Influence of Substrate Structure and Associated Properties on Endothelial Cell Behavior in the Context of Behaviors Associated with Laminar Flow Conditions.

In order to treat most vascular diseases, arterial grafts are commonly employed for replacing small-diameter vessels, yet they often cause thrombosis. The growth of endothelial cells along the interior surfaces of these grafts (substrates) is critical to mitigate thrombosis. Typically, endothelial cells are cultured inside these grafts under laminar flow conditions to emulate the native environment of blood vessels and produce an endothelium. Alternatively, the substrate structure could have a similar influence on endothelial cell behavior as laminar flow conditions. In this study, we investigated whether substrates with aligned fiber structures could induce responses in human umbilical vein endothelial cells (HUVECs) akin to those elicited by laminar flow. Our observations revealed that HUVECs on aligned substrates displayed significant morphological changes, aligning parallel to the fibers, similar to effects reported under laminar flow conditions. Conversely, HUVECs on random substrates maintained their characteristic cobblestone appearance. Notably, cell migration was more significant on aligned substrates. Also, we observed that while vWF expression was similar between both substrates, the HUVECs on aligned substrates showed more expression of platelet/endothelial cell adhesion molecule-1 (PECAM-1/CD31), laminin, and collagen IV. Additionally, these cells exhibited increased gene expression related to critical functions such as proliferation, extracellular matrix production, cytoskeletal reorganization, autophagy, and antithrombotic activity. These findings indicated that aligned substrates enhanced endothelial growth and behavior compared to random substrates. These improvements are similar to the beneficial effects of laminar flow on endothelial cells, which are well-documented compared to static or turbulent flow conditions.

Strategies for Development of Synthetic Heart Valve Tissue Engineering Scaffolds.

The current clinical solutions, including mechanical and bioprosthetic valves for valvular heart diseases, are plagued by coagulation, calcification, nondurability, and the inability to grow with patients. The tissue engineering approach attempts to resolve these shortcomings by producing heart valve scaffolds that may deliver patients a life-long solution. Heart valve scaffolds serve as a three-dimensional support structure made of biocompatible materials that provide adequate porosity for cell infiltration, and nutrient and waste transport, sponsor cell adhesion, proliferation, and differentiation, and allow for extracellular matrix production that together contributes to the generation of functional neotissue. The foundation of successful heart valve tissue engineering is replicating native heart valve architecture, mechanics, and cellular attributes through appropriate biomaterials and scaffold designs. This article reviews biomaterials, the fabrication of heart valve scaffolds, and their in-vitro and in-vivo evaluations applied for heart valve tissue engineering.

Fibrin gel enhanced trilayer structure in cell-cultured constructs.

Efficient cell seeding and subsequent support from a substrate ensure optimal cell growth and neotissue development during tissue engineering, including heart valve tissue engineering. Fibrin gel as a cell carrier may provide high cell seeding efficiency and adhesion property, improved cellular interaction, and structural support to enhance cellular growth in trilayer polycaprolactone (PCL) substrates that mimic the structure of native heart valve leaflets. This cell carrier gel coupled with a trilayer PCL substrate may enable the production of native-like cell-cultured leaflet constructs suitable for heart valve tissue engineering. In this study, we seeded valvular interstitial cells onto trilayer PCL substrates with fibrin gel as a cell carrier and cultured them for 1 month in vitro to determine if this gel can improve cell proliferation and production of extracellular matrix within the trilayer cell-cultured constructs. We observed that the fibrin gel enhanced cellular proliferation, their vimentin expression, and collagen and glycosaminoglycan production, leading to improved structure and mechanical properties of the developing PCL cell-cultured constructs. Fibrin gel as a cell carrier significantly improved the orientations of the cells and their produced tissue materials within trilayer PCL substrates that mimic the structure of native heart valve leaflets and, thus, may be highly beneficial for developing functional tissue-engineered leaflet constructs.

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

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.

Anisotropicity and flexibility in trilayered microfibrous substrates promote heart valve leaflet tissue engineering.

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 higherα-smooth muscle actin 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.

Strategies for development of decellularized heart valve scaffolds for tissue engineering.

Valvular heart diseases (VHDs) are currently treated using either mechanical or bioprosthetic heart valves. Unfortunately, mechanical valves require lifelong anticoagulation therapy, and bioprosthetic valves calcify and degrade over time, requiring subsequent valve replacement surgeries. Besides, both valves cannot grow with patients. Heart valve tissue engineering uses scaffolds as valve replacements with the potential to grow with patents, function indefinitely, and not require anticoagulation medication. These scaffolds provide three-dimensional supports for cellular adhesion and growth, leading to tissue formation and, finally, a new functional heart valve development. Heart valve scaffolds are made of either polymeric materials or decellularized tissue obtained from allogeneic or xenogeneic sources. This review discusses processes for preparing decellularized heart valve scaffolds, including decellularization, crosslinking, surface-coating, and sterilization. We also examine the predominant issues in scaffold development. Further, decellularized heart valve scaffold function in vitro and in vivo is evaluated.