Specimen Procurement and Selection
The Visible Heart® Laboratories received viable human hearts and heart-lung bloc specimens for research via LifeSource, a nonprofit organ procurement organization (Minneapolis, MN, USA). Donors gave consent for their organs to be used for scientific research purposes via LifeSource. The hearts used in this study were deemed non-viable for transplant due to advanced patient age, cardiac downtimes, identified poor cardiac function, and/or other reasons. All specimens were received as fresh viable tissues, along with donors’ relative cardiac-pulmonary clinical histories. We dissected lungs from the hearts while fresh, and each specimen was carefully cannulated for immediate reanimation, if specific criteria were met. Otherwise, hearts were placed within a formalin fixation apparatus14,15 for a 24-hour fixation period. The perfusion-fixation apparatus preserved hearts in their end diastolic shape, importantly keeping the aortas and coronaries dilated. After being perfusion fixed (including hearts initially reanimated), each specimen was placed in its own container and stored for future studies.
We performed PCIs and micro-CT scanning utilizing six human heart specimens (see Table 3 for detailed patient information). Three of the specimens exhibited adequate cardiac function prior to donation and were considered viable candidates for reanimation and eventual perfusion fixation, as described earlier. The remaining three hearts were received from LifeSource two to eleven years prior to this study. We selected these specimens due to noted histories of coronary artery disease and prior imaging indicating the LM coronaries were patent enough to perform PCIs.
Coronary Intervention in Isolated Hearts
We performed PCIs in three reanimated hearts (HH534, HH541, HH556) using Visible Heart® methodologies16; subsequently each heart was carefully removed from the apparatus and perfusion fixed so as not to damage the newly implanted stent(s). Previously fixed hearts (HH115, HH202, HH479) were rinsed for 24 hours, re-cannulated, and placed in an acrylic box where they were attached to a Langendorff static perfusion apparatus (Fig. 4a) which continuously perfused the aortas and coronaries with water. The advantages of utilizing Visible Heart® methodologies while performing PCIs were as follows: 1) endoscopic cameras enabled direct visualization of each procedural step; 2) there were no risks to any living patient, thus unlimited fluoroscopy could be used; and 3) OCT could be utilized as many times as desired as there was no need to expedite the procedures.
Each PCI procedure was guided and recorded simultaneously by 2.4mm and 4mm endoscopic cameras (Olympus, Tokyo, Japan), OEC Elite Fluoroscopy (GE, Boston, MA, USA), and episodic OCT imaging, as shown in Figure 4b. The combination of these imaging modalities nearly simultaneously would not be possible without the use of a clear perfusion solution (Krebs-Henseleit buffer for reanimated hearts and water for fixed hearts) that continuously circulated through the apparatus. All PCIs were conducted using Resolute Onyx drug-eluting stents, compliant and non-compliant Euphora balloons, and Cougar XT guidewires (Medtronic, Santa Rosa, CA, USA).
OCT Imaging during PCI
After stent implantations, we captured OCT scans using OPTIS Intravascular Imaging System and DragonflyTM Imaging Catheters (Abbott Vascular, Abbott Park, IL, USA). The automatic pullback system captured 540 frames over a scanning trajectory of 54mm with a 5mm penetration distance, to capture the highest resolutions possible (~100µm). Since specimens were continuously perfused using clear solutions in both setups, no contrast injections were needed or administered during OCT scanning.
Following ex vivo stenting, the three reanimated hearts were perfusion fixed and then placed in formalin containers for long-term preservation. All OCT image datasets were post-processed to identify both distal and proximal portions of the implanted stents and then stored as 2D images. Since intracoronary OCT is a widely accepted method of imaging implanted stents, the images collected were later used to compare to the micro-CT reconstructions.
Before micro-CT scanning each human heart, the specimens were rinsed in water for a minimum of 24 hours to remove traces of formalin before handling and/or transportation. Once thoroughly rinsed, each heart was placed within the specially constructed plexiglass container and then scanned using an X5000 micro-CT scanner (North Star Imaging, Rogers, MN, USA). All heart specimens were carefully perfusion fixed to elicit an end diastolic shape for all four chambers (maximal filled) prior to scanning because the internal space of the scanner did not readily allow for our perfusion system to be used during scanning. Each heart was placed in the scanner as shown in Figure 5, and imaging was performed utilizing the following parameters to achieve approximate isotropic voxel sizes of 20 x 20 x 20 µm: 170kV tube voltage, 144µA tube current, 24.5 isowatts, and 1,500 radiograph images captured throughout a ~15-min scanning duration. We selected these parameters after numerous iterations and scanning trials, all utilizing Resolute Onyx stents implanted in swine heart coronaries, to optimize scanning resolutions while minimizing streaking, shadowing, and/or scanning artifacts. Figure 6 shows the progression of scanning parameters trialed, until we finalized the optimal parameters used in this study. Once scans were completed, each heart specimen was returned to the laboratory and placed in its respective formalin container. Imaging datasets were then reconstructed using North Star Imaging’s reconstruction software, into 8-bit 2D images (.tiff), for future analyses as described below.
We imported .tiff files from micro-CT scans into the DICOM analysis software Mimics (Materialise, Leven, Belgium), where they were computationally “stacked” to form 3D volumes from 2D images17, followed by further post-processing. Using Mimics, for each heart’s image dataset, we generated a high-frequency “mask” to segment out the higher density portions of the scan, i.e., the cobalt alloy shell and platinum iridium core of the Resolute Onyx. We manually created additional masks to segment out the vessel blood volumes and tissues. For subsequent analyses, each generated model consisted of a portion of the aortic wall, left coronary ostia, LM vessels, coronary stent(s), and proximal portions of left circumflex artery (LCX) and left anterior descending (LAD) coronaries. Once these models were generated, we used assessment tools in Mimics to measure the relative lumen areas. The 3D models were then exported from Mimics to be rendered as virtual reality scenes using a video game design software (Unity, Unity Technologies, San Francisco, CA, USA) which allowed for further visual inspections of LM stenting outcomes.