Osirix software

Both knees of all pigs underwent MRI at 1, 2, 4, and 12 weeks after therapeutic cell implantation, using a clinical 3T MRI scanner (Signa HDxt, GE Healthcare, Milwaukee, Wisconsin) and a one-channel receive-only loop coil (GE Healthcare, Milwaukee, Wisconsin). The MRI protocol consisted of three clinical sequences: a fat-saturated PD-weighted fast spin-echo, a multi-echo spin-echo (MESE), and a three-dimensional fat-saturated spoiled gradient echo (SPGR) sequence. Parameters for the PD were TR = 3,345 ms, TE = 33 ms, FA = 111º, matrix size = 192 x 192 pixels, slice thickness (SL) = 1.5 mm, FOV = 8 cm and TA = 5 min. Parameters for T2 mapping (MESE) included TR = 41 ms, TE = 10/ 20/ 30/ 41/ 51/ 61 ms, FA = 90º, matrix size = 192 x 192 pixels, SL = 1.5 mm, FOV = 8 cm, TA = 12 min. T2* mapping (SPGR) was measured using TR = 100 ms, TE = 2.7/ 6.9/ 11.2/ 15.5/ 19.8/ 24.1/ 28.8/ 32.7 ms, FA = 80º, matrix size = 192 x 192 pixels, SL = 1.5 mm, FOV = 8 cm, TA = 6 min). For MRI data analysis, T2* relaxation times in each implant were measured by an operator-defined region of interest on a T2* map using Osirix Software (version 10.0, 64 bits; Pixmeo, Geneva, Switzerland).

A total of seven observers with at least five years of working experience and with CBCT working experience were selected for this investigation. All observers were trained on the OsiriX software (Version 7.0.2; Pixmeo SARL, Bernex, Switzerland) and calibrated by having them assess one CBCT case that was not entered into the evaluation. The order of the CBCTs was randomized for each observer and observation. To assess intra-operator-reproducibility, the procedure was repeated within six weeks again with a randomized order of CBCT images.

The volume of contrast agent uptake in the aorta and brachiocephalic arteries (BCA) was calculated from inversion recovery MRI images by manually segmenting the visually enhanced region of the vessel wall (OsiriX Foundation, Geneva, Switzerland). Cardiac function was assessed from Cine-FLASH images calculating left end-systolic and end-diastolic ventricle volumes and the ejection fraction through OsiriX software (Pixmeo SARL, Geneva, Switzerland).

After completion of control IgG, doxorubicin, CD47 mAb, CD47 mAb + doxorubicin therapy, all mice underwent MRI on a 7T MR scanner (Bruker‐Agilent Technologies‐General Electric Healthcare, Billerica, MA, USA) before intravenous injection of ferumoxytol and at 24 h after injection of ferumoxytol via the tail vein (Feraheme™; AMAG Pharmaceuticals, Waltham, MA, USA, 30 mg·kg⁻¹). The following pulse sequences were used: T2‐weighted fast spin echo sequences with a repetition time (TR) of 4500 ms, an echo time (TE) of 42 ms, and a flip angle α: 90° and T2‐weighted multislice multiecho (MSME) sequences with a TR of 3000 ms, a TE of 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, and 96 ms and α: 90°. A field of view of 2 cm × 2 cm and a slice thickness of 0.5 mm for the MRI acquisitions. MSME images were used to create T2 maps and measure T2 relaxation times of the whole tumor with osirix software (Pixmeo, Geneva, Switzerland).

Scanning and analyses followed published guidelines [29 (link)]. Ex vivo high-resolution acquisitions (VivaCT 40, Scanco, Brüttisellen, Switzerland) at 10.5 μm voxel size (55 kV, 145 μA, 347 ms integration time), were performed on post-extraction days 28 and immediately after drill preparation. Multiplanar reconstruction and volume rendering were carried out using OsiriX software (version 5.8, Pixmeo, Bernex, Switzerland).

The vascularized area was quantified in Osirix software (V.4.12, Pixmeo, Geneva) by manually selecting region of interests (ROIs) around tubular vessel-like structures on post-contrast T2*-w images. Care was taken to exclude areas that were hypointense on pre-contrast images and most likely represent microbleedings or calcifications. For histogram analysis, the tumor region and an outside region in the frontal white matter were manually segmented. Intensity values for histogram analysis were read out in Matlab (Release 2015a, The MathWorks, Inc., Natick, MA). Images were normalized using the following formula: (mean voxel intensity_tumor – intensity_outside)/standard deviation (SD).