Int J Nanomedicine. 2015 Dec 7;10:7307-17. doi: 10.2147/IJN.S93670. eCollection 2015.
Highly efficient mesenchymal stem cell proliferation on poly-ε-caprolactone nanofibers with embedded magnetic nanoparticles
Daňková J, Buzgo M, Vejpravová J, Kubíčková S, Sovková V, Vysloužilová L, Mantlíková A, Nečas A, Amler E.
Laboratory of Tissue Engineering, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic; Institute of Biophysics, Second Faculty of Medicine, Charles University in Prague, Prague, Czech Republic; Faculty of Biomedical Engineering, Czech Technical University in Prague, Kladno, Czech Republic; University Center for Energy Efficient Buildings, Czech Technical University in Prague, Bustehrad, Czech Republic; Department of Magnetic Nanosystems, Institute of Physics, Academy of Sciences of the Czech Republic, Prague, Czech Republic; Department of Nonwoven Textiles, Faculty of Textile Engineering, Technical University of Liberec, Liberec, Czech Republic; Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic.
In this study, we have developed a combined methodological approach for accelerating MSCs proliferation in vitro. A new composite nanofibrous scaffold formed by electrospinning from a mixture of poly-ε-caprolactone and magnetic particles was tested with porcine mesenchymal stem cells and showed great biocompatibility, ability to promote cellular adhesion, accelerate MSCs proliferation and support osteogenic differentiation. These results predetermine this scaffold, due to its cellular and physical attributes, very promising for the acceleration of MSCs proliferation and the regeneration of hard tissues.
KEYWORDS: magnetic particles; mesenchymal stem cells; nanofibers; tissue engineering
Mesenchymal stem cells (MSCs) are multipotent non-hematopoietic cells that can differentiate into cells of mesodermal origin tissues e.g. to bone, cartilage, muscle, fat or nerve cells. They are most often isolated from bone marrow. However, the number of isolated MSCs covers just a small fraction of the cells (between 0.001 % and 0.01 %), which is not enough to treat the tissue defects, so ex vivo expansion is a necessary step for clinical applications of MSCs.
In spite of the fact, that magnetic particles are extensively used in medicine (contrast agents for magnetic resonance imaging (MRI) or heating mediators for cancer therapy (hyperthermia)), their influence on cells and living organisms remains unclear. It leads researchers to investigate the way in which they influence cells and living organisms. Magnetic labelled MSCs increased their proliferation rate approximately five times , but the influence of embedded magnetic particles in nanofibrous material on MSCs proliferation and differentiation has not been fully examined in the past.
In the present study, we have produced composite scaffold made from poly-ε-caprolactone and magnetic particles (PCL-MNPs) by needleless electrospinning. Subsequently, the MSCs were cultured on this scaffold and their biological response was examined. No external magnetic field which could influence cell behaviour was applied during experiment.
Magnetic particles were commercially purchased from Sigma Aldrich. We characterized particles and produced scaffolds them by HR-SEM, powder X-ray diffraction, Mössbauer spectroscopy and magnetization measurements. From the results, magnetic particles showed clearly features of maghemite (g-Fe2O3). Based on the saturation magnetization values we estimated the content of MNPs in the composite as 7.9±0.1 wt %. The MNPs appeared as spherical objects with the size between 50 – 100 nm, forming irregular agglomerates in nanofibrous scaffold (Figure 1).
Figure 1: Electron microscopy of scaffold with magnetic particles.
After characterisation, in vitro testing with porcine MSCs was performed. The biological properties as MSCs metabolic activity, proliferation and alkaline phosphatase activity were monitored for a period of 21 days to test the biocompatibility of the scaffolds and the influence of the magnetic nanoparticles in the PCL on the cells. As shown in the graphs (Figure 2), the viability of the cells cultivated on PCL-MNPs was significantly higher on days 7 and 21 than for the cells seeded on the scaffolds made from PCL. A PicoGreen assay was performed to estimate the DNA values in the scaffolds in order to find out the difference in the cell proliferation on the samples with and without magnetic nanoparticles. Clearly, a significantly higher amount of DNA was recorded on PCL-MNPs on the first day of cultivation. Therefore, MNPs probably contributed to the better cell adhesion. Static cell proliferation on the PCL scaffold was observed over a period of 7 days. After that, the proliferation decreased between days 7 and 21 (P<0.05). Contrary, the cell number seeded on PCL-MNPs increased significantly from day 1 to day 21. This trend is also supported by confocal microscopy observations (Figure 3). To study the effect of magnetic particles incorporated into PCL nanofibers on osteogenic differentiation of MSCs, the alkaline phosphatase activity was measured using an ALP assay. Surprisingly, the cells seeded on the PCL scaffolds showed significantly higher alkaline phosphatase activity on day 1. After that, alkaline phosphatase activity was significantly increased in case of MSCs cultivated on the PCL-MNPs on days 7 and 21. To prove the previous results, the cell spreading and cell morphology were evaluated by confocal microscopy (Figure 3). Obviously, the cells on the PCL-MNPs were localized in larger colonies and had a well spread morphology. At the end of experiment, the cells were fully confluent on the PCL-MNPs, but not in case of PCL.
Figure 2: In vitro testing of the scaffold. The viability of cells was measured by MTS assay, proliferation was monitored via PicoGreen assay and the activity of ALP was determined by way of ALP assay.
One of the key factors for tissue engineering materials is their biocompatibility. Here we used polycaprolactone as a slowly degradable, biocompatible polymer to create nanofiber scaffold with incorporated magnetic particles. Its biocompatibility has been recently demonstrated with fibroblasts  or MSCs . Besides, the PCL with the molecular weight of 45 kDa enables the degradation in vivo in time frame of bone regeneration .
The study was focused on the influence of embedded magnetic nanoparticles in PCL scaffold on MSCs in vitro. Cells showed significantly better viability on days 7 and 21 when seeded on the PCL-MNPs scaffold than on PCL alone. The increase of viability of cells while cultivated on composite material with MNPs have been reported [5, 6], but there has been no evidence of such an increase in cellular viability without using external field until now. The proliferation of cells on PCL-MNPs increased gradually and there was the biggest difference between the PCL-MNPs group and the PCL group on day 21. This fact was clearly confirmed by confocal microscopy, where better cell adhesion and accelerated proliferation was obvious. These results are supported by other studies where good cell adhesion and proliferation was observed during testing of nanofiber scaffolds made from polymeric material with magnetic nanoparticles [7-10]. We also demonstrated that PCL-MNPs scaffolds did not restrict MSCs differentiation when ALP activity increased significantly in cells cultivated on PCL-MNPs. Other authors have also reported the beneficial effect of magnetic particles in polymeric scaffold on osteogenic differentiation of MSCs [11-14].
Nanofiber meshes from PCL are widely used for tissue engineering applications, due to their ability to mimic ECM and promote cellular adhesion, growth and proliferation. Scaffolds enriched by magnetic nanoparticles have shown even better properties for MSCs and their proliferation. Magnetic materials can positively influence cellular adhesion, proliferation and differentiation. Nevertheless, the reason has not been satisfactorily described so far. Magnetic scaffolds can generate a magnetic field to the surroundings, which consequently alters the microenvironment conditions of cells. If each magnetic nanoparticle is considered as a single magnetic domain on nanoscale level, it might affect ion channels and influence cellular processes . Moreover, cells are known to respond to mechanical stimuli, which initiate signalling pathways influencing dynamics of cell membrane. The signals can be transduced via direct activation of mechanosensitive ion channels or through the deformation of cell membrane. MNPs functionalized with specific peptides or antibodies to attach to ion channels or surface receptors of MCSs have been designed [16, 17]. The exposure to magnetic field produced by MNPs led to the membrane deformation of human MCSs and caused the polarization of the membrane, receptor activation and activation of downstream signals. Last but not least, superparamagnetic iron oxide nanoparticles were discovered to accelerate the cell cycle and promote the growth of human MSCs due to the ability to decrease intracellular H2O2 and to change the expression of cell cycle regulators through free iron (Fe) released from lysosomal degradation  .
More information has to be found out to discover the inductor of observed effects. The characteristics of cells cultured on magnetic scaffold were significantly improved in terms of cell adhesion, spreading, proliferation and differentiation, while it has been clearly shown that magnetic particles play a key role for providing such properties to the final composite. The mechanism how can the scaffold help to regenerate the tissue may be multi-factorial.
Figure 3: Cell morphology evaluated by confocal microscopy. Phalloidin-rhodamine was used to stain cytoplasm (red), DNA was stained by Hoechst 33342 (blue). (A) MSCs on PCL scaffold on day 1; (B) MSCs on PCL-MNPs scaffold on day 1; (C) MSCs on PCL scaffold on day 7; (D) MSCs on PCL-MNPs scaffold on day 7; (E) MSCs on PCL scaffold on day 21; (F) MSCs on PCL-MNPs scaffold on day 21.
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