Elsevier

Joint Bone Spine

Volume 75, Issue 4, July 2008, Pages 432-438
Joint Bone Spine

Original article
In vivo MR imaging tracking of magnetic iron oxide nanoparticle labeled, engineered, autologous bone marrow mesenchymal stem cells following intra-articular injection

https://doi.org/10.1016/j.jbspin.2007.09.013Get rights and content

Abstract

Objective

To track superparamagnetic iron oxide nanoparticle (SPIO)-labeled, bone-derived mesenchymal stem cells (MSCs) by in vivo magnetic resonance imaging (MRI) with a 1.5 T-system following injection of engineered autologous MSCs into the knee joint cavity in rabbit articular cartilage defect models.

Methods

Rabbit MSCs were labeled with SPIO and a transfection agent. Cell viability, proliferation and differentiation capacity were assessed in vitro using appropriate functional assays. Cells underwent GRE T2*-weighted MRI in vitro. The autologous MSCs seeded in chitosan and glycerophosphate (C-GP) gel were injected into the knee joint cavity of rabbit models for cartilage defects. All rabbits underwent GRE T2*-weighted MRI 1, 4, 8 and 12 weeks post-injection. MR imaging findings were compared histologically.

Results

Nanoparticles were stained with Prussian blue and observed by transmission electron microscopy inside the cells. Cell viability, proliferation, and differentiation were comparable between labeled and non-labeled cells. After intra-articular injection of labeled autologous MSCs, marked hypointense signal void areas representing the injected MSCs can be observed for at least 12 weeks on GRE T2*-weighted images. At 12 weeks post-injection, labeled MSCs migrated into the synovial fluid at the suprapatellar bursa, the popliteal space site and subchondral bone of the femur but no MSCs were detected in the defect. Histochemical staining confirmed the presence of Prussian blue-positive cells and BrdU-positive cells.

Conclusions

MRI would be an efficient noninvasive technique to visually track SPIO-labeled seed cells in vivo; the engineered autologous MSCs do not actively participate in the repair of articular cartilage defects 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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