An Injectable Oxygen Release System to Augment Cell Survival and Promote Cardiac Repair Following Myocardial Infarction

Abstract

Oxygen deficiency after myocardial infarction (MI) leads to massive cardiac cell death. Protection of cardiac cells and promotion of cardiac repair are key therapeutic goals. These goals may be achieved by re-introducing oxygen into the infarcted area. Yet current systemic oxygen delivery approaches cannot efficiently diffuse oxygen into the infarcted area that has extremely low blood flow. In this work, we developed a new oxygen delivery system that can be delivered specifically to the infarcted tissue, and continuously release oxygen to protect the cardiac cells. The system was based on a thermosensitive, injectable and fast gelation hydrogel, and oxygen releasing microspheres. The fast gelation hydrogel was used to increase microsphere retention in the heart tissue. The system was able to continuously release oxygen for 4 weeks. The released oxygen significantly increased survival of cardiac cells under the hypoxic condition (1% O₂) mimicking that of the infarcted hearts. It also reduced myofibroblast formation under hypoxic condition (1% O₂). After implanting into infarcted hearts for 4 weeks, the released oxygen significantly augmented cell survival, decreased macrophage density, reduced collagen deposition and myofibroblast density, and stimulated tissue angiogenesis, leading to a significant increase in cardiac function.

Figure 1

Oxygen release microsphere fabrication and oxygen release mechanism.

Figure 2

Core-shell structure of oxygen release microsphere and oxygen release kinetics. (A) PLGA shell; (B) PVP/H₂O₂ core; (C) combined core-shell structure. For imaging purpose, rhodamine and draq5 were added to the shell and core respectively before fabrication; (D) SEM image of microsphere; and (E) oxygen release kinetics during a 28-day release period. Scale bar = 5 µm.

Figure 3

HUVECs, cardiac fibroblasts, and cardiomyocytes survival under normal and hypoxic conditions. (A) dsDNA content. Three cell types were seeded in 3D collagen gel, respectively. Hydrogel (Gel) was then injected into the collagen gel and cultured under normal (Gel Normal group) or 1% O₂ (Gel Hypoxia group) conditions. Oxygen release system (Gel/O₂) was also injected into the collagen gel but cultured under 1% O₂ (Gel/O₂ Hypoxia group). dsDNA was measured 2 days after injection. dsDNA under hypoxia was normalized to the collagen gel injected with Gel and cultured under normal condition; and (B) live cell images of HUVECs, cardiac fibroblasts, and cardiomyocytes in the 3D collagen gel. Cells were labeled with live cell tracker CMFDA before seeding. *p < 0.05. Scale bar = 30 µm.

Figure 4

ROS content in HUVECs, cardiac fibroblasts, and cardiomyocytes seeded in collagen gels. The Gel and Gel/O₂ groups were injected into the collagen gels respectively and cultured under normal condition or hypoxia. ROS content was measured 2 days after injection. (A) HUVECs; (B) Cardiac fibroblasts; and (C) cardiomyocytes. *p < 0.05.

Figure 5

Gene expression of TGFβ1 (A) and TGFβRII (B) in cardiac fibroblasts when seeded in collagen gels. The Gel and Gel/O₂ groups were injected into the collagen gels respectively and cultured under normal condition or hypoxia (1% O₂). Gene expression was measured 2 days after injection. TGFβ1 (5 ng/mL) was added into the medium during culture. *p < 0.05.

Figure 6

Cardiac fibroblasts differentiation into myofibroblasts. (A) Gene expressions of αSMA, CTGF and Collagen 1A1; (B,C) immunostaining of αSMA; and (D) percentage of αSMA+ myofibroblasts. Cardiac fibroblasts were seeded in collagen gels. The Gel and Gel/O₂ groups were injected into the collagen gels respectively and cultured under hypoxia (1% O₂). Gene and protein expressions were measured 2 days after injection. TGFβ1 (5 ng/mL) was added into the medium during culture. *p < 0.05. Scale bar = 30 µm.

Figure 7

H&E staining and relative wall thickness of the infarcted hearts 4 weeks after injection. (A–D) H&E staining of Sham (A); MI (B); Gel (C); and Gel/O₂ (D) groups. Scale bar = 2 mm for whole heart view, and scale bar = 50 µm for higher magnification view. The higher magnification H&E images indicated that cells were infiltrated into the hydrogel; and (E) relative wall thickness. p < 0.05.

Figure 8

MHC staining and MHC+ cell density of the infarcted hearts 4 weeks after injection. (A–D) MHC staining of Sham (A); MI (B); Gel (C); and Gel/O₂ (D) groups; and (E) MHC+ cell density in the infarcted area. Sham, MI and Gel groups were used as controls. Scale bar = 30 µm. *p < 0.05.

Figure 9

Ki67 staining of the infarcted hearts harvested 4 weeks after injection. (A–C) Ki67 staining of MI (A); Gel (B); and Gel/O₂ (C) groups; and (D) Ki67+ cell density in the infarcted area. Scale bar = 30 µm. *p < 0.05.

Figure 10

Immunohistological analysis of the infarcted region and myofibroblast density after 4 weeks of injection. (A–C) Immunohistological staining of αSMA (green), vWF (red), and Hoechst (blue) for MI (A); Gel (B); and Gel/O₂ (C) groups; (D) myofibroblast density quantified from immunohistological images. and (E) Blood vessel density in the infarcted area. Scale bar = 60 μm. *p < 0.05.

Figure 11

Picrosirius red staining of the infarcted hearts harvested 4 weeks after injection. Views were taken at the infarcted region of the MI (A); Gel (B); and Gel/O₂ (C) groups. Total area of collagen (D) was analyzed from the images. Scale bar = 20 µm. *p < 0.05.

Figure 12

F4/80 staining and F4/80+ cell density in the infarcted area 4 weeks after injection. (A–C) F4/80 staining for MI (A); Gel (B); and Gel/O₂ (C) groups; and (D) F4/80+ cell density quantified from images. Scale bar = 30 μm. *p < 0.05.

Figure 13

ROS staining of the infarcted hearts harvested 4 weeks after injection. (A–C) CM-H2DCFDA staining for MI (A); Gel (B); and Gel/O₂ (C) groups; and (D) ROS content quantified from images. Scale bar = 50 μm. *p < 0.05.

Figure 14

Echocardiographic analysis of Sham, MI, Gel, and Gel/O₂ groups. (A) Ejection fraction; (B) Fractional shortening; (C) Fractional area change. *p < 0.05.