Injectable Peptide Hydrogels Loaded with Murine Embryonic Stem Cells Relieve Ischemia In Vivo after Myocardial Infarction

Abstract

Myocardial infarction (MI) is a major cause of morbidity and mortality worldwide, especially in aging and metabolically unhealthy populations. A major target of regenerative tissue engineering is the restoration of viable cardiomyocytes to preserve cardiac function and circumvent the progression to heart failure post-MI. Amelioration of ischemia is a crucial component of such restorative strategies. Angiogenic β-sheet peptides can self-assemble into thixotropic nanofibrous hydrogels. These syringe aspiratable cytocompatible gels were loaded with stem cells and showed excellent cytocompatibility and minimal impact on the storage and loss moduli of hydrogels. Gels with and without cells were delivered into the myocardium of a mouse MI model (LAD ligation). Cardiac function and tissue remodeling were evaluated up to 4 weeks in vivo. Injectable peptide hydrogels synergized with loaded murine embryonic stem cells to demonstrate enhanced survival after intracardiac delivery during the acute phase post-MI, especially at 7 days. This approach shows promise for post-MI treatment and potentially functional cardiac tissue regeneration and warrants large-scale animal testing prior to clinical translation.

Figure 1.

Schematic representation of the overall therapeutic approach: synergizing angiogenic self-assembling peptide hydrogels (SAPHs) and mESCs for treating myocardial infarction (MI). Angiogenic β-sheet peptides self-assemble into nanofibrous hydrogels that are thixotropic owing to noncovalent supramolecular interactions. These syringe/pipet aspiratable cytocompatible gels were loaded with murine embryonic stem cells (mESCs). Myocardial infarction was created in mice by the ligation of the left anterior descending coronary artery. Infarcted myocardium was injected with cells, scaffolds, or mESCs + scaffold. Cardiac function was evaluated through echocardiography, Kaplan–Meier survival analysis, histology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) at 7- and 28-day time points post-MI.

Figure 2.

Impact of cell addition on the angiogenic peptide hydrogel. (A) SLan samples formulated in 1–10 mM concentrations showed gelation at concentrations of 2.88 mM (1 wt %) or higher, resisting flow in the absence of mechanical shear. The storage moduli of gelled constructs, (B) 1 mM (poor gel), (C) 2.88 mM, (D) 5 mM, and (E) 10 mM, were 5–10× higher than that of the corresponding loss moduli at low shear strain (1%) when tested for 90 s, with near instantaneous shear recovery at high strain (100%) for 30 s. Similar rheometric testing revealed that (F) 1 wt % gels loaded with 1 M cells/mL mouse embryonic stem cells showed little change in storage or loss moduli of gels. A C2C12 cell line (which was used due to the cost and complicated expansion of mESCs) was loaded into scaffolds over 3 orders of magnitude, (G) 100 K cells/mL, (H) 1 M cells/mL, and (I) 10 M cells/mL, to determine the potential effects of cells on scaffold shear thinning and recovery properties, which indicated that at even 10 M cells/mL loading in scaffolds G′ was not significantly impacted.

Figure 3.

Cardiac functional assessment demonstrating the potential of SLan + mESCs to improve post-MI remodeling. (A) Kaplan–Meier analysis demonstrating enhanced post-MI survival in SLan+cell conditions compared to SLan alone, mESC alone, or PBS vehicle. (B) Echocardiography experiments demonstrated that SLan+cells significantly improved cardiac function (systolic function, LVEF%) at day 7 post-MI compared to PBS control and that SLan treatment (with or without mESC) did not compromise cardiac function at 28-day time points post-MI; animals in all groups were used for comparison. (C) Left ventricular chamber dilation (LVIDd) was significantly increased in the PBS group by 28 days post-MI but not in the combination (SLan + mESCs) treatment group. (D–K) Post-mortem heart weight to tibia length (HW/TL), left ventricle weight to tibia length (LV/TL), and individual cardiomyocyte cross-sectional area shown for each treatment group after 7 days post-MI (D–G) showed some differences between groups, but by 28 days post-MI (H–K), only SLan and PBS showed a significant difference from sham for the cardiomyocyte cross-sectional area. Scale bar, 50 μm, data are mean ± standard error of the mean (SEM) (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 4.

Histological evaluation of fibrosis, apoptosis, and post-MI myocardial remodeling. (A–C) Assessment of cardiac fibrosis using picrosirius red (PSR) staining of collagen deposition demonstrated a significant increase in PBS, SLan, and mESCs treatment groups at 7 and 28 days post-MI, compared to Sham group. SLan + mESCs treatment showed no significant increase in fibrosis compared to Sham and was significantly reduced compared to the PBS group (scale bar, 100 μm). (D–F) Apoptosis was determined by TUNEL staining in the border/remote region at 7 and 28 days post-MI. Prevalence of TUNEL-positive cells (arrows) was significantly higher in the PBS group at 7 days post-MI compared to sham, and mESCs and SLan + mESCs treatments had significantly reduced apoptosis compared to PBS control (scale bar, 100 μm). H&E staining at (G) 7 days and (H) 28 days, coupled with Masson’s Trichrome (MT) staining at (I) 7 days and (J) 28 days (scale bar, 1 mm), showed left ventricular wall thinning and fibrotic scarring, consistent with PSR staining and quantitation seen above. Quantified cellular infiltration density, average LV wall thickness, and blood vessel density did not significantly change over 28 days. Data are mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 5.

Evaluation of cytokine secretion from cells with scaffolds and gene expression in cardiac tissue. (A) Cytokine secretion due to the effect of SLan on mESCs was compared to the mESCs alone. After 24 h, in vitro cultures were screened (111 cytokines), wherein 16 were detected, notably upregulation of cytokines such as MMP3, VEGF, and IGFBP5 was observed. (n = 1, for screening only). (B) RT-qPCR results demonstrating the gene expression profile of cardiac tissue after 7 days post-MI observed upregulation of IGFBP5, LIX, VEGF, and MMP3, which suggests MI and remodeling responses for PBS and mESCs n = 5, 4 for the others (*p < 0.05, **p < 0.01, ***p < 0.001). (C) RT-qPCR results demonstrating the gene expression profile of cardiac tissue after 28 days post-MI for SLan+cells n = 6, 4 for the others (*p < 0.05). VEGF expression was highest in the SLan alone condition, while the SLan + mESCs group showed the highest LIX/CXCL5 expression, suggesting distinct roles of SLan alone and SLan + mESCs in angiogenic regulation. Additionally, LDLR, WISP-1, and PAI-1 showed increased expression levels, suggesting a potential effect of SLan + mESCs in promoting angiogenesis (*p < 0.05).