Substrates with Tunable Hydrophobicity for Optimal Cell Adhesion.

Electrospinning is a technique used to create nano/micro-fibrous materials from various polymers for biomedical uses. Polymers like polycaprolactone (PCL) are commonly used, but their hydrophobic properties can limit their applications. To enhance hydrophilicity, nonionic surfactants such as sorbitane monooleate (Span80) and poloxamer (P188) can be added to the PCL electrospinning solution without altering its net charge density. These additions enable the successful production of PCL/P188 and PCL/Span80 fibrous substrates. In this study, P188 and Span80 are incorporated into the PCL solutions; they are successfully electrospun into PCL/P188 and PCL/Span80 substrates, respectively. PCL/P188 substrates show that until a specific P188 concentration, fiber and pore sizes are similar to PCL substrates. However, exceeding 0.30% P188 concentration enlarges fibers, impacting fiber uniformity at higher concentrations. Conversely, higher concentrations of Span80 result in thicker, less uniform fibers, indicating potential disruptions in the electrospinning process. Notably, both surfactants significantly improve substrate hydrophilicity, enhancing the adhesion and proliferation of fibroblasts, endothelial cells, and smooth muscle cells. P188, in particular, shows superior efficacy in promoting cell adhesion and growth at concentrations optimized for different cell types. Therefore, precise surfactant concentrations in the electrospinning solution can lead to the optimization of electrospun substrates for tissue engineering applications.

Non-immune factors cause prolonged myofibroblast phenotype in implanted synthetic heart valve scaffolds.

The clinical application of heart valve scaffolds is hindered by complications associated with the activation of valvular interstitial cell-like (VIC-like) cells and their transdifferentiation into myofibroblasts. This study aimed to examine several molecular pathway(s) that may trigger the overactive myofibroblast phenotypes in the implanted scaffolds. So, we investigated the influence of three molecular pathways - macrophage-induced inflammation, the TGF-ß1-SMAD2, and WNT/ß-catenin ß on VIC-like cells during tissue engineering of heart valve scaffolds. We implanted electrospun heart valve scaffolds in adult sheep for up to 6 months in the right ventricular outflow tract (RVOT) and analyzed biomolecular (gene and protein) expression associated with the above three pathways by the scaffold infiltrating cells. The results showed a gradual increase in gene and protein expression of markers related to the activation of VIC-like cells and the myofibroblast phenotypes over 6 months of scaffold implantation. Conversely, there was a gradual increase in macrophage activity for the first three months after scaffold implantation. However, a decrease in macrophage activity from three to six months of scaffold tissue engineering suggested that immunological signal factors were not the primary cause of myofibroblast phenotype. Similarly, the gene and protein expression of factors associated with the TGF-ß1-SMAD2 pathway in the cells increased in the first three months but declined in the next three months. Contrastingly, the gene and protein expression of factors associated with the WNT/ß-catenin pathway increased significantly over the six-month study. Thus, the WNT/ß-catenin pathway could be the predominant mechanism in activating VIC-like cells and subsequent myofibroblast phenotype.

Innovative Substrate Design with Basement Membrane Components for Enhanced Endothelial Cell Function and Endothelization.

Enhancing endothelial cell growth on small-diameter vascular grafts produced from decellularized tissues or synthetic substrates is pivotal for preventing thrombosis. While optimized decellularization protocols can preserve the structure and many components of the extracellular matrix (ECM), the process can still lead to the loss of crucial basement membrane proteins, such as laminin, collagen IV, and perlecan, which are pivotal for endothelial cell adherence and functional growth. This loss can result in poor endothelialization and endothelial cell activation causing thrombosis and intimal hyperplasia. To address this, the basement membrane's ECM is emulated on fiber substrates, providing a more physiological environment for endothelial cells. Thus, fibroblasts are cultured on fiber substrates to produce an ECM membrane substrate (EMMS) with basement membrane proteins. The EMMS then underwent antigen removal (AR) treatment to eliminate antigens from the membrane while preserving essential proteins and producing an AR-treated membrane substrate (AMS). Subsequently, human endothelial cells cultured on the AMS exhibited superior proliferation, nitric oxide production, and increased expression of endothelial markers of quiescence/homeostasis, along with autophagy and antithrombotic factors, compared to those on the decellularized aortic tissue. This strategy showed the potential of pre-endowing fiber substrates with a basement membrane to enable better endothelization.

Differential expression of cellular prion protein (PrPC) in mouse hepatitis virus induced neuroinflammation.

The cellular prion protein (PrPC) is an extracellular cell membrane protein. Due to its diversified roles, a definite role of PrPC has been difficult to establish. During viral infection, PrPC has been reported to play a pleiotropic role. Here, we have attempted to envision the function of PrPC in the neurotropic m-CoV-MHV-RSA59-induced model of neuroinflammation in C57BL/6 mice. A significant upregulation of PrPC at protein and mRNA levels was evident in infected mouse brains during the acute phase of neuroinflammation. Furthermore, investigation of the effect of MHV-RSA59 infection on PrPC expression in specific neuronal, microglial, and astrocytoma cell lines, revealed a differential expression of prion protein during neuroinflammation. Additionally, siRNA-mediated downregulation of prnp transcripts reduced the expression of viral antigen and viral infectivity in these cell lines. Cumulatively, our results suggest that PrPC expression significantly increases during acute MHV-RSA59 infection and that PrPC also assists in viral infectivity and viral replication.

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.

Neutron-activated, plasmonically excitable Fe-Pt-Yb2O3 nanoparticles delivering anti-cancer radiation against human glioblastoma cells.

Magnetic nanoparticles can be functionalized in many ways for biomedical applications. Here, we combine four advantageous features in a novel Fe-Pt-Yb2O3 core-shell nanoparticle. (a) The nanoparticles have a size of 10 nm allowing them to diffuse through neuronal tissue. (b) The particles are superparamagnetic after synthesis and ferromagnetic after annealing, enabling directional control by magnetic fields, enhance NMRI contrast, and hyperthermia treatment. (c) After neutron-activation of the shell, they carry low-energetic, short half-life ß-radiation from 175Yb, 177Yb, and 177Lu. (d) Additionally, the particles can be optically visualized by plasmonic excitation and luminescence. To demonstrate the potential of the particles for cancer treatment, we exposed cultured human glioblastoma cells (LN-18) to non-activated and activated particles to confirm that the particles are internalized, and that the ß-radiation of the radioisotopes incorporated in the neutron-activated shell of the nanoparticles kills more than 98% of the LN-18 cancer cells, promising for future anti-cancer applications.

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.

Fibrous heart valve leaflet substrate with native-mimicked morphology.

Tissue-engineered heart valves are a promising alternative solution to prosthetic valves. However, long-term functionalities of tissue-engineered heart valves depend on the ability to mimic the trilayered, oriented structure of native heart valve leaflets. In this study, using electrospinning, we developed trilayered microfibrous leaflet substrates with morphological characteristics similar to native leaflets. The substrates were implanted subcutaneously in rats to study the effect of their trilayered oriented structure on in vivo tissue engineering. The tissue constructs showed a well-defined structure, with a circumferentially oriented layer, a randomly oriented layer and a radially oriented layer. The extracellular matrix, produced during in vivo tissue engineering, consisted of collagen, glycosaminoglycans, and elastin, all major components of native leaflets. Moreover, the anisotropic tensile properties of the constructs were sufficient to bear the valvular physiological load. Finally, the expression of vimentin and α-smooth muscle actin, at the gene and protein level, was detected in the residing cells, revealing their growing state and their transdifferentiation to myofibroblasts. Our data support a critical role for the trilayered structure and anisotropic properties in functional leaflet tissue constructs, and indicate that the leaflet substrates have the potential for the development of valve scaffolds for heart valve replacements.

Leaflet Tissue Generation from Microfibrous Heart Valve Leaflet Scaffolds with Native Characteristics.

Mechanical and bioprosthetic valves that are currently applied for replacing diseased heart valves are not fully efficient. Heart valve tissue engineering may solve the issues faced by the prosthetic valves in heart valve replacement. The leaflets of native heart valves have a trilayered structure with layer-specific orientations; thus, it is imperative to develop functional leaflet tissue constructs with a native trilayered, oriented structure. Its key solution is to develop leaflet scaffolds with a native morphology and structure. In this study, microfibrous leaflet scaffolds with a native trilayered and oriented structure were developed in an electrospinning system. The scaffolds were implanted for 3 months in rats subcutaneously to study the scaffold efficiencies in generating functional tissue-engineered leaflet constructs. These in vivo tissue-engineered leaflet constructs had a trilayered, oriented structure similar to native leaflets. The tensile properties of constructs indicated that they were able to endure the hydrodynamic load of the native heart valve. Collagen, glycosaminoglycans, and elastin─the predominant extracellular matrix components of native leaflets─were found sufficiently in the leaflet tissue constructs. The residing cells in the leaflet tissue constructs showed vimentin and α-smooth muscle actin expression, i.e., the constructs were in a growing state. Thus, the trilayered, oriented fibrous leaflet scaffolds produced in this study could be useful to develop heart valve scaffolds for successful heart valve replacements.

Trilayered tissue construct mimicking the orientations of three layers of a native heart valve leaflet.

A tissue-engineered heart valve can be an alternative to a prosthetic valve in heart valve replacement; however, it is not fully efficient in terms of long-lasting functionality, as leaflets in engineered valves do not possess the trilayered native leaflet structure. Previously, we developed a flat, trilayered, oriented nanofibrous (TN) scaffold mimicking the trilayered structure and orientation of native heart valve leaflets. In vivo tissue engineering-a practical regenerative medicine technology-can be used to develop an autologous heart valve. Thus, in this study, we used our flat, trilayered, oriented nanofibrous scaffolds to develop trilayered tissue structures with native leaflet orientations through in vivo tissue engineering in a rat model. After 2 months of in vivo tissue engineering, infiltrated cells and their deposited collagen fibrils were found aligned in the circumferential and radial layers, and randomly oriented in the random layer of the scaffolds, i.e., trilayered tissue constructs (TTCs) were developed. Tensile properties of the TTCs were higher than that of the control tissue constructs (without any scaffolds) due to influence of fibers of the scaffolds in tissue engineering. Different extracellular matrix proteins-collagen, glycosaminoglycans, and elastin-that exist in native leaflets were observed in the TTCs. Gene expression of the TTCs indicated that the tissue constructs were in growing stage. There was no sign of calcification in the tissue constructs. The TTCs developed with the flat TN scaffolds indicate that an autologous leaflet-shaped, trilayered tissue construct that can function as a native leaflet can be developed.

In vivo tissue engineering of a trilayered leaflet-shaped tissue construct.

Aim: We aimed to develop a leaflet-shaped trilayered tissue construct mimicking the morphology of native heart valve leaflets. Materials & methods: Electrospinning and in vivo tissue engineering methods were employed. Results: We developed leaflet-shaped microfibrous scaffolds, each with circumferentially, randomly and radially oriented three layers mimicking the trilayered, oriented structure of native leaflets. After 3 months in vivo tissue engineering with the scaffolds, the generated leaflet-shaped tissue constructs had a trilayered structure mimicking the orientations of native heart valve leaflets. Presence of collagen, glycosaminoglycans and elastin seen in native leaflets was observed in the engineered tissue constructs. Conclusion: Trilayered, oriented fibrous scaffolds brought the orientations of the infiltrated cells and their produced extracellular matrix proteins into the constructs.

Trilayered tissue structure with leaflet-like orientations developed through in vivo tissue engineering.

A tissue-engineered heart valve can be an alternative to current mechanical or bioprosthetic valves that face limitations, especially in pediatric patients. However, it remains challenging to produce a functional tissue-engineered heart valve with three leaflets mimicking the trilayered, oriented structure of a native valve leaflet. In our previous study, a flat, trilayered nanofibrous substrate mimicking the orientations of three layers in a native leaflet-circumferential, random and radial orientations in fibrosa, spongiosa and ventricularis layers, respectively, was developed through electrospinning. In this study, we sought to develop a trilayered tissue structure mimicking the orientations of a native valve leaflet through in vivo tissue engineering, a practical regenerative medicine technology that can be used to develop an autologous heart valve. Thus, the nanofibrous substrate was placed inside the closed trileaflet-shaped cavity of a mold and implanted subcutaneously in a rat model for in vivo tissue engineering. After two months, the explanted tissue construct had a trilayered structure mimicking the orientations of a native valve leaflet. The infiltrated cells and their deposited collagen fibrils were oriented along the nanofibers in each layer of the substrate. Besides collagen, presence of glycosaminoglycans and elastin in the construct was observed.

Optimization of polycaprolactone fibrous scaffold for heart valve tissue engineering.

Pore size is generally small in nanofibrous scaffolds prepared by electrospinning polymeric solutions. Increase of scaffold thickness leads to decrease in pore size, causing impediment to cell infiltration into the scaffolds during tissue engineering. In contrast, comparatively larger pore size can be realized in microfibrous scaffolds prepared from polymeric solutions at higher concentrations. Further, microfibrous scaffolds are conducive to infiltration of reparative M2 phenotype macrophages during in vivo/in situ tissue engineering. However, rise of mechanical properties of a fibrous scaffold with the increase of polymer concentration may limit the functionality of a scaffold-based, tissue-engineered heart valve. In this study, we developed microfibrous scaffolds from 14%, 16% and 18% (wt/v) polycaprolactone (PCL) polymer solutions prepared with chloroform solvent. Porcine valvular interstitial cells were cultured in the scaffolds for 14 d to investigate the effect of microfibers prepared with different PCL concentrations on the seeded cells. Further, fresh microfibrous scaffolds were implanted subcutaneously in a rat model for two months to investigate the effect of microfibers on infiltrated cells. Cell proliferation, and its morphologies, gene expression and deposition of different extracellular matrix proteins in the in vitro study were characterized. During the in vivo study, we characterized cell infiltration, and myofibroblast and M1/M2 phenotypes expression of the infiltrated cells. Among different PCL concentrations, microfibrous scaffolds from 14% solution were suitable for heart valve tissue engineering for their sufficient pore size and low but adequate tensile properties, which promoted cell adhesion to and proliferation in the scaffolds, and effective gene expression and extracellular matrix deposition by the cells in vitro. They also encouraged the cells in vivo for their infiltration and effective gene expression, including M2 phenotype expression.

Endothelialization of cardiovascular devices.

Blood-contacting surfaces of cardiovascular devices are not biocompatible for creating an endothelial layer on them. Numerous research studies have mainly sought to modify these surfaces through physical, chemical and biological means to ease early endothelial cell (EC) adhesion, migration and proliferation, and eventually to build an endothelial layer on the surfaces. The first priority for surface modification is inhibition of protein adsorption that leads to inhibition of platelet adhesion to the device surfaces, which may favor EC adhesion. Surface modification through surface texturing, if applicable, can bring some hopeful outcomes in this regard. Surface modifications through chemical and/or biological means may play a significant role in easy endothelialization of cardiovascular devices and inhibit smooth muscle cell proliferation. Cellular engineering of cells relevant to endothelialization can boost the positive outcomes obtained through surface engineering. This review briefly summarizes recent developments and research in early endothelialization of cardiovascular devices. STATEMENT OF SIGNIFICANCE: Endothelialization of cardiovascular implants, including heart valves, vascular stents and vascular grafts is crucial to solve many problems in our health care system. Numerous research efforts have been made to improve endothelialization on the surfaces of cardiovascular implants, mainly through surface modifications in three ways - physically, chemically and biologically. This review is intended to highlight comprehensive research studies to date on surface modifications aiming for early endothelialization on the blood-contacting surfaces of cardiovascular implants. It also discusses future perspectives to help guide endothelialization strategies and inspire further innovations.

Behavior of valvular interstitial cells on trilayered nanofibrous substrate mimicking morphologies of heart valve leaflet.

Heart valve tissue engineering could be an alternative to the current bioprosthetic heart valve that faces limitations especially in pediatric patients. However, heart valve tissue engineering has remained challenging because leaflets - the primary component of a heart valve - have three layers with three diverse orientations - circumferential, random and radial, respectively. In order to mimic the orientations, we first designed three novel collectors to fabricate three nanofibrous layers with those orientations from a polymeric biomaterial in an electrospinning system. Then, we devised a novel direct electrospinning technique to develop a unified trilayered nanofibrous (TN) substrate comprising those oriented layers. The TN substrate supported the growth and orientations of seeded porcine valvular interstitial cells (PVICs) and their deposited collagen fibrils. After one month culture, the obtained trilayered tissue construct (TC) exhibited increased tensile properties over its TN substrate. Most importantly, the developed TC did not show any sign of shrinkage. Gene expression pattern of the PVICs indicated the developing stage of the TC. Their protein expression pattern was quite similar to that of leaflets. STATEMENT OF SIGNIFICANCE: This manuscript talks about development of a novel trilayered nanofibrous substrate mimicking the morphologies of a heart valve leaflet. It also describes culturing of valvular interstitial cells that reside in a leaflet, in the substrate and compares the behavior of the cultured cells with that in native leaflets in terms cell morphology, protein deposition and its orientation, and molecular signature. This study builds the groundwork for our future trilayered, tissue-engineered leaflet development. This research article would be of great interest to investigators and researchers in the field of cardiovascular tissue engineering especially in cardiac valve tissue engineering through biomaterial-based tissue engineering.

Biomaterial characterization of off-the-shelf decellularized porcine pericardial tissue for use in prosthetic valvular applications.

Fixed pericardial tissue is commonly used for commercially available xenograft valve implants, and has proven durability, but lacks the capability to remodel and grow. Decellularized porcine pericardial tissue has the promise to outperform fixed tissue and remodel, but the decellularization process has been shown to damage the collagen structure and reduce mechanical integrity of the tissue. Therefore, a comparison of uniaxial tensile properties was performed on decellularized, decellularized-sterilized, fixed, and native porcine pericardial tissue versus native valve leaflet cusps. The results of non-parametric analysis showed statistically significant differences (p < .05) between the stiffness of decellularized versus native pericardium and native cusps as well as fixed tissue, respectively; however, decellularized tissue showed large increases in elastic properties. Porosity testing of the tissues showed no statistical difference between decellularized and decell-sterilized tissue compared with native cusps (p > .05). Scanning electron microscopy confirmed that valvular endothelial and interstitial cells colonized the decellularized pericardial surface when seeded and grown for 30 days in static culture. Collagen assays and transmission electron microscopy analysis showed limited reductions in collagen with processing; yet glycosaminoglycan assays showed great reductions in the processed pericardium relative to native cusps. Decellularized pericardium had comparatively low mechanical properties among the groups studied; yet the stiffness was comparatively similar to the native cusps and demonstrated a lack of cytotoxicity. Suture retention, accelerated wear, and hydrodynamic testing of prototype decellularized and decell-sterilized valves showed positive functionality. Sterilized tissue could mimic valvular mechanical environment in vitro, therefore making it a viable potential candidate for off-the-shelf tissue-engineered valvular applications.