Abstract
Nanotechnology plays a promising role in biomedical applications, particularly tissue engineering. Recently, the application of magnetic scaffolds and pulsed electromagnetic field (PEMF) exposure has been considered in bone tissue regeneration. In this study, 3rd generation dendrimer-modified superparamagnetic iron oxide nanoparticles (G3-SPIONs) are synthesized comprehensively characterized. Magnetic polycaprolactone (PCL) nanofibers are prepared by incorporating G3-SPIONs within the electrospinning process ,and physicochemical characteristics ,as well as cytocompatibility and cell attachment are assessed. Eventually, the osteogenic differentiation ability of adipocyte-derived mesenchymal stem cells (ADMSCs) cultured on the magnetic scaffold with and without PEMF exposure was investigated by measurement of alkaline phosphatase (ALP) activity and calcium content. The expression of specific bone markers was studied using the Real-time PCR method. According to the results, G3-SPIONs with mean size and zeta potential of 17.95 ± 3.57 nm and 22.7 mV, respectively, show a high saturation magnetization (57.75 emu/g). Adding G3-SPIONs to PCL significantly decrease nanofibers size to 495±144 nm and improves cell attachment and growth. The ADMSCs cultured on the G3-SPION-PCL scaffold in the presence of osteogenic media (OM) and exposure to PEMF expressed the highest Osteocalcin and Runx2 and showed higher calcium content as well as ALP activity. It can be concluded that the synthesized G3-SPION incorporated PCL nanofibers serve as a promising magnetic scaffold for bone regeneration. Also, utilizing the magnetic scaffold in the presence of OM and PEMF provides a synergistic effect toward osteogenic differentiation of ADMSCs.
Key Words: Superparamagnetic iron oxide nanoparticles, Dendrimer, Polycaprolactone, Pulsed electromagnetic field, Bone tissue engineering
Introduction
According to the American academy of orthopedic surgeons, there are 6.3 million fractures every year in the United States. Although surgery serves as a common treatment for bone fracture, in some cases (around 10%), infection and insufficient vascularization following the may result in incomplete healing (Ng, Spiller, Bernhard, & Vunjak-Novakovic, 2017; Stevens, Yang, Mohandas, Stucker, & Nguyen, 2008). The factors like aging populations and the high incidence of osteo-degenerative diseases significantly lead to growth in the artificial regeneration of bone tissue as a field of research (Joshi & Grinstaff, 2008). Autologous bone grafting is usually known as the gold standard for the treatment of bone defects. However, there are several disadvantages, including painful procedure, high cost, limited tissue supply, and extended hospitalization (Kohli et al., 2018; Polo-Corrales, Latorre-Esteves, & Ramirez-Vick, 2014). On the other hand, despite the abundance of resources for allograft based bone grafting, it has limited application due to the poor adaptation and the risk of disease transmission (Robbins, Lauryssen, & Songer, 2017; Stevens et al., 2008). In order to overcome such limitations, bone tissue engineering is becoming a promising treatment in which the combination of living cells, engineered scaffolds, and biochemical/biophysical factors can enhance tissue regeneration. Actullay, bone issue engineering ,in association with technologies in fracture stabilization, seems as innovative solutions (Campana et al., 2014).
Recently, it has been documented that in addition to physical stimulations including tensile and compressive stresses, fluid shear stresses, and heat showing a remarkable role in osteogenic differentiation, magnetic stimulations can also considerably improve bone regeneration (Xia et al., 2018). In this regards, magnetic nanoparticles (MNPs) incorporation into the bone tissue engineering scaffolds, with or without a magnetic field exposure, have enormous potential for bone tissue engineering applications (Bock et al., 2010; He et al., 2017). Previous studies have demonstrated that ion exchanging channels on the cell membrane and the corresponded biochemical pathway can be influenced by magnetic fields (Zhao et al., 2011). Magnetic scaffolds in which MNPs are incorporated have significant effects on adhesion, proliferation, and differentiation potency of stem cells. The force generated by magnetic nanoparticles under a magnetic field can significantly influence the micro-environment around the cells, leading to a series of changes in cell behavior via magneto-mechanical stimulations (Jiang et al., 2016; Xia et al., 2019; Xia et al., 2018). Among MNPs, iron oxide nanoparticles have attracted much interest in biomedical applications such as magnetic-activated cell sorting (MACS), hyperthermia, drug delivery, and contrast-enhanced MRI, because they belong to a group of non-toxic materials that exhibit good biocompatibility due to the presence of iron ions (Kandpal, Sah, Loshali, Joshi, & Prasad, 2014) (Chauhan et al., 2013; Ito, Shinkai, Honda, & Kobayashi, 2005). (Ayyappan et al., 2010; Koehler et al., 2009; Marinin, 2012). As a challenging issue, in biological media, MNPs usually tend to be agglomerated because of the high surface-area-to-volume ratio as well as magnetic attraction. To avoid this problem and to stabilize nanoparticles, the MNPs surface coating is preferred (Mascolo, Pei, & Ring, 2013). Ideally, these coatings should be non-immunogenic and hydrophilic enough to prevent the opsonization by plasma proteins resulting decrease in reticuloendothelial system clearance and an increase in the shelf-life within the body. The materials used for coating are commonly polymeric ones with improved physical-chemical properties and appropriate biocompatibility (Chauhan et al., 2013; Favela-Camacho, Samaniego-Benítez, Godínez-García, Avilés-Arellano, & Pérez-Robles, 2019). Dendrimers are highlighted by their unique properties, such as mono-dispersity, well-defined structure, having a large number of surface functional groups, and antimicrobial activities. Such characteristics make them an appropriate tool for biomedical applications, particularly tissue engineering (Abdel-Sayed et al., 2016; Gorain et al., 2017; Kesharwani, Gajbhiye, K Tekade, & K Jain, 2011; Kesharwani, Tekade, Gajbhiye, Jain, & Jain, 2011; Kesharwani, Tekade, & Jain, 2015). Incorporation of dendrimers into the scaffolds structure, particularly in the surface, may improve cell-substrate interactions. Besides, the dendrimers due to the porous structure could improve the interconnection in the scaffolds structure and consequently help to cell-cell interactions (Gorain et al., 2017; Joshi & Grinstaff, 2008). Poly (amidoamine) dendrimers, PAMAM, have received widespread attention because of the fundamental nature and their polar properties (Bosman, Janssen, & Meijer, 1999). PAMAM are hydrophilic, biocompatible, monodisperse, and cascade-branched macromolecules with highly flexible surface chemistry. In order to reduce MNPs agglomeration and increase their cationic surface charge, coating with PAMAM can be considered as an ideal option because PAMAM has plenty of peripheral functional groups and high hydrophilicity. PAMAM dendrimers can introduce a dense outer amine shell on the MNPs through a cascade-type generation (Boas, Christensen, & Heegaard, 2006; Khodadust, Unsoy, Yalcın, Gunduz, & Gunduz, 2013; Klajnert & Bryszewska, 2001). Polycaprolactone (PCL) is considered as an appropriate candidate for bone tissue engineering regarding its long-term degradation. the FDA approves PCL for some clinical applications. It is biodegradable polyester and appropriate for long-term bone implantation. Studies have shown that PCL act as a supportive role to keep living osteoblasts and fibroblasts cells. PCL maintains its primary structure in the biological fluids and tends to blend with other polymers. [31] Considering the inherent properties of MNPs, positive features of dendrimers as a right candidate for surface coating, this project aimed to synthesize a PCL-based magnetic scaffold by incorporation of dendrimers-modified MNPs. Iron oxide nanoparticles (SPION) were functionalized by 3rd generation of PAMAM dendrimer and comprehensively characterized. Dendrimerized SPIONs were incorporated into the PCL nanofibrous scaffold by blend electrospining. Osteogentic differentiation of MSCs cultured on the magnetic scaffolds was studied in the presence of pulsed electromagnetic field produced by bioreactor.
Materials and methods
2.1. Chemicals
Ferrous chloride hexahydrate (FeCl3.6H2O), ferric sulfate heptahydrate (FeSO4.7H2O), (3-aminopropyl) triethoxysilane (APTES), methanol (CH3OH), ethanol (C2H5OH), Chloroform (CHCl₃), dimethyl sulfoxide (DMSO) and beta-glycerol phosphate were purchased from Merck (Darmstadt, Germany). Methyl acrylate (MA), ethylenediamine (EDA), ammonium hydroxide (NH4OH), polycaprolactone (PCL, MW= 70000-90000 g/mol), dimethylformamide (DMF), 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide (MTT), dexamethasone, ascorbic acid-2-phosphate, glutaraldehyde, paraformaldehyde, and diarylpyrimidine (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin were purchased from Gibco (USA). Human adipose mesenchymal stem cells (ADMSCs ) obtained from Stem Cells Technology Research Centre cell bank (Tehran, Iran).
2.2. Synthesis of SPIONs
The co-precipitation method was used to synthesize SPIONs following the reported standard protocol. (Esmaeili, Khalili, et al., 2019; Tajabadi, Khosroshahi, & Bonakdar, 2013) A solution mixture of ferrous chloride hexahydrate (FeCl3.6H2O), 0.2 M, and ferric sulfate heptahydrate (FeSO4.7H2O), 0.1 M, was prepared in distilled water and used as a source of iron ions. The aqueous ammonia solution (5.4 M) was added dropwise to the solution of iron salts under nitrogen protection and stirred at 50 °C for 30 min. The obtained precipitation was washed five times with distilled water using magnetic separation to remove any excess unreacted materials and air-dried at room temperature.