Original articleIn vivo MR imaging tracking of magnetic iron oxide nanoparticle labeled, engineered, autologous bone marrow mesenchymal stem cells following intra-articular injection
Introduction
Clinically, articular cartilage defects occur commonly in association with different pathological situations such as trauma, osteoarthritis, and rheumatoid arthritis [1]. Clinical treatments for cartilage defects range historically from very conservative to invasive, such as debridement, microfracture, and mosaic plasty; however, these treatments elicit incomplete repair, e.g., fibrocartilage [2], [3]. Recently, tissue engineering procedures hold promise for the treatment of articular cartilage defects to achieve the regeneration to hyaline cartilage. At present, numerous studies have demonstrated that MSCs derived from bone marrow are pluripotent and can differentiate into osteocyte, chondrocyte, and adipocyte lineages [4], [5]. MSCs have been successfully used as seed cells to repair large full-thickness defects created in the femoral condyle of animals; however, there is still a lack of understanding regarding the characteristics of the seed cells in repairing defects. It is still unclear whether the enhancement in tissue repair is due to host cells recruited to the defects in response to the implant, or to re-population of the implanted MSCs [6], [7]. Moreover, the contribution of implanted cells to tissue repair, including the degree to which the implanted cells survive and integrate into the newly formed cartilage, remains uncertain. The development of tissue engineering therapies requires an efficient and noninvasive technique to monitor the in vivo behavior of implanted cells in host tissue and thus help understand the characteristics of the seed cells. Unlike the commonly used, invasive cell tracking by postmortem histological methods [8], [9], [10], MR imaging is valuable to visually monitor the in vivo dynamic biodistribution of implanted cells by using SPIO nanoparticles for magnetic labeling of cells [11], [12], [13]. Noninvasive imaging techniques to track cells will help understand cell functions, determine the efficacy of cell therapies, and optimize therapeutic protocols to increase the likelihood of cartilage regeneration.
Here, we tested SPIO nanoparticles as a label for in vivo monitoring of bone marrow-derived rabbit MSCs and investigated the influence of this technique on the biological properties of the MSCs. Then, in order to determine the fate of SPIO-labeled, autologous MSCs in vivo, we evaluated these cells following intra-articular injection into experimental rabbit articular cartilage defects by MR imaging using a conventional 1.5-T clinical system for 3 months.
Section snippets
MSC isolation and culture [14]
(Appendix S1; see the supplementary material associated with this article online).
Iron labeling of cells
As described previously [15], a commercially available ferumoxide suspension, Feridex IV (11.2 mg Fe/ml, Advanced. Mag. Co., USA), was diluted with culture medium to 50 μg/ml upon use. Protamine sulfate (Sigma) used as the transfection agent was prepared as a fresh stock solution of 10 mg/ml in distilled water. Then 6 μg/ml Protamine sulfate diluted from stock solutions was mixed on a rotating shaker with ferumoxides
Magnetic labeling of MSCs
After 12 h of incubation with SPIO nanoparticles, more than 90% of the cells turned positive upon Prussian blue staining. Prussian blue staining demonstrated iron-containing sites as blue spots in the cytoplasm (Fig. 1A and B). Trypan blue staining did not suggest a decreased viability of the labeled cells when compared with unlabeled cells. Transmission electron microscopy confirmed the presence of iron oxide nanoparticles inside the cytoplasm and vesicles (Fig. 1C).
In vitro labeling effect on MSC proliferation and differentiation capacity
Trypan blue exclusion assay
Discussion
Our results showed that MSCs can be efficiently labeled by 25 μg/ml SPIO and protamine sulfate as transfection agent with no effect on cell viability, proliferation and differentiation, which was consistent with a previous study [15]. It is still controversial whether magnetic labeling inhibits chondrogenic differentiation of MSCs [19], [20]. We think that the inconsistent observations were most likely due to different labeling techniques or transfection agents. In MRI detection of MSCs in
Acknowledgements
We thank Chen Wei for his help in obtaining the ferumoxide suspension. This work was supported by a grant from the National Natural Science Foundation of China (No. 30300079).
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