Šárka Kubinová

Nanotechnologies in regenerative medicine.

Abstract Nanotechnology offers promising perspectives in biomedical research as well as in clinical practice. To cover some of the latest nanotechnology trends in regenerative medicine, this review will focus on the use of nanomaterials for tissue engineering and cell therapy. Nanofibrous materials that mimic the native extracellular matrix and promote the adhesion of various cells are being developed as tissue-engineered scaffolds for the skin, bone, vasculature, heart, cornea, nervous system, and other tissues. A range of novel materials has been developed to enhance the bioactive or therapeutic properties of these nanofibrous scaffolds via surface modifications, including the immobilization of functional cell-adhesive ligands and bioactive molecules such as drugs, enzymes and cytokines. As a new approach, nanofibers prepared by using industrial scale needleless technology have been recently introduced, and their use as scaffolds to treat spinal cord injury or as cell carriers for the regeneration of the injured cornea is the subject of much current study. Cell therapy is a modern approach of regenerative medicine for the treatment of various diseases or injuries. To follow the migration and fate of transplanted cells, superparamagnetic iron oxide nanoparticles have been developed for cell labeling and non-invasive MRI monitoring of cells in the living organism, with successful applications in, e.g, the central nervous system, heart, liver and kidney and also in pancreatic islet and stem cell transplantation.

Treatment of Ocular Surface Injuries by Limbal and Mesenchymal Stem Cells Growing on Nanofiber Scaffolds

Stem cell (SC) therapy represents a promising approach to treat a wide variety of injuries, inherited diseases, or acquired SC deficiencies. One of the major problems associated with SC therapy remains the absence of a suitable matrix for SC growth and transfer. We describe here the growth and metabolic characteristics of mouse limbal stem cells (LSCs) and mesenchymal stem cells (MSCs) growing on 3D nanofiber scaffolds fabricated from polyamide 6/12 (PA6/12). The nanofibers were prepared by the original needleless electrospun Nanospider technology, which enables to create nanofibers of defined diameter, porosity, and a basis weight. Copolymer PA6/12 was selected on the basis of the stability of its nanofibers in aqueous solutions, its biocompatibility, and its superior properties as a matrix for the growth of LSCs, MSCs, and corneal epithelial and endothelial cell lines. The morphology, growth properties, and viability of cells grown on PA6/12 nanofibers were comparable with those grown on plastic. LSCs labeled with the fluorescent dye PKH26 and grown on PA6/12 nanofibers were transferred onto the damaged ocular surface, where their seeding and survival were monitored. Cotransfer of LSCs with MSCs, which have immunosuppressive properties, significantly inhibited local inflammatory reactions and supported the healing process. The results thus show that nanofibers prepared from copolymer PA6/12 represent a convenient scaffold for growth of LSCs and MSCs and transfer to treat SC deficiencies and various ocular surface injuries.

Cholesterol-modified superporous poly(2-hydroxyethyl methacrylate) scaffolds for tissue engineering

Modifications of poly(2-hydroxyethyl methacrylate) (PHEMA) with cholesterol and laminin have been developed to design scaffolds that promote cell-surface interaction. Cholesterol-modified superporous PHEMA scaffolds have been prepared by the bulk radical copolymerization of 2-hydroxyethyl methacrylate (HEMA), cholesterol methacrylate (CHLMA) and the cross-linking agent ethylene dimethacrylate (EDMA) in the presence of ammonium oxalate crystals to introduce interconnected superpores in the matrix. With the aim of immobilizing laminin (LN), carboxyl groups were also introduced to the scaffold by the copolymerization of the above monomers with 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA). Subsequently, the MCMEMA moiety in the resulting hydrogel was hydrolyzed to [2-(methacryloyloxy)ethoxy]acetic acid (MOEAA), and laminin was immobilized via carbodiimide and N-hydroxysulfosuccinimide chemistry. The attachment, viability and morphology of mesenchymal stem cells (MSCs) were evaluated on both nonporous and superporous laminin-modified as well as laminin-unmodified PHEMA and poly(2-hydroxyethyl methacrylate-co-cholesterol methacrylate) P(HEMA-CHLMA) hydrogels. Neat PHEMA and laminin-modified PHEMA (LN-PHEMA) scaffolds facilitated MSC attachment, but did not support cell spreading and proliferation; the viability of the attached cells decreased with time of cultivation. In contrast, MSCs spread and proliferated on P(HEMA-CHLMA) and LN-P(HEMA-CHLMA) hydrogels.

Nanotechnology for treatment of stroke and spinal cord injury

The use of nanotechnology in cell therapy and tissue engineering offers promising future perspectives for brain and spinal cord injury treatment. Stem cells have been shown to selectively target injured brain and spinal cord tissue and improve functional recovery. To allow cell detection, superparamagnetic iron-oxide nanoparticles can be used to label transplanted cells. MRI is then a suitable method for the in vivo tracking of grafted cells in the host organism. CNS, and particularly spinal cord, injury is accompanied by tissue damage and the formation of physical and biochemical barriers that prevent axons from regenerating. One aspect of nanomedicine is the development of biologically compatible nanofiber scaffolds that mimic the structure of the extracellular matrix and can serve as a permissive bridge for axonal regeneration or as a drug-delivery system. The incorporation of biologically active epitopes and/or the utilization of these scaffolds as stem cell carriers may further enhance their therapeutic efficacy.

Highly efficient magnetic targeting of mesenchymal stem cells in spinal cord injury.

The transplantation of mesenchymal stem cells (MSC) is currently under study as a therapeutic approach for spinal cord injury, and the number of transplanted cells that reach the lesioned tissue is one of the critical parameters. In this study, intrathecally transplanted cells labeled with superparamagnetic iron oxide nanoparticles were guided by a magnetic field and successfully targeted near the lesion site in the rat spinal cord. Magnetic resonance imaging and histological analysis revealed significant differences in cell numbers and cell distribution near the lesion site under the magnet in comparison to control groups. The cell distribution correlated well with the calculated distribution of magnetic forces exerted on the transplanted cells in the subarachnoid space and lesion site. The kinetics of the cells’ accumulation near the lesion site is described within the framework of a mathematical model that reveals those parameters critical for cell targeting and suggests ways to enhance the efficiency of magnetic cell delivery. In particular, we show that the targeting efficiency can be increased by using magnets that produce spatially modulated stray fields. Such magnetic systems with tunable geometric parameters may provide the additional level of control needed to enhance the efficiency of stem cell delivery in spinal cord injury.

Human Mesenchymal Stem Cells Modulate Inflammatory Cytokines after Spinal Cord Injury in Rat

Transplantation of mesenchymal stem cells (MSC) improves functional recovery in experimental models of spinal cord injury (SCI); however, the mechanisms underlying this effect are not completely understood. We investigated the effect of intrathecal implantation of human MSC on functional recovery, astrogliosis and levels of inflammatory cytokines in rats using balloon-induced spinal cord compression lesions. Transplanted cells did not survive at the lesion site of the spinal cord; however, functional recovery was enhanced in the MSC-treated group as was confirmed by the Basso, Beattie, and Bresnahan (BBB) and the flat beam test. Morphometric analysis showed a significantly higher amount of remaining white matter in the cranial part of the lesioned spinal cords. Immunohistochemical analysis of the lesions indicated the rearrangement of the glial scar in MSC-treated animals. Real-time PCR analysis revealed an increased expression of Irf5, Mrc1, Fgf2, Gap43 and Gfap. Transplantation of MSCs into a lesioned spinal cord reduced TNFα, IL-4, IL-1β, IL-2, IL-6 and IL-12 and increased the levels of MIP-1α and RANTES when compared to saline-treated controls. Intrathecal implantation of MSCs reduces the inflammatory reaction and apoptosis, improves functional recovery and modulates glial scar formation after SCI, regardless of cell survival. Therefore, repeated applications may prolong the beneficial effects induced by MSC application.

Regenerative medicine for the treatment of spinal cord injury: more than just promises?

Spinal cord injury triggers a complex set of events that lead to tissue healing without the restoration of normal function due to the poor regenerative capacity of the spinal cord. Nevertheless, current knowledge about the intrinsic regenerative ability of central nervous system axons, when in a supportive environment, has made the prospect of treating spinal cord injury a reality. Among the range of strategies under investigation, cell‐based therapies offer the most promising results, due to the multifactorial roles that these cells can fulfil. However, the best cell source is still a matter of debate, as are clinical issues that include the optimal cell dose as well as the timing and route of administration. In this context, the role of biomaterials is gaining importance. These can not only act as vehicles for the administered cells but also, in the case of chronic lesions, can be used to fill the permanent cyst, thus creating a more favourable and conducive environment for axonal regeneration in addition to serving as local delivery systems of therapeutic agents to improve the regenerative milieu. Some of the candidate molecules for the future are discussed in view of the knowledge derived from studying the mechanisms that facilitate the intrinsic regenerative capacity of central nervous system neurons. The future challenge for the multidisciplinary teams working in the field is to translate the knowledge acquired in basic research into effective combinatorial therapies to be applied in the clinic.

Biomaterials combined with cell therapy for treatment of spinal cord injury

Spinal cord injury (SCI) is a devastating traumatic injury resulting in paralysis or sensory deficits due to tissue damage and the poor ability of axons to regenerate across the lesion. Despite extensive research, there is still no effective treatment that would restore lost function after SCI. A possible therapeutic approach would be to bridge the area of injury with a bioengineered scaffold that would create a stimulatory environment as well as provide guidance cues for the re-establishment of damaged axonal connections. Advanced scaffold design aims at the fabrication of complex materials providing the concomitant delivery of cells, neurotrophic factors or other bioactive substances to achieve a synergistic effect for treatment. This review summarizes the current utilization of scaffolding materials for SCI treatment in terms of their physicochemical properties and emphasizes their use in combination with various cell types, as well as with other combinatorial approaches promoting spinal cord repair.

Cell death induced by ozone and various non-thermal plasmas: therapeutic perspectives and limitations

Non-thermal plasma has been recognized as a promising tool across a vast variety of biomedical applications, with the potential to create novel therapeutic methods. However, the understanding of the molecular mechanisms behind non-thermal plasma cellular effects remains a significant challenge. In this study, we show how two types of different non-thermal plasmas induce cell death in mammalian cell cultures via the formation of multiple intracellular reactive oxygen/nitrogen species. Our results showed a discrepancy in the superoxide accumulation and lysosomal activity in response to air and helium plasma, suggesting that triggered signalling cascades might be grossly different between different plasmas. In addition, the effects of ozone, a considerable component of non-thermal plasma, have been simultaneously evaluated and have revealed much faster and higher cytotoxic effects. Our findings offer novel insight into plasma-induced cellular responses, and provide a basis for better controlled biomedical applications.

Cyclosporine A-loaded and stem cell-seeded electrospun nanofibers for cell-based therapy and local immunosuppression

Cyclosporine A (CsA), a potent immunosuppressive drug with low water solubility, was dissolved in poly(L-lactic acid) (PLA) solution, and nanofibers were fabricated from this mixture by electrospinning technology. The addition of CsA into the PLA solution and the conditions of the electrospinning process did not influence the structure of the nanofibers nor affect the pharmacological activity of CsA. Study of the CsA release behavior in culture medium showed a release for at least 96 h. After the topical application of CsA-loaded nanofibers on skin allografts in vivo, the release was significantly slower and about 35% of the drug was still retained in the nanofibers on day 8. The addition of CsA-loaded nanofibers into cultures of mouse spleen cells stimulated with Concanavalin A selectively inhibited T cell functions; the activity of stimulated macrophages or the growth of non-T-cell populations was not suppressed in the presence of CsA-loaded nanofibers. The covering of skin allografts with CsA-loaded nanofibers significantly attenuated the local production of the proinflammatory cytokines IL-2, IFN-γ and IL-17. These results suggest that CsA-loaded electrospun nanofibers can serve as effective drug carriers for the local/topical suppression of an inflammatory reaction and simultaneously could be used as scaffolds for cell-based therapy.