Figure 1.
Viability and apoptosis of SPIO labeled human bone MSC (2D Culture).
A) Lactate dehydrogenase viability assay. B) Caspase 3 activity. Cell death and apoptosis were minimal in MSC cultured in media only (control) under different concentration of superparamagnetic iron oxide particles (SPIO from 12.5 to1600 µg ƒe/mL). All comparisons of labeled conditions (SPIO) were performed versus the control condition (0) with a 1-way ANOVA followed by a Bonferonni's test. Data are mean ± sd. *p<0.05, **p<0.01, ***p<0.001. The experiment was carried out twice in triplicate.
Figure 2.
Histological visualization of human bone MSC (2D culture) using Prussian Blue staining.
Staining was performed at 24(SPIO). Staining increases with SPIO concentration. It should be noted that the SPIO uptake by MSCs is amplified by the use of the Poly-L-Lysine (PLL) transfection agent.
Figure 3.
Normalized gene expression (TGF-β1 versus ITS) at D28 with various SPIO concentrations.
All comparisons of labeled conditions (SPIO) were performed versus the control condition (0) with a 1-way ANOVA followed by a Bonferonni's test. Data are mean ± sd. *p<0.05, **p<0.01, ***p<0.001. The experiment was carried out twice in triplicate.
Figure 4.
Normalized mitochondrial activity of sponges seeded with undifferentiated and differentiated MSCs performed at D28 using MTT assay normalized with DNA content of each sponge.
All comparisons of labeled conditions (SPIO) were performed versus the control condition (0) with a 2-ways ANOVA (SPIO concentration and medium, ITS vs TGFβ) followed by a Bonferonni's test. Data are mean ± sd. *p<0.05. The experiment was carried out four times. There is a significant interaction between the 2 factors (SPIO concentration and culture medium, ITS and TGFβ-1 conditions)
Figure 5.
Histological analysis of sponges seeded with undifferentiated and differentiated MSCs performed at D28 using Prussian Blue staining.
When compared to control (ITS 1%), TGF-β1 induced a greater and more homogeneous synthesis of the extracellular matrix. Non labeled cells do not contain any iron. Dose-dependent intracellular Prussian blue staining was observed in collagen sponge colonized by mesenchymal stem cells at 28 days after SPIO labeled different concentrations of iron: 0, 12.5, 25, 50, 100 and 200 µg Fe/ml. Bar scale corresponds to 100 µm.
Figure 6.
Histological and immuno-histochemical analysis of sponges seeded with differentiated MSCs performed at D28.
When compared to control (0) SPIO addition led to proteoglycan depletion beginning at 25 µg Fe/mL (Acian Blue, AB), which increased as the SPIO concentration was raised. Similarly, SPIO labeling exerted a deleterious effect on collagen synthesis, as illustrated by Sirius Red (SR) staining, most specifically on collagen II synthesis. No effect is depicted on cell viability assessed with Hematoxylin-Erythrosin-Saffron
Figure 7.
MR assessments of collagen sponges at 7 (A,B) and 3T (C, D).
A. 7T MR images of sponges (black arrow) seeded with undifferentiated (ITS 1%) or differentiated (TGFβ-1 (10 ng/mL)) human bone MSCs labeled with increasing SPIO concentration, after 28 days in vitro. SPIO are homogeneously distributed in the sponges and the T2 signal decreases as the SPIO concentration increases for both conditions. B. Representation of mean relaxation time values of functionalized biomaterials on D28 in vitro at 7 T. Both ITS (0) and TGFβ-1 (0) are significantly different (+++, p<0.001, Student's t test). For each condition, T2 values of labeled sponges were compared to their own control (ITS 0 and TGF respectively) with a 2 way ANOVA followed by a Bonferonni's test. Data are mean ± sd. *p<0.05, **p<0.01, ***p<0.001. The experiment was done twice in triplicate. There is a significant interaction between culture medium and SPIO concentrations. C. 3T MR images of sponges (white arrow) seeded with undifferentiated (ITS 1%) or differentiated (TGFβ-1 (10 ng/mL)) human bone MSCs labeled with increasing SPIO concentrations, after 28 days in vitro. D. Corresponding T2 values. ITS (0) and TGFβ-1 (0) are significantly different (+++, p<0.001, Student's t test). For each condition T2 values of labeled sponges were compared with their own control (ITS 0 and TGF respectively with a 2 way ANOVA followed by a Bonferonni's test. Data are mean ± sd. *p<0.05, **p<0.01, ***p<0.001. The experiment was carried out twice in triplicate. There is a significant interaction between culture medium and SPIO concentrations.