Acellular cardiac scaffolds enriched with MSC-derived extracellular vesicles limit ventricular remodelling and exert local and systemic immunomodulation in a myocardial infarction porcine model

Rationale: Extracellular vesicles (EVs) from mesenchymal stromal cell (MSC) are a potential therapy for cardiac healing after myocardial infarction (MI). Nevertheless, neither their efficient administration nor therapeutic mechanisms are fully elucidated. Here, we evaluate the preclinical efficacy of a tissue engineering approach to locally deliver porcine cardiac adipose tissue MSC-EV (cATMSC-EV) in an acute MI pig model. Methods: After MI by permanent ligation of the coronary artery, pigs (n = 24) were randomized to Untreated or treated groups with a decellularised pericardial scaffold filled with peptide hydrogel and cATMSC-EV purified by size exclusion chromatography (EV-Treated group) or buffer (Control group), placed over the post-infarcted myocardium. Results: After 30 days, cardiac MRI showed an improved cardiac function in EV-Treated animals, with significantly higher right ventricle ejection fraction (+20.8% in EV-Treated; p = 0.026), and less ventricle dilatation, indicating less myocardial remodelling. Scar size was reduced, with less fibrosis in the distal myocardium (-42.6% Col I in EV-Treated vs Untreated; p = 0.03), a 2-fold increase in vascular density (EV-Treated; p = 0.019) and less CCL2 transcription in the infarct core. EV-treated animals had less macrophage infiltration in the infarct core (-31.7% of CD163+ cells/field in EV-Treated; p = 0.026), but 5.8 times more expressing anti-inflammatory CD73 (p = 0.015). Systemically, locally delivered cATMSC-EV also triggered a systemic effect, doubling the circulating IL-1ra (p = 0.01), and reducing the PBMC rush 2d post-MI, the TNFα and GM-CSF levels at 30d post-MI, and modulating the CD73+ and CCR2+ monocyte populations, related to immunomodulation and fibrosis modulation. Conclusions: These results highlight the potential of cATMSC-EV in modulating hallmarks of ischemic injury for cardiac repair after MI.


EV delivery within cardiac scaffolds
Either mixture was laid over 2 cm 2 lyophilised, decellularised scaffolds, cut with a scalpel, to both rehydrate and fill the scaffolds with EV. After 15 min of seeding and scaffold rehydration at RT, 100 µL DMEM medium without phenol red (Gibco) was added over scaffolds to promote the salt-triggered peptide folding and consequent gel formation for 10 min. Then, scaffolds were washed twice with 2 mL Plasmalyte ® 148 (Viaflo, Baxter) with 15 min incubation in between for pH balancing, and left at RT from 15 min to up to 2 h before in vivo implantation.
Half of the produced EV-cardiac scaffolds were seeded with NIR815-labelled cATMSC-EV. To check the accuracy of the scaffold loading, they were scanned in the 800-nm channel in a Pearl Impulse Imager (Li-COR Biosciences -GmbH) just before implantation.

Non-invasive cardiac magnetic resonance imaging
All images were performed in a 3T state-of-the-art imaging system (Vantage Galan 3T, Canon Medical Systems) with all animals in prone position using a 16-element phased array coil (Atlas SPEEDER Body Coil) placed over the chest. Images were acquired during breath-holds with electrocardiographic gating. We used a segmented k-space steady state precession ( Inversion time was optimized to null the normal myocardium. All images were reviewed and analysed off-line using a cMRI dedicated analysis software (Medis) by a level 3 cMRI expert blinded to the clinical data. Left and right ventricular (LV and RV, respectively) endocardial borders (papillary muscles were excluded) were manually traced in all short-axis cine images at the end-diastolic and end-systolic frames to determine enddiastolic and end-systolic volumes (EDV and ESV), respectively, using QMass (Medis). LV mass was calculated by subtracting the endocardial volume from the epicardial volume at end diastole and then multiplying by tissue density (1.05 g/mL). Left and right ejection fraction (LVEF and RVEF, respectively) were calculated. Calculation of forward aortic volume was performed using QFlow (Medis) tracing ROI at the aorta in phase-contrast sequences.

Supplementary Material
Monguió-Tortajada et al. 2022 Background noise correction was performed at all images. The endocardial and epicardial contours on delayed enhancement images were also outlined manually. ROIs were then manually traced in the hyperenhanced area at place of maximum signal intensity and in the normal-appearing remote myocardium. As previously described, the areas of hyperenhanced myocardium were then automatically segmented by using a full-width at half-maximum (FWHM) algorithm with QMass. Two corrections were required for all automated ROIs. First, microvascular obstruction (defined as hypointensity within a hyperintense region in subjects with infarctions) was adjusted to be included as late gadolinium enhancement (LGE) if present.
Second, any obvious blood pool or pericardial partial voluming and artefacts were further removed. Scar volume for each slice was calculated as: scar area × slice thickness. The scar mass was expressed as total scar volume ×1.05 g. Scar size was also expressed as a percentage of the total myocardial volume: (scar volume / myocardium volume) × 100.
Height, weight and heart rate of pigs was recorded at every cMRI scan. Body surface area (BSA) was calculated as previously described (Kelley et al. 1973). Cardiac index was calculated as (forward aortic volume × heart rate)/ BSA.