Research ArticleBioengineering

An off-the-shelf artificial cardiac patch improves cardiac repair after myocardial infarction in rats and pigs

See allHide authors and affiliations

Science Translational Medicine  08 Apr 2020:
Vol. 12, Issue 538, eaat9683
DOI: 10.1126/scitranslmed.aat9683
  • Fig. 1 Histological and mechanical comparison of decellularized myoECM and NHT.

    (A) Schematic showing fabrication of artCP. (B) Histological comparison of NHT (top) and decellularized myoECM (bottom) including Alcian blue, Gormori’s Blue, Van Gieson’s, Masson’s trichrome, and H&E staining. Scale bars, 200 μm. (C and D) Uniaxial testing on NHT and myoECM (n = 5). (C) Plots of tension versus strain. (D) Plots of stress versus strain. (E) Thickness comparison of NHT and myoECM samples for uniaxial testing. (F) DNA quantification before and after myoECM decellularization (n = 8). All data are means ± SD. Comparisons between two groups were performed using two-tailed unpaired Student’s t test. NS indicates P > 0.05. ****P < 0.0001.

  • Fig. 2 Generation of artCP by embedding synCSCs into myoECM via vacuum filtration.

    (A) Schematic showing the vacuum filtration method. (B) Representative fluorescence images showing artCPs before and after PBS wash. synCSCs (red) were prelabeled with Texas Red succinimidyl ester, and myoECM (green) was prelabeled with anti–collagen I and FITC-conjugated secondary antibodies. Scale bars, 25 μm. (C) Quantitation of embedded synCSCs before and after wash as in (B) (n = 6). All data are means ± SD. Comparisons between two groups were performed using two-tailed unpaired Student’s t test. NS indicates P > 0.05. (D) Representative SEM images showing the cross-sectional view of myoECM and artCP. synCSCs are pseudocolored red.

  • Fig. 3 Cryostability of artCPs.

    (A) Schematic of the study design. (B) Hepatocyte growth factor (HGF), (C) insulin-like growth factor (IGF), and (D) vascular endothelial growth factor (VEGF) analyzed by ELISA (n = 4). myoECM and empty-artCP (myoECM embedded with empty-synCSCs) were used as controls. W, week. (E) Total protein amounts in different synCSC batches measured by the BCA Protein Assay Kit (n = 3). All data are means ± SD. Comparisons among groups were performed using one-way ANOVA followed by post hoc Bonferroni test. NS indicates P > 0.05. **P < 0.01 and ****P < 0.0001.

  • Fig. 4 Effects of artCP on cardiomyocytes in vitro.

    (A) Representative fluorescence micrographs showing Ki67+ expression (green) in NRCMs. Scale bars, 50 μm. α-SA, sarcomeric α-actinin. (B) Quantitation of Ki67+ cells in (A) (n = 3). (C) Representative fluorescence micrographs of LIVE/DEAD assay to determine the viability of NRCMs. Scale bars, 50 μm. (D) NRCM viability measured from (C) using ImageJ software (n = 3). (E) Representative fluorescence micrographs of cell apoptosis detected by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) expression (red). Scale bars, 50 μm. (F) TUNEL+ NRCM percentage determined from (E) using ImageJ software (n = 3). All data are means ± SD. Comparisons among groups were performed using one-way ANOVA followed by post hoc Bonferroni test. The comparisons between samples are indicated by lines, and the statistical significance is indicated by asterisks above the lines. *P < 0.05 and **P < 0.01.

  • Fig. 5 Transplantation of artCPs in rats with MI.

    (A) Schematic showing the study design. Cardiac function was assessed before MI, 4 hours after MI (baseline), and 21 days after MI (end point). (B) Representative M-mode echocardiography images at baseline and end point taken from one animal in each group. LVEF was analyzed before MI (C), 4 hours after MI (D), and 21 days after MI (E). Treatment effects were determined as the change in LVEF from 4 hours after MI to 21 days after MI (F). n = 5 in each group. LVFS was also analyzed before MI (G), 4 hours after MI (H), and 21 days after MI (I), and treatment effects were calculated as the change in LVFS from 4 hours after MI to 21 days after MI (J). n = 5 in each group. All data are means ± SD. Comparisons among groups were performed using one-way ANOVA followed by post hoc Bonferroni test. The comparisons between samples are indicated by lines, and the statistical significance is indicated by asterisks above the lines. NS indicates P > 0.05. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 6 Rat cardiac morphometry assessed through H&E and Masson’s trichrome staining.

    (A) H&E staining was performed on 10-μm cryosections of rat hearts 21 days after MI. Insets outlined by red dashed boxes are shown at higher magnification in the bottom row. Yellow dashed lines indicate the location of myoECM and infarcted tissue (middle) or viable tissue, infarcted tissue, and artCP (right). Scale bars, 250 μm. (B) Masson’s trichrome staining performed on 5-μm cryosections. Insets outlined by red dashed boxes are shown at higher magnification in the bottom row. Yellow dashed lines indicate the location of myoECM and infarcted tissue (middle) or viable tissue, infarcted tissue, and artCP (right). Scale bars, 500 μm. Morphometric parameters including infarct size (C), the percentage of viable myocardium at risk area (D), and infarct wall thickness (E) were measured from the Masson’s trichrome–stained slides via NIH ImageJ software. n = 5 in each group. All data are means ± SD. Comparisons among groups were performed using one-way ANOVA followed by post hoc Bonferroni test. The comparisons between samples are indicated by lines, and the statistical significance is indicated by asterisks above the lines. NS indicates P > 0.05. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 7 IHC of rat cardiac tissue to explore the potential therapeutic mechanism of artCP.

    (A) Endothelial cell marker von Willebrand factor (vWF) (green) was detected at day 21 in the heart-infarcted area that interfaced with transplanted artCP or myoECM. Scale bars, 200 μm. (B) The pooled data of vWF+ signal per high-power field (HPF) assessed by ImageJ software (n = 5). (C) Ki67+ expression (green) detected on the infarct periphery. Scale bars, 50 μm. Scale bars (zoomed snapshot), 10 μm. Insets are outlined by dashed white lines shown at higher magnification in the lower row. (D) Ki67+ cells per HPF assessed by ImageJ software (n = 5). (E) Phosphorylated histone H3 (pH3+) expression (green). Scale bars, 25 μm. Insets are outlined by dashed white lines shown at higher magnification in the lower row. Scale bars (higher magnification snapshots), 10 μm. (F) pH3+ cells and (G) pH3+ cardiomyocytes (nuclei inside of α-SA+ cells that overlaid with pH3+ signals) per HPF as assessed using ImageJ software (n = 5). CMs, cardiomyocytes. All data are means ± SD. Comparisons among groups were performed using one-way ANOVA followed by post hoc Bonferroni test. The comparisons between samples are indicated by lines, and the statistical significance is indicated by asterisks above the lines. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 8 Transplantation of artCP to porcine MI models.

    (A) Schematic showing the study design. The representative pictures show the porcine MI model creation via LAD ligation (left) and artCP transplantation (right). (B) Electrocardiogram (ECG) was collected before MI and 24 hours and 7 days after MI. MI was indicated by ST segment elevation. (C) Serum cardiac troponin I (cTnI) was measured before MI and 24 hours and 7 days after MI in each group of animals (n = 3). (D) Heart sectioning for gross assessment of infarct size. The top left image (MI control) shows the area of infarction due to successful MI creation (red dashed circle) and five sections (1 cm in thickness; dashed line) cut from apex to level of ligation. The bottom left image shows the artCP transplanted area (red dashed circle). The images on the right show the TTC staining of five heart sections from one heart in the MI-only group (top) and the artCP-treated group (bottom). The white area in the TTC-stained heart sections indicates infarction. The position of artCP was indicated with green arrows. (E) Infarction area percentage measured in heart slices 2, 3, and 4 using ImageJ software (n = 3). All data are means ± SD. Comparisons among groups were performed using one-way ANOVA followed by post hoc Bonferroni test. The comparisons between samples are indicated by lines, and the statistical significance is indicated by asterisks above the lines. *P < 0.05 and ***P < 0.001. (F) Masson’s trichrome staining of the infarct periphery performed to detect the myocardium integrity. Scale bars, 400 μm.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/538/eaat9683/DC1

    Materials and Methods

    Fig. S1. Characterization of artCP.

    Fig. S2. Effects of artCP on endothelial cells in vitro.

    Fig. S3. Effects of artCP on CPC differentiation in vitro.

    Fig. S4. synCSC retention and biodistribution in rats.

    Fig. S5. Immunogenicity of artCP in the post-MI rat heart.

    Fig. S6. Rat heart dimensions measured by echocardiography.

    Fig. S7. H&E staining of artCP and adjacent myocardium 21 days after transplantation in a rat.

    Fig. S8. Treatment effects from empty, cryopreserved, or fresh artCP transplantation in rats.

    Fig. S9. Heart morphometric analysis after patch transplantation in rats.

    Fig. S10. Liver and kidney functions 21 days after artCP transplantation in rats.

    Fig. S11. Percentage of pH3+ or Ki67+ cardiomyocytes in total cells 21 days after patch transplantation in rats.

    Fig. S12. Staining of proliferation marker Aurora B after artCP or myoECM transplantation in rats.

    Fig. S13. Analyses of myocardial proliferation and apoptosis 21 days after patch transplantation in rats.

    Fig. S14. Effects of artCP therapy on angiomyogenesis in rats.

    Fig. S15. Cardiac functional assessment of artCP therapy in a porcine MI model.

    Fig. S16. Effects of artCP transplantation on liver and kidney functions in the porcine MI model.

    Fig. S17. Immunogenicity analysis of artCP transplantation in the porcine MI model.

    Table S1. myoECM average tension/stress versus strain.

    Table S2. NHT average tension/stress versus strain.

    Reference (69)

  • This PDF file includes:

    • Materials and Methods
    • Fig. S1. Characterization of artCP.
    • Fig. S2. Effects of artCP on endothelial cells in vitro.
    • Fig. S3. Effects of artCP on CPC differentiation in vitro.
    • Fig. S4. synCSC retention and biodistribution in rats.
    • Fig. S5. Immunogenicity of artCP in the post-MI rat heart.
    • Fig. S6. Rat heart dimensions measured by echocardiography.
    • Fig. S7. H&E staining of artCP and adjacent myocardium 21 days after transplantation in a rat.
    • Fig. S8. Treatment effects from empty, cryopreserved, or fresh artCP transplantation in rats.
    • Fig. S9. Heart morphometric analysis after patch transplantation in rats.
    • Fig. S10. Liver and kidney functions 21 days after artCP transplantation in rats.
    • Fig. S11. Percentage of pH3+ or Ki67+ cardiomyocytes in total cells 21 days after patch transplantation in rats.
    • Fig. S12. Staining of proliferation marker Aurora B after artCP or myoECM transplantation in rats.
    • Fig. S13. Analyses of myocardial proliferation and apoptosis 21 days after patch transplantation in rats.
    • Fig. S14. Effects of artCP therapy on angiomyogenesis in rats.
    • Fig. S15. Cardiac functional assessment of artCP therapy in a porcine MI model.
    • Fig. S16. Effects of artCP transplantation on liver and kidney functions in the porcine MI model.
    • Fig. S17. Immunogenicity analysis of artCP transplantation in the porcine MI model.
    • Table S1. myoECM average tension/stress versus strain.
    • Table S2. NHT average tension/stress versus strain.
    • Reference (69)

    [Download PDF]

Stay Connected to Science Translational Medicine

Navigate This Article