NANOTECHNOLOGY AND BIOMATERIALS IN MEDICINE

Transcription

1 NANOTECHNOLOGY AND BIOMATERIALS IN MEDICINE Šárka KUBINOVÁ a, Pavla JENDELOVÁ a,b, Petr LESNÝ a,b, Vladimír HOLÁŇ c, Eva SYKOVÁ a,b a Institute of Experimental Medicine AS CR, v.v.i., Prague, Czech Republic, b Department of Neuroscience and Center for Cell Therapy and Tissue Repair, 2 nd Medical Faculty, Charles University, Prague, Czech Republic c Institute of Molecular Genetics AS CR, v.v.i., Prague, Czech Republic, Abstract Nanotechnology is a rapidly developing field that offers promising future perspectives for medical research. Our studies focus on the use of nanotechnology in cell therapy and tissue engineering for the treatment of brain and spinal cord injury, corneal defects and chronic wounds. Stem cells have been shown to selectively target injured brain and spinal cord tissue and improve functional recovery. To label transplanted cells and subsequently follow their fate in the host organism, superparamagnetic iron oxide (SPIO) nanoparticles visualized by magnetic resonance imaging (MRI) have been developed. Our studies have demonstrated that grafted adult as well as embryonic stem cells labeled with SPIO nanoparticles migrate into a lesion site in the brain as well as in the spinal cord, where they then elicit functional improvement. Spinal cord injury is accompanied by tissue damage and the formation of physical and biochemical barriers that prevent axons from regenerating. Biologically compatible nanofiber or hydrogel scaffolds can serve as a permissive bridge for axonal regeneration and the re-establishment of damaged connections. Electrospun nanofibers provide a three-dimensional cell culture surface that mimics the extracellular matrix of native tissue and can be used as carriers for stem cells or as a drug delivery system. The growth of mesenchymal and limbal stem cells was evaluated on various electrospun nanofibers prepared by the unique Nanospider TM technology based on industrial scale needleless technologies. The cell culture results showed that these nanofibers were suitable for cell attachment and proliferation. Nanofiber scaffolds were then used as cell carriers for the regeneration of the injured cornea and skin wounds. 1. CELL THERAPY IN THE CENTRAL NERVOUS SYSTEM 1.1. Mesenchymal stem cells Cell therapy is one strategy for treating a variety of central nervous system disorders, and some successful therapeutic approaches have already been transferred to clinical use [1]. The use of mesenchymal stem cells (MSC) presents several advantages that highlight their promising therapeutic potential in human medicine. MSC are present in adult tissue, primarily in the bone marrow, but they can be found in fat, skin, liver, peripheral blood, umbilical cord, etc; they are easy to isolate and expand, and their immunomodulatory properties offer potential for their use in cell therapy even in allogeneic settings. MSC are multipotent cells, i.e. they can differentiate not only into cells of mesenchymal origin, but also into nonmesenchymal cell phenotypes. MSC produce a variety of growth factors, chemokines and other bioactive factors supporting regeneration, and in addition, they can be genetically modified and can serve as carriers for drug delivery [2]. After transplantation into the neuronal tissue or intravenous administration, MSC have been shown to respond to intrinsic signals and migrate toward the site of injury, reduce the lesion volume and elicit functional improvement [1]. 1

2 1.2 Nanoparticles for labeling of stem cells A crucial aspect of successful cell transplantation is tracking and monitoring the grafted cells in the host organism. To label transplanted cells, superparamagnetic iron oxide (SPIO) nanoparticles visualized by magnetic resonance imaging (MRI) have been developed. SPIO nanoparticles are negative contrast agents that selectively shorten T2 relaxation time and thus can be detected in the tissue as a hypointense (dark) signal. MRI as a non-invasive method allows the tracking of grafted cells and the real-time monitoring of their migration and persistence in the targeted tissue. SPIO nanoparticles usually consist of a crystalline iron oxide core and a polymer shell. A commercial contrast agent based on dextran-coated SPIO nanoparticles, Endorem, has been shown to be a suitable contrast agent for labeling rat and human MSC or embryonic stem cells (ESC) [3]. Various modifications of SPIO nanoparticles have been developed to prevent their aggregation, to induce the efficient internalization of the contrast agent into the cell and to minimize any deleterious effects on cellular function. In collaboration with the Institute of Macromolecular Chemistry AS CR, we have developed and patented several types of iron oxide nanoparticles with poly-l-lysine, D-mannose and poly(n,n-dimethylacrylamide) coatings. Nanoparticles with these modified coatings exhibited better internalization into the cells and easier MRI detection with a lower concentration of iron within the cells when compared with Endorem [4]. Fig.1. (A) Scheme of an iron nanoparticle. The contrast agent Endorem consists of a superparamagnetic Fe 3O 4 core coated by a dextran shell. (B) Scheme of poly-l-lysine coating of iron oxide nanoparticle. (C) Rat MSC culture labeled with SPIO nanoparticles and stained for Prussian blue. (D) Transmission electron micrograph of a cluster of iron nanoparticles surrounded by a cell membrane. 2

3 1.3. In vivo tracking of stem cells in brain and spinal cord lesion The fate of ESC and bone marrow MSC labeled with Endorem was studied in rats with a cortical or spinal cord lesion, i.e., in models of stroke and spinal cord injury (SCI), respectively. Cells were either grafted intracerebrally, contralaterally to a cortical photochemical lesion, or injected intravenously. One week after grafting, a hypointense MR signal was found in the lesion, which intensified during the second and third weeks regardless of the route of administration; its intensity corresponded to iron (Prussian blue) staining and electron microscopy observation. In rats with a SCI, the intravenous injection of MSC significantly improved the recovery of hindlimb motor function. The fate of transplanted MSC labeled with Endorem was followed by ex vivo MRI; staining for Prussian blue revealed many cells labeled with nanoparticles in the lesion site, whereas the lesion cavities were significantly smaller than in control animals [3]. To replace or regenerate damaged tissue, transplanted cells have been applied directly in suspension or transported by a carrier or scaffold that additionally forms a tissue bridge. To facilitate the regeneration of injured spinal cord, a cell-polymer construct was developed using Endorem-labeled MSC seeded on a poly(2-hydroxyethylmethacrylate) hydrogel (PHEMA) and implanted into a spinal cord hemisection. Six weeks after implantation, the hydrogel re-established the anatomic continuity of the tissue with axonal ingrowth into the hydrogel. MRI and Prussian blue staining confirmed positively stained cells within the hydrogel [3]. Our studies demonstrated that SPIO nanoparticle labeling together with MRI visualization is a suitable method for the in vivo tracking of transplanted cells in the host organism. Fig.2 (A) Cortical photochemical lesion visible on MRI 2 weeks after induction as a hyperintensive (light) area. (B) Both the cell implant (MSCs in the hemisphere contralateral to the lesion) and the lesion are hypointense (dark) 2 weeks after implantation. (C) A few cells weakly stained for Prussian blue were found in the photochemical lesion in animals without implanted cells. (D and E) A hypointense signal in the lesion was seen 7 days after the i.v. injection of Endorem-labeled rat MSCs (D) and persisted for 7 weeks (E). Insets show a higher magnification view of the lesion. (F) Massive invasion of rat MSCs (Prussian blue staining counterstained with hematoxylin) into a photochemical lesion 7 weeks after i.v. injection. 3

4 2. NANOFIBERS AS SCAFFOLDS IN TISSUE ENGINEERING 2.1. Electrospun nanofibers As scaffolds in tissue engineering, electrospun nanofibers provide a new porous cell culture surface mimicking the extracellular matrix of native tissue and organs. The role of the scaffold is to support seeded cells before in vivo transplantation, thus the scaffold requirements include biocompatibility, controlled porosity and permeability, suitable mechanical properties comparable to the tissue and, additionally, support for cell attachment and proliferation. The micro-architecture of the scaffold should consequently improve tissue organization and function. In the electrospinning process, nanofibers are created as polymeric jets from the surface of a polymeric solution in a very high intensity electrostatic field (5-100kV). The nanofibers can be formed either at the tip of a capillary tube (needle or capillary spinners) [5] or from liquid surfaces on a rotating spinning electrode (needleless Nanospider TM technology, [6-7]. Compared to needle electrospinning, the Nanospider TM technology enables the production of nanofibers on an industrial scale. g Fig. 3. Principle of needleless electrospinning b e f d a c a - electrode metal roller as positive electrode b fiber-forming polymer layer c - reservoir of polymer solution d - textile substrate (supportive material) e - fiber formation direction f - electrode earthing shield A thin layer of polymer solution film (Fig.3b) is raised by a metal roller (Fig.3a), which is at the same time the positive electrode. This electrode is partially submerged in the polymer solution (Fig.3c), and nanofibers are created between the spinning electrode and the collector (Fig.3f, negative electrode) due to the very high intensity electrostatic field (Fig.3e). The solvent evaporates and the fibers stretch at room or elevated temperature and are collected by a polypropylene nonwoven fabric (Fig.3d) on the negative electrode (Fig.3f) Cell growth on nanofibers Nanofibers were prepared from chitosan, gelatin, polyamide (PA6/12), polyacrylonitrile (PAN), poly-εcaprolactone (PCL), polylactide (PLA) and polyurethane (PUR) by Elmarco, Ltd. The Nanospider TM technology enables a continual manufacturing process with guaranteed quality of the samples. A disadvantage of this technology when utilized on an industrial scale is the greater amount of undesired residual solvents and monomers remaining in the nanofibrous material, leading to cytotoxicity. Testing cell viability revealed that the toxicity of the nanofibers acquired during the manufacturing process was removed by a washing out procedure in distilled water. The viability of human MSC grown on nanofibers (except for chitosan) did not significantly differ from the viability of control cells grown on polystyrene tissue culture dishes (Fig.4). The morphology of the cytoskeleton and the adhesion of cells growing on nanofibers were evaluated using immunofluorescent staining for the cytoskeletal protein F-actin and the focal adhesion protein vinculin (Fig.5). Except for chitosan, all tested nanofibers supported the formation of F-actin cytoskeletal filaments and vinculin adhesion complexes. Our results showed that nanofibers produced by the 4

5 Nanospider TM technology support stem cell adhesion, growth and proliferation and have considerable potential for biomedical applications as stem cell carriers. Fig. 4. A proliferation assay performed after 1 and 3 days of human MSC culture. The viability of MSC grown on all nanofibers, except for chitosan, did not significantly differ from the control viability on polystyrene tissue culture dishes. Fig. 5. Representative confocal micrographs of human MSC morphology cultured on PA 6/12 nanofibers. Staining for F- actin cytoskeletal filaments on non-oriented PA6/12 (A, C) and oriented PA6/12 (B) and vinculin adhesion complexes (D). Fig. 6. SEM micrographs of human MSC cultured on gelatin (A), PCL (B), PLA (C) and PUR (D). The structure of gelatin and PLA enabled the cells to grow between the fibers inside the porous structure. On the other hand, the dense structure of PCL and PUR allowed the cells to spread and grow rather on the surface of the nanofibrous substrate. 5

6 2.3. Nanofibers as stem cell carriers for corneal regeneration Corneal defects or ocular surface injuries represent one of the most frequent causes of blindness. If the limbus, where stem cells for corneal renewal and regeneration reside, is damaged, the cornea cannot heal properly and is overgrown by cells originating from the conjunctiva; this process leads to the loss of vision. The only way to treat such limbal stem cell (LSC) deficiencies is limbal transplantation or a transfer of LSC. It has been shown that LSC can be grown in vitro in tissue culture [8]. Nanofibers have proven to be a promising matrix for the growth of LSC and their transfer onto the damaged ocular surface. From a panel of nanofibers prepared from various polymers, we selected nanofibers fabricated from PA6/12. We showed that mouse LSC grow comparably well on plastic and these nanofibers. The mouse LSC were labeled in vitro with a vital fluorescent dye, PKH26, co-cultured on nanofibers with MSC (1:1) and transferred onto the damaged ocular surface of mice. The nanofiber scaffolds with labelled cells were fixed with the cell side facing down onto the ocular surface. Three days after cell transfer, the nanofibers were removed and the seeding and survival of the labelled cells on the ocular surface were monitored (Fig.7). The local inflammatory reaction occuring after the injury to the ocular surface, and characterized by the expression of genes for IL-2, IFN- and IL-6, was significantly attenuated in the recipients of nanofibers with LSC and MSC (Fig.8). The results showed that LSC transferred using a nanofiber scaffold can be detected on the ocular surface 7 and 14 days after cell transfer and that the transfer of stem cell improved corneal healing and significantly inhibited the local inflammatory reaction. Fig.7. Transfer of PKH26-labelled LSC onto the damaged ocular surface and detection of the transferred cells (A). Control normal, undamaged cornea, D2 - damaged cornea covered with nanofibers with labelled cells (white line), 2 days after cell transfer, D7 - cornea 7 days after cell transfer, D14 cornea 14 days after cell transfer. Fig.8. The local inflammatory reaction, as detected by the expression of genes for IL-2, IFN- and IL-6, was significantly inhibited by nanofibers with LSC and MSC. 6

7 2.4. Nanofibers as wound dressings The efficacy of gelatin and PCL as scaffolds in wound healing was examined using a full thickness wound healing test on rats. Compared to control wounds covered with gauze, gelatin, but not PCL, significantly increased the wound closure rate (Fig.9A). Histological evaluation using PCNA (proliferating cell nuclei) staining revealed improved re-epithelization with both gelatin and PCL wound dressings (Fig.9B). Fig.9. (A) Percentage of wound closure after 5 and 10 days post-wounding. (B) Full thickness skin wound stained for PCNA after 10 days of healing covered with control gauze or a PCL dressing. 3. FURTHER PERSPECTIVES AND CONCLUSIONS The development of efficient paramagnetic labeling together with the noninvasive MRI technique enables researchers to follow the fate of transplanted cells in the host organism and advances cell therapy further towards clinical application. It allows investigators to evaluate the effect of cell therapy in patients with various disorders, brain or spinal cord injuries and thus to establish the optimal conditions in terms of the number of transplanted cells, the route of their administration and the defined therapeutic window. Electrospun nanofibers produced by the needleless Nanospider TM technology support cell adhesion and growth and have a promising potential for biomedical applications as cell-seeded scaffolds for regeneration of the injured cornea or as wound dressings. References: [1] Syková E., Jendelová P. Migration, fate and in vivo imaging of adult stem cells in the CNS. Cell Death Differ, 2007, 14, [2] Li Y, Chopp M. Marrow stromal cell transplantation in stroke and traumatic brain injury. Neurosci Lett, 2009, 456, [3] Syková E., Jendelová P. Magnetic resonance tracking of transplanted stem cells in rat brain and spinal cord. Neurodegener Dis, 2006, 3, [4] Horák D., Babič M., Jendelová P. et al. Effect of different magnetic nanoparticle coatings on the efficiency of stem cell labeling. J Magn Magn Mater, 2009, in press. 7

8 [5] Taylor G.F., m.d. V.D. Electrically Driven Jets. Proc. R. Soc. London Ser., 1969, A 313, [6] Lukáš D., Sarkar A., Pokorný P. Self-organization of jets in electrospinning from free liquid surface: A generalized approach. J Appl Phys, 2008,103, [7] Jirsák O., Sanetrník F., Lukáš D. et al. U.S. patent No. WO , (17 March) [8] Pellegrini G., Rama P., Mavilio F. et al. Epithelial stem cells in corneal regeneration and epidermal gene therapy. J Pathol, 2009, 217, Acknowledgements This study was supported by KAN , AVOZ , IAA , LCC554, 1M0538, and 1M