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F-FPRGD2 PET of osteoarthritis
Supplementary methods
18F-FPRGD2
radiosynthesis
The 18F-FPRGD2 productions were compliant with cGMP regulations. The radiosynthesis of
N-succinimidyl 4-18F-fluorobenzoate (18F-SFB) was automatically performed on a
commercial synthesis module (GE Healthcare FASTlab™) with a radiochemical yield (decay
corrected) of 32% and a radiochemical purity >96%. The coupling reaction between
18
F-SFB
and the FPRGD2 was performed using a second disposable cassette clamped onto the
FASTlab™ (Fig. S1) [1]. The crude product was purified on a HPLC system; the fraction
containing the pure
18
F-FPRGD2 was collected on the Fastlab, diluted with saline and then
concentrated on a C18 cartridge. The cartridge was eluted with ethanol and saline and the
eluted solution was sterilized through a 0.22 µm filter. The mean activity of the purified
18
F-
FPRGD2 obtained was in the range of 3750 MBq in 130 minutes with an overall
radiochemical yield (from the
18
F-fluoride) of 13% (decay corrected), radiochemical purity
98% and a specific activity of 140 ± 40 TBq/mmol.
18F-FPRGD2
PET/CT
All patients were fasted for 6 hours prior to
18
F-FPRGD2 injection. Whole-body (from the
skull base to mid-thigh) PET/CT images were acquired on a Gemini PET/CT system (Philips
Medical Systems, Cleveland, OH, USA) TF or a Big Bore and started 60 minutes (median: 60
min; range 59-86) after intravenous injection of a mean activity (± SD) of 309 (± 18) MBq of
18
F-FPRGD2. A low dose CT (5 mm slice thickness; tube voltage: 120 kV and tube current–
time product: 50 to 80 mAs depending on the patient’s weight) followed by the PET emission
scan of 3 minutes per bed position (pbp) were performed. Considering the whole body
acquisition, the uptake time per field of view therefore varied from 60 minutes at the skull
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F-FPRGD2 PET of osteoarthritis
base to 85 minutes at the proximal region of the thigh. Data were reconstructed using time of
flight including correction for decay, scatter, random and attenuation (CT data were used for
attenuation correction).
FDG PET/CT
All patients were fasted for 6 hours prior to FDG injection; the mean (± SD) blood glucose
level was 96 (± 15) mg/dl at time of injection. The images were acquired 60 minutes (median:
70 min; range 52-116) after injection of a mean activity (± SD) of 304 (± 155) MBq of FDG
(depending on the patient’s weight). The low dose CT parameters were similar to the
18
F-
FPRGD2 PET/CT. The PET emission scan duration was 1 to 2 minutes pbp (depending on the
patient’s body mass index). Data were reconstructed using the same algorithm as the
18
F-
FPRGD2 PET/CT. Out of the 37 patients for which the FDG was available for comparison,
the
18
F-FPRGD2 PET/CT was performed on the same machine in 30 patients (81%). The
difference of uptake time between the FDG PET and the
18
F-FPRGD2 PET/CT exceeded 15
minutes for 13/37 (35%) patients (median: 11 min; range 0-39).
Cells from OA patients
Cartilage tissues were obtained from OA patients during joint replacement surgery in order to
assess the expression of integrin αvβ3 in cells present in OA joints. The institutional
Committee on Ethics (CHU de Liege, Belgium) approved the study protocol and the use of
verbal informed consent to allow research procedures on collected tissues, as explained in the
institutional information booklet written by the hospital and provided to each patient.
Clinicians gave an oral statement about the research, informed the patients of authorizations
obtained from the local Research Ethics Committee and answered any additional questions.
Then, clinicians obtained verbal consent from the patient to participate in the research.
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F-FPRGD2 PET of osteoarthritis
Chondrocytes were isolated from human femoral head cartilage as described in [2]. Cells
were cultured in DMEM medium (Cambrex Bio Science, Walkersville, USA), 10% FCS
(Lonza, Basel, Switzerland), L-glutamine (2 mM), streptomycin (100 mg/ml) and penicillin
(100 U/ml) (BioWhittaker, Walkersville, USA). Briefly, primary chondrocytes (2x105
cells/0.5ml of medium) were seeded in 24-well plates (BD Biosciences, USA) in triplicate and
cultivated 1, 4 or 14 days prior to perform protein extraction.
Western Blotting
Whole cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis as explained earlier [3]. Membranes were incubated three hours at room
temperature with following primary antibodies: αv (#ab76609) and β3 (#ab131056) from
ABCAM and GAPDH (#G9545, Sigma-Aldrich). Anti-rabbit secondary antibody (Cell
Signaling) and ECL chemiluminescent reagents (Amersham Biosciences) were used for
revelation.
Fluorescence-activated cell sorting
Single stainings were performed with FITC-conjugated αvβ3 (LM609) (#FCMAB282F) or
isotype control IgG1 (#MABC002F) antibodies from Millipore. The phenotype was
performed on gated living chondrocytes after 1 and 14 days of monolayer culture. The
fluorescence intensity was analyzed on 2 lasers FACScalibur with Cell Quest Pro software
(BD Biosciences).
Immunohistochemistry
Healthy articular cartilage sections were obtained from the Biobank of the Pathology
Department of the CHU of Liege. OA femoral heads of patients undergoing hip replacement
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F-FPRGD2 PET of osteoarthritis
surgery were cut in slices using a bandsaw before fixation in 4% paraformaldehyde 24h at
4°C, decalcification in DC2 (Labonord) for 2 hours at 4°C. Slices were cut in pieces prior to
be embedded in paraffin, and cut into 5μm sections (Microtome Leica RM2165). Paraffinembedded sections were deparaffinized with xylene and then rehydrated in graded ethanol.
Endogenous peroxidase activity was blocked by incubation of the sections with Dual
Endogenous Enzyme Block (Dako #S2003) for 5 minutes at room temperature.
Glycosaminoglycans were removed by incubating the sections with 0.4 units/liter of
proteinase-free chondroitinase ABC (Sigma) in 0.1M Tris HCl, pH 8.0, OVN at 37°C.
Nonspecific binding was blocked by incubation of the sections with Protein Block Serum-free
(Dako #X0909) for 10min. After rinsing, sections were incubated OVN with specific antiαvβ3 antibody (clone LM609) (1:50), or irrelevant anti-prolactin antibody (Biogenex) (1:50)
diluted in Antibody Diluent (Dako #S2022) at 4°C in a humidified chamber. Rinsed sections
were incubated with Envision+ System-HRP Labeled Polymer anti-rabbit (Dako #K4003) in a
humidified chamber for 30 minutes at room temperature. Peroxidase was detected with Liquid
DAB+ Substrate Chromogen System (Dako #K3468) for 10 minutes in a humidified chamber.
Rinsed sections were counterstained for 30 seconds with Carazzy’s hematoxylin, dehydrated
and mounted. The sections were examined using bright field microscopy with FSX100
(Olympus) microscope and FSX-BSW software (Olympus). The sections were examined
using bright field microscopy with FSX100 (Olympus) microscope and FSX-BSW software
(Olympus).
Statistical analyses
The Mc Nemar test was performed to compare the prevalence of musculoskeletal uptake of
18
F-FPRGD2 or FDG in our population. The uptake (SUVs and ratios) of
18
F-FPRGD2 and
FDG were compared using the Student t test for paired samples. The Spearman’s correlation
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18
F-FPRGD2 PET of osteoarthritis
coefficient was used to estimate the association between
18
F-FPRGD2 and FDG uptake. The
generalized linear mixed model (GLMM) was applied to compare the intensity of
18
F-
FPRGD2 and FDG uptake in the different subtypes of osteoarticular processes (OA,
discopathy, enthesis and fracture). The Scheffé post-hoc test was performed to compare
subtypes 2 by 2. The relation between
18
F-FPRGD2 or FDG uptake and the OA grade was
assessed using generalized estimating equations. The Poisson regression and GLMM were
used to test the relation between the age or gender and the number of joints with 18F-FPRGD2
(or FDG) uptake or the intensity of uptake. The generalized estimating equations were used
for the estimation of the relationship between the prevalence of 18F-FPRGD2-positive joint or
disc and the CT morphologic changes grades, the difference in occurrence in grades 1 to 3
were compared to the grade 0. Results were considered to be significant at the 5% level (p <
0.05). Calculations were done using SAS version 9.3 (SAS Institute, Cary, NC, USA).
Supplementary references
1.
Thonon D, Goblet D, Goukens E, Kaisin G, Paris J, Aerts J, et al. Fully automated
preparation and conjugation of N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) with RGD
peptide using a GE FASTlab synthesizer. Mol Imaging Biol. 2011;13(6):1088-95.
2.
Relic B, Bentires-Alj M, Ribbens C, Franchimont N, Guerne PA, Benoit V, et al.
TNF-α protects human primary articular chondrocytes from nitric oxide-induced apoptosis via
nuclear factor-κB. Lab Invest. 2002;82(12):1661-72.
3.
Relic B, Zeddou M, Desoroux A, Beguin Y, de Seny D, Malaise MG. Genistein
induces adipogenesis but inhibits leptin induction in human synovial fibroblasts. Lab Invest.
2009;89(7):811-22.
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F-FPRGD2 PET of osteoarthritis
Supplementary figures
Fig. S1 Radiosynthesis of 18F-FPRGD2: coupling reaction between PRGD2 and 18F-SFB [1].
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F-FPRGD2 PET of osteoarthritis
Fig. S2 The PET/CT images show focal intense uptake of 18F-FPRGD2 (A; red arrows) in the
insertion of the joint capsule on the femoral neck bilaterally. The FDG PET/CT images (B;
green arrows) show also a focal mild FDG uptake in the same location, but FDG uptake
(SUVmax 2.61) is clearly lower than 18F-FPRGD2 (SUVmax 5.61).
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F-FPRGD2 PET of osteoarthritis
Fig. S3 The PET/CT images show a miscellaneous uptake of 18F-FPRGD2 (A; purple arrows)
around a right hip prosthesis; the FDG PET/CT (B) shows higher FDG uptake (SUVmax 8.32;
dark green arrows) than
18
F-FPRGD2 uptake (SUVmax 2.51). In contrast, in the left hip joint
with signs of severe osteoarthritis, the
18
F-FPRGD2 uptake (SUVmax 4.39; red arrows) is
higher than FDG uptake (SUVmax 3.65; light green arrows).
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F-FPRGD2 PET of osteoarthritis
Fig. S4
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F-FPRGD2 uptake (SUVmax and signal/BG ratios) according to the subtypes of
musculoskeletal disorders. The p value is indicated when the SUVmax difference was
statistically significant.
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F-FPRGD2 PET of osteoarthritis
Fig. S5
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F-FPRGD2 uptake (SUVmax and signal/BG ratios) according to the location of joint.
The p value is indicated when the SUVmax difference was statistically significant.
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F-FPRGD2 PET of osteoarthritis
Fig. S6 The PET/CT images show both FDG and 18F-FPRGD2 uptake in the insertion of the
tendons of the hamstring muscles on the left ischial tuberosity, the
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F-FPRGD2 uptake (A;
red arrows; SUVmax 1.69) was lower than FDG uptake (B; green arrows; SUVmax 3.05).
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