Figure 1.
In vitro detection of SPIO nanoparticle-labeled muscle progenitor cells by histology, MRI, and μCT.
A) Cells incubated with increasing concentrations of SPIO particles and PLL were fixed and stained with Prussian blue and pararosaniline to identify iron-labeled cells. Slides were imaged using brightfield microscopy. The graph shows the percentage of SPIO-labeled cells at each concentration (mean ± standard error of the mean) as determined by analysis from three blinded observers. B) MRI of SPIO-labeled cell standards incorporated within fibrin sealant in micro-centrifuge tubes. The graphs display T2* relaxation time versus SPIO concentration (left) and average pixel intensity versus SPIO concentration (right) as determined from mean values generated from three different labeling experiments. C) μCT images of the same SPIO-labeled cell standards incorporated within fibrin sealant as depicted in B). The graph displays the number of opaque voxels versus SPIO concentration as determined from values generated from three different labeling experiments. Data represents mean ± standard error of the mean for each.
Figure 2.
Localization of SPIO-labeled cells in implanted rat hearts by MRI.
A) Arrows indicate the location of hypointense regions resulting from the presence of SPIO nanoparticles contained in the implant. Coronal (left), axial (center), and sagittal (left) MRI slices from in vivo cardiac imaging acquisitions were obtained 6 months post-implantation using a 7T magnet. B) Arrows indicate the location of SPIO-labeled cells (hypointense region) in short-axis slices from in vivo cardiac acquisitions taken from two different rats at 6 months and 1 year after surgical implantation of engineered tissues, respectively. These images were obtained using a 4.7T magnet. C) Arrows indicate the location of iron-labeled cells within the implant in an isolated heart that had been implanted with engineered tissues for 1 year. These coronal (left) and axial (right) MRI slices correspond to the in vivo images shown in A).
Figure 3.
Localization of SPIO-labeled cells in whole animals by μCT.
A) Arrows indicate the location of the SPIO nanoparticle-labeled muscle progenitors contained within engineered tissues that were implanted in the AV groove of an adult rat heart for 1 year. Unlike MRI images, the iron presents as a positive contrast or hyperintense signal. Coronal (A), axial (B), and sagittal (C) μCT images are shown. The panels on the left show individual μCT image slices, while the panels on the right depict the corresponding volumetric renderings from all of the acquired slices. These renderings were set to a threshold level that permitted simultaneous visualization of bone and iron-labeled cells. This processing resulted in loss of some soft tissue detail.
Figure 4.
Localization of SPIO-labeled cells in excised hearts by μCT.
A) Arrows indicate the location of the SPIO nanoparticle-labeled muscle progenitors contained within engineered tissues that were implanted in the AV groove of an adult rat heart for 1 year. Signal from iron is shown as a positive contrast signal (white spot). The asterisk indicates the position of the aorta exiting the heart. Coronal (A), axial (B), and sagittal (C) μCT images are shown. Once again, the panels on the left show individual image slices and the panels on the right show corresponding volumetric renderings. In the absence of signal from bone, soft tissue detail becomes more apparent.
Figure 5.
Localization of SPIO-labeled cells within the area of implants by histology.
A) A photograph depicting the fibrin sealant-based implant in a heart excised one year after implantation (left panel), a low magnification image of a Masson's trichrome stained heart section (center panel), and a low magnification image from an adjacent serial section showing Prussian blue iron stain (right panel) of SPIO-labeled cells within the implant. The right atrium (RA), right ventricle (RV), and aorta are depicted along with an outline of the implant (dotted line). Scale bars equal 500 µm. B) Higher magnification brightfield images from the boxed region in A) showing an unprocessed serial section demonstrating iron (brown) can be detected in the cells of the implant and the same Masson's trichrome and Prussian blue stained sections from A). Scale bars equal 50 µm. C) The same section as the left panel from B) immunostained for the presence of macrophages (MAC387 in green), DNA (DAPI in blue), and striated muscle (ACTN in red) (left panel). Adjacent serial sections were immunostained for cardiac troponin T (cTnT in red), DNA (DAPI in blue), and striated muscle (ACTN in green) (middle panel). The right panel shows an image of ACTN, cTnT, and DAPI staining in the right ventricle. Scale bars equal 50 µm. D) Serial sections from a different heart stained for Masson's trichrome (left panel), Prussian blue with pararosaniline (middle panel), and immuno-fluorescence from secondary antibodies detecting binding of cTnT and ACTN antibodies in addition to DAPI staining of DNA. Iron is abundant in implanted cells, but is not apparent in the epicardium or myocardium of the right ventricle. The location of SPIO-labeled cells corresponds to the location of ACTN positive (red), cTnT negative (green) cells. Scale bars equal 50 µm.
Figure 6.
SPIO nanoparticle-labeled cells located within the implant in three different hearts.
A) Brightfield, fluorescence, and merged images showing iron deposits in progenitor cells contained within the implant (left panel), staining of filamentous actin with phalloidin (green) and DNA (blue) (middle panel) in the same section, and the merged image (right panel). Arrows indicate a few inflammatory cells that are not positive for iron. Scale bars equal 50 µm. B) A different heart showing heavily-labeled cells in the implant using the same staining as described for A). Scale bars equal 25 µm. C) Iron was also evident (brown) within a portion of the implant immediately adjacent to the right ventricular epicardium in a separate tissue section (left panel) that was immunostained for ACTN (red) and DAPI (blue) (middle panel). The right panel shows an overlay of the two images to the left. Scale bars equal 25 µm.