C57BL/6 wild type and green fluorescence protein (GFP) transgenic mice were purchased from Jackson Laboratories (Bar Harbor, ME).
All procedures were approved by the institutional animal care and use committee and were compliant with the humane use of animals. A fracture of the tibia was created as previously described  with minor modifications. In brief, the mice were anesthetized with isoflurane inhalation and the left hind limb surface was soaked with 10% Povidone-iodine solution (Purdue Pharma L.P., Stamford, CT). Using a surgical scalpel (WESTNET, Inc., Canton, MA), an approximately 1-mm-long incision was created in the medial side of the left distal tibia without cutting entirely through the bone. In some mice, the incision was made in the soft-tissue without reaching the tibia. Mice remained mobile after the surgery.
89ZrCl4 was produced at the institutional cyclotron facility. 89Zr-oxine complex was synthesized from oxine and 89ZrCl4 as previously described . Briefly, 4 μl of 20% tween 80 solution (Sigma-Aldrich, St. Louis, MO) and 102 μl of 20 mM oxine in 0.04 N HCl were mixed in a tube, and then 60 ul of 89ZrCl4 (37–74 kBq) was added and vortexed. The resulting solution was neutralized to pH 7.0–7.5 by adding 500 mM NaHCO3 in portions with incremental vortexing.
BM cell labeling with 89Zr-oxine
BM cells were flushed from femurs and tibias of donor mice. Red blood cells were lysed using ammonium-chloride-potassium (ACK) lysis solution (Thermo Fisher Scientific Inc., Waltham, MA) and the BM cells were washed in phosphate-buffered saline (PBS). Cell labeling with 89Zr-oxine complex was performed to achieve the labeling doses optimized for the BM cells in the previous study . Cells were incubated with 89Zr-oxine complex at 45 kBq/106 cells in PBS at 30:1 cell suspension-to-89Zr-oxine solution volume ratios for 20 min at room temperature. Labeled cells were washed twice in RPMI culture media (Life Technologies, Grand Island, NY) supplemented with 10% fetal calf serum (Gemini Bio Products, Sacramento, CA), 100 IU/mL penicillin, 100 μg/mL streptomycin (Life Technologies), and 50 μM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). 89Zr-oxine-labeled BM cells were transferred to mice intravenously (2–2.3 × 107 cells at 9.65–15.7 kBq/106 cells). BM cells collected from wild type were used unless specified that GFP transgenic mice were used as donors.
MicroPET/CT imaging of transferred 89 Zr-oxine-labeled BM cells
One group of mice received 89Zr-oxine-labeled BM cell administration followed 1 day later by tibial injury (d1-fracture group). MicroPET/CT imaging (BioPET, Bioscan, Washington, DC) was performed 1 day after the cell transfer, before and after the injury, and on days 2 and 5. Another group of mice received 89Zr-oxine-labeled BM cells immediately after the tibial injury (d0-fracture group) followed by microPET/CT imaging on days 0, 1, 2, and 5. A 5-min emission PET scan per bed position was performed for a total of two bed positions on day 0, using a 400–700 keV energy window. On days 1, 2, and 5, scan time per bed position was increased to 6, 7.5, and 12.5 min, respectively, to account for radioactive decay. The acquired images were reconstructed using a three-dimensional ordered-subsets expectation maximization algorithm. VivoQuant software (inviCRO LLC, Boston, MA) was used to fuse the maximum intensity projection PET images with CT images.
Quantitation of 89Zr-oxine-labeled BM cell migration on microPET/CT images
Volumes of interest were drawn on the acquired images using VivoQuant and MIMvista (MIM software, Cleveland, OH) software. The percentage of injected cells migrated to the injury and organs were obtained from the quantitated radioactivity in the tissue corrected for decay divided by the injected dose. For the tibial fracture, corresponding contralateral tibia was used as a control. Signal-to-background ratios of the fracture or the contralateral control were calculated from maximum standardized uptake value (SUV) of the fracture or contralateral control against mean SUV of the muscle. For the bone marrow uptake, the skull, forelimbs distal to the shoulder joint, and fracture site were excluded from the quantitation.
Mobilization of BM cells by plerixafor
D1-fracture mice received an intravenous injection of 5 mg/kg of plerixafor (Adooq Bioscience, Irvine, CA) at days 1–3 after the BM cell transfer (d1-fracture-plerixafor group). For d0-fracture mice, plerixafor was injected on day 0 at the time of BM cell transfer, and on days 1 and 2 for a total of 3 doses (d0-fracture-plerixafor group).
Flow cytometry analysis of BM cells migrated to the fracture sites and spleen
Thirty-three million BM cells collected from GFP transgenic mice were transferred to wild-type hosts, in the d1- and d0-fracture models. Two days after cell transfer, BM cells were flushed and collected from the fractured section of tibia and the corresponding section of the contralateral non-fractured tibia. A 70 μm filter was used to create a single cell suspension. The femur of the contralateral side was also isolated and BM cells were collected. Spleen was harvested, homogenized, and passed through a 70 μm filter; red cells were lysed using ACK lysis solution. Collected cells were stained with antibodies against CD45, CD29, CD105, CD11b, Ly6C, and Ly6G or with lineage markers (CD3, NK1.1, Ly6G, CD2, CD5, B220). All antibodies were purchased from eBioscience (San Diego, CA). Data collected by a flow cytometer (FACSCalibur, Becton Dickinson, San Jose, CA) was analyzed using FlowJo software (Tree Star, Inc., Ashland, OR).
89Zr-oxine-labeled GFP+ BM cells were transferred to wild-type recipient mice and fracture was generated 1 day later. Tibias were removed from both fracture and non-fractured contralateral control sites at 1.5 and 7 weeks after the cell transfer and placed in 4% paraformaldehyde in PBS. Tibias were then embedded in paraffin and sectioned into 5-μm-thick slices. Following deparaffinization and rehydration, the slides were stained with hematoxylin and eosin (H&E). Immunohistochemistry staining was performed with an anti-GFP antibody conjugated with horseradish peroxidase (Life Technologies) followed by 3,3′-diaminobenzidine staining (GFP-IHC). Tibias from non-fractured wild-type and GFP transgenic mice were processed as controls.
Two-way repeated measure analysis of variance (ANOVA) with Sidak’s multiple comparisons test was used to analyze the difference in BM cell accumulation at the fracture site compared to non-fractured tibia. Mann-Whitney test was used for comparing the peak BM cell accumulation values at the fracture with and without mobilization, and for analyzing the flow cytometry results between fractured- and non-fractured-tibias. P value, with adjustment for multiple comparison when comparing more than two groups, less than 0.05 was considered significant.