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- עצמיים אבי האנדותל Cell-מתזמן טכנולוגיה בדיקה biocompatibility עבור התקנים לב וכלי דם במודל חיה גדולה
- במקביל, צלחת תזרים לשכת ואת מעגל זרימה רציפה להערכת ובתאים אנדותל תחת מתח זרימה למינרית שאר
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- Annals of Biomedical Engineering
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- American Journal of Physiology. Cell Physiology
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- Journal of Biomedical Materials Research. Part A
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- Journal of Biomedical Materials Research. Part A
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- Biomaterials
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- Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference
- Biomaterials
- Proceedings of the National Academy of Sciences of the United States of America
- Biomaterials
- Journal of Biomedical Materials Research. Part B, Applied Biomaterials
- Annals of Biomedical Engineering
- American Journal of Physiology. Cell Physiology
- Annals of Biomedical Engineering
- Muscle & Nerve
- Biomaterials
- American Journal of Physiology. Heart and Circulatory Physiology
- Langmuir : the ACS Journal of Surfaces and Colloids
- Molecular and Cellular Biology
- Tissue Engineering. Part A
- Tissue Engineering. Part A
- Tissue Engineering. Part A
- Annals of Biomedical Engineering
- American Journal of Physiology. Heart and Circulatory Physiology
- Tissue Engineering. Part A
- Arteriosclerosis, Thrombosis, and Vascular Biology
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- International Journal of High Throughput Screening
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- Journal of Biomechanics
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- Biomaterials
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Articles by George A. Truskey in JoVE
JoVE Bioengineering
עצמיים אבי האנדותל Cell-מתזמן טכנולוגיה בדיקה biocompatibility עבור התקנים לב וכלי דם במודל חיה גדולה
1Department of Biomedical Engineering, Duke University, 2School of Medicine, Duke University, 3Department of Surgery, Duke University Medical Center, 4School of Medicine, University of Pennsylvania
שיטה טיטניום זריעת דם biomaterials ליצור קשר עם תאים עצמיים ו biocompatibility בדיקות מתואר. שיטה זו משתמשת ובתאים אנדותל צינורות טיטניום, זורעים בתוך דקות של ניתוח ההשתלה לתוך חזירי venae cavae. טכניקה זו ניתנת להתאמה מכשירים רבים אחרים ביו מושתלת.
JoVE Bioengineering
במקביל, צלחת תזרים לשכת ואת מעגל זרימה רציפה להערכת ובתאים אנדותל תחת מתח זרימה למינרית שאר
1Department of Surgery, Duke University Medical Center, 2Department of Biomedical Engineering, Duke University, 3School of Medicine, University of Pennsylvania, 4Department of Medicine, Division of Cardiology, Duke University Medical Center
אנחנו המתאר שיטה תאים חסיד נתונים ללחץ הזרימה למינרית גזירה במעגל תזרים סטרילי מתמשך. הדבקה של התאים, מורפולוגיה ניתן ללמוד דרך תא שקוף, דגימות שהתקבלו במעגל לניתוח המטבוליט ותאי שנקטפו לאחר חשיפה גזירה לניסויים עתידיים או תרבות.
Other articles by George A. Truskey on PubMed
Effect of Fluid Shear Stress on the Permeability of the Arterial Endothelium
The localization of atherosclerotic lesions is due, in part, to regional variations in the permeability of arterial endothelium to macromolecules. In turn, endothelial permeability may be influenced by fluid shear stresses. The spatial variation in endothelial permeability is reviewed and evidence for shear stress dependence upon permeability is presented. These results are examined in light of various signaling mechanisms that increase permeability by increasing the transport of water and macromolecules through the junctions separating endothelial cells. Signaling pathways cause a change in the dense peripheral band of actin and actin stress fibers or alter the phosphorylation of junction proteins which affects their ability to localize in junctions. Future directions to clarify the effect of shear stress on permeability are considered.
Factors Influencing the Nonuniform Localization of Monocytes in the Arterial Wall
Adhesion of monocytes to arterial endothelium may contribute to the asymmetric distribution of atherosclerotic lesions. Possible mechanisms for adhesion in the relatively high shear stress environment found in arteries include greater monocyte deformation and/or more frequent penetration of microvilli through steric and charge barriers. In vivo, secondary flows generate forces acting normal to the endothelial cell surface. These forces may cause compression of the microvilli or enable cells to overcome steric or electrostatic barriers, increasing adhesion. To investigate this, we examined monocyte adhesion to activated endothelium in recirculating flow. Adhesion was characterized by short arrests in a narrow region on either side of the reattachment line. The median arrest time was longer than that observed at comparable shear stresses in a linear shear flow. The lifetimes of adhesion were analyzed using a model for multiple bond formation. For cells adhering near the reattachment line, the bond number per cell was greater than the value found for similar shear stresses under shear flow. Thus, multiple bond formation arising from greater normal forces in recirculating flow permits monocytes to adhere at higher shear stresses.
Apparent Elastic Modulus and Hysteresis of Skeletal Muscle Cells Throughout Differentiation
The effect of differentiation on the transverse mechanical properties of mammalian myocytes was determined by using atomic force microscopy. The apparent elastic modulus increased from 11.5 +/- 1.3 kPa for undifferentiated myoblasts to 45.3 +/- 4.0 kPa after 8 days of differentiation (P < 0.05). The relative contribution of viscosity, as determined from the normalized hysteresis area, ranged from 0.13 +/- 0.02 to 0.21 +/- 0.03 and did not change throughout differentiation. Myosin expression correlated with the apparent elastic modulus, but neither myosin nor beta-tubulin were associated with hysteresis. Microtubules did not affect mechanical properties because treatment with colchicine did not alter the apparent elastic modulus or hysteresis. Treatment with cytochalasin D or 2,3-butanedione 2-monoxime led to a significant reduction in the apparent elastic modulus but no change in hysteresis. In summary, skeletal muscle cells exhibited viscoelastic behavior that changed during differentiation, yielding an increase in the transverse elastic modulus. Major contributors to changes in the transverse elastic modulus during differentiation were actin and myosin.
Effect of Streptavidin Affinity Mutants on the Integrin-independent Adhesion of Biotinylated Endothelial Cells
We have previously shown that the high-affinity streptavidin (SA)-biotin interaction enhanced the initial integrin-mediated adhesion of biotinylated endothelial cells to SA-coated surface by serving as an extrinsic bond to stabilize and enhance the intrinsic fibronectin-integrin binding between the cell and surface. However, the SA-biotin interaction produced considerable detachment by cohesive failure of the membrane. In this study, we examined the hypothesis that reducing the SA-biotin bond affinity could reduce cohesive failure without reducing overall cell detachment. Two mutants of SA, W120F and W120A in which the tryptophan residue at position 120 of the SA molecule was substituted by phenylalanine and alanine, respectively, were characterized and tested in cell adhesion experiments. The binding affinity (K(A)) of SA to adsorbed biotin-labeled bovine serum albumin (b-BSA) ranged from 5.2+/-0.1 x 10(10)M(-1) for wild-type to 3.3+/-0.2 x 10(9)M(-1) for W120F and 4.1+/-1.0 x 10(6)M(-1) for W120A. One hour after cell attachment, the critical shear stress was 26.8+/-2.9 dyn/cm(2) for WT, 26.6+/-3.0 dyn/cm(2) for W120F, and 15.4+/-3.0 dyn/cm(2) for W120A. The focal contact areas of adherent cells were greater for the WT and W120F than the lower affinity mutant, W120A. When shear flow was applied to detach adherent cells, adhesive failure (ligand bond breakage) was favored over cohesive failure (membrane rupture), as the SA binding affinity decreased. Thus, cell adhesion augmented by SA-biotin linkages is dependent on the affinity constants of the SA-biotin bonds, but the reduction in cohesive failure was offset by a reduced strength of adhesion.
Synergistic Effect of High-affinity Binding and Flow Preconditioning on Endothelial Cell Adhesion
The current study examined whether the combined introduction of high-affinity avidin-biotin bonds and fibronectin-integrin bonds (i.e., dual ligand treatment) would further augment the adhesion of flow-preconditioned endothelial cells to model substrates via contributions to the actin cytoskeleton and the formation of focal contacts. Human umbilical vein endothelial cells (HUVEC) were grown under static conditions or exposed to a flow-preconditioning regimen for 24 h. Cell retention was determined by exposure to 75 dynes/cm(2). The combination of flow preconditioning and the dual ligand treatment yielded higher cell retention under flow compared to the cells adherent via fibronectin-integrin bonds only. This increase in adhesion strength correlated with a greater focal contact area. Elongation of the HUVEC occurred after exposure to flow preconditioning; however, orientation of dual ligand adherent cells was restricted due to the presence of the high-affinity ligand. Flow-preconditioned cells showed increased stress fiber formation compared to nonconditioned cells although the stress fibers per cell for flow-preconditioned cells were the same on both the ligand systems employed. The results indicate that enhanced adhesion strength is due to a combination of increased focal contact area, stress fiber formation, and cell alignment.
Relation Between Near-wall Residence Times of Monocytes and Early Lesion Growth in the Rabbit Aorto-celiac Junction
Transient particle-hemodynamic simulations were conducted in a model of the rabbit aorto-celiac junction to investigate mechanisms responsible for localized monocyte attachment and subsequent lesion formation. We hypothesized that the probability for monocyte deposition is related to discrete near-wall particle stasis and/or elevated concentrations, as encapsulated by a new near-wall residence time (NWRT) parameter. A low wall shear stress (WSS) condition accounted for factors such as endothelial cell expression of adhesive molecules as well as a reduced probability of monocyte rolling and detachment. To accurately simulate particle transport, terms for the near-wall drag modification and lift were included. Low WSS and high oscillatory shear index parameters proved ineffective compared to localized in vivo results of monocyte accumulation and lesion initialization. The NWRT parameter, with a limiting WSS condition, identified the lateral flow divider as most susceptible to monocyte deposition, as observed in vivo. A representative quantitative correlation between monocyte deposition and NWRT occurrence was established (r2 = 0.77 and p<10(-4)) on a highly focal basis for an averaged data set. Results indicate that cell transport and conditions for hemodyamically induced surface reactivity are necessary components in formulating an effective model for monocyte adhesion in complex three-dimensional vessel configurations.
High-affinity Augmentation of Endothelial Cell Attachment: Long-term Effects on Focal Contact and Actin Filament Formation
Coadsorption of high-affinity avidin with lower affinity cell adhesion protein fibronectin has been shown to significantly augment short-term (1 h) adhesion and spreading of endothelial cells; however, the longer term persistence of avidin binding and its effect on endothelial cell adhesion have not been addressed. In this study, the presence of avidin-biotin bonds 24 h after cell adhesion to the dual ligand surfaces was verified by laser confocal microscopy of a fluorescent avidin analog, streptavidin. Total internal reflection microscopy showed that the focal contact area, focal contact density, and cell spreading all increased significantly at 24 h compared to fibronectin-treated control surfaces. Focal contact area was identical when measured with cells that were labeled with either the fluorescent streptavidin or a carbocyanine dye incorporated in the cell membrane. Confocal images of stress fibers formed in cells adherent to dual ligand surfaces after 24 h were thicker and more numerous compared to cells adherent to fibronectin controls. The results indicate that 24 h after initial attachment avidin-biotin is localized to focal contacts on the basal surface and affects cell spreading, actin filament organization, and focal contact density.
Stretch-induced Nitric Oxide Modulates Mechanical Properties of Skeletal Muscle Cells
In this study, we examined the hypothesis that stretch-induced (nitric oxide) NO modulates the mechanical properties of skeletal muscles by increasing accumulation of protein levels of talin and vinculin and by inhibiting calpain-induced proteolysis, thereby stabilizing the focal contacts and the cytoskeleton. Differentiating C(2)C(12) myotubes were subjected to a single 10% step stretch for 0-4 days. The apparent elastic modulus of the cells, E(app), was subsequently determined by atomic force microscopy. Static stretch led to significant increases (P < 0.01) in E(app) beginning at 2 days. These increases were correlated with increases in NO activity and neuronal NO synthase (nNOS) protein expression. Expression of talin was upregulated throughout, whereas expression of vinculin was significantly increased only on days 3 and 4. Addition of the NO donor l-arginine onto stretched cells further enhanced E(app), NOS activity, and nNOS expression, whereas the presence of the NO inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) reversed the effects of mechanical stimulation and of l-arginine. Overall, viscous dissipation, as determined by the value of hysteresis, was not significantly altered. For assessment of the role of vinculin and talin stability, cells treated with l-NAME showed a significant decrease in E(app), whereas addition of a calpain inhibitor abolished the effect. Thus our results show that NO inhibition of calpain-initiated cleavage of cytoskeleton proteins was correlated with the changes in E(app). Together, our data suggest that NO modulates the mechanical behavior of skeletal muscle cells through the combined action of increased talin and vinculin levels and a decrease in calpain-mediated talin proteolysis.
Effect of Streptavidin-biotin on Endothelial Vasoregulation and Leukocyte Adhesion
The current study examines whether the adhesion promoting arginine-glycine-aspartate-streptavidin mutant (RGD-SA) also affects two important endothelial cell (EC) functions in vitro: vasoregulation and leukocyte adhesion. EC adherent to surfaces via fibronectin (Fn) or Fn plus RGD-SA were subjected to laminar shear flow and media samples were collected over a period of 4h to measure the concentration of nitric oxide (NO), prostacyclin (PGI(2)), and endothelin-1 (ET-1). Western blot analysis was used to quantify the levels of endothelial-derived nitric oxide synthase (eNOS) and cyclooxygenase II (COX II). In a separate set of experiments, fluorescent polymorphonuclear leukocyte (PMN) adhesion to EC was quantified for EC with and without exposure to flow preconditioning. When cell adhesion was supplemented with the SA-biotin system, flow-induced production of NO and PGI(2) increased significantly relative to cells adherent on Fn alone. Previous exposure of EC to shear flow also significantly decreased PMN attachment to SA-biotin supplemented EC, but only after 2h of exposure to shear flow. The observed decrease in PMN-EC adhesion was negated by NG-nitro-L-arginine methyl ester (L-NAME), an antagonist of NO synthesis, but not by indomethacin, an inhibitor to PGI(2) synthesis, indicating the induced effect of PMN-EC interaction is primarily NO-dependent. Results from this study suggest that the use of SA-biotin to supplement EC adhesion encourages vasodilation and PMN adhesion in vitro under physiological shear-stress conditions. We postulate that the presence of SA-biotin more efficiently transmits the shear-stress signal and amplifies the downstream events including the NO and PGI(2) release and leukocyte-EC inhibition. These results may have ramifications for reducing thrombus-induced vascular graft failure.
Effect of Streptavidin RGD Mutant on the Adhesion of Endothelial Cells
Adhesion of endothelial cells (EC) to surfaces can be enhanced by supplementing the integrin-mediated adhesion with high-affinity streptavidin (SA) that links a biotinylated EC to a biotinylated surface. Biotin pullout from the EC membrane limits the effectiveness of this treatment, leading to a predominance of EC detachment by cohesive failure. In this study we investigated whether a RGD-SA mutant that links SA to EC integrin receptors, and eliminates EC biotinylation, improves EC adhesion. Suspended EC were incubated with the RGD-SA mutant prior to cell seeding, primarily via attachment to the RGD binding site on alpha(v)beta(3) integrin. RGD-SA-incubated EC were subsequently seeded onto a surface preadsorbed with a mixture of fibronectin (Fn) and biotinylated bovine serum albumin (b-BSA). Results showed EC adhesion supplemented with the RGD-SA-biotin system significantly increased cell retention under flow, critical shear stresses for detachment, focal contact area, and force per bond relative to SA used with biotinylated EC. These increases were accompanied by significant reductions in membrane fragments left behind following EC detachment, which suggested cohesive failure via cell membrane rupture was significantly reduced, and enhanced phosphorylation of focal adhesion kinase, which suggested activation and clustering of integrin receptors. Together, these results show that the integrin-independent augmentation of EC adhesion using SA-biotin can be further improved through use of an RGD-SA mutant.
A 3D Numerical Study of the Effect of Channel Height on Leukocyte Deformation and Adhesion in Parallel-plate Flow Chambers
The effect of channel height on leukocyte adhesion to a lower plate in a parallel-plate flow chamber is studied by direct numerical simulations in three dimensions. The numerical model takes into account deformability and viscoelasticity of the leukocyte, membrane ruffles (microvilli), and the presence of mechanically different regions inside the cell (nucleus and cytoplasm). Leukocyte adhesion is assumed to be mediated by interactions of adhesion molecules on the tips of microvilli with their counterparts on the lower plate. Results of this study indicate that an adherent leukocyte experiences much less drag than a rigid sphere due to its deformation and transient stress growth. While overall leukocyte deformation is modest at shear stresses encountered in the microcirculation, deformation in the contact region is significant. At fixed wall shear stress, the contact area of the cell membrane with the substrate increases with increasing the ratio of cell diameter to channel height, leading to greater adhesion. This suggests that in vitro flow chamber studies typically underestimate leukocyte adhesion that occurs in the microcirculation.
Linoleic Acid Increases Monocyte Deformation and Adhesion to Endothelium
Fatty acids have been implicated in having both anti- or pro-inflammatory actions, which may contribute to the progression and severity of atherosclerosis. Linoleic acid has been shown by others to decrease CD18 expression and leukocyte adhesion under static conditions. We investigated the effect of steric acid (18:0), oleic acid (18:1), and linoleic acid (18:2) on the cortical tension (a measure of cell membrane deformability) and adhesion characteristics of the monocytic cell line Mono Mac 6 (MM6) cells to TNF-alpha activated HUVEC under fluid flow. Linoleic acid concentrations up to 23 microM decreased cortical tension and increased adhesion frequencies. Increased adhesion was not due to altered cell morphology or adhesion kinetics and occurred despite decreases in receptor expression (CD18 and CD11a). At higher levels of linoleic acid (> or = 46 microM), cell dissociation constants significantly increased. Results show that decreasing cortical tension increased the probability that contact between MM6 cells and endothelium would produce an adhesive interaction, possibly due to increased deformation of the microvilli and the cell membrane cortex. However, more deformable cells rolled more erratically at low shear rates. The different behavior during initial contact and rolling suggest that adhesion is influenced by two force-dependent mechanisms, deformation of microvilli and a steric barrier. Incubation of MM6 with 23 microM steric or oleic acid did not significantly affect cortical tension. However, cells incubated with steric acid greatly increased their adherence to HUVEC and cells incubated with oleic acid showed no significant effect, indicating factors other than deformability may dominate.
Synergistic Effect of Shear Stress and Streptavidin-biotin on the Expression of Endothelial Vasodilator and Cytoskeleton Genes
Dual ligand treatment of streptavidin(SA)-biotin and fibronectin (Fn) enhances the adhesion of endothelial cells (EC) onto synthetic surfaces and promotes the quiescent phenotype of adherent EC. The current study investigates the effect of the dual ligand on the expression of endothelial genes in static culture and under shear stress (4 h at 10 dynes/cm2). Expression of 23 genes in the classes of signaling, cytoskeleton/ECM, vasoregulation, and shear-responsive were examined. Eight genes (argininosuccinate synthetase, K+ channel, TGFbeta, Mn-SOD, alpha-tubulin, t-PA, COX2, and eNOS) were significantly upregulated by shear stress. Two genes (caveolin-1 and ET-1) were downregulated by shear stress. Three genes (RhoA, elastin, alpha-actinin) were upregulated by the dual ligand treatment in static culture, and four genes (FAK, elastin, COX2, and eNOS) were upregulated when the dual ligand and shear stress were applied simultaneously. Northern blot analyses on FAK, RhoA, elastin, and alpha-actinin revealed similar results. The results suggest (1) the use of SA-biotin to supplement EC adhesion enhances the integrity of the EC cytoskeleton by upregulating the expression of cytoskeleton/ECM genes, and (2) a likely relationship between the expression of cytoskeleton/ECM genes and the downstream events, such as the shear-induced expression of eNOS and COX2 genes. Analyses presented in this study provide insights into the mechanism by which SA-biotin-supplemented EC mediate gene expression.
Real-time Theoretical Compartmental Model of Blood-brain Barrier Drug Delivery
This study developed upon the Mahar Doan and Boje simulations to create a novel pharmacokinetic/pharmacodynamic real-time compartmental model in Simulink. The model allowed for varying combinations of oral and intravenous drug dosing under steady and nonsteady-state endogenous transport inhibition methods. A joint pharmacodynamic model simulated an extracellular receptor binding process and an intracellular enzymatic process as drug effect sites. Initial simulations were compared with the Mahar Doan and Boje data. Further simulation demonstrated the freedom and broadened understanding of drug kinetics and dynamics the implemented real-time model provided.
A System for the Direct Co-culture of Endothelium on Smooth Muscle Cells
The development of a functional, adherent endothelium is one of the major factors limiting the successful development of tissue engineered vascular grafts (TEVGs). The adhesion and function of endothelial cells (ECs) on smooth muscle cells (SMCs) are poorly understood. The goal of this research was to optimize conditions for the direct culture of endothelium on SMCs, and to develop an initial assessment of co-culture on EC function. The co-culture consisted of a culture substrate, a basal adhesion protein, a layer of porcine SMCs, a medial adhesion protein, and a layer of porcine ECs. Conditions that led to successful co-culture were: a polystyrene culture substrate, a quiescent state for SMCs, subconfluent density for SMC seeding and confluent density for EC seeding, and fibronectin (FN) for the basal adhesion protein. EC adhesion was not enhanced by addition of FN, collagen I, collagen IV or laminin (LN) to the medial layer. 3-D image reconstruction by confocal microscopy indicated that SMCs did not migrate over ECs and the cells were present in two distinct layers. Co-cultures could be consistently maintained for as long as 10 days. After exposure to 5 dyne/cm(2) for 7.5 h, ECs remained adherent to SMCs. PECAM staining indicated junction formation between ECs, but at a lower level than that observed with EC monocultures. Co-culturing ECs with SMCs did not change the growth rate of ECs, but EC DiI-Ac-LDL uptake was increased. Thus, a confluent and adherent layer of endothelium can be directly cultured on quiescent SMCs.
Effects of Titanium Particle Size on Osteoblast Functions in Vitro and in Vivo
The formation of titanium (Ti)-wear particles during the lifetime of an implant is believed to be a major component of loosening due to debris-induced changes in bone cell function. Radiographic evidence indicates a loss of fixation at the implant-bone interface, and we believe that the accumulation of Ti particles may act on the bone-remodeling process and impact both long- and short-term implant-fixation strengths. To determine the effects of various sizes of the Ti particles on osteoblast function in vivo, we measured the loss of integration strength around Ti-pin implants inserted into a rat tibia in conjunction with Ti particles from one of four size-groups. Implant integration is mediated primarily by osteoblast adhesion/focal contact pattern, viability, proliferation and differentiation, and osteoclast recruitment at the implant site in vivo. This study demonstrates the significant attenuation of osteoblast function concurrent with increased expression of receptor activator of nuclear factor kappaB ligand (RANKL), a dominant signal for osteoclast recruitment, which is regulated differentially, depending on the size of the Ti particle. Zymography studies have also demonstrated increased activities of matrix metalloproteinases (MMP) 2 and 9 in cells exposed to larger Ti particles. In summary, all particles have adverse effects on osteoblast function, resulting in decreased bone formation and integration, but different mechanisms are elicited by particles of different sizes.
Mylar and Teflon-AF As Cell Culture Substrates for Studying Endothelial Cell Adhesion
The textured and opaque nature of Dacron and ePTFE has prevented the use of these fabrics in conventional cell culture techniques normally employed to optimize cell attachment and retention. This lack of optimization has led, in part, to the poor performance of endothelialization strategies for improving vascular graft patency. Here we show that thin, transparent films of Mylar and Teflon-AF are viable in vitro cell culture mimics of Dacron and ePTFE vascular graft materials, particularly for the study of protein mediated endothelial cell (EC) attachment, spreading and adhesion. Glass substrates were used as controls. X-ray photoelectron spectroscopy (XPS) and contact angle analysis showed that Mylar and Teflon-AF have surface chemistries that closely match Dacron and ePTFE. (125)I radiolabeling was used to quantify fibronectin (FN) adsorption, and FN and biotinylated-BSA "dual ligand" co-adsorption onto glass, Mylar and Teflon-AF substrates. Native human umbilical vein endothelial cells (HUVEC) and streptavidin-incubated biotinylated-HUVEC (SA-b-HUVEC) spreading was measured using phase contrast microscopy. Cell retention and adhesion was determined using phase contrast microscopy under laminar flow. All surfaces lacking protein pre-treatment, regardless of surface type, showed the lowest degree of cell spreading and retention. Dual ligand treated Mylar films showed significantly greater SA-b-HUVEC spreading up to 2 h, but were similar to HUVEC on FN treated Mylar at longer times; whereas SA-b-HUVEC spreading on dual ligand treated Teflon-AF was never significantly different from HUVEC on FN treated Teflon-AF at any time point. SA-b-HUVEC retention was significantly greater on dual ligand treated Mylar compared to HUVEC on FN treated Mylar over the entire range of shear stresses tested (3.54-28.3 dynes/cm(2)); whereas SA-b-HUVEC retention to dual ligand and HUVEC retention to FN treated Teflon-AF gave similar results at each shear stress, with only the mid-range of stresses showing significant difference in cell retention to Teflon-AF.
In Vivo Performance of Dual Ligand Augmented Endothelialized Expanded Polytetrafluoroethylene Vascular Grafts
In this study, we examined combinations of three approaches to improve the adhesion of endothelial cells (EC) onto expanded polytetrafluoroethylene (ePTFE) vascular grafts placed at the femoral artery of rats: (1) high-affinity receptor-ligand binding of RGD-streptavidin (SA) and biotin to supplement integrin-mediated EC adhesion; (2) cell sodding to pressurize the seeded EC into the interstices of the ePTFE grafts; and (3) longer postseeding attachment time from 1 to 24 h prior to implantation. An in vitro system, which accounts for cell loss due to both graft handling and shear stress, was designed to optimize conditions for in vivo experiments. Results suggest that longer in vitro attachment time enabled the adherent EC to endure mechanical stresses by forming strong adhesions to the underlying extracellular matrix substrates; cell sodding helped to retain the adherent EC by physically docking the cells against the graft interstices; and the SA-biotin interaction enhanced the early attachment of EC but did not lead to better cell retention or reduced surface coverage of blood clot in the current study. Mechanical manipulation of cells during implantation is a limiting factor in maintaining a confluent EC layer on synthetic vascular grafts.
Adhesion and Function of Human Endothelial Cells Co-cultured on Smooth Muscle Cells
To evaluate interactions between human endothelial cells (ECs) and smooth muscle cells (SMCs) for the development of tissue-engineered vessels, we examined the adhesion and key cell properties of human ECs grown on quiescent human aortic SMCs. ECs attached to SMCs spread more slowly than ECs attached to fibronectin surfaces, and ECs aligned along the direction of the SMCs. ECs attached firmly and less than 5% of the cells were removed by shear stresses as high as 300 dyn cm(-2). Unlike porcine SMCs and co-cultures, human SMCs or co-cultures do not contract under flow, and the human ECs and SMCs in co-culture align toward the direction of flow. A confluent endothelium could be maintained in co-culture for over 30 days, and some of the ECs reoriented perpendicular to the SMCs after 9 days in static culture. Surface tissue factor levels in ECs and SMCs were less in co-culture than in monoculture. Co-culture induced an increase in calponin expression in SMCs. These findings show that human co-cultures can be maintained for long culture periods, where the endothelium remains confluent and responds to long-term exposure to flow, and EC-SMC interactions lead to an increase in SMC differentiation and an EC surface that is less thrombotic.
Effect of Cyclic Stretch on Beta1D-integrin Expression and Activation of FAK and RhoA
Integrins play a pivotal role in proliferation, differentiation, and survival in skeletal and cardiac myocytes. The beta(1D)-isoform of the beta(1)-integrin is specifically expressed in striated skeletal muscle. However, little is known about the role and the mechanisms by which the splice variant beta(1D)-integrin regulates myogenesis and mechanotransduction. We observed that cyclic mechanical stretch increases beta(1D)-integrin protein levels and activates the downstream cytoskeletal signaling proteins focal adhesion kinase (FAK) and RhoA. Elimination of native beta(1D)-integrin expression by RNA interference in immature developing myoblasts abolished stretch-induced increases in FAK phosphorylation and further downregulated RhoA activity. Blocking of beta(1D)-integrin expression prevented myocellular fusion to form multinucleated mature myotubes. Restoration of human beta(1D)-integrin expression in beta(1D)-integrin-deficient cells partially restored myotube formation. The onset of myofusion also requires the generation of nitric oxide (NO). The release of NO affects cytoskeletal proteins by mediating RhoA activity and protein degradation. Our previous study demonstrated that stretch-induced NO positively modulates mechanical properties of differentiating skeletal myocytes. We found a significant decrease in NO production and apparent elastic modulus in beta(1D)-integrin-deficient cells, suggesting signaling interactions between beta(1D)-integrin and neuronal NO synthase to mediate mechanotransduction and myogenesis in skeletal myocytes. These results suggest that, in addition to regulating differentiation, the beta(1D)-integrin isoform plays a critical role in the response of skeletal myoblasts to cyclic stretch by activating the downstream components of FAK and RhoA activity and affecting NO release.
Flow and High Affinity Binding Affect the Elastic Modulus of the Nucleus, Cell Body and the Stress Fibers of Endothelial Cells
Cell mechanical properties are important in the adhesion of endothelial cells to synthetic vascular grafts exposed to shear flow. We hypothesized that the local apparent elastic modulus of the nucleus and the cell body would increase to a greater extent for cells adherent via the dual ligand (integrin-fibronectin/avidin-biotin) and exposed to flow, than for cells treated with either ligand alone. High affinity avidin-biotin bonds and in vitro flow exposure were used to improve adhesion to grafts thereby altering the mechanical properties of endothelial cells. Introduction of the dual ligand chemistry at the cell-substrate interface increased the apparent elastic modulus of the cells as compared to cells adherent with the fibronectin-integrin bonds only. Cells cultured on the dual ligand surface exhibited higher elastic moduli of the nucleus and cell body relative to cells cultured on fibronectin alone. Exposure of cells to flow increased the apparent elastic modulus of the cell body, nucleus, and stress fibers of cells adherent to the fibronectin surface. A similar effect was seen for cells adherent to the dual ligand surface, although there was little effect on the elastic modulus of the nucleus. While the dual ligand surface produces an increase in adhesion strength, focal contact area and elastic modulus, the change in elastic modulus after exposure to flow is due only to an increase in stress fibers and not an increase in contact area.
Morphology and Ultrastructure of Differentiating Three-dimensional Mammalian Skeletal Muscle in a Collagen Gel
Because previous studies of three-dimensional skeletal muscle cultures have shown limited differentiation, the goal of this study was to establish conditions that would produce mature sarcomeres in a mammalian-derived skeletal muscle construct. We evaluated the differentiation of bioartificial muscles generated from C(2)C(12) myoblasts in a collagen gel cultured under steady, passive tension for up to 36 days. Staining for alpha-actinin, myosin, and F-actin indicated the presence of striated fibers as early as 6 days post-differentiation. Electron microscopy at 16 days post-differentiation revealed multinucleated myotubes with ordered, striated myofibers. At 33 days, the cultures contained collagen fibers and showed localization of paxillin at the fiber termini, suggesting that myotendinous junctions were forming. The present study demonstrates mature muscle synthesis in a three-dimensional system using a pure mammalian myoblast cell line. Our results suggest that this culture model can be used to evaluate the effects of various mechanical and biochemical cues on muscle development under normal and pathological conditions.
The Use of Mild Trypsinization Conditions in the Detachment of Endothelial Cells to Promote Subsequent Endothelialization on Synthetic Surfaces
A necessary condition for endothelialization of small diameter grafts is rapid and firm adhesion of endothelial cells upon exposure to flow. To retain integrins on the cell surface, we assessed the effects of trypsin concentration, the duration of trypsin incubation, and trypsin neutralization methods on endothelial cell adhesion. Human umbilical vein endothelial cells which were detached using 0.025% trypsin for 5 min and seeded onto glass pretreated with fibronectin had close to 100% cell retention when shear stresses as high as 200 dyn/cm2 were applied for 2 min. An equivalent level of cell retention was observed on fibronectin coated Teflon-AF for shear stresses up to 60 dyn/cm2 applied for 4h. Using 0.025% trypsin, initial cell spreading and cell surface alpha5beta1 integrins were increased relative to cells treated with 0.5% trypsin. After 1h of attachment, focal adhesions formed when low trypsin concentrations were used but were less evident with high trypsin concentrations. These results showed that low trypsin concentrations produced faster spreading, a higher number of intact integrins, and rapid focal adhesion formation.
Smooth Muscle Cell Rigidity and Extracellular Matrix Organization Influence Endothelial Cell Spreading and Adhesion Formation in Coculture
Efforts to develop functional tissue-engineered blood vessels have focused on improving the strength and mechanical properties of the vessel wall, while the functional status of the endothelium within these vessels has received less attention. Endothelial cell (EC) function is influenced by interactions between its basal surface and the underlying extracellular matrix. In this study, we utilized a coculture model of a tissue-engineered blood vessel to evaluate EC attachment, spreading, and adhesion formation to the extracellular matrix on the surface of quiescent smooth muscle cells (SMCs). ECs attached to and spread on SMCs primarily through the alpha(5)beta(1)-integrin complex, whereas ECs used either alpha(5)beta(1)- or alpha(v)beta(3)-integrin to spread on fibronectin (FN) adsorbed to plastic. ECs in coculture lacked focal adhesions, but EC alpha(5)beta(1)-integrin bound to fibrillar FN on the SMC surface, promoting rapid fibrillar adhesion formation. As assessed by both Western blot analysis and quantitative real-time RT-PCR, coculture suppressed the expression of focal adhesion proteins and mRNA, whereas tensin protein and mRNA expression were elevated. When attached to polyacrylamide gels with similar elastic moduli as SMCs, focal adhesion formation and the rate of cell spreading increased relative to ECs in coculture. Thus, the elastic properties are only one factor contributing to EC spreading and focal adhesion formation in coculture. The results suggest that the softness of the SMCs and the fibrillar organization of FN inhibit focal adhesions and reduce cell spreading while promoting fibrillar adhesion formation. These changes in the type of adhesions may alter EC signaling pathways in tissue-engineered blood vessels.
Streptavidin Binding and Endothelial Cell Adhesion to Biotinylated Fibronectin
A dual ligand (DL) system that combines high affinity streptavidin-biotin binding with lower affinity fibronectin-integrin ligand binding was developed to augment endothelial cell adhesion to polymers. In this study, we examined the utility of biotinylated fibronectin (bFN) as an enhancement to the previously developed DL approach. The goal was to make the system more amenable to clinical studies by eliminating xenogenic bovine serum albumin (bBSA). Fibronectin (FN) biotinylation was achieved with Sulfo-NHS-LC-Biotin. The affinity of conjugated biotin for wild-type streptavidin (WT-SA) and a mutant strain streptavidin (RGD-SA) was measured using surface plasmon resonance (SPR) spectroscopy. Enzyme-Linked ImmunoSorbent Assay (ELISA) absorbance values confirmed the accessibility of the cell binding domain on mildly biotinylated bFN when compared to unmodified native protein. SPR binding analysis confirmed similar binding behavior to bFN with WT-SA and RGD-SA. Kinetic analysis, however, showed no increase in affinity due to increased biotins per FN, an indication of the absence of positive cooperativity in the system. We verified the essential utility of bFN in affinity binding by SPR and confirmed the potential for integrin-FN linkages by ELISA. Finally, Vinculin immunostaining was used to determine focal adhesion formation using bFN in the DL system. Significantly greater focal adhesion density was achieved with the bFN in the DL system than with FN alone.
Mice Lacking Homer 1 Exhibit a Skeletal Myopathy Characterized by Abnormal Transient Receptor Potential Channel Activity
Transient receptor potential (TRP) channels are nonselective cation channels, several of which are expressed in striated muscle. Because the scaffolding protein Homer 1 has been implicated in TRP channel regulation, we hypothesized that Homer proteins play a significant role in skeletal muscle function. Mice lacking Homer 1 exhibited a myopathy characterized by decreased muscle fiber cross-sectional area and decreased skeletal muscle force generation. Homer 1 knockout myotubes displayed increased basal current density and spontaneous cation influx. This spontaneous cation influx in Homer 1 knockout myotubes was blocked by reexpression of Homer 1b, but not Homer 1a, and by gene silencing of TRPC1. Moreover, diminished Homer 1 expression in mouse models of Duchenne's muscular dystrophy suggests that loss of Homer 1 scaffolding of TRP channels may contribute to the increased stretch-activated channel activity observed in mdx myofibers. These findings provide direct evidence that Homer 1 functions as an important scaffold for TRP channels and regulates mechanotransduction in skeletal muscle.
Comparison of Endothelial Cell Phenotypic Markers of Late-outgrowth Endothelial Progenitor Cells Isolated from Patients with Coronary Artery Disease and Healthy Volunteers
The lack of easily isolated autologous endothelial cell (EC) sources is one of the major challenges with vascular tissue engineering interventions. This article examines the isolation and expansion of late-outgrowth endothelial progenitor cells (EPCs) from 50-mL samples of peripheral blood drawn from patients with significant coronary artery disease (CAD) and healthy young adult volunteers. In cases in which late-outgrowth EPCs were successfully isolated, the cells were assayed in vitro for their expression of EC markers, proliferation potential and ability to endothelialize synthetic materials, form new blood vessels, and produce nitric oxide. Late-outgrowth EPCs from patients with CAD and healthy volunteers exhibited critical EC markers and morphological characteristics that were analogous to a control population of human aortic ECs. To our knowledge, this is the first study to examine the suitability of late-outgrowth EPCs from patients with CAD for autologous endothelialization applications.
Characterization of Umbilical Cord Blood-derived Late Outgrowth Endothelial Progenitor Cells Exposed to Laminar Shear Stress
Endothelial progenitor cells isolated from umbilical cord blood (CB-EPCs) represent a promising source of endothelial cells for synthetic vascular grafts and tissue-engineered blood vessels since they are readily attainable, can be easily isolated, and possess a high proliferation potential. The objective of this study was to compare the functional behavior of late outgrowth CB-EPCs with human aortic endothelial cells (HAECs). CB-EPCs and HAECs were cultured on either smooth muscle cells in a coculture model of a tissue-engineered blood vessels or fibronectin adsorbed to Teflon-AF-coated glass slides. Late outgrowth CB-EPCs expressed endothelial cell-specific markers and were negative for the monocytic marker CD14. CB-EPCs have higher proliferation rates than HAECs, but are slightly smaller in size. CB-EPCs remained adherent under supraphysiological shear stresses, oriented and elongated in the direction of flow, and expressed similar numbers of alpha(5)beta(1) and alpha(v)beta(3) integrins and antithrombotic genes compared to HAECs. There were some differences in mRNA levels of E-selectin and vascular cell adhesion molecule 1 between CB-EPCs and HAECs; however, protein levels were similar on the two cell types, and CB-EPCs did not support adhesion of monocytes in the absence of tumor necrosis factor-alpha stimulation. Although CB-EPCs expressed significantly less endothelial nitric oxide synthase protein after exposure to flow than HAECs, nitric oxide levels induced by flow were not significantly different. These results suggest that late outgrowth CB-EPCs are functionally similar to HAECs under flow conditions and are a promising cell source for cardiovascular therapies.
Porcine Endothelial Cells Cocultured with Smooth Muscle Cells Became Procoagulant in Vitro
Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surface on vascular grafts. In this study, we used a porcine EC/smooth muscle cell (SMC) coculture model that was previously developed to examine the efficacy of EC seeding. Expression of tissue factor (TF), a primary initiator in the coagulation cascade, and TF activity were used as indicators of thrombogenicity. Using immunostaining, primary cultures of porcine EC showed a low level of TF expression, but a highly heterogeneous distribution pattern with 14% of ECs expressing TF. Quiescent primary cultures of porcine SMCs displayed a high level of TF expression and a uniform pattern of staining. When we used a two-stage amidolytic assay, TF activity of ECs cultured alone was very low, whereas that of SMCs was high. ECs cocultured with SMCs initially showed low TF activity, but TF activity of cocultures increased significantly 7-8 days after EC seeding. The increased TF activity was not due to the activation of nuclear factor kappa-B on ECs and SMCs, as immunostaining for p65 indicated that nuclear factor kappa-B was localized in the cytoplasm in an inactive form in both ECs and SMCs. Rather, increased TF activity appeared to be due to the elevated reactive oxygen species levels and contraction of the coculture, thereby compromising the integrity of EC monolayer and exposing TF on SMCs. The incubation of cocultures with N-acetyl-cysteine (2 mM), an antioxidant, inhibited contraction, suggesting involvement of reactive oxygen species in regulating the contraction. The results obtained from this study provide useful information for understanding thrombosis in tissue-engineered vascular grafts.
Peptide Interfacial Biomaterials Improve Endothelial Cell Adhesion and Spreading on Synthetic Polyglycolic Acid Materials
Resorbable scaffolds such as polyglycolic acid (PGA) are employed in a number of clinical and tissue engineering applications owing to their desirable property of allowing remodeling to form native tissue over time. However, native PGA does not promote endothelial cell adhesion. Here we describe a novel treatment with hetero-bifunctional peptide linkers, termed "interfacial biomaterials" (IFBMs), which are used to alter the surface of PGA to provide appropriate biological cues. IFBMs couple an affinity peptide for the material with a biologically active peptide that promotes desired cellular responses. One such PGA affinity peptide was coupled to the integrin binding domain, Arg-Gly-Asp (RGD), to build a chemically synthesized bimodular 27 amino acid peptide that mediated interactions between PGA and integrin receptors on endothelial cells. Quartz crystal microbalance with dissipation monitoring (QCMD) was used to determine the association constant (K (A) 1 x 10(7) M(-1)) and surface thickness (~3.5 nm). Cell binding studies indicated that IFBM efficiently mediated adhesion, spreading, and cytoskeletal organization of endothelial cells on PGA in an integrin-dependent manner. We show that the IFBM peptide promotes a 200% increase in endothelial cell binding to PGA as well as 70-120% increase in cell spreading from 30 to 60 minutes after plating.
Direct-contact Co-culture Between Smooth Muscle and Endothelial Cells Inhibits TNF-alpha-mediated Endothelial Cell Activation
We used a direct-contact endothelial cell-smooth muscle cell (EC-SMC) co-culture to examine whether quiescent SMCs regulate the EC inflammatory response to tumor necrosis factor (TNF)-alpha. ECs were cultured under static and physiological flow conditions. Compared with TNF-alpha-treated ECs in monoculture, TNF-alpha-treated ECs in co-culture had less NF-kappaB nuclear translocation; less intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin surface protein expression; no change in TNF receptor expression, but greater Kruppel-like factor 2 (KLF2) gene expression. After flow preconditioning for 24 h at 15 dyne/cm(2), and exposure of ECs to flow and TNF-alpha for 4.5 h, ECs in co-culture had less ICAM-1, VCAM-1, and E-selectin surface protein expression. Exposure to flow greatly increased KLF2 gene expression levels in both EC cultures; as a result, ECs in co-culture and monoculture had similar levels of post-flow KLF2 gene expression. The reduced levels of TNF-alpha-induced adhesion molecule expression in co-culture required the presence of quiescent SMCs; adhesion to decellularized extracellular matrix (ECM) or co-culture with fibroblasts produced only a modest reduction in EC adhesion molecule expression. Furthermore, co-culture of quiescent SMCs and ECs on the opposite side of a 10-microm-thick porous membrane did not alter the TNF-alpha-mediated ICAM-1 surface protein expression. Although the ECM produced by SMCs plays some role in reducing TNF-alpha-mediated inflammation, these results suggest that the direct contact between ECs and quiescent SMCs is required to inhibit TNF-alpha-mediated activation.
Effect of MicroRNA Modulation on Bioartificial Muscle Function
Cellular therapies have recently employed the use of small RNA molecules, particularly microRNAs (miRNAs), to regulate various cellular processes that may be altered in disease states. In this study, we examined the effect of transient muscle-specific miRNA inhibition on the function of three-dimensional skeletal muscle cultures, or bioartificial muscles (BAMs). Skeletal myoblast differentiation in vitro is enhanced by inhibiting a proliferation-promoting miRNA (miR-133) expressed in muscle tissues. As assessed by functional force measurements in response to electrical stimulation at frequencies ranging from 0 to 20 Hz, peak forces exhibited by BAMs with miR-133 inhibition (anti-miR-133) were on average 20% higher than the corresponding negative control, although dynamic responses to electrical stimulation in miRNA-transfected BAMs and negative controls were similar to nontransfected controls. Immunostaining for alpha-actinin and myosin also showed more distinct striations and myofiber organization in anti-miR-133 BAMs, and fiber diameters were significantly larger in these BAMs over both the nontransfected and negative controls. Compared to the negative control, anti-miR-133 BAMs exhibited more intense nuclear staining for Mef2, a key myogenic differentiation marker. To our knowledge, this study is the first to demonstrate that miRNA mediation has functional effects on tissue-engineered constructs.
Human Umbilical Cord Blood-derived Endothelial Cells Reendothelialize Vein Grafts and Prevent Thrombosis
To accelerate vein graft reendothelialization and reduce vein graft thrombosis by infusing human umbilical cord blood-derived endothelial cells (hCB-ECs) because loss of endothelium contributes to vein graft thrombosis and neointimal hyperplasia.
Dynamic Adhesion of Umbilical Cord Blood Endothelial Progenitor Cells Under Laminar Shear Stress
Late outgrowth endothelial progenitor cells (EPCs) represent a promising cell source for rapid reendothelialization of damaged vasculature after expansion ex vivo and injection into the bloodstream. We characterized the dynamic adhesion of umbilical-cord-blood-derived EPCs (CB-EPCs) to surfaces coated with fibronectin. CB-EPC solution density affected the number of adherent cells and larger cells preferentially adhered at lower cell densities. The number of adherent cells varied with shear stress, with the maximum number of adherent cells and the shear stress at maximum adhesion depending upon fluid viscosity. CB-EPCs underwent limited rolling, transiently tethering for short distances before firm arrest. Immediately before arrest, the instantaneous velocity decreased independent of shear stress. A dimensional analysis indicated that adhesion was a function of the net force on the cells, the ratio of cell diffusion to sliding speed, and molecular diffusivity. Adhesion was not limited by the settling rate and was highly specific to α(5)β(1) integrin. Total internal reflection fluorescence microscopy showed that CB-EPCs produced multiple contacts of α(5)β(1) with the surface and the contact area grew during the first 20 min of attachment. These results demonstrate that CB-EPC adhesion from blood can occur under physiological levels of shear stress.
Endothelial Cell Vascular Smooth Muscle Cell Co-Culture Assay For High Throughput Screening Assays For Discovery of Anti-Angiogenesis Agents and Other Therapeutic Molecules
Drug development for many diseases would be aided greatly by accurate in vitro model systems that replicate key elements of in vivo physiology. The recent development of co-culture systems of endothelial cells and vascular smooth muscle cells can be extended to high throughput systems for the identification of compounds for angiogenesis, vascular repair and hypertension. In this review, the various co-culture systems are reviewed and biological interactions between endothelial cells and vascular smooth muscle cells are discussed. Key considerations in the design of high throughput systems are presented and selected examples are discussed.
Quantitative Microscopy and Nanoscopy of Sickle Red Blood Cells Performed by Wide Field Digital Interferometry
We have applied wide-field digital interferometry (WFDI) to examine the morphology and dynamics of live red blood cells (RBCs) from individuals who suffer from sickle cell anemia (SCA), a genetic disorder that affects the structure and mechanical properties of RBCs. WFDI is a noncontact, label-free optical microscopy approach that can yield quantitative thickness profiles of RBCs and measurements of their membrane fluctuations at the nanometer scale reflecting their stiffness. We find that RBCs from individuals with SCA are significantly stiffer than those from a healthy control. Moreover, we show that the technique is sensitive enough to distinguish classes of RBCs in SCA, including sickle RBCs with apparently normal morphology, compared to the stiffer crescent-shaped sickle RBCs. We expect that this approach will be useful for diagnosis of SCA and for determining efficacy of therapeutic agents.
Biomechanical Effects of Flow and Coculture on Human Aortic and Cord Blood-derived Endothelial Cells
Human endothelial cells derived from umbilical cord blood (hCB-ECs) represent a promising cell source for endothelialization of tissue engineered blood vessels. hCB-ECs cultured directly above human aortic smooth muscle cells (SMCs), which model native and tissue engineered blood vessels, produce a confluent endothelium that responds to flow like normal human aortic endothelial cells (HAECs). The objective of this study was to quantify the elastic modulus of hCB-ECs cocultured with SMCs under static and flow conditions using atomic force microscopy (AFM). Cytoskeleton structures were assessed by AFM cell surface imaging and immunofluorescence of F-actin. The elastic moduli of hCB-ECs and HAECs were similar and significantly smaller than the value for SMCs in monoculture under static conditions (p<0.05). In coculture, hCB-ECs and HAECs became significantly stiffer with moduli 160-180% larger than their corresponding values in monoculture. While the moduli of hCB-ECs and HAECs almost doubled in monoculture and flow condition, their corresponding values in coculture declined after exposure to flow. Both the number and diameter of cortical stress fiber per cell width increased in coculture and/or flow conditions, whereas the subcortical stress fiber density throughout the cell interior increased by a smaller amount. These findings indicate that changes to biomechanical properties in coculture and/or exposure to flow are correlated with changes in the cortical stress fiber density. For ECs, fluid shear stress appeared to have greater effect on the elastic modulus than the presence of SMCs and changes to the elastic modulus in coculture may be due to EC-SMC communication.
Use of Autologous Blood-derived Endothelial Progenitor Cells at Point-of-care to Protect Against Implant Thrombosis in a Large Animal Model
Titanium (Ti) is commonly utilized in many cardiovascular devices, e.g. as a component of Nitinol stents, intra- and extracorporeal mechanical circulatory assist devices, but is associated with the risk of thromboemboli formation. We propose to solve this problem by lining the Ti blood-contacting surfaces with autologous peripheral blood-derived late outgrowth endothelial progenitor cells (EPCs) after having previously demonstrated that these EPCs adhere to and grow on Ti under physiological shear stresses and functionally adapt to their environment under flow conditions ex vivo. Autologous fluorescently-labeled porcine EPCs were seeded at the point-of-care in the operating room onto Ti tubes for 30 min and implanted into the pro-thrombotic environment of the inferior vena cava of swine (n = 8). After 3 days, Ti tubes were explanted, disassembled, and the blood-contacting surface was imaged. A blinded analysis found all 4 cell-seeded implants to be free of clot, whereas 4 controls without EPCs were either entirely occluded or partially thrombosed. Pre-labeled EPCs had spread and were present on all 4 cell-seeded implants while no endothelial cells were observed on control implants. These results suggest that late outgrowth autologous EPCs represent a promising source of lining Ti implants to reduce thrombosis in vivo.
The Biocompatibility of Titanium Cardiovascular Devices Seeded with Autologous Blood-derived Endothelial Progenitor Cells: EPC-seeded Antithrombotic Ti Implants
Implantable and extracorporeal cardiovascular devices are commonly made from titanium (Ti) (e.g. Ti-coated Nitinol stents and mechanical circulatory assist devices). Endothelializing the blood-contacting Ti surfaces of these devices would provide them with an antithrombogenic coating that mimics the native lining of blood vessels and the heart. We evaluated the viability and adherence of peripheral blood-derived porcine endothelial progenitor cells (EPCs), seeded onto thin Ti layers on glass slides under static conditions and after exposure to fluid shear stresses. EPCs attached and grew to confluence on Ti in serum-free medium, without preadsorption of proteins. After attachment to Ti for 15 min, less than 5% of the cells detached at a shear stress of 100 dyne / cm(2). Confluent monolayers of EPCs on smooth Ti surfaces (Rq of 10 nm), exposed to 15 or 100 dyne/cm(2) for 48 h, aligned and elongated in the direction of flow and produced nitric oxide dependent on the level of shear stress. EPC-coated Ti surfaces had dramatically reduced platelet adhesion when compared to uncoated Ti surfaces. These results indicate that peripheral blood-derived EPCs adhere and function normally on Ti surfaces. Therefore EPCs may be used to seed cardiovascular devices prior to implantation to ameliorate platelet activation and thrombus formation.
