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Synergistic effect of strontium and silicon in strontium-substituted sub-micron bioactive glass for enhanced osteogenesis
Materials Science & Engineering C 89 (2018) 245–255
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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
Synergistic effect of strontium and silicon in strontium-substituted submicron bioactive glass for enhanced osteogenesis
T
Wen Zhanga,b,c,1, Deqiu Huangd,1, Fujian Zhaoa,c, Wendong Gaoa,c, Luyao Suna,c, Xian Lia,c, ⁎ Xiaofeng Chena,c, a
Department of Biomedical Engineering, School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, China. Institute for Brain Research and Rehabilitation, South China Normal University, Guangzhou, 510631, China c National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006, China d College of Biophotonics, South China Normal University, Guangzhou 510631, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Strontium-substituted sub-micron bioactive glasses Wnt/β-catenin NFATc Osteogenesis
Strontium-substituted sub-micron bioactive glasses (Sr-SBG) have been reported to have enhanced osteogenic differentiation capacity compared to sub-micron bioactive glasses (SBG) in our previous study. However, the underlying molecular mechanisms of such beneficial effect of Sr-SBG are still not fully understood. In this study, we synthesized Sr-SBG, studied the effects of Sr-SBG on proliferation and osteogenic differentiation of mouse mesenchymal stem cells (mMSCs), and identified the molecular mechanisms of the enhancement effect of Sr-SBG on mMSCs. The results demonstrated that Sr-SBG had more profound promotion effect on proliferation and osteogenic differentiation of mMSCs than SBG and SrCl2 group which containing identical Sr concentration with Sr-SBG group. RT-qPCR and western blot analysis showed that the mRNA expressions and protein expressions involved in NFATc and Wnt/β-catenin signaling pathways were all upregulated mediated by Sr-SBG, while only Wnt/β-catenin signaling pathway related genes upregulated in SBG group and only NFATc signaling pathway activated in SrCl2 group, suggesting that NFATc and Wnt/β-catenin signaling pathways played important roles in osteogenesis enhancement induced by Sr-SBG. To conform the above conclusion, cyclosporin A (CSA) was applied to inhibit NFATc signaling pathway. It was found that the enhanced osteogenic differentiation of mMSCs induced by Sr-SBG was partially abrogated and the activated Wnt/β-catenin signaling pathway was also inhibited in part. However, the effects of SBG on proliferation and osteogenesis of mMSCs were unimpaired, yet the effects of SrCl2 were greatly suppressed. Taken together, these results indicated that strontium activated NFATc signaling pathway and silicate activated Wnt/β-catenin signaling pathway might synergistically mediated the enhanced osteogenesis induced by Sr-SBG.
1. Introduction Multifunctional bioactive materials for the repair of bone defects caused by trauma, tumors, infections or genetic malformations, have attracted much attention in the past several years. In 1970s, Carlisle and Schwarz suggested that Silicon (Si) element might play an important role in skeletal development and bone repair [1,2]. Since then, many studies on silicate bioactive materials, including Si-substituted calcium phosphates [3–5], silicate-based bioceramics [6–9] and bioactive glasses [10–13], have been employed for bone regeneration. Bioactive glass is one of the typical bioactive materials possessing osteoinductive properties for its function of activating osteogenesis-related gene expression of bone marrow stromal cells (BMSCs) [14–16].
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Recently, micro-nano bioactive glass (MNBG) with controlled size and morphology was developed through the combination of sol-gel technique and template self-assembly method. Compared with conventional bioactive glasses, MNBG has superior apatite formation ability and biological activity due to their significantly increased surface area [17,18]. To achieve improved osteogenesis of bioactive glasses for bone repair, it was proved that the substitution of Sr ions into bioactive glasses could enhance the osteogenic differentiation of BMSCs [19–24]. Our previous work also demonstrated that Sr-substituted sub-micron bioactive glass (Sr-SBG) could improve osteogenesis than SBG [25]. Nevertheless, the underlying molecular mechanisms are still under investigation. To develop an ideal bioactive glass-based bone repair biomaterial, it is important to clarify the molecular mechanism through
Corresponding author. E-mail address: [email protected] (X. Chen). These authors contributed to the work equally and should be regarded as co-first authors.
https://doi.org/10.1016/j.msec.2018.04.012 Received 5 September 2017; Received in revised form 30 March 2018; Accepted 10 April 2018 Available online 11 April 2018 0928-4931/ © 2018 Published by Elsevier B.V.
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2.2. Synthesis of SBG and Sr-SBG and their ion dissolutions
which the biomaterial regulates osteogenic differentiation of BMSCs. During embryonic development, Wnt signaling plays a critical role in bone formation through regulating the determination of cell fate, and influencing the proliferation and differentiation of stem cells [26–28]. Previous researches have demonstrated that the bioactive Si ions released from BG and other silicate bioceramics could promote proliferation of BMSCs and induce osteogenic differentiation of BMSCs. The underlying mechanism of the enhanced proliferation and differentiation of BMSCs mediated by silicate was supposed to be related to the Wnt signaling pathway activation [7,29,30]. Sr is a trace element which could not only stimulate bone formation but also inhibit bone resorption during bone metabolism [31–33]. The mechanism was supposed to be related to the ability of Sr ions to promote the activity of alkaline phosphatase (ALP) and mRNA expression of osteogenesis-related gene of MSCs [34,35]. It was found that systemic administration of strontium ranelate could lead to increased bone mineral density (BMD) and decreased probability of bone fractures occurrence in osteoporotic patients [36–38]. Consequently, plenty of Sr-containing bone materials were developed due to their enhanced osteogenic capacity for rapid osseointegration [3,39–42]. Recently, Fromigue et al. showed that the Nuclear factor of activated T-cells (NFATc) signaling pathway and downstream canonical and non-canonical Wnt signaling pathways were involved in strontium ranelate induced proliferation and differentiation of murine osteoblasts [43]. NFATc are transcription factors that are highly phosphorylated and remain in the cytoplasm in unstimulated cells, and are involved in the differentiation of various cell types. When intracellular calcium level increases, calcineurin will be activated followed by dephosphorylation of NFATc1, leading to the translocation of NFATc1 to nucleus and binding to the promoter of target genes [44]. It was demonstrated that NFATc1 played an important role in both osteoblasts and osteoclasts, which is expressed during osteogenic differentiation and osteoclastogenesis. Based on the important role of Wnt/β-catenin signaling pathway mediated by silicate and NFATc signaling pathway induced by Sr element in osteogenesis, this study was conducted to investigate the effect of Wnt/β-catenin and NFATc signaling pathway on Sr-SBG stimulated osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (mMSCs) since that both Sr and Si ions are dominant components of Sr-SBG. We hypothesized that Sr-SBG could promote osteogenic differentiation of mMSCs through the synergistically effect of SBG activated Wnt/β-catenin pathway and Sr activated NFATc pathway. To test this hypothesis, we conducted three experimental groups: SBG group, Sr-SBG group and SrCl2 group, and investigated their effect on proliferation and osteogenesis of mMSCs and their influence on activation of the two signaling pathways above by RT-qPCR and western blot analysis. Meanwhile, NFATc signaling pathway was blocked by using cyclosporin A (CSA) to evaluate the osteogenic differentiation capacity of mMSCs induced by Sr-SBG. The results could further verify whether NFATc and Wnt/β-catenin signaling pathways played a synergistic effect on the enhanced osteogenesis of Sr-SBG or not. This study might clarify a key scientific problem before the application of Sr-SBG in clinic.
SBG and Sr-SBG were prepared according to the previous described method by the combination of sol-gel and template self-assembly technique using DDA as both catalyst and template agent [17,18,25]. The constituents ratio of SBG was SiO2: CaO: P2O5 = 60: 36: 4 (mol/ mol), and the constituents ratio of Sr-SBG was SiO2: CaO: SrO: P2O5 = 60: 30: 6: 4 (mol/mol). Typically, a given amount of ethanol was mixed with deionized water. Then, 4 g DDA was dissolved into the mixed solution above by stirring at 40 °C for 10 min. After that, two equal proportion of total 16 ml TEOS was added to the above mixture under vigorous stirring for 30 min interval. Finally, TEP and CN or SN was added under stirring in order with 30 min intervals. After another 3 h vigorous stirring, the resulting milky suspension was aged for 6 h and centrifuged at 4000 rpm to obtain white gel precipitate. The white precipitate was rinsed with ethanol and deionized water for three times each, and freeze-dried for 48 h. Eventually, SBG and Sr-SBG powders were obtained after sintering in air at 650 °C for 3 h. The ion dissolutions of SBG and Sr-SBG in Dulbecco modified Eagle medium (DMEM) was prepared as previously described. Briefly, SBG and Sr-SBG powders were autoclaved and then added into DMEM medium at a ratio of 1 mg/ ml. The suspension was maintained at 37 °C in a shaker with the speed set as 120 rpm for 48 h. The ion dissolutions of SBG and Sr-SBG were obtained by centrifugation and filtration using a 0.22 μm syringe filter. 10% fetal bovine serum and 1% (v/v) penicillin/streptomycin were added to the ion dissolutions for cell culture in the following experiments. 2.3. Characterization of SBG and Sr-SBG Scanning electron microscopy (SEM) (DSM 982-Gemini, Zeiss, Germany) was conducted to characterize the morphology of SBG and Sr-SBG. The particle size distribution was assayed on Zetasizer Nano ZS (Malvern Instruments, UK). X-ray photoelectron spectroscopy (Kratos Axis UlraDLD, UK) and Energy-dispersive X-ray spectroscopy were used to test the element composition of SBG and Sr-SBG. And the concentrations of ion dissolution of SBG and Sr-SBG were tested by inductively coupled plasma-atomic emission spectrometry (ICP-AES) (PS1000-AT, Leeman, USA). 2.4. Cell culture mMSCs was obtained from American Type Culture Collection (ATCC® CRL-12424™) and used in the subsequent experiments. mMSCs were cultured in DMEM with 10% fetal bovine serum and 1% (v/v) penicillin/streptomycin, under a 5% CO2 incubator at 37 °C. After the confluence reached to 90%, cells were passaged using 0.25% trypsinEDTA. The third to sixth passages of mMSCs were used for cell proliferation, osteogenic differentiation and activated/inactivation signaling pathway studies. Four groups (a. Control group with DMEM medium; b. SBG group with ion dissolution of SBG; c. Sr-SBG group with ion dissolution of Sr-SBG; d. SrCl2 group with equal Sr ion concentration with Sr-SBG group) were set up in the following experiments.
2. Materials and methods
2.5. Proliferation assay
2.1. Materials
Proliferation of mMSCs was carried out by a Cell Counting Kit-8 assay kit (CCK-8, Dojindo Laboratories, Kumamoto, Japan). mMSCs were seeded at a density of 3000 cells/well in a 96-well plate. After cells attached, the culture medium was replaced by ion dissolution of each group. The culture medium of each group was refreshed every 3 days. 1, 3 and 7 days later, cells were harvested for CCK-8 assay (five samples for each group). For each well, the medium was displaced by 100 μl DMEM containing 10% CCK-8 and maintained in the incubator at 37 °C for 1 h. The absorbance of each well was measured at 450 nm by
Ethanol (AR), tetraethyl orthosilicate (TEOS) (AR), calcium nitrate tetrahydrate (CN) (AR), and strontium nitrate (SN) (AR) were obtained from Guangzhou Chemical Reagent Factory, P.R. China. Triethylphophate (TEP) (AR) and Dodecylamine (DDA) were purchased from Aladdin (Shanghai, P.R. China). All chemical reagents were used directly without other treatment.
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Thermo Scientific Microplate Reader (Thermo 3001, USA).
Table 1 Primer sequences used for gene expression analysis.
2.6. Osteogenic differentiation of mMSCs
Genes
Primer sequences
To investigate the osteogenic differentiation of mMSCs, cells were seeded in 24-well plates (5 × 104 cells/well, three samples for each group). After attachment, the media were replaced by medium of each group supplemented with osteogenic inducing component (10 mM βglycerophosphate, 50 μM ascorbic acid, and 10 nM dexamethasone). Every 3 days, the culture medium was refreshed of each group. The osteogenic differentiation of mMSCs in each group was examined with alkaline phosphatase (ALP) staining and ALP activity assay, alizarin red-S staining and quantitative analysis, mRNA expressions of marker genes associated with osteogenesis.
ALP
Forward:5′-TGCCTACTTGTGTGGCGTGAA-3′ Reverse:5′-TCACCCGAGTGGTAGTCACAATG-3′ Forward:5′-GCAGTCTTCTGCGGCAGGCA-3′ Reverse:5′-CACCGGGAGGGAGGAGGCAA-3′ Forward:5′-AGCAGCTTGGCCCAGACCTA-3′ Reverse:5′-TAGCGCCGGAGTCTGTTCACTAC-3′ Forward:5′-ATGCCGCGACCTCAAGATG-3′ Reverse:5′-TGAGGCACAGACGGCTGAGTA-3′ Forward:5′-CACTGGCGGTGCAACAAGA-3′ Reverse:5′-TTTCATAACAGCGGAGGCATTTC-3′ Forward:5′-CTCCCACTCTTCCACCTTCG-3′ Reverse:5′-TTGCTGTAGCCGTATTCATT-3′ Forward:5′-GGTCCTCTGTGAACTTGC-3′ Reverse:5′-GTAATCCTGTGGCTTGTCC-3′ Forward:5′-GGTAACTCTGTCTTTCTAACCTTAAGCTC-3′ Reverse:5′-GTGATGACCCCAGCATGCACCAGTCACAG-3′ Forward:5′-ACCTCAAGTGCAAACTCTCACCCA-3′ Reverse:5′-AGCTGTTTCCGTGGATCTCACACT-3′ Forward:5′-AAGTTGAGGTTCCGCAGTCCAG-3′ Reverse:5′-CGCAAACAGGCAAAGGTCAGG-3′ Forward:5′-CCTCGGAGATGGTGGTAGA-3′ Reverse:5′-GTTAGGTTCGCAGAAGTTGG-3′
OPN OCN COLI Runx2 GAPDH β-catenin NFATc1
2.6.1. ALP staining and ALP activity assay of mMSCs When the incubation time of mMSCs with medium in each group supplemented with osteogenic inducing component reached to 7 and 14 days, ALP staining and ALP activity assay were performed using commercially available kits. BCIP/NBT ALP Color Development Kit (Beyotime) was used for the ALP staining of mMSCs cultured for 7 days. Typically, cells were fixed with 10% neutral formalin after rinsing with PBS for three times. Then, a mixture of BCIP and NBT in buffer solution were added to each well and stained for 30 min. After washing off the unbound dye, images were taken on an inverted microscope. To study the ALP activity mMSCs, cells incubated for 7 and 14 days were terminated and washed three times with pre-cold PBS, and then were lysed with lysis solution including 0.1% triton X-100 and 0.5 mM MgCl2 in 10 mM tris-HCl (pH 7.4) for 1 h on ice. After the lysates centrifuged at 16,000 g for 15 min at 4 °C, the supernatant were collected and stored in −20 °C. ALP activity assay were performed by using the ALP assay Kit (Beyotime) according to the instructions of the manufacturer. Total protein content was evaluated by the bicinchoninic acid protein assay kit (Thermo scientific). The results were presented by normalizing ALP activity to the total protein content.
Axin2 DKK1 Wnt3A
2.7. Activation of Wnt and NFATc signaling pathway For activation of Wnt and NFATc signaling pathway study, mMSCs were cultured in 6-well plate at a seeding density of 2 × 105 cells/well (three samples for each group) and incubated with media in each group for 7 days. Wnt/β-catenin signaling pathway related genes (β-catenin, Axin2, DKK1, Wnt3A) and NFATc signaling pathway related gene NFATc1 were investigated by RT-qPCR assay as described above, and primers used in this assay were listed in Table 1. Protein expressions of the two signaling pathway above (β-catenin and NFATc1) were analyzed by western blot. Generally, total protein of mMSCs were extracted by RIPA lysis buffer (Beyotime) with addition of PMSF and protease inhibitor cocktail (cOmplete ULTRA Tablets). After centrifugation, the supernatant of cell lysates were collected and stored in −80 °C or used immediately for western blot assay. SDS-PAGE gels were used to separate the total proteins, which were then transferred onto a PVDF membrane. The membranes were immersed in 5% skim milk for 1 h at room temperature to block nonspecific site, and subsequently incubated with a rabbit anti-β-catenin antibody (proteintech, 1:10000) and mouse anti-NFATc1 antibody (Santa Cruz Biotechnology, 1:1000) overnight at 4 °C. A rabbit monoclonal anti-GAPDH antibody (CST, 1:1000) was used as reference protein. After washed with TBST (Tris Buffered Saline, with Tween-20) for four times (5 min/time), the membranes were incubated with HRP-conjugated secondary antibodies (proteintech) for 1 h at room temperature. Eventually, the membranes were washed with TBST for four times (5 min/time) and the protein band were detected by using a chemiluminescent system (ECL-plus, Beyotime). AlphaEase FC software was used to analysis the quantitative densitometric of the images.
2.6.2. Alizarin red S staining and quantitative assay of mMSCs Mineralization of extracellular matrix evaluated by Alizarin red S staining was performed using mMSCs treated with medium in each group including osteogenic inducing supplements for 14 days. After washing with PBS for three times, cells were fixed with 4% paraformaldehyde for 20 min. After fixation, cells were rinsed three times with ultrapure water, and were stained with 0.5% Alizarin red S (pH 4.2) for 5 min at room temperature. Unbound Alizarin red S was removed by washing with ultrapure water until the cleaning water became colorless. The plate was air-dried at room temperature and images were captured by inverted microscopy. Alizarin Red S staining quantitative analysis was carried out by separate the combined stain from cells with 10% cetylpyridinium chloride for 1 h. The absorbance of eluent solution was measured on a microplate reader at 562 nm. 2.6.3. Osteogenesis-related gene expressions in mMSCs Osteogenesis-related gene (ALP, OPN, OCN, COL1, Runx2) expressions were evaluated by RT-qPCR after mMSCs were induced by medium in each group with osteogenic inducing supplement for 7 and 14 days. To extract the total RNA from mMSCs, cells incubated for 7 and 14 days were terminated and total RNA were isolated using HiPure Total RNA Micro Kit (Magen) according to the manufacturer's instruction. Then, Reverse Transcription Reagents Kit (Takara) was applied to synthesize the complementary DNA from 500 ng total RNA. The RTqPCR procedures were carried out on Quantstudio 6 Flex (Life technologies) using SYBR Green qPCR kit (Thermo Scientific). RT-qPCR primers used in this study were listed in Table 1. Target gene expressions were analyzed using the 2−ΔΔCTmethod and each gene was normalized with house-keeping gene GAPDH.
2.8. Effect of NFATc signaling pathway inactivation on osteogenic differentiation For inactivation of signaling pathway study, the calcineurin inhibitor cyclosporin A (CSA) was used to inhibit NFATc signaling pathway. mMSCs in each group was treated with respective medium containing osteogenic inducing component and CSA (100 ng/ml) for 7 and 14 days. Then the osteogenic differentiation of mMSCs, gene and protein expression of NFATc and β-catenin signaling pathways were measured as described in Sections 2.6 and 2.7.
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Fig. 1. SEM images of SBG (A) and Sr-SBG (B) and EDS analysis of SBG (C) and Sr-SBG (D).
2.9. Statistical analysis
3.2. Proliferation of mMSCs
All of the experiments were repeated three times and the data were presented as mean ± standard deviation (SD). One-way analysis of variance with LSD post-test was used to analyze the significant differences between groups. The values of P < 0.05 were considered as statistically significant. SPSS Statistics version 22 (IBM, USA) was used to carry out the statistical analysis.
To examine mMSCs proliferation affected by Sr-SBG, mMSCs were incubated with media in each group for 1, 3, and 7 days and CCK-8 assay was then conducted. As shown in Fig. 4, there was no significant difference in cell proliferation among each treatment groups on the first day. When the culture time increased to 3 days and 7 days, all of the three experiment groups revealed an enhanced cell proliferation ability. Statistically significant difference was detected between SBG group and Sr-SBG or between Sr-SBG group and SrCl2 group on day 7 (P < 0.05), demonstrating improved effect of Sr-SBG than SBG and SrCl2 in cell proliferation.
3. Results 3.1. The characterization of SBG and Sr-SBG SBG and Sr-SBG microspheres were synthesized successfully by the combination of sol-gel method and template self-assembly technique. The morphology of SBG and Sr-SBG particles were examined by SEM (Fig. 1A and B). It was shown that SBG and Sr-SBG had similar size, which was approximately 500 nm. Dynamic light scattering (DLS) analysis revealed that the average size of SBG and Sr-SBG was 447.3 nm and 567.8 nm, respectively (Fig. 2). The increased particle size of SrSBG than SBG could be ascribed to the larger ionic radius of Sr2+ than Ca2+, which may result in weaker field strength of Sr2+ than Ca2+, thus leading to much looser network structure and larger particle size of bioactive glass with the incorporation of Sr element. EDS (Fig. 1C and D) and XPS (Fig. 3) characterization was performed to examine the elemental compositions of SBG and Sr-SBG, which all demonstrated that the Sr ion was successfully incorporated into SBG. The detection of ion concentration in SBG and Sr-SBG dissolutions determined by ICPAES was shown in Table 2. The SBG and Sr-SBG ion dissolutions had similar Si and P ion concentration. To the contrary, the differences of Ca and Sr ion concentration between SBG and Sr-SBG dissolutions were distinct. There was a reduction of Ca ion concentration and increased Sr ion concentration of Sr-SBG compared with SBG.
3.3. Osteogenic differentiation of mMSCs Osteogenic differentiation assessment of mMSCs induced by medium from each group was performed by ALP activity assay, alizarin red S assay and RT-qPCR assay. mMSCs cultured for 7 and 14 days were used to detect the ALP activity. ALP staining results shown in Fig. 5A indicated that mMSCs in three experimental group were deeper stained than that in control group on day 7, while the Sr-SBG group showed the deepest stained ALP. Similarly, as plotted in Fig. 3C, the quantitative measurement of ALP activity of mMSCs showed that cells treated by SrSBG extracts expressed significant higher level of ALP than that with SBG and SrCl2 on both day 7 and 14. The formation of extracellular calcified deposits of mMSCs treated with medium in each groups for 14 days were examined by Alizarin Red S staining and quantitative analysis. As shown in Fig. 5B, more obvious mineralized nodules were generated in mMSCs after stimulated by Sr-SBG than that by SBG, SrCl2 and control medium. Quantitative analysis of alizarin red S showed similar trends with alizarin red S staining (Fig. 5D). RT-qPCR determination of mRNA expression of osteogenic marker genes was shown in Fig. 6. The mRNA expression of ALP, OPN, OCN, 248
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Fig. 2. Size distribution of SBG (A) and Sr-SBG (B).
COL I and Runx2 were significantly upregulated in all three experimental group on day 7 and 14, whereas there was an enhanced effect of mRNA expression of ALP on day 7, OPN on day 7 and 14, and OCN on day 14 in Sr-SBG group compared with SBG and SrCl2 group (P < 0.05).
Table 2 Si, Ca, P and Sr concentration of materials extracts.
DMEM SBG Sr-SBG SrCl2
3.4. Activation of Wnt and NFATc signaling pathway To investigate the activation of Wnt/β-catenin and NFATc signaling pathways during osteogenic differentiation of mMSCs mediated by SBG, Sr-SBG and SrCl2, cells were cultured with SBG, Sr-SBG and SrCl2 for 7 days. The effects of SBG, Sr-SBG and SrCl2 on activation of Wnt/βcatenin and NFATc signaling pathways determined by mRNA expression of β-catenin, Axin2, DKK1, Wnt3A, NFATc1 and western blot analysis of β-catenin and NFATc1 protein expression were shown in Figs.7 and 8, respectively. All the treatment of SBG, Sr-SBG and SrCl2 could increase the mRNA expression of β-catenin, Axin2, DKK1, Wnt3A and NFATc1 compared with control group. However, through comparison between any two experimental groups, there was an enhanced effect of Sr-SBG group in β-catenin, Axin2 and Wnt3A expression than SBG and SrCl2 group. There was also a significant increase in NFATc1 expression of Sr-SBG and SrCl2 group than SBG group. In addition, the upregulation of Wnt3A expression in SrCl2 group was more profound than that in SBG group. Western blot analysis indicated that the protein expression of NFATc1 in Sr-SBG and SrCl2 group were significantly higher than the other two groups, whereas there was no significant differences between SBG group and control group. The results also showed that significant differences in β-catenin expression were detected among three experimental groups, with Sr-SBG being the highest, and SrCl2 group being the lowest.
Si (mg/l)
Ca (mg/l)
P (mg/l)
Sr (mg/l)
0.02 31.04 32.35 0.02
51.85 44.29 41.02 57.76
27.93 12.32 11.43 27.61
0 0 6.518 6.525
Fig. 4. CCK-8 assay of mMSCs incubated with SBG, Sr-SBG and SrCl2. *Significant difference (P < 0.05) for SBG, Sr-SBG or SrCl2 group vs control group. #Significant difference (P < 0.05) for Sr-SBG or SrCl2 group vs SBG group. @Significant difference (P < 0.05) for SrCl2 group vs Sr-SBG group. Values were expressed as mean ± SD.
Fig. 3. XPS characterization of SBG (A) and Sr-SBG (B). 249
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Fig. 5. Osteogenic differentiation of mMSCs after treatment of SBG, Sr-SBG and SrCl2. ALP staining (A) and ALP activity (C) of mMSCs stimulated by SBG, Sr-SBG and SrCl2 with osteogenic supplements for 7 and 14 days. Alizarin red S staining (B) and Alizarin red S quantitative measurement (D) of mMSCs induced by SBG, Sr-SBG and SrCl2 with osteogenic supplements for 14 days. Scale bar = 100 μm. *Significant difference (P < 0.05) for SBG, Sr-SBG or SrCl2 group vs control group. #Significant difference (P < 0.05) for Sr-SBG or SrCl2 group vs SBG group. @Significant difference (P < 0.05) for SrCl2 group vs Sr-SBG group. Values were expressed as mean ± SD.
Fig. 6. mRNA expression of osteogenic related genes (ALP, OPN, OCN, COL1, Runx2) of mMSCs mediated by SBG, Sr-SBG and SrCl2 with osteogenic supplements for 7 and 14 days. *Significant difference (P < 0.05) for SBG, Sr-SBG or SrCl2 group vs control group. #Significant difference (P < 0.05) for Sr-SBG or SrCl2 group vs SBG group. @Significant difference (P < 0.05) for SrCl2 group vs Sr-SBG group. Values were expressed as mean ± SD. 250
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Fig. 7. Effect of SBG, Sr-SBG and SrCl2 on the expression of Wnt/β-catenin and NFATc signaling pathways. RT-qPCR analysis of Wnt/β-catenin signaling related genes (β-catenin, Axin2, DKK1, Wnt3A) and NFATc signaling related gene NFATc1 of mMSCs cultured with SBG, Sr-SBG and SrCl2 for 7 days. *Significant difference (P < 0.05) for SBG, Sr-SBG or SrCl2 group vs control group. #Significant difference (P < 0.05) for Sr-SBG or SrCl2 group vs SBG group. @Significant difference (P < 0.05) for SrCl2 group vs Sr-SBG group. Values were expressed as mean ± SD. Table 3 Si, Ca, P and Sr concentration of materials extracts cultured with mMSCs for 3 days.
DMEM SBG Sr-SBG SrCl2
Si (mg/l)
Ca (mg/l)
P (mg/l)
Sr (mg/l)
0.03 17.45 17.82 0.03
52.02 42.05 40.48 37.23
28.21 24.79 21.97 28.95
0 0 3.0245 3.275
3.5. Ion concentration changes after incubated with mMSCs The Si, Ca, P, and Sr ion concentration of media in four groups were determined by ICP-AES after their incubation with mMSCs for 3 days and was shown in Table 3. By contrasting ion concentration of each of the four groups in Table 2, there was a significant reduction of Si concentration in SBG and Sr-SBG group, and obvious declined Sr concentration in Sr-SBG and SrCl2 group. Also, there was a slight decrease in Ca concentration and increased P concentration in all four groups.
3.6. Effect of NFATc signaling pathway inactivation on proliferation and osteogenic differentiation of mMSCs To identify the effects of NFATc signaling pathway inactivation on proliferation and osteogenic differentiation of mMSCs induced by SBG, Sr-SBG and SrCl2, cells were treated with NFATc signaling pathway inhibitor CSA to block the pathway during incubated with SBG, Sr-SBG and SrCl2. As shown in Fig. 9, there was no significant difference between SBG and Sr-SBG group on cell proliferation, demonstrating the enhanced effect of cell replication induced by Sr-SBG were partially inhibited. However, the cell proliferation promotion effect of SrCl2 was greatly impaired. Fig. 10 A and C showed the ALP staining and ALP activity results of mMSCs on day 7 and 14 after NFATc pathway inhibited, and Fig. 10B and D displayed alizarin red S staining and quantitative assay results on day 14. Compared with the results of not being treated with CSA, the
Fig. 8. Protein expression of β-catenin and NFATc1 after 7 days' culture with SBG, Sr-SBG and SrCl2 by western blot analysis. *Significant difference (P < 0.05) for SBG, Sr-SBG or SrCl2 group vs control group. #Significant difference (P < 0.05) for Sr-SBG or SrCl2 group vs SBG group. @Significant difference (P < 0.05) for SrCl2 group vs Sr-SBG group. Values were expressed as mean ± SD.
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As shown in Fig. 11, after treatment of CSA to inactivate NFATc signaling pathway, there was little difference between SBG and Sr-SBG group in mRNA expression of ALP, OPN, OCN and COLIon day 7 and 14. However, there was significant difference between SrCl2 group and SBG or SrCl2 and Sr-SBG group in mRNA expression of OPN on day 14 and OCN, COLI on day 7 and 14. Furthermore, after NFATc pathway was inhibited, mRNA expression of β-catenin, Axin2, DKK1, Wnt3A, NFATc1 were carried out and the results were shown in Fig. 12. RT-qPCR analysis showed that the addition of NFATc signaling pathway inhibitor CSA in the culture medium inhibited the mRNA expression of NFATc1 and partially inhibited the upregulation of β-catenin in Sr-SBG group. There was no significant difference between Sr-SBG group and SBG group in β-catenin expression, while SrCl2 group showed significant downregulated β-catenin expression compared with SBG group and Sr-SBG group. 4. Discussion Fig. 9. Cell proliferation of mMSCs treated with SBG, Sr-SBG and SrCl2 with 100 ng/ml CSA for 1, 3 and 7 days. *Significant difference (P < 0.05) for SBG, Sr-SBG or SrCl2 group vs control group. #Significant difference (P < 0.05) for Sr-SBG or SrCl2 group vs SBG group. @Significant difference (P < 0.05) for SrCl2 group vs Sr-SBG group. Values were expressed as mean ± SD.
In this study, we investigated the stimulatory effects and related mechanisms of Sr-SBG on proliferation and osteogenic differentiation of mMSCs. As shown in the results, Sr-SBG had a more enhanced effect on cell proliferation and osteogenic differentiation of mMSCs compared with SBG and SrCl2. Furthermore, our study showed that Sr-SBG could induce the upregulation of Wnt/β-catenin signaling pathway and NFATc signaling pathway related genes. Though DKK1 as a negative regulator of Wnt/β-catenin signaling pathway was also upregulated in Sr-SBG group, the whole effect of β-catenin protein expression induced by Sr-SBG was upregulated. As shown in the results of gene and protein expression, the expression of NFATc1 was higher in SrCl2 group than that in SBG group, and β-catenin expression in SrCl2 group was much
increased ALP activity of mMSCs had declined in part in Sr-SBG group, with no significant difference between SBG and Sr-SBG group, and the increased ALP activity had a greatly reduction in SrCl2 group. Meanwhile, alizarin red S staining and quantitative assay conveyed identical mineralized nodules in Sr-SBG group with SBG group, while SrCl2 group showed much less mineralized nodules than Sr-SBG and SBG group.
Fig. 10. Effect of NFATc signaling pathway inactivation on osteogenic differentiation of mMSCs treated by SBG, Sr-SBG and SrCl2. ALP staining (A) and ALP activity (C) of mMSCs stimulated by SBG, Sr-SBG and SrCl2 with CSA (100 ng/ml) for 7 and 14 days. Alizarin red S staining (B) and Alizarin red S quantitative measurement (D) of mMSCs induced by SBG, Sr-SBG and SrCl2 with CSA (100 ng/ml) for 14 days. Scale bar = 100 μm. *Significant difference (P < 0.05) for SBG, Sr-SBG or SrCl2 group vs control group. #Significant difference (P < 0.05) for Sr-SBG or SrCl2 group vs SBG group. @Significant difference (P < 0.05) for SrCl2 group vs Sr-SBG group. Values were expressed as mean ± SD. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 252
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Fig. 11. mRNA expression of osteogenic related genes (ALP, OPN, OCN, COL1, Runx2) of mMSCs mediated by SBG, Sr-SBG and SrCl2 with CSA (100 ng/ml) for 7 and 14 days. *Significant difference (P < 0.05) for SBG, Sr-SBG or SrCl2 group vs control group. #Significant difference (P < 0.05) for Sr-SBG or SrCl2 group vs SBG group. @Significant difference (P < 0.05) for SrCl2 group vs Sr-SBG group. Values were expressed as mean ± SD.
Also, there was identical Sr concentration in Sr-SBG and SrCl2 group, while no Sr ion could be detected in control and SBG group. It could also be seen that after incubated with mMSCs for 3 days, the ion concentrations of each group were changed a lot, with Si and Sr concentration declined largely in Sr-SBG group and P concentration increased in all groups. This phenomenon demonstrated that Si and Sr were entered into interior of cells and started to executive function. Previous studies demonstrated that Si and Sr ion could promote cell
lower than that in SBG group, demonstrating that only NFATc signaling pathway in SrCl2 group and only Wnt/β-catenin signaling pathway in SBG group was activated. To further explore the ion roles in the osteogenic differentiation enhancement effect of Sr-SBG, the ion concentrations in Control, SBG, Sr-SBG and SrCl2 groups were detected. The major differences among four groups were that Sr-SBG and SBG contained identical amount of Si, whereas the control medium and SrCl2 contained a small amount of Si.
Fig. 12. Effect of NFATc signaling pathway inactivation on the expression of Wnt/β-catenin and NFATc signaling pathways induced by SBG, Sr-SBG and SrCl2. RTqPCR analysis of Wnt/β-catenin signaling related genes (β-catenin, Axin2, DKK1, Wnt3A) and NFATc signaling related gene NFATc1 of mMSCs cultured with SBG, SrSBG and SrCl2 with CSA (100 ng/ml) for 7 days. *Significant difference (P < 0.05) for SBG, Sr-SBG or SrCl2 group vs control group. #Significant difference (P < 0.05) for Sr-SBG or SrCl2 group vs SBG group. @Significant difference (P < 0.05) for SrCl2 group vs Sr-SBG group. Values were expressed as mean ± SD. 253
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Scheme 1. Proposed mechanism of enhanced cell proliferation and osteogenic differentiation of mMSCs induced by Sr-SBG. Strontium ion released from Sr-SBG could mediate activation of NFATc1 signaling pathway, and subsequent increased Wnt3A mRNA expression in mMSCs, eventually activate β-catanin dependent Wnt/βcatanin signaling pathway. Si ion released from Sr-SBG could activate Wnt/β-catanin signaling pathway. Sr and Si released from Sr-SBG might synergistically promote cell proliferation and osteogenesis of mMSCs.
studied.
proliferation and osteogenesis of MSCs or mouse osteoblasts through Wnt/β-catenin signaling pathway or NFATc signaling pathway, respectively [29,43]. So it is reasonable to hypothesize that Si ion and Sr ion released from Sr-SBG may play a synergistic effect in the proliferation and osteogenic differentiation of mMSCs through NFATc signaling pathway and Wnt/β-catenin signaling pathway. Having shown that Sr-SBG ion dissolutions activate NFATc signaling pathway in mMSCs, we further studied the effect of Sr-SBG on proliferation and osteogenesis of mMSCs when NFATc signaling pathway was blocked. The results showed that the enhanced cell proliferation and osteogenic differentiation was partially attenuated in Sr-SBG group and greatly declined in SrCl2 group after being treated with NFATc signaling pathway inhibitor CSA, indicating that NFATc signaling pathway was involved in Sr-SBG-induced cell replication and differentiation in vitro, which was consistent with the recent finding that NFATc signaling pathway activation induced by Sr directed mouse osteoblast proliferation and osteogenic differentiation [43]. Moreover, the results also showed that Sr-SBG and SrCl2 could increase the mRNA expression of Wnt3A, which implicated that Sr promotes cell proliferation in part via activation of Wnt/β-catenin signaling pathway. And the upregulation of Wnt3A was blunted in Sr-SBG and SrCl2 group after blocking NFATc signaling pathway, which was a proof that the activation of NFATc signaling pathway induced by SrSBG or SrCl2 lead to the upregulation of Wnt3A expression, thus activating Wnt/β-catenin signaling pathway and osteogenic related gene expression. Taken together, we supposed that the molecular mechanism of enhanced proliferation and osteogenesis effect induced by Sr-SBG might attribute to the synergistic effect of Si ion induced Wnt/β-catenin signaling pathway and Sr ion induced NFATc signaling pathway and Wnt3A triggered Wnt/β-catenin signaling pathway (Scheme 1). However, we only used Sr-SBG ion dissolution to evaluate its enhanced proliferation and osteogenesis effects and underlying molecular mechanism on mMSCs in vitro, the detailed mechanism of Sr-SBG particles with different size and various Sr incorporation ratio on osteogenic differentiation of MSCs in vitro and in vivo required to be further
5. Conclusions In the present study, strontium-substituted sub-micron bioactive glasses (Sr-SBG) were prepared and its effect on proliferation and osteogenic differentiation of mMSCs were investigated in vitro by a comparative study among SBG, Sr-SBG and SrCl2. The results indicated that Sr-SBG ion dissolution had enhanced effect on proliferation and osteogenic differentiation than SBG ion dissolution or SrCl2 containing identical Sr with Sr-SBG. The underlying molecular mechanism of this enhancement induced by Sr-SBG was proved to be related to the activation of both NFATc and downstream Wnt/β-catenin signaling pathway, while SBG only activated Wnt/β-catenin signaling pathway and SrCl2 only activated NFATc signaling pathway. Furthermore, blocking of NFATc signaling pathway could partially abolish the enhanced effect of Sr-SBG than SBG, yet the osteogenesis induced by SrCl2 was also largely declined. All these results suggested that NFATc signaling pathway activation mediated by Sr and Wnt/β-catenin signaling pathway activated by Si had synergistic effect on the enhanced proliferation and osteogenic differentiation of mMSCs induced by Sr-SBG. These results may provide further understanding of the molecular mechanisms of application of Sr-substituted bioactive glass for bone tissue engineering.
Acknowledgements This study was financially supported by The Joint Funds of the National Natural Science Foundation of China (Grant No. U1501245), the National Natural Science Foundation of China (grant no. 51672088), the Fundamental Research Funds for the Central Universities (grant no. 2015ZP020, 2014ZB0018), and the Natural Science Foundation of Guangdong Province (No. 2015A030310034).
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Synergistic effect of strontium and silicon in strontium-substituted submicron bioactive glass for enhanced osteogenesis
T
Wen Zhanga,b,c,1, Deqiu Huangd,1, Fujian Zhaoa,c, Wendong Gaoa,c, Luyao Suna,c, Xian Lia,c, ⁎ Xiaofeng Chena,c, a
Department of Biomedical Engineering, School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, China. Institute for Brain Research and Rehabilitation, South China Normal University, Guangzhou, 510631, China c National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006, China d College of Biophotonics, South China Normal University, Guangzhou 510631, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Strontium-substituted sub-micron bioactive glasses Wnt/β-catenin NFATc Osteogenesis
Strontium-substituted sub-micron bioactive glasses (Sr-SBG) have been reported to have enhanced osteogenic differentiation capacity compared to sub-micron bioactive glasses (SBG) in our previous study. However, the underlying molecular mechanisms of such beneficial effect of Sr-SBG are still not fully understood. In this study, we synthesized Sr-SBG, studied the effects of Sr-SBG on proliferation and osteogenic differentiation of mouse mesenchymal stem cells (mMSCs), and identified the molecular mechanisms of the enhancement effect of Sr-SBG on mMSCs. The results demonstrated that Sr-SBG had more profound promotion effect on proliferation and osteogenic differentiation of mMSCs than SBG and SrCl2 group which containing identical Sr concentration with Sr-SBG group. RT-qPCR and western blot analysis showed that the mRNA expressions and protein expressions involved in NFATc and Wnt/β-catenin signaling pathways were all upregulated mediated by Sr-SBG, while only Wnt/β-catenin signaling pathway related genes upregulated in SBG group and only NFATc signaling pathway activated in SrCl2 group, suggesting that NFATc and Wnt/β-catenin signaling pathways played important roles in osteogenesis enhancement induced by Sr-SBG. To conform the above conclusion, cyclosporin A (CSA) was applied to inhibit NFATc signaling pathway. It was found that the enhanced osteogenic differentiation of mMSCs induced by Sr-SBG was partially abrogated and the activated Wnt/β-catenin signaling pathway was also inhibited in part. However, the effects of SBG on proliferation and osteogenesis of mMSCs were unimpaired, yet the effects of SrCl2 were greatly suppressed. Taken together, these results indicated that strontium activated NFATc signaling pathway and silicate activated Wnt/β-catenin signaling pathway might synergistically mediated the enhanced osteogenesis induced by Sr-SBG.
1. Introduction Multifunctional bioactive materials for the repair of bone defects caused by trauma, tumors, infections or genetic malformations, have attracted much attention in the past several years. In 1970s, Carlisle and Schwarz suggested that Silicon (Si) element might play an important role in skeletal development and bone repair [1,2]. Since then, many studies on silicate bioactive materials, including Si-substituted calcium phosphates [3–5], silicate-based bioceramics [6–9] and bioactive glasses [10–13], have been employed for bone regeneration. Bioactive glass is one of the typical bioactive materials possessing osteoinductive properties for its function of activating osteogenesis-related gene expression of bone marrow stromal cells (BMSCs) [14–16].
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1
Recently, micro-nano bioactive glass (MNBG) with controlled size and morphology was developed through the combination of sol-gel technique and template self-assembly method. Compared with conventional bioactive glasses, MNBG has superior apatite formation ability and biological activity due to their significantly increased surface area [17,18]. To achieve improved osteogenesis of bioactive glasses for bone repair, it was proved that the substitution of Sr ions into bioactive glasses could enhance the osteogenic differentiation of BMSCs [19–24]. Our previous work also demonstrated that Sr-substituted sub-micron bioactive glass (Sr-SBG) could improve osteogenesis than SBG [25]. Nevertheless, the underlying molecular mechanisms are still under investigation. To develop an ideal bioactive glass-based bone repair biomaterial, it is important to clarify the molecular mechanism through
Corresponding author. E-mail address: [email protected] (X. Chen). These authors contributed to the work equally and should be regarded as co-first authors.
https://doi.org/10.1016/j.msec.2018.04.012 Received 5 September 2017; Received in revised form 30 March 2018; Accepted 10 April 2018 Available online 11 April 2018 0928-4931/ © 2018 Published by Elsevier B.V.
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2.2. Synthesis of SBG and Sr-SBG and their ion dissolutions
which the biomaterial regulates osteogenic differentiation of BMSCs. During embryonic development, Wnt signaling plays a critical role in bone formation through regulating the determination of cell fate, and influencing the proliferation and differentiation of stem cells [26–28]. Previous researches have demonstrated that the bioactive Si ions released from BG and other silicate bioceramics could promote proliferation of BMSCs and induce osteogenic differentiation of BMSCs. The underlying mechanism of the enhanced proliferation and differentiation of BMSCs mediated by silicate was supposed to be related to the Wnt signaling pathway activation [7,29,30]. Sr is a trace element which could not only stimulate bone formation but also inhibit bone resorption during bone metabolism [31–33]. The mechanism was supposed to be related to the ability of Sr ions to promote the activity of alkaline phosphatase (ALP) and mRNA expression of osteogenesis-related gene of MSCs [34,35]. It was found that systemic administration of strontium ranelate could lead to increased bone mineral density (BMD) and decreased probability of bone fractures occurrence in osteoporotic patients [36–38]. Consequently, plenty of Sr-containing bone materials were developed due to their enhanced osteogenic capacity for rapid osseointegration [3,39–42]. Recently, Fromigue et al. showed that the Nuclear factor of activated T-cells (NFATc) signaling pathway and downstream canonical and non-canonical Wnt signaling pathways were involved in strontium ranelate induced proliferation and differentiation of murine osteoblasts [43]. NFATc are transcription factors that are highly phosphorylated and remain in the cytoplasm in unstimulated cells, and are involved in the differentiation of various cell types. When intracellular calcium level increases, calcineurin will be activated followed by dephosphorylation of NFATc1, leading to the translocation of NFATc1 to nucleus and binding to the promoter of target genes [44]. It was demonstrated that NFATc1 played an important role in both osteoblasts and osteoclasts, which is expressed during osteogenic differentiation and osteoclastogenesis. Based on the important role of Wnt/β-catenin signaling pathway mediated by silicate and NFATc signaling pathway induced by Sr element in osteogenesis, this study was conducted to investigate the effect of Wnt/β-catenin and NFATc signaling pathway on Sr-SBG stimulated osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (mMSCs) since that both Sr and Si ions are dominant components of Sr-SBG. We hypothesized that Sr-SBG could promote osteogenic differentiation of mMSCs through the synergistically effect of SBG activated Wnt/β-catenin pathway and Sr activated NFATc pathway. To test this hypothesis, we conducted three experimental groups: SBG group, Sr-SBG group and SrCl2 group, and investigated their effect on proliferation and osteogenesis of mMSCs and their influence on activation of the two signaling pathways above by RT-qPCR and western blot analysis. Meanwhile, NFATc signaling pathway was blocked by using cyclosporin A (CSA) to evaluate the osteogenic differentiation capacity of mMSCs induced by Sr-SBG. The results could further verify whether NFATc and Wnt/β-catenin signaling pathways played a synergistic effect on the enhanced osteogenesis of Sr-SBG or not. This study might clarify a key scientific problem before the application of Sr-SBG in clinic.
SBG and Sr-SBG were prepared according to the previous described method by the combination of sol-gel and template self-assembly technique using DDA as both catalyst and template agent [17,18,25]. The constituents ratio of SBG was SiO2: CaO: P2O5 = 60: 36: 4 (mol/ mol), and the constituents ratio of Sr-SBG was SiO2: CaO: SrO: P2O5 = 60: 30: 6: 4 (mol/mol). Typically, a given amount of ethanol was mixed with deionized water. Then, 4 g DDA was dissolved into the mixed solution above by stirring at 40 °C for 10 min. After that, two equal proportion of total 16 ml TEOS was added to the above mixture under vigorous stirring for 30 min interval. Finally, TEP and CN or SN was added under stirring in order with 30 min intervals. After another 3 h vigorous stirring, the resulting milky suspension was aged for 6 h and centrifuged at 4000 rpm to obtain white gel precipitate. The white precipitate was rinsed with ethanol and deionized water for three times each, and freeze-dried for 48 h. Eventually, SBG and Sr-SBG powders were obtained after sintering in air at 650 °C for 3 h. The ion dissolutions of SBG and Sr-SBG in Dulbecco modified Eagle medium (DMEM) was prepared as previously described. Briefly, SBG and Sr-SBG powders were autoclaved and then added into DMEM medium at a ratio of 1 mg/ ml. The suspension was maintained at 37 °C in a shaker with the speed set as 120 rpm for 48 h. The ion dissolutions of SBG and Sr-SBG were obtained by centrifugation and filtration using a 0.22 μm syringe filter. 10% fetal bovine serum and 1% (v/v) penicillin/streptomycin were added to the ion dissolutions for cell culture in the following experiments. 2.3. Characterization of SBG and Sr-SBG Scanning electron microscopy (SEM) (DSM 982-Gemini, Zeiss, Germany) was conducted to characterize the morphology of SBG and Sr-SBG. The particle size distribution was assayed on Zetasizer Nano ZS (Malvern Instruments, UK). X-ray photoelectron spectroscopy (Kratos Axis UlraDLD, UK) and Energy-dispersive X-ray spectroscopy were used to test the element composition of SBG and Sr-SBG. And the concentrations of ion dissolution of SBG and Sr-SBG were tested by inductively coupled plasma-atomic emission spectrometry (ICP-AES) (PS1000-AT, Leeman, USA). 2.4. Cell culture mMSCs was obtained from American Type Culture Collection (ATCC® CRL-12424™) and used in the subsequent experiments. mMSCs were cultured in DMEM with 10% fetal bovine serum and 1% (v/v) penicillin/streptomycin, under a 5% CO2 incubator at 37 °C. After the confluence reached to 90%, cells were passaged using 0.25% trypsinEDTA. The third to sixth passages of mMSCs were used for cell proliferation, osteogenic differentiation and activated/inactivation signaling pathway studies. Four groups (a. Control group with DMEM medium; b. SBG group with ion dissolution of SBG; c. Sr-SBG group with ion dissolution of Sr-SBG; d. SrCl2 group with equal Sr ion concentration with Sr-SBG group) were set up in the following experiments.
2. Materials and methods
2.5. Proliferation assay
2.1. Materials
Proliferation of mMSCs was carried out by a Cell Counting Kit-8 assay kit (CCK-8, Dojindo Laboratories, Kumamoto, Japan). mMSCs were seeded at a density of 3000 cells/well in a 96-well plate. After cells attached, the culture medium was replaced by ion dissolution of each group. The culture medium of each group was refreshed every 3 days. 1, 3 and 7 days later, cells were harvested for CCK-8 assay (five samples for each group). For each well, the medium was displaced by 100 μl DMEM containing 10% CCK-8 and maintained in the incubator at 37 °C for 1 h. The absorbance of each well was measured at 450 nm by
Ethanol (AR), tetraethyl orthosilicate (TEOS) (AR), calcium nitrate tetrahydrate (CN) (AR), and strontium nitrate (SN) (AR) were obtained from Guangzhou Chemical Reagent Factory, P.R. China. Triethylphophate (TEP) (AR) and Dodecylamine (DDA) were purchased from Aladdin (Shanghai, P.R. China). All chemical reagents were used directly without other treatment.
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Thermo Scientific Microplate Reader (Thermo 3001, USA).
Table 1 Primer sequences used for gene expression analysis.
2.6. Osteogenic differentiation of mMSCs
Genes
Primer sequences
To investigate the osteogenic differentiation of mMSCs, cells were seeded in 24-well plates (5 × 104 cells/well, three samples for each group). After attachment, the media were replaced by medium of each group supplemented with osteogenic inducing component (10 mM βglycerophosphate, 50 μM ascorbic acid, and 10 nM dexamethasone). Every 3 days, the culture medium was refreshed of each group. The osteogenic differentiation of mMSCs in each group was examined with alkaline phosphatase (ALP) staining and ALP activity assay, alizarin red-S staining and quantitative analysis, mRNA expressions of marker genes associated with osteogenesis.
ALP
Forward:5′-TGCCTACTTGTGTGGCGTGAA-3′ Reverse:5′-TCACCCGAGTGGTAGTCACAATG-3′ Forward:5′-GCAGTCTTCTGCGGCAGGCA-3′ Reverse:5′-CACCGGGAGGGAGGAGGCAA-3′ Forward:5′-AGCAGCTTGGCCCAGACCTA-3′ Reverse:5′-TAGCGCCGGAGTCTGTTCACTAC-3′ Forward:5′-ATGCCGCGACCTCAAGATG-3′ Reverse:5′-TGAGGCACAGACGGCTGAGTA-3′ Forward:5′-CACTGGCGGTGCAACAAGA-3′ Reverse:5′-TTTCATAACAGCGGAGGCATTTC-3′ Forward:5′-CTCCCACTCTTCCACCTTCG-3′ Reverse:5′-TTGCTGTAGCCGTATTCATT-3′ Forward:5′-GGTCCTCTGTGAACTTGC-3′ Reverse:5′-GTAATCCTGTGGCTTGTCC-3′ Forward:5′-GGTAACTCTGTCTTTCTAACCTTAAGCTC-3′ Reverse:5′-GTGATGACCCCAGCATGCACCAGTCACAG-3′ Forward:5′-ACCTCAAGTGCAAACTCTCACCCA-3′ Reverse:5′-AGCTGTTTCCGTGGATCTCACACT-3′ Forward:5′-AAGTTGAGGTTCCGCAGTCCAG-3′ Reverse:5′-CGCAAACAGGCAAAGGTCAGG-3′ Forward:5′-CCTCGGAGATGGTGGTAGA-3′ Reverse:5′-GTTAGGTTCGCAGAAGTTGG-3′
OPN OCN COLI Runx2 GAPDH β-catenin NFATc1
2.6.1. ALP staining and ALP activity assay of mMSCs When the incubation time of mMSCs with medium in each group supplemented with osteogenic inducing component reached to 7 and 14 days, ALP staining and ALP activity assay were performed using commercially available kits. BCIP/NBT ALP Color Development Kit (Beyotime) was used for the ALP staining of mMSCs cultured for 7 days. Typically, cells were fixed with 10% neutral formalin after rinsing with PBS for three times. Then, a mixture of BCIP and NBT in buffer solution were added to each well and stained for 30 min. After washing off the unbound dye, images were taken on an inverted microscope. To study the ALP activity mMSCs, cells incubated for 7 and 14 days were terminated and washed three times with pre-cold PBS, and then were lysed with lysis solution including 0.1% triton X-100 and 0.5 mM MgCl2 in 10 mM tris-HCl (pH 7.4) for 1 h on ice. After the lysates centrifuged at 16,000 g for 15 min at 4 °C, the supernatant were collected and stored in −20 °C. ALP activity assay were performed by using the ALP assay Kit (Beyotime) according to the instructions of the manufacturer. Total protein content was evaluated by the bicinchoninic acid protein assay kit (Thermo scientific). The results were presented by normalizing ALP activity to the total protein content.
Axin2 DKK1 Wnt3A
2.7. Activation of Wnt and NFATc signaling pathway For activation of Wnt and NFATc signaling pathway study, mMSCs were cultured in 6-well plate at a seeding density of 2 × 105 cells/well (three samples for each group) and incubated with media in each group for 7 days. Wnt/β-catenin signaling pathway related genes (β-catenin, Axin2, DKK1, Wnt3A) and NFATc signaling pathway related gene NFATc1 were investigated by RT-qPCR assay as described above, and primers used in this assay were listed in Table 1. Protein expressions of the two signaling pathway above (β-catenin and NFATc1) were analyzed by western blot. Generally, total protein of mMSCs were extracted by RIPA lysis buffer (Beyotime) with addition of PMSF and protease inhibitor cocktail (cOmplete ULTRA Tablets). After centrifugation, the supernatant of cell lysates were collected and stored in −80 °C or used immediately for western blot assay. SDS-PAGE gels were used to separate the total proteins, which were then transferred onto a PVDF membrane. The membranes were immersed in 5% skim milk for 1 h at room temperature to block nonspecific site, and subsequently incubated with a rabbit anti-β-catenin antibody (proteintech, 1:10000) and mouse anti-NFATc1 antibody (Santa Cruz Biotechnology, 1:1000) overnight at 4 °C. A rabbit monoclonal anti-GAPDH antibody (CST, 1:1000) was used as reference protein. After washed with TBST (Tris Buffered Saline, with Tween-20) for four times (5 min/time), the membranes were incubated with HRP-conjugated secondary antibodies (proteintech) for 1 h at room temperature. Eventually, the membranes were washed with TBST for four times (5 min/time) and the protein band were detected by using a chemiluminescent system (ECL-plus, Beyotime). AlphaEase FC software was used to analysis the quantitative densitometric of the images.
2.6.2. Alizarin red S staining and quantitative assay of mMSCs Mineralization of extracellular matrix evaluated by Alizarin red S staining was performed using mMSCs treated with medium in each group including osteogenic inducing supplements for 14 days. After washing with PBS for three times, cells were fixed with 4% paraformaldehyde for 20 min. After fixation, cells were rinsed three times with ultrapure water, and were stained with 0.5% Alizarin red S (pH 4.2) for 5 min at room temperature. Unbound Alizarin red S was removed by washing with ultrapure water until the cleaning water became colorless. The plate was air-dried at room temperature and images were captured by inverted microscopy. Alizarin Red S staining quantitative analysis was carried out by separate the combined stain from cells with 10% cetylpyridinium chloride for 1 h. The absorbance of eluent solution was measured on a microplate reader at 562 nm. 2.6.3. Osteogenesis-related gene expressions in mMSCs Osteogenesis-related gene (ALP, OPN, OCN, COL1, Runx2) expressions were evaluated by RT-qPCR after mMSCs were induced by medium in each group with osteogenic inducing supplement for 7 and 14 days. To extract the total RNA from mMSCs, cells incubated for 7 and 14 days were terminated and total RNA were isolated using HiPure Total RNA Micro Kit (Magen) according to the manufacturer's instruction. Then, Reverse Transcription Reagents Kit (Takara) was applied to synthesize the complementary DNA from 500 ng total RNA. The RTqPCR procedures were carried out on Quantstudio 6 Flex (Life technologies) using SYBR Green qPCR kit (Thermo Scientific). RT-qPCR primers used in this study were listed in Table 1. Target gene expressions were analyzed using the 2−ΔΔCTmethod and each gene was normalized with house-keeping gene GAPDH.
2.8. Effect of NFATc signaling pathway inactivation on osteogenic differentiation For inactivation of signaling pathway study, the calcineurin inhibitor cyclosporin A (CSA) was used to inhibit NFATc signaling pathway. mMSCs in each group was treated with respective medium containing osteogenic inducing component and CSA (100 ng/ml) for 7 and 14 days. Then the osteogenic differentiation of mMSCs, gene and protein expression of NFATc and β-catenin signaling pathways were measured as described in Sections 2.6 and 2.7.
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Fig. 1. SEM images of SBG (A) and Sr-SBG (B) and EDS analysis of SBG (C) and Sr-SBG (D).
2.9. Statistical analysis
3.2. Proliferation of mMSCs
All of the experiments were repeated three times and the data were presented as mean ± standard deviation (SD). One-way analysis of variance with LSD post-test was used to analyze the significant differences between groups. The values of P < 0.05 were considered as statistically significant. SPSS Statistics version 22 (IBM, USA) was used to carry out the statistical analysis.
To examine mMSCs proliferation affected by Sr-SBG, mMSCs were incubated with media in each group for 1, 3, and 7 days and CCK-8 assay was then conducted. As shown in Fig. 4, there was no significant difference in cell proliferation among each treatment groups on the first day. When the culture time increased to 3 days and 7 days, all of the three experiment groups revealed an enhanced cell proliferation ability. Statistically significant difference was detected between SBG group and Sr-SBG or between Sr-SBG group and SrCl2 group on day 7 (P < 0.05), demonstrating improved effect of Sr-SBG than SBG and SrCl2 in cell proliferation.
3. Results 3.1. The characterization of SBG and Sr-SBG SBG and Sr-SBG microspheres were synthesized successfully by the combination of sol-gel method and template self-assembly technique. The morphology of SBG and Sr-SBG particles were examined by SEM (Fig. 1A and B). It was shown that SBG and Sr-SBG had similar size, which was approximately 500 nm. Dynamic light scattering (DLS) analysis revealed that the average size of SBG and Sr-SBG was 447.3 nm and 567.8 nm, respectively (Fig. 2). The increased particle size of SrSBG than SBG could be ascribed to the larger ionic radius of Sr2+ than Ca2+, which may result in weaker field strength of Sr2+ than Ca2+, thus leading to much looser network structure and larger particle size of bioactive glass with the incorporation of Sr element. EDS (Fig. 1C and D) and XPS (Fig. 3) characterization was performed to examine the elemental compositions of SBG and Sr-SBG, which all demonstrated that the Sr ion was successfully incorporated into SBG. The detection of ion concentration in SBG and Sr-SBG dissolutions determined by ICPAES was shown in Table 2. The SBG and Sr-SBG ion dissolutions had similar Si and P ion concentration. To the contrary, the differences of Ca and Sr ion concentration between SBG and Sr-SBG dissolutions were distinct. There was a reduction of Ca ion concentration and increased Sr ion concentration of Sr-SBG compared with SBG.
3.3. Osteogenic differentiation of mMSCs Osteogenic differentiation assessment of mMSCs induced by medium from each group was performed by ALP activity assay, alizarin red S assay and RT-qPCR assay. mMSCs cultured for 7 and 14 days were used to detect the ALP activity. ALP staining results shown in Fig. 5A indicated that mMSCs in three experimental group were deeper stained than that in control group on day 7, while the Sr-SBG group showed the deepest stained ALP. Similarly, as plotted in Fig. 3C, the quantitative measurement of ALP activity of mMSCs showed that cells treated by SrSBG extracts expressed significant higher level of ALP than that with SBG and SrCl2 on both day 7 and 14. The formation of extracellular calcified deposits of mMSCs treated with medium in each groups for 14 days were examined by Alizarin Red S staining and quantitative analysis. As shown in Fig. 5B, more obvious mineralized nodules were generated in mMSCs after stimulated by Sr-SBG than that by SBG, SrCl2 and control medium. Quantitative analysis of alizarin red S showed similar trends with alizarin red S staining (Fig. 5D). RT-qPCR determination of mRNA expression of osteogenic marker genes was shown in Fig. 6. The mRNA expression of ALP, OPN, OCN, 248
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Fig. 2. Size distribution of SBG (A) and Sr-SBG (B).
COL I and Runx2 were significantly upregulated in all three experimental group on day 7 and 14, whereas there was an enhanced effect of mRNA expression of ALP on day 7, OPN on day 7 and 14, and OCN on day 14 in Sr-SBG group compared with SBG and SrCl2 group (P < 0.05).
Table 2 Si, Ca, P and Sr concentration of materials extracts.
DMEM SBG Sr-SBG SrCl2
3.4. Activation of Wnt and NFATc signaling pathway To investigate the activation of Wnt/β-catenin and NFATc signaling pathways during osteogenic differentiation of mMSCs mediated by SBG, Sr-SBG and SrCl2, cells were cultured with SBG, Sr-SBG and SrCl2 for 7 days. The effects of SBG, Sr-SBG and SrCl2 on activation of Wnt/βcatenin and NFATc signaling pathways determined by mRNA expression of β-catenin, Axin2, DKK1, Wnt3A, NFATc1 and western blot analysis of β-catenin and NFATc1 protein expression were shown in Figs.7 and 8, respectively. All the treatment of SBG, Sr-SBG and SrCl2 could increase the mRNA expression of β-catenin, Axin2, DKK1, Wnt3A and NFATc1 compared with control group. However, through comparison between any two experimental groups, there was an enhanced effect of Sr-SBG group in β-catenin, Axin2 and Wnt3A expression than SBG and SrCl2 group. There was also a significant increase in NFATc1 expression of Sr-SBG and SrCl2 group than SBG group. In addition, the upregulation of Wnt3A expression in SrCl2 group was more profound than that in SBG group. Western blot analysis indicated that the protein expression of NFATc1 in Sr-SBG and SrCl2 group were significantly higher than the other two groups, whereas there was no significant differences between SBG group and control group. The results also showed that significant differences in β-catenin expression were detected among three experimental groups, with Sr-SBG being the highest, and SrCl2 group being the lowest.
Si (mg/l)
Ca (mg/l)
P (mg/l)
Sr (mg/l)
0.02 31.04 32.35 0.02
51.85 44.29 41.02 57.76
27.93 12.32 11.43 27.61
0 0 6.518 6.525
Fig. 4. CCK-8 assay of mMSCs incubated with SBG, Sr-SBG and SrCl2. *Significant difference (P < 0.05) for SBG, Sr-SBG or SrCl2 group vs control group. #Significant difference (P < 0.05) for Sr-SBG or SrCl2 group vs SBG group. @Significant difference (P < 0.05) for SrCl2 group vs Sr-SBG group. Values were expressed as mean ± SD.
Fig. 3. XPS characterization of SBG (A) and Sr-SBG (B). 249
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Fig. 5. Osteogenic differentiation of mMSCs after treatment of SBG, Sr-SBG and SrCl2. ALP staining (A) and ALP activity (C) of mMSCs stimulated by SBG, Sr-SBG and SrCl2 with osteogenic supplements for 7 and 14 days. Alizarin red S staining (B) and Alizarin red S quantitative measurement (D) of mMSCs induced by SBG, Sr-SBG and SrCl2 with osteogenic supplements for 14 days. Scale bar = 100 μm. *Significant difference (P < 0.05) for SBG, Sr-SBG or SrCl2 group vs control group. #Significant difference (P < 0.05) for Sr-SBG or SrCl2 group vs SBG group. @Significant difference (P < 0.05) for SrCl2 group vs Sr-SBG group. Values were expressed as mean ± SD.
Fig. 6. mRNA expression of osteogenic related genes (ALP, OPN, OCN, COL1, Runx2) of mMSCs mediated by SBG, Sr-SBG and SrCl2 with osteogenic supplements for 7 and 14 days. *Significant difference (P < 0.05) for SBG, Sr-SBG or SrCl2 group vs control group. #Significant difference (P < 0.05) for Sr-SBG or SrCl2 group vs SBG group. @Significant difference (P < 0.05) for SrCl2 group vs Sr-SBG group. Values were expressed as mean ± SD. 250
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Fig. 7. Effect of SBG, Sr-SBG and SrCl2 on the expression of Wnt/β-catenin and NFATc signaling pathways. RT-qPCR analysis of Wnt/β-catenin signaling related genes (β-catenin, Axin2, DKK1, Wnt3A) and NFATc signaling related gene NFATc1 of mMSCs cultured with SBG, Sr-SBG and SrCl2 for 7 days. *Significant difference (P < 0.05) for SBG, Sr-SBG or SrCl2 group vs control group. #Significant difference (P < 0.05) for Sr-SBG or SrCl2 group vs SBG group. @Significant difference (P < 0.05) for SrCl2 group vs Sr-SBG group. Values were expressed as mean ± SD. Table 3 Si, Ca, P and Sr concentration of materials extracts cultured with mMSCs for 3 days.
DMEM SBG Sr-SBG SrCl2
Si (mg/l)
Ca (mg/l)
P (mg/l)
Sr (mg/l)
0.03 17.45 17.82 0.03
52.02 42.05 40.48 37.23
28.21 24.79 21.97 28.95
0 0 3.0245 3.275
3.5. Ion concentration changes after incubated with mMSCs The Si, Ca, P, and Sr ion concentration of media in four groups were determined by ICP-AES after their incubation with mMSCs for 3 days and was shown in Table 3. By contrasting ion concentration of each of the four groups in Table 2, there was a significant reduction of Si concentration in SBG and Sr-SBG group, and obvious declined Sr concentration in Sr-SBG and SrCl2 group. Also, there was a slight decrease in Ca concentration and increased P concentration in all four groups.
3.6. Effect of NFATc signaling pathway inactivation on proliferation and osteogenic differentiation of mMSCs To identify the effects of NFATc signaling pathway inactivation on proliferation and osteogenic differentiation of mMSCs induced by SBG, Sr-SBG and SrCl2, cells were treated with NFATc signaling pathway inhibitor CSA to block the pathway during incubated with SBG, Sr-SBG and SrCl2. As shown in Fig. 9, there was no significant difference between SBG and Sr-SBG group on cell proliferation, demonstrating the enhanced effect of cell replication induced by Sr-SBG were partially inhibited. However, the cell proliferation promotion effect of SrCl2 was greatly impaired. Fig. 10 A and C showed the ALP staining and ALP activity results of mMSCs on day 7 and 14 after NFATc pathway inhibited, and Fig. 10B and D displayed alizarin red S staining and quantitative assay results on day 14. Compared with the results of not being treated with CSA, the
Fig. 8. Protein expression of β-catenin and NFATc1 after 7 days' culture with SBG, Sr-SBG and SrCl2 by western blot analysis. *Significant difference (P < 0.05) for SBG, Sr-SBG or SrCl2 group vs control group. #Significant difference (P < 0.05) for Sr-SBG or SrCl2 group vs SBG group. @Significant difference (P < 0.05) for SrCl2 group vs Sr-SBG group. Values were expressed as mean ± SD.
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As shown in Fig. 11, after treatment of CSA to inactivate NFATc signaling pathway, there was little difference between SBG and Sr-SBG group in mRNA expression of ALP, OPN, OCN and COLIon day 7 and 14. However, there was significant difference between SrCl2 group and SBG or SrCl2 and Sr-SBG group in mRNA expression of OPN on day 14 and OCN, COLI on day 7 and 14. Furthermore, after NFATc pathway was inhibited, mRNA expression of β-catenin, Axin2, DKK1, Wnt3A, NFATc1 were carried out and the results were shown in Fig. 12. RT-qPCR analysis showed that the addition of NFATc signaling pathway inhibitor CSA in the culture medium inhibited the mRNA expression of NFATc1 and partially inhibited the upregulation of β-catenin in Sr-SBG group. There was no significant difference between Sr-SBG group and SBG group in β-catenin expression, while SrCl2 group showed significant downregulated β-catenin expression compared with SBG group and Sr-SBG group. 4. Discussion Fig. 9. Cell proliferation of mMSCs treated with SBG, Sr-SBG and SrCl2 with 100 ng/ml CSA for 1, 3 and 7 days. *Significant difference (P < 0.05) for SBG, Sr-SBG or SrCl2 group vs control group. #Significant difference (P < 0.05) for Sr-SBG or SrCl2 group vs SBG group. @Significant difference (P < 0.05) for SrCl2 group vs Sr-SBG group. Values were expressed as mean ± SD.
In this study, we investigated the stimulatory effects and related mechanisms of Sr-SBG on proliferation and osteogenic differentiation of mMSCs. As shown in the results, Sr-SBG had a more enhanced effect on cell proliferation and osteogenic differentiation of mMSCs compared with SBG and SrCl2. Furthermore, our study showed that Sr-SBG could induce the upregulation of Wnt/β-catenin signaling pathway and NFATc signaling pathway related genes. Though DKK1 as a negative regulator of Wnt/β-catenin signaling pathway was also upregulated in Sr-SBG group, the whole effect of β-catenin protein expression induced by Sr-SBG was upregulated. As shown in the results of gene and protein expression, the expression of NFATc1 was higher in SrCl2 group than that in SBG group, and β-catenin expression in SrCl2 group was much
increased ALP activity of mMSCs had declined in part in Sr-SBG group, with no significant difference between SBG and Sr-SBG group, and the increased ALP activity had a greatly reduction in SrCl2 group. Meanwhile, alizarin red S staining and quantitative assay conveyed identical mineralized nodules in Sr-SBG group with SBG group, while SrCl2 group showed much less mineralized nodules than Sr-SBG and SBG group.
Fig. 10. Effect of NFATc signaling pathway inactivation on osteogenic differentiation of mMSCs treated by SBG, Sr-SBG and SrCl2. ALP staining (A) and ALP activity (C) of mMSCs stimulated by SBG, Sr-SBG and SrCl2 with CSA (100 ng/ml) for 7 and 14 days. Alizarin red S staining (B) and Alizarin red S quantitative measurement (D) of mMSCs induced by SBG, Sr-SBG and SrCl2 with CSA (100 ng/ml) for 14 days. Scale bar = 100 μm. *Significant difference (P < 0.05) for SBG, Sr-SBG or SrCl2 group vs control group. #Significant difference (P < 0.05) for Sr-SBG or SrCl2 group vs SBG group. @Significant difference (P < 0.05) for SrCl2 group vs Sr-SBG group. Values were expressed as mean ± SD. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 252
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Fig. 11. mRNA expression of osteogenic related genes (ALP, OPN, OCN, COL1, Runx2) of mMSCs mediated by SBG, Sr-SBG and SrCl2 with CSA (100 ng/ml) for 7 and 14 days. *Significant difference (P < 0.05) for SBG, Sr-SBG or SrCl2 group vs control group. #Significant difference (P < 0.05) for Sr-SBG or SrCl2 group vs SBG group. @Significant difference (P < 0.05) for SrCl2 group vs Sr-SBG group. Values were expressed as mean ± SD.
Also, there was identical Sr concentration in Sr-SBG and SrCl2 group, while no Sr ion could be detected in control and SBG group. It could also be seen that after incubated with mMSCs for 3 days, the ion concentrations of each group were changed a lot, with Si and Sr concentration declined largely in Sr-SBG group and P concentration increased in all groups. This phenomenon demonstrated that Si and Sr were entered into interior of cells and started to executive function. Previous studies demonstrated that Si and Sr ion could promote cell
lower than that in SBG group, demonstrating that only NFATc signaling pathway in SrCl2 group and only Wnt/β-catenin signaling pathway in SBG group was activated. To further explore the ion roles in the osteogenic differentiation enhancement effect of Sr-SBG, the ion concentrations in Control, SBG, Sr-SBG and SrCl2 groups were detected. The major differences among four groups were that Sr-SBG and SBG contained identical amount of Si, whereas the control medium and SrCl2 contained a small amount of Si.
Fig. 12. Effect of NFATc signaling pathway inactivation on the expression of Wnt/β-catenin and NFATc signaling pathways induced by SBG, Sr-SBG and SrCl2. RTqPCR analysis of Wnt/β-catenin signaling related genes (β-catenin, Axin2, DKK1, Wnt3A) and NFATc signaling related gene NFATc1 of mMSCs cultured with SBG, SrSBG and SrCl2 with CSA (100 ng/ml) for 7 days. *Significant difference (P < 0.05) for SBG, Sr-SBG or SrCl2 group vs control group. #Significant difference (P < 0.05) for Sr-SBG or SrCl2 group vs SBG group. @Significant difference (P < 0.05) for SrCl2 group vs Sr-SBG group. Values were expressed as mean ± SD. 253
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Scheme 1. Proposed mechanism of enhanced cell proliferation and osteogenic differentiation of mMSCs induced by Sr-SBG. Strontium ion released from Sr-SBG could mediate activation of NFATc1 signaling pathway, and subsequent increased Wnt3A mRNA expression in mMSCs, eventually activate β-catanin dependent Wnt/βcatanin signaling pathway. Si ion released from Sr-SBG could activate Wnt/β-catanin signaling pathway. Sr and Si released from Sr-SBG might synergistically promote cell proliferation and osteogenesis of mMSCs.
studied.
proliferation and osteogenesis of MSCs or mouse osteoblasts through Wnt/β-catenin signaling pathway or NFATc signaling pathway, respectively [29,43]. So it is reasonable to hypothesize that Si ion and Sr ion released from Sr-SBG may play a synergistic effect in the proliferation and osteogenic differentiation of mMSCs through NFATc signaling pathway and Wnt/β-catenin signaling pathway. Having shown that Sr-SBG ion dissolutions activate NFATc signaling pathway in mMSCs, we further studied the effect of Sr-SBG on proliferation and osteogenesis of mMSCs when NFATc signaling pathway was blocked. The results showed that the enhanced cell proliferation and osteogenic differentiation was partially attenuated in Sr-SBG group and greatly declined in SrCl2 group after being treated with NFATc signaling pathway inhibitor CSA, indicating that NFATc signaling pathway was involved in Sr-SBG-induced cell replication and differentiation in vitro, which was consistent with the recent finding that NFATc signaling pathway activation induced by Sr directed mouse osteoblast proliferation and osteogenic differentiation [43]. Moreover, the results also showed that Sr-SBG and SrCl2 could increase the mRNA expression of Wnt3A, which implicated that Sr promotes cell proliferation in part via activation of Wnt/β-catenin signaling pathway. And the upregulation of Wnt3A was blunted in Sr-SBG and SrCl2 group after blocking NFATc signaling pathway, which was a proof that the activation of NFATc signaling pathway induced by SrSBG or SrCl2 lead to the upregulation of Wnt3A expression, thus activating Wnt/β-catenin signaling pathway and osteogenic related gene expression. Taken together, we supposed that the molecular mechanism of enhanced proliferation and osteogenesis effect induced by Sr-SBG might attribute to the synergistic effect of Si ion induced Wnt/β-catenin signaling pathway and Sr ion induced NFATc signaling pathway and Wnt3A triggered Wnt/β-catenin signaling pathway (Scheme 1). However, we only used Sr-SBG ion dissolution to evaluate its enhanced proliferation and osteogenesis effects and underlying molecular mechanism on mMSCs in vitro, the detailed mechanism of Sr-SBG particles with different size and various Sr incorporation ratio on osteogenic differentiation of MSCs in vitro and in vivo required to be further
5. Conclusions In the present study, strontium-substituted sub-micron bioactive glasses (Sr-SBG) were prepared and its effect on proliferation and osteogenic differentiation of mMSCs were investigated in vitro by a comparative study among SBG, Sr-SBG and SrCl2. The results indicated that Sr-SBG ion dissolution had enhanced effect on proliferation and osteogenic differentiation than SBG ion dissolution or SrCl2 containing identical Sr with Sr-SBG. The underlying molecular mechanism of this enhancement induced by Sr-SBG was proved to be related to the activation of both NFATc and downstream Wnt/β-catenin signaling pathway, while SBG only activated Wnt/β-catenin signaling pathway and SrCl2 only activated NFATc signaling pathway. Furthermore, blocking of NFATc signaling pathway could partially abolish the enhanced effect of Sr-SBG than SBG, yet the osteogenesis induced by SrCl2 was also largely declined. All these results suggested that NFATc signaling pathway activation mediated by Sr and Wnt/β-catenin signaling pathway activated by Si had synergistic effect on the enhanced proliferation and osteogenic differentiation of mMSCs induced by Sr-SBG. These results may provide further understanding of the molecular mechanisms of application of Sr-substituted bioactive glass for bone tissue engineering.
Acknowledgements This study was financially supported by The Joint Funds of the National Natural Science Foundation of China (Grant No. U1501245), the National Natural Science Foundation of China (grant no. 51672088), the Fundamental Research Funds for the Central Universities (grant no. 2015ZP020, 2014ZB0018), and the Natural Science Foundation of Guangdong Province (No. 2015A030310034).
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