MRI-Tracking of Dental Pulp Stem Cells In Vitro and In Vivo Using Dextran-Coated Superparamagnetic Iron Oxide Nanoparticles

Abstract

The aim of this study was to track dental pulp stem cells (DPSCs) labeled with dextran-coated superparamagnetic iron oxide nanoparticles (SPIONs) using magnetic resonance imaging (MRI). Dental pulp was isolated from male Sprague Dawley rats and cultured in Dulbecco's modified Eagle's medium F12 (DMEM-F12) and 10% fetal bovine serum. Effects of SPIONs on morphology, viability, apoptosis, stemness, and osteogenic and adipogenic differentiation of DPSCs were assessed. Prussian blue staining and MRI were conducted to determine in vitro efficiency of SPIONs uptake by the cells. Both non-labeled and labeled DPSCs were adherent to culture plates and showed spindle-shape morphologies, respectively. They were positive for osteogenic and adipogenic induction and expression of cluster of differentiation (CD) 73 and CD90 biomarkers, but negative for expression of CD34 and CD45 biomarkers. The SPIONs were non-toxic and did not induce apoptosis in doses less than 25 mg/mL. Internalization of the SPIONs within the DPSCs was confirmed by Prussian blue staining and MRI. Our findings revealed that the MRI-based method could successfully monitor DPSCs labeled with dextran-coated SPIONs without any significant effect on osteogenic and adipogenic differentiation, viability, and stemness of DPSCs. We provided the in vitro evidence supporting the feasibility of an MRI-based method to monitor DPSCs labeled with SPIONs without any significant reduction in viability, proliferation, and differentiation properties of labeled cells, showing that internalization of SPIONs within DPSCs were not toxic at doses less than 25 mg/mL. In general, the SPION labeling does not seem to impair cell survival or differentiation. SPIONs are biocompatible, easily available, and cost effective, opening a new avenue in stem cell labeling in regenerative medicine.

Keywords: MRI; dental pulp stem cells; iron oxide; labeling; nanoparticle; tracking.

Figure 1

Comparison of cell morphology of dental pulp stem cells (DPSCs) ((A) non-labeled and (E) labeled DPSCs), osteogenic induction measurement using Alizarin Red staining ((B) non-labeled and (F) labeled DPSCs), adipogenic induction measurement using Oil Red-O staining ((C) non-labeled and (G) labeled DPSCs), and RT-PCR to characterize the cell differentiation ((D) non-labeled and (H) labeled DPSCs). Superparamagnetic iron oxide nanoparticles (SPIONs); cluster of differentiation (CD).

Figure 2

(A) MTT assay comparing the viability and proliferation capacity of different DPSCs. 1: Non-labeled cells, 2: Labeled cells with 1.5 mg/mL of SPIONs, 3: Labeled cells with 2.5 mg/mL of SPIONs, 4: Labeled cells with 3.5 mg/mL of SPIONs, 5: Labeled cells with 4.5 mg/mL of SPIONs, 6: Labeled cells with 5.5 mg/mL of SPIONs, 7: Labeled cells with 12 mg/mL of SPIONs, 8: Labeled cells with 25 mg/mL of SPIONs, 9: DMSO. The assay indicated that the SPIONs did not induce any significant decrease in cell viability at doses less than 25 mg/mL compared to non-labeled cells (mean ± SEM, * p < 0.05). B: The number of non-labeled and labeled DPSCs with 3.5 mg/mL of SPIONs.

Figure 3

The effect of SPIONs on the expression level of the pro-apoptotic gene in labeled DPSCs assessed by RT-PCR ((A) Bax), anti-apoptotic genes ((B) Bcl-2), and Bax:Bcl-2 ratio (C) (mean ± SEM, no statistical difference was noted).

Figure 4

(A) Dead cells were scored as necrotic (Annexin V-negative/7-AAD-positive, upper left quadrants, Q1), late apoptotic (Annexin V-positive/7-AAD-positive, upper right quadrants, Q2), early apoptotic (Annexin V-positive/7-AAD-negative, lower right quadrants, Q3), and a gating on the normal cells that are considered viable, are PE Annexin V and 7-AAD negative (lower left quadrants, Q4). B. Apoptosis percentage was indicated as a bar chart (mean ± SEM, no statistical difference was noted).

Figure 5

A: The T2-weighted MRI of in vitro samples. 1: DPSCs labeled with 3.5 mg/mL of SPIONs showing a hypointense signal of the iron oxide contrast, 2: Non-labeled DPSCs without any hypointense signal for the iron oxide contrast, 3: 15% agarose gel lacking the hypointense signal of the iron oxide contrast, 4: 5 mg/mL of SPIONs in 15% gel revealing the hypointense signal of the iron oxide contrast, 5: 15% gel without any hypointense signal of the iron oxide contrast, 6: 5 mg/mL SPIONs demonstrating the hypointense signal of the iron oxide contrast, 7: DPSCs labeled with 3.5 mg/mL SPIONs denoting the hypointense signal of the iron oxide contrast, 8: DPSCs labeled with 0.35 mg/mL SPIONs in absence of any hypointense signal of the iron oxide contrast, 9: H₂O lacking the hypointense signal of the iron oxide contrast, and 10: Non-coated iron oxide particle exhibiting an extremely hypointense signal of the iron oxide contrast. (B) The T2-weighted images of DPSCs labeled with 3.5 mg/mL SPIONs injected intraperitoneally with hypointense signal of the iron oxide contrast, in the right picture, compared to non-labeled DPSCs, in the left picture, lacking the hypointense signal (rats in supine position).