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In JoVE (1)
Other Publications (19)
- Annals of Biomedical Engineering
- Annals of Biomedical Engineering
- The Annals of Thoracic Surgery
- Annals of Biomedical Engineering
- Journal of Biomechanical Engineering
- The Journal of Heart Valve Disease
- Journal of Biomechanical Engineering
- The Journal of Heart Valve Disease
- Biotechnology and Applied Biochemistry
- Cytotechnology
- Tissue Engineering
- The Journal of Heart Valve Disease
- Biorheology
- The Journal of Heart Valve Disease
- Biomechanics and Modeling in Mechanobiology
- The Journal of Heart Valve Disease
- Methods in Molecular Biology (Clifton, N.J.)
- Biomechanics and Modeling in Mechanobiology
- International Journal of Inflammation
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Articles by James N. Warnock in JoVE
JoVE Bioengineering
Devirli Basınç Biyoreaktör Tasarım Ex vivo Eğitim
Department of Agricultural and Biological Engineering, Mississippi State University
Basınç koşulları fizyolojik ve patolojik kalp kapakçığı dokusu tabi yapabilen bir siklik basınç biyoreaktör dizayn edilmiştir. LabVIEW programı, kullanıcıların basınç büyüklüğü, genlik ve frekans kontrol etmenizi sağlar. Bu cihaz, kalp kapakçığı dokusu veya izole hücrelerin mechanobiology incelemek için kullanılabilir.
Other articles by James N. Warnock on PubMed
Effects of Constant Static Pressure on the Biological Properties of Porcine Aortic Valve Leaflets
An understanding of how mechanical forces impact cells within valve leaflets would greatly benefit the development of a tissue-engineered heart valve. In this study, the effect of constant ambient pressure on the biological properties of heart valve leaflets was evaluated using a custom-designed pressure system. Native porcine aortic valve leaflets were exposed to static pressures of 100, 140, or 170 mmHg for 48 h. Collagen synthesis, DNA synthesis, sulfated glycoaminoglycan (sGAG) synthesis, alpha-SMC actin expression, and extracellular matrix (ECM) structure were examined. Results showed that elevated pressure caused an increase in collagen synthesis. This increase was not statistically significant at 100 mmHg, but at 140 mmHg and 170 mmHg collagen synthesis increased by 37.5 and 90%, respectively. No significant difference in DNA or sGAG synthesis was observed at elevated pressures, with the exception that DNA synthesis at 100 mmHg decreased. A notable decline in alpha-SMC actin was observed over the course of the experiments although no significant difference was observed between the pressure and control groups. It was concluded that elevated pressure caused a proportional increase in collagen synthesis of porcine aortic valve leaflets, but was unable to preserve alpha-SMC actin immunoreactive cells.
Cyclic Pressure Affects the Biological Properties of Porcine Aortic Valve Leaflets in a Magnitude and Frequency Dependent Manner
An understanding of how mechanical forces impact cells within valve leaflets would greatly benefit the development of a tissue-engineered heart valve. Previous studies by this group have shown that exposure to constant static pressure leads to enhanced collagen synthesis in porcine aortic valve leaflets. In this study, the effect of cyclic pressure was evaluated using a custom-designed pressure system. Different pressure magnitudes (100, 140, and 170 mmHg) as well as pulse frequencies (0.5, 1.167, and 2 Hz) were studied. Collagen synthesis, cell proliferation, sGAG synthesis, alpha-SMC actin expression, and extracellular matrix (ECM) structure were chosen as markers for valvular biological responses. Results showed that aortic valve leaflets responded to cyclic pressure in a magnitude and frequency-dependent manner. Increases in pressure magnitude (with the frequency fixed at 1.167 Hz) resulted in significant increases in both collagen and sGAG synthesis, while DNA synthesis remained unchanged. Responses to pulse frequency (with the magnitude fixed at 100 mmHg) were more complex. Collagen and sGAG synthesis were increased by 25 and 14% respectively at 0.5 Hz; but were not affected at 1.167 and 2 Hz. In contrast, DNA synthesis increased by 72% at 2 Hz, but not at 0.5 and 1.167 Hz. Under extreme pressure conditions (170 mmHg, 2 Hz), collagen and sGAG synthesis were increased but to a lesser degree than at 170 mmHg, and 1.167 Hz. Cell proliferation was not affected. A notable decline in a-SMC actin was observed over the course of the experiments, although no significant difference was observed between the cyclic pressure and control groups. It was concluded that cyclic pressure affected biosynthetic activity of aortic valve leaflets in a magnitude and frequency dependent manner. Collagen and sGAG synthesis were positively correlated and more responsive to pressure magnitude than pulse frequency. DNA synthesis was more responsive to pulse frequency than pressure magnitude. However, when combined, pressure magnitude and pulse frequency appeared to have an attenuating effect on each other. The number of alpha-SMC actin positive cells did not vary with cyclic pressure, regardless of pulse frequency and pressure magnitude.
Structural Characterization of the Chordae Tendineae in Native Porcine Mitral Valves
This study was aimed to characterize the different mitral valve chordae tendineae to provide additional understanding of their function.
Normal Physiological Conditions Maintain the Biological Characteristics of Porcine Aortic Heart Valves: an Ex Vivo Organ Culture Study
The aortic valve functions in a complex mechanical environment which leads to force-dependent cellular and tissue responses. Characterization of these responses provides a fundamental understanding of valve pathogenesis. The aim of this work was to study the biological characteristics of native porcine aortic valves cultured in an ex vivo pulsatile organ culture system capable of maintaining physiological pressures (120/80 mmHg) and cardiac output (4.2 l/min). Collagen, sGAG and elastin contents of the valve leaflets were measured and cusp morphology, cell phenotype, cell proliferation and apoptosis were examined. Presence of endothelial cells (ECs) on the leaflet surface was also evaluated. The differences in collagen, sGAG and elastin contents were not significant (p > 0.05) between the cultured and fresh valve leaflets. The cultured valves maintained the native ECM composition of the leaflets while preserving the morphology and cell phenotype. Cell phenotype in leaflets incubated statically under atmospheric conditions decreased compared to fresh and cultured valve leaflets, indicating the importance of mechanical forces in maintaining the natural biology of the valve leaflets. ECs were retained on the surfaces of cultured leaflets with no remodeling of the leaflets. The number of apoptotic cells in the cultured leaflets was significantly (p < 0.05) less than in the statically incubated leaflets and comparable to fresh leaflets. The sterile ex vivo organ culture system thus maintained the viability and native biological characteristics of the aortic valves that were cultured under dynamic conditions for a period of 48 h.
Design of a Sterile Organ Culture System for the Ex Vivo Study of Aortic Heart Valves
The biological response of valves to mechanical forces is not well understood. The aim of this study was to design a pulsatile system to enable the ex vivo study of aortic valves when subjected to various hemodynamic conditions. A bioreactor was designed to subject porcine aortic valves to physiological and pathophysiological pressure and flow conditions, while maintaining viability and sterility. Pressure and flow rate could be independently controlled to produce clinically relevant mechanical conditions. The oxygen transfer rate was characterized and sterile operation was achieved over 96 hours. The oxygenation capabilities ensure sufficient oxygen transport to valves, allowing operation for extended periods.
Differential Immediate-early Gene Responses to Elevated Pressure in Porcine Aortic Valve Interstitial Cells
Cardiovascular risk factors are believed to play a role in the pathogenesis of aortic valve disease. In the present study the hypothesis was proposed that elevated pressure would cause a change in the expression of prototypical pro-inflammatory genes. Hence, the expression of MCP-1, osteopontin (OPN), VCAM-1, GM-CSF and PAI-1 was examined using semi-quantitative real-time RT-PCR.
Flow and Thrombosis at Orifices Simulating Mechanical Heart Valve Leakage Regions
While it is established that mechanical heart valves (MHVs) damage blood elements during leakage and forward flow, the role in thrombus formation of platelet activation by high shear flow geometries remains unclear. In this study, continuously recalcified blood was used to measure the effects of blood flow through orifices, which model MHVs, on the generation of procoagulant thrombin and the resulting formation of thrombus. The contribution of platelets to this process was also assessed.
Cyclic Aortic Pressure Affects the Biological Properties of Porcine Pulmonary Valve Leaflets
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.
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.
Cell Culture Processes for the Production of Viral Vectors for Gene Therapy Purposes
Gene therapy is a promising technology for the treatment of several acquired and inherited diseases. However, for gene therapy to be a commercial and clinical success, scalable cell culture processes must be in place to produce the required amount of viral vectors to meet market demand. Each type of vector has its own distinct characteristics and consequently its own challenges for production. This article reviews the current technology that has been developed for the efficient, large-scale manufacture of retrovirus, lentivirus, adenovirus, adeno-associated virus and herpes simplex virus vectors.
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.
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.
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.
Cyclic Strain Regulates Pro-inflammatory Protein Expression in Porcine Aortic Valve Endothelial Cells
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.
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.
Vasoactive Agents Alter the Biomechanical Properties of Aortic Heart Valve Leaflets in a Time-dependent Manner
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.
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.
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-1β 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.
