Victor V. Nikolaychik

A new method for continual quantitation of viable cells on endothelialized polyurethanes

Many of the segmented polyurethanes currently used in cardiovascular prostheses undergo either modification of their surface structure or are lined with a confluent monolayer of endothelial cells to improve their hemocompatibility. During the establishment of an endothelial cell lining on these biopolymers it is necessary to continually monitor the number of viable cells that are covering the substrate. Yet, not all of the conventional cell enumeration techniques are suitable for assessing the growth of endothelial cells on polyurethanes. Methods, such as direct cell counting, dye uptake, or DNA or protein staining require either a transparent scaffold or lead to termination of the culturing process prior to measurement. In addition, some of the spectroscopic assays are often hampered by interaction of the dyes and/or solubilizers with the various constituents (e.g., catalyzers, antioxidants) and/or functional groups in the polyurethane formulations. In addressing these problems, we adapted a novel, highly reproducible fluorescent assay which is based on reduction by viable cells of an electrochemically sensitive compound, Alamar Blue. The bioreduced product is soluble and stable in culture media and noncytotoxic. In addition, the assay is independent of the geometry or physicochemical properties of the polymeric surfaces. In the present study we focus on the implementation of this assay to monitoring attachment and growth of various endothelial cell types on segmented polyurethanes.

GTSF-2: A NEW, VERSATILE CELL CULTURE MEDIUM FOR DIVERSE NORMAL AND TRANSFORMED MAMMALIAN CELLS

SummaryThe aim of this study was to test the versatility of a new basal cell culture medium, GTSF-2. In addition to traditional growth-factors, GTSF-2 contains a blend of three sugars (glucose, galactose, and fructose) at their physiological levels. For these studies, we isolated normal endothelial cells from human, bovine, and rat large blood vessels and microvessels. In addition, GTSF-2 was also tested as a replacement for high-glucose-containing medium for PC12 pheochromocytoma cells and for other, transformed cell lines. The cell growth characteristics were assessed with a novel cell viability and proliferation assay, which is based on the bioreduction of the fluorescent dye, Alamar Blue. After appropriate calibration, the Alamar Blue assay was found to be equivalent to established cell proliferation assays. Alamar Blue offers the advantage that cell proliferation can be measured in the same wells over an extended period of time. For some of the cell types (e.g., endothelial cells isolated from the bovine aorta, the rat adrenal medulla, or the transformed cells), proliferation in unmodified GTSF-2 was equivalent to that in the original culture media. For others cell types (e.g., human umbilical vein endothelial cells and PC12 cells), GTSF-2 proved to be a superior growth medium, when supplemented with simple additives, such as endothelial cell growth supplement (bFGF) or horse serum. Our results suggest that GTSF-2 is a versatile basal medium that will be useful for studying organ-specific differentiation in heterotypic coculture studies.

Photoremodeling of Arterial Wall Reduces Restenosis After Balloon Angioplasty in an Atherosclerotic Rabbit Model

OBJECTIVESnThis study evaluated the long-term impact of endoluminal low power red laser light (LPRLL) on restenosis in an atherosclerotic rabbit model.nnnBACKGROUNDnDespite widespread application of balloon angioplasty for treatment of coronary artery disease, restenosis limits its clinical benefits. Restenosis is a complex process and may be partly attributed to the inability of the vascular endothelium to regenerate and cover the denuded area at the site of arterial injury. We previously demonstrated that LPRLL stimulates endothelial cell proliferation in vitro and contributes to rapid endothelial regeneration after balloon injury in nonatherosclerotic rabbits.nnnMETHODSnRabbit abdominal aortas (n = 12) were treated in separate zones with balloon dilation and balloon dilation plus laser illumination. Endoluminal laser therapy was performed using a laser-balloon catheter delivering a single dose of 10 mW for 3 min from a helium-neon laser (632 nm). Angiography was performed before and after treatment and was repeated 8 weeks before harvesting the aortas.nnnRESULTSnQuantitative angiographic analysis demonstrated no differences in the minimal lumen diameter (MLD) between the two zones before treatment; an increase in the MLD in both zones after balloon angioplasty and a significant versus slight reduction of the MLD in the balloon treatment versus balloon plus laser zones at 8 weeks. Histologic examination showed a very high level of myointimal hyperplasia in the balloon treatment zones but a minimal level in the LPRLL-treated zones. Morphometric analysis revealed a statistically significant difference in the lumen area, intimal area and intima/media ratio between the balloon versus balloon plus laser treatment sites.nnnCONCLUSIONSnOur experimental data indicate that endoluminal irradiation with LPRLL prevents restenosis after balloon angioplasty in an atherosclerotic rabbit model.

A new, cryoprecipitate based coating for improved endothelial cell attachment and growth on medical grade artificial surfaces.

Monoprotein coatings of biomaterials with either natural adhesion molecules or genetically designed analogs have been used to facilitate attachment and spreading of endothelial cells. However, such treatments were found insufficient to maintain the integrity of the endothelial surface under turbulent flow conditions. In addition, when brought into contact with blood, these coatings were susceptible to plasma and cell proteinases that could readily destroy their structure and weaken cell adherence to the surface. In addressing these problems, we developed a cryoprecipitate-based coating that can firmly bind to any nonporous, prosthetic surface and interact with endothelial cells. The primary structure of the coating consisted of an autologous fibrin meshwork. It was refined by various compositions of the fibrinogen containing mixture and secured to polystyrene or polyurethane surfaces by dry-heat treatment. Further modulation of the coating was achieved by physically immobilizing various doses of heparin and insulin into the three dimensional matrix of the meshwork. Endothelial cells attached and grew much better on polyurethanes coated with this autologous protein complex than on a polystyrene tissue culture surface. With proper use of its capacity to mimic the properties of basal membrane, and absence of immunologic complications, the resulting coating may become an ideal multifunctional interface between cells and prosthetic materials.

In Vitro Testing of Endothelial Cell Monolayers Under Dynamic Conditions Inside a Beating Ventricular Prosthesis

Thromboembolic complications remain a major problem associated with the long-term clinical use of cardiac prostheses. A promising approach toward resolving this predicament is lining the blood contacting surfaces with a functional monolayer of endothelial cells (EC). In developing an endothelialized cardiac prosthesis, the authors in the past focused on establishing a confluent EC monolayer on the luminal surface of ventricular blood sacs. In this study, the authors concentrated on exposing the post confluent monolayers to the dynamic conditions inside a beating ventricle. The cells, derived from either bovine aortae or jugular veins, were grown to post confluence inside fully assembled ventricles on fibronectin or plasma cryoprecipitate coated, textured surfaces. After 11 days of culturing under static conditions, the endothelialized ventricles were connected to a mock loop that was run for 6 and 24 hr at 60 bpm and mean flow rate of 3.2 L/min. The status of the monolayer was evaluated by Alamar Blue assay before and after each run, and the extent of surface coverage was determined visually using bright field microscopic study after cell staining with KMnO4 and toluidine blue. In addition, morphometric information on cells/polyurethane surface was obtained with a scanning electron microscope. After 6 hr of pumping, cell staining revealed signs of moderate cell loss in fibronectin coated blood sacs, whereas in cryoprecipitate coated bladders the signs of denudation were marginal. In seven ventricles operated for 24 hr, Alamar Blue measurements indicated 35 +/- 16% of cell loss from monolayers established on fibronectin coating, but only 4.8 +/- 6.25% on cryoprecipitate. Thus, the current study demonstrates the feasibility of maintaining an intact endothelial surface in a beating ventricular prosthesis and indicates that the integrity of the endothelial lining is dependent upon a proper choice of surface macrostructure and protein coating.

Endothelial Cell Seeding with Rotation of a Ventricular Blood Sac

Successful establishment of a durable endothelial cell (EC) monolayer inside a ventricular blood sac requires homogeneous coverage of the entire luminal surface with attached cells. For this purpose, a new device was developed that slowly rotates a fully assembled cardiac prosthesis with three degrees of freedom. We seeded ECs derived from human adipose tissue at a density of approximately 3.5 x 10(4) cells/cm2 onto the surfaces of polyurethane-made blood sacs and ersatz bladders (consisting of T-25 tissue culture flasks). The kinetics of cell attachment, spreading, and proliferation were determined using video microscopy combined with image analysis and cell viability assays. After 60 min of seeding at 5-10 rotations/hr, the plating efficiency inside the blood sacs was 35.7 +/- 11%, with cell viability remaining approximately 90 +/- 5%. After 3 hr, when the plating efficiency reached a plateau (approximately 70%), the rotation was stopped and the ECs were allowed to spread and proliferate under static conditions. Within 48 hr, the entire luminal surface was evenly covered by a confluent EC monolayer. Our long-term studies show that with a proper feeding schedule, such an EC monolayer can be maintained intact in vitro for more than 2 weeks.

Long-term follow-up after coronary stenting and intravascular red laser therapy

A high restenosis rate remains a limiting factor for coronary angioplasty and stenting. Recently, use of intravascular red light therapy (IRLT) has been shown to be effective in different animal models and in humans in reducing the restenosis rate. Sixty-eight patients were treated with IRLT in conjunction with coronary stenting procedures. Mean age was 64 +/- 9 years. Treated lesions were type A (11), type B (42), and type C (18) with a mean lesion length of 16.5 +/- 2.4 mm. Reference vessel diameter and minimal lumen diameter (MLD) before therapy were 2.90 +/- 0.15 and 1.12 +/- 0.36 mm, respectively. After stenting and laser irradiation, MLD was 2.76 +/- 0.39 mm. No procedural complications or in-hospital adverse events occurred. All patients were followed up as depicted in the protocol. Sixty-one patients underwent angiographic restudy, which revealed restenosis in 9 patients (14.7%). Observed restenosis rate by artery size was > 3 mm (n = 21, 0%), 2.5 to 3.0 mm (n = 28, 14.2%), and <2.5 mm (n = 12, 41.6%). We conclude that IRLT is safe and feasible and reduces the expected restenosis rate in patients after coronary stenting in arteries of >2.5 mm.

Successful Dynamic Cardiomyoplasty with Pharmaceutical Support

Dynamic cardiomyoplasty is an attractive alternative to heart transplantation. We used fibrin sealant to facilitate the intraoperative bonding of skeletal muscle to the myocardial wall, focusing on prevention of ischemia-reperfusion injuries in the skeletal muscle flap, and enhancement of angiogenesis in the “repaired” heart. In a sheep model, we used autologous fibrin sealant to join the tissues, to create a provisional matrix for angiogenesis, and to act as a depot for the delivery of agents aimed at minimizing ischemia-reperfusion lesion formation. Coadministered with the fibrin sealant were the following pharmaceuticals: deferoxamine (an iron chelator), pyrrolostatin (a free radical scavenger), and aprotinin (a protease inhibitor). Five days after cardiomyoplasty, the skeletal muscle was stimulated with a progressive electrical regimen. After two months, the skeletal muscle showed none of the signs of necrosis or ischemia-reperfusion damage seen in the controls. The layer of fibrin sealant rapidly (<2 weeks) became densely vascularized with capillaries. The expedited angiogenesis provided an organic bridge between skeletal muscle and myocardium. By contrast, in controls there was poor contact between the tissues, with evidence of fiber deterioration and loss of vascular network integrity in the transposed muscle flap. Even greater angiogenic stimulation was seen when pharmaceuticals were included into the fibrin meshwork, which minimized the formation of ischemia-reperfusion lesions. Over time, these agents promoted much more extensive vascularization than did fibrin sealant alone. This therapeutic strategy, using a pharmacologically-enhanced fibrin sealant, is clearly beneficial for countering muscle flap postoperative injury, and may open promising pathways for the design of other biomechanical assist devices.

Photobiomodulation of vascular endothelial and smooth muscle cells in vitro with red laser light

Numerous reports suggest that low power red laser light (LPRLL) is capable of affecting cellular processes in the absence of significant thermal effect. The objective of the present study was to determine the effect of LPRLL on viability, growth, and attachment characteristics of rabbit and human aortic endothelial cells (EC) and smooth muscle cells (SMC) in vitro. All cell cultures were irradiated with single dose LPRLL using a He-Ne continuous wave laser with different energy densities. Assessment of effect on cell viability, growth, and attachment was performed utilizing Alamar Blue assay. Based upon our experiments, we conclude that: 1) stimulation and/or inhibition of cell growth and death can be obtained with LPRLL by varying the energy level, 2) LPRLL increases EC attachment, and 3) EC are more sensitive to photobiomodulation with LPRLL than SMC. These data may have significant importance leading to the establishment of new methods for phototherapy of atherosclerosis and restenosis.

Durability of Endothelial Cell Monolayers Inside a Beating Cardiac Prosthesis

Thromboembolic complications associated with the use of cardiac prostheses might be alleviated by lining the blood-contacting surfaces of these devices with a functional monolayer of endothelial cells. In the current study, we tested our hypothesis that precoating textured surfaces of artificial ventricles with various plasma proteins could enhance the resistance of endothelial cell monolayers to hemodynamic forces generated within an in vitro mock circulatory loop system. Bovine jugular vein endothelial cells were grown to confluence on the luminal surface of artificial ventricles constructed of textured, medical grade polyurethane (Biospan), which had been precoated with either fibronectin or plasma cryoprecipitate. Following 7 days of culturing under static conditions, the endothelialized ventricles were connected to a mock loop system, and exposed to pulsatile flow for 6 and 24h (60bpm, 3.21/min mean flow rate, 150mmHg ejection pressure). Retention of endothelial cells was evaluated by Alamar Blue assay before and after each run. Monolayer integrity and additional morphometric parameters were also assessed by direct visualization, employing various light and electron microscopic techniques. In ventricles which had been precoated with fibronectin, Alamar Blue assay indicated cellular retention to be 77% ± 4% and 72% ± 5% of static controls, after 6 and 24h, respectively. In marked contrast, cryoprecipitate-coated ventricles retained over 90% of their endothelial cell lining through 24 h of exposure to physiological hemodynamic conditions. These findings were confirmed by visual inspection. Our study demonstrates the feasibility of maintaining an intact endothelial surface in a beating ventricular prosthesis, and that the durability of the cell layer is highly dependent upon the selection of biomaterial surface topography and protein coating.