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Biogenic silica nanostructures derived from Sorghum bicolor induced osteogenic differentiation through BSP, BMP-2 and BMP-4 gene expression
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Biogenic silica nanostructures derived from Sorghum bicolor induced osteogenic differentiation through BSP, BMP-2 and BMP-4 gene expression Saleh Ahmed Atiah Hamad Jaafari, Jegan Athinarayanan, Vaiyapuri Subbarayan Periasamy, Ali A. Alshatwi* Nanobiotechnology and Molecular Biology Research Laboratory, Department of Food Science and Nutrition, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia
A R T I C LE I N FO
A B S T R A C T
Keywords: Biogenic silica Sorghum bicolor Human mesenchymal stem cell Osteogenic differentiation
Development of scaffolds from naturally available biomaterials for bone tissue engineering is an interesting research area. Among the different scaffolds established for this purpose, biogenic silica nanostructure (BSN) serves as a promising naturally available inorganic and biocompatible material. A few studies have demonstrated the intrinsic biological activity of synthetic silica nanoparticles. The virtually infinite promising applications of these structures rely on their erratic physicochemical properties. We have derived BSNs from Sorghum bicolor seed head using a progressive approach. The intrinsic biological activities were analyzed using human mesenchymal stem cells (hMSCs) as an in vitro model with MTT assay and acridine orange/ethidium bromide staining. We also studied the role of BSNs in the osteogenic differentiation of hMSCs using alkaline phosphatase staining, alizarin red staining, and gene expression analysis. BSNs increased the formation of calcium nodules and stimulated alkaline phosphatase (ALP) activity. Significant changes and/or upregulation in the expression of osteogenic prominent markers such as ALP, bone morphogenetic protein-2 (BMP-2), BMP-4, bone sialoprotein (BSP), collagen-1 (Col-1), and Runt-related transcription factor 2 (RUNX2) genes were observed. Taken together, these results suggest that BSNs exhibited biocompatibility and induced osteogenic differentiation of hMSCs, indicative of their potential applications for bone tissue engineering.
1. Introduction Bone is a natural organic-inorganic hybrid architecture comprising hydroxyapatite (Ca5[PO4]3OH2) nanostructures embedded in an extracellular collagenous matrix. Bone is composed of 10–20% water, 20–30% organic molecules, and 60–70% inorganic materials [1]. Among natural nanocomposites, bone is an important nanocomposite that acts as a skeleton of the human body. Bone health is measured based on bone mineral density [2]. Osteoporosis is a key communal disease that induces weakness in bone strength by decreasing the bone mineral density, resulting in hip, spine, and wrist bone fractures [3,4]. Previous epidemic studies have indicated that 150 million people are affected by osteoporosis worldwide [5]. Postmenopausal women easily suffer from bone degenerative diseasesas do men with low bone mineral density at old age [6]. In particular, primary and secondary osteoporosis are triggered through estrogen deficiency and hormonal disorders [7]. Nutritional deficiency also promotes the development of osteoporotic disorders [8]. Pattern of lifestyle plays a vital role in the
incidence of osteoporosis, including low physical activity, lack of calcium intake, and vitamin D deficiency. Bone health issues have become a major concern worldwide [9]. Tissue engineering is a rapidly growing research filed that involves development of tissue or organ mimics under controlled environment for the repair or replacement of damaged tissues [10]. Development of biomimetic scaffolds with unique physical, chemical, and biological characteristics is the main goal of tissue engineering [11]. Bone tissue engineering requires scaffolds that are non-toxic and biodegradable and that induce cell adhesion, proliferation, and differentiation [12]. At present, nanostructures serve as a platform to improve the physical, chemical, and biological properties of scaffolds. Several studies have demonstrated the different types of nanostructures, and nanocomposite-based scaffolds have been exploited for bone tissue engineering. For instance, hydroxyapatite nanocomposites derived from cellulose, silk, chitosan, alginate, graphene, and carbon nanotubes have been applied for bone tissue engineering [12–16]. Aside from hydroxyapatite, silicate- and silica-based nanocomposites are also promising inorganic
⁎ Corresponding author at: Department of Food Science and Nutrition, College of Food Sciences and Agriculture, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia. E-mail addresses: [email protected], [email protected] (A.A. Alshatwi).
https://doi.org/10.1016/j.procbio.2019.12.015 Received 28 November 2019; Accepted 23 December 2019 1359-5113/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Saleh Ahmed Atiah Hamad Jaafari, et al., Process Biochemistry, https://doi.org/10.1016/j.procbio.2019.12.015
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Table 1 QuantiTect primer assays. Genes
Cat. No
Accession Number
Unicode
BGLAP (bone gamma-carboxyglutamate protein) NANOG (Homo sapiens Nanog homeobox) POUG5F1 (POU domain, class 5, transcription factor 1) BMP-2 (Bone morphogenetic protein 2) BMP-4 (Bone morphogenetic protein 4) RUNX2 (Runt-related transcription factor 2) BSP (Bone sialoprotein) ALP (Alkaline phosphatase) COL-I (Collagen I) GAPDH (Glyceraldehyde-3-phosphate dehydrogenase)
QT00232771 QT01025850 QT01664257 QT00012544 QT00997647 QT00020517 QT00093709 QT00012957 QT01947309 QT01192646
NM_199173 NM_024865 NM_203289 NM_001200 NM_001202 NM_001015051 NM_004967 NM_001127501 NM_077365 NM_002046
Hs.654541 Hs.635882 Hs.249184 Hs.73853 Hs.68879 Hs.535845 Hs.518726 Hs.75431 Hs.172928 Hs.544577
Fig. 1. FTIR spectra of (a) Sorghum bicolor seed head, (b) Sorghum bicolor seed head ash derived sodium silicate, and (c) biogenic silica nanostructures obtained from Sorghum bicolor biomass.
substances for scaffold generation for bone tissue engineering. Jurkic et al. demonstrated that silicon plays a vital role in skin aging, collagen synthesis, bone mineralization, and appearance of nails and hair [17]. Studies have reported that silicon deficiency promotes damage to hair, wrinkles in skin and fragility to nails and bones and induces cartilaginous tissue abnormalities [18–20]. Jugdaohsingh et al. revealed that the increase in silicon intake resulted in the improvement in bone mineral density of men and younger women [20]. Ha et al. demonstrated that 50 nm-sized spherical silica nanoparticles enhanced in vitro osteoblast differentiation and mineralization [21]. In addition, mesoporous silica/gelatin sandwich-type three-dimensional scaffold induced osteogenic differentiation of human osteoblast-like MG63 cells [22]. The study by Wiens et al. revealed that biosilica treatment triggers the expression of osteoprotegerin in association with the increase in cytokine level, which inhibits osteoclast differentiation through the elimination of receptor activator of nuclear factor kappa-B ligand (RANKL) function [7]. Recently, Ha et al. reported that silica nanoparticles osteogenic differentiation potential is varied based on their size, surface functionalization, and chemical composition of silica nanoparticles [23]. These studies increase our attention to analyze the role of biogenic silica effect on osteogenic differentiation. In addition, the role of Sorghum bicolor derived biogenic silica nanostructures in osteogenic differentiation is not explored well. Sorghum bicolor is an important cereal crop mainly cultivated in Asia and Africa [24]. The generation of agricultural residue from S. bicolor has steadily increased every year. Agricultural residues are enriched with various chemicals and materials. In particular, biosilicification of S. bicolor results in the generation of large quantity of silica; this process may act as a silica bioprecursor [25,26]. Unfortunately, only a small amount of agricultural residue is exploited for different purposes, while the remaining amount is not appropriately used [27]. These residues are burned in open air or dumped in open sites, thereby causing environmental problems [28]. Therefore, the proper utilization of S. bicolor agricultural residue is necessary to avoid environmental pollution
Fig. 2. X-ray diffractograms of (a) Sorghum bicolor seed head, (b) Sorghum bicolor seed head ash, (c) sodium silicate, and (d) biogenic silica nanostructures obtained from Sorghum bicolor biomass.
and increase its value. Balamurugan and Saravanan demonstrate that silica nanoparticles fabricated using sorghum seed head [29]. However, the biological behavior of the sorghum bicolor derived silica nanostructures is unknown. In the present study, we have valorized S. bicolor seed head with the production of a biocompatible biogenic silica nanostructure (BSN). Furthermore, we have assessed the BSN role on osteogenic differentiation using bone marrow human mesenchymal stem cells (hMSCs) as in vitro model due to their potential to differentiate into various lineages including chondrocytes, adipocytes, neurons, myocytes, osteoblasts, hepatocytes and cardiomyocytes. 2. Materials and methods 2.1. Materials S. bicolor seed heads were acquired from a farming field in Jazan, 2
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Fig. 3. Elemental composition of (a–b) sodium silicate and (c–d) biogenic silica nanostructures obtained from Sorghum bicolor biomass.
Fig. 4. Scanning electron microscopy images of (a–b) sodium silicate and (c–d) biogenic silica nanostructures obtained from Sorghum bicolor biomass.
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye, propidium iodide (PI), acridine orange (AO), and ethidium bromide (EB) were supplied by Thermo Fisher Scientific (Waltham, MA, USA).
Kingdom of Saudi Arabia. Sodium hydroxide (NaOH) and hydrochloric acid (HCl) were purchased from Merck (Kenilworth, NJ, USA). Fetal bovine serum (FBS), vitamin D, dexamethasone, β-glycerophosphate disodium salt hydrate, Dulbecco’s modified Eagle’s medium (DMEM), 33
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Fig. 5. Transmission electron microscopy images of (a–b) biogenic silica nanostructures obtained from Sorghum bicolor biomass.
Tokyo, Japan).
2.4. Cell culture The bone marrow hMSCs were cultured in DMEM supplemented with 10% FBS and antibiotics (streptomycin/penicillin) in T25 or T75 flasks. The cell culture flasks were incubated in a CO2 incubator with 5% CO2.
2.5. Analysis of cell viability The effects of BSN on the viability of hMSCs were evaluated after BSN treatment using MTT assay [30–33]. The BSNs were dispersed in cell culture media using sonication process. The MTT cell viability assay determines the mitochondrial function of live cells through the reduction of the MTT dye into a purple formazan. Around 10,000 hMSCs were plated in each well of 96-well plates and incubated at 37 °C in CO2 incubator for 24 h. Cells were exposed to different doses of BSNs (0, 12.5, 25, 50, 100 and 200 μg/mL) for 24 and 48 h. After incubation, the medium was carefully removed and each well was incubated with 20 μL MTT dye (concentration of 5 mg/mL). The plates were incubated in the dark at 37 °C for 4 h to allow for the formation of insoluble formazan crystals. The supernatant solution was carefully discarded and the intracellular formazan crystals were dissolved in 100 μL dimethyl sulfoxide (DMSO). The absorbance of the resulting solution was measured using a microplate reader at 570 nm (measurement filter) and 630 nm (reference filter) (GloMax, Promega, USA).
Fig. 6. Influence of sodium silicate and biogenic silica nanostructures on human mesenchymal stem cell viability. This data are obtained from four independent experiments (n = 4) and presented as the mean of standard deviation (SD).
2.2. Synthesis of silica nanostructures S. bicolor seed head powder was incubated in a muffle furnace at 600 °C for 2 h. The obtained ash (10 g) was treated with 2 M NaOH and mixed well. The mixture was transferred to a hydrothermal reactor and incubated at 120 °C for 3 h. To extract sodium silicate, the resultant slurry was filtered and washed with hot water. Sodium silicate was neutralized at pH 7.0 using 2.5 N sulfuric acid (H2SO4; added dropwise). After neutralization, the mixture was incubated overnight under constant stirring and the silica was allowed to precipitate. The precipitate was filtered, washed with distilled water, dried, and used for further studies.
Cell viability(%) = 2.3. Characterization Infrared spectra of S. bicolor seed head and derived BSNs were obtained with Nicolette Nexus 470 Fourier transformation infrared spectroscopy (FTIR). The crystalline nature of the sample was investigated with an X-ray diffractometer (PANalyticalX’Pert) with Cu Kα radiation =1.5406 Å. The elemental composition and surface morphology of the synthesized BSN were analyzed with field-emission scanning electron microscopy (SEM; JEOL, JSM-7600 F, Japan) associated with an energy dispersive X-ray spectrometer (EDX). The structural features were examined using transmission electron microscopy (TEM; 2010 F, JEOL,
Mean OD of treated cells × 100 Mean OD of untreated cells(control)
2.6. Analysis of cellular morphology The hMSCs were seeded at a density of 50,000 cells/well in six-well cell culture plates and incubated for 24 h. The cells were treated with different doses (0, 25, 50 and 100 μg/mL) of BSNs for 24 h. Following incubation, the medium was carefully removed and the cells were washed with phosphate-buffered saline (PBS), followed by observation of cellular morphology under an inverted microscope. 4
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Fig. 7. Effects of sodium silicate and biogenic silica nanostructures on cellular and nuclear morphologies in human mesenchymal stem cells. The cells treated with different concentration (0, 25, 50 and 100 μg/mL) of sodium silicate and biogenic silica nanostructures. The cellular morphology was examined under bright-field microscope. The elongated and healthy cells were observed in untreated and treated cells. To distinguish live and dead cells depend on their morphological behaviors, untreated and treated cells were stained with Acridine orange/Ethidium bromide and examined under fluorescent microscopy. The untreated and treated cells showed intact green nuclear architecture. This result intensely reveals that no apoptotic cells were found in the untreated and treated cells. (Scale bar =100 μm).
The cells untreated and treated with different doses (0, 25, 50 and 100 μg/mL) of BSNs were washed twice with PBS and stained with 50 μL of AO/EB dual stain. The cellular morphology was examined under a fluorescence microscope (Axiovert 40 CFL, Carl Zeiss, Germany).
synthesized BSNs (0, 25, 50 and 100 μg/mL) was analyzed using alkaline phosphatase staining. After 7 and 14 days of incubation, the wells were rinsed with PBS and the cells were fixed with acetone/citrate buffer. ALP substrate solution was added and the cells were treated with naphthol AS-TR phosphate in 0.1 M Tris buffer, pH 9.0 (ALP substrate) at room temperature for 1 h. After incubation, the cells were observed under a microscope.
2.8. Osteogenic differentiation
2.10. Alizarin red S staining
The hMSCs were seeded at a density of 5 × 104 cells/well in 24-well plates and cultured in normal DMEM. After 24 h, the cells cultured in the osteogenic induction medium (DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, 10 nM dexamethasone [Sigma-Aldrich], 10 mM β-glycerophosphate, 50 μg/mL l-ascorbic acid, and 10 nM calcitriol [1α, 25-dihydroxyvitamin D3; Sigma]). The medium was replaced with fresh medium every 3 days. To evaluate the osteogenic potential, cells were cultured in the presence of different doses of BSNs in osteogenic differentiation medium. The control cells were cultured in osteogenic differentiation medium without BSNs.
After incubation with BSNs (0, 25, 50 and 100 μg/mL), cells were washed with PBS and fixed with 4% paraformaldehyde. After fixation, the cells were washed with distilled water and stained with 2% alizarin red S at room temperature for 30 min. Excess stain was washed with water and the cells were examined under phase contrast microscope (Axiovert 40 CFL, Carl Zeiss, Germany).
2.7. AO/EB staining
2.11. Gene expression analysis Complementary DNA (cDNA) was obtained from the differentiating cells using Fastlane cell cDNA kit (Qiagen, Switzerland). The cDNA was quantified using Nanodrop spectrophotometer (NanoDrop 2000 spectrophotometer, Thermo Fisher Scientific). As per the manufacturer’s guidelines, relative quantification is used to detect an expression
2.9. Alkaline phosphatase assay Alkaline phosphatase activity of the cells treated with the 5
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Fig. 8. Alkaline phosphatase assay. Alkaline phosphatase activity of human mesenchymal stem cells after 7 and 14 days of cultivation with various concentrations (0, 25, 50 and 100 μg/mL) of sodium silicate and biogenic silica nanostructures. The color intensity increased by time- and dose-dependent manner in biogenic silica nanostructures. When compare with sodium silicate, biogenic silica nanostructures significantly increase the ALP activity.
ash contain silica in alkali medium. Previously, Balamurugan et al. fabricated silica nanoparticles from sorghum seed by dissolve the ash in alkali using conventional method [29].
changes in the mRNA level of target genes (Table 1) relative to a reference gene adopting real-time polymerase chain reaction (PCR; Applied Biosystem Real-Time PCR Detection System) using SYBR Green Master kit (Applied Biosystems, UK). Glyceraldehyde-3-phoshate dehydrogenase (GAPDH) was used as a reference gene to quantify the gene expression level using a previously described method [34]. The following formula were used to determine the expression ratio of reference gene to target gene and relative expression levels: ΔCt = Ct (target genes) − Ct (GAPDH) and ΔΔCt = ΔCt (Treated) − ΔCt (Control), respectively. The value was used to plot the expression of target genes using the method of 2−ΔΔCt.
SiO2 + 2NaOH → Na2SiO3 + H2O The transparent sodium silicate solution was neutralized to pH 7 using H2SO4 solution. Due to neutralization, sodium silicate was converted to silica nanostructures, which slowly precipitated out. Na2SiO3 + H2SO4 → SiO2 + H2O The physicochemical properties of the obtained silica nanostructures were analyzed. Fig. 1 shows the FTIR absorption spectra of BSNs derived from S. bicolor seed head. The seed head showed several absorption peaks around 3454, 2433, 1827, 1664, 1521, 1272, 1172, 1111, 1062, 901, and 724 cm−1, corresponding to water molecules and organic substances such as cellulose, hemicellulose, and lignin (Fig. 1a). The seed head ash-derived sodium silicate exhibited bands at 1446, 897, 862, and 689 cm−1 that were attributed to the SieO band of silicate. Fig. 1c shows the FTIR spectra of the BSNs derived from S. bicolor biomass. We observed two peaks around 1046 and 797 cm−1, assigned to the asymmetric stretching of SieOeSi and symmetric bending of SieO, respectively. The results clearly indicate that the BSN FTIR spectra were highly similar to those of commercial silica reported in earlier studies [35]. The X-ray diffraction (XRD) spectra of S. bicolor ash, sodium silicate, and silica are shown in Fig. 2. We observed several peaks for sodium silicate samples at 2θ value of 17.05, 27.8. 30.3, 31.9, 32.4, 33.4, 34.3, 35.4, 36.4, 38.1, 40.1, 43.9, 45.2, 46.7, 48.4, 51.18, 53.7, 55.2, 59.1, and 62.7°, corresponding to sodium silicate. The XRD pattern of BSN showed a broad peak around 22.7°, indicative of the amorphous nature
2.12. Statistical analysis All data were acquired from three independent experiments. The results are presented as the mean ± standard deviation using SPSS software (IBM Corporation, USA). For all comparisons, differences were considered statistically significant at p < 0.05. 3. Results and discussion 3.1. Synthesis of silica nanostructures Highly pure silica nanostructures were derived from S. bicolor seed husk using sequential process, including calcination, alkaline treatment, and acid precipitation. The S. bicolor seed head was calcinated at 600 °C, which resulted in the removal of organic substances and formation of ash. The ash contained a large quantity of silica and trace amounts of unwanted metals. The successive alkali treatment dissolved the silica in the ash to obtain sodium silicate, which was transparent and clear. In this study, we used hydrothermal approach to dissolve the 6
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Fig. 9. Alizarin red S staining. The human mesenchymal stem cells after 7 days of cultivation with various concentrations (0, 25, 50 and 100 μg/mL) of sodium silicate and biogenic silica nanostructures. The cells were stained with Alizarin Red S. Red color accumulation indicates calcium deposits in the cells. We observed calcium deposits in sodium silicate and biogenic silica nanostructures treated cells. The high-level calcium deposits observed in 100 μg/mL biogenic silica nanostructures treated cells.
sodium silicate. In contrast, BSNs showed uniform clusters of sphereshaped particles that were 30–80 nm in size. Fig. 5 shows TEM images of BSNs (Fig. 5a–b). The derived BSNs had spherical agglomerated shape with a dimeter of 30–80 nm. BSNs showed clusters of spherical particles. Thus, the results of TEM were in line with those of SEM.
of BSNs (Fig. 2c). The absence of any peaks related to impurities such as alkaline earth metals and sodium sulfate confirmed the high purity of BSNs. The elemental composition analysis of sodium silicate and BSN is shown in Fig. 3. The energy dispersive spectroscopy (EDS) spectra of sodium silicate showed peaks of Na, Si, O, and C. This result indicates the absence of any undesirable elemental impurities in sodium silicate (Fig. 3a). In BSNs, we observed the peaks corresponding to C, Si, and O, with no other metal impurities. The metal impurities were eliminated during the sequential process. The results of FTIR, EDS, and XRD analyses provide a strong evidence of the high purity of BSNs. SEM and TEM analyses were performed to evaluate the morphologies of sodium silicate and BSNs. Fig. 4 shows the SEM images of sodium silicate and BSNs that suggests flaky irregular morphology for
3.2. Biocompatibility assessment The biocompatibility of the synthesized BSN and its influence on osteogenesis were assessed in hMSCs as an in vitro model. The effects of BSN on the viability of hMSCs were determined with MTT assay. hMSCs were cultured in the presence of BSNs for 24 and 48 h, and the biocompatibility of BSN was compared with that of the ash-derived sodium silicate. As shown in Fig. 6, the exposure to BSN had no significant 7
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Fig. 10. Quantitative RT-PCR analysis of osteogenic markers in human mesenchymal stem cells. Comparison of the change in the expression level, expressed as fold change (i.e., the ratio of the target gene to the reference gene, GAPDH), in hMSCs after exposure to sodium silicate and biogenic silica nanostructures for 7 and 14 days. Data represent the mean ± SD of three determinations, each performed in triplicates (p < 0.05).
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deposition and ALP activity in hMSCs and found that BSN stimulated hMSC differentiation into osteoblasts. Earlier studies have demonstrated the role that silicon plays in bone generation and bone mineral density [17]. A few studies have suggested the potential role of silicon in the enhancement of osteogenic differentiation [23,36–39]. Gaharwar et al. showed that cytocompatible silicate nanoplatelets stimulated the osteogenic differentiation of cells through the upregulation in the expression of ALP, RUNX2, osteocalcin, and osteopontinproteins [40]. Mihaila et al. reported that silicate nanostructures stimulated the osteogenic differentiation of SSEA-4 subpopulation of human adiposederived stem cells by increasing the expression of osteogenic early and late markers associated with high ALP activity [41]. Ha et al. recently demonstrated the silica nanoparticle-induced osteoblast differentiation based on their sizes (50–450 nm), surface functional groups (CO2H, mNH2, OH, NR4+), and chemical composition [23]. The soluble form of silica promotes ALP activity and increases Col-1 synthesis in human osteoblast-like cells [42]. Some studies have demonstrated the role and mechanism of silica in osteogenesis. For instance, Shie et al. suggested that silicon improves Col-1synthesis and activates extracellular signalregulated kinase (ERK) pathway in MG63 cells [43]. Furthermore, silica nanoparticles were shown to induce osteoblast formation by increasing calcium deposition, stimulating ALP activity, and synthesizing BSP and Col-1 [21,36,44]. The present study demonstrates that S. bicolor-derived BSNs were internalized by hMSCs and regulated cell proliferation and metabolism. S. bicolor-derived BSNs stimulated the osteogenic differentiation of hMSCs by increasing the expression level of ALP, enhancing calcium deposition, and upregulating the expression of osteogenic marker genes. Thus, S. bicolor-derived BSNs may serve as promising agents to repair and regenerate the bone tissue.
effect on the number of viable hMSCs as compared to control cells. Sodium silicate was biocompatible with hMSCs. BSN at doses as high as 200 μg/mL showed 90% hMSC viability in 24 h. Thus, BSNs are practically harmless at high concentrations after exposure for 48 h. These results revealed the absence of any toxicity of SNs and sodium silicate. The cytocompatibility was more distinct for BSNs as compared with sodium silicate. We performed cellular and nuclear morphological analyses of cells following exposure to sodium silicate and BSNs to understand the cytological features. hMSCs were exposed to three different doses of BSNs and sodium silicate for 24 h. As shown in Fig. 7, the cells exposed to BSNs showed no change in morphology and adherence to plate and grew well. The nuclear architecture of BSN-treated hMSCs was intact and healthy without any alterations in the nuclear structure. These results are very similar to those obtained with control cells. Overall microscopic studies revealed that sodium silicate and BSNs are nontoxic and may be useful for biomedical applications. 3.3. Osteogenic differentiation We analyzed the effects of the biocompatible BSNs on the osteoblastic differentiation of hMSCs. To analyze whether BSNs could promote osteogenic differentiation of hMSCs, we evaluated the activity of ALP in hMSCs after osteogenic induction and sodium silicate and BSN exposure. ALP is an important phenotypic marker of osteoblasts owing to its presence in their cellular membranes. The cells were treated with BSNs for 7 and 14 days in osteoblastic induction medium. After 7 days, we observed higher intensity of ALP staining in BSN-treated cells than in control cells (Fig. 8). The ALP activity was higher in BSN-treated cells than sodium silicate-treated cells. These observations suggest that BSNs enhanced the osteogenic differentiation of hMSCs. However, the activity of ALP considerably increased with an increase in BSN dose (25 and 100 μg/mL). After osteogenic induction, hMSC mineralization was assessed using alizarin red S staining (Fig. 9). Alizarin red stain reacts with calcium to form a chelate and produces an orange red color. The cells treated with sodium silicate and BSNs showed different levels of matrix mineralization. Osteogenesis-induced cells showed nodule formation. In particular, BSN-treated cells had more calcium nodules after 7 days of incubation as compared with sodium silicate-treated cells. These results suggest that the treatment with high concentrations of BSNs showed around high-level increase in nodule formation as compared with the control and sodium silicate-treated cells. Our study results suggest that BSNs may promote bone-like nodule formation in hMSCs after osteoinduction.
4. Conclusion BSNs with 30–70 nm diameter were derived from S. bicolor biomass. The biocompatibility of BSNs was studied using in vitro assays, including MTT assay and AO/EB staining. These results suggest that the synthesized BSNs were non-toxic and may be used for biomedical applications. We analyzed the role of BSNs in osteogenesis of hMSCs. BSNs induced calcium deposition and increased ALP activity. The expression levels of osteogenic markers ALP, BSP, BMP-2, BMP-4, Runx2, and Col-1 were upregulated by BSN treatment at high concentrations. Our study results clearly indicate that BSNs trigger the osteogenic differentiation of hMSCs in a dose- and time-dependent manner. BSNs may stimulate osteoblast formation and BSN-based scaffolds may be used for bone tissue engineering. Declaration of Competing Interest
3.4. Osteogenic gene expression None. The expression of osteogenic differentiation markers including ALP, bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-4 (BMP-4), bone sialoprotein (BSP), collagen-1 (Col-1), Runt-related transcription factor 2 (RUNX2), bone gamma-carboxyglutamate protein (BGLAP), NANOG, and POUG5F1 was assessed after osteogenic induction following sodium silicate and BSN treatment at 7 and 14 days (Fig. 10). The gene expression level of ALP, BMP-2, BMP-4, Col-1, RUNX2, and BSP increased with an increase in the incubation time and dose of BSNs. ALP gene expression pattern suggests that BSN treatment showed a 3.5-fold increase in ALP expression as compared with control cells. The expression of BMP-2 was eight-fold higher in BSN-treated cells than in control cells. RUNX2 expression pattern (2.5-fold increase) was similar to that of BMP-2 in the cells treated with BSNs for 7 and 14 days. After 14 days, Col-1 expression level was significantly higher (P < 0.05) in the BSN-treated cells than sodium silicate-treated and control cells. We observed a tremendous upregulation in the expression of BMP-4 after 7 and 14 days in BSN-treated cells as compared with control cells. We investigated the influence of BSNs on calcium
Acknowledgements The authors would like to thank Deanship of scientific research for funding and supporting this research through the initiative of DSR Graduate Students Research Support (GSR). Also, we thank the RSSU at King Saud University for their technical support. References [1] C. Liu, Collagen–hydroxyapatite composite scaffolds for tissue engineering, Hydroxyapatite (HAp) for Biomedical Applications, (2015), pp. 211–234, https:// doi.org/10.1016/B978-1-78242-033-0.00010-9. [2] P. Lips, Epidemiology and predictors of fractures associated with osteoporosis, Am. J. Med. 103 (1997) 3S–11S, https://doi.org/10.1016/S0002-9343(97)90021-8. [3] R. Bernabei, A.M. Martone, E. Ortolani, F. Landi, E. Marzetti, Screening, diagnosis and treatment of osteoporosis: a brief review, Clin. Cases Miner. Bone Metab. 11 (2014) 201–207. [4] M. Gass, B. Dawson-Hughes, Preventing osteoporosis-related fractures: an overview, Am. J. Med. 119 (2006) S3–S11, https://doi.org/10.1016/j.amjmed.2005.12.
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Biogenic silica nanostructures derived from Sorghum bicolor induced osteogenic differentiation through BSP, BMP-2 and BMP-4 gene expression Saleh Ahmed Atiah Hamad Jaafari, Jegan Athinarayanan, Vaiyapuri Subbarayan Periasamy, Ali A. Alshatwi* Nanobiotechnology and Molecular Biology Research Laboratory, Department of Food Science and Nutrition, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia
A R T I C LE I N FO
A B S T R A C T
Keywords: Biogenic silica Sorghum bicolor Human mesenchymal stem cell Osteogenic differentiation
Development of scaffolds from naturally available biomaterials for bone tissue engineering is an interesting research area. Among the different scaffolds established for this purpose, biogenic silica nanostructure (BSN) serves as a promising naturally available inorganic and biocompatible material. A few studies have demonstrated the intrinsic biological activity of synthetic silica nanoparticles. The virtually infinite promising applications of these structures rely on their erratic physicochemical properties. We have derived BSNs from Sorghum bicolor seed head using a progressive approach. The intrinsic biological activities were analyzed using human mesenchymal stem cells (hMSCs) as an in vitro model with MTT assay and acridine orange/ethidium bromide staining. We also studied the role of BSNs in the osteogenic differentiation of hMSCs using alkaline phosphatase staining, alizarin red staining, and gene expression analysis. BSNs increased the formation of calcium nodules and stimulated alkaline phosphatase (ALP) activity. Significant changes and/or upregulation in the expression of osteogenic prominent markers such as ALP, bone morphogenetic protein-2 (BMP-2), BMP-4, bone sialoprotein (BSP), collagen-1 (Col-1), and Runt-related transcription factor 2 (RUNX2) genes were observed. Taken together, these results suggest that BSNs exhibited biocompatibility and induced osteogenic differentiation of hMSCs, indicative of their potential applications for bone tissue engineering.
1. Introduction Bone is a natural organic-inorganic hybrid architecture comprising hydroxyapatite (Ca5[PO4]3OH2) nanostructures embedded in an extracellular collagenous matrix. Bone is composed of 10–20% water, 20–30% organic molecules, and 60–70% inorganic materials [1]. Among natural nanocomposites, bone is an important nanocomposite that acts as a skeleton of the human body. Bone health is measured based on bone mineral density [2]. Osteoporosis is a key communal disease that induces weakness in bone strength by decreasing the bone mineral density, resulting in hip, spine, and wrist bone fractures [3,4]. Previous epidemic studies have indicated that 150 million people are affected by osteoporosis worldwide [5]. Postmenopausal women easily suffer from bone degenerative diseasesas do men with low bone mineral density at old age [6]. In particular, primary and secondary osteoporosis are triggered through estrogen deficiency and hormonal disorders [7]. Nutritional deficiency also promotes the development of osteoporotic disorders [8]. Pattern of lifestyle plays a vital role in the
incidence of osteoporosis, including low physical activity, lack of calcium intake, and vitamin D deficiency. Bone health issues have become a major concern worldwide [9]. Tissue engineering is a rapidly growing research filed that involves development of tissue or organ mimics under controlled environment for the repair or replacement of damaged tissues [10]. Development of biomimetic scaffolds with unique physical, chemical, and biological characteristics is the main goal of tissue engineering [11]. Bone tissue engineering requires scaffolds that are non-toxic and biodegradable and that induce cell adhesion, proliferation, and differentiation [12]. At present, nanostructures serve as a platform to improve the physical, chemical, and biological properties of scaffolds. Several studies have demonstrated the different types of nanostructures, and nanocomposite-based scaffolds have been exploited for bone tissue engineering. For instance, hydroxyapatite nanocomposites derived from cellulose, silk, chitosan, alginate, graphene, and carbon nanotubes have been applied for bone tissue engineering [12–16]. Aside from hydroxyapatite, silicate- and silica-based nanocomposites are also promising inorganic
⁎ Corresponding author at: Department of Food Science and Nutrition, College of Food Sciences and Agriculture, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia. E-mail addresses: [email protected], [email protected] (A.A. Alshatwi).
https://doi.org/10.1016/j.procbio.2019.12.015 Received 28 November 2019; Accepted 23 December 2019 1359-5113/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Saleh Ahmed Atiah Hamad Jaafari, et al., Process Biochemistry, https://doi.org/10.1016/j.procbio.2019.12.015
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Table 1 QuantiTect primer assays. Genes
Cat. No
Accession Number
Unicode
BGLAP (bone gamma-carboxyglutamate protein) NANOG (Homo sapiens Nanog homeobox) POUG5F1 (POU domain, class 5, transcription factor 1) BMP-2 (Bone morphogenetic protein 2) BMP-4 (Bone morphogenetic protein 4) RUNX2 (Runt-related transcription factor 2) BSP (Bone sialoprotein) ALP (Alkaline phosphatase) COL-I (Collagen I) GAPDH (Glyceraldehyde-3-phosphate dehydrogenase)
QT00232771 QT01025850 QT01664257 QT00012544 QT00997647 QT00020517 QT00093709 QT00012957 QT01947309 QT01192646
NM_199173 NM_024865 NM_203289 NM_001200 NM_001202 NM_001015051 NM_004967 NM_001127501 NM_077365 NM_002046
Hs.654541 Hs.635882 Hs.249184 Hs.73853 Hs.68879 Hs.535845 Hs.518726 Hs.75431 Hs.172928 Hs.544577
Fig. 1. FTIR spectra of (a) Sorghum bicolor seed head, (b) Sorghum bicolor seed head ash derived sodium silicate, and (c) biogenic silica nanostructures obtained from Sorghum bicolor biomass.
substances for scaffold generation for bone tissue engineering. Jurkic et al. demonstrated that silicon plays a vital role in skin aging, collagen synthesis, bone mineralization, and appearance of nails and hair [17]. Studies have reported that silicon deficiency promotes damage to hair, wrinkles in skin and fragility to nails and bones and induces cartilaginous tissue abnormalities [18–20]. Jugdaohsingh et al. revealed that the increase in silicon intake resulted in the improvement in bone mineral density of men and younger women [20]. Ha et al. demonstrated that 50 nm-sized spherical silica nanoparticles enhanced in vitro osteoblast differentiation and mineralization [21]. In addition, mesoporous silica/gelatin sandwich-type three-dimensional scaffold induced osteogenic differentiation of human osteoblast-like MG63 cells [22]. The study by Wiens et al. revealed that biosilica treatment triggers the expression of osteoprotegerin in association with the increase in cytokine level, which inhibits osteoclast differentiation through the elimination of receptor activator of nuclear factor kappa-B ligand (RANKL) function [7]. Recently, Ha et al. reported that silica nanoparticles osteogenic differentiation potential is varied based on their size, surface functionalization, and chemical composition of silica nanoparticles [23]. These studies increase our attention to analyze the role of biogenic silica effect on osteogenic differentiation. In addition, the role of Sorghum bicolor derived biogenic silica nanostructures in osteogenic differentiation is not explored well. Sorghum bicolor is an important cereal crop mainly cultivated in Asia and Africa [24]. The generation of agricultural residue from S. bicolor has steadily increased every year. Agricultural residues are enriched with various chemicals and materials. In particular, biosilicification of S. bicolor results in the generation of large quantity of silica; this process may act as a silica bioprecursor [25,26]. Unfortunately, only a small amount of agricultural residue is exploited for different purposes, while the remaining amount is not appropriately used [27]. These residues are burned in open air or dumped in open sites, thereby causing environmental problems [28]. Therefore, the proper utilization of S. bicolor agricultural residue is necessary to avoid environmental pollution
Fig. 2. X-ray diffractograms of (a) Sorghum bicolor seed head, (b) Sorghum bicolor seed head ash, (c) sodium silicate, and (d) biogenic silica nanostructures obtained from Sorghum bicolor biomass.
and increase its value. Balamurugan and Saravanan demonstrate that silica nanoparticles fabricated using sorghum seed head [29]. However, the biological behavior of the sorghum bicolor derived silica nanostructures is unknown. In the present study, we have valorized S. bicolor seed head with the production of a biocompatible biogenic silica nanostructure (BSN). Furthermore, we have assessed the BSN role on osteogenic differentiation using bone marrow human mesenchymal stem cells (hMSCs) as in vitro model due to their potential to differentiate into various lineages including chondrocytes, adipocytes, neurons, myocytes, osteoblasts, hepatocytes and cardiomyocytes. 2. Materials and methods 2.1. Materials S. bicolor seed heads were acquired from a farming field in Jazan, 2
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Fig. 3. Elemental composition of (a–b) sodium silicate and (c–d) biogenic silica nanostructures obtained from Sorghum bicolor biomass.
Fig. 4. Scanning electron microscopy images of (a–b) sodium silicate and (c–d) biogenic silica nanostructures obtained from Sorghum bicolor biomass.
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye, propidium iodide (PI), acridine orange (AO), and ethidium bromide (EB) were supplied by Thermo Fisher Scientific (Waltham, MA, USA).
Kingdom of Saudi Arabia. Sodium hydroxide (NaOH) and hydrochloric acid (HCl) were purchased from Merck (Kenilworth, NJ, USA). Fetal bovine serum (FBS), vitamin D, dexamethasone, β-glycerophosphate disodium salt hydrate, Dulbecco’s modified Eagle’s medium (DMEM), 33
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Fig. 5. Transmission electron microscopy images of (a–b) biogenic silica nanostructures obtained from Sorghum bicolor biomass.
Tokyo, Japan).
2.4. Cell culture The bone marrow hMSCs were cultured in DMEM supplemented with 10% FBS and antibiotics (streptomycin/penicillin) in T25 or T75 flasks. The cell culture flasks were incubated in a CO2 incubator with 5% CO2.
2.5. Analysis of cell viability The effects of BSN on the viability of hMSCs were evaluated after BSN treatment using MTT assay [30–33]. The BSNs were dispersed in cell culture media using sonication process. The MTT cell viability assay determines the mitochondrial function of live cells through the reduction of the MTT dye into a purple formazan. Around 10,000 hMSCs were plated in each well of 96-well plates and incubated at 37 °C in CO2 incubator for 24 h. Cells were exposed to different doses of BSNs (0, 12.5, 25, 50, 100 and 200 μg/mL) for 24 and 48 h. After incubation, the medium was carefully removed and each well was incubated with 20 μL MTT dye (concentration of 5 mg/mL). The plates were incubated in the dark at 37 °C for 4 h to allow for the formation of insoluble formazan crystals. The supernatant solution was carefully discarded and the intracellular formazan crystals were dissolved in 100 μL dimethyl sulfoxide (DMSO). The absorbance of the resulting solution was measured using a microplate reader at 570 nm (measurement filter) and 630 nm (reference filter) (GloMax, Promega, USA).
Fig. 6. Influence of sodium silicate and biogenic silica nanostructures on human mesenchymal stem cell viability. This data are obtained from four independent experiments (n = 4) and presented as the mean of standard deviation (SD).
2.2. Synthesis of silica nanostructures S. bicolor seed head powder was incubated in a muffle furnace at 600 °C for 2 h. The obtained ash (10 g) was treated with 2 M NaOH and mixed well. The mixture was transferred to a hydrothermal reactor and incubated at 120 °C for 3 h. To extract sodium silicate, the resultant slurry was filtered and washed with hot water. Sodium silicate was neutralized at pH 7.0 using 2.5 N sulfuric acid (H2SO4; added dropwise). After neutralization, the mixture was incubated overnight under constant stirring and the silica was allowed to precipitate. The precipitate was filtered, washed with distilled water, dried, and used for further studies.
Cell viability(%) = 2.3. Characterization Infrared spectra of S. bicolor seed head and derived BSNs were obtained with Nicolette Nexus 470 Fourier transformation infrared spectroscopy (FTIR). The crystalline nature of the sample was investigated with an X-ray diffractometer (PANalyticalX’Pert) with Cu Kα radiation =1.5406 Å. The elemental composition and surface morphology of the synthesized BSN were analyzed with field-emission scanning electron microscopy (SEM; JEOL, JSM-7600 F, Japan) associated with an energy dispersive X-ray spectrometer (EDX). The structural features were examined using transmission electron microscopy (TEM; 2010 F, JEOL,
Mean OD of treated cells × 100 Mean OD of untreated cells(control)
2.6. Analysis of cellular morphology The hMSCs were seeded at a density of 50,000 cells/well in six-well cell culture plates and incubated for 24 h. The cells were treated with different doses (0, 25, 50 and 100 μg/mL) of BSNs for 24 h. Following incubation, the medium was carefully removed and the cells were washed with phosphate-buffered saline (PBS), followed by observation of cellular morphology under an inverted microscope. 4
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Fig. 7. Effects of sodium silicate and biogenic silica nanostructures on cellular and nuclear morphologies in human mesenchymal stem cells. The cells treated with different concentration (0, 25, 50 and 100 μg/mL) of sodium silicate and biogenic silica nanostructures. The cellular morphology was examined under bright-field microscope. The elongated and healthy cells were observed in untreated and treated cells. To distinguish live and dead cells depend on their morphological behaviors, untreated and treated cells were stained with Acridine orange/Ethidium bromide and examined under fluorescent microscopy. The untreated and treated cells showed intact green nuclear architecture. This result intensely reveals that no apoptotic cells were found in the untreated and treated cells. (Scale bar =100 μm).
The cells untreated and treated with different doses (0, 25, 50 and 100 μg/mL) of BSNs were washed twice with PBS and stained with 50 μL of AO/EB dual stain. The cellular morphology was examined under a fluorescence microscope (Axiovert 40 CFL, Carl Zeiss, Germany).
synthesized BSNs (0, 25, 50 and 100 μg/mL) was analyzed using alkaline phosphatase staining. After 7 and 14 days of incubation, the wells were rinsed with PBS and the cells were fixed with acetone/citrate buffer. ALP substrate solution was added and the cells were treated with naphthol AS-TR phosphate in 0.1 M Tris buffer, pH 9.0 (ALP substrate) at room temperature for 1 h. After incubation, the cells were observed under a microscope.
2.8. Osteogenic differentiation
2.10. Alizarin red S staining
The hMSCs were seeded at a density of 5 × 104 cells/well in 24-well plates and cultured in normal DMEM. After 24 h, the cells cultured in the osteogenic induction medium (DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, 10 nM dexamethasone [Sigma-Aldrich], 10 mM β-glycerophosphate, 50 μg/mL l-ascorbic acid, and 10 nM calcitriol [1α, 25-dihydroxyvitamin D3; Sigma]). The medium was replaced with fresh medium every 3 days. To evaluate the osteogenic potential, cells were cultured in the presence of different doses of BSNs in osteogenic differentiation medium. The control cells were cultured in osteogenic differentiation medium without BSNs.
After incubation with BSNs (0, 25, 50 and 100 μg/mL), cells were washed with PBS and fixed with 4% paraformaldehyde. After fixation, the cells were washed with distilled water and stained with 2% alizarin red S at room temperature for 30 min. Excess stain was washed with water and the cells were examined under phase contrast microscope (Axiovert 40 CFL, Carl Zeiss, Germany).
2.7. AO/EB staining
2.11. Gene expression analysis Complementary DNA (cDNA) was obtained from the differentiating cells using Fastlane cell cDNA kit (Qiagen, Switzerland). The cDNA was quantified using Nanodrop spectrophotometer (NanoDrop 2000 spectrophotometer, Thermo Fisher Scientific). As per the manufacturer’s guidelines, relative quantification is used to detect an expression
2.9. Alkaline phosphatase assay Alkaline phosphatase activity of the cells treated with the 5
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Fig. 8. Alkaline phosphatase assay. Alkaline phosphatase activity of human mesenchymal stem cells after 7 and 14 days of cultivation with various concentrations (0, 25, 50 and 100 μg/mL) of sodium silicate and biogenic silica nanostructures. The color intensity increased by time- and dose-dependent manner in biogenic silica nanostructures. When compare with sodium silicate, biogenic silica nanostructures significantly increase the ALP activity.
ash contain silica in alkali medium. Previously, Balamurugan et al. fabricated silica nanoparticles from sorghum seed by dissolve the ash in alkali using conventional method [29].
changes in the mRNA level of target genes (Table 1) relative to a reference gene adopting real-time polymerase chain reaction (PCR; Applied Biosystem Real-Time PCR Detection System) using SYBR Green Master kit (Applied Biosystems, UK). Glyceraldehyde-3-phoshate dehydrogenase (GAPDH) was used as a reference gene to quantify the gene expression level using a previously described method [34]. The following formula were used to determine the expression ratio of reference gene to target gene and relative expression levels: ΔCt = Ct (target genes) − Ct (GAPDH) and ΔΔCt = ΔCt (Treated) − ΔCt (Control), respectively. The value was used to plot the expression of target genes using the method of 2−ΔΔCt.
SiO2 + 2NaOH → Na2SiO3 + H2O The transparent sodium silicate solution was neutralized to pH 7 using H2SO4 solution. Due to neutralization, sodium silicate was converted to silica nanostructures, which slowly precipitated out. Na2SiO3 + H2SO4 → SiO2 + H2O The physicochemical properties of the obtained silica nanostructures were analyzed. Fig. 1 shows the FTIR absorption spectra of BSNs derived from S. bicolor seed head. The seed head showed several absorption peaks around 3454, 2433, 1827, 1664, 1521, 1272, 1172, 1111, 1062, 901, and 724 cm−1, corresponding to water molecules and organic substances such as cellulose, hemicellulose, and lignin (Fig. 1a). The seed head ash-derived sodium silicate exhibited bands at 1446, 897, 862, and 689 cm−1 that were attributed to the SieO band of silicate. Fig. 1c shows the FTIR spectra of the BSNs derived from S. bicolor biomass. We observed two peaks around 1046 and 797 cm−1, assigned to the asymmetric stretching of SieOeSi and symmetric bending of SieO, respectively. The results clearly indicate that the BSN FTIR spectra were highly similar to those of commercial silica reported in earlier studies [35]. The X-ray diffraction (XRD) spectra of S. bicolor ash, sodium silicate, and silica are shown in Fig. 2. We observed several peaks for sodium silicate samples at 2θ value of 17.05, 27.8. 30.3, 31.9, 32.4, 33.4, 34.3, 35.4, 36.4, 38.1, 40.1, 43.9, 45.2, 46.7, 48.4, 51.18, 53.7, 55.2, 59.1, and 62.7°, corresponding to sodium silicate. The XRD pattern of BSN showed a broad peak around 22.7°, indicative of the amorphous nature
2.12. Statistical analysis All data were acquired from three independent experiments. The results are presented as the mean ± standard deviation using SPSS software (IBM Corporation, USA). For all comparisons, differences were considered statistically significant at p < 0.05. 3. Results and discussion 3.1. Synthesis of silica nanostructures Highly pure silica nanostructures were derived from S. bicolor seed husk using sequential process, including calcination, alkaline treatment, and acid precipitation. The S. bicolor seed head was calcinated at 600 °C, which resulted in the removal of organic substances and formation of ash. The ash contained a large quantity of silica and trace amounts of unwanted metals. The successive alkali treatment dissolved the silica in the ash to obtain sodium silicate, which was transparent and clear. In this study, we used hydrothermal approach to dissolve the 6
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Fig. 9. Alizarin red S staining. The human mesenchymal stem cells after 7 days of cultivation with various concentrations (0, 25, 50 and 100 μg/mL) of sodium silicate and biogenic silica nanostructures. The cells were stained with Alizarin Red S. Red color accumulation indicates calcium deposits in the cells. We observed calcium deposits in sodium silicate and biogenic silica nanostructures treated cells. The high-level calcium deposits observed in 100 μg/mL biogenic silica nanostructures treated cells.
sodium silicate. In contrast, BSNs showed uniform clusters of sphereshaped particles that were 30–80 nm in size. Fig. 5 shows TEM images of BSNs (Fig. 5a–b). The derived BSNs had spherical agglomerated shape with a dimeter of 30–80 nm. BSNs showed clusters of spherical particles. Thus, the results of TEM were in line with those of SEM.
of BSNs (Fig. 2c). The absence of any peaks related to impurities such as alkaline earth metals and sodium sulfate confirmed the high purity of BSNs. The elemental composition analysis of sodium silicate and BSN is shown in Fig. 3. The energy dispersive spectroscopy (EDS) spectra of sodium silicate showed peaks of Na, Si, O, and C. This result indicates the absence of any undesirable elemental impurities in sodium silicate (Fig. 3a). In BSNs, we observed the peaks corresponding to C, Si, and O, with no other metal impurities. The metal impurities were eliminated during the sequential process. The results of FTIR, EDS, and XRD analyses provide a strong evidence of the high purity of BSNs. SEM and TEM analyses were performed to evaluate the morphologies of sodium silicate and BSNs. Fig. 4 shows the SEM images of sodium silicate and BSNs that suggests flaky irregular morphology for
3.2. Biocompatibility assessment The biocompatibility of the synthesized BSN and its influence on osteogenesis were assessed in hMSCs as an in vitro model. The effects of BSN on the viability of hMSCs were determined with MTT assay. hMSCs were cultured in the presence of BSNs for 24 and 48 h, and the biocompatibility of BSN was compared with that of the ash-derived sodium silicate. As shown in Fig. 6, the exposure to BSN had no significant 7
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Fig. 10. Quantitative RT-PCR analysis of osteogenic markers in human mesenchymal stem cells. Comparison of the change in the expression level, expressed as fold change (i.e., the ratio of the target gene to the reference gene, GAPDH), in hMSCs after exposure to sodium silicate and biogenic silica nanostructures for 7 and 14 days. Data represent the mean ± SD of three determinations, each performed in triplicates (p < 0.05).
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deposition and ALP activity in hMSCs and found that BSN stimulated hMSC differentiation into osteoblasts. Earlier studies have demonstrated the role that silicon plays in bone generation and bone mineral density [17]. A few studies have suggested the potential role of silicon in the enhancement of osteogenic differentiation [23,36–39]. Gaharwar et al. showed that cytocompatible silicate nanoplatelets stimulated the osteogenic differentiation of cells through the upregulation in the expression of ALP, RUNX2, osteocalcin, and osteopontinproteins [40]. Mihaila et al. reported that silicate nanostructures stimulated the osteogenic differentiation of SSEA-4 subpopulation of human adiposederived stem cells by increasing the expression of osteogenic early and late markers associated with high ALP activity [41]. Ha et al. recently demonstrated the silica nanoparticle-induced osteoblast differentiation based on their sizes (50–450 nm), surface functional groups (CO2H, mNH2, OH, NR4+), and chemical composition [23]. The soluble form of silica promotes ALP activity and increases Col-1 synthesis in human osteoblast-like cells [42]. Some studies have demonstrated the role and mechanism of silica in osteogenesis. For instance, Shie et al. suggested that silicon improves Col-1synthesis and activates extracellular signalregulated kinase (ERK) pathway in MG63 cells [43]. Furthermore, silica nanoparticles were shown to induce osteoblast formation by increasing calcium deposition, stimulating ALP activity, and synthesizing BSP and Col-1 [21,36,44]. The present study demonstrates that S. bicolor-derived BSNs were internalized by hMSCs and regulated cell proliferation and metabolism. S. bicolor-derived BSNs stimulated the osteogenic differentiation of hMSCs by increasing the expression level of ALP, enhancing calcium deposition, and upregulating the expression of osteogenic marker genes. Thus, S. bicolor-derived BSNs may serve as promising agents to repair and regenerate the bone tissue.
effect on the number of viable hMSCs as compared to control cells. Sodium silicate was biocompatible with hMSCs. BSN at doses as high as 200 μg/mL showed 90% hMSC viability in 24 h. Thus, BSNs are practically harmless at high concentrations after exposure for 48 h. These results revealed the absence of any toxicity of SNs and sodium silicate. The cytocompatibility was more distinct for BSNs as compared with sodium silicate. We performed cellular and nuclear morphological analyses of cells following exposure to sodium silicate and BSNs to understand the cytological features. hMSCs were exposed to three different doses of BSNs and sodium silicate for 24 h. As shown in Fig. 7, the cells exposed to BSNs showed no change in morphology and adherence to plate and grew well. The nuclear architecture of BSN-treated hMSCs was intact and healthy without any alterations in the nuclear structure. These results are very similar to those obtained with control cells. Overall microscopic studies revealed that sodium silicate and BSNs are nontoxic and may be useful for biomedical applications. 3.3. Osteogenic differentiation We analyzed the effects of the biocompatible BSNs on the osteoblastic differentiation of hMSCs. To analyze whether BSNs could promote osteogenic differentiation of hMSCs, we evaluated the activity of ALP in hMSCs after osteogenic induction and sodium silicate and BSN exposure. ALP is an important phenotypic marker of osteoblasts owing to its presence in their cellular membranes. The cells were treated with BSNs for 7 and 14 days in osteoblastic induction medium. After 7 days, we observed higher intensity of ALP staining in BSN-treated cells than in control cells (Fig. 8). The ALP activity was higher in BSN-treated cells than sodium silicate-treated cells. These observations suggest that BSNs enhanced the osteogenic differentiation of hMSCs. However, the activity of ALP considerably increased with an increase in BSN dose (25 and 100 μg/mL). After osteogenic induction, hMSC mineralization was assessed using alizarin red S staining (Fig. 9). Alizarin red stain reacts with calcium to form a chelate and produces an orange red color. The cells treated with sodium silicate and BSNs showed different levels of matrix mineralization. Osteogenesis-induced cells showed nodule formation. In particular, BSN-treated cells had more calcium nodules after 7 days of incubation as compared with sodium silicate-treated cells. These results suggest that the treatment with high concentrations of BSNs showed around high-level increase in nodule formation as compared with the control and sodium silicate-treated cells. Our study results suggest that BSNs may promote bone-like nodule formation in hMSCs after osteoinduction.
4. Conclusion BSNs with 30–70 nm diameter were derived from S. bicolor biomass. The biocompatibility of BSNs was studied using in vitro assays, including MTT assay and AO/EB staining. These results suggest that the synthesized BSNs were non-toxic and may be used for biomedical applications. We analyzed the role of BSNs in osteogenesis of hMSCs. BSNs induced calcium deposition and increased ALP activity. The expression levels of osteogenic markers ALP, BSP, BMP-2, BMP-4, Runx2, and Col-1 were upregulated by BSN treatment at high concentrations. Our study results clearly indicate that BSNs trigger the osteogenic differentiation of hMSCs in a dose- and time-dependent manner. BSNs may stimulate osteoblast formation and BSN-based scaffolds may be used for bone tissue engineering. Declaration of Competing Interest
3.4. Osteogenic gene expression None. The expression of osteogenic differentiation markers including ALP, bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-4 (BMP-4), bone sialoprotein (BSP), collagen-1 (Col-1), Runt-related transcription factor 2 (RUNX2), bone gamma-carboxyglutamate protein (BGLAP), NANOG, and POUG5F1 was assessed after osteogenic induction following sodium silicate and BSN treatment at 7 and 14 days (Fig. 10). The gene expression level of ALP, BMP-2, BMP-4, Col-1, RUNX2, and BSP increased with an increase in the incubation time and dose of BSNs. ALP gene expression pattern suggests that BSN treatment showed a 3.5-fold increase in ALP expression as compared with control cells. The expression of BMP-2 was eight-fold higher in BSN-treated cells than in control cells. RUNX2 expression pattern (2.5-fold increase) was similar to that of BMP-2 in the cells treated with BSNs for 7 and 14 days. After 14 days, Col-1 expression level was significantly higher (P < 0.05) in the BSN-treated cells than sodium silicate-treated and control cells. We observed a tremendous upregulation in the expression of BMP-4 after 7 and 14 days in BSN-treated cells as compared with control cells. We investigated the influence of BSNs on calcium
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