Noninvasive Assessment of an Engineered Bioactive Graft in Myocardial Infarction: Impact on Cardiac Function and Scar Healing

Abstract

Cardiac tissue engineering, which combines cells and biomaterials, is promising for limiting the sequelae of myocardial infarction (MI). We assessed myocardial function and scar evolution after implanting an engineered bioactive impedance graft (EBIG) in a swine MI model. The EBIG comprises a scaffold of decellularized human pericardium, green fluorescent protein-labeled porcine adipose tissue-derived progenitor cells (pATPCs), and a customized-design electrical impedance spectroscopy (EIS) monitoring system. Cardiac function was evaluated noninvasively by using magnetic resonance imaging (MRI). Scar healing was evaluated by using the EIS system within the implanted graft. Additionally, infarct size, fibrosis, and inflammation were explored by histopathology. Upon sacrifice 1 month after the intervention, MRI detected a significant improvement in left ventricular ejection fraction (7.5% ± 4.9% vs. 1.4% ± 3.7%; p = .038) and stroke volume (11.5 ± 5.9 ml vs. 3 ± 4.5 ml; p = .019) in EBIG-treated animals. Noninvasive EIS data analysis showed differences in both impedance magnitude ratio (-0.02 ± 0.04 per day vs. -0.48 ± 0.07 per day; p = .002) and phase angle slope (-0.18° ± 0.24° per day vs. -3.52° ± 0.84° per day; p = .004) in EBIG compared with control animals. Moreover, in EBIG-treated animals, the infarct size was 48% smaller (3.4% ± 0.6% vs. 6.5% ± 1%; p = .015), less inflammation was found by means of CD25⁺ lymphocytes (0.65 ± 0.12 vs. 1.26 ± 0.2; p = .006), and a lower collagen I/III ratio was detected (0.49 ± 0.06 vs. 1.66 ± 0.5; p = .019). An EBIG composed of acellular pericardium refilled with pATPCs significantly reduced infarct size and improved cardiac function in a preclinical model of MI. Noninvasive EIS monitoring was useful for tracking differential scar healing in EBIG-treated animals, which was confirmed by less inflammation and altered collagen deposit. Stem Cells Translational Medicine 2017;6:647-655.

Keywords: Angiogenesis; Bioimpedance; Magnetic resonance imaging; Myocardial infarction; Progenitor cells.

Figure 1

Study design. Abbreviations: EBIG, engineered bioactive impedance graft; EIS, electrical impedance spectroscopy; MI, myocardial infarction; MRI, magnetic resonance imaging; n, number of pigs.

Figure 2

Engineered bioactive impedance graft (EBIG). Photographs show the different steps of EBIG creation and implantation. (A, B): Native human (A) and decellularized and lyophilized pericardium (B). Scale bars = 1 cm. (C, D): Metallic electrodes placement 1 cm spaced. (E): Rehydration of the scaffold with or without cells. (F, G): Bioimpedance device coated with silicone (F) and its implantation in a subcutaneous pocket in the left supraescapular zone (G). (H, I): Adhesion of the engineered construct to the myocardial infarction (H) and the connection with the electrical impedance spectroscopy system (I). (J): Image showing the EBIG implanted in a swine. (K): Animal housing under an antenna noninvasively receiving and transmitting the bioimpedance signal.

Figure 3

Noninvasive cardiac function and morphometric analysis. (A): T1 short‐axis delayed enhancement images from control and EBIG‐treated animal after MI and after 30 days of follow‐up. (B, C): LVEF and SV at baseline and 30 days after MI in control and EBIG‐treated animals. Data for individual pigs (dots) and the Δ (±SEM) are shown. ∗, p = .038; †, p = .019. Dotted red lines indicate the mean evolution over time. (D, E): Representative heart sections from control and EBIG‐treated pigs showing the infarcted area of the LV. (F): Percentage of the LV infarct area measured in EBIG‐treated pig hearts compared with the control group after 30 days of follow‐up. ‡, p = .015. Data represent mean ± SEM. Abbreviations: EBIG, engineered bioactive impedance graft; LV, left ventricle; LVEF, left ventricular ejection fraction; MI, myocardial infarction; SV, stroke volume.

Figure 4

Green fluorescent protein‐adipose tissue‐derived progenitor cell (GFP‐ATPC) endothelial and cardiac differentiation. (A): Immunohistochemistry images from border, infarct, and EBIG zones of treated animals showing positive GFP‐ATPCs (green) and IsoB4, SMA, vWF, and CD31 (red) endothelial antibodies. (B): Representative images from border, infarct, and EBIG zones of treated animals showing positive GFP‐ATPCs (green) and NKX2.5, cKit, and circulating troponin I (white), and MEF2 (red) and cardiac troponin T (white), and cardiac markers. Nuclei are counterstained with 4′,6‐diamidino‐2‐phenylindole (blue). Scale bars = 50 μm. Abbreviations: EBIG, engineered bioactive impedance graft; IsoB4, GSLI B4 Isolectin; SMA, smooth muscle actin; vWF, von Willebrand factor.

Figure 5

Electrical impedance spectroscopy‐derived tissue‐state evolution estimators. Time course of the impedance magnitude ratio (A) and phase angle difference (B) in the control, EBIG‐treated, and sham groups is shown. The curves show 1 of every 100 measurement points for sake of clarity and represent the average curve for each group (control, circles, average of n = 4; EBIG‐treated, triangles, average of n = 4; sham, squares, average of n = 3). The three curves have been normalized to have the same origin to make the comparison easier. The dotted regions around the regression lines represent the 95% confidence interval for the regression. Both the magnitude ratio and phase angle difference show a clearly decreasing slope for the control group, whereas they remain almost constant for the EBIG‐treated and sham groups. These results are consistent with the transition to scar tissue for the control group and the existence of normal‐like tissue in the EBIG‐treated and sham groups. (A): At the bottom is the box plot of the magnitude ratio slope estimator. ∗, p = .002; †, p = .013. (B): At the bottom appears the box plot of the phase angle difference slope estimator, which also displayed a significant difference between controls and the other two groups. ‡, p = .004; §, p = .03. The means and confidence intervals can be found in Results. Abbreviations: EBIG, engineered bioactive impedance graft; HF, high frequency; LF, low frequency.

Figure 6

Myocardial fibrosis and inflammation. (A, B): Sirius red‐stained images exhibiting collagen (red) and healthy muscle (yellow) in the infarct core from control and EBIG‐treated animals. (C, D): Polarized light microscopy images show fibrils of collagen I (red/yellow) and collagen III (green) in the same sections. Scale bars = 50 μm. (E): Collagen I/III ratio measured on polarized light images. ∗, p = .019. (F): Percentage of collagen I and III measured on polarized light images. †, p < .001. (G–J): Representative images of CD3 (green) and CD25 (red) at ×400 (G, H) and ×630 (I, J) in the infarct core from control and EBIG‐treated groups. Scale bars = 50 μm (G, H) and 20 μm (I, J). (K): Percentage of CD3‐ and CD25‐positive cells and CD25/CD3 ratio. ‡, p = .001; §, p < .001. Data represent the mean ± SEM measurement. Abbreviations: ColI, collagen I; Col III, collagen III; CVF, collagen volume fraction; EBIG, engineered bioactive impedance graft.