Bioinspired ultra-low fouling coatings on medical devices to prevent device-associated infections and thrombosis.

Addressing thrombosis and biofouling of indwelling medical devices within healthcare institutions is an ongoing problem. In this work, two types of ultra-low fouling surfaces (i.e., superhydrophobic and lubricant-infused slippery surfaces) were fabricated to enhance the biocompatibility of commercial medical grade silicone rubber (SR) tubes that are widely used in clinical care. The superhydrophobic (SH) coatings on the tubing substrates were successfully created by dip-coating in superhydrophobic paints consisting of polydimethylsiloxane (PDMS), perfluorosilane-coated hydrophobic zinc oxide (ZnO) and copper (Cu) nanoparticles (NPs) in tetrahydrofuran (THF). The SH surfaces were converted to lubricant-infused slippery (LIS) surfaces through the infusion of silicone oil. The anti-biofouling properties of the coatings were investigated by adsorption of platelets, whole blood coagulation, and biofilm formation in vitro. The results revealed that the LIS tubes possess superior resistance to clot formation and platelet adhesion than uncoated and SH tubes. In addition, bacterial adhesion was investigated over 7 days in a drip-flow bioreactor, where the SH-ZnO-Cu tube and its slippery counterpart significantly reduced bacterial adhesion and biofilm formation of Escherichia coli relative to control tubes (>5 log10 and >3 log10 reduction, respectively). The coatings also demonstrated good compatibility with fibroblast cells. Therefore, the proposed coatings may find potential applications in high-efficiency on-demand prevention of biofilm and thrombosis formation on medical devices to improve their biocompatibility and reduce the risk of complications from medical devices.

Fabrication of Bacteria- and Blood-Repellent Superhydrophobic Polyurethane Sponge Materials.

Biofilm and thrombus formation on surfaces results in significant morbidity and mortality worldwide, which highlights the importance of the development of efficacious fouling-prevention approaches. In this work, novel highly robust and superhydrophobic coatings with outstanding multiliquid repellency, bactericidal performance, and extremely low bacterial and blood adhesion are fabricated by a simple two-step dip-coating method. The coatings are prepared combining 1H,1H,2H,2H-perfluorooctyltriethoxysilane (FAS-17)-coated hydrophobic zinc oxide and copper nanoparticles to construct hierarchical micro/nanostructures on commercial polyurethane (PU) sponges followed by polydimethylsiloxane (PDMS) treatment that is used to improve the binding degree between the nanoparticles and the sponge surface. The micro/nanotextured samples can repel various liquids including water, milk, coffee, juice, and blood. Relative to the original PU, the superhydrophobic characteristics of the fabricated sponge cause a significant reduction in the adhesion of bacteria (Staphylococcus aureus) by up to 99.9% over a 4-day period in a continuous drip-flow bioreactor. The sponge is also highly resistant to the adhesion of fibrinogen and activated platelets with ∼76 and 64% reduction, respectively, hence reducing the risk of blood coagulation and thrombus formation. More importantly, the sponge can sustain its superhydrophobicity even after being subjected to different types of harsh mechanical damage such as finger-wiping, knife-scratching, tape-peeling, hand-kneading, hand-rubbing, bending, compress-release (1000 cycles) tests, and 1000 cm sandpaper abrasion under 250 g of loading. Hence, this novel hybrid surface with robustness and the ability to resist blood adhesion and bacterial contamination makes it an attractive candidate for use in diverse application areas.

Author Correction: Mechanosensation of cyclical force by PIEZO1 is essential for innate immunity.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mechanosensation of cyclical force by PIEZO1 is essential for innate immunity.

Direct recognition of invading pathogens by innate immune cells is a critical driver of the inflammatory response. However, cells of the innate immune system can also sense their local microenvironment and respond to physiological fluctuations in temperature, pH, oxygen and nutrient availability, which are altered during inflammation. Although cells of the immune system experience force and pressure throughout their life cycle, little is known about how these mechanical processes regulate the immune response. Here we show that cyclical hydrostatic pressure, similar to that experienced by immune cells in the lung, initiates an inflammatory response via the mechanically activated ion channel PIEZO1. Mice lacking PIEZO1 in innate immune cells showed ablated pulmonary inflammation in the context of bacterial infection or fibrotic autoinflammation. Our results reveal an environmental sensory axis that stimulates innate immune cells to mount an inflammatory response, and demonstrate a physiological role for PIEZO1 and mechanosensation in immunity.

Left Ventricular Hemodynamics with an Implanted Assist Device: An In Vitro Fluid Dynamics Study.

Left ventricle assist devices (VADs) aid the heart pumping blood into the systemic circulation and grant the required cardiac output (CO) when the heart itself cannot provide it. However, it is unclear how effective these devices are at restoring not only physiological CO values but also normal intraventricular hemodynamics. In this work, the modified hemodynamics due to a VAD implantation is studied in vitro using an elastic ventricle made of silicone, which is incorporated into a pulse-duplicator setup prescribing a realistic pulsatile flow. Thereafter, a continuous axial pump is connected at the ventricle apex to mimic a VAD and its effect on the ventricular hemodynamics is investigated as a function of the pump flow suction. Using particle image velocimetry (PIV), we observe that the continuous pump flow effectively provides unloading on the ventricle and yields an increased CO. Conversely, the continuous blood suction from the ventricle apex deeply alters the hemodynamics and, in addition, the VAD obstruction in the ventricle behaves as a bluff body that affects the vorticity distribution in the LV thus creating a stagnant region at the ventricle apex. This phenomenon is rationalized by measuring in a modified set-up the benefits on the hemodynamics of a flush-mounted device. Additionally, the suction operated by the VAD reduces the ventricular pressure and yields an increase in the swirling motion around the ventricle axis, in a similar fashion as the bath-tub vortex effect, thus further modifying the intraventricular hemodynamics with respect to healthy conditions.

Quantitative Characterization of Aortic Valve Endothelial Cell Viability and Morphology In Situ Under Cyclic Stretch.

Current protocols for mechanical preconditioning of tissue engineered heart valves have focused on application of pressure, flexure and fluid flow to stimulate collagen production, ECM remodeling and improving mechanical performance. The aim of this study was to determine if mechanical preconditioning with cyclic stretch could promote an intact endothelium that resembled the viability and morphology of a native valve. Confocal laser scanning microscopy was used to image endothelial cells on aortic valve strips subjected to static incubation or physiological strain regimens. An automated image analysis program was designed and implemented to detect and analyze live and dead cells in images captured of a live aortic valve endothelium. The images were preprocessed, segmented, and quantitatively analyzed for live/dead cell ratio, minimum neighbor distance and circularity. Significant differences in live/dead cellular ratio and the minimum distance between cells were observed between static and strained endothelia, indicating that cyclic strain is an important stimulus for maintaining a healthy endothelium. In conclusion, in vitro application of physiological levels of cyclic strain to tissue engineered heart valves seeded with autologous endothelial cells would be advantageous.

Cyclic pressure and angiotensin II influence the biomechanical properties of aortic valves.

Hypertension is a known risk factor for aortic stenosis. The elevated blood pressure increases the transvalvular load and can elicit inflammation and extracellular matrix (ECM) remodeling. Elevated cyclic pressure and the vasoactive agent angiotensin II (Ang II) both promote collagen synthesis, an early hallmark of aortic sclerosis. In the current study, it was hypothesized that elevated cyclic pressure and/or angiotensin II decreases extensibility of aortic valve leaflets due to an increase in collagen content and/or interstitial cell stiffness. Porcine aortic valve leaflets were exposed to pressure conditions of increasing magnitude (static atmospheric pressure, 80, and 120 mmHg) with and without 10−6 M Ang II. Biaxial mechanical testing was performed to determine extensibility in the circumferential and radial directions and collagen content was determined using a quantitative dye-binding method at 24 and 48 h. Isolated aortic valve interstitial cells exposed to the same experimental conditions were subjected to atomic force microscopy to assess cellular stiffness at 24 h. Leaflet tissue incubated with Ang II decreased tissue extensibility in the radial direction, but not in the circumferential direction. Elevated cyclic pressure decreased extensibility in both the radial and circumferential directions. Ang II and elevated cyclic pressure both increased the collagen content in leaflet tissue. Interstitial cells incubated with Ang II were stiffer than those incubated without Ang II while elevated cyclic pressure caused a decrease in cell stiffness. The results of the current study demonstrated that both pressure and Ang II play a role in altering the biomechanical properties of aortic valve leaflets. Ang II and elevated cyclic pressure decreased the extensibility of aortic valve leaflet tissue. Ang II induced direction specific changes in extensibility, demonstrating different response mechanisms. These findings help to provide a better understanding of the responses of aortic valves to mechanical and biochemical changes that occur under hypertensive conditions.

Side-specific characterization of aortic valve endothelial cell adhesion molecules under cyclic strain.

BACKGROUND AND AIM OF THE STUDY: Aortic valve ectopic calcification occurs exclusively on the fibrosa surface. This may be due to the distinct mechanical environments on either side of the valve, or to the existence of unique, side-specific endothelial sub-phenotypes. The study aim was to determine if side-specific endothelial cells (ECs) would differentially express cell-cell and cell-matrix adhesion molecules in response to elevated levels of equibiaxial tensile strain. METHODS: Side-specific porcine aortic valve ECs were isolated and strained at 10% or 20% using a Flexcell 4000T for 24 h, and compared to static controls. The quantity and pattern of distribution of adhesion proteins was then assessed using ELISA and fluorescence microscopy, respectively. The adhesion proteins of interest were platelet endothelial cell adhesion molecule-1 (PECAM-1), beta1-integrin, VE-cadherin, and vinculin. RESULTS: Overall, ventricular ECs were more reactive to changes in cyclic strain, with significant increases in VE-cadherin and vinculin at 20% strain. However, the expression of beta1-integrin was significantly increased at 20% strain in fibrosa ECs. Expression of PECAM-1 was not significantly changed at all strain levels for both sub-populations of ECs. CONCLUSION: Endothelial cells isolated from the fibrosa and ventricularis surfaces of porcine aortic valves showed significantly different expression profiles of cell-cell and cell-extracellular matrix adhesion molecules under elevated tensile strain. These differences in response to cyclic strain suggest that different endothelial sub-phenotypes exist on the fibrosa and ventricularis surfaces of the aortic valve.

Live en face imaging of aortic valve leaflets under mechanical stress.

Soft tissues, such as tendons, skin, arteries, or lung, are constantly subject to mechanical stresses in vivo. None more so than the aortic heart valve that experiences an array of forces including shear stress, cyclic pressure, strain, and flexion. Anisotropic biaxial cyclic stretch maintains valve homeostasis; however, abnormal forces are implicated in disease progression. The response of the valve endothelium to deviations from physiological levels has not been fully characterized. Here, we show the design and validation of a novel stretch apparatus capable of applying biaxial stretch to viable heart valve tissue, while simultaneously allowing for live en face endothelial cell imaging via confocal laser scanning microscopy (CLSM). Real-time imaging of tissue is possible while undergoing highly characterized mechanical conditions and maintaining the native extracellular matrix. Thus, it provides significant advantages over traditional cell culture or in vivo animal models. Planar biaxial tissue stretching with simultaneous live cell imaging could prove useful in studying the mechanobiology of any soft tissue.

Gene Profiling of Aortic Valve Interstitial Cells under Elevated Pressure Conditions: Modulation of Inflammatory Gene Networks.

The study aimed to identify mechanosensitive pathways and gene networks that are stimulated by elevated cyclic pressure in aortic valve interstitial cells (VICs) and lead to detrimental tissue remodeling and/or pathogenesis. Porcine aortic valve leaflets were exposed to cyclic pressures of 80 or 120 mmHg, corresponding to diastolic transvalvular pressure in normal and hypertensive conditions, respectively. Linear, two-cycle amplification of total RNA, followed by microarray was performed for transcriptome analysis (with qRT-PCR validation). A combination of systems biology modeling and pathway analysis identified novel genes and molecular mechanisms underlying the biological response of VICs to elevated pressure. 56 gene transcripts related to inflammatory response mechanisms were differentially expressed. TNF-α, IL-1α, and IL-1ß were key cytokines identified from the gene network model. Also of interest was the discovery that pentraxin 3 (PTX3) was significantly upregulated under elevated pressure conditions (41-fold change). In conclusion, a gene network model showing differentially expressed inflammatory genes and their interactions in VICs exposed to elevated pressure has been developed. This system overview has detected key molecules that could be targeted for pharmacotherapy of aortic stenosis in hypertensive patients.

Design of a cyclic pressure bioreactor for the ex vivo study of aortic heart valves.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage(1-3). Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease(4). The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve(5). Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves(6-9). Pulsatile bioreactors have also been developed to study a range of tissues including cartilage(10), bone(11) and bladder(12). The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Introduction to viral vectors.

Viral vector is the most effective means of gene transfer to modify specific cell type or tissue and can be manipulated to express therapeutic genes. Several virus types are currently being investigated for use to deliver genes to cells to provide either transient or permanent transgene expression. These include adenoviruses (Ads), retroviruses (γ-retroviruses and lentiviruses), poxviruses, adeno-associated viruses, baculoviruses, and herpes simplex viruses. The choice of virus for routine clinical use will depend on the efficiency of transgene expression, ease of production, safety, toxicity, and stability. This chapter provides an introductory overview of the general characteristics of viral vectors commonly used in gene transfer and their advantages and disadvantages for gene therapy use.

Vasoactive agents alter the biomechanical properties of aortic heart valve leaflets in a time-dependent manner.

BACKGROUND AND AIM OF THE STUDY: Although the vasoactive agents, angiotensin II (Ang II) and 5-hydroxytryptamine (5-HT) are implicated in aortic heart valve disease, it is unclear how these compounds alter the biomechanical properties of valve leaflet tissue. The study aim was to characterize temporal changes in the elastic modulus of tissues incubated with these compounds. METHODS: Valve leaflets were excised from fresh porcine aortic heart valves. Leaflet tissue was incubated with 10(-6) M 5-HT, or 10(-6) M Ang II. The stress and elongation of the tissue in the circumferential and radial directions was measured using a stepper motor-driven micromechanical testing machine at 0.5, 6, and 24 h, followed by calculations of strain and elastic modulus of each sample. RESULTS: Tissue samples incubated with Ang II showed a significant increase in stiffness with time in the radial direction, but not in the circumferential direction. Regression analysis showed a correlation between time and elastic modulus for the tissue (R2 = 0.84). Conversely, leaflets incubated in 5-HT did not show any significant change in elastic modulus over time in the radial direction; however, significant increases in stiffness were observed after 24 h in the circumferential direction. A strong correlation between the elastic modulus in the circumferential direction and time was also noted (R2 = 0.99). CONCLUSION: The study results showed that vasoactive agents are capable of increasing the elastic modulus of aortic valve tissue in a time-dependent manner. Furthermore, the biomechanical changes induced by vasoactive agents are direction-specific, indicating different modes of action.

Cyclic strain inhibits acute pro-inflammatory gene expression in aortic valve interstitial cells.

Mechanical in vitro preconditioning of tissue engineered heart valves is viewed as an essential process for tissue development prior to in vivo implantation. However, a number of pro-inflammatory genes are mechanosensitive and their elaboration could elicit an adverse response in the host. We hypothesized that the application of normal physiological levels of strain to isolated valve interstitial cells would inhibit the expression of pro-inflammatory genes. Cells were subjected to 0, 5, 10, 15 and 20% strain. Expression of VCAM-1, MCP-1, GM-CSF and OPN was then measured using qRT-PCR. With the exception of OPN, all genes were significantly up regulated when no strain was applied. MCP-1 expression was significantly lower in the presence of strain, although strain magnitude did not affect the expression level. VCAM-1 and GM-CSF had the lowest expression levels at 15% strain, which represent normal physiological conditions. These findings were confirmed using confocal microscopy. Additionally, pSMAD 2/3 and IkappaBalpha expression were imaged to elucidate potential mechanisms of gene expression. Data showed that 15% strain increased pSMAD 2/3 expression and prevented phosphorylation of IkappaBalpha. In conclusion, cyclic strain reduces expression of pro-inflammatory genes, which may be beneficial for the in vitro pre-conditioning of tissue engineered heart valves.

Cyclic strain regulates pro-inflammatory protein expression in porcine aortic valve endothelial cells.

BACKGROUND AND AIM OF THE STUDY: The endothelium of diseased heart valves is known to express the adhesion molecules VCAM-1, ICAM-1 and E-selectin, while healthy valves lack these pro-inflammatory proteins. The study aim was to determine if mechanical forces were responsible for the pro-inflammatory reaction in aortic valve endothelial cells. METHODS: Isolated porcine aortic valve endothelial cells (PAVEC) were cultured and seeded onto BioFlexTM culture plates. The cells were exposed to equibiaxial cyclic strains of 5, 10 and 20% for 24 h in a Flexcell FX-4000T Tension Plus system at 1 Hz. Pro-inflammatory protein expression was detected through the use of monoclonal antibodies via fluorescence-assisted cell sorting (FACS) and confocal laser scanning microscopy (CLSM). RESULTS: Pro-inflammatory protein expression was evident at cyclic strains of 5 and 20%, while a 10% strain did not elicit an inflammatory response. Confocal images indicated a disrupted endothelial monolayer, evidence of significant cell death, and the presence of all adhesion molecules at 5% strain. PAVEC exposed to 10% cyclic strain failed to express any of the pro-inflammatory proteins, while the cellular monolayer appeared near-confluent and characteristically similar to cellular images captured prior to cyclic stretching. CLSM images of PAVEC cyclically stretched by 20% demonstrated a similar proinflammatory reaction to those with 5% strain, while the cellular environment also showed decreased monolayer integrity. FACS data showed a significant up-regulation of the membrane-bound VCAM-1-, ICAM-1- and E-selectin-positive cells at 5% and 20% strain, compared to 10% strain and controls. CONCLUSION: The finding that equibiaxial cyclic strain regulates the pro-inflammatory response in PAVEC suggests that alterations in the mechanical environment of heart valves may contribute to valve pathogenesis.

Influence of hydrostatic and distortional stress on chondroinduction.

Undifferentiated connective tissue that arises during embryonic development and some healing processes contains pluripotent mesenchymal stem cells. It is becoming increasingly evident that the mechanical environment is an important differentiation factor for these cells. In our laboratory, we have focused on the potential for mechanical signals to induce chondrogenic differentiation of mesenchymal stem cells. Using C3H10T1/2 cells as a model, we have investigated the influence of hydrostatic pressure, equibiaxial contraction, and centrifugal pressure on chondroinduction. Cells responded to cyclic hydrostatic compression (5 MPa at 1 Hz) and cyclic contractile strain (15% at 1 Hz) by upregulating aggrecan and collagen type II gene expression. In addition, a preliminary study of the effects of centrifugal pressure (4.1 MPa for 30 min) suggests that it may increase cell proliferation and stimulate proteoglycan and collagen type II production. We speculate that compression, whether it is distortional or hydrostatic in nature, applied to undifferentiated connective tissue triggers differentiation toward a chondrocyte-like phenotype and production of a less permeable extracellular matrix which is capable of sustaining increasingly higher hydrostatic fluid pressure for compressive load support.

Mechanobiology of the aortic heart valve.

The aortic heart valve is a complex and sophisticated structure that functions in a mechanically challenging environment. With each cardiac cycle, blood flow exerts shear stresses, bending stress and tensile and compressive forces on the valve tissue. These forces determine a plethora of biological responses, including gene expression, protein activation and cell phenotype. Consequently, mechanical forces may influence valve remodeling or pathological changes. Understanding the mechanobiology of heart valves is a vast task. Herein, some of the recent studies that have increased current knowledge of endothelial and interstitial cell interactions with physical forces are examined. Additionally, experimental co-culture models are described that are being developed to further improve the understanding of endothelial-interstitial cell interactions. Finally, the means by which organ culture systems are being utilized to study heart valve biology, thereby providing a complementary approach to in vivo experimentation, are described.

Evaluation of porcine aortic valve interstitial cell activity using different serum types in two- and three-dimensional culture.

Unlike established cell lines used in the biotechnology industry, primary cells used in tissue engineering require culture media to be supplemented with serum. The most common serum is fetal bovine serum (FBS); however, FBS is expensive, negatively affecting process economics. Less-costly alternative sera are commercially available, but their efficacy has not been documented. Therefore, bovine calf serum (BCS), bovine growth serum (BGS), and newborn calf serum (NCS) were compared with FBS. Porcine aortic valve interstitial cells (VICs) were cultured as 2-dimensional (2-D) monolayers or as 3-dimensional (3-D) collagen gels using medium supplemented with 10% FBS, BGS, BCS, or NCS. No significant difference was seen in cellular activity between VICs cultured in BCS and those cultured in FBS in 2-D cultures, whereas cells cultured in BGS and NCS had significantly lower specific growth rates coupled with markedly higher metabolic activity than cells cultured in FBS. No statistically significant differences were seen in cellular activity between any of the sera when cells were cultured in 3-D constructs. In conclusion, BCS is a suitable alternative to FBS for the 2-D and 3-D culture of VICs, which may be used to develop a tissue-engineered valve.

Bioreactor systems for the production of biopharmaceuticals from animal cells.

The demand for biopharmaceutical products is set to see a significant increase over the next few years. As a consequence, the processes used to produce these products must be able to meet market requirements. The present paper reviews the current technologies available for animal cell culture and highlights the advantages and disadvantages of each method, while also providing details of recent case studies. Processes are described for both suspension and anchorage-dependent cell lines.

Cyclic aortic pressure affects the biological properties of porcine pulmonary valve leaflets.

BACKGROUND AND AIM OF THE STUDY: Native pulmonary valve leaflets (PVL) are exposed to lower pressures compared to aortic valve leaflets. Knowledge of the biology of PVL exposed to aortic pressures is limited. Hence, the study's aim was to investigate the biological properties of PVL subjected to normal aortic pressures. METHODS: Porcine PVL were exposed to mean pulsatile pressures of 30 mmHg or 100 mmHg for 48 h in vitro. Subsequently, PVL were subjected to a mean pulsatile pressure of 30 mmHg for 48 h, followed by increased pressure (100 mmHg) for additional 48 h. Leaflets were evaluated by measuring collagen, DNA and sGAG contents in pressure-exposed and control PVL. Cusp morphology and cell phenotype were examined using hematoxylin and eosin staining (H and E) and alpha-smooth muscle actin (alpha-SMA) immunohistochemistry, respectively. RESULTS: PVL exposed to 30 mmHg showed no significant difference (p > 0.05) in collagen, DNA or sGAG contents compared to statically incubated PVL. However, PVL exposed to 100 mmHg showed a significant increase (p < 0.05) in both collagen and sGAG contents. Collagen content was also significantly increased (p < 0.05) in PVL exposed to varying pressures for 96 h compared to PVL exposed to 30 mmHg. The morphology of PVL exposed to cyclic pressures was comparable to that of both fresh and static leaflets, while alpha-SMA expression was decreased in PVL exposed to cyclic pressures when compared to fresh PVL. CONCLUSION: PVL have the ability to withstand elevated mechanical conditions by increasing the total collagen and sGAG content of the leaflets. The structural integrity of the PVL is unaltered by changes in extracellular matrix composition. However, pulsatile pressures on the PVL did not preserve the native cell phenotype.