately 9.8%. The presence of oxygen in the implanted layer, which may have been derived from residual gas, indicated that magnesium oxide formed during ion implantation. The roughness of SLA Ti and Mg-SLA Ti specimens was also measured. A SLA Ti specimen with an average roughness (Ra) of 2.0 ± 0.5 μm was used for Mg ion
3.2. Effect of Mg ion implantation on cell proliferation To evaluate the effect on cell proliferation and toxicity of Mg ion implantation into SLA-treated Ti specimens, hMSCs were seeded and cultured on the specimens. As shown in Fig. 4A, the proliferation of cells on SLA Ti and Mg-SLA Ti gradually increased with cultivation time. At day 1, proliferation of cells cultured on SLA Ti (0.37 ± 0.03) was lower than that of cells cultured on Mg-SLA Ti (0.62 ± 0.06) and this difference was continuously observed until 15 days. To evaluate the effect of Mg ion implantation on initial cell adhesion, adherent cells were stained 1 h after seeding. The number of adherent cells on Mg-SLA Ti was 2.15-fold higher than that on SLA Ti (Fig. 4B and C). These results suggest that Mg ion implantation affects the initial cell adhesion. To evaluate the potential cytotoxic effects of Mg ion implantation, hMSCs were cultured on the Mg-SLA Ti substrate, and live/dead staining was performed after 1 day and 5 days. Although a few dead or unhealthy cells were observed, the fluorescent images showed
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Fig. 5. Live/dead fluorescence imaging of human mesenchymal stem cells (hMSCs) cultured on sandblasting and acid etching (SLA)-treated titanium (SLA Ti) and Mg ion-implanted SLA Ti (Mg-SLA Ti) specimens in normal growth media. The viability/cytotoxicity assay was performed after 1 day and 5 days of culture. Live cells were stained green by calcein acetoxymethyl (calcein AM) and dead cells were stained red by ethidium homodimer-1 (EthD-1). The size bar represents 500 μm.
that nearly all cells were viable and that they maintained a fibroblastic morphology during cultivation (Fig. 5). 3.3. Effect of Mg ion implantation on cell morphology To observe the cell adhesion morphology, SEM analysis was performed after 1 h and 72 h to evaluate initial and growth adhesion behavior, respectively. Fig. 6 demonstrates the interaction between the cells and the Ti surface as observed by SEM. After 1 h of
incubation, almost all cells maintained a spherical morphology, and only a few cells presented a slight extracellular membrane bridge for attachment onto the surface of SLA Ti. However, numerous cells presented a wide cellular membrane bridge and flattened morphology on Mg-SLA Ti. These initial cell adhesion results were consistent with the fluorescent image analysis at 1 h (Fig. 4B) and 1 day (Fig. 5). Furthermore, after 72 h of cultivation, these cells were remarkably elongated and formed a fibroblast-like morphology on both SLA Ti and Mg-SLA Ti.
Fig. 6. Cell adhesion and growth morphology determined by scanning electron microscopy (SEM) of human mesenchymal stem cells (hMSCs) cultured on sandblasting and acid etching (SLA)-treated titanium (SLA Ti) and Mg ion-implanted SLA Ti (Mg-SLA Ti) specimens. hMSCs were seeded and cultured, and the initial cell adhesion and growth morphology were observed at 1 h and 72 h.
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These results agree with those obtained for ALP activity shown in Fig. 7.
4. Discussion
Fig. 7. Alkaline phosphatase (ALP) activity of human mesenchymal stem cells (hMSCs) cultured on sandblasting and acid etching (SLA)-treated titanium (SLA Ti) and Mg ion-implanted SLA Ti (Mg-SLA Ti) specimens. Cells were cultured in media in the presence and absence of an osteoblast differentiation stimulator (OS) for 7 days, and ALP activity was measured. In the presence of OS, the ALP activity was increased by Mg ion implantation. Values are expressed as the mean ± SD. Significance was defined as **P b 0.01 when compared with the SLA Ti specimen.
3.4. Effect of Mg ion implantation on osteoblast differentiation To determine the effect of Mg ion implantation on osteoblast differentiation of cultured hMSCs, OS-containing medium was used as described in the Materials and methods. When the cells on SLA Ti were treated with OS medium, ALP activity increased from 0.23 ± 0.07 nmol/30 min per mg of protein to 1.28 ± 0.07 nmol/30 min per mg of protein. When the cells were cultured on Mg-SLA Ti specimens with OS medium, ALP activity increased from 0.33 ± 0.08 nmol/ 30 min per mg of protein to 2.33 ± 0.11 nmol/30 min per mg of protein. Although, ALP activity increased on both specimen types, ALP activity of cells cultured on Mg-SLA Ti was approximately 1.8-folds higher than that of cells cultured on SLA Ti (Fig. 7). In addition, calcium accumulation was assessed to evaluate osteoblast differentiation after culture for 21 days with OS medium, and AR-S staining was performed (Fig. 8). When treated with OS medium, cells cultured on SLA Ti and Mg-SLA Ti demonstrated calcium accumulation. However, calcium accumulation on Mg-SLA Ti was significantly higher than that on SLA Ti (approximately 1.5-fold higher).
To enhance osseointegration, the topography, free energy, crystal structure, and chemical composition of the surface of dental implants have been modified. Various studies have attempted ion implantation in dental implants using nitrogen, oxygen, calcium, and Mg [8,12,23]. To modify the surface roughness, resorbable blast media (RBM) such as hydroxyapatite and calcium phosphate have been widely used [24]. The SLA technique has also been used to modify the surface roughness [25]. Although Cho et al. [10] recently reported that Mg ions implanted in RBM-treated Ti surfaces using the PSII technique provided enhanced osseointegration capacity, no reports have addressed the effect of Mg ion implantation on SLA-treated Ti. Therefore, in this study, Mg ions were implanted in the surface of SLA-treated commercial pure Ti using the PSII technique. A previous study reported an enhanced cell response after Mg ion implantation and suggested that the response would improve with increasing concentrations of Mg ions [26]. However, the strongest osseointegration was observed in a range of 8.36% to 9.33% for the Mg ion content, which suggested that an optimal surface concentration exists [15]. Cho et al. recommended implantation with a concentration of less than 10% [10]. Therefore, in this study, we used a Mg ion concentration of less than 10%, and Mg ion-implanted specimens were prepared at a final Mg concentration of 2.3 × 10 16 ions/cm2 using the vacuum arc method for the PSII technique. Several methods can be used to produce plasma from a metal, e.g., vaporization, radio-frequency discharge, microwave, sputtering, and vacuum arc, but the vacuum arc technique is simpler than the other methods. After Mg ion implantation, the surface roughness was maintained and the Ra was unchanged compared with SLA Ti; these results were consistent with a previous report [27]. To determine the effect of Mg ion on hMSCs, the cells were observed to proliferate over time. In bone healing and implant osseointegration, MSCs are the key repair cells, and their cellular response is important because successful osseointegration of implants depends on the attachment of MSCs onto the implant surface [28]. Therefore, hMSCs were used in this study to evaluate biocompatibility. In this study, we showed that Mg ion implantation enhanced MSC attachment and proliferation. The results for the initial cell number after 1 h of incubation suggests that the observed differences resulted from the initial cell adhesion to the surface. Our results show that the number of hMSCs that initially attached was higher on Mg-SLA Ti than on SLA Ti, which indicates that Mg ion implantation enhanced the initial cell attachment onto the surface
Fig. 8. Calcium accumulation determined by alizarin red staining of human mesenchymal stem cells (hMSCs) cultured on sandblasting and acid etching (SLA)-treated titanium (SLA Ti) and Mg ion-implanted SLA Ti (Mg-SLA Ti) specimens. Cells were cultured in media in the presence and absence of an osteoblast differentiation stimulator (OS) for 3 weeks, and the stained portions were photographed using a digital camera (A). The stained portion was solubilized using a cetylpyridinium chloride (CPC) solution, and the optical density was then measured (B). In the presence of OS, calcium accumulation was increased by Mg ion implantation. Values are expressed as the mean ± SD. Significance was defined as *P b 0.05 when compared with the SLA Ti specimen.
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of Mg-SLA Ti. These findings may explain the relationship between in vivo osseointegration and MSCs in terms of Mg ion implantation. Several types of ions have been used for surface modification of dental implants [8,12,23]. However, negative physiological and cellular effects have been reported to result from the release of metal ions [29]. In contrast, Klein et al. demonstrated that a low concentration of released metal ions can lead to enhanced cellular adhesion [30]. These reports therefore suggest that the metal ion release pattern is important for successful dental implantation. In this study, the pattern of Mg ion release from Mg-SLA Ti was analyzed in vitro. Although Mg ions were rapidly released within the first 7 days, the released Mg ions were not cytotoxic to hMSCs. Furthermore, our SEM analysis showed the morphology of the interaction between the cells and the Ti surface. After 1 h of incubation, almost all cells maintained a spherical morphology on the surface of SLA Ti. In contrast, numerous cells presented a wide extracellular membrane bridge and flattened morphology on Mg-SLA Ti. Furthermore, spreading cells showed fibroblast-like morphology on the surface of SLA Ti and Mg-SLA Ti. These morphological changes are typical phenomena during the cell adhesion process and they only reflect the differences in the timing of cell adhesion. Therefore, these results indicated that implanted Mg ions facilitate the fast initial cell adhesion of MSCs. Several studies have shown that Mg ions can enhance cell attachment and provide a beneficial effect during the initial stage of the bone response [19,31] and play an essential role in the binding interactions between the integrin family of cell surface receptors and their ligand proteins [31]. Sul et al. [15] reported that Mg ions promote integrin–ligand interactions to provide an improved bone response. However, in the present study of the interaction between hMSCs and Mg ions implanted in a Ti surface, the Mg ions may have played a role in the binding interaction between MSCs and the titanium surface. To evaluate the effect of Mg ion implantation on osteoblast differentiation, ALP activity and calcium accumulation were measured. ALP activity is one of the most commonly used early markers of osteogenesis, and calcium accumulation is used as a late marker [32,33]. In the present study, ALP activity was higher on Mg-SLA Ti than on SLA Ti when the cells were cultured in OS-containing medium. These results suggest that Mg ions increase ALP activity. However, ALP activity was not significantly affected when the cells were cultured in the absence of OS. Therefore, we consider that Mg ion implantation does not directly affect osteoblast differentiation. Similar results for osteoblast differentiation were obtained when AR-S staining was used as a late marker. Intracellular communication is an important factor for cellular differentiation, including osteoblast differentiation [34]. In our results, Mg ion implantation improved the initial cell attachment onto the surface of specimens. Therefore, the observed enhancement of differentiation may have been the result of earlier confluence of cells cultured on Mg-SLA Ti when compared with cells cultured on SLA Ti, which could have resulted in earlier differentiation. 5. Conclusion This study examined the cellular response to SLA-treated Ti implanted with Mg ions using the PSII method. The biocompatibility was evaluated using hMSC cells. Distinct cytotoxicity was not observed, and the initial cellular attachment was improved, with a resulting enhancement of osteoblast differentiation. These findings suggest that
Mg ion implantation into SLA Ti dental implants using the PSII technique may be a useful modification to improve osseointegration. However, further studies are necessary to determine the bone response in an in vivo model.
Acknowledgments This work was supported by a research grant from Wonkwang University in 2012 and Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (2012-040526).
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