Functionalised polyurethane scaffolds mimicking cardiac primitive cell niche microenvironment by additive manufacturing

INTRODUCTION: Human cardiac fibroblasts (CFs) have been found to deposit in vitro a 2D “biomatrix” (BM) with similar composition to the natural cardiac extracellular matrix (ECM). The study of the behaviour of human cardiac primitive cells (CPCs) in contact with BM showed that laminin-1 (LN1) promotes the adhesion, viability, proliferation and differentiation of CPCs. Recently, a polyurethane (PU) with elastomeric-like properties was synthesised and scaffolds were prepared by melt-extrusion additive manufacturing (AM). In this work, PU scaffolds were surface functionalised with LN1 and BM with the aim to reproduce CPC-niche microenvironment. Gelatin (G) was also grafted as a control. METHODS: PU was synthesized from poly(ε-caprolactone) diol (Mn = 2000 Da), 1,4-budandiisocyanate and L-lysine ethyl ester dihydrochloride. Bi-layered scaffolds with 0°/90° lay-down pattern were prepared by additive-manufacturing technique. Functionalisation was performed by two steps: 1) acrylic acid grafting/polymerization following Argon Plasma treatment; 2) carbodiimide mediated grafting of LN1, G or solubilised BM (produced by in vitro culture of CFs, followed by decellularisation and further BM solubilisation in a pepsin solution). Physicochemical characterisation of the surface coating was performed by XPS, FTIR-ATR, colorimetric tests, static contact angle measurements and ELISA assay. In vitro cell tests were performed using CPCs. RESULTS: PU scaffolds showed a mean fibre diameter of 152±5 µm and mean spacing of 505±5 µm. FITR-ATR analysis of coated scaffolds showed higher intensity of the absorption bands at 3370 cm-1 (-OH and –NH stretching) and 1650 cm-1 (amide I). Contact angle decreased from 90° for PU to 60-70° for LN1, G and BM coated PU. XPS analysis confirmed the successful surface functionalisation. CPC proliferation on PU-LN1 scaffolds was higher than on PU scaffolds, increasing from 8.2 % on day 7 to 11.8% on day 14. LN1-functionalization also stimulated CPC differentiation into cardiomyocytes, smooth muscle cells and endothelial cells (RT-PCR analysis). Scaffolds were found to slowly degrade in vitro by hydrolytic mechanism, whereas using an enzyme (lipase) degradation occurred in 3 weeks. Subcutaneous implantation of scaffolds in mice showed their tissue compatibility, low inflammatory response and low degradation rate (they were stable after 1 month). DISCUSSION & CONCLUSIONS: PU scaffolds fabricated by AM and surface grafted with LN1 or BM were developed as 3D substrates mimicking CPC niche microenvironment. They could be used as cellularised patches for in vivo implantation in myocardial tissue engineering or as in vitro models of CPC niches to study CPC behaviour in both normal and pathological conditions.