J Mater Sci: Mater Med DOI 10.1007/s10856-014-5227-z
A comparative study on the effects of pristine and functionalized single-walled carbon nanotubes on osteoblasts: ultrastructural and biochemical properties Qiu Tong • Wu Qingzhi • Dai Honglian • Wang Xinyu • Wang Youfa • Li Shipu • Li Junli
Received: 6 May 2013 / Accepted: 21 April 2014 Ó Springer Science+Business Media New York 2014
Abstract A comparative study was performed to investigate the ultrastructural and biomolecular properties of osteoblasts induced by three types of single-walled carbon nanotubes (SWNTs). The results on cellular uptake and ultrastructural alteration indicate that SWNTs enter osteoblasts by endocytosis. SWNTs-COOH and SWNTs-OH particles were freely dispersed in the cytoplasm, while pristine SWNTs were localized to the periphery of the cell. Both SWNTs-OH and SWNTs-COOH promoted cell changes in cell activity regarding mRNA expression at doses of 50 and 100 lg/mL in the first 24 h. When treated with 50 lg/mL SWNTs-COOH for 48 h, the expression of type I collagen increased by 6.3-fold (for MG63) or 9.1fold (for primary osteoblasts) compared with the control group. The present study observed for the first time that SWNTs-COOH initiated the prompt and the maximum upregulation of type I collagen gene expression, and simultaneously induced the expansion of the endoplasmic reticulum for increased protein synthesis, which in turn accelerated the mineralization process. However, impaired cell properties and mitochondrial injury were
detected following treatment with SWNTs at 100 lg/mL after 48 h. In conclusion, we believe that SWNTs-COOH is a good candidate for the fabrication of biomedical scaffolds for bone regeneration.
Abbreviation CNTs Carbon nanotubes MWNTs Multi-walled carbon nanotubes PCL Polycaprolacton SWNTs Single-walled carbon nanotubes ER Endoplasmic reticulum MTS Metabolic activity test TEM Transmission electron microscope COL-I Type I collagen ON Osteonectin Alpase Alkaline phosphatase MEM Minimum essential medium
1 Introduction Q. Tong W. Qingzhi D. Honglian W. Xinyu W. Youfa L. Shipu Biomedical Materials and Engineering Center, Wuhan University of Technology, Wuhan 430070, People’s Republic of China D. Honglian W. Xinyu W. Youfa L. Shipu State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Wuhan 430070, People’s Republic of China L. Junli (&) College of Science, Wuhan University of Technology, Wuhan 430070, People’s Republic of China e-mail: [email protected]
Carbon nanotubes (CNTs) have attracted considerable attention due to their promising applications in biomedical fields, such as biosensors, drug and vaccine delivery vehicles, and scaffold materials. The use of CNT-based scaffolds for bone regeneration is of particular interest because of their suitable dimensions that are similar to type I collagen, the major organic component of bone. Balani et al. [1] and Xu et al. [2] prepared multi-walled carbon nanotube (MWNT)-reinforced hydroxyapatite coating and confirmed its biocompatibility with different human osteoblast cell lines. Hirata et al. [3, 4] reported that
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MWNT-coated 3D collagen scaffolds induced bone formation in vivo, and MWCNT-coated polylactic acid (PLLA) scaffolds initiated cell attachment and enhanced the DNA content of Saos-2 cells. Furthermore, the unique effect of CNTs alone on cell biology was also examined. It was reported that single-walled carbon nanotube (SWNTs) regulated the metabolic activity of Saos-2 cells and significantly promoted cell proliferation [5, 6]. Zhang et al. [7] reported that the surface modification of MWCNTs promoted the adhesion and cell viability of osteoblasts. Furthermore, Li et al. [8] demonstrated that the MWNTs induced ectopic bone formation in the dorsal musculature of oddy mice. Osteoclasts and osteoblasts are the two primary cell types used to create and maintain bones. They are highly specialized cells that must work in perfect synchronization to maintain the skeletal system. Osteoclasts resorb bone tissue back into the body, whereas osteoblasts create bone. Osteogenesis is induced by osteoblastic cells and is accompanied by a sequence of characteristic events, involving cell attachment, cell proliferation and the expression of osteoblastic phenotype. So far, few investigations have been performed to illustrate the molecular mechanisms on the morphological and biochemical alterations of osteoblasts induced by SWNTs [9]. Herein, we investigated the physiological and bimolecular responses of osteoblasts induced by different SWNTs (pristine SWNTs, SWNTs-OH and SWNTs-COOH), using MG-63 cells and primary osteoblasts as the model system. We measured cell viability using metabolic activity test (MTS) and examined cellular uptake and intracellular distribution of SWNTs with transmission electron microscopy (TEM). To examine the molecular responses of cells exposed to different SWNTs, the expression levels of several genes were determined by quantitative real-time PCR, including the attachment-related gene: actin, the proliferation-related gene: c-jun, and the osteoblastic marker genes: type I collagen, COL-I, osteonectin, ON, alkaline phosphatase, ALPase.
solution and deionized water several times before use. To obtain a uniform solution, SWCNTs were dispersed in cell culture medium without serum by ultrasonication at a concentration of 1 mg/mL, and the supernatant was collected after centrifugation (5009g for 20 min). Cells were exposed to the supernatant at a final concentration of 25, 50 and 100 lg/mL. 2.2 Cell culture and treatments
2 Methods
Osteoblast-like MG-63 cells, originally isolated from a human sarcoma, were obtained from China Center for Type Culture Collection (Wuhan). MG-63 cells are relatively immature osteoblasts that have been well characterized and widely used for biomaterials testing, and show several similarities with isolated human bone-derived cells. MG-63 cells were grown in 25 cm2 flasks with minimum essential medium (MEM) supplemented with 10 % fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. At confluence, adherent cells were detached with 0.25 % trypsin, and seeded in 6-well plates at a density of 3 9 105 cells/mL. MG-63 cells were cultured at 37 °C in a humidified CO2 (5 %) atmosphere for 24 h. Primary rat calvarial osteoblasts were prepared by serial collagenase digestion. In brief, new born rat calvariae (day 1) were removed from soft tissues and digested five times with 0.1 % collagenase and 0.2 % dispase in Dulbecco’s Modified Eagle’s Medium (DMEM) for 10 min at 37 °C. After centrifugation and washing with the medium, cells were resuspended in DMEM supplemented with 10 % FBS in a humidified CO2 (5 %) atmosphere for 48 h. Then, nonadherent cells were removed, and adherent cells were harvested after treatment with 0.25 % trypsin/EDTA and successively passaged. All procedures undertaken in this study were approved by the Animal Care and Use Committees of Wuhan University of Technology and conformed to NIH guidelines. For exposure, culture medium was removed and fresh medium with or without SWNTs was added to the wells. 6 ml medium is for the 6-well plate, while 150ul medium is for the 96-well plate. Every 3 wells are set for one treatment. Cells were further cultured for 24 or 48 h.
2.1 Materials
2.3 Transmission electron microscopy
Three types of SWNTs, purified pristine SWNTs, hydroxyl purified SWNTs (SWNTs-OH) and carboxyl purified SWNTs (SWNTs-COOH), were purchased from Chengdu Organic Chemicals Co. Ltd (Chinese Academy of Science, China). All nanotubes were 1–2 nm in diameter and 1–3 lm in length. The hydroxyl content in SWNTs-OH was 3.96 wt%, and the carboxyl content in SWNTs-COOH was 2.73 wt%. The SWNTs were washed in dilute H2SO4
The ultrastructural alterations of MG63 and the internalization of SWNTs were observed through TEM. Briefly, MG-63 cells were incubated with different SWNTs at a dose of 50 lg/mL. Subsequently, the cells were washed with PBS three times, and prefixed in 4 % paraformaldehyde for 2 h and in 1 % osmium tetroxide at 4 °C for 1 h. Cells were then washed with 0.1 M sodium cacodylate buffer (pH 7.4) three times, dehydrated and embedded in
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resin. Ultrathin sections were made and stained with uranyl acetate and lead citrate. The samples were examined using a transmission electron microscope (HITACHI H-1000FA). 2.4 Metabolic activity test Metabolic activity test (MTS-Promega) was performed according to the manufacturer’s protocol. For this assay, 105 cells were exposed to different SWNTs (raw SWNTs, SWNTs-OH and SWNTs-COOH) and incubated for 24 h or 48 h. The spectrophotometric absorbance was quantified using Multiskan Spectrum (Thermo Scientific, Brussels, Belgium). The results were presented directly proportional to the control group. 2.5 Total RNA isolation and quantitative real-time PCR (Q-PCR) Total RNA was isolated from cells using trizol reagent (Invitrogen, USA) and reverse transcribed into cDNA using first strand cDNA synthesis kit (Fermentas, USA). Q-PCR was conducted with the SYBR Green qPCR kit (Toyota, Japan) on a CFX96 Real-Time System (Bio-Rad, USA). The reactions were performed in a 20 lL volume mix containing 10 lL SYBR Green I mixture, 2 lL primers (10 lM/L), 2 lL cDNA and 6 lL sterile water. Cycling conditions were set as follows: 3 min at 95 °C, 40 cycles of 10 s at 95 °C, 30 s at 61–69 °C, and 10 s at 72 °C. Melting curve analysis of amplification products was performed at the end of each PCR reaction to confirm that a single PCR product was detected. Each sample was run in three tubes, and the PCR reactions without the template were used as the blank. After PCR amplification, data were analyzed with the Bio-Rad CFX manager software version 1.0 and the fold difference was calculated using the 2-44CT method. The sequences of PCR primers were used as follows for MG63, with GAPDH as the internal control gene: GAPDH 50 -GAGCCACATCG CTCAGACAC-30 (forward), 50 -CATGTAGTTGAGGTCA ATGAAGG-30 (reverse); Actin 50 -GATGCAGAAGGAGA TCACTG-30 (forward), 50 -GGGTGTAACGACACTAAGT C-30 (reverse); C-jun 50 -GCCAACATGCTCAGGGAACA GGTG-30 (forward), 50 -GCCAACATGCTCAGGGAACA GGTG-30 (reverse); COL-I 50 -AACGCGTGTCAATCCCT TGT-30 (forward), 50 -GAACGAGGTAGTCTTTCAGCAA CA-30 (reverse); ALPase 50 -CTCGTCGACACCTGGAAG AGCTTCAAACCG-30 (forward), 50 -GGATCCGTCACGT TGTTCCTGTTCAGC-30 (reverse); ON 50 -TCACATTAGG CTGTTGGTTCAAA-30 (forward), 50 -GCGTGACCACTT CCCAGAGA-30 (reverse). The sequences of PCR primers were used as follows for primary osteoblast, with GAPDH as the internal control gene: GAPDH 50 -ATGGAGAAGGCTG GGGCTCACCT-30 (forward), 50 -AGCCCTTCCACGATG
CCAAAGTTGT-30 (reverse); Actin 50 -GACCTCTATGCC AACACAGTGCTGT-30 (forward), 50 -CTAGAAGCATTT GCGGTGCACGATG-30 (reverse); C-jun 50 -GCCAACAT GCTCAGGGAACAGGTG-30 (forward), 50 -GCCAACAT GCTCAGGGAACAGGTG-30 (reverse); COL-I 50 -CCTAC AGCACGCTTGTGGATG-30 (forward), 50 -AGATTGGGA TGGAGGGAGTTTAC-30 (reverse); ALPase 50 -TGCCCT GAAACTCCAAAAACT-30 (forward), 50 -ATCTCCAGCC GTGTCTCCTC-30 (reverse); ON 50 -GAAGAGATGGTGG CGGAG-30 (forward), 50 -ACAGGCAGGGGGCAATGTAT TTG-30 (reverse). 2.6 Collagen (COL) production and alkaline phosphatase (ALP) activity of cells The levels of the COL and the ALP activity were determined by ELISA, following the manufacturer’s instructions. Briefly, at culture times of 24 and 48 h, the cells were collected, washed with PBS, homogenized with 1 ml tris buffer (pH 7.4) and then sonicated. The cell lysate was applied in the following measurements. After standards and samples were added into each 96-well plate pre-coated with antigen, the first antibody was added into each well and incubated for 30 min at 37 °C. After aspirating and washing 5 times, the second antibody was added into each well and incubated for 20 min at room temperature. A stop solution was added into each well, and the absorbance value was read by a microplate reader at a 450 nm wavelength within 15 min. The measured concentration and activity were normalized by cell number to take into account the differences in cell growth. 2.7 Statistical analysis All values were expressed as mean ± standard deviation and significant differences between the treatment groups and the control group were analyzed using one-way ANOVA (P \ 0.05 or P \ 0.01).
3 Results and discussion The evaluation of cell uptake and intracellular location of CNTs is crucial for understanding the mechanisms of their biological effects. In our experiment, the internalization of SWNTs in MG63 cells was observed by TEM after 24 h and 48 h exposure (Fig. 1). We found that SWNTs were incorporated into the cytosol after 24 h of treatment (Fig. 1b, c, e, h). Previous studies have also described the capacity of CNTs to penetrate transformed cell lines from different origins, including A549, HeLa, and Jurkat cells [10]. Mooney et al. [11] reported the uptake of SWNTs by human mesenchymal stem cells (MSCs). Zhang et al. [12]
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Fig. 1 Ultrastructure changes of MG-63 cells exposed to SWNTs. a Cells without stimulation in control group (2,5009). b–d Cells treated with raw SWNTs-COOH for 24 h (B 2,5009 , C 5,0009) or 48 h (D 5,0009). e–g Cells treated with SWNTs-OH for 24 h (E 2,5009, F 5,0009) or 48 h (G 5,0009). h–j Cells treated with raw
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SWNTs for 24 h (H 2,5009, I 5,0009) or 48 h (J 5,0009). Serious expansion of ER is indicated by white arrows, while the SWNTs in the cells are indicated by black arrows. Mild swelling of the mitochondria is indicated by asterisk
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Fig. 2 Metabolic activity of MG-63 and primary osteoblasts in the presence of SWNTs. a, b Are for MG63 at 24 and 48 h respectively, while c, d are for primary osteoblasts at 24 and 48 h respectively. The
x-axis represents the types of SWNTs, while y-axis represents the viability ratio compared with the control ones. (Asterisk) indicates significant change (*P \ 0.05, **P \ 0.01)
observed the distribution of SWNTs-COOH in primary cells (osteoblasts) after exposure to a dose of 50 lg/mL for 24 h. More rapidly, Mu et al. [13] identified that SWNTsCOOH were translocated into cytoplasmic vesicles as soon as 1 h after treatment in HEK293 cells. TEM observation of fluorescently-labeled CNTs was used to verify that the CNTs were localized primarily within the cytoplasm and perinuclear region of the cell [7, 13, 14]. We also found that several particles were incorporated into cellular lysosomes, especially when cells were co-cultured with SWNTs-COOH for 48 h (Fig. 1d) and raw SWNTs for 24 h (Fig. 1i). No particles were found in the nucleus and neither apoptosis nor necrosis features were observed in cells during the experiment. A similar phenomenon was also reported by Giorgio et al. [14] and Zhang et al. [7], who showed that SWCNTs or MWCNTs induced the formation of abundant phagolysosomes containing internalized CNTs in RAW264.7 cells. In vitro studies, normal human osteoblasts and osteoblast-like cell lines have been shown to combine with various particles, including titanium, Ti–6Al–4 V, cobalt, chromium, cobalt–chromium, and ultrahigh molecular weight polyethylene (UHMWPE)
[15–17]. In addition, human osteoblasts were shown to express CD68, a protein associated with macrophages, when exposed to Ti particles [16]. Therefore, the identification of these CNT-containing endosome-like vesicles suggested a possible cell-uptake mechanism by endocytosis in osteoblasts [18]. There was no evidence that SWCNTs were in the nucleus, which agrees with previous studies [7, 13]. However, Mooney et al. [11] reported the SWNTsCOOH primarily occupied a cytoplasmic location for the first 24 h, and then assumed a nuclear location after 6 days. Therefore, nanotubes might interact with proteins located on the cell membrane or in the cytoplasm during the initial internalization steps. In this study, raw SWNTs, SWNTsCOOH and SWNTs-OH showed different behavior in their distribution in the cell: raw SWNTs were observed at the periphery of the cells, while SWNTs-COOH and SWNTsOH particles were freely dispersed in the cytoplasm (Fig. 1b, e, h). The results from RAW264.7 cells treated with CNTs also revealed that the negatively-charged MWCNTs-COOH may facilitate transport of MWCNTs through the cell membrane [7]. Furthermore, it was verified that carboxyl-functionalized surfaces enhanced cellular
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Fig. 3 Gene expression of MG63 exposed to SWNTs for 24 or 48 h. a, b, c are for the 24 h treatments, while d, e, f are for the 48 h treatments. In a and d, SWNTS are at the concentration of 25 lg/ml. In b and e, SWNTS are at the concentration of 50 lg/ml. In c and f,
SWNTS are at the concentration of 100 lg/ml. The x-axis represents the types of genes, while y-axis represents the expression ratio compared with the control ones. (Asterisk) indicates significant change (*P \ 0.05, **P \ 0.01)
uptake, and the amount of nanoparticles internalized could be correlated to the degree of carboxyl functionalization on the nanoparticle surface [19]. Remarkable changes were observed in cells treated with SWNTs. We found some secondary lysosomes and fast growing endoplasmic reticulum (ER) in cells treated with SWNTs-COOH and SWNTs-OH (Fig. 1c, d, f, g). Ultrastructurally, osteoblasts feature a complement of organelles characteristic of cells actively involved in protein synthesis. They have abundant ER, numerous ribosomes, and the Golgi apparatus and mitochondria are quite prominent. In the present study, ER expansion was prominent (Fig. 1c, d), especially in cells treated with SWNTs-COOH, suggesting the occurrence of ER stress. Hamamura et al. [20] demonstrated that ER stress from thapsigargin and tunicamycin led to two distinctive consequences, apoptosis or regulation of osteoblastogenesis. This bi-phasic response was dependent on exposure time to ER stress: 1 h treatment favored the pathway toward transcriptional activation of selected genes, such as Runx2 and type I collagen, as opposed to 24 h for the apoptotic pathway. Furthermore, recent research revealed that both bone morphogenetic protein-2 (BMP-2) and old astrocyte specifically induced
substance (OASIS) increased ER stress to stimulate differentiation of osteoblasts [21, 22]. Meanwhile, we observed increased cell metabolic activity (Fig. 2) and significantly increased COL-I expression in both SWNTsCOOH-treated cells types at 24 h (Figs. 3, 4). The activation of COL-I was prominent in ER stress by thapsigargin and tunicamycin at the first hours and by Old Astrocyte Specifically Induced Substance (OASIS) cases [20, 22]. Moreover, raw SWNTs did not induce significant ER expansion (Fig. 1h), while the expression of differentiation-related genes was not altered at 24 h (Fig. 3). Therefore, in the present study, ER expansion was related to active protein synthesis. After 48 h exposure to raw SWNTs and SWNTs-OH, we observed the formation of myeloid bodies in the lysosome and swollen mitochondria cristae in many cells (Fig. 1g, j). Similarly, nuclear degeneration and mitochondrial enlargement were seen in RAW 264.7 cells exposed to tau-MWCNTs [23]. Zhong et al. [24] also demonstrated that SWCNTs preferentially accumulated in lysosomes of the target organelles, and impaired mitochondrial activity as a result of cytotoxicity. Therefore, SWNTs may impair osteoblast activity through the
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Fig. 4 Gene expression of primary osteoblasts exposed to SWNTs for 24 or 48 h. a, b, c are for the 24 h treatments, while d, e, f are for the 48 h treatments. In a and d, SWNTS are at the concentration of 25 lg/ml. In b and e, SWNTS are at the concentration of 50 lg/ml. In
c and f, SWNTS are at the concentration of 100 lg/ml. The x-axis represents the types of genes, while y-axis represents the expression ratio compared with the control ones. (Asterisk) indicates significant change (*P \ 0.05, **P \ 0.01)
mitochondrial pathway. With increased concentration (100 lg/mL) and exposure time (48 h) to SWNTs, there was a decrease in metabolic activity and obvious suppression of gene expression in both cell models (Figs. 2, 3, 4). Osteoblasts are attachment-dependent cells that interact with their substrate via cytoskeletal alterations. The interaction of bone cells with biomaterials influences both their proliferation and differentiation. Some previous studies found enhanced proliferation and adherence of osteoblasts exposed to SWNT-based substrates or their composites [5, 6, 25]. At the molecular level, we verified increases in c-jun and actin mRNA expression after 24 h treatment with SWNTs-COOH at all doses, with SWNTs-OH at doses of 50 and 100 lg/mL, and with raw SWNTs at a dose of 50 lg/mL in MG63 and at doses of 25 and 50 lg/mL in primary osteoblasts (Figs. 3, 4). The expression of c-jun was the highest in proliferating osteoblasts [26]. The transcriptional regulation of actin mRNA was accompanied by reorganization of the actin cytoskeleton [27]. Previously, Kalbacova et al. [5] demonstrated thinner and diffused actin stress fibers in cells on SWCNTs films, which was attributed to rapid reorganization during cell migration across the nano-phase surface. Bacakova et al. [25]
detected distinct b-actin filament bundles in MG63 cells on PTFE/PVDF/PP mixed with single-wall carbon nanohorns (SWNH) or MWNT-A. Hinz et al. [28] indicated that an increased a-smooth muscle actin (a-SMA) expression was responsible for the enhancement of fibroblast contractile activity. Therefore, upregulation of actin expression in the present study is probably related to changes in cell activity, such as attachment and migration. Osteoblast differentiation and extracellular matrix production are critical processes for implant osseointegration or bone tissue engineering. Col-I is the most abundant protein in the bone matrix, which is synthesized by active osteoblasts [29, 30], mesenchymal cells, and fibroblasts [31], and its structure is conductive to mineral deposition. It also binds noncollagenous matrix proteins that initiate and control mineralization. As to MG63, after 24 h treatment with SWNTs-COOH, enhanced transcription of COLI occurred in all three concentrations, while enhanced ON and ALP transcription only occurred at the higher concentrations (Fig. 3). SWNTs-OH treatment also elevated the transcription of ON and ALP at 50 lg/mL and the transcriptions of COL-I, ON and ALP at 100 lg/mL (Fig. 3). Similar trends were recorded in primary osteoblast, that is, SWNTs-COOH treatment enhanced
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Fig. 5 Collagen (COL) production and alkaline phosphatase (ALP) activity of cells. a, b are for MG63 at 24 and 48 h respectively, while c, d are for primary osteoblasts at 24 and 48 h respectively. The x-axis
represents the types of proteins, while y-axis represents the ratio compared with the control ones. (Asterisk) indicates significant change (*P \ 0.05, **P \ 0.01)
transcription of COL-I in all three concentrations. Meanwhile, SWNTs-OH treatment enhanced the transcriptions of COL-I, ON and ALP in all three concentrations (Fig. 4). The maximum increments in the expression of COL-1 were 6.3-fold (for MG63) and 9.1-fold (for primary osteoblasts) that of the control group, when cells were co-cultured with 50 lg/mL SWNTs-COOH (Figs. 3, 4). Elisa results further confirmed the enhanced production in COL (Fig. 5). The results revealed that SWNTs-COOH induced a prompt increase in COL-I expression at 24 h with the lowest dose and the biggest increase throughout the experiment. Armentano et al. [32] demonstrated that cell differentiation was controlled on a SWNTs-COOH film, which promoted hydroxyapatite deposition by acting as an effective nucleation surface to induce the formation of a biomimetic apatite coating. In the present study, the results indicated that COL-I is a sensitive molecular marker of maturation of MG63 cells following SWNTs–COOH exposure, and the phagocytosis of SWNTs-COOH facilitates mineralization by increasing COL-I expression.
4 Conclusion
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In conclusion, we demonstrated that SWNTs can enter osteoblast cells by endocytosis, and carboxyl functionalization promotes increased cellular uptake. SWNTs-COOH initiated the prompt and the maximum upregulation of COL-I genes and simultaneously induced ER expansion for the increased needs of protein synthesis, which accelerates the mineralization process. However, impaired cell markers of mitochondria injury were detected upon interaction with all SWNTs at the highest dose after 48 h. Therefore, SWNTs-COOH is a good candidate for the fabrication of biomedical scaffolds for bone regeneration. Acknowledgments This study was supported by the major program of the National Natural Science Foundation of China (Grant No. 81190133), the National Natural Science Foundation of China (Grant No. 31300791), the National Basic Science Research Program of China (973 Program) (Grant No. 2011CB606205), the National Natural Science Foundation of China (Grant No. 51172172), and the Fundamental Research Funds for the Central Universities (2012-IV-069).
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A comparative study on the effects of pristine and functionalized single-walled carbon nanotubes on osteoblasts: ultrastructural and biochemical properties Qiu Tong • Wu Qingzhi • Dai Honglian • Wang Xinyu • Wang Youfa • Li Shipu • Li Junli
Received: 6 May 2013 / Accepted: 21 April 2014 Ó Springer Science+Business Media New York 2014
Abstract A comparative study was performed to investigate the ultrastructural and biomolecular properties of osteoblasts induced by three types of single-walled carbon nanotubes (SWNTs). The results on cellular uptake and ultrastructural alteration indicate that SWNTs enter osteoblasts by endocytosis. SWNTs-COOH and SWNTs-OH particles were freely dispersed in the cytoplasm, while pristine SWNTs were localized to the periphery of the cell. Both SWNTs-OH and SWNTs-COOH promoted cell changes in cell activity regarding mRNA expression at doses of 50 and 100 lg/mL in the first 24 h. When treated with 50 lg/mL SWNTs-COOH for 48 h, the expression of type I collagen increased by 6.3-fold (for MG63) or 9.1fold (for primary osteoblasts) compared with the control group. The present study observed for the first time that SWNTs-COOH initiated the prompt and the maximum upregulation of type I collagen gene expression, and simultaneously induced the expansion of the endoplasmic reticulum for increased protein synthesis, which in turn accelerated the mineralization process. However, impaired cell properties and mitochondrial injury were
detected following treatment with SWNTs at 100 lg/mL after 48 h. In conclusion, we believe that SWNTs-COOH is a good candidate for the fabrication of biomedical scaffolds for bone regeneration.
Abbreviation CNTs Carbon nanotubes MWNTs Multi-walled carbon nanotubes PCL Polycaprolacton SWNTs Single-walled carbon nanotubes ER Endoplasmic reticulum MTS Metabolic activity test TEM Transmission electron microscope COL-I Type I collagen ON Osteonectin Alpase Alkaline phosphatase MEM Minimum essential medium
1 Introduction Q. Tong W. Qingzhi D. Honglian W. Xinyu W. Youfa L. Shipu Biomedical Materials and Engineering Center, Wuhan University of Technology, Wuhan 430070, People’s Republic of China D. Honglian W. Xinyu W. Youfa L. Shipu State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Wuhan 430070, People’s Republic of China L. Junli (&) College of Science, Wuhan University of Technology, Wuhan 430070, People’s Republic of China e-mail: [email protected]
Carbon nanotubes (CNTs) have attracted considerable attention due to their promising applications in biomedical fields, such as biosensors, drug and vaccine delivery vehicles, and scaffold materials. The use of CNT-based scaffolds for bone regeneration is of particular interest because of their suitable dimensions that are similar to type I collagen, the major organic component of bone. Balani et al. [1] and Xu et al. [2] prepared multi-walled carbon nanotube (MWNT)-reinforced hydroxyapatite coating and confirmed its biocompatibility with different human osteoblast cell lines. Hirata et al. [3, 4] reported that
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MWNT-coated 3D collagen scaffolds induced bone formation in vivo, and MWCNT-coated polylactic acid (PLLA) scaffolds initiated cell attachment and enhanced the DNA content of Saos-2 cells. Furthermore, the unique effect of CNTs alone on cell biology was also examined. It was reported that single-walled carbon nanotube (SWNTs) regulated the metabolic activity of Saos-2 cells and significantly promoted cell proliferation [5, 6]. Zhang et al. [7] reported that the surface modification of MWCNTs promoted the adhesion and cell viability of osteoblasts. Furthermore, Li et al. [8] demonstrated that the MWNTs induced ectopic bone formation in the dorsal musculature of oddy mice. Osteoclasts and osteoblasts are the two primary cell types used to create and maintain bones. They are highly specialized cells that must work in perfect synchronization to maintain the skeletal system. Osteoclasts resorb bone tissue back into the body, whereas osteoblasts create bone. Osteogenesis is induced by osteoblastic cells and is accompanied by a sequence of characteristic events, involving cell attachment, cell proliferation and the expression of osteoblastic phenotype. So far, few investigations have been performed to illustrate the molecular mechanisms on the morphological and biochemical alterations of osteoblasts induced by SWNTs [9]. Herein, we investigated the physiological and bimolecular responses of osteoblasts induced by different SWNTs (pristine SWNTs, SWNTs-OH and SWNTs-COOH), using MG-63 cells and primary osteoblasts as the model system. We measured cell viability using metabolic activity test (MTS) and examined cellular uptake and intracellular distribution of SWNTs with transmission electron microscopy (TEM). To examine the molecular responses of cells exposed to different SWNTs, the expression levels of several genes were determined by quantitative real-time PCR, including the attachment-related gene: actin, the proliferation-related gene: c-jun, and the osteoblastic marker genes: type I collagen, COL-I, osteonectin, ON, alkaline phosphatase, ALPase.
solution and deionized water several times before use. To obtain a uniform solution, SWCNTs were dispersed in cell culture medium without serum by ultrasonication at a concentration of 1 mg/mL, and the supernatant was collected after centrifugation (5009g for 20 min). Cells were exposed to the supernatant at a final concentration of 25, 50 and 100 lg/mL. 2.2 Cell culture and treatments
2 Methods
Osteoblast-like MG-63 cells, originally isolated from a human sarcoma, were obtained from China Center for Type Culture Collection (Wuhan). MG-63 cells are relatively immature osteoblasts that have been well characterized and widely used for biomaterials testing, and show several similarities with isolated human bone-derived cells. MG-63 cells were grown in 25 cm2 flasks with minimum essential medium (MEM) supplemented with 10 % fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. At confluence, adherent cells were detached with 0.25 % trypsin, and seeded in 6-well plates at a density of 3 9 105 cells/mL. MG-63 cells were cultured at 37 °C in a humidified CO2 (5 %) atmosphere for 24 h. Primary rat calvarial osteoblasts were prepared by serial collagenase digestion. In brief, new born rat calvariae (day 1) were removed from soft tissues and digested five times with 0.1 % collagenase and 0.2 % dispase in Dulbecco’s Modified Eagle’s Medium (DMEM) for 10 min at 37 °C. After centrifugation and washing with the medium, cells were resuspended in DMEM supplemented with 10 % FBS in a humidified CO2 (5 %) atmosphere for 48 h. Then, nonadherent cells were removed, and adherent cells were harvested after treatment with 0.25 % trypsin/EDTA and successively passaged. All procedures undertaken in this study were approved by the Animal Care and Use Committees of Wuhan University of Technology and conformed to NIH guidelines. For exposure, culture medium was removed and fresh medium with or without SWNTs was added to the wells. 6 ml medium is for the 6-well plate, while 150ul medium is for the 96-well plate. Every 3 wells are set for one treatment. Cells were further cultured for 24 or 48 h.
2.1 Materials
2.3 Transmission electron microscopy
Three types of SWNTs, purified pristine SWNTs, hydroxyl purified SWNTs (SWNTs-OH) and carboxyl purified SWNTs (SWNTs-COOH), were purchased from Chengdu Organic Chemicals Co. Ltd (Chinese Academy of Science, China). All nanotubes were 1–2 nm in diameter and 1–3 lm in length. The hydroxyl content in SWNTs-OH was 3.96 wt%, and the carboxyl content in SWNTs-COOH was 2.73 wt%. The SWNTs were washed in dilute H2SO4
The ultrastructural alterations of MG63 and the internalization of SWNTs were observed through TEM. Briefly, MG-63 cells were incubated with different SWNTs at a dose of 50 lg/mL. Subsequently, the cells were washed with PBS three times, and prefixed in 4 % paraformaldehyde for 2 h and in 1 % osmium tetroxide at 4 °C for 1 h. Cells were then washed with 0.1 M sodium cacodylate buffer (pH 7.4) three times, dehydrated and embedded in
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resin. Ultrathin sections were made and stained with uranyl acetate and lead citrate. The samples were examined using a transmission electron microscope (HITACHI H-1000FA). 2.4 Metabolic activity test Metabolic activity test (MTS-Promega) was performed according to the manufacturer’s protocol. For this assay, 105 cells were exposed to different SWNTs (raw SWNTs, SWNTs-OH and SWNTs-COOH) and incubated for 24 h or 48 h. The spectrophotometric absorbance was quantified using Multiskan Spectrum (Thermo Scientific, Brussels, Belgium). The results were presented directly proportional to the control group. 2.5 Total RNA isolation and quantitative real-time PCR (Q-PCR) Total RNA was isolated from cells using trizol reagent (Invitrogen, USA) and reverse transcribed into cDNA using first strand cDNA synthesis kit (Fermentas, USA). Q-PCR was conducted with the SYBR Green qPCR kit (Toyota, Japan) on a CFX96 Real-Time System (Bio-Rad, USA). The reactions were performed in a 20 lL volume mix containing 10 lL SYBR Green I mixture, 2 lL primers (10 lM/L), 2 lL cDNA and 6 lL sterile water. Cycling conditions were set as follows: 3 min at 95 °C, 40 cycles of 10 s at 95 °C, 30 s at 61–69 °C, and 10 s at 72 °C. Melting curve analysis of amplification products was performed at the end of each PCR reaction to confirm that a single PCR product was detected. Each sample was run in three tubes, and the PCR reactions without the template were used as the blank. After PCR amplification, data were analyzed with the Bio-Rad CFX manager software version 1.0 and the fold difference was calculated using the 2-44CT method. The sequences of PCR primers were used as follows for MG63, with GAPDH as the internal control gene: GAPDH 50 -GAGCCACATCG CTCAGACAC-30 (forward), 50 -CATGTAGTTGAGGTCA ATGAAGG-30 (reverse); Actin 50 -GATGCAGAAGGAGA TCACTG-30 (forward), 50 -GGGTGTAACGACACTAAGT C-30 (reverse); C-jun 50 -GCCAACATGCTCAGGGAACA GGTG-30 (forward), 50 -GCCAACATGCTCAGGGAACA GGTG-30 (reverse); COL-I 50 -AACGCGTGTCAATCCCT TGT-30 (forward), 50 -GAACGAGGTAGTCTTTCAGCAA CA-30 (reverse); ALPase 50 -CTCGTCGACACCTGGAAG AGCTTCAAACCG-30 (forward), 50 -GGATCCGTCACGT TGTTCCTGTTCAGC-30 (reverse); ON 50 -TCACATTAGG CTGTTGGTTCAAA-30 (forward), 50 -GCGTGACCACTT CCCAGAGA-30 (reverse). The sequences of PCR primers were used as follows for primary osteoblast, with GAPDH as the internal control gene: GAPDH 50 -ATGGAGAAGGCTG GGGCTCACCT-30 (forward), 50 -AGCCCTTCCACGATG
CCAAAGTTGT-30 (reverse); Actin 50 -GACCTCTATGCC AACACAGTGCTGT-30 (forward), 50 -CTAGAAGCATTT GCGGTGCACGATG-30 (reverse); C-jun 50 -GCCAACAT GCTCAGGGAACAGGTG-30 (forward), 50 -GCCAACAT GCTCAGGGAACAGGTG-30 (reverse); COL-I 50 -CCTAC AGCACGCTTGTGGATG-30 (forward), 50 -AGATTGGGA TGGAGGGAGTTTAC-30 (reverse); ALPase 50 -TGCCCT GAAACTCCAAAAACT-30 (forward), 50 -ATCTCCAGCC GTGTCTCCTC-30 (reverse); ON 50 -GAAGAGATGGTGG CGGAG-30 (forward), 50 -ACAGGCAGGGGGCAATGTAT TTG-30 (reverse). 2.6 Collagen (COL) production and alkaline phosphatase (ALP) activity of cells The levels of the COL and the ALP activity were determined by ELISA, following the manufacturer’s instructions. Briefly, at culture times of 24 and 48 h, the cells were collected, washed with PBS, homogenized with 1 ml tris buffer (pH 7.4) and then sonicated. The cell lysate was applied in the following measurements. After standards and samples were added into each 96-well plate pre-coated with antigen, the first antibody was added into each well and incubated for 30 min at 37 °C. After aspirating and washing 5 times, the second antibody was added into each well and incubated for 20 min at room temperature. A stop solution was added into each well, and the absorbance value was read by a microplate reader at a 450 nm wavelength within 15 min. The measured concentration and activity were normalized by cell number to take into account the differences in cell growth. 2.7 Statistical analysis All values were expressed as mean ± standard deviation and significant differences between the treatment groups and the control group were analyzed using one-way ANOVA (P \ 0.05 or P \ 0.01).
3 Results and discussion The evaluation of cell uptake and intracellular location of CNTs is crucial for understanding the mechanisms of their biological effects. In our experiment, the internalization of SWNTs in MG63 cells was observed by TEM after 24 h and 48 h exposure (Fig. 1). We found that SWNTs were incorporated into the cytosol after 24 h of treatment (Fig. 1b, c, e, h). Previous studies have also described the capacity of CNTs to penetrate transformed cell lines from different origins, including A549, HeLa, and Jurkat cells [10]. Mooney et al. [11] reported the uptake of SWNTs by human mesenchymal stem cells (MSCs). Zhang et al. [12]
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Fig. 1 Ultrastructure changes of MG-63 cells exposed to SWNTs. a Cells without stimulation in control group (2,5009). b–d Cells treated with raw SWNTs-COOH for 24 h (B 2,5009 , C 5,0009) or 48 h (D 5,0009). e–g Cells treated with SWNTs-OH for 24 h (E 2,5009, F 5,0009) or 48 h (G 5,0009). h–j Cells treated with raw
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SWNTs for 24 h (H 2,5009, I 5,0009) or 48 h (J 5,0009). Serious expansion of ER is indicated by white arrows, while the SWNTs in the cells are indicated by black arrows. Mild swelling of the mitochondria is indicated by asterisk
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Fig. 2 Metabolic activity of MG-63 and primary osteoblasts in the presence of SWNTs. a, b Are for MG63 at 24 and 48 h respectively, while c, d are for primary osteoblasts at 24 and 48 h respectively. The
x-axis represents the types of SWNTs, while y-axis represents the viability ratio compared with the control ones. (Asterisk) indicates significant change (*P \ 0.05, **P \ 0.01)
observed the distribution of SWNTs-COOH in primary cells (osteoblasts) after exposure to a dose of 50 lg/mL for 24 h. More rapidly, Mu et al. [13] identified that SWNTsCOOH were translocated into cytoplasmic vesicles as soon as 1 h after treatment in HEK293 cells. TEM observation of fluorescently-labeled CNTs was used to verify that the CNTs were localized primarily within the cytoplasm and perinuclear region of the cell [7, 13, 14]. We also found that several particles were incorporated into cellular lysosomes, especially when cells were co-cultured with SWNTs-COOH for 48 h (Fig. 1d) and raw SWNTs for 24 h (Fig. 1i). No particles were found in the nucleus and neither apoptosis nor necrosis features were observed in cells during the experiment. A similar phenomenon was also reported by Giorgio et al. [14] and Zhang et al. [7], who showed that SWCNTs or MWCNTs induced the formation of abundant phagolysosomes containing internalized CNTs in RAW264.7 cells. In vitro studies, normal human osteoblasts and osteoblast-like cell lines have been shown to combine with various particles, including titanium, Ti–6Al–4 V, cobalt, chromium, cobalt–chromium, and ultrahigh molecular weight polyethylene (UHMWPE)
[15–17]. In addition, human osteoblasts were shown to express CD68, a protein associated with macrophages, when exposed to Ti particles [16]. Therefore, the identification of these CNT-containing endosome-like vesicles suggested a possible cell-uptake mechanism by endocytosis in osteoblasts [18]. There was no evidence that SWCNTs were in the nucleus, which agrees with previous studies [7, 13]. However, Mooney et al. [11] reported the SWNTsCOOH primarily occupied a cytoplasmic location for the first 24 h, and then assumed a nuclear location after 6 days. Therefore, nanotubes might interact with proteins located on the cell membrane or in the cytoplasm during the initial internalization steps. In this study, raw SWNTs, SWNTsCOOH and SWNTs-OH showed different behavior in their distribution in the cell: raw SWNTs were observed at the periphery of the cells, while SWNTs-COOH and SWNTsOH particles were freely dispersed in the cytoplasm (Fig. 1b, e, h). The results from RAW264.7 cells treated with CNTs also revealed that the negatively-charged MWCNTs-COOH may facilitate transport of MWCNTs through the cell membrane [7]. Furthermore, it was verified that carboxyl-functionalized surfaces enhanced cellular
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Fig. 3 Gene expression of MG63 exposed to SWNTs for 24 or 48 h. a, b, c are for the 24 h treatments, while d, e, f are for the 48 h treatments. In a and d, SWNTS are at the concentration of 25 lg/ml. In b and e, SWNTS are at the concentration of 50 lg/ml. In c and f,
SWNTS are at the concentration of 100 lg/ml. The x-axis represents the types of genes, while y-axis represents the expression ratio compared with the control ones. (Asterisk) indicates significant change (*P \ 0.05, **P \ 0.01)
uptake, and the amount of nanoparticles internalized could be correlated to the degree of carboxyl functionalization on the nanoparticle surface [19]. Remarkable changes were observed in cells treated with SWNTs. We found some secondary lysosomes and fast growing endoplasmic reticulum (ER) in cells treated with SWNTs-COOH and SWNTs-OH (Fig. 1c, d, f, g). Ultrastructurally, osteoblasts feature a complement of organelles characteristic of cells actively involved in protein synthesis. They have abundant ER, numerous ribosomes, and the Golgi apparatus and mitochondria are quite prominent. In the present study, ER expansion was prominent (Fig. 1c, d), especially in cells treated with SWNTs-COOH, suggesting the occurrence of ER stress. Hamamura et al. [20] demonstrated that ER stress from thapsigargin and tunicamycin led to two distinctive consequences, apoptosis or regulation of osteoblastogenesis. This bi-phasic response was dependent on exposure time to ER stress: 1 h treatment favored the pathway toward transcriptional activation of selected genes, such as Runx2 and type I collagen, as opposed to 24 h for the apoptotic pathway. Furthermore, recent research revealed that both bone morphogenetic protein-2 (BMP-2) and old astrocyte specifically induced
substance (OASIS) increased ER stress to stimulate differentiation of osteoblasts [21, 22]. Meanwhile, we observed increased cell metabolic activity (Fig. 2) and significantly increased COL-I expression in both SWNTsCOOH-treated cells types at 24 h (Figs. 3, 4). The activation of COL-I was prominent in ER stress by thapsigargin and tunicamycin at the first hours and by Old Astrocyte Specifically Induced Substance (OASIS) cases [20, 22]. Moreover, raw SWNTs did not induce significant ER expansion (Fig. 1h), while the expression of differentiation-related genes was not altered at 24 h (Fig. 3). Therefore, in the present study, ER expansion was related to active protein synthesis. After 48 h exposure to raw SWNTs and SWNTs-OH, we observed the formation of myeloid bodies in the lysosome and swollen mitochondria cristae in many cells (Fig. 1g, j). Similarly, nuclear degeneration and mitochondrial enlargement were seen in RAW 264.7 cells exposed to tau-MWCNTs [23]. Zhong et al. [24] also demonstrated that SWCNTs preferentially accumulated in lysosomes of the target organelles, and impaired mitochondrial activity as a result of cytotoxicity. Therefore, SWNTs may impair osteoblast activity through the
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Fig. 4 Gene expression of primary osteoblasts exposed to SWNTs for 24 or 48 h. a, b, c are for the 24 h treatments, while d, e, f are for the 48 h treatments. In a and d, SWNTS are at the concentration of 25 lg/ml. In b and e, SWNTS are at the concentration of 50 lg/ml. In
c and f, SWNTS are at the concentration of 100 lg/ml. The x-axis represents the types of genes, while y-axis represents the expression ratio compared with the control ones. (Asterisk) indicates significant change (*P \ 0.05, **P \ 0.01)
mitochondrial pathway. With increased concentration (100 lg/mL) and exposure time (48 h) to SWNTs, there was a decrease in metabolic activity and obvious suppression of gene expression in both cell models (Figs. 2, 3, 4). Osteoblasts are attachment-dependent cells that interact with their substrate via cytoskeletal alterations. The interaction of bone cells with biomaterials influences both their proliferation and differentiation. Some previous studies found enhanced proliferation and adherence of osteoblasts exposed to SWNT-based substrates or their composites [5, 6, 25]. At the molecular level, we verified increases in c-jun and actin mRNA expression after 24 h treatment with SWNTs-COOH at all doses, with SWNTs-OH at doses of 50 and 100 lg/mL, and with raw SWNTs at a dose of 50 lg/mL in MG63 and at doses of 25 and 50 lg/mL in primary osteoblasts (Figs. 3, 4). The expression of c-jun was the highest in proliferating osteoblasts [26]. The transcriptional regulation of actin mRNA was accompanied by reorganization of the actin cytoskeleton [27]. Previously, Kalbacova et al. [5] demonstrated thinner and diffused actin stress fibers in cells on SWCNTs films, which was attributed to rapid reorganization during cell migration across the nano-phase surface. Bacakova et al. [25]
detected distinct b-actin filament bundles in MG63 cells on PTFE/PVDF/PP mixed with single-wall carbon nanohorns (SWNH) or MWNT-A. Hinz et al. [28] indicated that an increased a-smooth muscle actin (a-SMA) expression was responsible for the enhancement of fibroblast contractile activity. Therefore, upregulation of actin expression in the present study is probably related to changes in cell activity, such as attachment and migration. Osteoblast differentiation and extracellular matrix production are critical processes for implant osseointegration or bone tissue engineering. Col-I is the most abundant protein in the bone matrix, which is synthesized by active osteoblasts [29, 30], mesenchymal cells, and fibroblasts [31], and its structure is conductive to mineral deposition. It also binds noncollagenous matrix proteins that initiate and control mineralization. As to MG63, after 24 h treatment with SWNTs-COOH, enhanced transcription of COLI occurred in all three concentrations, while enhanced ON and ALP transcription only occurred at the higher concentrations (Fig. 3). SWNTs-OH treatment also elevated the transcription of ON and ALP at 50 lg/mL and the transcriptions of COL-I, ON and ALP at 100 lg/mL (Fig. 3). Similar trends were recorded in primary osteoblast, that is, SWNTs-COOH treatment enhanced
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Fig. 5 Collagen (COL) production and alkaline phosphatase (ALP) activity of cells. a, b are for MG63 at 24 and 48 h respectively, while c, d are for primary osteoblasts at 24 and 48 h respectively. The x-axis
represents the types of proteins, while y-axis represents the ratio compared with the control ones. (Asterisk) indicates significant change (*P \ 0.05, **P \ 0.01)
transcription of COL-I in all three concentrations. Meanwhile, SWNTs-OH treatment enhanced the transcriptions of COL-I, ON and ALP in all three concentrations (Fig. 4). The maximum increments in the expression of COL-1 were 6.3-fold (for MG63) and 9.1-fold (for primary osteoblasts) that of the control group, when cells were co-cultured with 50 lg/mL SWNTs-COOH (Figs. 3, 4). Elisa results further confirmed the enhanced production in COL (Fig. 5). The results revealed that SWNTs-COOH induced a prompt increase in COL-I expression at 24 h with the lowest dose and the biggest increase throughout the experiment. Armentano et al. [32] demonstrated that cell differentiation was controlled on a SWNTs-COOH film, which promoted hydroxyapatite deposition by acting as an effective nucleation surface to induce the formation of a biomimetic apatite coating. In the present study, the results indicated that COL-I is a sensitive molecular marker of maturation of MG63 cells following SWNTs–COOH exposure, and the phagocytosis of SWNTs-COOH facilitates mineralization by increasing COL-I expression.
4 Conclusion
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In conclusion, we demonstrated that SWNTs can enter osteoblast cells by endocytosis, and carboxyl functionalization promotes increased cellular uptake. SWNTs-COOH initiated the prompt and the maximum upregulation of COL-I genes and simultaneously induced ER expansion for the increased needs of protein synthesis, which accelerates the mineralization process. However, impaired cell markers of mitochondria injury were detected upon interaction with all SWNTs at the highest dose after 48 h. Therefore, SWNTs-COOH is a good candidate for the fabrication of biomedical scaffolds for bone regeneration. Acknowledgments This study was supported by the major program of the National Natural Science Foundation of China (Grant No. 81190133), the National Natural Science Foundation of China (Grant No. 31300791), the National Basic Science Research Program of China (973 Program) (Grant No. 2011CB606205), the National Natural Science Foundation of China (Grant No. 51172172), and the Fundamental Research Funds for the Central Universities (2012-IV-069).
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