Development of trilayer leaflets for heart valve tissue engineering
Authors:   , Snyder Yuriy 
Published: November 2024

Approximately 2.5 percent of the US population is affected by valvular heart diseases (VHDs), typically managed through complex valve-sparing surgeries or heart valve replacement with mechanical or bioprosthetic valves. These strategies, however, can lead to complications such as the need for continuous anticoagulation, calcification, and degradation. Tissue engineering offers a promising alternative to current treatments by using biodegradable polymeric heart valve substrates that support cell growth and remodeling, facilitating the development of tissue-engineered heart valves. However, the clinical application of these substrates is limited by issues related to the leaflets caused by valvular interstitial cells (VICs). Most leaflet substrates lack native-like cues, such as the structural, mechanical, flexural, and anisotropic properties that VICs require. Therefore, developing leaflet substrates with an anisotropic structure and native-like mechanical properties could provide the appropriate biomechanical environment to reduce adverse effects in tissue-engineered leaflets. This dissertation research aims to develop novel leaflet substrates with trilayer structures and mechanical properties similar to those of native leaflets. It explores new material combinations, including PCL, PLCL, poly(trimethylene carbonate-co-L-lactide) (PTMC-LA), and poly(trimethylene carbonate-co-caprolactone) (PTMC-CL), which are electrospun and optimized to enhance the native-like properties of the substrates. The research also validates the benefits of mimicking these properties by comparing substrates with native-like characteristics to those with non-native properties. In the first study, we developed trilayer leaflet substrates from PCL and PLLA with varying anisotropic properties. We evaluated the effects of these properties, along with flexural characteristics, on VICs cultured on the substrates. The PCL substrates exhibited lower unidirectional tensile properties, but a higher flexural modulus compared to the PLLA substrates. Both substrates demonstrated extensive cellular adhesion and proliferation when seeded with VICs and cultured for one month. However, the tissue-engineered PLLA leaflet substrates showed greater cellular growth, higher hydrophilicity, lower crystallinity, and increased anisotropy, which enhanced interactions with the VICs. Gene expression analysis revealed higher levels of [alpha]-smooth muscle actin and collagen type 1 in the tissue-engineered PLLA leaflet substrates, suggesting that the differences in anisotropic and flexural properties influenced cellular behavior. In the second study, we hypothesized that trilayer leaflet substrates with anisotropic mechanical properties could enhance VIC behavior in tissue-engineered leaflets. To test this, we produced trilayer anisotropic fibrous PCL substrates and randomly oriented isotropic fibrous PCL substrates as controls. The random PCL substrates exhibited higher radial and lower circumferential tensile properties but similar flexural properties compared to the trilayer PCL substrates. VICs cultured on these substrates resulted in constructs with distinct properties. The trilayer constructs showed more cellular proliferation and extracellular matrix production, as well as superior gene and protein expression compared to the random constructs. Additionally, the trilayer constructs demonstrated more anisotropic mechanical properties and higher vimentin, collagen, TGF-[beta]1, and TGF-[beta]3 gene expression in their random and radial layers than in the circumferential layer. This study confirms that the trilayer structure and anisotropic properties significantly benefit VIC response compared to random structure and isotropic properties in leaflet substrates. In the third study, we developed and optimized novel trilayer PCL/PLCL leaflet substrates designed to replicate the structural, tensile, flexural, and anisotropic properties of native leaflets in porcine and ovine animal models. Unlike traditional trilayer leaflet substrates used in heart valve tissue engineering, which lack elastomeric properties, the incorporation of PLCL provides these new substrates with elastomeric properties. The novel trilayer PCL/PLCL substrates were compared to trilayer PCL substrates. The PCL/PLCL substrates exhibited native-like tensile, flexural, and anisotropic properties, along with lower crystallinity and hydrophobicity, but higher anisotropy and flexibility than the PCL substrates. These attributes resulted in increased cell proliferation, infiltration, extracellular matrix production, and superior gene expression in the tissue-engineered PCL/PLCL constructs. Additionally, the tissue-engineered PCL/PLCL constructs demonstrated better resistance to calcification. This study suggests that trilayer PCL/PLCL leaflet substrates with native-like mechanical and flexural properties could significantly advance heart valve tissue engineering. In the fourth study, we made leaflet substrates that accurately mimic the trilayered structure, anisotropic tensile characteristics, and elastomeric features of native human heart valve leaflets by using a novel blend of PCL with poly-(trimethylene carbonate-co-L-lactide) (PTMC-LA) and poly-(trimethylene carbonate-co-caprolactone) (PTMC-CL) to produce an elastomeric trilayer PCL/PTMC leaflet substrates. The novel trilayer PCL/PTMC substrates were compared to trilayer PCL substrates. VICs were cultured on these substrates for one month to develop trilayer PCL and PCL/PTMC constructs. The PCL/PTMC substrates, with lower crystallinity and hydrophobicity, showed better cell adhesion, infiltration, and significantly greater cell proliferation, extracellular matrix production, and favorable gene and protein expression compared to PCL substrates. Additionally, the PCL/PTMC substrates demonstrated superior resistance to calcification in an osteogenic environment. This study highlights that trilayer PCL/PTMC substrates with native-like properties enhance leaflet tissue engineering.