Synopsis

To enhance MR image contrast of calcium phosphate cement (CPC) to bone, CPC was tagged with ¹⁹F loaded nanoparticles. This ¹⁹F labeled CPC material, after implantation into bone defects in rat legs, was monitored in vivo at 11.7T by longitudinal ¹⁹F zero echo time (ZTE) MR imaging. We demonstrate that the overlay of ¹⁹F ZTE images on ¹H images allows for an excellent qualitative view of CPC with no background. By a quantitative evaluation of the ¹⁹F signal the in vivo degradation of CPC over weeks can be followed by MRI.

Introduction

Calcium phosphate cement (CPC), a widely used material for bone defect restoration, has poor MRI contrast relative to bone, due to similar T₁ and T₂ relaxation properties.¹ Thus，to enhance contrast CPCs have been tagged with gadolinium or iron-oxide agents, but these unfavorably affect the properties of CPC and MRI of iron-oxide particles suffers from a blooming effect.¹ Previously, we reported that incorporation of ¹⁹F loaded nanoparticles² into CPC material allowed for a better MRI visualization of CPC in bone.³ Results from ¹⁹F MRI of in vitro and ex vivo samples with these nanoparticles demonstrated the possibility of qualitative and quantitative evaluation of the presence of CPC.

In this study we assessed the real potential of this approach in a longitudinal study with in vivo ¹H and ¹⁹F MRI of ¹⁹F labeled CPC implanted into rat legs. We investigated whether ¹⁹F labeled CPC can be measured qualitatively and quantitatively by a comparison with the volumes of CPC implants estimated by computer tomography (CT), and whether the in vivo degradation of CPC can be traced over time by quantifying the ¹⁹F signal.

Methods

CPC material consisted of 59.1wt% alpha-tricalcium phosphate, 1.5wt% carboxymethyl cellulose and 39.4wt% cryo-grinded poly-lactic-co-glycolic acid (PLGA) particles. As ¹⁹F tracer we selected perfluoro-15-crown-5-ether (PFCE)² embedded into PLGA nanoparticles through a single emulsion reaction.⁴ This was incorporated into CPC at a concentration of 16.7%. After incorporated with the ¹⁹F nanoparticles CPC did not show different injectability and cohesion properties.

In vitro sample

20mg PFCE nanoparticles were dissolved into distilled water in a 200μl eppendorf tube as a control sample for ¹⁹F quantifications.

In vivo rat legs

All animal surgeries were performed with approval of the ethics committee for the care and use of laboratory animals. Three healthy male Wistar rats (weight: 250-300g) were included in this study. A cylindrical defect (diameter:2.5mm; depth:3mm) was drilled in both right and left posterior legs of each rat. Each defect was then filled with injectable CPC material mixed with PFCE nanoparticles. Both legs of each rat, along with the control sample, were separately imaged by MR at day 0, at 4 weeks and at 8 weeks.

Experiments

Measurements were performed on an 11.7T MR-system (BioSpec, Bruker) using a ¹H-¹⁹F dual-channel volume coil with 40mm inner diameter. ¹H/¹⁹F ZTE images for each rat leg were acquired with TR=2ms, image resolution=0.31mm³/1.25mm³, 1averages/32averages and FA=2°/4° in less than 7mins/14mins.

Data Analysis

The ¹⁹F signals in rat legs as well as in the control sample were quantified in MRIcro software. Regions of interest (ROIs) were manually outlined. ¹⁹F signal per ROI was quantified through multiplying the mean pixel intensity by the pixel number of the ROI and slice thickness.⁵ The total ¹⁹F amount was then summed over slices and expressed in arbitrary units (a.u.). For each leg, the ¹⁹F signals at three time points were corrected by normalizing the quantified ¹⁹F signals of the control sample at 4 and 8 weeks to the one at day 0.

Pearson correlation (SPSS) was used to evaluate the correlation between the normalized ¹⁹F signals and the CPC volumes in each rat leg across three time points. These volumes were estimated from corresponding images acquired by CT (details not shown).

Results

On ¹H ZTE images, CPC cannot be differentiated from bone in the rat legs in vivo (Fig.1A,B). However, in ¹⁹F ZTE images the PFCE in CPC becomes visible and overlaying the ¹⁹F on ¹H ZTE images enables a proper sample evaluation (Fig.1C,D).

The ¹⁹F signals in CPC decreased from day 0 to 8 weeks, reflecting the in vivo degradation of CPC over time (Fig.2A). A strong linear relationship between the normalized ¹⁹F signals and the estimated CPC volumes across three time points was found (Fig.2B). This strong relationship was confirmed for CPC implants in the other rat legs by high Pearson correlation coefficients (Table 1; mean: 0.957±0.05).

Discussion and conclusion

Acknowledgements

References

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3. Dou, W. et al. 19F ZTE MR Imaging of 19F labeled calcium phosphate cement at 11.7T. Proc. ISMRM Congress. 2016;p. 3877.

4. Srinivas, M. et al. Customizable, multi-functional fluorocarbon nanoparticles for quantitative in vivo imaging using 19F MRI and optical imaging. Biomaterials. 2010;31(27):7070-7.

5. Srinivas, M. et al. In vivo 19F MRI for cell tracking. J.Vis.Exp. 2013;(81):e50802.