Ofra Ziv-Polat

Novel magnetic fibrin hydrogel scaffolds containing thrombin and growth factors conjugated iron oxide nanoparticles for tissue engineering

Novel tissue-engineered magnetic fibrin hydrogel scaffolds were prepared by the interaction of thrombin-conjugated iron oxide magnetic nanoparticles with fibrinogen. In addition, stabilization of basal fibroblast growth factor (bFGF) was achieved by the covalent and physical conjugation of the growth factor to the magnetic nanoparticles. Adult nasal olfactory mucosa (NOM) cells were seeded in the transparent fibrin scaffolds in the absence or presence of the free or conjugated bFGF-iron oxide nanoparticles. The conjugated bFGF enhanced significantly the growth and differentiation of the NOM cells in the fibrin scaffolds, compared to the same or even five times higher concentration of the free bFGF. In the presence of the bFGF-conjugated magnetic nanoparticles, the cultured NOM cells proliferated and formed a three-dimensional interconnected network composed mainly of tapered bipolar cells. The magnetic properties of these matrices are due to the integration of the thrombin- and bFGF-conjugated magnetic nanoparticles within the scaffolds. The magnetic properties of these scaffolds may be used in future work for various applications, such as magnetic resonance visualization of the scaffolds after implantation and reloading the scaffolds via magnetic forces with bioactive agents, eg, growth factors bound to the iron oxide magnetic nanoparticles.

The Role of Neurotrophic Factors Conjugated to Iron Oxide Nanoparticles in Peripheral Nerve Regeneration: In Vitro Studies

Local delivery of neurotrophic factors is a pillar of neural repair strategies in the peripheral nervous system. The main disadvantage of the free growth factors is their short half-life of few minutes. In order to prolong their activity, we have conjugated to iron oxide nanoparticles three neurotrophic factors: nerve growth factor (βNGF), glial cell-derived neurotrophic factor (GDNF), and basic fibroblast growth factor (FGF-2). Comparative stability studies of free versus conjugated factors revealed that the conjugated neurotrophic factors were significantly more stable in tissue cultures and in medium at 37°C. The biological effects of free versus conjugated neurotrophic factors were examined on organotypic dorsal root ganglion (DRG) cultures performed in NVR-Gel, composed mainly of hyaluronic acid and laminin. Results revealed that the conjugated neurotrophic factors enhanced early nerve fiber sprouting compared to the corresponding free factors. The most meaningful result was that conjugated-GDNF, accelerated the onset and progression of myelin significantly earlier than the free GDNF and the other free and conjugated factors. This is probably due to the beneficial and long-acting effect that the stabilized conjugated-GDNF had on neurons and Schwann cells. These conclusive results make NVR-Gel enriched with conjugated-GDNF, a desirable scaffold for the reconstruction of severed peripheral nerve.

Magnetic Scaffolds Enriched with Bioactive Nanoparticles for Tissue Engineering

Novel magnetic fibrin hydrogel scaffolds for cell implantation and tissue engineering are reported. The magnetic scaffolds are produced by the interaction between thrombin-conjugated maghemite nanoparticles of narrow size distribution and fibrinogen. These scaffolds, enriched with growth factor conjugated fluorescent maghemite nanoparticles, provide a supporting 3D environment for massive proliferation of various cell types, and can be successfully visualized by MRI.

Enhancement of the growth and differentiation of nasal olfactory mucosa cells by the conjugation of growth factors to functional nanoparticles.

Growth factors are critical components in the tissue engineering approach. Basic fibroblast growth factor (bFGF), a representative growth factor, stimulates the cellular functions of various cells and has been used extensively for the repair and regeneration of tissues. The in vivo half-life time of free bFGF is short, about 3-10 min, due to rapid enzymatic degradation. Stabilization of the bFGF was accomplished by the covalent or physical conjugation of this factor to fluorescent maghemite (γ-Fe(2)O(3)) nanoparticles. In the present study, nasal olfactory mucosa (NOM) cells from adult rats were cultured in suspension on chitosan microcarriers (MCs) in the presence of the nonconjugated or bFGF-conjugated nanoparticles, or the free factor. The floating cells/nonconjugated, conjugated, or free bFGF/MCs aggregates were then seeded in a viscous gel. In this manuscript, we are the first to report that the stabilization of the factor by its conjugation to these nanoparticles significantly improved NOM cell-proliferation properties (migration, growth, and differentiation), compared to the same concentration, or even five times higher, of the free factor. This novel approach may significantly contribute to the advancement of the tissue engineering field.

Nanotechnology versus stem cell engineering: in vitro comparison of neurite inductive potentials.

Purpose Innovative nerve conduits for peripheral nerve reconstruction are needed in order to specifically support peripheral nerve regeneration (PNR) whenever nerve autotransplantation is not an option. Specific support of PNR could be achieved by neurotrophic factor delivery within the nerve conduits via nanotechnology or stem cell engineering and transplantation. Methods Here, we comparatively investigated the bioactivity of selected neurotrophic factors conjugated to iron oxide nanoparticles (np-NTFs) and of bone marrow-derived stem cells genetically engineered to overexpress those neurotrophic factors (NTF-BMSCs). The neurite outgrowth inductive activity was monitored in culture systems of adult and neonatal rat sensory dorsal root ganglion neurons as well as in the cell line from rat pheochromocytoma (PC-12) cell sympathetic culture model system. Results We demonstrate that np-NTFs reliably support numeric neurite outgrowth in all utilized culture models. In some aspects, especially with regard to their long-term bioactivity, np-NTFs are even superior to free NTFs. Engineered NTF-BMSCs proved to be less effective in induction of sensory neurite outgrowth but demonstrated an increased bioactivity in the PC-12 cell culture system. In contrast, primary nontransfected BMSCs were as effective as np-NTFs in sensory neurite induction and demonstrated an impairment of neuronal differentiation in the PC-12 cell system. Conclusion Our results evidence that nanotechnology as used in our setup is superior over stem cell engineering when it comes to in vitro models for PNR. Furthermore, np-NTFs can easily be suspended in regenerative hydrogel matrix and could be delivered that way to nerve conduits for future in vivo studies and medical application.

Synthesis, fluorescence and biodistribution of a bone-targeted near-infrared conjugate

Enhanced imaging of early-stage bone abnormalities, such as primary tumors or metastases is highly required as the widely-used bone scan frequently lacks the desired sensitivity. Near IR (NIR) fluorescence imaging affords high contrast and enhanced sensitivity, as body tissue expresses minimal autofluorescence at NIR range (600-1200 nm). Indocyanine green (ICG), a biocompatible NIR dye, is widely used in the imaging of various organs, such as liver, heart and blood circulation. We report the preparation and in-vivo testing of a bone-targeting ICG derivative, in comparison to the parent molecule(s). Since ICG itself is chemically unreactive, and could not form conjugates, we prepared two novel ICG conjugatable derivatives. The overall ICG structure was maintained while only a replacement of one or two sulfonate groups with carboxylic acids resulted in new linkers for covalent binding to biomolecules. These derivatives were evaluated for their fluorescence and biodistribution in comparison to ICG and were found to be comparable. One of the novel ICG-derivatives was conjugated to a bone-targeting moiety and this new compound was found to bind to growing regions of the skeleton, and emit fluorescence for as long as two weeks in young mice.

Application of iron oxide anoparticles in neuronal tissue engineering

Since the introduction of nanotechnology, nanoscale materials have developed rapidly and have been applied in various fields including in the pharmaceutical industry, medicine, and tissue engineering. Among a variety of nanomaterials, magnetic iron oxide nanoparticles (IONPs) have been intensively investigated for numerous in vivo applications such as gene and drug delivery, diagnostics, cell labeling and sorting. Compared to nanoparticles made from other materials, IONPs have several additional unique applications due to their magnetic properties, for example, magnetic cell separation, magnetic resonance imaging (MRI) and X-ray contrast agents, tumor hyperthermia, and in the targeting of bioactive agents immobilized on magnetic materials with the presence of an external magnetic field (Gupta and Gupta, 2005). IONPs are considered biocompatible and biodegradable (reviewed by Wang et al. (2009)). Indeed, some IONPs preparations have been approved for clinical applications by the Food and Drug Administration (FDA) and/or the European Commission (EC) and are commercially available, such as Lumirem (as a contrast agent for MRI for the gastrointestinal tract) and Feraheme (for the treatment of iron deficiency anemia) (Cortajarena et al., 2014).

Neuronal and Glial Growth in Organotypic Cultures after Vitrification

Fetal rat dorsal root ganglia (DRG) and spinal cords (SC) slices from rat fetuses were vitrified in a new semiautomatic vitrification system, cooled in sterile slush liquid air (SLA) and stored in a special sterile sealed container in liquid nitrogen (LN). Upon warming organotypic stationary cultures were performed in using NVR-Gel (composed mainly from hyaluronic acid and laminin) and enriched with neuronal factors conjugated to iron oxide nanoparticles. Evaluation of cultures was made by daily phase-contrast microscopy observations and by immune fluorescent staining. Results revealed that SC neurons maintained their multipolar shape and regrew dendrites and axons. The round shape DRG neurons exhibited euchromatic nuclei with prominent nucleoli and an active regeneration of nerve processes. Migration of both neurons and flat cells (fibroblasts and glia Schwann cells) started within 48 hours after seeding and intensified in the upcoming days. In conclusion, it can be said that the using a semi-automatic vitrification, sterile vitrification and sterile storage of neuronal tissues from the CNS and the PNS is a successful advanced technology for the preservation of neurons and glial cells, as shown in the regain of a full regular growth pattern in culture. This may an important step towards clinical use in the reconstruction of severe peripheral nerves and spinal cord injuries.