Biodegradable Cardiac Occluder with Surface Modification by Gelatin-Peptide Conjugate to Promote Endogenous Tissue Regeneration

Abstract

Transcatheter intervention has been the preferred treatment for congenital structural heart diseases by implanting occluders into the heart defect site through minimally invasive access. Biodegradable polymers provide a promising alternative for cardiovascular implants by conferring therapeutic function and eliminating long-term complications, but inducing in situ cardiac tissue regeneration remains a substantial clinical challenge. PGAG (polydioxanone/poly (l-lactic acid)-gelatin-A5G81) occluders are prepared by covalently conjugating biomolecules composed of gelatin and layer adhesive protein-derived peptides (A5G81) to the surface of polydioxanone and poly (l-lactic acid) fibers. The polymer microfiber-biomacromolecule-peptide frame with biophysical and biochemical cues could orchestrate the biomaterial-host cell interactions, by recruiting endogenous endothelial cells, promoting their adhesion and proliferation, and polarizing immune cells into anti-inflammatory phenotypes and augmenting the release of reparative cytokines. In a porcine atrial septal defect (ASD) model, PGAG occluders promote in situ tissue regeneration by accelerating surface endothelialization and regulating immune response, which mitigate inflammation and fibrosis formation, and facilitate the fusion of occluder with surrounding heart tissue. Collectively, this work highlights the modulation of cell-biomaterial interactions for tissue regeneration in cardiac defect models, ensuring endothelialization and extracellular matrix remodeling on polymeric scaffolds. Bioinspired cell-material interface offers a highly efficient and generalized approach for constructing bioactive coatings on medical devices.

Keywords: biodegradable polymers; cell-material interface; congenital heart disease; occluder; tissue regeneration.

Figure 1

Schematic illustration for the preparation and application of PGAG occluder. A) PGAG occluder is composed of a PGAG frame and a PGAG flow‐blocking membrane. The gelatin‐peptide structure interacts with integrin receptor, promotes endothelial cell (EC) adhesion and proliferation, and mitigates pro‐inflammatory macrophage activation and fibrosis. B) Follow‐up of a porcine ASD model demonstrates cardiac repair induced by implantation of a PGAG occluder.

Figure 2

Characterization of surface structure and mechanical properties of PGAG. A) Cross‐sectional and lateral SEM micrographs of PGAG monofilaments. B,C) Surface optical profile images of PGAG monofilaments with longitudinal grooves. D) Fluorescent images of GA and A5GB1 of PGAG labeled with Cy5 and FITC, respectively. E) XPS survey scan, C 1s and N 1s spectra of PGAG. F) FTIR spectra of PGAG. G) Morphology of the PGAG occluder under compression and tension. H) The maximum stress, fracture strain for PGAG and PDO (n = 3). Data are presented as mean ± SD. p‐values are calculated using unpaired t‐test. N.S. means no significant.

Figure 3

PGAG promoted survival, adhesion, and proliferation of endothelial cells. A) HUVECs cocultured on PDO, PDGA, PDAG and PGAG membranes for 24 and 48 h. B) Representative SEM images of HUVECs adhering on membranes. C) Representative immunofluorescence images of HUVECs cocultured on PDO, PDGA, PDAG and PGAG membranes (blue: nuclear; green: integrin α3; red: FAK). D) Western blot analysis of FAK, PI3K, AKT, Erk1/2, MEK1/2 protein expression by treated HUVECs. E) Quantitative expression of FAK, F) PI3K, and G) Erk1/2 (n = 4 for each test). H) Schematic diagram of PGAG promoting survival, adhesion, and proliferation of endothelial cells. Data are presented as mean ± SD. p‐values are calculated using one‐way ANOVA with Bonferroni correction. ns = no significance, *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4

PGAG mitigated inflammation and promoted release of reparative cytokines. A) Representative images of BMDMs treated on PDO, PDGA, PDAG, and PGAG membranes (blue: nuclear; red: F4/80⁺; green: CD86⁺). B) Quantification of percentage of CD86⁺ expression within F4/80⁺ BMDMs (n = 3). The box plot indicates the range from min to max. Relative protein expression of C) TGF‐β, D) VEGF, E) IL‐4, F) IL‐10, G) TNF‐α and H) IFN‐γ in supernate of BMDMs determined by ELISA (n = 4 for each test). I) Relative protein expression of MMP9 in BMDMs determined by western blotting (n = 3). Data are presented as mean ± SD. p‐values are calculated using one‐way ANOVA with Bonferroni correction. ns = no significance, *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 5

PGAG occluder was safe and effective in a porcine ASD model. A) Macroscopic views of implanted PDO and PGAG occluders in an ASD model at 1, 3, 6 months. B) Quantification of endothelium coverage area of discs (n = 3 pigs at each time point, left and right discs for each pig). C) Quantification of disc residual area (n = 3 pigs at each time point). D) Cardiac MRI for occluders at 3 months. (E) TTE images of occluders at 1, 3, and 6 months. F) Occluder thickness determined by TTE (n = 9 at 1 month, n = 6 at 3 month and n = 3 at 6 month). G) Frequency of arrhythmia (n = 9 at 1 month, n = 6 at 3 month and n = 3 at 6 month). H) Quantification of blood examination at 6 months. The square indicates the mean value (n = 3). WBC: white blood cell; HGB: hemoglobin; PLT: platelet; CREA: creatinine; ALT: albumin; ALT: alanine transaminase; AST: aspartate transaminase. Data are presented as mean ± SD. p‐values are calculated using unpaired t test. ns = no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6

PGAG occluder alleviated inflammation and fibrosis during the regeneration process. A,B) Masson and H&E staining of cardiac tissue surrounding occluders. C) Immunohistochemical staining of COLA1. D,E) Inflammation score and fiber disorder score of surrounding fibers, with score 0 = normal, 1 = slight, 2 = mild, 3 = moderate, 4 = severe (n = 20 at each time point). F) Diameter of monofilament (n = 20 at each time point). G) Representative SEM image of monofilament at 1 month. Data are presented as mean ± SD. p‐values are calculated using unpaired t test. ns = no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 7

PGAG occluder promoted endothelialization and mitigated polarization of pro‐inflammatory macrophages. A) Immunofluorescence staining of CD31 (green) and integrin α3 (red). The white line indicates 100 µm. B) Immunofluorescence staining of CD68 (green) and CD86 (red). The white line indicates 200 µm. Statistical data of average fluorescence intensity of C) CD31, D) integrin α3, E) CD68, and F) CD86 (n = 5 for each test). Data are presented as mean ± SD. p‐values are calculated using unpaired t test. ns = no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 8

Immunomodulation and proendothelialization revealed by multiomics. A) Cluster heatmap of significantly upregulated and downregulated DEGs by transcriptomics. B) Volcano plots. The red dots indicate the proteins selected based on adjusted P value < 0.05 and log₂FC > 2. C) KEGG pathway‐enrichment analysis of DEGs. D) GO pathway‐enrichment analysis of DEGs. E) GO pathway‐enrichment analysis of DEPs by proteomics. BP: biological process; CC: cellular component; MF: molecular function.