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Heart valve leaflet substrates with native trilayer and anisotropic structures are crucial for successful heart valve tissue engineering. In this study, we used the electrospinning technique to produce trilayer microfibrous leaflet substrates using two biocompatible and biodegradable polymers - poly (L-lactic acid) (PLLA) and polycaprolactone (PCL), separately. Different polymer concentrations for each layer were applied to bring a high degree of mechanical and structural anisotropy to the substrates. PCL leaflet substrates exhibited lower unidirectional tensile properties than PLLA leaflet substrates. However, the PLLA substrates exhibited a lower flexural modulus than the PCL substrates. These substrates were seeded with porcine valvular interstitial cells (PVICs) and cultured for one month in static conditions. Both substrates exhibited cellular adhesion and proliferation, resulting in the production of tissue-engineered constructs. The PLLA tissue-engineered constructs had more cellular growth than the PCL tissue-engineered constructs. The PLLA substrates showed higher hydrophilicity, lower crystallinity, and more significant anisotropy than PCL substrates, which may have enhanced their interactions with PVICs. Analysis of gene expression showed higher α-SMA and collagen type 1 expression in PLLA tissue-engineered constructs than in PCL tissue-engineered constructs. The differences in anisotropic and flexural properties may have accounted for the different cellular behaviors in these two individual polymer substrates.

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.

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Creating leaflet substrates that accurately mimic the trilayered structure, anisotropic tensile characteristics, and elastomeric features of native human heart valve leaflets is essential to the success of heart valve tissue engineering. Earlier leaflet substrates have successfully replicated the mechanical properties of animal (porcine) heart valve leaflets. However, not one of them has been able to simultaneously replicate the trilayered structure and the highly anisotropic and elastomeric mechanical properties of human aortic heart valve leaflets. In this study, we employed a combination of polymers − polycaprolactone (PCL), poly-(trimethylene carbonate-co-L-lactide) (PTMC-LA), and poly-(trimethylene carbonate-co-caprolactone) (PTMC-CL) − in an electrospinning system to create elastomeric trilayer PCL/PTMC leaflet substrates that possess mechanical, flexural, and anisotropic properties similar to those of human native heart valve leaflets. We then compared these substrates with non-elastomeric trilayer PCL leaflet substrates to determine their effect on leaflet tissue engineering. Porcine valvular interstitial cells (PVICs) were cultured on these substrates for 1 month in static conditions to develop trilayer PCL and PCL/PTMC cell-cultured constructs. The PCL/PTMC substrates exhibited lower crystallinity and hydrophobicity than the PCL substrate, resulting in better cell adhesion and infiltration. The PCL/PTMC substrates demonstrated significantly greater cell proliferation, extracellular matrix production, and preferable gene and protein expression than the PCL substrates. Furthermore, in an osteogenic environment, the PCL/PTMC substrates showed superior resistance to calcification compared to the PCL substrates. The development of trilayer PCL/PTMC substrates with mechanical and structural characteristics resembling native tissue enhances leaflet tissue engineering.