Surface functionalization of polyurethane scaffolds mimicking the myocardial microenvironment to support cardiac primitive cells

Abstract

Scaffolds populated with human cardiac progenitor cells (CPCs) represent a therapeutic opportunity for heart regeneration after myocardial infarction. In this work, square-grid scaffolds are prepared by melt-extrusion additive manufacturing from a polyurethane (PU), further subjected to plasma treatment for acrylic acid surface grafting/polymerization and finally grafted with laminin-1 (PU-LN1) or gelatin (PU-G) by carbodiimide chemistry. LN1 is a cardiac niche extracellular matrix component and plays a key role in heart formation during embryogenesis, while G is a low-cost cell-adhesion protein, here used as a control functionalizing molecule. X-ray photoelectron spectroscopy analysis shows nitrogen percentage increase after functionalization. O1s and C1s core-level spectra and static contact angle measurements show changes associated with successful functionalization. ELISA assay confirms LN1 surface grafting. PU-G and PU-LN1 scaffolds both improve CPC adhesion, but LN1 functionalization is superior in promoting proliferation, protection from apoptosis and expression of differentiation markers for cardiomyocytes, endothelial and smooth muscle cells. PU-LN1 and PU scaffolds are biodegraded into non-cytotoxic residues. Scaffolds subcutaneously implanted in mice evoke weak inflammation and integrate with the host tissue, evidencing a significant blood vessel density around the scaffolds. PU-LN1 scaffolds show their superiority in driving CPC behavior, evidencing their promising role in myocardial regenerative medicine.

Fig 1. Scaffold design and micrograph.

(A) Overall scaffold dimensions (top left) and a zoomed view of the two overlaying layers (bottom right) detailing fiber diameter (μm) and fiber-to-fiber distance (μm). (B) Representative FEG-SEM micrograph of an additively manufactured PU scaffold.

Fig 2. High resolution XPS spectra.

(A) O1s of PU scaffolds; (B) O1s of plasma-treated PU scaffolds; (C) C1s of PU scaffolds; (D) C1s of plasma-treated scaffolds; (E) C1s of PU-G scaffolds and (F) C1s of PU-LN1 scaffolds.

Fig 3. Characterization of functionalization steps.

(A) Static contact angle values of PU (PU), plasma-treated PU (plasma-treated PU) and protein functionalized PU (PU-G and PU-LN1) films (n = 3; **** p<0.0001; *** p<0.001). (B) Colorimetric quantification of–COOH surface density in PU and plasma-treated PU scaffolds by TBO assay (n = 3, **** p<0.0001). (C) Quantification of LN1 grafting by ELISA assay: mean values of deduced LN1 concentrations measured on PU and PU-LN1 scaffolds (n = 3). Unpaired two-tailed t test was used for statistical analysis of data (**p < 0.01; ****p<0.0001).

Fig 4. CPCs cultured on bare and surface-functionalized PU scaffolds: Cell morphology, proliferation, apoptosis and gene expression.

SEM micrographs of PU-based scaffolds cultured with human CPCs for 7 (on the left) and 14 days (on the right): (A, B) PU, (C, D) PU-G, (E, F) PU-LN1. Scale bar: 100 μm. (G) Proliferation, (H) apoptosis and (I) gene expression of CPCs on PU scaffolds (control, white bars), PU-G scaffolds (grey bars) and PU-LN1 scaffolds (black bars) at different time points. *p<0.05, **p<0.01, ***p<0.001 vs. control, #p<0.05, ##p<0.01, ###p<0.001 vs. PU-G scaffolds.

Fig 5. Hydrolytic and enzymatic degradation of bare and LN1-functionalized PU scaffolds: Weight loss, changes in morphology, loss of molecular weight and cytotoxicity of degradation products.

Weight loss profiles of PU and PU-LN1 scaffolds undergoing (A) hydrolytic and (B) enzymatic degradation. (C) M_n loss profiles of PU scaffolds during hydrolytic and enzymatic degradation. (D) SEM micrographs of PU-LN1 scaffolds during hydrolytic and enzymatic degradation. (E) Viability of NIH-3T3 cells in the presence of Dulbecco’s Modified Eagle’s Medium (DMEM) containing PU degradation products (0.1 mg/mL) compared to control conditions, as evaluated by Cell Titer Blue assay (n = 4).

Fig 6. Results from in vivo tests carried out in mice: Histological analysis and quantification of blood vessels in the tissues surrounding the implants.

Histological analysis by hematoxylin and eosin staining of PU and PU-LN1 scaffolds and surrounding tissues explanted 15 (A) and 30 (B) days following subcutaneous implantation in mice. Arrows indicate blood vessels (scale bar: 50 μm). (C) Number of blood vessels per mm² in the tissues surrounding PU and PU-LN1 scaffolds, and explanted 15 and 30 days after subcutaneous implantation in mice. Unpaired two-tailed t test was used for statistical analysis.