Research ArticleCELL BIOLOGY

Cardiac cell–integrated microneedle patch for treating myocardial infarction

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Science Advances  28 Nov 2018:
Vol. 4, no. 11, eaat9365
DOI: 10.1126/sciadv.aat9365
  • Fig. 1 Characterization of MN-CSCs.

    (A) Schematic showing the overall design used to test the therapeutic benefits of MN-CSCs on infarcted heart. (B) SEM image of MN. Scale bar, 500 μm. (C) Representative fluorescent image indicating that DiO-labeled CSCs (green) were encapsulated in fibrin gel and then integrated onto the top surface of MN array (red). Scale bar, 500 μm.

  • Fig. 2 Effects of MN-CSCs on NRCMs function in vitro.

    (A) Schematic showing the study design used to test the effects of MN-CSCs on NRCMs in vitro. (B) Calcein (live)/EthD (dead) staining revealed the viability and morphology of CSCs cultured on the MN patch and the quantitative analysis of CSC viability on days 1, 3, and 7. n = 3 for each group at each time points. Scale bar, 200 μm. (C) Confocal image indicating CSCs (green) migration from the fibrin gel into the MN cavity after 3 days in culture. (D) Releases of various CSC factors (namely VEGF, IGF-1, and HGF). n = 6 for each group at each time point. (E) Calcein (live)/EthD (dead) staining revealed the morphology and viability of NRCMs 3 days in culture with MN-CSC patch. Scale bar, 200 μm. (F) Quantitative analysis of NRCM morphology cultured alone or cocultured with MN or MN-CSC patch at day 3. n = 6 for each group. (G) Quantitative analysis of cell viability for NRCMs cultured alone or cocultured with MN or MN-CSC patch at day 3. n = 3 for each group. HPF, high power field. (H) Time-lapse videos revealed coculture with MN-CSC patch significantly increased cardiomyocyte contractility at day 3. n = 6 for each group. (I and J) Representative fluorescent micrographs and quantitative analysis of NRCMs stained with proliferation marker Ki67 (red) and α-sarcomeric actin (α-SA) (green). NRCMs were cultured alone or with MN or MN-CSC patch. n = 3 for each group. Scale bars, 50 μm. All data are presented as means ± SD. A two-tailed, unpaired Student’s t test was used to compare between any two groups. One-way analysis of variance (ANOVA), followed by post hoc Bonferroni test, was used to compare between three or more groups. *P < 0.05. DAPI, 4′,6-diamidino-2-phenylindole.

  • Fig. 3 MN-CSC patch reduces apoptosis and promotes angiomyogenesis in the post-MI heart.

    (A) Schematic showing the overall animal study design used to test the therapeutic benefits of MN-CSCs in a rat model of MI. (B) Placement of a MN-CSC patch on the rat heart. (C) H&E staining indicating the presence of MN-CSC patch on the infarcted heart. Scale bar, 1 mm. (D) Fluorescent image showing Cy5.5-labeled MNs (red) could be readily detected on the heart (green) 7 days after the transplantation. Scale bar, 400 μm. (E) Representative fluorescent micrographs showing the presence of CD68+ cells (green) in the control MI heart or hearts treated with MN or MN-CSC patch at day 7. The numbers of CD68+ cells were quantified. n = 3 hearts for each group. Scale bar, 200 μm. (F) Representative fluorescent micrographs showing the presence of TUNEL+ (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling–positive) apoptotic cells (red) in the MI hearts treated alone or treated with MN or MN-CSC patch at day 7. The numbers of TUNEL+ apoptotic cells were quantified. n = 3 for each group. Scale bars, 100 μm. (G) Representative fluorescent micrograph showing the presence of Ki67+ cardiomyocyte nuclei (red) in the MI hearts treated with MN-CSC on day 7. The numbers of Ki67+ nuclei were quantified in MI-, MI + MN-, or MI + MN-CSC–treated hearts. n = 3 for each group. Scale bar, 200 μm. (H) Representative fluorescent micrograph showing the presence of α–smooth muscle actin (α-SMA; green) in the MI hearts treated with MN-CSC on day 7. The numbers of α-SMA+ vasculatures were quantified in MI-, MI + MN-, or MI + MN-CSC–treated hearts. n = 3 for each group. Scale bar, 200 μm. All data are presented as means ± SD. Comparisons between three or more groups were performed using one-way ANOVA, followed by post hoc Bonferroni test. *P < 0.05.

  • Fig. 4 MN-CSC ameliorates ventricular dysfunction and promotes cardiac repair in a rat model of heart attack.

    (A) Representative Masson’s trichrome–stained myocardial sections 3 weeks after MI. Blue, scar tissue; red, viable myocardium; snapshots, high-magnification images of the black box area. (B) Representative M-mode echocardiographic images showing the LV wall motion of the hearts. (C and D) Quantitative analyses of (C) infarct wall thickness and (D) viable tissue in the risk area based on Masson’s trichrome staining. n = 6 animals per group. (E and F) LVEFs measured by echocardiography at 4 hours (baseline) and 3 weeks (endpoint) after MI. n = 6 animals per group. All data are presented as means ± SD. Comparisons between three or more groups were performed using one-way ANOVA, followed by post hoc Bonferroni test. *P < 0.05 when compared with the MI group; #P < 0.05 when compared with the MI + MN group; &P < 0.05 when compared with the MI + CSC group.

  • Fig. 5 Induction of MI in the swine by LAD ligation.

    (A) Representative images of LAD ligation (left) and MN-CSC patch placement on the heart (right). (B) Serum concentration of cTnl before MI and 24 hours/48 hours after MI. All data are presented as means ± SD. n = 3 animals per group. ***P < 0.001 when compared with baseline (before MI) and 48 hours after MI. Black bar, MI-only group; red bar, MN-CSC patch–transplanted group. (C) Quantitative analyses of infarct size at 48 hours after MI through calculation. All data are presented as means ± SD. n = 3 animals per group. NS (not significant) indicates P > 0.05 when compared between two groups. Black bar, MI-only group; red bar, MN-CSC patch–transplanted group. (D) Macroscopic TTC staining images representing infarct area on multiple slices of an infarcted pig heart.

  • Fig. 6 MN-CSC therapy ameliorates ventricular dysfunction and promotes cardiac repair in a swine model of MI.

    (A to C) LVEFs determined by echocardiography at baseline (A) (4 hours after infarct) and endpoint (B) (48 hours after infarct). The treatment effects are calculated as the change of LVEFs from endpoint to baseline (C). (D to F) Fractional shortenings (FSs) determined by echocardiography at baseline (D) (4 hours after infarct) and endpoint (E) (48 hours after infarct). The treatment effects are calculated as the change of FSs from baseline to endpoint (F). All data are presented as means ± SD. n = 3 animals per group. **P < 0.01 when compared between two groups. Black bars, MI-only group; red bars, MN-CSC patch–transplanted group. (G to J) ALT (G), AST (H), creatinine (I), and BUN (J) are evaluated at baseline (before MI) and endpoint (48 hours after MI). Black, MI-only group; red, MN-CSC patch–transplanted group. All data are presented as means ± SD. n = 3 animals per group. NS indicates P > 0.05.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/11/eaat9365/DC1

    Fig. S1. Characterization of PVA MN.

    Fig. S2. Characterization of NRCMs cultured with MN.

    Fig. S3. Local T cell immune response in immunocompetent rat treated with a MN-CSC patch.

    Fig. S4. MN-CSC therapy protects cardiac morphology and reduces fibrosis in a rat model of MI.

    Fig. S5. Cardiac functions at baseline and 3 weeks after MI + No–MN-CSC or MI + MN-CSC treatment.

    Fig. S6. Effects of PVA patches on kidney and liver functions 21 days after transplantation.

    Fig. S7. Changes in ECG parameters from pre-LAD ligation to post-LAD ligation in swine study.

    Movie S1. Beating NRCMs cultured alone on TCP.

    Movie S2. Beating NRCMs cultured with a MN patch on TCP.

    Movie S3. Beating NRCMs cultured with a MN patch loaded with CSCs (MN-CSCs) on TCP.

    Movie S4. MN patch loaded with CSCs (MN-CSCs) placed on the surface of an infarcted rat heart.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Characterization of PVA MN.
    • Fig. S2. Characterization of NRCMs cultured with MN.
    • Fig. S3. Local T cell immune response in immunocompetent rat treated with a MN-CSC patch.
    • Fig. S4. MN-CSC therapy protects cardiac morphology and reduces fibrosis in a rat model of MI.
    • Fig. S5. Cardiac functions at baseline and 3 weeks after MI + No–MN-CSC or MI + MN-CSC treatment.
    • Fig. S6. Effects of PVA patches on kidney and liver functions 21 days after transplantation.
    • Fig. S7. Changes in ECG parameters from pre-LAD ligation to post-LAD ligation in swine study.
    • Legends for Movies S1 to S4

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Beating NRCMs cultured alone on TCP.
    • Movie S2 (.mp4 format). Beating NRCMs cultured with a MN patch on TCP.
    • Movie S3 (.mp4 format). Beating NRCMs cultured with a MN patch loaded with CSCs (MN-CSCs) on TCP.
    • Movie S4 (.mp4 format). MN patch loaded with CSCs (MN-CSCs) placed on the surface of an infarcted rat heart.

    Files in this Data Supplement:

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