Velocity Measurement of In Vitro Blood Flow in Microchip Cultured Endothelial Cells

2008 ◽  
Author(s):  
Naoki Segawa ◽  
Yasuhiko Sugii
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Blood Vessel ◽  
Cell Attachment ◽  
Three Dimensional ◽  
Vascular Diseases ◽  
Culture Method ◽  
Measured Velocity ◽  
Micro Piv

In order to investigate vascular diseases such as cause of atherosclerosis and myocardial infarction, relationships of endothelial cells (ECs) covered with surface blood vessels and blood flow stimulation have been experimentally studied. In the study, a blood vessel model for in vitro experiment made from polydimethylsiloxane (PDMS) microchip with a straight microchannel with 400 μm width and 100 μm depth was developed. By optimizing cells cultured condition such as the liquid introduction method and the surface coating for enhancement of cell attachment on the microchannel wall, cell culture method in the microchip were developed. Velocity distributions on the ECs surface in the blood vessel model were measured using micro PIV technique. Measured velocity vectors on the ECs surface were fluctuated caused by the three dimensional effect of the cell shape.

scholarly journals A hydrogel platform for in vitro three dimensional assembly of human stem cell-derived islet cells and endothelial cells

2019 ◽  
Vol 97 ◽  
pp. 272-280 ◽  
Author(s):  
Punn Augsornworawat ◽  
Leonardo Velazco-Cruz ◽  
Jiwon Song ◽  
Jeffrey R. Millman
Keyword(s):  
Endothelial Cells ◽  
Stem Cell ◽  
Islet Cells ◽  
Three Dimensional ◽  
Human Stem Cell

scholarly journals Osteogenic Properties of 3D-Printed Silica-Carbon-Calcite Composite Scaffolds: Novel Approach for Personalized Bone Tissue Regeneration

2021 ◽  
Vol 22 (2) ◽  
pp. 475
Author(s):  
Parastoo Memarian ◽  
Francesco Sartor ◽  
Enrico Bernardo ◽  
Hamada Elsayed ◽  
Batur Ercan ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Bone Tissue ◽  
Cell Attachment ◽  
Three Dimensional ◽  
Bone Tissue Regeneration ◽  
Hemolysis Assay ◽  
Novel Approach ◽  
Related Transcription Factor ◽  
Diphenyl Tetrazolium Bromide

Carbon enriched bioceramic (C-Bio) scaffolds have recently shown exceptional results in terms of their biological and mechanical properties. The present study aims at assessing the ability of the C-Bio scaffolds to affect the commitment of canine adipose-derived mesenchymal stem cells (cAD-MSCs) and investigating the influence of carbon on cell proliferation and osteogenic differentiation of cAD-MSCs in vitro. The commitment of cAD-MSCs to an osteoblastic phenotype has been evaluated by expression of several osteogenic markers using real-time PCR. Biocompatibility analyses through 3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), lactate dehydrogenase (LDH) activity, hemolysis assay, and Ames test demonstrated excellent biocompatibility of both materials. A significant increase in the extracellular alkaline phosphatase (ALP) activity and expression of runt-related transcription factor (RUNX), ALP, osterix (OSX), and receptor activator of nuclear factor kappa-Β ligand (RANKL) genes was observed in C-Bio scaffolds compared to those without carbon (Bio). Scanning electron microscopy (SEM) demonstrated excellent cell attachment on both material surfaces; however, the cellular layer on C-Bio fibers exhibited an apparent secretome activity. Based on our findings, graphene can improve cell adhesion, growth, and osteogenic differentiation of cAD-MSCs in vitro. This study proposed carbon as an additive for a novel three-dimensional (3D)-printable biocompatible scaffold which could become the key structural material for bone tissue reconstruction.

scholarly journals Effect of aminaphtone on in vitro vascular permeability and capillary-like maintenance

2017 ◽  
Vol 33 (9) ◽  
pp. 592-599 ◽  
Author(s):  
Francesca Felice ◽  
Ester Belardinelli ◽  
Alessandro Frullini ◽  
Tatiana Santoni ◽  
Egidio Imbalzano ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Chronic Venous Insufficiency ◽  
Venous Insufficiency ◽  
Umbilical Vein ◽  
Three Dimensional ◽  
Endothelial Permeability ◽  
Human Umbilical Vein ◽  
Pre Treatment ◽  
Umbilical Vein Endothelial Cells

Objectives Aminaphtone, a naphtohydrochinone used in the treatment of capillary disorders, may affect oedema in chronic venous insufficiency. Aim of study is to investigate the effect of aminaphtone on vascular endothelial permeability in vitro and its effects on three-dimensional capillary-like structures formed by human umbilical vein endothelial cells. Method Human umbilical vein endothelial cells were treated with 50 ng/ml VEGF for 2 h and aminaphtone for 6 h. Permeability assay, VE-cadherin expression and Matrigel assay were performed. Results VEGF-induced permeability was significantly decreased by aminaphtone in a range concentration of 1–20 µg/ml. Aminaphtone restored VE-cadherin expression. Finally, 6 h pre-treatment with aminaphtone significantly preserved capillary-like structures formed by human umbilical vein endothelial cells on Matrigel up to 48 h compared to untreated cells. Conclusions Aminaphtone significantly protects endothelium permeability and stabilises endothelial cells organised in capillary-like structures, modulating VE-cadherin expression. These data might explain the clinical benefit of aminaphtone on chronic venous insufficiency.

scholarly journals Hydrogel-Coated Textile Scaffolds as Three-Dimensional Growth Support for Human Umbilical Vein Endothelial Cells (HUVECs): Possibilities as Coculture System in Liver Tissue Engineering

2002 ◽  
Vol 11 (4) ◽  
pp. 369-377 ◽  
Author(s):  
Makarand V. Risbud ◽  
Erdal Karamuk ◽  
René Moser ◽  
Joerg Mayer
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Three Dimensional ◽  
Mesh Size ◽  
Cell Types ◽  
Cell Number ◽  
Human Umbilical Vein ◽  
Hydrogel Matrix ◽  
Umbilical Vein Endothelial Cells

Three-dimensional (3-D) scaffolds offer an exciting possibility to develop cocultures of various cell types. Here we report chitosan–collagen hydrogel-coated fabric scaffolds with defined mesh size and fiber diameter for 3-D culture of human umbilical vein endothelial cells (HUVECs). These scaffolds did not require pre-coating with fibronectin and they supported proper HUVEC attachment and growth. Scaffolds preserved endothelial cell-specific cobblestone morphology and cells were growing in compartments defined by the textile mesh. HUVECs on the scaffold maintained the property of contact inhibition and did not exhibit overgrowth until the end of in vitro culture (day 6). MTT assay showed that cells had preserved mitochondrial functionality. It was also noted that cell number on the chitosan-coated scaffold was lower than that of collagen-coated scaffolds. Calcein AM and ethidium homodimer (EtD-1) dual staining demonstrated presence of viable and metabolically active cells, indicating growth supportive properties of the scaffolds. Actin labeling revealed absence of actin stress fibers and uniform distribution of F-actin in the cells, indicating their proper attachment to the scaffold matrix. Confocal microscopic studies showed that HUVECs growing on the scaffold had preserved functionality as seen by expression of von Willebrand (vW) factor. Observations also revealed that functional HUVECs were growing at various depths in the hydrogel matrix, thus demonstrating the potential of these scaffolds to support 3-D growth of cells. We foresee the application of this scaffold system in the design of liver bioreactors wherein hepatocytes could be cocultured in parallel with endothelial cells to enhance and preserve liver-specific functions.

Abstract 14820: Sirt7 Deficiency in Blood Vessel Components Impairs Vascular Function by Inhibiting Cell Cycle- and Inflammatory-Related Protein Expression

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Yuichi Kimura ◽  
Yasuhiro Izumiya ◽  
Satoshi Araki ◽  
Satoru Yamamura ◽  
Yoshiro Onoue ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Blood Flow ◽  
Vascular Function ◽  
Vascular Dysfunction ◽  
Vascular Diseases ◽  
Tube Formation ◽  
Hindlimb Ischemia ◽  
Proliferation Assay

Introduction: Aging is a well-established cardiovascular risk factor and associated with vascular dysfunction. Sirt7, one of the members of mammalian sirtuin family, is thought to be involved in age-related diseases. However, little is known about the relative contribution of Sirt7 in vascular dysfunction. Hypothesis: Sirt7 maintains vascular cell functions and its deficiency plays a critical role in vascular diseases. Methods: Sirt7 loss- and gain-of-function experiments were performed with human aortic smooth muscle cells (HAoSMCs) and human umbilical vein endothelial cells (HUVECs). In vivo, blood flow recovery was evaluated by hindlimb ischemia model in homozygous Sirt7 deficient (Sirt7-/-) and wild-type (WT) mice. Irradiated WT mice were intravenously received bone marrow (BM) cells from WT or Sirt7 -/- mouse to achieve BM transfer. Results: An RNAi-medicated Sirt7 knockdown resulted in a significant inhibition of HAoSMCs proliferation following serum or Platelet-derived growth factor BB (PDGF-BB) stimulation as determined by cell count, BrdU cell proliferation assay and MTS proliferation assay. Knockdown of endogenous Sirt7 also reduced cell migration as revealed by Boyden chamber migration assay. The Cyclin D1 and Cyclin dependent kinase 2 (CDK2) protein levels were significantly decreased in Sirt7 siRNA-treated HAoSMCs in response to serum or PDGF-BB stimulation. In endothelial cells, knockdown of Sirt7 attenuated tube formation, proliferation and migration. These changes were accompanied by reduced ERK activation and VCAM-1 mRNA and protein expression in Sirt7 siRNA-treated HUVECs. Conversely, overexpression of Sirt7 by adenovirus enhanced tube formation and cell proliferation. In vivo, blood flow recovery in response to hindlimb ischemia was significantly attenuated in Sirt7-/- mice compared with WT mice. There was no difference in blood flow recovery between WT mice transplanted with WT or Sirt7-/- BM cells suggesting that Sirt7 deficiency in vascular cells have a predominant effect on attenuated blood flow recovery in response to hindlimb ischemia. Conclusions: Sirt7 in blood vessel components have an important role in maintenance of vascular function. Sirt7 could be a promising therapeutic target for vascular diseases.

Interaction between vascular endothelial cells and vascular intimal spindle-shaped cells in vitro

1983 ◽  
Vol 60 (1) ◽  
pp. 89-102
Author(s):  
D de Bono ◽  
C. Green
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Three Dimensional ◽  
Vascular Endothelial ◽  
Pure Cultures ◽  
Species Specific ◽  
Endothelial Growth Factor

The interactions between human or bovine vascular endothelial cells and fibroblast-like vascular intimal spindle-shaped cells have been studied in vitro, using species-specific antibodies to identify the different components in mixed cultures. Pure cultures of endothelial cells grow as uniform, nonoverlapping monolayers, but this growth pattern is lost after the addition of spindle cells, probably because the extracellular matrix secreted by the latter causes the endothelial cells to modify the way they are attached to the substrate. The result is a network of tubular aggregates of endothelial cells in a three-dimensional ‘polylayer’ of spindle-shaped cells. On the other hand, endothelial cells added to growth-inhibited cultures of spindle-shaped cells will grow in sheets over the surface of the culture. Human endothelial cells grown in contact with spindle-shaped cells have a reduced requirement for a brain-derived endothelial growth factor. The interactions of endothelial cells and other connective tissue cells in vitro may be relevant to the mechanisms of endothelial growth and blood vessel formation in vivo, and emphasize the potential importance of extracellular matrix in controlling endothelial cell behaviour.

Abstract 1103: Integrin-linked Kinase and Laminin alpha 4 Mutations Cause Human Cardiomyopathy

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Ralph Knöll ◽  
Ruben Postel ◽  
Ralph Krätzner ◽  
Gerrit Hennecke ◽  
Andrei M Vacaru ◽  
...  
Keyword(s):  
Heart Failure ◽  
Endothelial Cells ◽  
Dilated Cardiomyopathy ◽  
Cell Attachment ◽  
Dissociation Constants ◽  
Binding Capacity ◽  
Myocardial Dysfunction ◽  
Protein Interaction Data ◽  
Integrin Linked Kinase

Background - Dilated Cardiomyopathy (DCM) is a syndrome characterized by ventricular dilation, contractile dysfunction and symptoms of congestive heart failure. Extracellular matrix proteins such as laminins as well as endothelial cells are known to influence cardiomyocyte performance, however a molecular link between mutations in the ECM and DCM has not been provided. Methods and Results - Using a forward genetic screen in zebrafish to identify novel genes required for myocardial function we were able to identify the lost-contact (loc) mutant, encoding a nonsense mutation in the integrin-linked kinase (ILK) gene. This loc/ilk mutant is associated with both, a severe defect in cardiomyocytes as well as in endothelial cells leading to severe myocardial dysfunction. Additional experiments revealed the epistatic regulation between the ECM component laminin alpha 4 (LAMA4), integrin and ILK, which led us to screen for mutations in the human ILK and LAMA4 genes in patients with severe dilated cardiomyopathy (DCM). We identified two novel amino acid residue altering mutations (2828C>T (Pro943Leu), 3217C>T (Arg1073X)) in the integrin interacting domain of the LAMA4 gene and one mutation (785C>T (Ala262Val)) in the ILK gene. BIAcore quantitative protein/protein interaction data, which have been used to determine the equilibrium dissociation constants, point to the loss of integrin binding capacity in case of the Pro943Leu (KD=5 +/− 3 uM) and Arg1073X lama 4 (KD=1 +/− 0.2 uM) mutants in comparison to the wildtype lama 4 protein (KD = 440 +/− 20 nM). Cell attachment assays point to a defect in endothelial cell adhesion when LAMA4 mutants have been expressed in vitro and used as a substrate. 785C>T (Ala262Val) ILK did not change its affinity, as measured by GST pull down assays as well as by a yeast 2 hybrid analysis, to a well known interacting protein such as b-parvin but revealed a 63% decrease in kinase activity. Additional functional data point to the loss of endothelial cells in affected patients as a direct consequence of the mutant genes, leading ultimately to heart failure. Conclusions - We report the first human mutations in the LAMA4, integrin and ILK system and provide a new mechanistic basis for DCM in man, which involves endothelial cells as well as cardiomyocytes.

Limitations of In-Vitro Labeling of Endothelial Cells with Indium-111 Oxine

1995 ◽  
Vol 4 (3) ◽  
pp. 291-296 ◽  
Author(s):  
H.M.H. Carr ◽  
J.V. Smyth ◽  
O.B. Rooney ◽  
P.D. Dodd ◽  
H. Sharma ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Attachment ◽  
Control Group ◽  
Vascular Prostheses ◽  
Long Term Effect ◽  
Indium 111 ◽  
Suitable Technique

Indium-111 oxine labeling is widely used as a marker of endothelial cell attachment to vascular prostheses. The long term effect of labeling human adult endothelial cells (HAECs) with this isotope has not been determined. In this study the viability of labeled HAECs, leakage of isotope from labeled cells and adherence of circulating isotope to fibronectin coated prostheses were investigated over 24 h. The effect of incubation time on labeling efficiency was also assessed. There were significant differences in cell viability between the labeled and unlabeled groups beyond 4 h (p < 0.005, 2-tailed, unpaired t-test). In the control group cell numbers increased by 42% while in the labeled group this had decreased by 20% at 24 h. Spontaneous leakage increased with time but was maximal in the first 2 h. Adherence of circulating isotope to fibronectin coated expanded polytetrafluoroethylene (ePTFE) grafts was minimal but was significantly greater to gelatin impregnated Dacron (GEL-SEAL) beyond 1 hour (p < 0.05). Incubation times greater than 5 minutes during labeling do not significantly improve labeling efficiency, and may contribute to toxicity by prolonging exposure to oxine. Indium-111 oxine labeling of HAECs is a suitable technique for acute studies of endothelial cell kinetics up to 4 h, but its use in chronic studies may lead to significant underestimations of cell retention.

scholarly journals Improved Bioactivity of 3D Printed Porous Titanium Alloy Scaffold with Chitosan/Magnesium-Calcium Silicate Composite for Orthopaedic Applications

Materials ◽  
2019 ◽  
Vol 12 (2) ◽  
pp. 203 ◽  
Author(s):  
Chun-Hao Tsai ◽  
Chih-Hung Hung ◽  
Che-Nan Kuo ◽  
Cheng-Yu Chen ◽  
Yu-Ning Peng ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Calcium Silicate ◽  
Cell Attachment ◽  
Three Dimensional ◽  
Bone Defects ◽  
Metal Alloys ◽  
Potential Candidate ◽  
Natural Bone

Recently, cases of bone defects have been increasing incrementally. Thus, repair or replacement of bone defects is gradually becoming a huge problem for orthopaedic surgeons. Three-dimensional (3D) scaffolds have since emerged as a potential candidate for bone replacement, of which titanium (Ti) alloys are one of the most promising candidates among the metal alloys due to their low cytotoxicity and mechanical properties. However, bioactivity remains a problem for metal alloys, which can be enhanced using simple immersion techniques to coat bioactive compounds onto the surface of Ti–6Al–4V scaffolds. In our study, we fabricated magnesium-calcium silicate (Mg–CS) and chitosan (CH) compounds onto Ti–6Al–4V scaffolds. Characterization of these surface-modified scaffolds involved an assessment of physicochemical properties as well as mechanical testing. Adhesion, proliferation, and growth of human Wharton’s Jelly mesenchymal stem cells (WJMSCs) were assessed in vitro. In addition, the cell attachment morphology was examined using scanning electron microscopy to assess adhesion qualities. Osteogenic and mineralization assays were conducted to assess osteogenic expression. In conclusion, the Mg–CS/CH coated Ti–6Al–4V scaffolds were able to exhibit and retain pore sizes and their original morphologies and architectures, which significantly affected subsequent hard tissue regeneration. In addition, the surface was shown to be hydrophilic after modification and showed mechanical strength comparable to natural bone. Not only were our modified scaffolds able to match the mechanical properties of natural bone, it was also found that such modifications enhanced cellular behavior such as adhesion, proliferation, and differentiation, which led to enhanced osteogenesis and mineralization downstream. In vivo results indicated that Mg–CS/CH coated Ti–6Al–4V enhances the bone regeneration and ingrowth at the critical size bone defects of rabbits. These results indicated that the proposed Mg–CS/CH coated Ti–6Al–4V scaffolds exhibited a favorable, inducive micro-environment that could serve as a promising modification for future bone tissue engineering scaffolds.

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