An Anisotropic Natural-Synthetic Composite Scaffold for Heart Valve Tissue Engineering

Introduction: : Valvular heart disease affects millions globally, with current treatment options, mechanical and bioprosthetic valves, remaining fundamentally limited. These prosthetics are associated with significant complications, including thrombogenicity, structural degeneration, calcification, lack of somatic growth potential, and the need for lifelong anticoagulation or immunosuppressive therapy. To address these limitations, we developed a novel anisotropic fibrous composite scaffold composed of fibrinogen, a patient-derived, biologically active protein, and polycarbonate polyurethane (PCNU), a biocompatible, mechanically robust, biodegradable synthetic polymer. The scaffold is fabricated using a dual-electrospinning technique designed to mimic the aligned fibrous architecture, biaxial mechanical behavior, and biochemical milieu of native heart valve tissue.
We hypothesized that incorporating fibrinogen into the scaffold would enhance cellular integration and neo-tissue formation due to its inherent biocompatibility, native extracellular matrix-like properties, and patient-specific origin. In contrast, PCNU provides tunable mechanical support and controlled degradation kinetics for early-stage durability and eventual host-mediated remodeling. This composite strategy aims to leverage the biological functionality of fibrinogen with the structural resilience of PCNU, ultimately yielding a next-generation heart valve scaffold with the potential for in situ regeneration, growth adaptability, and long-term physiological performance.

Materials and

Methods: : Composite scaffolds were fabricated using electrospinning of fibrinogen and polycarbonate polyurethane. We first established an optimized solvent mixture to yield a homogeneous fibrinogen solution (100 mg/mL). Subsequently, the electrospinning parameters were systematically determined to produce anisotropic fibrinogen sheets with minimal bead formation and no needle clotting during the fabrication. The resulting sheets were cross-linked using glutaraldehyde vapor for durations of 2, 4, and 8 hours, followed by a comprehensive rinsing protocol to determine the optimal crosslinking condition. In parallel, PCNU was dissolved in hexafluoroisopropanol (HFIP) at ~15% w/v and electrospun under conditions tailored to yield anisotropic fibrous sheets. Both fibrinogen and PCNU sheets were independently characterized for their morphological and mechanical properties.
To fabricate the composite scaffolds, fibrinogen and PCNU were concurrently electrospun using a dual-spinneret electrospinning device with the optimal parameters per component, i.e., polymer concentration, fibrinogen to PCNU ratio, applied voltages (positive and negative), nozzle-to-collector distance, collector rotation speed, and individual flow rates. Composite scaffolds were cross-linked (4 hours) and subjected to extensive rinsing to eliminate residual solvents and sacrificial components. Scaffolds were then characterized using scanning electron microscopy (SEM) for morphological properties, Raman spectroscopy and Fourier Transform Infrared spectroscopy (FTIR) for chemical composition analysis, and biaxial mechanical testing under physiological conditions (pH 7.4, 37 °C) for mechanical performance. Statistical analysis was conducted using GraphPad PRISM.

Results, Conclusions, and Discussions:: Scanning electron microscopy (SEM) revealed highly aligned fibrous networks within the composite scaffolds (Fig. 1A-C), with fiber diameters ranging from 0.5 to 2 µm and pore areas between 2 to 1,500 µm², depending on polymer composition and post-processing steps. Raman spectroscopy confirmed the effective removal of sacrificial polymers and minimal residual glutaraldehyde, while maintaining the characteristic spectral peaks of both fibrinogen and PCNU, indicating the chemical integrity of the composite components. Biaxial mechanical testing demonstrated anisotropic biaxial mechanical behavior closely mimicking that of native aortic valve tissue (Fig. 1D). The scaffolds exhibited low tangent moduli of approximately 314 N/m (fiber direction) and 29.8 N/m (cross-fiber direction) in the low-strain region, transitioning to high tangent moduli of 609 N/m and 112 N/m in the respective directions at high-strain region. Corresponding extensibility values were 0.057 (fiber direction) and 0.33 (cross-fiber direction), aligning well with mechanical benchmarks reported for native aortic valve leaflets. In contrast, fibrinogen-only scaffolds lacked sufficient structural integrity to undergo mechanical testing, highlighting the importance of PCNU integration for mechanical reinforcement. Statistical analysis of fiber diameter and pore area distributions demonstrated reproducibility of scaffold fabrication, with coefficients of variation below 15% across independent batches.
In conclusion, the engineered fibrinogen-PCNU composite scaffold demonstrates significant potential for heart valve tissue engineering by combining bioactivity, structural alignment, and physiologically relevant mechanical behavior and integrity. Future research will investigate the scaffold degradation kinetics under enzymatic and hydrolytic conditions, conduct extensive in vitro cell culture assays to evaluate cell integration, response, and tissue synthesis, and implement dynamic mechanical conditioning in a heart valve bioreactor to assess functional performance under physiologically relevant hemodynamic loading. These evaluations are critical steps toward the scaffold’s translation into clinically viable heart valve replacements.

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