Boron nitride nanotube-enhanced osteogenic differentiation of mesenchymal stem cells Xia Li,1 Xiupeng Wang,2 Xiangfen Jiang,1 Maho Yamaguchi,1 Atsuo Ito,2 Yoshio Bando,1 Dmitri Golberg1 1
World Premier International Center for Materials Nanoarchitectonics (WPI-MANA), National Institute for Materials Science (NIMS), Namiki 1-1, Tsukuba, Ibaraki 305-0044, Japan 2 Human Technology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan Received 27 September 2014; revised 8 January 2015; accepted 29 January 2015 Published online 12 March 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33391 Abstract: The interaction between boron nitride nanotubes (BNNTs) layer and mesenchymal stem cells (MSCs) is evaluated for the first time in this study. BNNTs layer supports the attachment and growth of MSCs and exhibits good biocompatibility with MSCs. BNNTs show high protein adsorption ability, promote the proliferation of MSCs and increase the secretion of total protein by MSCs. Especially, BNNTs enhance the alkaline phosphatase (ALP) activity as an early marker of osteoblasts, ALP/total protein and osteocalcin (OCN) as a late marker of osteogenic differentiation, which
shows that BNNTs can enhance osteogenesis of MSCs. The release of trace boron and the stress on cells exerted by BNNTs with a fiber structure may account for the enhanced differentiation of MSCs into osteoblasts. Therefore BNNTs are potentially useful for bone regeneration in orthopedic C 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part applications. V B: Appl Biomater, 104B: 323–329, 2016.
Key Words: boron nitride nanotubes, mesenchymal stem cells, proliferation, osteogenic differentiation
How to cite this article: Li X, Wang X, Jiang X, Yamaguchi M, Ito A, Bando Y, Golberg D. 2016. Boron nitride nanotubeenhanced osteogenic differentiation of mesenchymal stem cells. J Biomed Mater Res Part B 2016:104B:323–329.
INTRODUCTION
Boron nitride nanotubes (BNNTs),1 as a structural analogue of carbon nanotubes (CNTs), recently attract increasing attention in the biomedical field including bone tissue engineering,2,3 drug delivery,4–7 and boron neutron capture cancer therapy.8 For example, BNNTs are used to improve mechanical properties of conventional bone substitute materials, such as polylactide–polycaprolactone copolymer2 and hydroxyapatite.3 BNNTs induce apatite formation in a simulated body fluid environment, which shows feasibility in orthopedic applications.9 They can also deliver DNA oligomers to the interior of cells with no apparent toxicity.10 As a whole, BNNTs show good biocompatibility,2,3,10,11 except for the morphological cytotoxicity associated with too long BNNTs.12 The interaction between BNNTs and several types of cells, such as human embryonic kidney 293 cells,10 osteoblast,2 macrophages,2 neuronal-like PC12 cells,11 human neuroblastoma cells13 and so on, have been studied. However, up to now, there have been no reports in regards of BNNTs effects on mesenchymal stem cells (MSCs). MSCs are multipotent stem cells that can differentiate into a variety of cell types, including chondrocytes, osteo-
blasts, adipocytes, fibroblasts, marrow stroma and so on.14 MSCs have an enormous therapeutic potential for regenerative medicine owing to their plentiful source in bone marrow and high capacity for self-renewal while maintaining multipotency. For instance, MSCs (preferably the patient’s own) are of essential importance for tissue engineered implants, as MSCs will endow metabolic activity and biological integration with the tissue engineered implants.15 MSCs can differentiate into osteogenic cells under appropriate extracellular matrix (ECM).14 BNNTs have the following possible merits in bone tissue engineering: (i) Boron is an essential trace element which is beneficial to the bone growth and maintenance, arthritis alleviation or risk reduction and so on16,17; (ii) BNNTs are light in weight having excellent mechanical properties with bending stress values of 100–260 MPa and elastic modulus of 0.5–0.6 TPa1,18; (iii) BNNTs show good biocompatibility; (iv) White color of BNNTs is aesthetically advantageous in a biomaterial. A very low level of boron ions, even at 0.1–100 ng mL21, significantly increases osteogenic differentiation by enhancing ALP activity, stimulates osteogenic differentiation-related marker gene expression and increases the formation of mineralized nodules in osteoblasts and human bone marrow stromal
Correspondence to: X. Li; e-mail: [email protected] (or) D. Golberg; e-mail: [email protected] Contract grant sponsors: World Premier International (WPI) Center for Materials Nanoarchitectonics (MANA) of the National Institute for Materials Science (NIMS), Tsukuba, Japan
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cells.16,19 Because bone is a load-bearing tissue in a human body, the excellence of BNNTs mechanical properties is attractive for orthopedic applications. Based on the above reasons, the interaction between a BNNTs layer and MSCs is for the first time evaluated in this study. And herein, we report that the BNNTs layer supports attachment and growth of MSCs and enhances osteogenic differentiation of MSCs. This work suggests that BNNTs are promising as a tissue engineering scaffold for bone regeneration.
MSCs culture on BNNTs layer About 1 mL of MSCs suspension, about 104 cells mL21 in culture medium (MEME supplemented with 10% FBS, 10 mM sodium b-glycerophosphate, 10 nM dexamethasone and 82 lg mL21 vitamin C), was poured on the BNNT layer coated plate. The MSCs attached to the BNNT layer coated plate were cultured in a humidified atmosphere of 5% CO2 at 37 C.
MATERIALS AND METHODS
MSCs proliferation, ALP activity, total protein content, and OCN protein content tests The MSCs attached to BNNTs layer were cultured for 7 and 14 days. In this case, the culture medium was replaced with a fresh one every 3 days. After culturing, the MSCs on BNNTs layer were mildly washed twice with phosphate buffered saline (PBS(-)) for the following tests. The number of MSCs on BNNTs layer was determined by the WST-8 method using a CCK-8 kit (Dojindo Laboratories, Japan) in accordance with the manufacturer’s instructions. As for the total protein test and alkaline phosphatase (ALP) activity test, after culturing, the cells were washed twice with PBS(-). An aliquot of 300 mL of 0.05% Triton X was added to the culture well and the mixture was incubated at 4 C for 2 h. The resulting supernatant was assayed for total protein, OCN content and ALP activity as follows. Total protein content was assayed by the Bradford method using a BioRad protein assay reagent kit (Bio-Rad Laboratories, Japan) in accordance with the manufacturer’s instructions. The ALP activity of the MSCs was assayed using a LaboassayTM ALP kit (Wako Pure Chemicals, Japan) in accordance with the manufacturer’s instructions. The OCN protein content of the MSCs was assayed using a rat OCN EIA kit (Biomedical Technologies, USA) in accordance with the manufacturer’s instructions. The boron release from BNNTs in the Tris-HCl buffer was quantitatively analyzed using an inductively coupled plasma atomic emission spectrometer (ICP: SPS7800, Seiko Instruments, Japan).
Preparation of BNNTs layer BNNTs used in this study were synthesized by a chemical vapor deposition method using boron and metal oxide as precursors. The detailed growth procedure was reported elsewhere.20,21 The as-grown BNNTs were purified at temperatures up to 1900 C under the protection of argon gas to remove impurities. Purified BNNTs are “snow-white” in color. The purified BNNTs were further shortened, as was previously reported.22 The BNNTs were put in an alumina crucible and oxidized at 1000 C for 5 h. The oxidized BNNTs were put into a bottle with a sufficient amount of water and sonicated for 5 h. The BNNTs were filtered off after sonication. Then, BNNTs were suspended in alcohol and sonicated for 10 h. A certain amount of BNNTs suspension was dropped onto cover glasses treated beforehand with the piranha solution (H2SO4:H2O2 5 3:1 at 80oC for 40 min), surrounded with a parafilm in the 24well culture plate and dried in the clean bench. The cover glass coated with the BNNTs layer in the plate was sterilized by UV light overnight and used for the following cell culture study. Protein adsorption The cover glass coated with BNNTs layer at 25 lg mL21 and without BNNTs layer were added into 1 mL 100 lg mL21 fibrinogen, laminin, or fibronectin in a PBS buffer, respectively. After 4 h of incubation at 37 C, the protein adsorption was quantified using a QuantiPro bicinchoninic acid (BCA) assay kit (Sigma, USA). Bone marrow MSCs harvest MSCs were obtained from bilateral femora from Fischer 344/N syngeneic rats.23 Both ends of the rat femora were cut away from the epiphysis. Bone marrow was flushed out with 15 mL of culture medium, minimal essential medium eagle (MEME) containing 10% FBS and 1% antibiotics (100 U mL21 penicillin G, 100 mg mL21 streptomycin sulfate, and 0.25 mg mL21 amphotericin B). The bone marrow suspension was poured in a 75 cm2 tissue culture polystyrene flask and incubated at 37 C in a humidified atmosphere containing 5% CO2. The culture medium was replaced with a fresh one every 3 days until 90% confluence was reached. All the animal experiments were permitted by the Ethical Committee of the National Institute for Materials Science (NIMS), Japan. All the animal experiments and feeding were carried out in accordance with the guidelines of the Ethical Committee of NIMS, Japan.
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Actin staining For actin staining, cells were fixed in 4% formaldehyde solution, permeabilized with 0.1% Triton X-100, and then stained by fluorescein isothiocyanate (FITC)—conjugated phalloidin (Sigma, Germany). After counterstaining the nuclei with DAPI (Vector Labs), fluorescence images were obtained using fluorescence microscopy (BX51, Olympus, Japan). Statistical analysis All statistical comparisons were conducted using a 95% confidence interval (p < 0.05). Single-factor analysis of variance (ANOVA) was performed to identify significant differences among the groups. When significant differences were found, the Tukey’s post hoc multiple-comparison test was used to determine the significant differences between the mean values of the groups. The experimental data were expressed as means 6 standard deviation.
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FIGURE 1. SEM (A) and TEM (B) images of BNNTs; AFM images of BNNTs layer on a cover glass with different amounts of BNNTs for the subsequent cell culture (C, 2 lg mL21; D, 5 lg mL21; E, 10 lg mL21; F, 25 lg mL21).
RESULTS
The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of shorten BN nanotubes (around 1–2 lm in length and 80 nm in diameter) synthesized by the chemical vapor deposition (CVD) method, oxidized in air at 1000 C for 5 h and sonicated in water are shown in Figure 1(A,B). Atomic force microscopy (AFM) analysis [Figure 1(C–F)] revealed that the BNNTs are discretely distributed on the glass substrate treated with the piranha solution.
BNNT layer can enhance the protein adsorption and MSCs attachment. Protein adsorption is critical for regulating cellular behaviors on materials surfaces such as adhesion and proliferation. Therefore, the adsorption of individual proteins such as fibrinogen, laminin and fibronectin was examined for BNNTs. Quantitative measurements indicated that BNNTs layer on cover glass had showed higher proteins adsorption ability than cover glass control (Table I). Cellular morphology after 1 day culturing was evaluated by
FIGURE 2. Left: Fluorescent images of MSCs after 24 h of culture with control (A–C) and BNNTs layer at 2 lg mL21 (D–F); Right: Quantification of cell attachment area of MSCs after 24 h of culture with control and BNNTs layer at 2 lg mL21 (n 5 80).
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TABLE I. Different Protein Adsorption Amounts for BNNTs Layer Coated Cover Glass and Control
Proteins Fibrinogen Laminin Fibronectin
Protein Adsorption Amounts for BNNTs layer (lg)
Protein adsorption amounts for control (lg)
23.3 6 1.2 9.1 6 3.7 18.2 6 1.7
5.2 6 0.5 4.9 6 1.0 5.9 6 1.4
staining the F-actin fibers with phalloidin. Fluorescent images illustrated that the shapes of MSCs cultured on control and BN nanotubes layer had been distinctly different (Figure 2). For control, MSCs displayed spindle-shape or irregular morphology, most probably due to poor adhesion to glass substrate. Whereas, cells cultivated on the BNNTs layer were larger, more widespread, more flat and more stretched out. BNNTs layer promoted proliferation, total protein and osteogenic differentiation of MSCs. Figure 3 shows the MSCs proliferation on the BNNTs layer after 7 and 14 days of culture. With an increase in amount of BNNTs, the number of MSCs increases and then decreases compared with control. The number of MSCs on BNNTs layer with 5 lg mL21 concentration is the highest among the data for each group after 14 days of culture. Figure 4 depicts the total protein of MSCs on the BNNTs layer after 7 and 14 days of culture. Totally, the MSCs on the BNNTs layer at a concentration lower than 10 lg mL21 display higher protein secretion than the controls after 7 or 14 days of culture. Measurements of specific ALP activity and OCN protein content were taken as indicators of osteogenic differentiation. ALP is an early osteoblastic marker of the protein level that does not rise in uninduced MSC, but displays a strong expression during osteogenic differentiation.24 Figure 5 illustrates the ALP activities of MSCs on the BNNTs layer after 7 and 14 days of culture. MSCs on the BNNTs layer with a low BNNT content show higher ALP activity than the controls after 7 days of culture. The ALP activity of the MSCs cultured on the BNNTs layer is significantly increased after 14 days of culture compared with that after 7 days of culture. On the BNNTs layer with a 1–10 lg mL21 concentration, the MSCs exhibit markedly increased ALP activity compared with the controls after 14 days of culture. The MSCs on the BNNTs layer with 2 lg mL21 concentration show the highest ALP
activity among all the other BNNTs layer. The ALP activity of MSCs on BNNTs layer with 2 lg mL21 concentration was about two times higher as compared with that without BNNTs layers after 14 days of culture. ALP per total protein after 14 days of culture reveals the same tendency, as seen in Figure 5(C), with the highest ALP per total protein being at 2 lg mL21 concentration. This means that the BNNTs layer enhanced osteogenic differentiation of the MSCs compared with the control. Similar to the results on ALP activity, MSCs on the BNNTs layer showed significantly increased OCN protein concentration, a late marker of osteogenic differentiation, as compared with control after 14 days of culture [Figure 5(D)]. The MSCs on the BNNTs layer with 10 lg mL21 concentration show the highest OCN amount. Boron release from BNNTs layer after 1 week was detected by the ICP method (Figure 6). With an increase in BNNTs amounts, boron release was gradually enhanced. The boron concentrations for BNNTs layer (at 1–25 lg mL21) after 1 week immersion in Tris-HCl buffer were about 0.1 3.2 ng/mL. In addition, with the increase in time, BNNTs in Tris-HCl buffer exhibit gradual boron release (Figure 6). DISCUSSION
BNNTs are promising tissue engineering scaffolds for bone tissue regeneration due to not only their active effects on the osteogenesis but also due to their excellent mechanical properties. In this study, we used naked BNNTs to check their effects on MSCs and avoid any other possible interference, such as dispersing agents or surface modification. The present BNNTs show good biocompatibility, exhibit high protein adsorption ability and promote the proliferation of MSCs and secretion of total protein. BNNTs enhance ALP activity, an early marker of osteoblasts, which implies that BNNTs can enhance the osteogenesis of MSCs. The observed effect is certainly due to a complex interplay between chemical and mechanical properties of BNNTs, and the interactions between BNNTs and cells. This study confirmed that BNNTs had enhanced the differentiation of MSCs into osteoblasts and had positive effects on osteogenesis (Figures 3–5). Our results are consistent with the previous reports about positive effects of BNNTs-polymer/hydroxyapatite composites on osteoblasts.2,3 It was demonstrated that the addition of BNNTs into polymers had showed higher osteoblast cell viability
FIGURE 3. Effects of BNNTs layer on the MSCs proliferation after 7 days (A) and 14 days (B) of culture (n 5 8).
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FIGURE 4. Effects of BNNTs layer on the total protein content of MSCs after 7 days (A) and 14 days (B) of culture (n 5 8).
and a seven times increase in the levels of expression of Runx2, a master regulator of osteoblastogenesis.2 Moreover, the addition of BNNTs into hydroxyapatite ceramics even exhibited slightly higher osteoblast cell viability than pure hydroxyapatite, the inorganic component of the bone.3 The ability of BNNTs to enhance differentiation of MSCs into osteoblasts further broadens feasibility of BNNT applications in bone tissue engineering. Cell attachment and spreading involve a series of steps starting from adsorption of proteins on the culturing surface, through the attachment of cells onto the protein layer, and to the cytoplasm spreading. FBS in the culture medium contains great varieties and amounts of proteins, such as collagen, fibronectin, vitronectin and laminin, and so forth. It is known that the nanotubular morphologies of BNNTs provide such structures with an increased chance of adsorb-
ing or entrapping biomolecules within their inner channels as well as on their external surfaces.25 This study also confirms the high protein adsorption efficiency for BNNTs (Table I). So, the proteins in FBS-containing cell culture medium will be adsorbed on the BNNTs layer during MSCs culture. Through this adsorbed protein layer, the MSCs sense and attach to the foreign surfaces. Thus, the attachment, spreading, cytoskeletal organization, proliferation and differentiation of MSCs can be affected. We also document, that the BNNTs layer made the morphology of MSCs to be more stretched out, compared with the random shape on the control. The morphology of MSCs has been reported to be related to their differentiation capacity for multipotentiality. MSCs with spindle-like morphology have a higher potential for adipogenesis, while those with flat and spreading shape have a higher potential for osteogenesis.26
FIGURE 5. Effects of BNNTs layer on the ALP activity of MSCs after 7 days (A) and 14 days (B) of culture (n 5 8); Effects of BNNTs layer on the ratio of ALP activity to total protein of MSCs after 14 days of culture (C); effects of BNNTs layer on OCN protein concentration of MSCs after 14 days of culture (n 5 3) (D).
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FIGURE 6. Boron concentration after immersing BNNTs layer in Tris-HCl for 7 days (A); Boron release curve after immersing BNNTs particles at 100 lg mL21 in Tris-HCl buffer at 37 C (B).
One reason for the enhanced osteogenesis is a release of boron from BNNTs due to hydrolysis of BNNTs. Boron nitride can be hydrolyzed into boric acid and ammonia, which then transformed into ammonium borate hydrates.27 Although the as-prepared BNNTs are structurally perfect and chemically very stable, there are structural defects and hydroxyl groups on their edges after oxidation and sonication.28 During the culture of the obtained BNNTs with MSCs in the culture medium, the boron–nitrogen bonds near some defect sites might be prone to the attacks of oxygen atoms of water molecules.29 The defect further propagates when adjacent units are hydrolyzed.29 The hydrolysis of BNNTs may result in the slow release of boron. Boron is an essential trace element in a human body, which is beneficial to the bone growth and maintenance, arthritis alleviation or risk reduction and so on.16,17 A very low level of boron ions, even at 0.1–100 ng mL21, significantly enhances bonerelated gene expression, protein levels and the formation of mineralized nodules in osteoblasts (MC3T3-E1).16 Similarly, for human bone marrow stromal cells (BMSCs), boron increased osteogenic differentiation by enhancing ALP activity, stimulating osteogenic differentiation-related marker gene expression and increasing calcium deposition at low concentrations of boron (1, 10, and 100 ng mL21), and inhibited proliferation of BMSCs at a high concentration of boron (1000 ng mL21).19 The safety tolerance level of boron in drinking water is estimated to be 40–150 lg mL21 and no acute toxicity has been reported even at levels reaching
300 lg mL21.30 Our parallel experiments also confirm that boric oxide as a source of boron can promote the proliferation of MSCs and can stimulate ALP activity (Figure 7). In this study, BNNTs at 1–10 lg mL21, that shows a boron release to a level of 0.1–1 ng mL21 in Tris-HCl after 1 week, exhibited enhanced ALP activity of MSCs in the culture medium. This result is consistent with the previous reports about the effective boron concentration to enhance osteogenesis.16,19 It should be mentioned that, in case of the dense BNNTs layer (such as 25 lg mL21), too much free BNNTs particles may detach, be endocytosized by MSCs, and influence the viability of MSCs and the ALP activity. However, it is possible to fabricate, using BNNTs, some forms of composites that can avoid detachment of BNNT particles from the composite substrate for bone regeneration in orthopedic application. Another explanation for the enhanced osteogenesis is the stress or strain effects on MSCs, which are caused by the enhanced cell spreading31,32 or the surface morphology and stiffness of the substrate.14,33 MSCs on the control show random morphology, while those on BNNTs layer exhibit stretched morphology, as marked in Figure 5. The stress to stretch the MSCs generated tension on actin filaments and eventually enhanced the osteogenesis. Moreover, MSCs differentiation depends strongly on ECM’s stiffness.14,33 Soft matrices that mimic brain are neurogenic, whereas stiffer matrices that mimic muscle are myogenic, and, comparatively, rigid matrices that mimic collagenous bone are
FIGURE 7. Effects of boric oxide at 10 ng mL21 on the proliferation (A) and the ALP activity (B) of MSCs after 14 days of culture (n 5 3).
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proved to be osteogenic.14 Naive MSCs are shown to specify lineage and commit to phenotypes with extreme sensitivity to tissue level elasticity. BNNTs possess high elastic modulus of 0.5–0.6 TPa,1,18 which contributes to stem cells’ osteogenetic differentiation. It is considered that there are synergetic effects between trace boron release from BNNTs and the stress of BNNTs layer on MSCs, since MSCs on such layer exhibited much higher ALP content compared with those in the presence of equivalent elemental boron amount [Figs. 5(B) and 7(B)]. CONCLUSIONS
’Overall, boron nitride nanotubes (BNNTs) exhibit good biocompatibility with mesenchymal stem cells (MSCs). They show high protein adsorption ability, promote the proliferation of MSCs and increase secretion of total protein. Especially, BNNTs enhance the alkaline phosphatase (ALP) activity, an early marker of osteoblasts, secretion of total protein, and ALP/total protein, all of which suggest that BNNTs can enhance osteogenesis of MSCs. The release of trace boron and the stress on cells due to the fiber structure of BNNTs may enhance osteogenic differentiation of mesenchymal stem cells. Therefore, it is envisaged that BNNTs are potentially useful for bone regeneration in orthopedic applications. ACKNOWLEDGMENT
The authors are grateful to Drs. K. Iiyama, N. Kawamoto, and A. Nukui for their generous technical assistance. REFERENCES 1. Golberg D, Bando Y, Huang Y, Terao T, Mitome M, Tang CC, et al. Boron nitride nanotubes and nanosheets. ACS Nano 2010;4: 2979–2993. 2. Lahiri D, Rouzaud F, Richard T, Keshri AK, Bakshi SR, Kos L, et al. Boron nitride nanotube reinforced polylactide-polycaprolactone copolymer composite: Mechanical properties and cytocompatibility with osteoblasts and macrophages in vitro. Acta Biomater 2010;6:3524–3533. 3. Lahiri D, Singh V, Benaduce AP, Seal S, Kos L, Agarwal A. Boron nitride nanotube reinforced hydroxyapatite composite: Mechanical and tribological performance and in-vitro biocompatibility to osteoblasts. J Mech Behav Biomed 2011;4:44–56. 4. Li X, Zhi C, Hanagata N, Yamaguchi M, Bando Y, Golberg D. Boron nitride nanotubes functionalized with mesoporous silica for intracellular delivery of chemotherapy drugs. Chem Commun 2013;49:7337–7339. 5. Ciofani G, Danti S, Genchi GG, Mazzolai B, Mattoli V. Boron nitride nanotubes: Biocompatibility and potential spill-over in nanomedicine. Small 2013;9:1672–1685. 6. Li X, Hanagata N, Wang XB, Yamaguchi M, Yi W, Bando Y, et al. Multimodal luminescent-magnetic boron nitride nanotubes@NaGdF4:Eu structures for cancer therapy. Chem Commun 2014; 50:4371–4374. 7. Weng Q, Wang B, Wang X, Hanagata N, Li X, Liu D, et al. Highly water-soluble, porous, and biocompatible boron nitrides for anticancer drug delivery. ACS Nano 2014;8:6123–6130. 8. Buzatu DA, Wilkes JG, Miller D, Darsey JA, Heinze T, Biris A, et al. Nanotubes for cancer therapy and diagnostics. US Pat, 7608240, 2009. 9. Lahiri D, Singh V, Keshri AK, Seal S, Agarwal A. Apatite formability of boron nitride nanotubes. Nanotechnology 2011;22:205601 (8pp). 10. Chen X, Wu P, Rousseas M, Okawa D, Gartner Z, Zettl A, et al. Boron nitride nanotubes are noncytotoxic and can be functionalized for interaction with proteins and cells. J Am Chem Soc 2009; 131:890–891.
11. Ciofani G, Danti S, D’Alessandro D, Ricotti L, Moscato S, Bertoni G, et al. Enhancement of neurite outgrowth in neuronal-like cells following boron nitride nanotube-mediated stimulation. ACS Nano 2010;4:6267–6277. 12. Horvath L, Magrez A, Golberg D, Zhi CY, Bando Y, Smajda R, et al. In vitro investigation of the cellular toxicity of boron nitride nanotubes. ACS Nano 2011;5:3800–3810. 13. Ciofani G, Raffa V, Menciassi A, Dario P. Preparation of boron nitride nanotubes aqueous dispersions for biological applications. J Nanosci Nanotechnol 2008;8:6223–6231. 14. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell 2006;126:677–689. 15. Mendes SC, Tibbe JM, Veenhof M, Bakker K, Both S, Platenburg PP, et al. Bone tissue-engineered implants using human bone marrow stromal cells: Effect of culture conditions and donor age. Tissue Eng 2002;8:911–920. 16. Hakki SS, Bozkurt BS, Hakki EE. Boron regulates mineralized tissue-associated proteins in osteoblasts (MC3T3-E1). J Trace Elem Med Biol 2010;24:243–250. 17. Newnham RE. Essentiality of boron for healthy bones and joints. Environ Health Persp 1994;102:83–85. 18. Zhi C, Bando Y, Tang C, Golberg D. Boron nitride nanotubes. Mater Sci Eng R 2010;70:92–111. 19. Ying XZ, Cheng SW, Wang W, Lin ZQ, Chen QY, Zhang W, et al. Effect of boron on osteogenic differentiation of human bone marrow stromal cells. Biol Trace Elem Res 2011;144:306–315. 20. Tang C, Bando Y, Sato T, Kurashima K. A novel precursor for synthesis of pure boron nitride nanotubes. Chem Commun 2002: 1290–1291. 21. Zhi C, Bando Y, Tan C, Golberg D. Effective precursor for high yield synthesis of pure BN nanotubes. Solid State Commun 2005; 135:67–70. 22. Zhi C, Hanagata N, Bando Y, Golberg D. Dispersible shortened boron nitride nanotubes with improved molecule-loading capacity. Chem Asian J 2011;6:2530–2535. 23. Wang X, Li X, Ito A, Sogo Y. Synthesis and characterization of hierarchically macroporous and mesoporous CaO-MO-SiO2-P2O5 (M=Mg, Zn, Sr) bioactive glass scaffolds. Acta Biomater 2011;7: 3638–3644. 24. Kasten P, Beyen I, Niemeyer P, Luginbuhl R, Bohner M, Richter W. Porosity and pore size of beta-tricalcium phosphate scaffold can influence protein production and osteogenic differentiation of human mesenchymal stem cells: An in vitro and in vivo study. Acta Biomater 2008;4:1904–1915. 25. Zhi C, Bando Y, Tang C, Golberg D. Immobilization of proteins on boron nitride nanotubes. J Am Chem Soc 2005;127:17144–17145. 26. Sekiya I, Larson BL, Smith JR, Pochampally R, Cui JG, Prockop DJ. Expansion of human adult stem cells from bone marrow stroma: Conditions that maximize the yields of early progenitors and evaluate their quality. Stem Cells 2002;20:530–541. 27. Cao F, Liu K, Fang ZY, Wang SQ. Hydrolysis mechanism of borazine-derived boron nitride pyrolysized below 1200 oC. J Mater Sci Technol 2012;28:956–960. 28. Zhi C, Bando Y, Terao T, Tang C, Kuwahara H, Golberg D. Chemically activated boron nitride nanotubes. Chem Asian J 2009;4: 1536–1540. 29. Lin Y, Williams TV, Xu TB, Cao W, Elsayed-Ali HE, Connell JW. Aqueous dispersions of few-layered and monolayered hexagonal boron nitride nanosheets from sonication-assisted hydrolysis: Critical role of water. J Phys Chem C 2011;115:2679–2685. 30. Wu CT, Miron R, Sculean A, Kaskel S, Doert T, Schulze R, et al. Proliferation, differentiation and gene expression of osteoblasts in boron-containing associated with dexamethasone deliver from mesoporous bioactive glass scaffolds. Biomaterials 2011;32:7068– 7078. 31. Kilian KA, Bugarija B, Lahn BT, Mrksich M. Geometric cues for directing the differentiation of mesenchymal stem cells. Proc Natl Acad Sci USA 2010;107:4872–4877. 32. McBeath R, Pirone DM, Nelson CM, Bhadriraju K, Chen CS. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell 2004;6:483–495. 33. Discher DE, Janmey P, Wang YL. Tissue cells feel and respond to the stiffness of their substrate. Science 2005;310:1139–1143.
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World Premier International Center for Materials Nanoarchitectonics (WPI-MANA), National Institute for Materials Science (NIMS), Namiki 1-1, Tsukuba, Ibaraki 305-0044, Japan 2 Human Technology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan Received 27 September 2014; revised 8 January 2015; accepted 29 January 2015 Published online 12 March 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33391 Abstract: The interaction between boron nitride nanotubes (BNNTs) layer and mesenchymal stem cells (MSCs) is evaluated for the first time in this study. BNNTs layer supports the attachment and growth of MSCs and exhibits good biocompatibility with MSCs. BNNTs show high protein adsorption ability, promote the proliferation of MSCs and increase the secretion of total protein by MSCs. Especially, BNNTs enhance the alkaline phosphatase (ALP) activity as an early marker of osteoblasts, ALP/total protein and osteocalcin (OCN) as a late marker of osteogenic differentiation, which
shows that BNNTs can enhance osteogenesis of MSCs. The release of trace boron and the stress on cells exerted by BNNTs with a fiber structure may account for the enhanced differentiation of MSCs into osteoblasts. Therefore BNNTs are potentially useful for bone regeneration in orthopedic C 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part applications. V B: Appl Biomater, 104B: 323–329, 2016.
Key Words: boron nitride nanotubes, mesenchymal stem cells, proliferation, osteogenic differentiation
How to cite this article: Li X, Wang X, Jiang X, Yamaguchi M, Ito A, Bando Y, Golberg D. 2016. Boron nitride nanotubeenhanced osteogenic differentiation of mesenchymal stem cells. J Biomed Mater Res Part B 2016:104B:323–329.
INTRODUCTION
Boron nitride nanotubes (BNNTs),1 as a structural analogue of carbon nanotubes (CNTs), recently attract increasing attention in the biomedical field including bone tissue engineering,2,3 drug delivery,4–7 and boron neutron capture cancer therapy.8 For example, BNNTs are used to improve mechanical properties of conventional bone substitute materials, such as polylactide–polycaprolactone copolymer2 and hydroxyapatite.3 BNNTs induce apatite formation in a simulated body fluid environment, which shows feasibility in orthopedic applications.9 They can also deliver DNA oligomers to the interior of cells with no apparent toxicity.10 As a whole, BNNTs show good biocompatibility,2,3,10,11 except for the morphological cytotoxicity associated with too long BNNTs.12 The interaction between BNNTs and several types of cells, such as human embryonic kidney 293 cells,10 osteoblast,2 macrophages,2 neuronal-like PC12 cells,11 human neuroblastoma cells13 and so on, have been studied. However, up to now, there have been no reports in regards of BNNTs effects on mesenchymal stem cells (MSCs). MSCs are multipotent stem cells that can differentiate into a variety of cell types, including chondrocytes, osteo-
blasts, adipocytes, fibroblasts, marrow stroma and so on.14 MSCs have an enormous therapeutic potential for regenerative medicine owing to their plentiful source in bone marrow and high capacity for self-renewal while maintaining multipotency. For instance, MSCs (preferably the patient’s own) are of essential importance for tissue engineered implants, as MSCs will endow metabolic activity and biological integration with the tissue engineered implants.15 MSCs can differentiate into osteogenic cells under appropriate extracellular matrix (ECM).14 BNNTs have the following possible merits in bone tissue engineering: (i) Boron is an essential trace element which is beneficial to the bone growth and maintenance, arthritis alleviation or risk reduction and so on16,17; (ii) BNNTs are light in weight having excellent mechanical properties with bending stress values of 100–260 MPa and elastic modulus of 0.5–0.6 TPa1,18; (iii) BNNTs show good biocompatibility; (iv) White color of BNNTs is aesthetically advantageous in a biomaterial. A very low level of boron ions, even at 0.1–100 ng mL21, significantly increases osteogenic differentiation by enhancing ALP activity, stimulates osteogenic differentiation-related marker gene expression and increases the formation of mineralized nodules in osteoblasts and human bone marrow stromal
Correspondence to: X. Li; e-mail: [email protected] (or) D. Golberg; e-mail: [email protected] Contract grant sponsors: World Premier International (WPI) Center for Materials Nanoarchitectonics (MANA) of the National Institute for Materials Science (NIMS), Tsukuba, Japan
C 2015 WILEY PERIODICALS, INC. V
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cells.16,19 Because bone is a load-bearing tissue in a human body, the excellence of BNNTs mechanical properties is attractive for orthopedic applications. Based on the above reasons, the interaction between a BNNTs layer and MSCs is for the first time evaluated in this study. And herein, we report that the BNNTs layer supports attachment and growth of MSCs and enhances osteogenic differentiation of MSCs. This work suggests that BNNTs are promising as a tissue engineering scaffold for bone regeneration.
MSCs culture on BNNTs layer About 1 mL of MSCs suspension, about 104 cells mL21 in culture medium (MEME supplemented with 10% FBS, 10 mM sodium b-glycerophosphate, 10 nM dexamethasone and 82 lg mL21 vitamin C), was poured on the BNNT layer coated plate. The MSCs attached to the BNNT layer coated plate were cultured in a humidified atmosphere of 5% CO2 at 37 C.
MATERIALS AND METHODS
MSCs proliferation, ALP activity, total protein content, and OCN protein content tests The MSCs attached to BNNTs layer were cultured for 7 and 14 days. In this case, the culture medium was replaced with a fresh one every 3 days. After culturing, the MSCs on BNNTs layer were mildly washed twice with phosphate buffered saline (PBS(-)) for the following tests. The number of MSCs on BNNTs layer was determined by the WST-8 method using a CCK-8 kit (Dojindo Laboratories, Japan) in accordance with the manufacturer’s instructions. As for the total protein test and alkaline phosphatase (ALP) activity test, after culturing, the cells were washed twice with PBS(-). An aliquot of 300 mL of 0.05% Triton X was added to the culture well and the mixture was incubated at 4 C for 2 h. The resulting supernatant was assayed for total protein, OCN content and ALP activity as follows. Total protein content was assayed by the Bradford method using a BioRad protein assay reagent kit (Bio-Rad Laboratories, Japan) in accordance with the manufacturer’s instructions. The ALP activity of the MSCs was assayed using a LaboassayTM ALP kit (Wako Pure Chemicals, Japan) in accordance with the manufacturer’s instructions. The OCN protein content of the MSCs was assayed using a rat OCN EIA kit (Biomedical Technologies, USA) in accordance with the manufacturer’s instructions. The boron release from BNNTs in the Tris-HCl buffer was quantitatively analyzed using an inductively coupled plasma atomic emission spectrometer (ICP: SPS7800, Seiko Instruments, Japan).
Preparation of BNNTs layer BNNTs used in this study were synthesized by a chemical vapor deposition method using boron and metal oxide as precursors. The detailed growth procedure was reported elsewhere.20,21 The as-grown BNNTs were purified at temperatures up to 1900 C under the protection of argon gas to remove impurities. Purified BNNTs are “snow-white” in color. The purified BNNTs were further shortened, as was previously reported.22 The BNNTs were put in an alumina crucible and oxidized at 1000 C for 5 h. The oxidized BNNTs were put into a bottle with a sufficient amount of water and sonicated for 5 h. The BNNTs were filtered off after sonication. Then, BNNTs were suspended in alcohol and sonicated for 10 h. A certain amount of BNNTs suspension was dropped onto cover glasses treated beforehand with the piranha solution (H2SO4:H2O2 5 3:1 at 80oC for 40 min), surrounded with a parafilm in the 24well culture plate and dried in the clean bench. The cover glass coated with the BNNTs layer in the plate was sterilized by UV light overnight and used for the following cell culture study. Protein adsorption The cover glass coated with BNNTs layer at 25 lg mL21 and without BNNTs layer were added into 1 mL 100 lg mL21 fibrinogen, laminin, or fibronectin in a PBS buffer, respectively. After 4 h of incubation at 37 C, the protein adsorption was quantified using a QuantiPro bicinchoninic acid (BCA) assay kit (Sigma, USA). Bone marrow MSCs harvest MSCs were obtained from bilateral femora from Fischer 344/N syngeneic rats.23 Both ends of the rat femora were cut away from the epiphysis. Bone marrow was flushed out with 15 mL of culture medium, minimal essential medium eagle (MEME) containing 10% FBS and 1% antibiotics (100 U mL21 penicillin G, 100 mg mL21 streptomycin sulfate, and 0.25 mg mL21 amphotericin B). The bone marrow suspension was poured in a 75 cm2 tissue culture polystyrene flask and incubated at 37 C in a humidified atmosphere containing 5% CO2. The culture medium was replaced with a fresh one every 3 days until 90% confluence was reached. All the animal experiments were permitted by the Ethical Committee of the National Institute for Materials Science (NIMS), Japan. All the animal experiments and feeding were carried out in accordance with the guidelines of the Ethical Committee of NIMS, Japan.
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Actin staining For actin staining, cells were fixed in 4% formaldehyde solution, permeabilized with 0.1% Triton X-100, and then stained by fluorescein isothiocyanate (FITC)—conjugated phalloidin (Sigma, Germany). After counterstaining the nuclei with DAPI (Vector Labs), fluorescence images were obtained using fluorescence microscopy (BX51, Olympus, Japan). Statistical analysis All statistical comparisons were conducted using a 95% confidence interval (p < 0.05). Single-factor analysis of variance (ANOVA) was performed to identify significant differences among the groups. When significant differences were found, the Tukey’s post hoc multiple-comparison test was used to determine the significant differences between the mean values of the groups. The experimental data were expressed as means 6 standard deviation.
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FIGURE 1. SEM (A) and TEM (B) images of BNNTs; AFM images of BNNTs layer on a cover glass with different amounts of BNNTs for the subsequent cell culture (C, 2 lg mL21; D, 5 lg mL21; E, 10 lg mL21; F, 25 lg mL21).
RESULTS
The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of shorten BN nanotubes (around 1–2 lm in length and 80 nm in diameter) synthesized by the chemical vapor deposition (CVD) method, oxidized in air at 1000 C for 5 h and sonicated in water are shown in Figure 1(A,B). Atomic force microscopy (AFM) analysis [Figure 1(C–F)] revealed that the BNNTs are discretely distributed on the glass substrate treated with the piranha solution.
BNNT layer can enhance the protein adsorption and MSCs attachment. Protein adsorption is critical for regulating cellular behaviors on materials surfaces such as adhesion and proliferation. Therefore, the adsorption of individual proteins such as fibrinogen, laminin and fibronectin was examined for BNNTs. Quantitative measurements indicated that BNNTs layer on cover glass had showed higher proteins adsorption ability than cover glass control (Table I). Cellular morphology after 1 day culturing was evaluated by
FIGURE 2. Left: Fluorescent images of MSCs after 24 h of culture with control (A–C) and BNNTs layer at 2 lg mL21 (D–F); Right: Quantification of cell attachment area of MSCs after 24 h of culture with control and BNNTs layer at 2 lg mL21 (n 5 80).
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TABLE I. Different Protein Adsorption Amounts for BNNTs Layer Coated Cover Glass and Control
Proteins Fibrinogen Laminin Fibronectin
Protein Adsorption Amounts for BNNTs layer (lg)
Protein adsorption amounts for control (lg)
23.3 6 1.2 9.1 6 3.7 18.2 6 1.7
5.2 6 0.5 4.9 6 1.0 5.9 6 1.4
staining the F-actin fibers with phalloidin. Fluorescent images illustrated that the shapes of MSCs cultured on control and BN nanotubes layer had been distinctly different (Figure 2). For control, MSCs displayed spindle-shape or irregular morphology, most probably due to poor adhesion to glass substrate. Whereas, cells cultivated on the BNNTs layer were larger, more widespread, more flat and more stretched out. BNNTs layer promoted proliferation, total protein and osteogenic differentiation of MSCs. Figure 3 shows the MSCs proliferation on the BNNTs layer after 7 and 14 days of culture. With an increase in amount of BNNTs, the number of MSCs increases and then decreases compared with control. The number of MSCs on BNNTs layer with 5 lg mL21 concentration is the highest among the data for each group after 14 days of culture. Figure 4 depicts the total protein of MSCs on the BNNTs layer after 7 and 14 days of culture. Totally, the MSCs on the BNNTs layer at a concentration lower than 10 lg mL21 display higher protein secretion than the controls after 7 or 14 days of culture. Measurements of specific ALP activity and OCN protein content were taken as indicators of osteogenic differentiation. ALP is an early osteoblastic marker of the protein level that does not rise in uninduced MSC, but displays a strong expression during osteogenic differentiation.24 Figure 5 illustrates the ALP activities of MSCs on the BNNTs layer after 7 and 14 days of culture. MSCs on the BNNTs layer with a low BNNT content show higher ALP activity than the controls after 7 days of culture. The ALP activity of the MSCs cultured on the BNNTs layer is significantly increased after 14 days of culture compared with that after 7 days of culture. On the BNNTs layer with a 1–10 lg mL21 concentration, the MSCs exhibit markedly increased ALP activity compared with the controls after 14 days of culture. The MSCs on the BNNTs layer with 2 lg mL21 concentration show the highest ALP
activity among all the other BNNTs layer. The ALP activity of MSCs on BNNTs layer with 2 lg mL21 concentration was about two times higher as compared with that without BNNTs layers after 14 days of culture. ALP per total protein after 14 days of culture reveals the same tendency, as seen in Figure 5(C), with the highest ALP per total protein being at 2 lg mL21 concentration. This means that the BNNTs layer enhanced osteogenic differentiation of the MSCs compared with the control. Similar to the results on ALP activity, MSCs on the BNNTs layer showed significantly increased OCN protein concentration, a late marker of osteogenic differentiation, as compared with control after 14 days of culture [Figure 5(D)]. The MSCs on the BNNTs layer with 10 lg mL21 concentration show the highest OCN amount. Boron release from BNNTs layer after 1 week was detected by the ICP method (Figure 6). With an increase in BNNTs amounts, boron release was gradually enhanced. The boron concentrations for BNNTs layer (at 1–25 lg mL21) after 1 week immersion in Tris-HCl buffer were about 0.1 3.2 ng/mL. In addition, with the increase in time, BNNTs in Tris-HCl buffer exhibit gradual boron release (Figure 6). DISCUSSION
BNNTs are promising tissue engineering scaffolds for bone tissue regeneration due to not only their active effects on the osteogenesis but also due to their excellent mechanical properties. In this study, we used naked BNNTs to check their effects on MSCs and avoid any other possible interference, such as dispersing agents or surface modification. The present BNNTs show good biocompatibility, exhibit high protein adsorption ability and promote the proliferation of MSCs and secretion of total protein. BNNTs enhance ALP activity, an early marker of osteoblasts, which implies that BNNTs can enhance the osteogenesis of MSCs. The observed effect is certainly due to a complex interplay between chemical and mechanical properties of BNNTs, and the interactions between BNNTs and cells. This study confirmed that BNNTs had enhanced the differentiation of MSCs into osteoblasts and had positive effects on osteogenesis (Figures 3–5). Our results are consistent with the previous reports about positive effects of BNNTs-polymer/hydroxyapatite composites on osteoblasts.2,3 It was demonstrated that the addition of BNNTs into polymers had showed higher osteoblast cell viability
FIGURE 3. Effects of BNNTs layer on the MSCs proliferation after 7 days (A) and 14 days (B) of culture (n 5 8).
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FIGURE 4. Effects of BNNTs layer on the total protein content of MSCs after 7 days (A) and 14 days (B) of culture (n 5 8).
and a seven times increase in the levels of expression of Runx2, a master regulator of osteoblastogenesis.2 Moreover, the addition of BNNTs into hydroxyapatite ceramics even exhibited slightly higher osteoblast cell viability than pure hydroxyapatite, the inorganic component of the bone.3 The ability of BNNTs to enhance differentiation of MSCs into osteoblasts further broadens feasibility of BNNT applications in bone tissue engineering. Cell attachment and spreading involve a series of steps starting from adsorption of proteins on the culturing surface, through the attachment of cells onto the protein layer, and to the cytoplasm spreading. FBS in the culture medium contains great varieties and amounts of proteins, such as collagen, fibronectin, vitronectin and laminin, and so forth. It is known that the nanotubular morphologies of BNNTs provide such structures with an increased chance of adsorb-
ing or entrapping biomolecules within their inner channels as well as on their external surfaces.25 This study also confirms the high protein adsorption efficiency for BNNTs (Table I). So, the proteins in FBS-containing cell culture medium will be adsorbed on the BNNTs layer during MSCs culture. Through this adsorbed protein layer, the MSCs sense and attach to the foreign surfaces. Thus, the attachment, spreading, cytoskeletal organization, proliferation and differentiation of MSCs can be affected. We also document, that the BNNTs layer made the morphology of MSCs to be more stretched out, compared with the random shape on the control. The morphology of MSCs has been reported to be related to their differentiation capacity for multipotentiality. MSCs with spindle-like morphology have a higher potential for adipogenesis, while those with flat and spreading shape have a higher potential for osteogenesis.26
FIGURE 5. Effects of BNNTs layer on the ALP activity of MSCs after 7 days (A) and 14 days (B) of culture (n 5 8); Effects of BNNTs layer on the ratio of ALP activity to total protein of MSCs after 14 days of culture (C); effects of BNNTs layer on OCN protein concentration of MSCs after 14 days of culture (n 5 3) (D).
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FIGURE 6. Boron concentration after immersing BNNTs layer in Tris-HCl for 7 days (A); Boron release curve after immersing BNNTs particles at 100 lg mL21 in Tris-HCl buffer at 37 C (B).
One reason for the enhanced osteogenesis is a release of boron from BNNTs due to hydrolysis of BNNTs. Boron nitride can be hydrolyzed into boric acid and ammonia, which then transformed into ammonium borate hydrates.27 Although the as-prepared BNNTs are structurally perfect and chemically very stable, there are structural defects and hydroxyl groups on their edges after oxidation and sonication.28 During the culture of the obtained BNNTs with MSCs in the culture medium, the boron–nitrogen bonds near some defect sites might be prone to the attacks of oxygen atoms of water molecules.29 The defect further propagates when adjacent units are hydrolyzed.29 The hydrolysis of BNNTs may result in the slow release of boron. Boron is an essential trace element in a human body, which is beneficial to the bone growth and maintenance, arthritis alleviation or risk reduction and so on.16,17 A very low level of boron ions, even at 0.1–100 ng mL21, significantly enhances bonerelated gene expression, protein levels and the formation of mineralized nodules in osteoblasts (MC3T3-E1).16 Similarly, for human bone marrow stromal cells (BMSCs), boron increased osteogenic differentiation by enhancing ALP activity, stimulating osteogenic differentiation-related marker gene expression and increasing calcium deposition at low concentrations of boron (1, 10, and 100 ng mL21), and inhibited proliferation of BMSCs at a high concentration of boron (1000 ng mL21).19 The safety tolerance level of boron in drinking water is estimated to be 40–150 lg mL21 and no acute toxicity has been reported even at levels reaching
300 lg mL21.30 Our parallel experiments also confirm that boric oxide as a source of boron can promote the proliferation of MSCs and can stimulate ALP activity (Figure 7). In this study, BNNTs at 1–10 lg mL21, that shows a boron release to a level of 0.1–1 ng mL21 in Tris-HCl after 1 week, exhibited enhanced ALP activity of MSCs in the culture medium. This result is consistent with the previous reports about the effective boron concentration to enhance osteogenesis.16,19 It should be mentioned that, in case of the dense BNNTs layer (such as 25 lg mL21), too much free BNNTs particles may detach, be endocytosized by MSCs, and influence the viability of MSCs and the ALP activity. However, it is possible to fabricate, using BNNTs, some forms of composites that can avoid detachment of BNNT particles from the composite substrate for bone regeneration in orthopedic application. Another explanation for the enhanced osteogenesis is the stress or strain effects on MSCs, which are caused by the enhanced cell spreading31,32 or the surface morphology and stiffness of the substrate.14,33 MSCs on the control show random morphology, while those on BNNTs layer exhibit stretched morphology, as marked in Figure 5. The stress to stretch the MSCs generated tension on actin filaments and eventually enhanced the osteogenesis. Moreover, MSCs differentiation depends strongly on ECM’s stiffness.14,33 Soft matrices that mimic brain are neurogenic, whereas stiffer matrices that mimic muscle are myogenic, and, comparatively, rigid matrices that mimic collagenous bone are
FIGURE 7. Effects of boric oxide at 10 ng mL21 on the proliferation (A) and the ALP activity (B) of MSCs after 14 days of culture (n 5 3).
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proved to be osteogenic.14 Naive MSCs are shown to specify lineage and commit to phenotypes with extreme sensitivity to tissue level elasticity. BNNTs possess high elastic modulus of 0.5–0.6 TPa,1,18 which contributes to stem cells’ osteogenetic differentiation. It is considered that there are synergetic effects between trace boron release from BNNTs and the stress of BNNTs layer on MSCs, since MSCs on such layer exhibited much higher ALP content compared with those in the presence of equivalent elemental boron amount [Figs. 5(B) and 7(B)]. CONCLUSIONS
’Overall, boron nitride nanotubes (BNNTs) exhibit good biocompatibility with mesenchymal stem cells (MSCs). They show high protein adsorption ability, promote the proliferation of MSCs and increase secretion of total protein. Especially, BNNTs enhance the alkaline phosphatase (ALP) activity, an early marker of osteoblasts, secretion of total protein, and ALP/total protein, all of which suggest that BNNTs can enhance osteogenesis of MSCs. The release of trace boron and the stress on cells due to the fiber structure of BNNTs may enhance osteogenic differentiation of mesenchymal stem cells. Therefore, it is envisaged that BNNTs are potentially useful for bone regeneration in orthopedic applications. ACKNOWLEDGMENT
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