Table 1.
Primers designed with Primer3 software.
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.
(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.
Table 2.
C, O and N percentages and O/C and N/C ratios, obtained from XPS analysis.
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) Mn 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.