UHMWPE-induced calvarial osteolysis
UHMWPE-induced calvarial osteolysis was performed according to published protocols (16
). Briefly, mice in each group (C57BL/6 female 8–12 wk old) were sedated with isoflurane gas and anesthetized with ketamine (20% ketamine HCl, 15% xylazine, and 65% saline at 0.1 ml per 20 g body weight). A 10-mm incision was made over the sagittal midline suture of the calvarium and a 1.0 × 1.0 cm area of the periosteum exposed. In sham control mice, the incision was closed without any further intervention, whereas experimental mice received 20 μl dried UHMWPE particles with an average size of 53–75 μm (Sigma-Aldrich, St. Louis, MO). The particles were distributed over the periosteum using a sterile sharp surgical spatula and the incision sutured. Twelve days postoperation, a microcomputed tomography (micro-CT) scan was performed to confirm and verify the development of particle-induced osteolysis. Micro-CT scans were performed on the Aloka LaTheta laboratory CT scan machine (Aloka, System Engineering Co., Tokyo, Japan). The x-ray voltage was set at high. For each skull, 162 slices were scanned with an average section-to-section distance of 0.03 nm at a slow acquisition speed using a facedown head-front orientation. Data were analyzed using LaTheta software (Aloka, System Engineering, Tokyo, Japan). All experiments were conducted in accordance with the Institute for Animal Care and Use Committee of Albert Einstein College of Medicine (New York, NY).
H&E staining of mouse calvaria
Post-CT scan animals were euthanized utilizing CO2. Calvaria were excised and fixed in 4% paraformaldehyde in PBS (pH 7.4) and placed at 4°C. After 24 h, samples were placed in 10% EDTA in PBS (pH 7.4) for 48–72 h at 4°C until decalcified. The calvaria were then processed for paraffin embedding, sectioned to 5 μm, and stained with H&E.
In vitro bone marrow cell differentiation
Bone marrow cells were purified using the lineage-negative magnetic bead kit, which depletes cells expressing the following lineage Ags: CD5, B220, CD11b, Gr-1, CD-3, and Ter-119 (Miltenyi Biotec, Auburn, CA). Lineage negative (Lin–) precursors were then cultured in DMEM (Life Technologies, Rockville, MD), supplemented with 50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM l-glutamine, 0.1 mM nonessential amino acids, 1 mM pyruvate, and 10 mM HEPES containing either GM-CSF (10 ng/ml) for 8 d to obtain immature conventional DCs or further differentiated with RANK-L (5 ng/ml) and M-CSF (10 ng/ml) for 8 d to obtain osteoclasts.
Intravenous injection of GFP+ DCs and FACS analysis of cells recovered at sites of osteolysis
In some experiments, 2 wk postsurgery, mice (8–12-wk-old female C57BL/6) were injected i.v. with 107 bone marrow-derived GFP+-CD11c+ DCs prepared from C57BL/6-transgenic (Tg) (CAG-EGFP)10sb/J mice (The Jackson Laboratory, Bar Harbor, ME). The CD11c+ cells were purified from bone marrow cells that had been cultured in GM-CSF for 7 to 8 d, using CD11c+ Ab conjugated magnetic beads (Miltenyi Biotec). Cell purity following magnetic bead separation was assessed by FACS. Only preparations with a purity ≥99% were used. In some experiments, 96 h after DC injection, calvaria were isolated from sham and UHMWPE-implanted mice and directly observed as a whole mount using differential contrast imaging and fluorescence microscopy. In other experiments, 96 h following GFP+-DC injection, cells were retrieved from the calvaria by extensively washing the bone in DMEM and gently scraping the bone surface. Retrieved cells were either analyzed directly by FACS (to quantify the percentage of GFP+ cells) or stained for cathepsin K. For staining, cells were fixed for 10 min in 2% paraformaldehyde, washed in 10 mM glycine, and permeabilized for 30 min in 0.05% saponin. Cells were incubated on ice for 30 min with 10 μg/ml Ab to mouse cathepsin K (Santa Cruz Biotechnology, Santa Cruz, CA) followed by an anti-mouse IgG conjugated to Alexa Fluor 568. Cells were analyzed on an FACScan flow cytometer (BD Biosciences, San Jose, CA).
Western blot analysis
Cultured cells (DC or osteoclasts) were collected, washed in PBS, and lysed in 150 mM Tris-HCl (pH 8), 150 mM NaCl, 5 mM EDTA, and 1% Nonidet P-40 supplemented with a mixture of protease inhibitors (Roche, Basel, Switzerland). Eighty micrograms protein was run on a 12% SDS-PAGE. After blotting, membranes were probed with the following primary mAbs: cathepsin F, K, L, S, and TRAP (Santa Cruz Biotechnology). Secondary Abs were HRP-labeled, and detection was performed using the Super Signal West Pico Chemiluminesent substrate (Pierce, Rockford, IL).
The calvaria from sham and UHMWPE-implanted mice were extracted, decalcified as described above, and embedded in either OCT-compound (Tissue-Tek, Sakura, Torrance, CA) for frozen sections or in paraffin according to standard procedures. Slides were stained for 30 min at room temperature with 10 μg/ml Ab to mouse cathepsin K (Santa Cruz Biotechnology) followed by Alexa Fluor 568-conjugated goat anti-mouse secondary Ab. Stained slides were examined using a scanning confocal microscope (Leica AOBS system, Leica Microsystems, Deerfield, IL). All images were collected using identical fluorescence settings.
Fluorescence in situ hybridization
Fluorescence in situ hybridization was performed using a painting probe specific for the murine Y chromosome. DNA for the Y chromosome (M. A. Ferguson-Smith, University of Cambridge, Cambridge, U.K.) was labeled by degenerate oligonucleotide-primed PCR with Spectrum Orange-dUTP (Vysis, Abbott Molecular, Berkshire, U.K.). Five-micron formalin-fixed paraffin embedded tissue sections were baked overnight at 56°C, dipped twice in xylene for 5 min at room temperature, and rehydrated in ethanol (100–90–70%). Slides were then washed in 4× SSC/0.5% Tween 20 at room temperature for 30 min in a rotating shaker. Pretreatment to digest cytoplasm was performed with pepsin (final concentration 0.4 mg/ml) at 37°C for 30 min. Slides were denatured in formamide for 1 min and 45 s, and then a probe was applied and incubated overnight at 37°C in a humidified chamber. After washing, nuclei were counterstained with DAPI for 10 min. Interphase cells were imaged with an Olympus BX61 microscope equipped with a Cooke SensicamQE camera with IPLab for image acquisition (Olympus, Melville, NY). An IPLab script was generated to acquire images of interphase cells for the Spectrum Orange dye and for a differential contrast image. Multiple focal planes (13
) were acquired to ensure that signals on different focal planes were included. Images were analyzed with tools available through ImageJ (National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/
Generation and subcloning of the CD11c promoter
Two hundred nanograms genomic DNA from a 129 mouse was amplified using the following primers: forward primer 5′-CATGCTCGAGGATCCCAGAGCTGGCCTGCTG-3′ (designed with XhoI and BamHI restriction sites at the 5′); reverse primer 5′-CATGACCGGTCAGAAGCAGGCTCTGAGCAA-3′ (designed with the AgeI restriction site at the 3′). The amplified 2013-bp CD11c promoter corresponds to bases 3790–5802 of the published CD11c promoter sequence (Gene Bank DQ658851.1). Following PCR amplification, the promoter was digested with XhoI and AgeI restriction enzymes and cloned into the pCCL.PPT.PGK.GFP.PRE lentiviral vector upon removal of the PGK promoter (17
Lentiviral vector production and concentration
Lentiviral particles expressing either the non–tissue-specific PGK promoter-GFP construct or the tissue-specific mCD11c promoter-GFP construct were generated by calcium phosphate-mediated cotransfection of 293T cells with four plasmids: 1) a CMV promoter-driven packaging construct expressing the gag and pol genes; 2) a Rous sarcoma virus promoter-driven construct expressing rev; 3) a CMV promoter-driven construct expressing the VSV-g envelope; and 4) the previously generated 2013-bp CD11c promoter controlling GFP expression. Thirty hours following transfection, the culture supernatant containing the packaged viral particles was collected and concentrated by ultracentrifugation. Collected viral particles were titrated on bone marrow DCs using limiting dilution analysis. Briefly, 100,000 DCs were cultured in DMEM with progressively lower dilutions of either the PGK promoter-GFP or the murine CD11c prom-GFP lentivirus (1:10, 1:100, 1:1000, and 1:10,000). For each dilution, positively transduced DCs were quantified by FACS as a percentage of GFP+ cells. Calculation from the titration analysis indicated 1 × 109 transducing viral particles per milliliter.
Bone marrow transplant experiments
Six to 8-wk-old male mice (Csf1r–/– on an outbred C57BL/6J, C3Heb/FeJ-a/a, 129SvJ background) were killed with CO2 and the bone marrow collected by flushing femurs and tibia. Lineage negative (Lin–) cells were purified as described above (Miltenyi Biotec MACS Lin– cells). Cells were transduced with either the PGK prom-GFP lentiviral vector or the mCD11c prom-GFP lentiviral vector at a concentration of 108 TU/ml for 106 cells. Transduced cells were cultured for 24 h in StemSpan media (StemCell Technologies, Vancouver, British Columbia, Canada) supplemented with IL-3, IL-6, stem cell factor, and Flt-3 ligand cytokines. For bone-marrow transfer experiments, transduced cells were injected into the tail vein (106 cells/mouse) of 8-wk-old mice (C57BL/6 or Csf1r+/–) that had been previously lethally irradiated (2 × 6 Gy). Full engraftment of the transplanted hematopoietic stem cells was observed 2 mo following bone marrow transplantion.
Characterization of GFP+ DCs in blood, bone marrow, and spleen of the transplanted mice
Spleen, blood, and bone marrow samples were harvested from each mouse 2 mo following bone marrow transplantion. Single-cell suspensions were prepared from each organ and RBCs removed by lysis. For immunostaining, 105 cells were incubated in staining buffer (PBS, 2% FBS) for 30 min at 4°C with 1 to 2 μg/ml of the following PE-conjugated primary Abs: CD19, CD11b, CD11c, Gr-1, CD3, and NK1.1 (BD Pharmingen, San Diego, CA). Following incubation, cells were washed three times in staining buffer and analyzed on an FACScan flow cytometer (BD Biosciences).