Elastomeric Trilayer Substrates with Native-like Mechanical Properties for Heart Valve Leaflet Tissue Engineering

Abstract

Heart valve leaflets have a complex trilayered structure with layer-specific orientations, anisotropic tensile properties, and elastomeric characteristics that are difficult to mimic collectively. Previously, trilayer leaflet substrates intended for heart valve tissue engineering were developed with nonelastomeric biomaterials that cannot deliver native-like mechanical properties. In this study, by electrospinning polycaprolactone (PCL) polymer and poly­(l-lactide-co-ε-caprolactone) (PLCL) copolymer, we created elastomeric trilayer PCL/PLCL leaflet substrates with native-like tensile, flexural, and anisotropic properties and compared them with trilayer PCL leaflet substrates (as control) to find their effectiveness in heart valve leaflet tissue engineering. These substrates were seeded with porcine valvular interstitial cells (PVICs) and cultured for 1 month in static conditions to produce cell-cultured constructs. The PCL/PLCL substrates had lower crystallinity and hydrophobicity but higher anisotropy and flexibility than PCL leaflet substrates. These attributes contributed to more significant cell proliferation, infiltration, extracellular matrix production, and superior gene expression in the PCL/PLCL cell-cultured constructs than in the PCL cell-cultured constructs. Further, the PCL/PLCL constructs showed better resistance to calcification than PCL constructs. Trilayer PCL/PLCL leaflet substrates with native-like mechanical and flexural properties could significantly improve heart valve tissue engineering.

Electrospun Elastomeric Leaflet Substrates that Structurally and Mechanically Mimic Human Aortic Valve Leaflets

“…These observations could be due to the PCL/PTMC solutions being electrospun at higher voltages than PCL solutions, leading to a more scattered deposition of fibers that resulted in larger pores . VICs typically exhibit cell diameters of 8.44 ± 2.5 μm; therefore, the pore sizes of both substrates are sufficient for cell infiltration …”

“…For chemical analysis of the electrospun substrates, ATR-FTIR spectroscopy was utilized over a range of 500–3000 cm –1 for PCL, R-PCL/PTMC-CL, PTMC-CL, PTMC-LA, and C-PCL/PTMC-LA substrates. The ATR-FTIR spectra of the samples were acquired using a Nicolet iS10 with an Everest Diamond ATR spectrometer system from (Thermo Scientific, USA) based on a previous protocol . The Spectragryph software was employed to determine the significant peaks and the area under the curve (AUC) of the FTIR data.…”

“…Prior to imaging, the trilayer PCL and PCL/PTMC substrates ( n = 2 of each) as well as the cell-cultured constructs ( n = 2 of each) were cut, mounted onto metal stubs, and sputter-coated (150TES, EMS) with 25 nm of platinum as previously described . Scanning electron microscopy (SEM) was conducted using the Quanta 600F Microscope (FEI, USA) to characterize the substrate structures.…”

“…The total collagen and GAG contents of the PCL and PCL/PTMC cell-cultured constructs ( n = 5 of each type) were determined using a Sircol collagen assay and GAG assay kits, respectively, using a published protocol . Briefly, the cell-cultured constructs were trimmed and weighted.…”

“…Radial or circumferential strips cut from the PCL and PCL/PTMC cell-cultured constructs were fixed, embedded in the O.C.T. compound, and sectioned as previously described . The sections were thawed at 25 °C for 15 min and then stained with H&E, Alcian blue, Masson’s trichrome, or von Kossa.…”