Effect of labeling on adipogenic differentiation of ASC.

<p>After cell labeling with (A) BNF starch nanoparticles and (B) nanomag-D-spio nanoparticles, adipogenic differentiation conditions were provided for 21 days and lipid droplet deposition was measured. BNF starch labeling of ASC resulted in a dose-dependent reduction of adipogenic differentiation potential (n = 4; boxplots, Mann-Whitney U-test; *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001).</p

Proliferation of nanoparticle-labeled ASC.

<p>Cells were treated with (A) BNF starch nanoparticles and (B) nanomag-D-spio nanoparticles and cell numbers were determined up to 10 days after labeling. Cell treatment with both nanoparticle types resulted generally in a higher proliferation rate compared to control cells (n = 4; median, error bars represent 25^th and 75^th percentiles).</p

Viability/Cytotoxicity test after nanoparticle labeling.

<p>After treatment of ASC with BNF starch nanoparticles (10/25/50 µg Fe/ml) and nanomag-D-spio nanoparticles, a viability/cytotoxicity assay was performed up to 14 days following labeling. Viable cells and nuclei of apoptotic cells were stained with calcein AM (green) and ethidium homodimer (red), respectively. Cells were counterstained with Hoechst 33342 (blue). No cytotoxic effects were detected due to nanoparticle labeling (Axio Observer, Carl Zeiss Microscopy GmbH, Jena, Germany; scale bars  = 50 µm).</p

Physico-chemical characterization of nanoparticles.

<p>Both nanoparticle types including their redF-labeled and PDL-coated variants were characterized by determination of hydrodynamic diameter, polydispersity index, and zeta potential.</p><p>Physico-chemical characterization of nanoparticles.</p

Effect of labeling on chondrogenic differentiation of ASC.

<p>(A) Unlabeled, (B) BNF starch- and (C, E, F) nanomag-D-spio-labeled cells were chondrogenically stimulated for 21 d. Pellets were analyzed using Heidenhain's AZAN trichrome staining showing a reduced collagen type II-positive extracellular matrix (light blue) due to nanoparticle labeling. BNF-labeled cells (25 and 50 µg Fe/ml) failed to generate compact pellets (not shown) (Axio Imager M2, Carl Zeiss Microscopy GmbH, Jena, Germany; scale bars  = 100 µm). (D) Collagen type II was analyzed using Real-Time quantitative PCR revealing a diminished mRNA expression of collagen type II due to nanoparticle labeling (2∧ΔΔCT ± %CV; n = 2, normalized to β-actin).</p

Activity of mitochondrial dehydrogenases.

<p>Cells were treated with (A) BNF starch nanoparticles and (B) nanomag-D-spio nanoparticles. The activity of mitochondrial dehydrogenases was determined at day 7 after labeling. Only BNF starch labeling of ASC at the highest concentration of 50 µg Fe/ml diminished the activity of mitochondrial dehydrogenases significantly compared to control cells (n = 4; (A) mean ± SD, ANOVA/Tukey's-test; (B) boxplot, Mann-Whitney U-test; *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001).</p

Nanoparticle internalization verified by Prussian Blue staining.

<p>After incubation of ASC with BNF starch nanoparticles (10/25/50 µg Fe/ml) and nanomag-D-spio nanoparticles (25/50/100 µg Fe/ml), iron oxide of internalized particles was visualized by Prussian Blue staining (Zeiss Axiovert 40 CFL, Carl Zeiss Microscopy GmbH, Jena, Germany; scale bars  = 50 µm).</p

Nanoparticle uptake of labeled ASC.

<p>Uptake of nanoparticles was determined using a ferrozin assay and displayed as number of particles per cell. Shown is the maximal and minimal as well as mean ± SD number of particles per cell.</p>a,b<p>no significant difference between cellular uptake of BNF starch and nanomag-D-spio nanoparticles (Mann-Whitney U-test).</p><p>Nanoparticle uptake of labeled ASC.</p

Comparative <i>In Vitro</i> Study on Magnetic Iron Oxide Nanoparticles for MRI Tracking of Adipose Tissue-Derived Progenitor Cells

<div><p>Magnetic resonance imaging (MRI) using measurement of the transverse relaxation time (R2*) is to be considered as a promising approach for cell tracking experiments to evaluate the fate of transplanted progenitor cells and develop successful cell therapies for tissue engineering. While the relationship between core composition of nanoparticles and their MRI properties is well studied, little is known about possible effects on progenitor cells. This <i>in vitro</i> study aims at comparing two magnetic iron oxide nanoparticle types, single vs. multi-core nanoparticles, regarding their physico-chemical characteristics, effects on cellular behavior of adipose tissue-derived stem cells (ASC) like differentiation and proliferation as well as their detection and quantification by means of MRI. Quantification of both nanoparticle types revealed a linear correlation between labeling concentration and R2* values. However, according to core composition, different levels of labeling concentrations were needed to achieve comparable R2* values. Cell viability was not altered for all labeling concentrations, whereas the proliferation rate increased with increasing labeling concentrations. Likewise, deposition of lipid droplets as well as matrix calcification revealed to be highly dose-dependent particularly regarding multi-core nanoparticle-labeled cells. Synthesis of cartilage matrix proteins and mRNA expression of collagen type II was also highly dependent on nanoparticle labeling. In general, the differentiation potential was decreased with increasing labeling concentrations. This <i>in vitro</i> study provides the proof of principle for further <i>in vivo</i> tracking experiments of progenitor cells using nanoparticles with different core compositions but also provides striking evidence that combined testing of biological and MRI properties is advisable as improved MRI properties of multi-core nanoparticles may result in altered cell functions.</p></div

Effect of labeling on osteogenic differentiation of ASC.

<p>Cells were labeled with (A) BNF starch nanoparticles and (B) nanomag-D-spio nanoparticles. Osteogenic differentiation conditions were provided for 28 days following measurement of matrix calcification. BNF starch labeling of ASC resulted in a dose-dependent reduction of osteogenic differentiation potential whereas only nanomag-D-spio labeling at the highest concentration (100 µg Fe/ml) had a diminishing effect on the matrix calcification (n = 5; boxplots, Mann-Whitney U-test; *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001).</p