Intramyocardial delivery of mesenchymal stem cell-seeded hydrogel preserves cardiac function and attenuates ventricular remodeling after myocardial infarction

Abstract

Background: To improve the efficacy of bone marrow-derived mesenchymal stem cell (MSC) therapy targeted to infarcted myocardium, we investigated whether a self-setting silanized hydroxypropyl methylcellulose (Si-HPMC) hydrogel seeded with MSC (MSC+hydrogel) could preserve cardiac function and attenuate left ventricular (LV) remodeling during an 8-week follow-up study in a rat model of myocardial infarction (MI).

Methodology/principal finding: Si-HPMC hydrogel alone, MSC alone or MSC+hydrogel were injected into the myocardium immediately after coronary artery ligation in female Lewis rats. Animals in the MSC+hydrogel group showed an increase in cardiac function up to 28 days after MI and a mid-term prevention of cardiac function alteration at day 56. Histological analyses indicated that the injection of MSC+hydrogel induced a decrease in MI size and an increase in scar thickness and ultimately limited the transmural extent of MI. These findings show that intramyocardial injection of MSC+hydrogel induced short-term recovery of ventricular function and mid-term attenuation of remodeling after MI.

Conclusion/significance: These beneficial effects may be related to the specific scaffolding properties of the Si-HPMC hydrogel that may provide the ability to support MSC injection and engraftment within myocardium.

Figure 1. MSC characterization and viability in 3D culture within the Si-HPMC hydrogel.

(A and B) Flow cytometric analysis of MSC for CD29, CD54, CD90, CD34, CD45 and CD86 expression. 10,000 events were scored. Results are expressed as % of positive cells in the whole population on representative histogram plots. (C and D) MSC were cultured in 3D Si-HPMC hydrogel for the indicated times. (C) Labeling cells with calcein-AM (green color) and with EthD-1 (red color) revealed living and dead cells, respectively. Representative samples of MSC cultures visualized by confocal microscopy. (D) As described in the Materials section, the percentages of living and dead MSC cultured in 3D within hydrogel over 7 days (p = NS as compared between time points, one-way ANOVA). All values represent mean ± SEM. Scale bar = 100 µm.

Figure 2. Evaluation of MSC engraftment 24 hours and 14 days after in vivo injection with Si-HPMC hydrogel into cardiac tissue.

MSC engraftment 24 hours and 14 days after in vivo MSC+Si-HPMC hydrogel injection into cardiac tissue is shown on representative transversal histology heart sections. (A, B) Cell nuclei were labeled with To-Pro-3 (red fluorescence). MSC were labeled prior to injection with a fluorescent dye, CFSE (green fluorescence) and visualized 24 h after the implantation. (C) CD90 staining allowed identification of implanted MSC in left ventricle 24 h after injection. (D) PKH26 labeled MSC (red fluorescence) in heart wall with DAPI for cell nuclei (blue fluorescence), 14 days after implantation. (A) Scale bar = 1.5 mm. (B, C and D) scale bar Scale bar = 0.5 mm.

Figure 3. Evaluation of cardiac function by echocardiography in rats after myocardial infarction (MI).

Measurements were performed at baseline before MI and 1, 7, 28 and 56 days after MI as indicated. (A) Left ventricular end-diastolic diameter (LVEDD). (B) Left ventricular end-systolic diameter (LVESD). (C) Left ventricular fractional shortening (LVFS). (D) Left ventricular ejection fraction (LVEF). ^¥ p<0.05 compared to day 1 post-MI in the same group, one-way repeated measures ANOVA.*p<0.001 vs. the PBS group at the same time-point, ^$p<0.05 vs. the hydrogel group at the same time-point and ⁺p<0.05 vs. the MSC group at the same time-point, one-way ANOVA. All values represent mean ± SEM.

Figure 4. Evaluation of myocardial infarction size and left ventricular fibrosis.

(A) Representative transversal histology sections of heart and Masson trichrome staining for infarct size measurement at day 56 after MI. Collagen-rich areas (scar tissue) are colored in blue and healthy myocardium in red. Scale bar = 1.5 mm. (B) Percentage of circumferential infarct size (MI size) divided by total LV tissue, and (C) percentage of fibrosis in total LV tissue. For (B) and (C): *p<0.05 and **p<0.001 vs. the PBS group, ^$ p<0.05 and ^$$ p<0.001 vs. the hydrogel group, one-way ANOVA. LV, left ventricle; RV, right ventricle. All values represent mean ± SEM.

Figure 5. Evaluation of scar thickness and infarct expansion.

(A) Representative photomicrographs of Masson trichrome staining of the scar area (collagen-rich areas in blue and healthy myocardium in red). The double arrow depicts the LV wall (epi, epicardium; endo, endocardium). The arrows show chondroid metaplasia of the endocardium. Scale bar = 0.5 mm. (B) Relative scar thickness (scar thickness/wall thickness). (C) Infarct expansion index ([LV cavity area/whole LV area]/relative scar thickness). For (B) and (C): *p<0.05 and **p<0.001, one-way ANOVA. All values represent mean ± SEM.