MegaPro, a clinically translatable nanoparticle for in vivo tracking of stem cell implants in pig cartilage defects.

Rationale: Efficient labeling methods for mesenchymal stem cells (MSCs) are crucial for tracking and understanding their behavior in regenerative medicine applications, particularly in cartilage defects. MegaPro nanoparticles have emerged as a potential alternative to ferumoxytol nanoparticles for this purpose. Methods: In this study, we employed mechanoporation to develop an efficient labeling method for MSCs using MegaPro nanoparticles and compared their effectiveness with ferumoxytol nanoparticles in tracking MSCs and chondrogenic pellets. Pig MSCs were labeled with both nanoparticles using a custom-made microfluidic device, and their characteristics were analyzed using various imaging and spectroscopy techniques. The viability and differentiation capacity of labeled MSCs were also assessed. Labeled MSCs and chondrogenic pellets were implanted into pig knee joints and monitored using MRI and histological analysis. Results: MegaPro-labeled MSCs demonstrated shorter T2 relaxation times, higher iron content, and greater nanoparticle uptake compared to ferumoxytol-labeled MSCs, without significantly affecting their viability and differentiation capacity. Post-implantation, MegaPro-labeled MSCs and chondrogenic pellets displayed a strong hypointense signal on MRI with considerably shorter T2* relaxation times compared to adjacent cartilage. The hypointense signal of both MegaPro- and ferumoxytol-labeled chondrogenic pellets decreased over time. Histological evaluations showed regenerated defect areas and proteoglycan formation with no significant differences between the labeled groups. Conclusion: Our study demonstrates that mechanoporation with MegaPro nanoparticles enables efficient MSC labeling without affecting viability or differentiation. MegaPro-labeled cells show enhanced MRI tracking compared to ferumoxytol-labeled cells, emphasizing their potential in clinical stem cell therapies for cartilage defects.

An intravascular magnetic wire for the high-throughput retrieval of circulating tumour cells in vivo.

The detection and analysis of rare blood biomarkers is necessary for early diagnosis of cancer and to facilitate the development of tailored therapies. However, current methods for the isolation of circulating tumour cells (CTCs) or nucleic acids present in a standard clinical sample of only 5-10 ml of blood provide inadequate yields for early cancer detection and comprehensive molecular profiling. Here, we report the development of a flexible magnetic wire that can retrieve rare biomarkers from the subject's blood in vivo at a much higher yield. The wire is inserted and removed through a standard intravenous catheter and captures biomarkers that have been previously labelled with injected magnetic particles. In a proof-of-concept experiment in a live porcine model, we demonstrate the in vivo labelling and single-pass capture of viable model CTCs in less than 10 s. The wire achieves capture efficiencies that correspond to enrichments of 10-80 times the amount of CTCs in a 5-ml blood draw, and 500-5,000 times the enrichments achieved using the commercially available Gilupi CellCollector.

Differences in Vascular Response between Balloon Overstretch and Stent Overexpansion in Nonatherosclerotic Porcine Coronary Arteries.

Which preclinical models are best suited for restenosis research remains uncertain. Here we compared the restenotic responses after balloon or stent overstretch injury in a porcine coronary artery. A total of 30 coronary lesions in 5 pigs were treated by balloon overdilatation or oversized stent implantation at various balloon-to-artery (B:A) ratios. Four weeks later, the lesions were examined in vivo by using coronary angiography, intravascular ultrasound, and optical coherence tomography (OCT). At follow-up, the lumen area stenosis and plaque burden at the minimal lumen area site were greater in stented sites than in balloon injury site (lumen area stenosis, 21.7 ± 8.9% compared with 32.8 ± 12.1%; plaque burden, 30.1% ± 10.1% compared with 44.7% ± 10.1%, respectively). The remodeling index was significantly smaller for the balloon-injury group than the stent group (0.86 ± 0.11 compared with 1.00 ±0.04). Only the stent group that was dilated at a high B:A ratio resulted in increased plaque burden. In the balloon-injury sites, high B:A ratios were significantly associated with greater negative remodeling. Tissue morphology assessment by OCT revealed that the predominant pattern in balloon injury sites was homogeneous, whereas that in stented sites was a layered to heterogeneous pattern. Neointimal proliferation was significantly greater after oversized stenting than after balloon overstretch injury. Together these findings suggest that stent overexpansion of porcine coronary arteries might be appropriate for researching restenosis than is the balloon overstretch injury model.

Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part II. In Vivo Imaging of Bone Marrow Stromal Cells in Swine with PET/CT and MR Imaging.

Purpose To quantitatively determine the limit of detection of marrow stromal cells (MSC) after cardiac cell therapy (CCT) in swine by using clinical positron emission tomography (PET) reporter gene imaging and magnetic resonance (MR) imaging with cell prelabeling. Materials and Methods Animal studies were approved by the institutional administrative panel on laboratory animal care. Seven swine received 23 intracardiac cell injections that contained control MSC and cell mixtures of MSC expressing a multimodality triple fusion (TF) reporter gene (MSC-TF) and bearing superparamagnetic iron oxide nanoparticles (NP) (MSC-TF-NP) or NP alone. Clinical MR imaging and PET reporter gene molecular imaging were performed after intravenous injection of the radiotracer fluorine 18-radiolabeled 9-[4-fluoro-3-(hydroxyl methyl) butyl] guanine ((18)F-FHBG). Linear regression analysis of both MR imaging and PET data and nonlinear regression analysis of PET data were performed, accounting for multiple injections per animal. Results MR imaging showed a positive correlation between MSC-TF-NP cell number and dephasing (dark) signal (R(2) = 0.72, P = .0001) and a lower detection limit of at least approximately 1.5 × 10(7) cells. PET reporter gene imaging demonstrated a significant positive correlation between MSC-TF and target-to-background ratio with the linear model (R(2) = 0.88, P = .0001, root mean square error = 0.523) and the nonlinear model (R(2) = 0.99, P = .0001, root mean square error = 0.273) and a lower detection limit of 2.5 × 10(8) cells. Conclusion The authors quantitatively determined the limit of detection of MSC after CCT in swine by using clinical PET reporter gene imaging and clinical MR imaging with cell prelabeling. (©) RSNA, 2016 Online supplemental material is available for this article.

Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part I. Reporter Gene Design, Characterization, and Optical in Vivo Imaging of Bone Marrow Stromal Cells after Myocardial Infarction.

Purpose To use multimodality reporter-gene imaging to assess the serial survival of marrow stromal cells (MSC) after therapy for myocardial infarction (MI) and to determine if the requisite preclinical imaging end point was met prior to a follow-up large-animal MSC imaging study. Materials and Methods Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. Mice (n = 19) that had experienced MI were injected with bone marrow-derived MSC that expressed a multimodality triple fusion (TF) reporter gene. The TF reporter gene (fluc2-egfp-sr39ttk) consisted of a human promoter, ubiquitin, driving firefly luciferase 2 (fluc2), enhanced green fluorescent protein (egfp), and the sr39tk positron emission tomography reporter gene. Serial bioluminescence imaging of MSC-TF and ex vivo luciferase assays were performed. Correlations were analyzed with the Pearson product-moment correlation, and serial imaging results were analyzed with a mixed-effects regression model. Results Analysis of the MSC-TF after cardiac cell therapy showed significantly lower signal on days 8 and 14 than on day 2 (P = .011 and P = .001, respectively). MSC-TF with MI demonstrated significantly higher signal than MSC-TF without MI at days 4, 8, and 14 (P = .016). Ex vivo luciferase activity assay confirmed the presence of MSC-TF on days 8 and 14 after MI. Conclusion Multimodality reporter-gene imaging was successfully used to assess serial MSC survival after therapy for MI, and it was determined that the requisite preclinical imaging end point, 14 days of MSC survival, was met prior to a follow-up large-animal MSC study. (©) RSNA, 2016 Online supplemental material is available for this article.

Manganese-Enhanced Magnetic Resonance Imaging Enables In Vivo Confirmation of Peri-Infarct Restoration Following Stem Cell Therapy in a Porcine Ischemia-Reperfusion Model.

BACKGROUND: The exact mechanism of stem cell therapy in augmenting the function of ischemic cardiomyopathy is unclear. In this study, we hypothesized that increased viability of the peri-infarct region (PIR) produces restorative benefits after stem cell engraftment. A novel multimodality imaging approach simultaneously assessed myocardial viability (manganese-enhanced magnetic resonance imaging [MEMRI]), myocardial scar (delayed gadolinium enhancement MRI), and transplanted stem cell engraftment (positron emission tomography reporter gene) in the injured porcine hearts. METHODS AND RESULTS: Twelve adult swine underwent ischemia-reperfusion injury. Digital subtraction of MEMRI-negative myocardium (intrainfarct region) from delayed gadolinium enhancement MRI-positive myocardium (PIR and intrainfarct region) clearly delineated the PIR in which the MEMRI-positive signal reflected PIR viability. Human amniotic mesenchymal stem cells (hAMSCs) represent a unique population of immunomodulatory mesodermal stem cells that restored the murine PIR. Immediately following hAMSC delivery, MEMRI demonstrated an increased PIR viability signal compared with control. Direct PIR viability remained higher in hAMSC-treated hearts for >6 weeks. Increased PIR viability correlated with improved regional contractility, left ventricular ejection fraction, infarct size, and hAMSC engraftment, as confirmed by immunocytochemistry. Increased MEMRI and positron emission tomography reporter gene signal in the intrainfarct region and the PIR correlated with sustained functional augmentation (global and regional) within the hAMSC group (mean change, left ventricular ejection fraction: hAMSC 85±60%, control 8±10%; P<0.05) and reduced chamber dilatation (left ventricular end-diastole volume increase: hAMSC 24±8%, control 110±30%; P<0.05). CONCLUSIONS: The positron emission tomography reporter gene signal of hAMSC engraftment correlates with the improved MEMRI signal in the PIR. The increased MEMRI signal represents PIR viability and the restorative potential of the injured heart. This in vivo multimodality imaging platform represents a novel, real-time method of tracking PIR viability and stem cell engraftment while providing a mechanistic explanation of the therapeutic efficacy of cardiovascular stem cells.

Preclinical derivation and imaging of autologously transplanted canine induced pluripotent stem cells.

Derivation of patient-specific induced pluripotent stem cells (iPSCs) opens a new avenue for future applications of regenerative medicine. However, before iPSCs can be used in a clinical setting, it is critical to validate their in vivo fate following autologous transplantation. Thus far, preclinical studies have been limited to small animals and have yet to be conducted in large animals that are physiologically more similar to humans. In this study, we report the first autologous transplantation of iPSCs in a large animal model through the generation of canine iPSCs (ciPSCs) from the canine adipose stromal cells and canine fibroblasts of adult mongrel dogs. We confirmed pluripotency of ciPSCs using the following techniques: (i) immunostaining and quantitative PCR for the presence of pluripotent and germ layer-specific markers in differentiated ciPSCs; (ii) microarray analysis that demonstrates similar gene expression profiles between ciPSCs and canine embryonic stem cells; (iii) teratoma formation assays; and (iv) karyotyping for genomic stability. Fate of ciPSCs autologously transplanted to the canine heart was tracked in vivo using clinical positron emission tomography, computed tomography, and magnetic resonance imaging. To demonstrate clinical potential of ciPSCs to treat models of injury, we generated endothelial cells (ciPSC-ECs) and used these cells to treat immunodeficient murine models of myocardial infarction and hindlimb ischemia.

Dual manganese-enhanced and delayed gadolinium-enhanced MRI detects myocardial border zone injury in a pig ischemia-reperfusion model.

BACKGROUND: Gadolinium (Gd)-based delayed-enhancement MRI (DEMRI) identifies nonviable myocardium but is nonspecific and may overestimate nonviable territory. Manganese (Mn(2+))-enhanced MRI (MEMRI) denotes specific Mn(2+) uptake into viable cardiomyocytes. We performed a dual-contrast myocardial assessment in a porcine ischemia-reperfusion (IR) model to test the hypothesis that combined DEMRI and MEMRI identifies viable infarct border zone (BZ) myocardium in vivo. METHODS AND RESULTS: Sixty-minute left anterior descending coronary artery IR injury was induced in 13 adult swine. Twenty-one days post-IR, 3-T cardiac MRI was performed. MEMRI was obtained after injection of 0.7 mL/kg Mn(2+) contrast agent. DEMRI was then acquired after injection of 0.2 mmol/kg Gd. Left ventricular (LV) mass, infarct, and function were analyzed. Subtraction of MEMRI defect from DEMRI signal identified injured BZ myocardium. Explanted hearts were analyzed by 2,3,5-triphenyltetrazolium chloride stain and tissue electron microscopy to compare infarct, BZ, and remote myocardium. Average LV ejection fraction was reduced (30±7%). MEMRI and DEMRI infarct volumes correlated with 2,3,5-triphenyltetrazolium chloride stain analysis (MEMRI, r=0.78; DEMRI, r=0.75; P<0.004). MEMRI infarct volume percentage was significantly lower than that of DEMRI (14±4% versus 23±4%; P<0.05). BZ MEMRI signal-to-noise ratio (SNR) was intermediate to remote and core infarct SNR (7.5±2.8 versus 13.2±3.4 and 2.9±1.6; P<0.0001), and DEMRI BZ SNR tended to be intermediate to remote and core infarct SNR (8.4±5.4 versus 3.3±0.6 and 14.3±6.6; P>0.05). Tissue electron microscopy analysis exhibited preserved cell structure in BZ cardiomyocytes despite transmural DEMRI enhancement. CONCLUSIONS: The dual-contrast MEMRI-DEMRI detects BZ viability within DEMRI infarct zones. This approach may identify injured, at-risk myocardium in ischemic cardiomyopathy.

Intriguing peri-strut low-intensity area detected by optical coherence tomography after coronary stent deployment.

BACKGROUND: Although peri-strut low-intensity area (PLIA) is frequently observed on post-stenting optical coherence tomography (OCT) images, the histology associated with PLIA is undocumented. METHODS AND RESULTS: The 36 porcine coronary lesions treated with bare-metal (BMS: n=16) or drug-eluting (DES: n=20) stents were assessed by OCT and histology at 28 days. DES showed a significantly higher incidence of PLIA than BMS. Also, +PLIA stents had greater neointima than PLIA stents. Histological analysis revealed the existence of fibrinoid and proteoglycans at the site of PLIA. CONCLUSIONS: PLIA might be represented by the presence of fibrinoid and proteoglycans, and associated with neointimal proliferation after stenting.

Development of animal model for calcified chronic total occlusion.

Chronic total coronary occlusion (CTO) remains a major problem for percutaneous revascularization, with relatively low primary success rates and a high incidence of restenosis and reocclusion compared with those of subtotal stenoses. No reproducible animal model simulating human CTOs has previously been developed. We hypothesized that an apatite-coated bioabsorbable polymer sponge could be implanted to produce calcified CTO lesions in animal coronary arteries/peripheral arteries. A total of 10 swine and six rabbits were used for this study. The apatite-coated bioabsorbable polymer sponges were implanted into a preselected segment of coronary and peripheral arteries. Four weeks after implantation, both angiography and histopathology were performed to document the presence or absence of CTO lesions. We could reproducibly develop CTO lesions in animal coronary/peripheral arteries that mimic human CTO lesions. These lesions were found to have microvascular channels and microcalcification similar to those of human older CTO lesions and demonstrate the development of adventitial arterioles, a consistent finding in human CTO. This CTO model might provide a platform for evaluating future CTO technologies as well as contributing to a better understanding of CTOs in both educational and practical terms.

The porcine restenosis model using thermal balloon injury: comparison with the model by coronary stenting.

BACKGROUND: Percutaneous coronary intervention (PCI) continues to revolutionize the treatment of coronary atherosclerosis and technologic advances require a preclinical coronary stenosis model. The purpose of this study was to systematically evaluate a porcine restenosis model of thermal balloon injury compared to stent overstretching. METHODS: To evaluate this injury model, 22 swine were utilized. For the induction of coronary stenoses, the thermal balloon-to-artery ratio was equal to the range of 1.2-1.3 and was placed at a desired location in the coronary arteries, inflated with 2 atm, and heated to 80 degrees C for 80 seconds. Quantitative coronary angiography was analyzed at baseline, immediately postprocedure, and 4 weeks at harvest. Quantitative coronary ultrasound analysis and histopathologic evaluation were also performed at 4 weeks postprocedure. RESULTS: A total of 54 coronary arteries (thermal balloon injury [Thermo]; n = 43, coronary stenting [Stent]; n = 11) from a total of 18 animals were analyzed for this study. At 4 weeks postprocedure, significantly greater coronary stenoses were observed in the Thermo Group versus the Stent Group (minimum lumen diameter: 1.00 % 0.63 mm vs. 1.58 % 0.44 mm; p = 0.009, % diameter stenosis [DS]: 66.2 +/- 21.6% vs. 48.1 +/- 11.4%; p = 0.02). There were significant linear correlations between the balloon-to-artery ratio, post %DS and %DS at 4 weeks (balloon-to-artery ratio; r = 0.538; p = 0.0012, post %DS; r = -0.744; p < 0.0001, respectively). CONCLUSION: This methodology may provide reproducible and consistent coronary stenoses. This model can be useful fnot only for the evaluation of medical devices, but also for technical training in PCI and development of coronary imaging technologies.

In vivo porcine model of reperfused myocardial infarction: in situ double staining to measure precise infarct area/area at risk.

OBJECTIVES: The aim of this study is to evaluate a catheter-based porcine model for reperfused myocardial infarction and investigate the appropriate location and duration of the occlusion. MATERIAL AND METHODS: A balloon catheter was placed in the left descending coronary artery (LAD) in 78 swine, and used to occlude the LAD. To evaluate this model, left ventricular ejection fraction (LVEF), infarct size, incidence of ventricular fibrillation (VF), and mortality was compared among three groups: 60-min proximal LAD occlusion (60P), 60-min mid LAD occlusion (60M), and 30-min proximal LAD occlusion (30P). RESULTS: In 72 of the 78 pigs, the procedures were successfully completed. Both mortality and incidence of VF were highest in the 60P group (66.7% and 91.7%, respectively). Myocardial infarction was successfully induced in all 72 animals and in situ double-staining with Evans blue dye and 2,3,5-triphenyltetrazolium chloride was performed to delineate area at risk for ischemia and infarcted myocardium. There was no difference in infarct size, expressed as a percentage of the area at risk, between the 60P and 60M groups (49.5% +/- 3.9% vs. 45.4% +/- 13.3%, respectively). Serial changes in LVEF of the 60M group demonstrated that until 14 days after reperfusion, LVEF improved naturally over time (36.4% +/- 6.6% at 24 hr, and 47.3% +/- 10.1% at 14 days). CONCLUSION: This model and methodology could provide a reproducible and consistent infarct size. The current study demonstrated that 60-min mid LAD occlusion can be the most feasible to serve as a porcine reperfused myocardial infarction model.

Novel stent system for accurate placement in aorto-ostial renal artery disease: preclinical study in porcine renal artery model.

BACKGROUND: Treatment of aorto-ostial renal artery stenosis has been associated with a lower procedural success and higher complication and restenosis rate, as compared to nonostial lesions. The design and delivery of currently available stent systems in ostial lesions can result in inaccurate stent positioning and placement leading to stent protrusion into the parent vessel lumen or geographic miss. A novel stent system (SquareOne Inc., Campbell, CA, USA) has been designed specifically for aorto-ostial lesions in the renal artery. This stent system aims to provide both tactile and visual confirmation of the ostium at the aorta, allow for improved accuracy during stent positioning and placement, provide complete scaffolding of the lesion at the aortic junction to the native vessel, and enable future vessel reaccess. METHODS: Stents (n=12) were implanted in both renal arteries of six swine. For histology, two animals were euthanized immediately after stent implantation, and each two animals were then followed up at 2 and 4 weeks, respectively. Intravascular ultrasound (IVUS) studies were performed immediately after stent implantation and at follow-up. RESULTS: Proper stent positioning and implantation was obtained in all animals. Angiographic and IVUS assessments indicated no dissection or thrombus formation. Histology demonstrated good apposition and endothelialization of the stent strut surface. CONCLUSION: The unique flared shape of this novel ostial stent system allows for improved accuracy during stent positioning and placement, as well as complete apposition and coverage/scaffolding of the similarly-shaped luminal ostium. Future studies will determine if this novel stent system fulfills the unmet clinical need in aorto-ostial stenoses.

Arteriotomy closure device application following percutaneous coronary intervention may prevent bleeding complications in patients with acute myocardial infarction.

The current study was performed to determine whether application of arteriotomy closure devices (ACDs) affect bleeding complications as compared to manual compression in patients with ST-elevated acute myocardial infarction (STEMI) who undergo primary or rescue percutaneous intervention (PCI). 314 consecutive cases of STEMI treated with PCI were retrospectively evaluated. Overall, 82.8% of patients received ACDs with total bleeding rate of 4.2% vs. 11.1% in patients who had manual compression, p=0.042. This difference in bleeding rates did not correlate with any clinical characteristic or utilization of GPIIb/IIIa inhibitors. Accordingly, ACDs can improve the acute results of PCI in STEMI patients.

The conversion in application of percutaneous coronary intervention following the introduction of drug eluting stents.

In order to determine the changes in the pattern of techniques applied during elective coronary intervention, data from 688 patients prior to Sirolimus. Drug-Eluting Stents (DES) approval was compared to 438 patients who underwent coronary intervention after DES approval. There was increased intervention to higher risk lesions, including smaller vessels and re-stenotic lesions after DES approval. Total number of stents per patient significantly decreased, despite longer stent length per patient or per lesion after DES approval. No significant difference was found in multivessel interventions.