Effect of Graphitic Layers Encapsulating Single-Crystal Apatite

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Effect of Graphitic Layers Encapsulating Single-Crystal Apatite Nanowire on the Osteogenesis of Human Mesenchymal Stem Cells Namjo Jeong, Yun Chang Park, Kyung-Mee Lee, Jae Hyup Lee, and Misun Cha J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 10 Oct 2014 Downloaded from http://pubs.acs.org on October 11, 2014

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The Journal of Physical Chemistry

Effect of Graphitic Layers Encapsulating SingleCrystal Apatite Nanowire on the Osteogenesis of Human Mesenchymal Stem Cells Namjo Jeong,+,§ Yun Chang Park,§ Kyung-Mee Lee,¶ Jae Hyup Lee,¶,* Misun Cha†,* +

Energy Materials and Convergence Research Department, Korea Institute of Energy Research,

71-2 Jang-dong, Yuseong-gu, Daejeon 305-343, Republic of Korea §

Jeju Global Research Center, Korea Institute of Energy Research, 200, Haemajihaean-ro,

Gujwa-eup, Jeju Special Self-Governing Province, 695-971, Republic of Korea ‡

Measurement and Analysis Division, National Nanofab Center, 291 Daehak-ro, Yuseong-gu,

Daejeon 305-806, Republic of Korea ¶

Department of Orthopedic Surgery, College of Medicine, Seoul National University, SMG-SNU

Boramae Medical Center, 39, Boramae Gil, Dongjak-Gu, Seoul 156-707, Republic of Korea †

Department of Chemical and Biological Engineering, Korea University, 145 Anam-ro,

Seongbuk-gu, Seoul, Republic of Korea *

Tel: +82-2-3290-3686, Fax: +82-2-926-6102, [email protected]; [email protected].

ACS Paragon Plus Environment

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ABSTRACT Ideally designed scaffold for tissue engineering must be able to provide an environment that recapitulates the physiological conditions to control stem cell function. Here, we compared vertically aligned single-crystal apatite nanowires sheathed in graphitic layers (SANGs) with single-crystal apatite nanowires (SANs), which had the same geometric properties as, but differing nanotopographic surface chemistry from SANGs, in order to evaluate the effect of the graphitic layer on the behavior of human mesenchymal stem cells (hMSCs). The difference in nanotopographic surface chemistry did not affect hMSC adhesion, growth, or morphology. However, hMSCs were more effectively differentiated into bone cells on SANGs through interaction with graphitic layers, which later degraded and thereby allowed the cells to continue differentiation on the bare apatite nanowires. Thus, SANGs provide an excellent microenvironment for the osteogenic differentiation of hMCS.

Keywords: apatite, graphitic layer, hMSC, nanowires, osteogenesis


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1. INTRODUCTION Human mesenchymal stem cells (hMSCs) of various origins are a potential cell source for many tissue-engineering applications,1,2 as they can self-renew, proliferate, and differentiate into multiple cell lineages.3–6 However, these hMSC properties are seen only within a specific extracellular environment that activates complex intracellular signalling systems. Engineering the cellular microenvironment has great potential to create a platform technology toward engineering of tissue and organs.7-20 Various synthetic scaffolds are used for effective hMSCbased tissue engineering; however, several factors pertaining to the scaffold design, such as surface chemical nature, physical structure, and the method of fabrication of the scaffolding material must be considered.21-29 Research in the field of tissue engineering is being directed towards mimicking the architectural and functional properties of the native ECM, which plays a key role in the regulation of cellular behavior such as adhesion, proliferation, differentiation, migration and apoptosis by influencing cells with biochemical signals and topological cues. A number of different materials and methods have been used for developing scaffolds for ECM mimicking, which include electrospinning, drawing, template synthesis, phase separation, and selfassembly.9,10, 27, 30 Cell adhesion to biomaterials induces signal transduction, which regulates the activity of transcription factors and consequently modulates gene expression. Cell interaction with biomaterials influences their capacity to proliferate and differentiate. In this context, graphitic surfaces with a wide range of characteristics have shown a variety of desirable properties as a biomaterial, including maintenance of cell integrity and lack of cytotoxicity and inflammatory reactivity.31 Among nanomaterials with a graphitic surface,


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vertically aligned carbon nanotubes (CNTs) are particularly promising for tissue regeneration because their diameters are similar to the physical dimensions of extracellular matrix proteins.32,33 Scaffold nanotopography has been found to affect behavioral changes in cells and tissues. Several recent studies have investigated the influence of nanostructures on MSC proliferation and differentiation into specific cell lineages.12, 15-17, 28 In addition, surface nanotopography is one of the most important parameters that can influence the cell/tissue-material interaction.7, 20, 29 Apatite composites with graphitic structures such as CNTs or graphenes have recently been suggested as bone grafting materials that provide biocompatibility and nanoscale reinforcement.34-38 In a previous study, we accomplished a chemical vapor deposition (CVD) synthesis of vertically aligned single-crystal apatite nanowire sheathed in graphitic layers (SANGs).39 Because of the unique nanotopographical structure, SANGs demonstrated excellent characteristics compared to plastic culture dishes with regard to induction of osteogenesis. However, the effect of graphitic layers on hMSC osteogenenic differentiation was not fully explored. Here, we have directly compared the SANGs with single-crystal apatite nanowires (SANs) as control, which have the same geometric properties as, but differing nanotopographic surface chemistry from SANGs, in order to evaluate the effect of graphitic layers on hMSC behavior. These experimental models are the concept of holding the geometric properties of the nanomorphology, while the different nanotopographic surface chemistries.

2. EXPERIMENTAL METHODS 2.1. Material and Scaffold Preparation


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E-glass fibers (Owens Corning) with approximately 25 wt% of CaO were used as starting materials for SANG growth. Glass fibers were assembled on a 10 × 10 × 1 mm3 (W × L × t) quartz plate and placed in the center of a quartz tube installed in a horizontal furnace. The furnace was heated to 750 °C at a rate of 10°C/min and maintained for 1 h at the set temperature. Ar of 5N-purity was used as a carrier gas at 1000 ml/min. C2H2 (99.9%, 50 ml/min) and PH3/Ar mixture gas (PH3 of 100 PPM in Ar, 99.999%, 50 ml/min) were used as the reactant gases for SANG growth. SANs, which had geometric properties identical to those of SANGs, were prepared by oxidizing SANGs at 650 °C for 20 min. During the oxidation process performed under atmospheric conditions, only the graphitic layers on SANGs were selectively removed. Thin films of glass fiber SANGs and SANs were prepared as scaffolds for hMSC culture. The scaffolds prepared (10 × 10 mm2, W × L) were installed in a circular polycarbonate frame, placed in 12-well plates, and sterilized with ethylene oxide gas. 2.2. Material Characterization Nanowires were characterized using scanning electron microscopy (SEM; S-4700, HITACHI; accelerating voltage of 10–15 keV), transmission electron microscopy (TEM; Tecnai F30 Supertwin; accelerating voltage of 200–300 kV), and time-of-flight secondary ion mass spectroscopy (TOF-SIMS; TOF, SIMS5 (ion TOF)). TOF-SIMS analysis was conducted with a Bi+ ion source and an accelerating voltage of 25 keV at 10-9 Torr. The spot area was 100 × 100 µm2, and the exposure time was 60 s. Spreading of water droplets on the surfaces of SANGs and SANs was measured using a Low Bond Axisymmetric Drop Shape Analyzer. The measurements (n = 3 of each) were carried out in air at room temperature by using deionized water as a probe liquid. Application of the droplet (approximately 20 µl) onto the surface was performed manually using a syringe, and the


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contact angle was measured immediately. The progression of spreading was recorded with a CCD camera connected to an image analyzer. The surface morphology of the substrate-attached cells and the features of the as-formed minerals were analyzed by SEM. The cells cultured on scaffolds were washed with phosphatebuffered saline (PBS) and fixed with 2% glutaraldehyde and then with 1% osmium tetraoxide. The samples were then dehydrated in a series of increasing concentrations of ethanol (50%, 70%, 80%, 90%, 95%, and 100%) and isoamyl acetate following critical-point drying. After coating with Os to avoid electron charging, samples were observed by SEM at an accelerating voltage of 10 keV. Cell-nanowire interactions were analyzed by TEM at an accelerating voltage of 200 keV. 2.3 hMSCs Culture Cryopreserved hMSCs were obtained from Lonza (USA). The cells were suspended in MSCGM culture medium (Lonza) and cultured at 37 °C in a humidified atmosphere containing 95% air and 5% CO2. Confluent cells were detached with 0.05% trypsin-EDTA and subcultured 4 times. The expanded hMSCs were then seeded on the surface of the prepared scaffolds at a density of 2.5 × 104 cells/scaffold and cultured for 7 and 21 days in the osteogenic medium (DMEM supplemented with 100 M dexamethasone, 10 mM β-glycerophosphate, and 100 M ascorbic acid-2-phosphate) (Sigma, St. Louis, MO, USA), which was changed twice a week. 2.4 Biocompatibility The biocompatibility of the scaffolds was evaluated by assessing the viability and proliferation of adhered cells using the MTS assay as described by the manufacturer (CellTiter 96 AQueous One solution, Promega, USA). Briefly, the samples were rinsed with PBS to remove unattached cells and incubated with 20% MTS reagent in a serum-free medium for 3 h at 37 °C. CellTiter 96 AQueous One Solution Reagent was added to each well, and the plates were incubated for 1–4 h


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at 37 °C in a humidified 5% CO2 atmosphere; the absorbance was measured at 490 nm using a plate reader (Thermo Fisher Scientific Inc., USA). The assay (4 wells per group) was performed as at least two independent experiments. Cell viability was determined as a percentage of that of control cells (100%). 2.5 Alkaline Phosphatase (ALP) Activity After osteogenic differentiation for 7 and 21 days, the differentiated cells were washed twice with PBS and lysed with 0.2% Triton X-100 in PBS. The cell lysates were centrifuged at 13,000 rpm for 10 min at 4 °C, and the supernatants were assayed for ALP activity using pnitrophenylphosphate as a substrate. The activity was defined as the amount of p-nitrophenol released after incubation for 20 min at room temperature and normalized by the total protein concentration. 2.6 Calcium Measurement The amount of calcium deposited by the scaffold-cultured cells in the matrix was determined with a calcium quantification kit (BioAssay Systems Inc, USA). The samples were solubilized in 500 µl of 0.1% HCl for 5 min at room temperature and centrifuged to remove any insoluble particles. The supernatants (5 µl) were mixed with phenolsulphonephthalein dye and assayed in triplicate using 96-well plates. The color intensity was measured at 612 nm. 2.7 Real-time Polymerase Chain Reaction (PCR) Total RNA was extracted from cells with the Easy-Blue RNA isolation reagent (Intron Bio, Korea) according to the manufacturer’s protocol. Complementary DNA was synthesized from 0.5 µg of total RNA using the iScript cDNA Synthesis Kit (Invitrogen, USA). Quantitative realtime PCR was performed using the LightCycler 480 SYBR Green I master kit and LightCycler


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480 instrument. Relative mRNA levels were normalized to that of GAPDH mRNA for each reaction.

Table 1. Gene-Specific Primers for Reverse Transcription PCR (RT-PCR) Analysis Gene Runx-2

Osteocalcin GAPDH

Sequence (F) CCG CAC GAC AAC CGC ACC AT (R) CGC TCC GGC CCA CAA ATC TC (F) CGA AGA CAA CAA CCT CTC CAA ATG (R) ACC ATC ATA GCC ATC GTA GCC TTG (F) GCC GTA GAA GCG CCG ATA GGC (R) ATG AGA GCC CTC ACA CTC CTC (F) CCA GAA CAT CAT CCC TGC CTC TAC (R) GGT CTC TCT CTT CCT CTT GTG C

Anneal temp. (℃)

size (bp)

57

530

51

257

51

297

54

554

3. RESULTS AND DISCUSSION 3.1. Scaffold Preparation and Characterization. Figure 1a shows the thin glass-fiber film assembled on a 10 × 10 mm2 (W × L) quartz plate. First, glass fibers of 10 mm in length were prepared on the quartz plate and wetted by the falling deionized water droplets; tweezers were used to flatten the wetted glass fibers. The assembled thin glass films were dried at 100 °C. SANG synthesis was performed by CVD.39 Photographs in Figure 1b show that the color of the glass fibers that grew changed to black (Figure 1b). Nanowires grew with dense and uniform coverage on the surface of glass fibers (Figure 1c and d) resembling a “tooth-to-tooth” structure between glass fibers (Figure 1e). Both positive and negative ions in the SANG TOF-SIMS spectra were obtained in the m/z range of 0–400, but peaks with significant intensity were mainly detected in the m/z range of 0– 100 for both positive and negative spectra (Figure 2). In order to investigate the surface chemistry of the core-shell nanostructure, we performed detection of positive and negative ions


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before and after ion-beam sputtering on SANG surface. Positive and negative ion SIMS spectra recorded before beam sputtering (Figure 2a and c) demonstrated characteristic peaks involving positive ions of Ca+ (m/z = 40), CaO+ (m/z = 56), and CaOH+ (m/z = 57) and negative ions of (m/z = 16),

(m/z = 17),

(m/z = 47),

(m/z = 63), and

(m/z = 79).

Figure 1. (a) Growth process of SANGs on amorphous calcium-rich glass fibers. (b) A photograph taken before and one taken after the growth of SANGs. (c, d) Low-magnification


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SEM images of SANGs synthesized on glass fibers. (e) The “tooth-to-tooth” shape of SANGs observed between glass fibers. These peaks were related to apatite even though their intensity differed from previously reported results because of the differences in the experimental materials and beam ion sources.40 ions (X = 1, 2, 3,… and Y = 0, 1, 2,…), such as such as

, and

,

ions (X = 1, 2, 3,… and Y = 0, 1, 2,…), such as

,

,

,

,

,

,

, and

, and

with high signal intensity were also detected. It is well known that these positive and negative ions can be observed in SIMS spectra for the surface of aligned carbon nanotubes.41

Figure 2. TOF-SIMS of SANGs. The spectra show positive (a, b) and negative (c, d) ion results before and after ion-beam sputter, respectively. We observed that the ions related to the graphitic layers were more prominent than those related to the apatite core before beam sputter (Figure 2a and c); this is because the graphitic layers encapsulating the apatite core were unimpaired before beam sputtering.42 After sputtering


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SANGs for 1 s, stronger positive ( and

,

, and

) and negative (

,

,

,

,

) ions corresponding to hydroxyapatite were detected, whereas the intensity of

ions relatively decreased (Figure 2b and d). These results imply that the graphitic layers sheathing the apatite core were significantly damaged during beam sputtering.24 In addition, peaks corresponding to

(m/z=19),

(m/z=23), and

(m/z=60) were observed at

relatively low intensity.

Figure 3. HRTEM images and FFT patterns of (a) SANGs and (b) SANs. (c) Photographic images of the scaffold for hMSC culture. (scale bar = 10 nm) The TOF-SIMS spectra strongly suggested the core-shell configuration of SANGs shown in the TEM images provided as Figure 3a and b. High-resolution TEM (HRTEM) images showed that the SANGs were surrounded by discontinuous graphitic layers, while no layers were


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observed on the radial surface of SANs (Figure 3a and b). SANs were prepared by SANG oxidation to investigate the osteogenic response of hMSCs to scaffold surface nanotopography (Figure 1a). The FFT images showed that SANGs and SAN had a lattice structure of the core nanowire, which had a diameter of approximately 15 nm corresponding to a single hexagonal apatite crystal (insets of Figure 3), indicating that no significant changes occurred after the oxidation of SANGs. Thin films of glass-fiber SANGs and SANs were prepared as scaffolds for hMSC culture (Figure 3c).

Figure 4. Water contact angle measurement in (a) culture dishes (θ = 70.2°), (b) SANs (θ = 66.5°), and (c) SANGs (θ = 144.7°). (d) High-magnification image of SANGs. Water contact angle measurements showed very similar results for culture dishes and SANs; however, the contact angle of SANGs indicated their hydrophobic nature due to the graphitic shell (θ = 144.7; Figure 4).

3. 2 Cell Attachment and Proliferation. SANGs and SANs were able to promote hMSC attachment and spreading with similar patterns (Figure 5). The attached cells had similar morphology in both cases and had spread well on both scaffolds. However, cell spreading and orientation were slightly different for SANGs


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and SANs. On SANG scaffolds (Fig 5a), hMSCs were more angular and appeared to spread in multiple directions, whereas on SAN scaffolds, the cells were more flattened and were oriented in one direction (Figure 5b). These results are consistent with SEM images showing the morphology and cell organization of hMSCs on SANG and SAN scaffolds (Figure 5c and d).

Figure 5. Morphology and behavior of hMSCs on the scaffolds (7-day culture). SEM images of (a) SANGs, (b) SANs, (scale bar = 10 um (upper), 1 um (lower)) and secondary extensions of hMSCs on the scaffold of (c) SANGs (d) SANs (scale bar = 5 um (left), 1 um (right), (e) MTS assay for hMSC growth.

MSCs were stretched on the surface of the scaffolds with their secondary extensions spreading on the matrix. These extensions had a diameter of approximately 200 nm and sprouted near the tip of the cytoplasmic extension. Detailed observation showed remarkable spreading of cell secondary extensions across the tips of SANGs and SANs (yellow arrows in Figure 5c and


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d). This finding implies that the tips of SANGs and SANs can provide focal adhesion points with little sagging for cell extension through their amoeboid movements. The MTS assay performed after 7 days of hMSC culture on the prepared scaffolds indicated that SANGs and SANs showed nearly identical biocompatibility with hMSCs and did not inhibit cell proliferation compared to that seen for culture dishes (Figure 5e).

Cell behavior on scaffold surface is very complex, being affected by many factors such as roughness, surface chemistry and wettability of scaffold.11-19,27-29 Some research has shown that the early adhesive behavior of MSCs is largely dependent on both the texture and the surface chemistry of the substrate.9, 13, 15, 27, 29 Interestingly, although the graphitic shell increased the hydrophobicity of the scaffolds (Figure 4), SANGs could support cell attachment and growth. Previous studies showed that hydrophilic scaffold surfaces were better in supporting cell adhesion.43,44 However, the SANG hydrophobic surface with appropriate nanotopography favored protein adsorption and may successfully support cell proliferation and differentiation.4547

Our results suggest that SANGs with the appropriate surface nanotopography can counteract

unfavorable hydrophobic effects and promote cell attachment. Photographs of SANG and SAN scaffolds after 7 days of hMSC culture support the successful cell attachment and growth (Figure 6a). The details of cell attachment to the surfaces of SANG and SAN was confirmed by HTEM images (Figure 6b and c). Cell was well-attached to graphitic layers, and no outstanding changes in both graphitic layers and apatite core was observed after 7-days-culture (Figure 6b and its FFT image). This was the same pattern through SAN (a FFT image of Figure 6c). In addition, some debris that is presumed to be occurred by electron beam were formed on the surface of SAN (Figure 6c), which indicates that SAN was more damageable than SANG by high electron beam energy during HRTEM observation.


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Figure 6. (a) Photographic images of the SANG and SAN scaffolds after 7 days hMSC culture. Corresponding HRTEM images and FFT patterns of (a) SANG and (b) SANs attached to cell (scale bar = 10 nm). 3.3 Osteogenesis of hMSCs. Most fabricated bone grafts usually mimic bone structure and topography at the microlevel; however recently, researchers are focusing on designing bone constructs with appropriate biomechanical properties and biomimetic behaviors at the nano-level.8-10 It was reported that the surface properties of nanomaterials such as chemical characteristics, stiffness and nanotopography has a tremendous impact on cell attachment and differentiation.7, 10, 16, 28, 29, 48 We investigated whether the nanotopographic surface chemistry of the scaffold had an effect on the osteogenic differentiation of hMSCs. After 7 days of culture, the SANGs appeared to have induced a higher deposition of bone-like mineral particles, and a dramatic course of


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mineralization was observed over 21 days of culture; remarkably, the surface of SANG scaffold was completely covered with a layer of mineral particles (Figure 7a). In contrast, we found that a relatively low amount of mineral particles was scattered on the surface of cells cultured on SAN scaffold (Figure 7b). These results indicate that SANGs constitute a suitable substrate for deposition of a mineralized matrix. We think that graphitic layers of SANGs with lower crystal quality may not withstand the oxidation process in an intracellular environment.39 In this oxidation process, negatively charged graphitic layer attributed to electrostatic attraction of Ca2+ ions to the SANGs surface.

Figure 7. Production of mineral particles by hMSCs cultured for 7 and 21 days on the scaffolds of (a) SANGs and (b) SANs. (scale bar = 100 um (left), 10 um (right))


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TEM images also confirmed the yield difference of bone mineral particles in types of scaffold (Figure 8a and b). Nanowires was well-attached to cell and bone mineral particles was observed on the cell surface (Figure 8a). Figure 8b clearly showed that cell could attach to the nanowire surfaces through their secondary expansion (Figure 8b). As-formed bone mineral particles seemed like a lump of the longitudinally grown crystals (Figure 8c). The crystal had a hexagonal apatite structure that can be assigned to {002} and {300} families of reflections (Figure 8d).

Figure 8. TEM images of (a) SAN and (b) SANG scaffolds after 21 days hMSCs culture (scale bar = 50 nm), (c) TEM (scale bar =100 nm) and (d) HRTEM images of bone mineral particles showing the alignment of apatite crystals (scale bar =10 nm), Inset of (d) shows corresponding FFT pattern. ALP activity, which is an early marker of osteogenic differentiation, and calcium content measured after 7 and 21 days of culture suggested that SANGs could support the differentiation of hMSCs to osteoblasts by accelerating osteogenic differentiation and promoting active


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deposition of bone-like mineral structures (Figure 9a). SANGs induced ALP activity in hMSCs at an earlier time point (7 days) than SANs, suggesting that hMSCs on SANG scaffolds started mineralization earlier than those on SANs. As shown in Figure 9b, the calcium content in hMSCs growing on SANGs was significantly higher than that in SAN-growing cells at an early stage and further increased after 21 days of culture. This effect is likely the result of high ALP activity in young osteoblasts, which is indicative of differentiation.48

Figure 9. Alkaline phosphatase activity (a) and calcium content (b) measured in hMSCs cultured for 7 and 21 days on SANG and SAN scaffolds. Gene expression was analyzed by quantitative real-time RT-PCR to further evaluate the osteogenic differentiation of hMSCs on the surfaces of SANGs and SANs (Figure 10). The expression level of runt-related transcription factor 2 (Runx 2) is controlled by external signals,49 and has been shown to strongly correlate with the early phase of hMSC differentiation into


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osteoblasts and upregulation of ALP, osteocalcin (OCN), and bone sialoprotein (BSP).50 After 7 days of culture, Runx-2 expression was higher in hMSCs on SANG scaffolds than in SANgrowing cells; however, it significantly increased in SAN-growing cells after 21 days of culture (Figure 10a).

Figure 10. Quantitative RT-PCR results for osteogenic gene expression: (a) Runx-2, (b) BSP, and (c) OCN.


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Expression of BSP, which is associated with hydroxyapatite deposition and matrix mineralization, was significantly upregulated in the cells on SANGs, peaking at day 7 and remaining higher than that in the cells on SANs over the course of culture (21 days) (Figure 10b). The level of OCN, which was expressed at a later stage of osteogenic differentiation, was also higher in hSMCs cultured on SANGs than in those on SANs at all points in time (Figure 10c). This pattern of mRNA expression correlated with the calcium content in SANG- and SANcultured cells. These data strongly indicate that SANGs with a graphitic surface promoted osteogenesis more effectively than SANs with an apatite surface.

Figure 11. Effect of graphitic layers encapsulating single-crystal apatite nanowire on the osteogenesis of human mesenchymal stem cells


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Our findings imply that the high observed efficiency of initial osteogenic differentiation resulted from the graphitic structure surrounding the apatite core, and the utility of the inner core apatite of the SANGs for contributing to continued growth may thus be questioned. Previously, we tried to observe the fate of the graphitic layers in SANGs after the differentiation process over 28 days.39 Interestingly, the graphitic layers in contact with the cell were degraded partially. These features showed an example of intelligent material behavior, the graphitic layers would be degraded through the interaction with the hMSCs and the exposed apatite core would continuously induce cell differentiation (Figure 11). At first, the morphological effects of the SANGs specific to the graphitic structure should stimulate the osteogenic differentiation in hMSCs. This mechanism strongly supported why the SANGs scaffold with a graphitic layers exhibited more excellent cell differentiation characteristics and produced a lot of mineral particles, compared with SANs scaffolds.

4. CONCLUSION The results of our study demonstrate that the promotion of osteogenesis by SANGs is specific to their unique nanotopograpic surface chemistry. SANG scaffolds with graphitic layers provided a significantly better supportive structure for the differentiation of hMSCs into osteoblasts than did SAN scaffolds with an apatite surface. The nanotopographic surface structure of SANGs reproduces the conditions in bone tissue, which presents a more alkaline environment rich in ceramic/mineral components. Therefore, the SANG surface graphitic structure is beneficial for the initial growth and differentiation of hMSCs,33,51 which would be followed by degradation of graphitic shells,39 and the bare apatite core would provide a


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biologically compatible environment for the later stages of osteogenesis. We believe that our SANGs with a unique core-shell nanostructure provide a useful scaffolding material for hMSCbased tissue engineering strategies because they reflect the dynamic nature of many biological processes. ACKNOWLEDGMENT This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A3010079). REFERENCES (1)

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SYNOPSIS


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Effect of Graphitic Layers Encapsulating Single-Crystal Apatite

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