Ferumoxide (Endorem, Guerbet, Aulnaysous-Bois, France) consists of superparamagnetic iron oxide (SPIO) particles with a nonstoichiometric magnetite core coated with dextran T-10.22
Ferumoxide has an r1
relaxivity of 40.0 mM−1
, an r2
relaxivity of 160 mM−1
(at 37°C and 0.47 T), and a hydrodynamic diameter of 80 to 150 nm.23
Ferumoxide is approved by the Food and Drug Administration (FDA) as a magnetic resonance contrast agent for liver imaging. Ferumoxide is taken up by cells of the reticuloendothelial system via endocytosis and stored in secondary lysosomes within the cytoplasm.24
Lipofectin (Invitrogen, Carlsbad, CA) is a reagent consisting of the cationic lipids N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoyl phophotidylethanolamine (DOPE) in a 1:1. mixture.25
The positively charged lipid molecules form complexes with the negatively charged contrast agent. The complexes then fuse with the cell membrane and deliver the contents into the cytosol.26
Protamine sulfate (American Pharmaceutical Partners, Schaumberg, IL) is a cationic peptide with a high arginine content and a molecular weight of approximately 4,000 Da.27
It is FDA approved to reverse heparin anticoagulation and is used in NPH insulin preparations. It has also been investigated as a transfection agent for cell labeling with ferumoxide.16
We investigated hMSCs (Lonza, Walkersville, MD), which were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) high-glucose medium (Invitrogen), supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT) and 1% penicillin-streptomycin in a humidified 5% CO2 atmosphere at 37°C. The medium was changed every other day. Cells were trypsinized at 90% confluency with 0.05% trypsin (Invitrogen). To preclude the possibility of senescence, experiments were performed at cell passages 6 to 12.
MSCs were labeled with ferumoxide by simple incubation (protocol 1), protamine transfection (protocol 2), or Lipofectin transfection (protocol 3) using established methods.16–18,28
Briefly, cells were plated at 90% confluency and allowed to adhere overnight at standard cell culture conditions. Culture medium was replaced with labeling medium (DMEM without FBS or penicillin-streptomycin) as specified below and incubated for 1, 6, 12, or 24 hours. Cells were then trypsinized and washed two times with phosphate-buffered saline (PBS) by centrifugation (400 rcf, 5 minutes, 25°C) to remove residual contrast agent. Cell viability was determined by the trypan blue exclusion test. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis was performed to quantify internalized iron content. We used the following protocols:
- Simple incubation: 20 mL of DMEM was mixed with ferumoxide at a concentration of 100 μg/mL and was added to the culture flasks
- Protamine transfection: ferumoxide and protamine were diluted in DMEM to a concentration of 100 μg/mL and 5 μg/mL, respectively. The solution was then shaken vigorously and added to an equal amount of full culture medium (final incubation concentration = 50 μg/mL).
- Lipofectin transfection: 178 μL Lipofectin reagent and 178 μL ferumoxide were dissolved in 1 mL of DMEM. Both solutions were let to sit for 30 minutes, shaken, and let to sit for another 30 minutes. The solution was then diluted in DMEM to a final volume of 20 mL (50 μg/mL) and added to the cell culture for labeling.
Fluorescence Microscopy of Ferumoxide-Labeled Cells
hMSCs were examined via confocal fluorescence microscopy for intracellular localization of contrast agent. Cells were plated onto multichamber glass slides (Nunc, Rochester, NY), allowed overnight to attach, and fixed at room temperature with Carnoy’s solution. Labeled cells were stained with anti-dextran fluorescein isothiocyanate (FITC; Stem Cell Technologies, Tukwila, WA) for 1 hour at room temperature, washed three times with PBS, and counterstained with DAPI. Samples were analyzed by confocal microscopy at 40× magnification (LSM 510, Zeiss, Thornwood, NY).
Electron Microscopy of Ferumoxide-Labelled Cells
hMSCs were plated onto Thermanox coverslips (Electron Microscopy Sciences, Hatfield, PA) and allowed to adhere. Fixation with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer was performed, and cells were postfixed with 1% osmium tetroxide followed by 2% aqueous uranyl acetate. Samples were dehydrated with a graded ethanol series and embedded in epoxy resin. Ultrathin sections were stained with 2% uranyl acetate and Reynolds lead citrate and examined at 80 kV in a JEOL 100CX II (Jeol, Tokyo, Japan) transmission electron microscope.
In Vitro MRI of Ferumoxide-Labeled Cells
Samples of 150,000 hMSCs, labeled with the protocols above, were embedded in 500 μL of 2% gelatin and underwent MRI on a 3 T clinical magnetic resonance scanner (Signa EXCITE, GE, Milwaukee, WI) using a quadrature knee coil (Clinical MR Solutions, Brookfield, WI). Cell samples were scanned in a water bath to avoid susceptibility artifacts. For determination of T2 relaxation times, spin echo (SE) sequences were obtained with a fixed repetition time (TR) of 2,000 ms and multiple echo time (TE) (60, 45, 30, 15 ms) values. T1 relaxation times were determined using a fixed TE of 15 ms and multiple TR (250, 500, 1,000, 4,000 ms) values. To determine T2* relaxation times, gradient echo images were obtained with a flip angle of 30°, a fixed TR of 500 ms, and varying TE (28.8, 14.4, 7.2, 3.7 ms) values. All sequences were acquired with a field of view (FOV) of 160 × 160 mm, a matrix of 256 × 256 pixels, a slice thickness of 3 mm, and one acquisition. These scans were performed in triplicate.
Ex Vivo MRI of MASIs in Pig Knee Specimens
Subsequently, we investigated the MRI characteristics of labeled hMSCs in Surgifoam scaffold (Johnson & Johnson, New Brunswick, NJ), an absorbable gelatin sponge. Surgifoam pads were immersed into liquid agarose (Type IX Ultra Low, 1.5% in PBS, Sigma-Aldrich, St. Louis, MO) at 37°C; 250,000 labeled cells were injected into the scaffold and cooled to 15°C to induce gelling. Scaffolds were then cut into cubic samples of 3 mm3 and implanted into artificially created full-thickness cartilage defects of pig knee joint specimens supplied by a local meat market. The following experimental groups were evaluated: scaffold only, scaffold with unlabeled hMSCs, scaffold with ferum-oxide-labeled hMSCs, scaffold with ferumoxide and Lipofectin, and scaffold with ferumoxide and protamine (n = 6 each). To remove trapped air, knee joints were filled with ultrasound gel (diluted 1:3 in PBS) after MASI.
MRI of pig knee specimens with MASIs was performed using a clinical 1.5 T magnetic resonance scanner (Signa EXCITE, GE) and a quadrature knee coil. T1-weighted SE sequences (TR 500 ms, TE 15 ms, band width [BW] 15.63 Hz, FOV 12 cm, matrix 512 × 192, two acquisitions, 3:16 minutes), moderately T2-weighted fat-saturated fast spin echo (FSE) sequences (4,300/25/31.25/15/512×256/2/4:14, echo train length 9), T1-weighted three-dimensional spoiled gradient recalled (SPGR) sequences (17/8.5/16/512×512/0.75/10:44, alpha 12), and T2*-weighted gradient echo sequences (500/14/15.63/12/512 × 192/2/3:16, alpha 30) were obtained with a 1 mm slice thickness.
All MRIs were analyzed using DICOM imaging software (OsiriX, UCLA, Los Angeles, CA). The signal intensities (SIs) of the cell suspension, chondrogenic pellet, or implanted scaffold were determined via user-defined regions of interest (ROI) and divided by the background noise to obtain the signal to noise ratio (SNR).
In Vivo MRI of MASIs in Osteochondral Defects of Rat Knee Joints
Preparation of MASI Constructs
hMSCs labeled with ferumoxide by simple incubation were used for in vivo experiments. Nonlabeled hMSCs served as controls. Subsets of labeled and unlabeled hMSCs underwent apoptosis induction via established techniques.21,29
In brief, hMSCs were incubated for 6 hours with mitomycin C at a concentration of 0.5 mg/mL at standard cell culture conditions. The cells were carefully washed three times with PBS and used for in vivo experiments.
Eighty milligrams of agarose powder (Sigma) was added to 2 mL of PBS for a final concentration of 40 mg/mL. This solution was gently shaken and auto-claved. After approximately 40 minutes, the fluid agarose solution was taken out of the autoclave and refrigerated for 24 hours before use. MASI constructs were prepared by combining 60% sterile agarose solution with 40% of the hMSC solution at a concentration of 15 million cells/mL.
The study was approved by the institutional animal care and use committee. In 10 nude athymic female Harlan rats, cartilage defects were created in the distal femur of both knee joints under inhalation anesthesia with 1.5 to 2% isoflurane in oxygen. A medial patellar skin incision was made, the patella was dislocated laterally, and a circular osteochondral defect (diameter: 1.5 mm, depth: 1.5 mm) was created in the distal femoral trochlear groove with a surgical drill. Hemostasis was achieved using cotton tips. MASI constructs of 5 μL total volume were transplanted into the defect. Six athymic rats received implantations of viable, ferumoxide-labeled hMSCs into cartilage defects of the right knee joint and mitomycin-pretreated, apoptotic ferumoxide-labeled hMSCs into the left knee joint. Four additional control animals received implantations of unlabeled viable and unlabeled apoptotic MSCs in each knee joint (n = 2 animals) or implantations of scaffold only in both knee joints (n = 2 animals). Then the patella was repositioned and the skin incision was closed by a suture.
Magnetic Resonance Imaging
All knee joints were evaluated with MRI on the day of implantation and then at weekly intervals for up to 12 weeks in rats with labeled hMSC transplants and up to 4 weeks for control rats (7 T magnetic resonance scanner, Varian, Palo Alto, CA). Animals were anesthetized with 2% isoflurane inhalation and placed supine on a custom-made animal imaging bed. Sagittal MRIs of the rat knee joints were obtained using a dedicated Helmholtz knee coil with T2-weighted SE sequences (TR 3,000 ms, TE 30 ms, two acquisitions). All sequences were obtained with an FOV of 25.6 mm, a matrix of 128 × 128 pixels, a slice thickness of 0.75 mm, and a flip angle of 90°/80°.
MRIs were analyzed using dedicated image processing software (ImageJ, National Institutes of Health, Bethesda, MD). The total area of the transplant was accessed by summation of the total number of pixels with a signal void on each image covering the transplant.
The SI of the transplant (SIMAST
) within the cartilage defect and the SI of background noise (SInoise
) in front of the knee joint (phase encoding direction) were measured using dedicated ROI. The minimum size of ROI was 15 pixels and the maximum size was 30 pixels. Measured SI values were normalized to the background noise and expressed as SNR = SIMAST
/standard deviation of Sinoise
Histopathology of Knee Joint Specimen
Following the last MRI, the animals were sacrificed, the knee joints were explanted, and specimens were placed in Cal-Ex II (a mixture of formaldehyde and formic acid; Fisher Scientific, Fair Lawn, NJ) for 48 to 72 hours. This decalcified and fixed the tissue simultaneously. Then the specimens were dissected parasagitally, dehydrated through graded alcohol washes, and embedded in paraffin. Five-micrometer sections were stained with hematoxylin and eosin (H&E), alcian blue, and Prussian blue. For immunohistochemistry, sections were deparaffinized and fixed with 4% formaldehyde followed by antigen retrieval with proteinase K and blocking of endogenous peroxidases with 1% H2O2 in methanol. The primary antibody used was specific for human CD44 antigen, which is a known marker for hMSCs (CD44 antibody, 1:150 dilution; Abcam, Cambridge, MA). The secondary antibody used was secondary biotinylated antimouse IgGs (Vector Laboratories, Burlingame, CA). For chromogenic staining, slides were blocked with 6% normal horse serum and developed with a chromogenic VECTASTAIN Elite ABC Kit (Vector Laboratories, Inc.) and a VIP Substrate Purple Kit (Vector Laboratories).
For fluorescent staining, slides were blocked with an avidin-biotin blocking kit (Molecular Probes, Eugene, OR) followed by blocking with 6% normal horse serum. Steptavidin-peroxidase conjugate coupled with biotinylated secondary antibody was visualized by Alexa Fluor 594 dye using tyramide amplification technique (TSA HRP-streptavidin kit, Molecular Probes).
T2 relaxation times of cell pellets and SNR data of MASIs in pig knee specimen were tested for significant differences between different labeling techniques using a t-test and a 5% level of significance. For comparison of multiple experimental groups, p values were adjusted with the Bonferroni correction.
For in vivo studies, SNR data and area of transplants with ferumoxide-labeled viable hMSCs and apoptotic hMSCs were tested for significant differences using a t-test. For all analyses, a p value of less than .05 was considered significant.