Noninvasive in vivo imaging techniques (bioluminescence and microCT) were used to investigate initial macrophage migration systemically from a remote injection site to polyethylene wear particles continuously infused into the femoral canal. To confirm the localization of the migrated reporter macrophages in the UHMWPE particles infused femora and to detect bone remodeling related cellular markers, histologic and immunohistologic staining were used. In total, 30 successful animals were obtained out of 41 operated nude mice and distributed into four groups: (1) 10 mice had UHMWPE particles in the pump and were injected intravenously with reporter cells; (2) 10 mice had saline in the pump and were injected with reporter cells intravenously; (3) five mice had UHMWPE in the pump and injected intravenously with the carrier solution Hanks’ balanced salt solution (HBSS); and (4) five mice had saline in the pump and injected intravenously with HBSS. Another 10 animals received no surgery and therefore served as untreated controls (Fig. ).
A diagram of the experimental design is shown. (A) Animals were divided into five groups; (B) schedule of experiment. *The number of animals for histologic analysis; ¤the number of animals for microCT scanning.
We obtained 12-week-old male immunodeficient nude mice from Charles River Laboratory Inc (Wilmington, MA) and they were maintained in the university’s animal facility. Animals were divided into five groups (Groups 1–5) randomly (Fig. ). The murine macrophage cell line RAW264.7 was transfected with the lentiviral vector to express the bioluminescent optical reporter gene, firefly luciferase (fluc), and a fluorescence reporter gene, green fluorescent protein (gfp), as previously described [7
]. Prior power analysis was based on published data using a similar experimental design [25
]. To detect a difference of 1.5 standard deviations from the mean for BLI and bone mineral density (BMD) with a power of 80% (α = 0.05, β = 0.20), nine animals would be needed in each group. We strictly followed Stanford University’s guidelines for the care and use of laboratory animals. The murine continuous femoral intramedullary infusion model used in this study was modified from a previously described rat model [22
] and validated by successfully pumping UHMWPE and blue polystyrene particles into murine femoral medullary canals [25
A Model 2006 Alzet osmotic pump was used in the present study and has a mean loading volume of 243 μL and a mean pumping rate of 0.15 μL/hour. According to the pumping rate and duration of the experiment, approximately 100 μL of the pump contents could be pumped out during 4 weeks. Based on the volume and density of the isolated UHMWPE particles, approximately 3.0 × 109
particles were infused into the femoral medullary cavity. Based on previous studies, we presumed the size and the number of particles infused would induce an inflammatory reaction [11
]. Conventional (nonhighly crosslinked) UHMWPE particles from mechanical testing simulator studies of metal-on-conventional polyethylene bearings were isolated by ultracentrifugation [5
]. The size of the UHMWPE particles was 1.0 ± 0.1 μm (mean ± SE) in length measured by scanning electron microscopy (Hitachi S-3400N; Hitachi High-Tech, Tokyo, Japan). Approximately 70% of the particles were submicron and the shape factor of more than 90% of the particles was greater than 2. The particles tested negative for endotoxin using a Limulus Amebocyte Lysate kit (BioWhittaker, Walkersville, MD). The particles were suspended in sterile saline at a concentration of 15 mg/mL (based on the volume and density of the UHMWPE particles isolated, 15 mg/mL equals approximately 3.1 × 1010
particles/mL). Before implantation, we placed particles suspended in saline (15 mg/mL) or saline alone into the pump; particle suspension or saline prefilled and prelinked silicon tubing (6 cm) and a hollow titanium rod (6 mm long, 23 gauge) were connected to the outlet of the pump flow modulator before implantation. Thus, in each osmotic pump, approximately 7.5 × 109
particles were loaded. During the 3-week period of implantation, approximately 80 μL of the particle suspension could be pumped out theoretically.
Under inhalation anesthesia of 3% to 5% isoflurane in 100% oxygen, animals were operated on a warmed small animal surgery station. Using a sterile technique, a series of needles from 25 gauge to 21 gauge were used to manually drill through the intercondylar notch to access the medullary cavity of the left femur progressively. The osmotic pump was implanted subcutaneously in the interscapular region through a separate incision. The titanium rod was press-fit into the distal femur, and the connecting tubing was passed through a subcutaneous tunnel. After implant insertion, the quadriceps-patellar complex was repositioned and the medial parapatellar arthrotomy and the dorsal incision for the pump were repaired with sutures and biocompatible glue. Buprenorphine (0.1 mg/kg; Ben Venue Laboratories, Bedford, OH) was given subcutaneously immediately after surgery and 4 hours later postoperatively for pain control. We checked the animals each day postoperatively. Only the data from the 30 animals with intact connections from the pump to the implanted rod in the femoral cavity during the whole experimental period were included in the final analysis.
Macrophage injections began 10 to 14 days postoperatively when wound healing was satisfactory. We injected reporter macrophages (5 × 105 cells) suspended in 0.1 mL HBSS (Invitrogen, Carlsbad, CA) intravenously through the lateral tail vein of mice. Luciferase substrate D-luciferin (Biosynth International, Itasca, IL) was administered by intraperitoneal injection (3 mg/mouse). Five minutes after substrate injection, bioluminescence images were taken of the entire mouse using an in vivo imaging system (Caliper LifeSciences, Hopkinton, MA) in the Stanford Small Animal Imaging Facility. We obtained prone and lateral images from each animal at each time point to better determine the origin of photon emission. Animals were imaged at 2-day intervals postmacrophage injection for 10 days. Bioluminescence images were quantified by drawing uniformly sized rectangle regions of interest (ROIs, 0.5 cm × 1.5 cm) over the implanted and contralateral thighs. The data were collected and expressed as photon/second/cm2/steradian (p/s/cm2/sr). The ratios of BLI (infused versus the contralateral nonoperated limb) for each group were calculated.
We presumed the systemic migration of macrophages to the femur resulting from both UHMWPE particle infusion and remote macrophage injection would increase osteolysis locally in the femur. A previous in vitro study showed resorption pits on bone slices incubated with inflammatory cells exposed to particles for 4 days [3
]. We performed pre- and postexperimental microCT scans in vivo to detect changes in BMD. Before the original surgical procedure while under inhalational anesthesia, animals were scanned using an eXplore RS microCT scanner (GE Medical Systems, Raleigh, NC) with 49-μm resolution. To minimize motion artifact, the femora and body were fixed by tape during scanning. After all the BLI studies were completed, animals were euthanized with CO2
and the implanted titanium rods were removed from the limbs and then microCT scanning was performed again. The image acquisition and reconstruction were completed using eXplore Evolver and eXplore Reconstruction interface software (GE Medical Systems), respectively. The thresholded bone mineral density (TBMD) was quantified based on Hounsfield unit (HU) calibration by using GEMS MicroView software (GE Medical Systems). The TBMD was normalized by subtracting the pretreatment values from postexperimental values of each animal. BMD was analyzed in GEMS MicroView (threshold: 1700 HU) with a phantom calibration for each scan and recorded in milligrams per milliliter of hydroxyapatite. Within the distal part of the femur, a three-dimensional ROI (4 mm × 4 mm × 3 mm) was created, which contained only the diaphysis proceeding proximally beginning 3 mm from the end of the femoral condyles along the femur. For Groups 1 and 2, six animals were randomly selected for microCT scanning and analysis; five mice in Groups 3 and 4 were also scanned. The data from these animals were analyzed blindly.
After completion of the imaging experiments, we collected all femora from the experimental and control groups. Femora from three animals in both the UHMWPE particle and saline groups were randomly chosen for histologic study. Frozen sections were collected from the distal to the middle portion of each femur and used for immunostaining. We used rabbit anti-GFP monoclonal antibody (Chemicon International, Temecula, CA) to detect exogenous macrophages tagged with green fluorescent protein (GFP). Rat antimouse MOMA-2 (AbD Serotec, Raleigh, NC) was used to detect total macrophages (both injected and noninjected). Mouse antihuman vitronectin receptor αVβ3 antibody (Chemicon International) and anti-osteocalcin antibody (LifeSpan Biosciences, Seattle, WA) were used to identify osteoclasts and osteoblasts, respectively. The secondary antibody used was goat antimouse/rabbit/rat IgG conjugated with Alexa Fluor 488/594 (Invitrogen, Carlsbad, CA). Briefly, phosphate-buffered saline (PBS) buffered paraldehyde solution (4%)-fixed frozen sections (6 μm thick) were blocked by Image-iT™ FX Signal Enhance (Invitrogen) for 30 minutes followed by modified Hank’s solution with 10% normal donkey serum for 1 hour at room temperature. Primary antibodies were incubated with sections at 4°C overnight; then the sections were incubated with secondary antibodies for 1 hour at room temperature. ProLong® Gold Antifade Reagent with DAPI (Invitrogen) was used for mounting. We performed washing with PBS between steps. After mounting, images were taken using Nuance multispectral imaging systems (Cri, Woburn, MA). Background autofluorescence was erased by examining the light spectra for specific fluorophores. For each randomly selected sample, we imaged six to eight sections by using the same setting of the program to reduce or erase the autofluorescence.
Hematoxylin and eosin (H&E) staining (Sigma, Steinheim, Germany) was performed to show the general morphology of the femoral section collected as mentioned previously. A leukocyte acid phosphatase kit (Sigma) was used to stain tartrate-resistant acid phosphatase (TRAP) for identification of osteoclasts.
The ratio of the bioluminescence of operated divided by the nonoperated femora within the ROI and TBMD of the microCT analysis of the femora was analyzed by the nonparametric Kruskal-Wallis and Mann-Whitney U tests (two-tailed) between groups (statistiXL, Broadway-Nedlands, Australia).