PLoS One. 2014 May 30;9(5):e98451.

Dose-response of superparamagnetic iron oxide labeling on mesenchymal stem cells chondrogenic differentiation: a multi-scale in vitro study.

Roeder E1, Henrionnet C1, Goebel JC2, Gambier N1, Beuf O2, Grenier D2, Chen B3, Vuissoz PA3, Gillet P1, Pinzano A1.


1Ingénierie Moléculaire et Physiopathologie Articulaire – Unité Mixte de Recherches 7365 Centre National de la Recherche Scientifique – Université de Lorraine, Vandoeuvre Lès Nancy, France.
2Centre de Recherche en Acquisition et Traitement de l’Image pour la Santé, Centre National de la Recherche Scientifique 5220, Institut National de la Santé et de la Recherche Médicale U1044, Université de Lyon, Institut National des Sciences Appliquées de Lyon, Villeurbanne, France.
3Imagerie Adaptative Diagnostique Interventionelle, Institut National de la Santé et de la Recherche Médicale U947, Vandoeuvre-Lès-Nancy, France.



AIM: The aim of this work was the development of successful cell therapy techniques for cartilage engineering. This will depend on the ability to monitor non-invasively transplanted cells, especially mesenchymal stem cells (MSCs) that are promising candidates to regenerate damaged tissues.

METHODS: MSCs were labeled with superparamagnetic iron oxide particles (SPIO). We examined the effects of long-term labeling, possible toxicological consequences and the possible influence of progressive concentrations of SPIO on chondrogenic differentiation capacity.

RESULTS: No influence of various SPIO concentrations was noted on human bone marrow MSC viability or proliferation. We demonstrated long-term (4 weeks) in vitro retention of SPIO by human bone marrow MSCs seeded in collagenic sponges under TGF-β1 chondrogenic conditions, detectable by Magnetic Resonance Imaging (MRI) and histology. Chondrogenic differentiation was demonstrated by molecular and histological analysis of labeled and unlabeled cells. Chondrogenic gene expression (COL2A2, ACAN, SOX9, COL10, COMP) was significantly altered in a dose-dependent manner in labeled cells, as were GAG and type II collagen staining. As expected, SPIO induced a dramatic decrease of MRI T2 values of sponges at 7T and 3T, even at low concentrations.

CONCLUSIONS: This study clearly demonstrates (1) long-term in vitro MSC traceability using SPIO and MRI and (2) a deleterious dose-dependence of SPIO on TGF-β1 driven chondrogenesis in collagen sponges. Low concentrations (12.5-25 µg Fe/mL) seem the best compromise to optimize both chondrogenesis and MRI labeling.

PMID: 24878844


Supplement :

SPIO1Hyaline cartilage is a conjunctive tissue covering articular surfaces of bone into the joints. Cartilage doesn’t display any vascular system neither innervation. This explains the limited or non-existent repair capacities of this tissue. So, when lesions occur, generally they worsen and turn into osteoarthritis overtime. Due to the frequency of articular diseases and aging of the population, the regeneration of damaged cartilage appears as a major challenge in regenerative medicine. Various surgical techniques were developed to repair injuries in cartilage without success to reproduce a repair tissue similar to native cartilage. Autologous chondrocyte implantation (ACI) shows good results at improving quality life of the patients. But the non load-carrying cartilage access, the limited number of chondrocytes and their fast loss of phenotype during the culture phases are some limitations of this technique. Tissue engineering is more and more studied. This methods is based on the production of a functionalized transplant in vitro thanks to a biomaterial seeded with cells and cultivated in adjusted culture conditions and aims to synthetize a neo-tissue having similar features as the original tissue. Due to their capacities of differentiation, the mesenchymal stem cells (MSC) provide a promising avenue to regenerate damaged tissues in articular diseases. With the in vivo transplantation in mind, the tracking of the transplant and particularly of the cells participating to the repair is essential. The development of an imaging technique allowing the diagnostic pre- and post-transplantation of the engineered implant is a serious topic. Magnetic Resonance Imaging (MRI) is a non-invasive technique and assures the measurement of the defect extent, the state of the surrounding tissues and the progressive healing at a tissue, cell and even molecular level. MRI appears as a good device to visualize the regeneration of the cartilage lesions. To trace the MSC within the repair tissue, some contrast agents might be used, such as the superparamagnetic iron oxide (SPIO) particles. This SPIO, internalized in the cells by endocytosis, decreases dramatically the signal and the T2 (Transversal relaxation time) and appear as dark spots in T2-weighted MRI pictures.

SPIO2While the safety of these various types of SPIO is a general consensus, their influence on the MSC differentiation is discussed within the scientific community. We proposed to evaluate the effect of a range concentration labeling of SPIO on MSC chondrogenic differentiation in this multi-scale study. The cells internalized the SPIO by endocytosis (Figure 1). This phenomenon requires the intrusion of the particles through the cell membranes. Our SPIO are coated with negatively charged dextran and as the cell membrane is negatively charged too, the uptake can be facilitated by the addition of a transfection agent, such as the Poly-L-Lysine (PLL). The PLL is positively charged and so improve endocytosis. From the internalization of exogenous particles, might result inflammation, apoptosis and/or reactive oxygen species (ROS) formation, for example. Then, we decided to investigate these various parameters to assess the safety of the SPIO we used. Cytotoxicity (Lactate deshydrogenase activity measurement), apoptosis (Caspase 3 activity measurement), inflammation (PGE2 assay) and ROS production (nitric oxide (NO) formation) of the MSC were minimal even at the highest concentration of SPIO (1600 mg Fe/mL). By these results, our study supported the previous published data on this subject.

As the safety of the particles was ensured, the next step was to explore the influence of our SPIO on the MSC differentiation capacities. MSC chondrogenic differentiation was induced by the addition of a well-known chondro-inductor, Transforming Growth Factor b1 (TGF-b1). The cells need a three-dimension scaffold (a type I and III collagen sponge) to support their adhesion, differentiation and matrix synthesis. We tested a lowered concentration range of SPIO (12.5 to 200 mg Fe/mL). After 28 days of differentiation, chondrogenic genes expressions were down-regulated in a concentration-dependent manner starting with the lowest concentration of 12.5 mg Fe/mL (Figure 2). The matrix synthesis is also impaired by the SPIO labeling. The depletion of proteoglycans (Alcian Blue) is particularly noticed and the production of collagen (Sirius Red) is affected (Figure 3). Prussian blue staining confirmed persisting intra-cellular SPIO internalization on D28 (Figure 4)



SPIO5The MRI might be used as a diagnostic tool of the lesion repair after transplantation of the engineered tissue. But, this device can also be an attractive feature in the context of a diagnostic of the engineered tissue quality in vitro previously to the implantation. We evaluated the MSC labeling by the SPIO in our sponges after 28 days of differentiation in vitro. After 28 days, the SPIO still decrease the signal in our biomaterial. T2 measurement indicates a reduction of this transversal relaxation time with the lowest concentration (12.5 mg Fe/mL), which increases, with the elevation of the SPIO concentration (Figure 5). This proves that the SPIO labeling can be use as a tracker for the detection of differentiated MSC after the biomaterial transplantation. What is interesting? MRI allows the discrimination between the two tissues synthetized from non-differentiated MSC and from MSC differentiated by addition of TGF-b1. Based on this work, the MRI might be use to appreciate the quality and to monitor the future of the implant before and after the transplantation in vivo.

The interest of our study is in 3 fold: First, this multi-scale work underlines the possibility to use low concentrations of SPIO to label cells without dramatically inhibiting the chondrogenic differentiation. Second, these low concentrations of SPIO allow MSC tracking within the neo-tissue without any indicator of saturation phenomenon and the labeling remains in the cells at least after 28 days post-exposure. And third, MRI appears as a promising technique to monitor SPIO labeled cells in pre- and post-transplantation contexts and the analysis of the T2 might enable the discrimination between a poor and good quality tissue.





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