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When size matters: Biological response to strontium- and cobalt-substituted bioactive glass particles
Available online at www.sciencedirect.com
ScienceDirect Materials Today: Proceedings 5 (2018) 15768–15775
www.materialstoday.com/proceedings
NTNM2017
When size matters: Biological response to strontium- and cobaltsubstituted bioactive glass particles Saeid Kargozara,*, Francesco Bainob , Nasrin Lotfibakhshaieshc, Robert G. Hilld, Peiman Brouki Milane, Sepideh Hamzehlouf, Mohammad Taghi Joghataeig, Masoud Mozafarih a Department of Modern Sciences and Technologies, School of Medicine, Mashhad University of Medical Sciences,Mashhad, Iran Institute of Materials Physics and Engineering, Department of Applied Science and Technology (DISAT),Politecnico di Torino, Torino, Italy c Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran d Unit of Dental Physical Sciences, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Mile End Road, London E1 4NS, United Kingdom e Department of Tissue Engineering and Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran f Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran g Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran h Bioengineering Research Group, Nanotechnology and Advanced Materials Department, Materials and Energy Research Center (MERC), P.O. Box 14155-4777, Tehran, Iran
b
Abstract In this study, the biological effects of two different particle sizes of strontium- and cobalt-substituted bioactive glasses (BGs) were compared to reveal the particle size effect. For this aim, the dissolution products of the BG particles with the mean diameter of 9 and 725 µm were added to cell culture media of osteosarcoma cell line SaOS-2. The results of alkaline phosphatase activity assay, alizarin red staining, and real time PCR showed that the smaller particles (9 µm) can be more effective than the larger ones (725 µm) in terms of osteogenesis and angiogenesis. However, 9-micron particles had higher cytotoxicity than the 725-µm samples. © 2018 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of INN International Conference/Workshop on “Nanotechnology and Nanomedicine’’ NTNM2017. Keywords: Nanotechnology; Nanomedicine; Bioactive glass; Nanofabrication; Tissue engineering
*
Corresponding author. Tell: +98-912-0882404 E-mail address: [email protected] 2214-7853 © 2018 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of INN International Conference/Workshop on “Nanotechnology and Nanomedicine’’ NTNM2017.
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1. Introduction The global demand for bone substitutions has been increasing annually, which is due to increased aging population, accidents, congenital genetic abnormalities and obesity [1, 2]. With the emergence of tissue engineering and its following approaches, scientists were able to fabricate and optimize different bone substitutes without the limitations of conventional grafts (autografts, allografts, and xenografts) like the shortage of donor tissue, transmission of infection, and zoonoses [3, 4]. Up to now, a large number of biomaterials have been developed to use in bone regeneration strategies [5-7]. Among different types of natural and synthetic substitutions, bioactive glasses (BGs) have gained a great reputation with respect to bone repair and regeneration applications [8-10]. This is due to their inherent properties such as excellent biocompatibility and the ability to promote osteogenesis and angiogenesis [11, 12]. Some kinds of both sol-gel and melt-derived BGs are being used in the clinical setting in order to meet the needs of diverse patient populations. Compared to the sol-gel materials, there are some merits of melt-derived BGs including easier processing and higher bioactivity [13]. More recently, a number of inorganic ionic species have been incorporated into the initial composition of BGs glasses (SiO2-CaO-Na2O-P2O5 system) to increase their biological properties [14]. In this regard, two therapeutic metal cations, i.e. strontium (Sr2+) and cobalt (Co2+), have attracted much attention because they are able to improve osteogenesis and angiogenesis to some extent [15, 16]. Strontium can increase bone density and thereby reduce fracture risk through enhancement of the osteoblast activity and inhibition of osteoclasts function [17]. Cobalt can improve angiogenesis via the activation of hypoxia-inducible factor 1-alpha (HIF-1α) pathway and thereby induce the gene expression of vascular endothelial growth factor (VEGF) [18]. However, it has been previously documented that these beneficial effects may vary based on the particle size of BGs [19]. In the present study, we evaluated the biological effects of two different sizes of as-prepared BGs containing strontium and cobalt ions on cell lines SaOS-2 and HUVEC, which were used for screening the osteogenetic and angiogenic potential, respectively. For this aim, we compared the biological effects of BG particles with two different sizes of 9 and 725 µm (mean diameter) using the assays cytotoxicity, alkaline phosphatase (ALP) activity, bone-like nodule formation, and real time PCR. 2. Materials and Methods 2.1 Materials To conduct the study, we used four different groups of as-prepared BGs of two different sizes [20, 21]. The BGs had been prepared via the melt-quenching route based on a multi-component SiO2-P2O5-CaO-SrO-Na2O-MgO-ZnOK2O system (Table 1). Briefly, all the high-purity oxides and carbonates were well-mixed and then placed in a platinum crucible. The mixtures were exposed to a temperature of 1400°C for 1 hour via an electric furnace (Lenton, Hope Valley, UK). After being melted, the BGs were immediately quenched into water to avoid crystallization. The resulted “frits” were then crushed by a vibratory puck mill (Gyro Mill, Glen Creston, London, UK) for 15 min. The obtained glasses were sieved to obtain particles with the mean diameter of 9 and 725 μm.
Table 1. Nominal compositions of the as-prepared BGs (mol.%). Sample
SiO2
P2O5
CaO
SrO
Na2O
MgO
K2O
CoO
Ca-glass Sr-glass Ca-Co-glass Sr-Co-glass
41.2 41.2 41.2 41.2
5.06 5.06 5.06 5.06
36.14 30.14 35.64 29.64
0 6 0 6
7.17 7.17 7.17 7.17
3.26 3.26 3.26 3.26
7.17 7.17 7.17 7.17
0 0 0.5 0.5
Size (mean diameter) 9 and 725 μm 9 and 725 μm 9 and 725 μm 9 and 725 μm
The cells used in this study were incubated with conditioned media containing 4 mg/ml of each sample in sizes 9 and 725 µm (RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin) at 37°C for different time points, as described in the following sections.
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2.2 Alkaline phosphatase (ALP) activity assay ALP activity assay was used to determine the osteogenic potential of the BGs. For this aim, SaOS-2 cells were grown in 6-well plates and then incubated with the conditioned medium containing 4 mg/ml of the glasses at three time points of 7, 14 and 21 days. At the end of each time period, all the supernatants were removed and the cells were immediately lysed by Tris-Triton buffer (20 mM Tris, 0.5% Triton X100). After that, the lysates were incubated with ALP solution (para-Nitrophenylphosphate (pNPP) Liquid Substrate System, Sigma, USA) at 37 °C for 30 min. Then, optical density of each sample was recorded at 450 nm using a microplate spectrophotometer (Synergy HT, BioTek, USA). Finally, a standard curve plotted by p-nitrophenol (pNP) was used to determine ALP concentrations (the concentration of ALP was normalized to the cell densities obtained using the WST-8 assay). 2.3 Alizarin red staining Alizarin red S staining was carried out to survey the formation of bone-like nodules in the treated cells. Based on the manufacturer’s protocol, the cells were firstly cultured in the conditioned medium (4 mg/ml of each sample) at 37 °C in a humidified air with 5% CO2. After 14-day incubation, the treated cells were washed by PBS three times and then fixed in 4% paraformaldehyde solution for 30 min. After the fixation, the cells were stained by 40 mM alizarin red S solution (pH = 4.2 – 4.4) for 45 min at room temperature in the dark. Finally, the cells were washed three times using distilled water and viewed by an inverted microscope (Olympus, Japan). 2.4 Cytotoxicity evaluation Cytocompatibility of the samples were evaluated using the standard colorimetric 3-(4,5-dimethyl-2 thiazolyl)2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. For this purpose, SaOS-2 cells were incubated with the conditioned media (containing 4 mg/ml of each sample) at 37°C for 3, 5 and 7 days. After the incubation intervals, the MTT solution (5 mg/ml) (Sigma-Aldrich, UK) was added to the cultures and incubated for 4 h. Then, all the supernatants were removed and dimethyl sulfoxide solution (DMSO) (Sigma-Aldrich, UK) was added to the culture plates. Finally, optical density (OD) of each plate was measured using a spectrophotometer (Synergy HT, BioTek, USA) at 570 nm wavelength. 2.5 Real time PCR Real time PCR assay was performed to evaluate the osteogenic and angiogenic potential of the glasses at the molecular level. For this aim, two cell lines (SaOS-2 and HUVECs) were cultured with the as-prepared conditioned media for 14 and 21 days. After the end of incubation time points, total RNA of the treated cells was isolated by the RNAeasy Mini Kit (Qiagen, USA). For conducting quantitative PCR (qPCR) assay, single strand cDNA was firstly synthesized from the isolated RNAs using a RevertAid First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania). The synthesized cDNAs were then used as the template for the qPCR reaction. The RotorGeneTMSYBER®Green PCR Kit (Qiagen, USA) was used to perform the assay with the specific primers for genes βactin, COL1A1, ALP, ON, and OCN (Table 2). The thermal cycling conditions included an initial denaturation step at 95ºC for 10 min, followed by 40 cycles at 95ºC for 30 s, 60ºC for 30 s and 72ºC for 30 s. The 2-ΔΔCt method was used to calculate relative quantification in gene expression. All the fold changes in gene expression were normalized to β-actin.
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Table 2. The characteristics of primers used for real-time PCR assay
Target Gene β-Actin ALP ON OCN COL1A1 VEGF
Forward primer sequences 5'-TCAGAGCAAGAGAGGCATCC-3' 5'-CACGTCGATTGCATCTCTGG-3' 5'-GAGGGCCTGGATCTTCTTTCTCCT-3' 5'-ACCATGAGAGCCCTCACACTCCT-3' 5'-TGTTCAGCTTTGTGGACCTCCG-3' 5'-ACCATGAACTTTCTGCTGTCTTG-3'
Reverse primer sequences 5'-GGTCATCTTCTCCACGGTTGG-3' 5'-GCCAGTACTTGGGGTCTTTC-3' 5'-CTCAGTCACCTCTGCCACAGTT-3' 5'-GTCTCTTCACTACCTCGCTGCC-3' 5'-CTGAGGCCGTTCTGTACGCA-3' 5'-ACATCCATGAACTTCACCACTTC-3'
3. Results and Discussion 3.1 ALP activity The ALP activity of the treated osteosarcoma cells with the dissolution products of 9- and 725- µm glasses is presented in Fig 1. All the samples increased the ALP activity of the cells at the defined time points to some extent. These results are in line with the previously published reports indicating that BGs are able to enhance the osteogenic activity via the induction of osteoblast markers such ALP [22]. With reference to the obtained results, it can be claimed that the smaller particles (9- µm glasses) have better efficiency than the larger ones in terms of osteogenic activity of the treated cells.
Fig. 1. ALP activity analysis for human osteosarcoma cells (SaoOS-2) cultured in the conditioned medium (4 mg/ml of each glass) of two different particle sizes (-A: 9 and -B: 725 µm).
3.2 Alizarin red staining As shown in Fig. 2, calcium nodule deposition was clearly observed in the groups treated with the Ca and CaCo glasses in both different sizes. However, the size of bone-like nodules for the treated cells with the 9- µm particles is slightly larger compared to the 725- µm grains. This data are consistent with the results of ALP activity assay clarifying the BGs with the mean diameter of 9 µm are more effective than 725 µm particles for induction of bone-like nodule formation in vitro.
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Fig. 2. Light micrograph of SaOS-2 cells cultured in the conditioned media (4 mg/ml of each glass) of two different particle sizes (A: 9 and B: 725 µm). (Scale bar: 200 μm).
3.3 MTT assay The results of MTT assay revealed that all the glasses have no cytotoxicity effect on the treated cells. As can be seen in Fig. 3, the best results were associated with Sr-containing samples, i.e. Sr- and Sr-Co-substituted glasses. However, it can be observed that there is a difference between the groups treated with dissolution products of 9- µm BGs in comparison to 725 µm samples. The higher cytotoxicity of the smaller particles may relate to create more alkaline conditions due to the more probable release of ions from their structure. This effect has been previously shown in the literature [23].
Fig. 3. MTT activity of SaOS-2, treated with dissolution ions from the glasses with two different particle sizes (4 mg/ml) (-A: 9 and -B: 725 µm).
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3.4 Real time PCR The effect of the different particles on up-regulation of the osteogenic and angiogenic genes is presented in Fig. 4. The results of q-PCR revealed that both the osteogenesis- and angiogenesis-related genes were overexpressed in the cells treated with the conditioned media of 9- and 725- µm particles. These results are in line with previously reported studies showing the effectiveness of BGs regarding over-expression of osteogenic and angiogenic genes. However, the expression of target genes was higher in the groups treated with the smaller grain size compared to larger particles. This over-expression is also related to the better release of ions from the smaller BG particles, which is in line with the other obtained data (ALP activity assay, alizarin red staining, and MTT assay). a
b
Fig. 4: Gene expression results: mRNA expression of (a) the osteogenic
and (b) angiogenic genes in SaOS-2 cells after 21 days
of incubation with the conditioned media (4 mg/mL of each glass) of two different particle sizes (-A: 9 and -B: 725 µm).
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4. Conclusions During the past few years, different types of BGs have been proposed for tissue engineering applications. Although it is clear that the chemical composition of glass materials could play an important role in tissue regeneration, it is expected that the particle size of particles could stimulate specific functions to the target tissues. This study shows that Sr/Co-doped BG particles with finer size (9 µm) can be more effective than larger particles (725 µm) in promoting osteogenesis and angiogenesis. However, it was observed that the smaller particles had higher cytotoxicity compared to the larger ones. These results further demonstrate the importance of both composition and particle size for the postoperative success of BG implants in a particulate form.
References [1] K. Nazemi, F. Moztarzadeh, N. Jalali, S. Asgari, M. Mozafari, Synthesis and characterization of poly (lactic-co-glycolic) acid nanoparticlesloaded chitosan/bioactive glass scaffolds as a localized delivery system in the bone defects, BioMed research international, 2014 (2014). [2] A.G. Nazari, A. Tahari, F. Moztarzadeh, M. Mozafari, M. Bahrololoom, Ion exchange behaviour of silver-doped apatite micro-and nanoparticles as antibacterial biomaterial, Micro & Nano Letters, 6 (2011) 713-717. [3] S. Kargozar, S.J. Hashemian, M. Soleimani, P.B. Milan, M. Askari, V. Khalaj, A. Samadikuchaksaraie, S. Hamzehlou, A.R. Katebi, N. Latifi, M. Mozafari, F. Baino, Acceleration of bone regeneration in bioactive glass/gelatin composite scaffolds seeded with bone marrow-derived mesenchymal stem cells over-expressing bone morphogenetic protein-7, Materials Science and Engineering: C, 75 (2017) 688-698. [4] B. Johari, M. Kadivar, S. Lak, M. Gholipourmalekabadi, A.M. Urbanska, M. Mozafari, M. Ahmadzadehzarajabad, A. Azarnezhad, S. Afshari, J. Zargan, Osteoblast-seeded bioglass/gelatin nanocomposite: a promising bone substitute in critical-size calvarial defect repair in rat, The International journal of artificial organs, 39 (2016) 524-533. [5] A. Samadikuchaksaraei, M. Gholipourmalekabadi, E. Erfani Ezadyar, M. Azami, M. Mozafari, B. Johari, S. Kargozar, S.B. Jameie, A. Korourian, A.M. Seifalian, Fabrication and in vivo evaluation of an osteoblast‐conditioned nano‐hydroxyapatite/gelatin composite scaffold for bone tissue regeneration, Journal of Biomedical Materials Research Part A, 104 (2016) 2001-2010. [6] A. Hamlekhan, M. Mozafari, N. Nezafati, M. Azami, H. Hadipour, A proposed fabrication method of novel PCL-GEL-HAp nanocomposite scaffolds for bone tissue engineering applications, Advanced Composites Letters, 19 (2010) 123-130. [7] B. Johari, M. Ahmadzadehzarajabad, M. Azami, M. Kazemi, M. Soleimani, S. Kargozar, S. Hajighasemlou, M.M. Farajollahi, A. Samadikuchaksaraei, Repair of rat critical size calvarial defect using osteoblast ‐ like and umbilical vein endothelial cells seeded in gelatin/hydroxyapatite scaffolds, Journal of Biomedical Materials Research Part A, 104 (2016) 1770-1778. [8] S. Kargozar, M. Mozafari, S.J. Hashemian, P. Brouki Milan, S. Hamzehlou, M. Soleimani, M.T. Joghataei, M. Gholipourmalekabadi, A. Korourian, K. Mousavizadeh, A.M. Seifalian, Osteogenic potential of stem cells-seeded bioactive nanocomposite scaffolds: A comparative study between human mesenchymal stem cells derived from bone, umbilical cord Wharton's jelly, and adipose tissue, Journal of Biomedical Materials Research Part B: Applied Biomaterials, DOI 10.1002/jbm.b.33814(2016) n/a-n/a. [9] R. Kamalian, A. Yazdanpanah, F. Moztarzadeh, R. Ravarian, Z. Moztarzadeh, M. Tahmasbi, M. Mozafari, Synthesis and characterization of bioactive glass/forsterite nanocomposites for bone implants, Ceramics-Silikáty, 56 (2012) 331-340. [10] F. Baino, M.A. Montealegre, G. Orlygsson, G. Novajra, C. Vitale-Brovarone, Bioactive glass coatings fabricated by laser cladding on ceramic acetabular cups: a proof-of-concept study, Journal of Materials Science, 52 (2017) 9115-9128. [11] G. Kaur, O.P. Pandey, K. Singh, D. Homa, B. Scott, G. Pickrell, A review of bioactive glasses: their structure, properties, fabrication and apatite formation, Journal of Biomedical Materials Research Part A, 102 (2014) 254-274. [12] L.A.H. Durand, G.E. Vargas, N.M. Romero, R. Vera-Mesones, J.M. Porto-López, A.R. Boccaccini, M.P. Zago, A. Baldi, A. Gorustovich, Angiogenic effects of ionic dissolution products released from a boron-doped 45S5 bioactive glass, Journal of Materials Chemistry B, 3 (2015) 1142-1148. [13] M. Lombardi, L. Gremillard, J. Chevalier, L. Lefebvre, I. Cacciotti, A. Bianco, L. 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Brandi, How strontium ranelate, via opposite effects on bone resorption and formation, prevents osteoporosis, Osteoporosis International, 22 (2011) 1659-1667. [18] S. Rey, G.L. Semenza, Hypoxia-inducible factor-1-dependent mechanisms of vascularization and vascular remodelling, Cardiovascular research, 86 (2010) 236-242.
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[19] E. Tamjid, R. Bagheri, M. Vossoughi, A. Simchi, Effect of particle size on the in vitro bioactivity, hydrophilicity and mechanical properties of bioactive glass-reinforced polycaprolactone composites, Materials Science and Engineering: C, 31 (2011) 1526-1533. [20] S. Kargozar, N. Lotfibakhshaiesh, J. Ai, A. Samadikuchaksaraie, R.G. Hill, P.A. Shah, P.B. Milan, M. Mozafari, M. Fathi, M.T. Joghataei, Synthesis, physico-chemical and biological characterization of strontium and cobalt substituted bioactive glasses for bone tissue engineering, Journal of Non-Crystalline Solids, 449 (2016) 133-140. [21] S. Kargozar, N. Lotfibakhshaiesh, J. Ai, M. Mozafari, P. Brouki Milan, S. Hamzehlou, M. Barati, F. Baino, R.G. Hill, M.T. Joghataei, Strontium- and cobalt-substituted bioactive glasses seeded with human umbilical cord perivascular cells to promote bone regeneration via enhanced osteogenic and angiogenic activities, Acta Biomaterialia, 58 (2017) 502-514. [22] G.C. Reilly, S. Radin, A.T. Chen, P. Ducheyne, Differential alkaline phosphatase responses of rat and human bone marrow derived mesenchymal stem cells to 45S5 bioactive glass, Biomaterials, 28 (2007) 4091-4097. [23] O. Tsigkou, S. Labbaf, M.M. Stevens, A.E. Porter, J.R. Jones, Monodispersed bioactive glass submicron particles and their effect on bone marrow and adipose tissue‐derived stem cells, Advanced healthcare materials, 3 (2014) 115-125.
ScienceDirect Materials Today: Proceedings 5 (2018) 15768–15775
www.materialstoday.com/proceedings
NTNM2017
When size matters: Biological response to strontium- and cobaltsubstituted bioactive glass particles Saeid Kargozara,*, Francesco Bainob , Nasrin Lotfibakhshaieshc, Robert G. Hilld, Peiman Brouki Milane, Sepideh Hamzehlouf, Mohammad Taghi Joghataeig, Masoud Mozafarih a Department of Modern Sciences and Technologies, School of Medicine, Mashhad University of Medical Sciences,Mashhad, Iran Institute of Materials Physics and Engineering, Department of Applied Science and Technology (DISAT),Politecnico di Torino, Torino, Italy c Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran d Unit of Dental Physical Sciences, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Mile End Road, London E1 4NS, United Kingdom e Department of Tissue Engineering and Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran f Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran g Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran h Bioengineering Research Group, Nanotechnology and Advanced Materials Department, Materials and Energy Research Center (MERC), P.O. Box 14155-4777, Tehran, Iran
b
Abstract In this study, the biological effects of two different particle sizes of strontium- and cobalt-substituted bioactive glasses (BGs) were compared to reveal the particle size effect. For this aim, the dissolution products of the BG particles with the mean diameter of 9 and 725 µm were added to cell culture media of osteosarcoma cell line SaOS-2. The results of alkaline phosphatase activity assay, alizarin red staining, and real time PCR showed that the smaller particles (9 µm) can be more effective than the larger ones (725 µm) in terms of osteogenesis and angiogenesis. However, 9-micron particles had higher cytotoxicity than the 725-µm samples. © 2018 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of INN International Conference/Workshop on “Nanotechnology and Nanomedicine’’ NTNM2017. Keywords: Nanotechnology; Nanomedicine; Bioactive glass; Nanofabrication; Tissue engineering
*
Corresponding author. Tell: +98-912-0882404 E-mail address: [email protected] 2214-7853 © 2018 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of INN International Conference/Workshop on “Nanotechnology and Nanomedicine’’ NTNM2017.
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1. Introduction The global demand for bone substitutions has been increasing annually, which is due to increased aging population, accidents, congenital genetic abnormalities and obesity [1, 2]. With the emergence of tissue engineering and its following approaches, scientists were able to fabricate and optimize different bone substitutes without the limitations of conventional grafts (autografts, allografts, and xenografts) like the shortage of donor tissue, transmission of infection, and zoonoses [3, 4]. Up to now, a large number of biomaterials have been developed to use in bone regeneration strategies [5-7]. Among different types of natural and synthetic substitutions, bioactive glasses (BGs) have gained a great reputation with respect to bone repair and regeneration applications [8-10]. This is due to their inherent properties such as excellent biocompatibility and the ability to promote osteogenesis and angiogenesis [11, 12]. Some kinds of both sol-gel and melt-derived BGs are being used in the clinical setting in order to meet the needs of diverse patient populations. Compared to the sol-gel materials, there are some merits of melt-derived BGs including easier processing and higher bioactivity [13]. More recently, a number of inorganic ionic species have been incorporated into the initial composition of BGs glasses (SiO2-CaO-Na2O-P2O5 system) to increase their biological properties [14]. In this regard, two therapeutic metal cations, i.e. strontium (Sr2+) and cobalt (Co2+), have attracted much attention because they are able to improve osteogenesis and angiogenesis to some extent [15, 16]. Strontium can increase bone density and thereby reduce fracture risk through enhancement of the osteoblast activity and inhibition of osteoclasts function [17]. Cobalt can improve angiogenesis via the activation of hypoxia-inducible factor 1-alpha (HIF-1α) pathway and thereby induce the gene expression of vascular endothelial growth factor (VEGF) [18]. However, it has been previously documented that these beneficial effects may vary based on the particle size of BGs [19]. In the present study, we evaluated the biological effects of two different sizes of as-prepared BGs containing strontium and cobalt ions on cell lines SaOS-2 and HUVEC, which were used for screening the osteogenetic and angiogenic potential, respectively. For this aim, we compared the biological effects of BG particles with two different sizes of 9 and 725 µm (mean diameter) using the assays cytotoxicity, alkaline phosphatase (ALP) activity, bone-like nodule formation, and real time PCR. 2. Materials and Methods 2.1 Materials To conduct the study, we used four different groups of as-prepared BGs of two different sizes [20, 21]. The BGs had been prepared via the melt-quenching route based on a multi-component SiO2-P2O5-CaO-SrO-Na2O-MgO-ZnOK2O system (Table 1). Briefly, all the high-purity oxides and carbonates were well-mixed and then placed in a platinum crucible. The mixtures were exposed to a temperature of 1400°C for 1 hour via an electric furnace (Lenton, Hope Valley, UK). After being melted, the BGs were immediately quenched into water to avoid crystallization. The resulted “frits” were then crushed by a vibratory puck mill (Gyro Mill, Glen Creston, London, UK) for 15 min. The obtained glasses were sieved to obtain particles with the mean diameter of 9 and 725 μm.
Table 1. Nominal compositions of the as-prepared BGs (mol.%). Sample
SiO2
P2O5
CaO
SrO
Na2O
MgO
K2O
CoO
Ca-glass Sr-glass Ca-Co-glass Sr-Co-glass
41.2 41.2 41.2 41.2
5.06 5.06 5.06 5.06
36.14 30.14 35.64 29.64
0 6 0 6
7.17 7.17 7.17 7.17
3.26 3.26 3.26 3.26
7.17 7.17 7.17 7.17
0 0 0.5 0.5
Size (mean diameter) 9 and 725 μm 9 and 725 μm 9 and 725 μm 9 and 725 μm
The cells used in this study were incubated with conditioned media containing 4 mg/ml of each sample in sizes 9 and 725 µm (RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin) at 37°C for different time points, as described in the following sections.
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2.2 Alkaline phosphatase (ALP) activity assay ALP activity assay was used to determine the osteogenic potential of the BGs. For this aim, SaOS-2 cells were grown in 6-well plates and then incubated with the conditioned medium containing 4 mg/ml of the glasses at three time points of 7, 14 and 21 days. At the end of each time period, all the supernatants were removed and the cells were immediately lysed by Tris-Triton buffer (20 mM Tris, 0.5% Triton X100). After that, the lysates were incubated with ALP solution (para-Nitrophenylphosphate (pNPP) Liquid Substrate System, Sigma, USA) at 37 °C for 30 min. Then, optical density of each sample was recorded at 450 nm using a microplate spectrophotometer (Synergy HT, BioTek, USA). Finally, a standard curve plotted by p-nitrophenol (pNP) was used to determine ALP concentrations (the concentration of ALP was normalized to the cell densities obtained using the WST-8 assay). 2.3 Alizarin red staining Alizarin red S staining was carried out to survey the formation of bone-like nodules in the treated cells. Based on the manufacturer’s protocol, the cells were firstly cultured in the conditioned medium (4 mg/ml of each sample) at 37 °C in a humidified air with 5% CO2. After 14-day incubation, the treated cells were washed by PBS three times and then fixed in 4% paraformaldehyde solution for 30 min. After the fixation, the cells were stained by 40 mM alizarin red S solution (pH = 4.2 – 4.4) for 45 min at room temperature in the dark. Finally, the cells were washed three times using distilled water and viewed by an inverted microscope (Olympus, Japan). 2.4 Cytotoxicity evaluation Cytocompatibility of the samples were evaluated using the standard colorimetric 3-(4,5-dimethyl-2 thiazolyl)2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. For this purpose, SaOS-2 cells were incubated with the conditioned media (containing 4 mg/ml of each sample) at 37°C for 3, 5 and 7 days. After the incubation intervals, the MTT solution (5 mg/ml) (Sigma-Aldrich, UK) was added to the cultures and incubated for 4 h. Then, all the supernatants were removed and dimethyl sulfoxide solution (DMSO) (Sigma-Aldrich, UK) was added to the culture plates. Finally, optical density (OD) of each plate was measured using a spectrophotometer (Synergy HT, BioTek, USA) at 570 nm wavelength. 2.5 Real time PCR Real time PCR assay was performed to evaluate the osteogenic and angiogenic potential of the glasses at the molecular level. For this aim, two cell lines (SaOS-2 and HUVECs) were cultured with the as-prepared conditioned media for 14 and 21 days. After the end of incubation time points, total RNA of the treated cells was isolated by the RNAeasy Mini Kit (Qiagen, USA). For conducting quantitative PCR (qPCR) assay, single strand cDNA was firstly synthesized from the isolated RNAs using a RevertAid First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania). The synthesized cDNAs were then used as the template for the qPCR reaction. The RotorGeneTMSYBER®Green PCR Kit (Qiagen, USA) was used to perform the assay with the specific primers for genes βactin, COL1A1, ALP, ON, and OCN (Table 2). The thermal cycling conditions included an initial denaturation step at 95ºC for 10 min, followed by 40 cycles at 95ºC for 30 s, 60ºC for 30 s and 72ºC for 30 s. The 2-ΔΔCt method was used to calculate relative quantification in gene expression. All the fold changes in gene expression were normalized to β-actin.
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Table 2. The characteristics of primers used for real-time PCR assay
Target Gene β-Actin ALP ON OCN COL1A1 VEGF
Forward primer sequences 5'-TCAGAGCAAGAGAGGCATCC-3' 5'-CACGTCGATTGCATCTCTGG-3' 5'-GAGGGCCTGGATCTTCTTTCTCCT-3' 5'-ACCATGAGAGCCCTCACACTCCT-3' 5'-TGTTCAGCTTTGTGGACCTCCG-3' 5'-ACCATGAACTTTCTGCTGTCTTG-3'
Reverse primer sequences 5'-GGTCATCTTCTCCACGGTTGG-3' 5'-GCCAGTACTTGGGGTCTTTC-3' 5'-CTCAGTCACCTCTGCCACAGTT-3' 5'-GTCTCTTCACTACCTCGCTGCC-3' 5'-CTGAGGCCGTTCTGTACGCA-3' 5'-ACATCCATGAACTTCACCACTTC-3'
3. Results and Discussion 3.1 ALP activity The ALP activity of the treated osteosarcoma cells with the dissolution products of 9- and 725- µm glasses is presented in Fig 1. All the samples increased the ALP activity of the cells at the defined time points to some extent. These results are in line with the previously published reports indicating that BGs are able to enhance the osteogenic activity via the induction of osteoblast markers such ALP [22]. With reference to the obtained results, it can be claimed that the smaller particles (9- µm glasses) have better efficiency than the larger ones in terms of osteogenic activity of the treated cells.
Fig. 1. ALP activity analysis for human osteosarcoma cells (SaoOS-2) cultured in the conditioned medium (4 mg/ml of each glass) of two different particle sizes (-A: 9 and -B: 725 µm).
3.2 Alizarin red staining As shown in Fig. 2, calcium nodule deposition was clearly observed in the groups treated with the Ca and CaCo glasses in both different sizes. However, the size of bone-like nodules for the treated cells with the 9- µm particles is slightly larger compared to the 725- µm grains. This data are consistent with the results of ALP activity assay clarifying the BGs with the mean diameter of 9 µm are more effective than 725 µm particles for induction of bone-like nodule formation in vitro.
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Fig. 2. Light micrograph of SaOS-2 cells cultured in the conditioned media (4 mg/ml of each glass) of two different particle sizes (A: 9 and B: 725 µm). (Scale bar: 200 μm).
3.3 MTT assay The results of MTT assay revealed that all the glasses have no cytotoxicity effect on the treated cells. As can be seen in Fig. 3, the best results were associated with Sr-containing samples, i.e. Sr- and Sr-Co-substituted glasses. However, it can be observed that there is a difference between the groups treated with dissolution products of 9- µm BGs in comparison to 725 µm samples. The higher cytotoxicity of the smaller particles may relate to create more alkaline conditions due to the more probable release of ions from their structure. This effect has been previously shown in the literature [23].
Fig. 3. MTT activity of SaOS-2, treated with dissolution ions from the glasses with two different particle sizes (4 mg/ml) (-A: 9 and -B: 725 µm).
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3.4 Real time PCR The effect of the different particles on up-regulation of the osteogenic and angiogenic genes is presented in Fig. 4. The results of q-PCR revealed that both the osteogenesis- and angiogenesis-related genes were overexpressed in the cells treated with the conditioned media of 9- and 725- µm particles. These results are in line with previously reported studies showing the effectiveness of BGs regarding over-expression of osteogenic and angiogenic genes. However, the expression of target genes was higher in the groups treated with the smaller grain size compared to larger particles. This over-expression is also related to the better release of ions from the smaller BG particles, which is in line with the other obtained data (ALP activity assay, alizarin red staining, and MTT assay). a
b
Fig. 4: Gene expression results: mRNA expression of (a) the osteogenic
and (b) angiogenic genes in SaOS-2 cells after 21 days
of incubation with the conditioned media (4 mg/mL of each glass) of two different particle sizes (-A: 9 and -B: 725 µm).
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4. Conclusions During the past few years, different types of BGs have been proposed for tissue engineering applications. Although it is clear that the chemical composition of glass materials could play an important role in tissue regeneration, it is expected that the particle size of particles could stimulate specific functions to the target tissues. This study shows that Sr/Co-doped BG particles with finer size (9 µm) can be more effective than larger particles (725 µm) in promoting osteogenesis and angiogenesis. However, it was observed that the smaller particles had higher cytotoxicity compared to the larger ones. These results further demonstrate the importance of both composition and particle size for the postoperative success of BG implants in a particulate form.
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