Currently used prosthetic heart valves show multiple limitations, including a reduced ability to regenerate. In this study we developed a three-layered electrospun heart valve using a dual electrospinning setup with a special 3D printed collector. In this manner, not only the microscopic but also the macroscopic structure of native heart valves was imitated. Biocompatibility of the used material constitutes an important property for clinical application. Re-seeding with human cells could allow for a regenerative approach.
A heart valve shaped template was designed in commercial computer aided design (CAD) software, subsequently 3D printed and used for dual electrospinning. The polymers polycaprolactone (PCL) and polyurethane (PU) were electrospun from opposite sides onto a rotating collector (voltage = 15kV; flow rate = 3 ml/h; rotation speed for aligned fibers = 1520 rpm and 38 rpm for unaligned fibers). In a multistep approach scaffolds consisting of different layers with aligned or unaligned fibers were fabricated. Quality, morphology and orientation of the fibers were evaluated with fluorescence and scanning electron microscopy (SEM). Percentual porosity was assessed with gravimetric measurement. Biomechanical properties were determined by uniaxial tensile tests. Pseudomonas cepacia lipase was used for PCL degradation. Evaluation of the biocompatibility was achieved by static seeding of aligned and unaligned scaffolds with human fibroblasts. Cellular behavior was analyzed with SEM, histological and immunofluorescence microscopy.
By CAD and 3D printing, it was possible to create an individual electrospinning collector, which precisely reproduces the macroscopic shape of a native heart valve. Thus, three-dimensional heart valve leaflets could be fabricated by using the collector in a dual electrospinning setup. To recreate the three layers (fibrosa, spongiosa, ventricularis) of the native valve, fibers were aligned circumferentially, randomly and radially. Homogenous, highly aligned (angle between fibers = 5.79 ± 1.61°) and unaligned fibers (no correlation possible) could be fabricated. Aligned fibers showed significantly higher tensile strength along the fiber direction than against it (15.72 ± 4.66 N/mm² vs. 1.83 ± 0.67 N/mm²; p<0.001). Unaligned layers had an overall tensile strength of 6.48 ± 2.3 N/mm². High percentual porosity (85.81 ± 1.59% for aligned and 83.49 ± 1.74% for unaligned fibers) in all layers of the scaffold could be demonstrated. Especially within the unaligned dual spun scaffolds the percentual porosity could be significantly enhanced (89.3 ± 2.95%; p<0.001) by dissolving the PCL using enzymatic degradation. A homogenous monolayer of adherent fibroblasts on the surface of the scaffolds was observed in SEM, histological and immunofluorescence staining. Furthermore, evaluation with SEM showed the formation of fibrin nets. This confirmed the biocompatibility of the material and its appropriate surface for cellular adhesion.
We established the development process of a biocompatible three-layered composite heart valve that replicates the fiber morphology as well as the geometry of a native aortic valve. The dual electrospun material was successfully seeded with fibroblasts, making it suitable for a regenerative approach. This method allows for individualized heart valve replacement by adjusting inserts of the 3D printed collector using personalized data e.g., CT scans.