Synopsis

Multipotent mesenchymal stem cells (MSCs) can be labeled with superparamagnetic iron-oxide nanoparticles (SPION) particles to track single cells with MRI, and thereby follow MSC infiltration. However, a limitation with conventional MR sequences is that their long echo times are unable to measure fast signal decays, which occur in dense bone tissue and with high SPION infiltration. Ultra-short echo time (UTE) MRI can capture these rapidly decaying signals. In this work, we use 3D cones to track tissue development after injection of SPION labeled MSCs in an ovine model.

Introduction

Chondral and osteochondral defects in joints are known contributors towards the development of osteoarthritis (OA))[1]. Current treatments for joint surface defects rely primarily on surgical intervention, such as autologous chondrocyte transplantation, mosaicplasty, and microfracture, which are limited in their ability to repair or stop progression of the disease.

The use of stem cells to treat joint surface defects is an attractive therapeutic option. Multipotent mesenchymal stem cells (MSCs) have been widely used in the treatment of joint surface defects and, like other stem cells, have the potential for self-renewal, differentiation, and integration [2]. Whilst a structural role in repair was first proposed as the mechanism of action of MSC i.e. differentiation into primary ‘building block’ for repair, more recently MSC have been proposed to have an indirect secretory role of action. Identifying the mechanism of action of these cells remains elusive and it remains unclear which mechanism is primary in MSC repair of joint tissues.

One strategy to identify how MSC are working is to track them in target tissues. MSCs can be labeled with superparamagnetic iron-oxide nanoparticles (SPION) particles to track single cells with Magnetic Resonance Imaging (MRI), and thereby follow stem cell infiltration [3-5]. However, a limitation to tracking is that conventional MR sequences have long echo times that are unable to measure fast signal decays, which occur in dense bone tissue and with high SPION infiltration [6]. Ultra-short echo time (UTE) MRI can capture these rapidly decaying signals. In this work, we use 3D cones [7] for UTE acquisition to track tissue development after injection of SPION labeled MSCs in an ovine model.

Methods

This study included ten mature female Welsh Mountain Sheep with approval from both Local Ethics and the UK Home Office. The right stifle joints of each animal were opened via a parapatellar approach with the animals under general anaesthesia and strict asepsis. A full thickness, 8 mm diameter, osteochondral defect was created in the medial femoral condyle (MFC) in the left stifle joints of each animal.

MSCs were labeled using a ferumoxide suspension, Feridex IV (11.2 mg Fe/ml, Advanced. Mag. Co., USA), diluted with culture medium to 50 μg/ml. The fresh stock solution was prepared with protamine sulphate (sigma aldritch) as the transfection agent in distilled water (10 mg/ml). Further diluted transfection agent (6ug/mL) was mixed at room temperature with ferumoxides in cell culture medium for 60 minutes at room temperature. The animals were then injected with labeled MSCs into the medial femoro-tibial joint. Animals received MSC injections either one week post surgery or 4 weeks post surgery, and were sacrificed one week post MSC injection.

Imaging was performed with a 12-channel receive-only head coil on a 3.0T MRI system (MR750 GE Healthcare, Waukesha, WI, USA). Multi-echo sagittal UTE images were acquired with 3D cones gradient echo using: field-of-view=180x180x108mm3, matrix=320x320x36, TR=23.0ms, TEs = 0.03,4.0,8.1,12.1,16.1ms, TR=23.0ms, flip angle=15 deg, BW=62.50kHz, averages = 1, scan time = 6:06min for all echoes. Relaxation maps were calculated using linear least-squares regression.

After imaging, the joints were opened and the osteochondral defect sites were decalcified in formic acid/sodium citrate over 4 weeks, prior to paraffin processing for histological imaging. Sections through the central portions of the defect were made 10 um thick and stained with Toluidine Blue and Safranin O/Fast Green for SPION localization. Individual components of the sections were evaluated for clotting, infiltration, hyaline cartilage, and structural characteristics of bonding or degeneration.

Results

Figure 1 shows a high R2* in the joint capsule, where SPIONs are observed to accumulate in the Prussian blue staining. Figure 2 shows no major observable differences between joints with SPION labeled and unlabeled cells, particularly in the site of the defect. No SPIONs were observed in the defect with staining. Figure 3 shows images at multiple echo times and histological images. Figure 4 shows multiple tissue types identified on both the subtracted echo time image and histology.

Discussion and Conclusion

We used UTE MRI to track iron-oxide labeled stem cells that were injected near the site of chondral defects in sheep knees. We did not observe R2* attenuation consistent with the presence of stem cells in the defects, and confirmed these findings with histology and Prussian blue staining. UTE imaging provided anatomical detail that correlated with histological findings of bone and vessels in the sites of defect.

Acknowledgements

This was a University of Cambridge sponsored study with research support from Arthritis Research UK, GlaxoSmithKline, the National Institute of Health Research Cambridge Biomedical Research Centre, and Addenbrooke's Charitable Trust.

References

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