In this study, spherical or hexagonal NaYF4:Yb,Er nanoparticles (UCNPs) with sizes of 25 nm (S-UCNPs) and 120 nm (L-UCNPs) were synthesized by high-temperature coprecipitation and subsequently modified with three kinds of polymers. These included poly(ethylene glycol) (PEG) and poly(N,N-dimethylacrylamide-co-2-aminoethylacrylamide) [P(DMA-AEA)] terminated with an alendronate anchoring group, and poly(methyl vinyl ether-co-maleic acid) (PMVEMA). The internalization of nanoparticles by rat mesenchymal stem cells (rMSCs) and C6 cancer cells (rat glial tumor cell line) was visualized by electron microscopy and the cytotoxicity of the UCNPs and their leaches was measured by the real-time proliferation assay. The comet assay was used to determine the oxidative damage of the UCNPs. An in vivo study on mice determined the elimination route and potential accumulation of UCNPs in the body. The results showed that the L- and S-UCNPs were internalized into cells in the lumen of endosomes. The proliferation assay revealed that the L-UCNPs were less toxic than S-UCNPs. The viability of rMSCs incubated with particles decreased in the order S-UCNP@Ale-(PDMA-AEA) > S-UCNP@Ale-PEG > S-UCNPs > S-UCNP@PMVEMA. Similar results were obtained in C6 cells. The oxidative damage measured by the comet assay showed that neat L-UCNPs caused more oxidative damage to rMSCs than all coated UCNPs while no difference was observed in C6 cells. An in vivo study indicated that L-UCNPs were eliminated from the body via the hepatobiliary route; L-UCNP@Ale-PEG particles were almost eliminated from the liver 96 h after intravenous application. Pilot fluorescence imaging confirmed the limited in vivo detection capabilities of the nanoparticles.

• Klíčová slova
• biological applications, toxicity, upconverting nanoparticles,
• MeSH
• krysa rodu Rattus MeSH
• mezenchymální kmenové buňky * metabolismus   účinky léků   cytologie   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nanočástice chemie   MeSH
• oxidační stres účinky léků   MeSH
• polyethylenglykoly chemie   MeSH
• velikost částic MeSH
• viabilita buněk účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• polyethylenglykoly MeSH

Preclinical and clinical studies with various stem cells, their secretomes, and extracellular vesicles (EVs) indicate their use as a promising strategy for the treatment of various diseases and tissue defects, including neurodegenerative diseases such as spinal cord injury (SCI) and amyotrophic lateral sclerosis (ALS). Autologous and allogenic mesenchymal stem cells (MSCs) are so far the best candidates for use in regenerative medicine. Here we review the effects of the implantation of MSCs (progenitors of mesodermal origin) in animal models of SCI and ALS and in clinical studies. MSCs possess multilineage differentiation potential and are easily expandable in vitro. These cells, obtained from bone marrow (BM), adipose tissue, Wharton jelly, or even other tissues, have immunomodulatory and paracrine potential, releasing a number of cytokines and factors which inhibit the proliferation of T cells, B cells, and natural killer cells and modify dendritic cell activity. They are hypoimmunogenic, migrate toward lesion sites, induce better regeneration, preserve perineuronal nets, and stimulate neural plasticity. There is a wide use of MSC systemic application or MSCs seeded on scaffolds and tissue bridges made from various synthetic and natural biomaterials, including human decellularized extracellular matrix (ECM) or nanofibers. The positive effects of MSC implantation have been recorded in animals with SCI lesions and ALS. Moreover, promising effects of autologous as well as allogenic MSCs for the treatment of SCI and ALS were demonstrated in recent clinical studies.

• Klíčová slova
• amyotrophic lateral sclerosis, biomaterials, cell therapy, conditioned medium, exosomes, mesenchymal stem cells, neurodegenerative diseases, spinal cord injury,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Superparamagnetic iron oxide nanoparticles (SPIOn) are widely used as a contrast agent for cell labeling. Macrophages are the first line of defense of organisms in contact with nanoparticles after their administration. In this study we investigated the effect of silica-coated nanoparticles (γ-Fe2O3-SiO2) with or without modification by an ascorbic acid (γ-Fe2O3-SiO2-ASA), which is meant to act as an antioxidative agent on rat peritoneal macrophages. Both types of nanoparticles were phagocytosed by macrophages in large amounts as confirmed by transmission electron microscopy and Prusian blue staining, however they did not substantially affect the viability of exposed cells in monitored intervals. We further explored cytotoxic effects related to oxidative stress, which is frequently documented in cells exposed to nanoparticles. Our analysis of double strand breaks (DSBs) marker γH2AX showed an increased number of DSBs in cells treated with nanoparticles. Nanoparticle exposure further revealed only slight changes in the expression of genes involved in oxidative stress response. Lipid peroxidation, another marker of oxidative stress, was not significantly affirmed after nanoparticle exposure. Our data indicate that the effect of both types of nanoparticles on cell viability, or biomolecules such as DNA or lipids, was similar; however the presence of ascorbic acid, either bound to the nanoparticles or added to the cultivation medium, worsened the negative effect of nanoparticles in various tests performed. The attachment of ascorbic acid on the surface of nanoparticles did not have a protective effect against induced cytotoxicity, as expected.

• Klíčová slova
• Cytotoxicity, Macrophages, Nanoparticles, Oxidative stress,
• MeSH
• antioxidancia metabolismus   toxicita   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• kyselina askorbová metabolismus   toxicita   MeSH
• magnetické nanočástice toxicita   MeSH
• peritoneální makrofágy účinky léků   metabolismus   MeSH
• potkani Wistar MeSH
• synergismus léků MeSH
• viabilita buněk účinky léků   fyziologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antioxidancia MeSH
• kyselina askorbová MeSH
• magnetické nanočástice MeSH

Manganese-zinc ferrite nanoparticles were synthesized by using a hydrothermal treatment, coated with silica, and then tested as efficient cellular labels for cell tracking, using magnetic resonance imaging (MRI) in vivo. A toxicity study was performed on rat mesenchymal stem cells and C6 glioblastoma cells. Adverse effects on viability and cell proliferation were observed at the highest concentration (0.55 mM) only; cell viability was not compromised at lower concentrations. Nanoparticle internalization was confirmed by transmission electron microscopy. The particles were found in membranous vesicles inside the cytoplasm. Although the metal content (0.42 pg Fe/cell) was lower compared to commercially available iron oxide nanoparticles, labeled cells reached a comparable relaxation rate R 2, owing to higher nanoparticle relaxivity. Cells from transgenic luciferase-positive rats were used for in vivo experiments. Labeled cells were transplanted into the muscles of non-bioluminescent rats and visualized by MRI. The cells produced a distinct hypointense signal in T2- or T2*-weighted MR images in vivo. Cell viability in vivo was verified by bioluminescence.

• Klíčová slova
• cell labeling, cell transplantation, doping, magnetic resonance imaging, nanoparticles,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: In vitro labelling of cells and small cell structures is a necessary step before in vivo monitoring of grafts. We modified and optimised a procedure for pancreatic islet labelling using bimodal positively charged poly(lactic-co-glycolic acid) nanoparticles with encapsulated perfluoro crown ethers and indocyanine green dye via microporation and compared the method with passive endocytosis. RESULTS: Pancreatic islets were microporated using two pulses at various voltages. We tested a standard procedure (poration in the presence of nanoparticles) and a modified protocol (pre-microporation in a buffer only, and subsequent islet incubation with nanoparticles on ice for 10 min). We compared islet labelling by microporation with labelling by endocytosis, i.e. pancreatic islets were incubated for 24 h in a medium with suspended nanoparticles. In order to verify the efficiency of the labelling procedures, we used 19F magnetic resonance imaging, optical fluorescence imaging and confocal microscopy. The experiment confirmed that microporation, albeit fast and effective, is invasive and may cause substantial harm to islets. To achieve sufficient poration and to minimise the reduction of viability, the electric field should be set at 20 kV/m (two pulses, 20 ms each). Poration in the presence of nanoparticles was found to be unsuitable for the nanoparticles used. The water suspension of nanoparticles (which served as a surfactant) was slightly foamy and microbubbles in the suspension were responsible for sparks causing the destruction of islets during poration. However, pre-microporation (poration of islets in a buffer only) followed by 10-min incubation with nanoparticles was safer. CONCLUSIONS: For labelling of pancreatic islets using poly(lactic-co-glycolic acid) nanoparticles, the modified microporation procedure with low voltage was found to be safer than the standard microporation procedure. The modified procedure was fast, however, efficiency was lower compared to endocytosis.

• Klíčová slova
• 19F magnetic resonance imaging, Bimodal nanoparticles, Cell labelling, Confocal microscopy, Endocytosis, Fluorescence imaging, Microporation, Pancreatic islets,
• Publikační typ
• časopisecké články MeSH

INTRODUCTION: Magnetic nanoparticles (NPs) represent a tool for use in magnetic resonance imaging (MRI)-guided thermoablation of tumors using an external high-frequency (HF) magnetic field. To avoid local overheating, perovskite NPs with a lower Curie temperature (T c) were proposed for use in thermotherapy. However, deposited power decreases when approaching the Curie temperature and consequently may not be sufficient for effective ablation. The goal of the study was to test this hypothesis. METHODS: Perovskite NPs (T c =66°C-74°C) were characterized and tested both in vitro and in vivo. In vitro, the cells suspended with NPs were exposed to a HF magnetic field together with control samples. In vivo, a NP suspension was injected into a induced tumor in rats. Distribution was checked by MRI and the rats were exposed to a HF field together with control animals. Apoptosis in the tissue was evaluated. RESULTS AND DISCUSSION: In vitro, the high concentration of suspended NPs caused an increase of the temperature in the cell sample, leading to cell death. In vivo, MRI confirmed distribution of the NPs in the tumor. The temperature in the tumor with injected NPs did not increase substantially in comparison with animals without particles during HF exposure. We proved that the deposited power from the NPs is too small and that thermoregulation of the animal is sufficient to conduct the heat away. Histology did not detect substantially higher apoptosis in NP-treated animals after ablation. CONCLUSION: Magnetic particles with low T c can be tracked in vivo by MRI and heated by a HF field. The particles are capable of inducing cell apoptosis in suspensions in vitro at high concentrations only. However, their effect in the case of extracellular deposition in vivo is questionable due to low deposited power and active thermoregulation of the tissue.

• Klíčová slova
• MRI, high-frequency magnetic field, hyperthermia, perovskite nanoparticles, tumor ablation,
• MeSH
• ablace přístrojové vybavení   metody   MeSH
• indukovaná hypertermie metody   MeSH
• kontrastní látky * chemie   farmakokinetika   MeSH
• magnetická rezonanční tomografie přístrojové vybavení   metody   MeSH
• magnety MeSH
• nádorové buněčné linie MeSH
• nanočástice * chemie   MeSH
• oxid křemičitý chemie   MeSH
• oxidy chemie   MeSH
• potkani Wistar MeSH
• sloučeniny vápníku chemie   MeSH
• suspenze MeSH
• teplota MeSH
• titan chemie   MeSH
• xenogenní modely - testy protinádorové aktivity MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kontrastní látky * MeSH
• oxid křemičitý MeSH
• oxidy MeSH
• perovskite MeSH Prohlížeč
• sloučeniny vápníku MeSH
• suspenze MeSH
• titan MeSH

The manipulation of brain nerve terminals by an external magnetic field promises breakthroughs in nano-neurotechnology. D-Mannose-coated superparamagnetic nanoparticles were synthesized by coprecipitation of Fe(II) and Fe(III) salts followed by oxidation with sodium hypochlorite and addition of D-mannose. Effects of D-mannose-coated superparamagnetic maghemite (γ-Fe2O3) nanoparticles on key characteristics of the glutamatergic neurotransmission were analysed. Using radiolabeled L-[(14)C]glutamate, it was shown that D-mannose-coated γ-Fe2O3 nanoparticles did not affect high-affinity Na(+)-dependent uptake, tonic release and the extracellular level of L-[(14)C]glutamate in isolated rat brain nerve terminals (synaptosomes). Also, the membrane potential of synaptosomes and acidification of synaptic vesicles was not changed as a result of the application of D-mannose-coated γ-Fe2O3 nanoparticles. This was demonstrated with the potential-sensitive fluorescent dye rhodamine 6G and the pH-sensitive dye acridine orange. The study also focused on the analysis of the potential use of these nanoparticles for manipulation of nerve terminals by an external magnetic field. It was shown that more than 84.3 ± 5.0% of L-[(14)C]glutamate-loaded synaptosomes (1 mg of protein/mL) incubated for 5 min with D-mannose-coated γ-Fe2O3 nanoparticles (250 µg/mL) moved to an area, in which the magnet (250 mT, gradient 5.5 Т/m) was applied compared to 33.5 ± 3.0% of the control and 48.6 ± 3.0% of samples that were treated with uncoated nanoparticles. Therefore, isolated brain nerve terminals can be easily manipulated by an external magnetic field using D-mannose-coated γ-Fe2O3 nanoparticles, while the key characteristics of glutamatergic neurotransmission are not affected. In other words, functionally active synaptosomes labeled with D-mannose-coated γ-Fe2O3 nanoparticles were obtained.

• Klíčová slova
• D-mannose, extracellular level, glutamate uptake and release, manipulation by an external magnetic field, membrane potential, nanoparticles, rat brain nerve terminals, synaptic vesicle acidification, γ-Fe2O3,
• Publikační typ
• časopisecké články MeSH

OBJECT: Metabolite changes in an experimental lesion in the rat cortex and in the contralateral hemisphere after the intravenous administration of mesenchymal stem cells (MSCs) were assessed by proton MR spectroscopy to verify the impact of the cell treatment on the brain tissue. MATERIALS AND METHODS: Wistar rats with a photochemical cortical lesion and transplanted MSCs or sham transplanted rats were examined. Proton spectra were obtained from the lesion and from the contralateral cortex. RESULTS: Magnetic resonance spectroscopy revealed a gradual recovery of the damaged tissue; however, we found no significant differences in metabolite concentrations in the lesioned hemisphere between treated and untreated animals. Higher concentrations of glutamate and N-acetyl aspartate were found in the contralateral hemisphere in cell-treated animals compared to untreated ones. Lesioned animals showed neurogenesis in the contralateral hemisphere; the number of newly generated cells in stem cell-treated animals was 50% higher than those observed in untreated animals. CONCLUSION: No direct impact of cell transplantation was observed in the lesion. However, changes in the contralateral hemisphere suggest that the transplanted MSCs might stimulate repair processes and plasticity resulting in the generation of newborn cells, which might enable the faster adoption of the damaged tissue's function by healthy tissue.

• MeSH
• časové faktory MeSH
• femur patologie   MeSH
• ferrokyanidy farmakologie   MeSH
• krysa rodu Rattus MeSH
• kyselina asparagová analogy a deriváty   metabolismus   MeSH
• kyselina glutamová metabolismus   MeSH
• magnetická rezonanční spektroskopie metody   MeSH
• mezenchymální kmenové buňky cytologie   MeSH
• mozek metabolismus   patologie   MeSH
• potkani Wistar MeSH
• transplantace mezenchymálních kmenových buněk metody   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ferric ferrocyanide MeSH Prohlížeč
• ferrokyanidy MeSH
• kyselina asparagová MeSH
• kyselina glutamová MeSH
• N-acetylaspartate MeSH Prohlížeč

The growth of bone marrow stromal cells was assessed in vitro in macroporous hydrogels based on 2-hydro- xyethyl methacrylate (HEMA) copolymers with different electric charges. Copolymers of HEMA with sodium methacrylate (MA(-)) carried a negative electric charge, copolymers of HEMA with [2-(methacryloyloxy)ethyl] trimethylammonium chloride (MOETA(-)) carried a positive electric charge and terpolymers of HEMA, MA(-) and MOETA(+) carried both, positive and negative electric charges. The charges in the polyelectrolyte complexes were shielded by counter-ions. The hydrogels had similar porosities, based on a comparison of their diffusion parameters for small cations as measured by the real-time tetramethylammonium iontophoretic method of diffusion analysis. The cell growth was studied in the peripheral and central regions of the hydrogels at 2 hours and 2, 7, 14 and 28 days after cell seeding. Image analysis revealed the highest cellular density in the HEMA-MOETA(+) copolymers; most of the cells were present in the peripheral region of the hydrogels. A lower density of cells but no difference between the peripheral and central regions was observed in the HEMA-MA(-) copolymers and in polyelectrolyte complexes. This study showed that positively charged functional groups promote the adhesion of cells.

• MeSH
• biokompatibilní materiály chemie   MeSH
• buňky kostní dřeně cytologie   MeSH
• buňky stromatu cytologie   MeSH
• časové faktory MeSH
• difuze MeSH
• elektrolyty chemie   MeSH
• femur metabolismus   MeSH
• krysa rodu Rattus MeSH
• methakryláty chemie   MeSH
• PEG-DMA hydrogel * MeSH
• počítačové zpracování obrazu MeSH
• potkani Wistar MeSH
• povrchové vlastnosti MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biokompatibilní materiály MeSH
• elektrolyty MeSH
• hydroxyethyl methacrylate MeSH Prohlížeč
• methakryláty MeSH
• PEG-DMA hydrogel * MeSH

1. Emerging clinical studies of treating brain and spinal cord injury (SCI) led us to examine the effect of autologous adult stem cell transplantation as well as the use of polymer scaffolds in spinal cord regeneration. We compared an intravenous injection of mesenchymal stem cells (MSCs) or the injection of a freshly prepared mononuclear fraction of bone marrow cells (BMCs) on the treatment of an acute or chronic balloon-induced spinal cord compression lesion in rats. Based on our experimental studies, autologous BMC implantation has been used in a Phase I/II clinical trial in patients (n=20) with a transversal spinal cord lesion. 2. MSCs were isolated from rat bone marrow by their adherence to plastic, labeled with iron-oxide nanoparticles and expanded in vitro. Macroporous hydrogels based on derivatives of 2-hydroxyethyl methacrylate (HEMA) or 2-hydroxypropyl methacrylamide (HPMA) were prepared, then modified by their copolymerization with a hydrolytically degradable crosslinker, N,O-dimethacryloylhydroxylamine, or by different surface electric charges. Hydrogels or hydrogels seeded with MSCs were implanted into rats with hemisected spinal cords. 3. Lesioned animals grafted with MSCs or BMCs had smaller lesions 35 days postgrafting and higher scores in BBB testing than did control animals and also showed a faster recovery of sensitivity in their hind limbs using the plantar test. The functional improvement was more pronounced in MSC-treated rats. In MR images, the lesion populated by grafted cells appeared as a dark hypointense area and was considerably smaller than in control animals. Morphometric measurements showed an increase in the volume of spared white matter in cell-treated animals. In the clinical trial, we compared intraarterial (via a. vertebralis, n=6) versus intravenous administration of BMCs (n=14) in a group of subacute (10-33 days post-SCI, n=8) and chronic patients (2-18 months, n=12). For patient follow-up we used MEP, SEP, MRI, and the ASIA score. Our clinical study revealed that the implantation of BMCs into patients is safe, as there were no complications following cell administration. Partial improvement in the ASIA score and partial recovery of MEP or SEP have been observed in all subacute patients who received cells via a. vertebralis (n=4) and in one out of four subacute patients who received cells intravenously. Improvement was also found in one chronic patient who received cells via a. vertebralis. A much larger population of patients is needed before any conclusions can be drawn. The implantation of hydrogels into hemisected rat spinal cords showed that cellular ingrowth was most pronounced in copolymers of HEMA with a positive surface electric charge. Although most of the cells had the morphological properties of connective tissue elements, we found NF-160-positive axons invading all the implanted hydrogels from both the proximal and distal stumps. The biodegradable hydrogels degraded from the border that was in direct contact with the spinal cord tissue. They were resorbed by macrophages and replaced by newly formed tissue containing connective tissue elements, blood vessels, GFAP-positive astrocytic processes, and NF-160-positive neurofilaments. Additionally, we implanted hydrogels seeded with nanoparticle-labeled MSCs into hemisected rat spinal cords. Hydrogels seeded with MSCs were visible on MR images as hypointense areas, and subsequent Prussian blue histological staining confirmed positively stained cells within the hydrogels. 4. We conclude that treatment with different bone marrow cell populations had a positive effect on behavioral outcome and histopathological assessment after SCI in rats; this positive effect was most pronounced following MSC treatment. Our clinical study suggests a possible positive effect in patients with SCI. Bridging the lesion cavity can be an approach for further improving regeneration. Our preclinical studies showed that macroporous polymer hydrogels based on derivatives of HEMA or HPMA are suitable materials for bridging cavities after SCI; their chemical and physical properties can be modified to a specific use, and 3D implants seeded with different cell types may facilitate the ingrowth of axons.

• MeSH
• autologní transplantace MeSH
• buňky kostní dřeně fyziologie   MeSH
• hydrogely terapeutické užití   MeSH
• komprese míchy terapie   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• mezenchymální kmenové buňky fyziologie   MeSH
• monocyty transplantace   MeSH
• pohyb buněk MeSH
• polymery terapeutické užití   MeSH
• poranění míchy terapie   MeSH
• regenerace nervu MeSH
• transplantace kostní dřeně metody   MeSH
• transplantace mezenchymálních kmenových buněk metody   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• hydrogely MeSH
• polymery MeSH