Gadolinium-based contrast agents (GBCAs) are applied worldwide to enhance magnetic resonance imaging.1 As free gadolinium (Gd) is toxic (LD50 GdCl3.6H2O: 100–200 mg/kg, intravenous in mice),2 chelating ligands are applied. These belong to 2 classes, macrocyclic and kinetically less stable linear complexes.1 Although GBCAs were thought to have a convincing safety profile (clinical dose for tissue enhancement: 0.1 mmol/kg), reports about Gd deposits in tissue in the past few years have triggered new animal research, investigating Gd retention after single or repeated administration of GBCAs in healthy animals and disease models with a focus on brain tissue.1 Gadolinium's fate in visceral organs and bone was investigated to a lesser extent, although bone was known to show the largest Gd content among studied tissues from animal studies in the 1990s.3 Depending on the delay, the concentration in the kidneys may exceed that in the bone. The particularity of bone lies in its very low rate of washout.

Bone Gd content was investigated in animal studies4–7 and in patients undergoing hip replacement surgery,8 as well as in autopsies9 preponderantly by bulk analysis. In many cases, inductively coupled plasma–mass spectrometry (ICP-MS) technique was used after dissolution of the sample material. It has been shown that Gd was retained in bone tissue for more than 8 years after injection, suggesting that Gd might be released from the chelating complex and incorporated into bone matrix due to its similarity to calcium.5 Speciation analysis elucidated that Gd was mainly dechelated in rat femur.10

Little is known about the spatial distribution of Gd in bone. One study investigated gadopentetate dimeglumine spatial distribution in bovine patellae after diffusion into the tissue,11 but the transferability of the results to intravenous injection remains questionable. Another study showed that Gd was initially located close to the periosteum and bone cavities in the femur of young rats, but after 1 year, Gd deposits moved toward the interior of the femur with bone remodeling processes.12 Only 1 investigation was performed using x-ray fluorescence spectroscopy, assigning Gd deposits to cement lines (CLs) and vascular channels,13 but the GBCA applied remained unclear.

Most animal studies investigated Gd deposits in rodent bone tissue.7 Here, 1 clinical dose of different GBCAs, comparable to the concentration of GBCAs applied to patients, was applied to sheep, as their anatomy and metabolism are more similar to those of humans than rodents.14,15

We aimed to determine the allocation of Gd in the femur of sheep that were exposed to a single clinical dose of Gd, and we used an analytical method (laser ablation [LA]–ICP-MS) to determine its spatial distribution 10 weeks after application of GBCAs.

MATERIALS AND METHODS

This prospective animal study was conducted from March to May 2018. Bone tissue analysis started in August 2021. Swiss-Alpine sheep (n = 36; all female; age range, 4–10 years) with a mean body weight of 80.8 ± 19.6 kg were prospectively investigated. The study was approved by local governmental authorities (animal license number ZH235/17) and conducted according to the Swiss Animal Welfare Act. Our study was added to a long-term feeding experiment leading to the following tooth wear- and rumen washing-related publications16–21 and GBCA-associated publications.14,15 The study design followed 3R requirements (replace, reduce, refine).

Sheep were randomly assigned to 6 groups of 6 animals each and injected intravenously with 1 of the GBCAs at a dose of 0.1 mmol/kg, or with an equal volume of saline (0.2 ml/kg NaCl 0.9%). Gadolinium-based contrast agents applied were gadodiamide (Omniscan; GE Healthcare AG, Wädenswil, Switzerland), gadobenate dimeglumine (MultiHance; Bracco Imaging Deutschland GmbH, Konstanz, Germany), gadoteridol (ProHance; Bracco Imaging Deutschland GmbH, Konstanz, Germany), gadobutrol (Gadovist; Bayer Vital GmbH, Leverkusen, Germany), and gadoterate meglumine (Dotarem; Guerbet AG, Paris, France). Injections and euthanasia were performed by 2 veterinarians (4 and 11 years of experience, respectively) blinded to the assigned study group as described before.14 Immediately after euthanasia, bone tissue samples collected from 1 randomly selected sheep femur of each group were harvested for further analysis. Cryopreserved samples included epiphysis (sample 2) and diaphysis including bone marrow (sample 1) as shown in Figure 1. Disposable equipment was used to avoid cross-contamination.

FIGURE 1:

Diagram showing the composition of the femur (A) with the trabecular bone of the epiphysis and articular cartilage (B), the compact bone of the diaphysis (C), and the medullary cavity with yellow bone marrow (fat cells) (D). Positioning of sample 1 (diaphysis) and sample 2 (epiphysis) is visualized accordingly.

Sample Preparation and Standard Preparation for LA-ICP-MS

Cryopreserved tissue of 1 randomly selected animal per group (epiphysis and diaphysis) was sawn, formalin-fixed, and methacrylate (MMA; Technovit 7200 VLC; Heraeus Kulzer, Wehrheim, Germany) embedded without decalcification according to standard protocols, cut (10 μm), and placed on quartz slides.

For the preparation of standard solutions of gadolinium chloride (Alfa Aesar, Haverhill, MA), a concentration range of 0 to 1 mg/g was prepared, and 900 μL of those was mixed with 100 mg dry gelatin (Carl Roth GmbH & Co. KG, Karlsruhe, Germany). After heating the mixtures to 50°C, they were homogenized using the vortex mixer Vortex-Genie 2 (Scientific Industries, Bohemia, NY). A drop of the mixture was placed on a cryo carrier, cooled to −20°C, and cut into 10 μm thin sections using the Cryostar NX70 cryostat (Thermo Fisher Scientific, Waltham, MA). The Gd concentration of the standards was validated by ICP-MS after acidic digestion with HNO3 (VWR Chemicals, Radnor, PA).

LA-ICP-MS Analysis

For spatially resolved analysis, the hyphenation with a dual concentric injector (Elemental Scientific Lasers, Bozeman, MT) of a 266-nm Nd:YAG laser ablation system (imageBIO266, Elemental Scientific Lasers) equipped with a 2-volume ablation cell (TV2; Elemental Scientific Lasers) and an inductively coupled plasma–triple quadrupole–mass spectrometer (iCAP TQ; Thermo Fisher Scientific, Waltham, MA) with oxygen as a reaction gas was used. Q3-analytes were 158Gd16O+, 43Ca16O+, 56Fe16O+, 66Zn+, and 32S16O+. The tissue was ablated at a laser fluence of 2.2 J/cm2. For analysis, spot sizes of 30 μm and 6 μm with a line spacing of 25 μm and 5 μm, respectively, were used at a scan speed of 100 μm/s and 20 μm/s, respectively, resulting in actual spot sizes of 25 μm and 5 μm.

The data were analyzed with the in-house developed software imajar3.64 (written by Robin Schmid). For quantification, 11 lines with a length of 20 seconds of each gelatin standard were ablated, and the first line was discarded for comparability reasons. The averaged intensity was plotted against concentration and evaluated using weighted linear regression according to.12 The limit of quantification (LOQ) was calculated according to Boumans.22 For the analysis with a spot size of 25 μm, an LOQ of 34 ng/g was obtained, and for the analysis with a smaller spot size of 5 μm, an LOQ of 380 ng/g was calculated. To allow a better assignment of the Gd deposits with the structures of the bone, overlay images were created. Details of quantification are provided as Supplemental Material, https://links.lww.com/RLI/A855.

Tissue Preparation and Histology Examination

Bone tissue (n = 36) was formalin-fixed, decalcified, paraffin-embedded, and hematoxylin-eosin (HE)–stained according to standard protocols. Histological evaluation was performed based on the INHAND (International Harmonization of Nomenclature and Diagnostic Criteria) guidelines for laboratory animals,23 using a microscope (Leica DMR system; Leica Biosystems, Nussloch, Germany) by a veterinary pathologist (12 years of experience). Histology of diaphysis, epiphysial cartilage, subchondral, and trabecular bone, as well as bone marrow, was scored according to a semiquantitative grading system (0, normal; 1, minimal; 2, mild; 3, moderate; 4, marked; 5 severe). The distribution of histological changes (F, focal; MF, multifocal; C, coalescing; and D, diffuse) was examined. Bone remodeling was analyzed based on bone activity level (bone resorption vs bone formation) and osteoclasts per microscopic field 100× (0, none; 1, rare, 1–4 cells; 2, moderate, 5–10 cells; 3, abundant, >11 cells).

RESULTS

Thirty-six Swiss-Alpine sheep (range, 4–10 years, female) were included in the study and received a single injection (0.1 mmol/kg bodyweight) of macrocyclic (gadobutrol, gadoteridol, and gadoterate meglumine), linear (gadodiamide and gadobenate dimeglumine) GBCAs, or saline. To clarify which parts of sheep femur were investigated in this study, a diagram with representations of tissues from femoral bone (epiphysis, diaphysis, and bone marrow) is shown in Figures 1A to D.

Spatial Distribution of Gd in Bone Tissue After GBCA Exposure

For one sheep of each treatment group, Gd deposits were investigated in the epiphysis and diaphysis as well as in bone marrow using LA-ICP-MS. Microscopic images as well as quantitative Gd elemental distribution maps are provided (Figs. 2A–X). Ten weeks after single injection of a clinically relevant dose in adult sheep, both linear species of GBCA resulted in considerably higher accumulation than macrocyclic GBCAs.

FIGURE 2:

Light microscopic images and elemental bioimages of thin sections of epiphysis and diaphysis after injection of gadodiamide, gadobenate, gadobutrol, gadoteridol, gadoterate, and saline. Each panel depicts a light microscopic image of the analyzed tissue to visualize mineralized bone structures and bone marrow cavities (left) and the quantitative gadolinium distribution (right). The dashed line marks the mineralized bone tissue outline. Arrows mark the bone cartilage junction (A, B, E, F) and the osteal surfaces (C, D, G, H). Bone marrow is marked by asterisks (O, P, W, X).

The approach to compare Gd concentrations is described in detail in Supplemental Material, https://links.lww.com/RLI/A855. Gadolinium deposits were highest in animals exposed to gadodiamide (Figs. 2A–D; 98th percentile B: 5800 ng/g, D: 5100 ng/g), followed by those injected with gadobenate (Fig. 2E–H; 98th percentile F: 720 ng/g, H: 710 ng/g), and less in those administered with the macrocyclic agents gadobutrol, gadoteridol, and gadoterate (Figs. 2I–T; 98th percentile J: 230 ng/g, L: 400 ng/g, N: 92 ng/g, P: 51 ng/g, R: 36 ng/g, T: 65 ng/g), whereas only extremely low Gd concentrations were detected in control animals (Figs. 2U–X; 98th percentile V: <LOQ, X: 35 ng/g).

Gadolinium retention after application of linear and macrocyclic GBCAs was seen within specific restricted tissue compartments: foremost in the endosteum of the epiphysis (Figs. 2B, F, J, N, R, ring-shaped structures), endosteum covering Haversian and Volkmann channels of the diaphysis (Figs. 2 D, H, L, P, T, dots and lines in the compact bone), as well as the endosteum of the bone marrow cavity and the periosteum (Figs. 2 D, H, L, P, T, white arrows). In addition, after injection of linear GBCAs and gadobutrol, Gd is found at the bone cartilage junction (BCJ) (Figs. 2B, F, J, white arrows). In bone marrow of trabecular bone (Figs. 2B, F, J, N, R) and yellow bone marrow of the medullary cavity (Figs. 2P, X; asterisk), Gd deposits were almost not detectable for all GBCAs applied. In addition, Gd was not found in osteons of epiphysis and diaphysis.

To further narrow down the exact sites of Gd deposits and to determine colocalization with other metals, bone exposed to gadodiamide was investigated with a spatial resolution of 5 μm, which was enabled by sufficient Gd tissue concentrations after application of gadodiamide. Three regions were selected for further investigation: the BCJ (Fig. 3A), the epiphysis (Fig. 3B), and the diaphysis (Fig. 3C). Overlays with calcium, iron, and zinc distributions are shown. Gadolinium deposits are detectable at the BCJ, more specifically at the border of calcified and uncalcified cartilage, the “tidemark” (Fig. 3D, asterisk), ring-shaped structures in trabecular and compact bone belonging to vascular channels (Figs. 3D–F, green arrows), the endosteum (Fig. 3E, red arrow), as well as in CLs, separating osteons and extraosteonal bone matrix (Fig. 3E, white arrows). Partial overlay of Gd with calcium (Figs. 3G–I), iron (Figs. 3J–L), and zinc (Figs. 3M–O) is seen mainly in ring-shaped structures and at endosteal surfaces (white arrows) and for Gd, zinc, and calcium also at the “tidemark” (Figs. 3G, M, asterisk). Zinc is found mainly in a layered pattern in the calcified cartilage zone neighboring the “tidemark” (Fig. 3M), as well as in a patchy pattern in lamellar bone structures (Figs. 3N, O).

FIGURE 3:

Light microscopic images and elemental bioimages of thin sections of sheep femur after injection of gadodiamide. The first row depicts the microscopic image of the analyzed tissue: bone cartilage junction (A), epiphysis (B), and diaphysis (C), the respective quantitative gadolinium distribution (D–F), an overlay of gadolinium distribution (green) with calcium (blue) (G–I), iron (magenta) (J–L), and zinc distribution (red) (M–O). Gd deposits at the tidemarks are indicated with asterisk (D, G, M), vascular channels with green arrows (D–F), endosteum with red arrows (E), and cement lines with white arrows (E, G–O).

Histological Analysis of Bone After GBCA Application

Formalin-fixed, paraffin-embedded, and HE-stained sections of epiphysis, diaphysis, as well as bone marrow were evaluated histologically from all 36 sheep. Exemplary photomicrographs are shown in Figures 4A–F. Analysis of the tissue response and bone remodeling was performed for epiphysis (cartilage, subchondral, and trabecular bone; Fig. 4 B), femur diaphysis (Fig. 4C), and yellow bone marrow (Fig. 4D) according to INHAND-based criteria. On all examined sections, no pathological alterations were detectable (N = within normal limits). No inflammatory or degenerative changes were present. Bone remodeling was static (bone formation = bone resorption) with 1–4 osteoclasts per microscopic field 100× (1, rare), which was interpreted as physiological. Between the groups of animals injected with different GBCAs, no significant changes could be detected with respect to the examined parameters.

FIGURE 4:

Representative photomicrographs from light microscopy (HE stain; original magnification 200×) of (A and D) the epiphysis (subchondral zone), (B and E) diaphysis (compact bone), and (C and F) yellow bone marrow (fat cells) are shown for gadodiamide-exposed animals (gadodiamide) (upper line, left to right) and control animals (saline) (lower line, left to right). No histological alterations are seen.

DISCUSSION

In this study, elemental imaging of adult sheep femur after single-dose injection of linear and macrocyclic GBCAs resulted in Gd deposition in bone linings and additionally in a subset of GBCAs in CLs and the BCJ. Histological examination of bone, cartilage, and bone marrow in the 36 sheep under study revealed no tissue abnormalities.

Since the 1980s, bone is known to be a Gd reservoir with Gd retention observed up to 8 years after GBCA application.5 Gadolinium bone concentration was mainly investigated by bulk analyses using ICP-MS–based techniques.4–8,24,25 Thus, there is only sparse information available about the distinct localization of Gd in bone tissue. In a study by Turyanskaya et al,13 a small bone sample exposed to an unknown GBCA the CLs and the vascular pore walls were discussed to be prone to Gd retention. Supposing that GBCAs will be distributed mainly by vasculature, it is conceivable that spatial proximity of blood vessels in Haversian and Volkmann channels enable incorporation of Gd into bone, potentially by entering the mineral phase after being laid down in CLs. This concept is in line with observations of Funke et al,12 describing Gd deposits after 5 weekly injections of gadobutrol and gadodiamide into 9-week-old young rats at endosteal surfaces and in small tissue pores, and the Gd deposits moved toward the interior of the femur over 1 year. In contrast to Fretellier et al7 and Funke et al,12 we do not detect substantial Gd deposits in bone marrow of epiphysis and diaphysis most likely due to the fact that in adult sheep only yellow bone marrow is present. In adult sheep, 10 weeks after single application of GBCAs, Gd deposits are found in the same locations as described by Turyanskaya et al13 and additionally at endosteal surfaces; for linear GBCAs and gadobutrol, Gd was also found at the BCJ. The Gd distribution pattern described in the literature12,13 and in this study assigns Gd deposits to matrix mineralization sites of bone and articular cartilage.26,27

To investigate the composition of Gd deposits and the mechanism of retention, Gd was coregistered with calcium, zinc, and iron. In recent years, there were fewer studies on Gd-loaded bone with respect to metal composition than ones on Gd-containing skin tissue. Groups investigating cutaneous Gd deposits found a correlation of Gd, phosphorus, and calcium, indicating insoluble Gd phosphate28,29 or deposits with Gd, phosphorus, calcium, and zinc.30 It is conceivable that ionic Gd3+ is released from its chelator (transmetalation)10 by competing ions such as calcium or zinc,31 but this is not proven yet. The mechanism of Gd incorporation in bone or the form in which Gd is deposited is far from being understood. The first question that could be addressed in the future is if Gd is deposited intracellularly, for example, in osteoblasts, but it is still unknown which transport mechanisms could be used. It is in principle possible to use channels such as aquaporins. It could be hypothesized that zinc-related processes are involved in deposits in CLs as proposed by Turyanskaya et al.13 We see spotted overlap of Gd with calcium, zinc, and iron. Also Funke et al12 reported Gd colocalization with calcium and iron at all time points. To provide further evidence on the mechanism and form of Gd deposition, more studies need to be performed. To ascertain intracellularity of Gd, for example, in osteoblasts, colocalization studies with lanthanide-spiked antibodies could be interesting to perform.

Although bone is known as a long-term Gd reservoir, to our knowledge, there are no other studies analyzing morphological changes of bone and cartilage tissue after application of GBCAs. In our study, using conventional histological stainings, we did not find any histological changes in the samples, which can be traced back to Gd injection.

LIMITATIONS

One limitation of the study is that assessment of Gd-related toxicity in phases of normal bone remodeling is still pending as speciation analysis determining the exact chemical form of Gd deposits is lacking—the same holds true for increased release from bone tissue into circulation (eg, pregnancy, lactation, and osteoporosis)5 or increased uptake into bone (eg, after in utero exposure, young age, or septicemia).7,32,33 It has been discussed for long time that bone might serve as a long-term reservoir, allowing for Gd redistribution into other tissues over time.4,34–36 Although Funke et al12 did not observe complete Gd clearance over the observation period of 12 months, this does not exclude slow release of Gd and redistribution on other bone surfaces.

Because of the lack of precise information regarding the age of the sheep from which the samples were obtained, we cannot entirely rule out any age-related effects. However, we consider this factor to be negligible because all the sheep used in the study were adults. In addition, we have to state about a potential loss of Gd during sample preparation, as well as about the limited sample size for analyzing spatial distribution of Gd by LA-ICP-MS.

Another drawback is that euthanization had to take place after 10 weeks and gives us only a snapshot of the Gd distribution pattern of adult bone. Therefore, no conclusions can be drawn about time aspects of Gd deposition in bone tissue or about application of GBCAs to developing bone and cartilage. Nevertheless, our study design reflects the most relevant clinical situation with a single dose of GBCAs applied.

Although PMMA-embedding is a standard preparation for bone, it cannot be excluded that this embedding medium influences tissue element composition. Skedros et al26 compared the calcium content of PMMA-embedded and native bone by quantitative energy dispersive x-ray analysis and found slight but no significant differences.

Taken together, the current study provides evidence that after application of a single dose of GBCAs in sheep, Gd retention is restricted to the bone linings, and in a subset of GBCAs found in CLs and the BCJ. No histological changes were detectable.

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