James N. Warnock

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, α-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 α-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 α-SMC actin positive cells did not vary with cyclic pressure, regardless of pulse frequency and pressure magnitude.

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.

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.

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, α–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 α–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 α-SMC actin immunoreactive cells.

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.

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.

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 IκBα expression were imaged to elucidate potential mechanisms of gene expression. Data showed that 15% strain increased pSMAD 2/3 expression and prevented phosphorylation of IκBα. 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.

Flow and thrombosis at orifices simulating mechanical heart valve leakage regions

BACKGROUND 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. METHOD OF APPROACH 200, 400, 800, and 1200 microm orifices simulated the hinge region of bileaflet MHVs, and 200, 400, and 800 microm wide slits modeled the centerline where the two leaflets meet when the MHV is closed. To assess activation of coagulation during blood recirculation, samples were withdrawn over 0-47 min and the plasmas assayed for thrombin-antithrombin-llI (TAT) levels. Model geometries were also inspected visually. RESULTS The 200 and 400 microm round orifices induced significant TAT generation and thrombosis over the study interval. In contrast, thrombin generation by the slit orifices, and by the 800 and 1200 microm round orifices, was negligible. In additional experiments with nonrecalcified or platelet-depleted blood, TAT levels were markedly reduced versus the studies with fully anticoagulated whole blood (p < 0.05). CONCLUSIONS Using the present method, a significant increase in TAT concentration was found for 200 and 400 microm orifices, but not 800 and 1200 microm orifices, indicating that these flow geometries exhibit a critical threshold for activation of coagulation and resulting formation of thrombus. Markedly lower TAT levels were produced in studies with platelet-depleted blood, documenting a key role for platelets in the thrombotic process.

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.

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.