Assessment of cytocompatibility and mechanical properties of detergent-decellularized ovine pericardial tissue

2019 ◽  
Vol 42 (11) ◽  
pp. 628-635
Author(s):  
Alexandru Mogaldea ◽  
Karolina Theodoridis ◽  
Tobias Goecke ◽  
Igor Tudorache ◽  
Axel Haverich ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Endothelial Cells ◽  
Fluorescent Protein ◽  
Fluorescent Microscopy ◽  
Burst Pressure ◽  
In Vitro Cultivation ◽  
Detergent Treatment ◽  
Decellularized Tissue ◽  
Retention Strength

Background: Autologous pericardium is widely used for the repair of different sized cardiovascular defects. However, its use is limited especially in redo cardiac surgery. We developed an engineered tissue based on decellularized pericardium reseeded with blood-derived endothelial cells. Materials and Methods: Decellularization of ovine pericardium was performed using detergent treatment. Ovine outgrowth blood-derived and green fluorescent protein–labeled endothelial cells were used to reseed the decellularized ovine pericardium on the mesothelial side. The cell adhesion was assessed using fluorescent microscopy up to 15 days of in vitro cultivation. The mechanical properties of the pericardium were evaluated using suturability, burst pressure, and suture retention strength tests. Results: After decellularization the pericardial sheets appeared cell-free and repopulation using ovine blood-derived endothelial cells was successful by forming a robust monolayer. Detergent treatment did not affect the extracellular matrix. The thickness of decellularized tissue was similar to native ovine pericardium (285.3 ± 28.2 µm, respective 276.9 ± 23.8 µm, p = 0.48). Decellularized patch showed similar suturability comparable to the native ovine pericardium. Resulted burst pressure was not significantly different (native/decellularized: 312.5 ± 13.6/304.2 ± 16, p = 0.35). The suture retention strength of native pericardium was 638.33 ± 90.2 gr and comparable to decellularized tissue (622.2 ± 89.9 gr, p = 0.76). No differences were observed concerning elongation of native and decellularized pericardium (8.33 ± 1.5 and 8.5 ± 0.84 mm, respectively; p = 0.82). Conclusion: Mesothelial surface of decellularized ovine pericardium is suitable for reseeding with ovine blood-derived endothelial cells. The mechanical properties of detergent-treated pericardium were comparable to native tissue.

scholarly journals Novel Infectivity-Enhanced Oncolytic Adenovirus with a Capsid-Incorporated Dual-Imaging Moiety for Monitoring Virotherapy in Ovarian Cancer

2009 ◽  
Vol 8 (5) ◽  
pp. 7290.2009.00025 ◽  
Author(s):  
Kristopher J. Kimball ◽  
Angel A. Rivera ◽  
Kurt R. Zinn ◽  
Mert Icyuz ◽  
Vaibhav Saini ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Fluorescent Protein ◽  
Imaging Modality ◽  
Fluorescent Microscopy ◽  
Oncolytic Adenovirus ◽  
Ovarian Cancer Cells ◽  
Microscopy Imaging ◽  
Dual Imaging

We sought to develop a cancer-targeted, infectivity-enhanced oncolytic adenovirus that embodies a capsid-labeling fusion for non-invasive dual-modality imaging of ovarian cancer virotherapy. A functional fusion protein composed of fluorescent and nuclear imaging tags was genetically incorporated into the capsid of an infectivity-enhanced conditionally replicative adenovirus. Incorporation of herpes simplex virus thymidine kinase (HSV-tk) and monomeric red fluorescent protein 1 (mRFP1) into the viral capsid and its genomic stability were verified by molecular analyses. Replication and oncolysis were evaluated in ovarian cancer cells. Fusion functionality was confirmed by in vitro gamma camera and fluorescent microscopy imaging. Comparison of tk-mRFP virus to single-modality controls revealed similar replication efficiency and oncolytic potency. Molecular fusion did not abolish enzymatic activity of HSV-tk as the virus effectively phosphorylated thymidine both ex vivo and in vitro. In vitro fluorescence imaging demonstrated a strong correlation between the intensity of fluorescent signal and cytopathic effect in infected ovarian cancer cells, suggesting that fluorescence can be used to monitor viral replication. We have in vitro validated a new infectivity-enhanced oncolytic adenovirus with a dual-imaging modality-labeled capsid, optimized for ovarian cancer virotherapy. The new agent could provide incremental gains toward climbing the barriers for achieving conditionally replicated adenovirus efficacy in human trials.

scholarly journals Biological Atomic Force Microscopy for Imaging Gold-Labeled Liposomes on Human Coronary Artery Endothelial Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Ana-María Zaske ◽  
Delia Danila ◽  
Michael C. Queen ◽  
Eva Golunski ◽  
Jodie L. Conyers
Keyword(s):  
Atomic Force Microscopy ◽  
Endothelial Cells ◽  
Coronary Artery ◽  
Cell Membrane ◽  
Fluorescent Microscopy ◽  
Human Coronary Artery ◽  
Force Microscopy ◽  
Cell Internalization ◽  
Atomic Force

Although atomic force microscopy (AFM) has been used extensively to characterize cell membrane structure and cellular processes such as endocytosis and exocytosis, the corrugated surface of the cell membrane hinders the visualization of extracellular entities, such as liposomes, that may interact with the cell. To overcome this barrier, we used 90 nm nanogold particles to label FITC liposomes and monitor their endocytosis on human coronary artery endothelial cells (HCAECs) in vitro. We were able to study the internalization process of gold-coupled liposomes on endothelial cells, by using AFM. We found that the gold-liposomes attached to the HCAEC cell membrane during the first 15–30 min of incubation, liposome cell internalization occurred from 30 to 60 min, and most of the gold-labeled liposomes had invaginated after 2 hr of incubation. Liposomal uptake took place most commonly at the periphery of the nuclear zone. Dynasore monohydrate, an inhibitor of endocytosis, obstructed the internalization of the gold-liposomes. This study showed the versatility of the AFM technique, combined with fluorescent microscopy, for investigating liposome uptake by endothelial cells. The 90 nm colloidal gold nanoparticles proved to be a noninvasive contrast agent that efficiently improves AFM imaging during the investigation of biological nanoprocesses.

scholarly journals Endothelial JAK3 Expression Enhances Pro-Hematopoietic Angiocrine Function of Sinusoidal Endothelial Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2488-2488 ◽  
Author(s):  
José Gabriel Barcia Durán
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Fluorescent Microscopy ◽  
Hematopoietic Stem ◽  
Interleukin Receptors ◽  
Sinusoidal Endothelium

Unlike Jak1, Jak2, and Tyk2, Jak3 is the only member of the Jak family of secondary messengers that signals exclusively by binding the common gamma chain of interleukin receptors IL2, IL4, IL7, IL9, IL15, and IL21. Jak3-null mice display defective T and NK cell development, which results in a mild SCID phenotype. Still, functional Jak3 expression outside the hematopoietic system remains unreported. Our data show that Jak3 is expressed in endothelial cells across hematopoietic and non-hematopoietic organs, with heightened expression in the bone marrow and spleen. Increased arterial zonation in the bone marrow of Jak3-null mice further suggests that Jak3 is a marker of sinusoidal endothelium, which is confirmed by fluorescent microscopy staining and single-cell RNA-sequencing. We also show that the Jak3-null niche is deleterious for the maintenance of long-term repopulating hematopoietic stem and progenitor cells (LT-HSCs) and that Jak3-overexpressing endothelial cells have increased potential to expand LT-HSCs in vitro. In addition, we identify the soluble factors downstream of Jak3 that provide endothelial cells with this functional advantage and show their localization to the bone marrow sinusoids in vivo. Our work serves to identify a novel function for a non-promiscuous tyrosine kinase in the bone marrow vascular niche and further characterize the hematopoietic stem cell niche of sinusoidal endothelium. Disclosures No relevant conflicts of interest to declare.

scholarly journals Modifications of the mechanical properties of in vivo tissue-engineered vascular grafts by chemical treatments for a short duration

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248346
Author(s):  
Tomoya Inoue ◽  
Keiichi Kanda ◽  
Masashi Yamanami ◽  
Daisuke Kami ◽  
Satoshi Gojo ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Young’S Modulus ◽  
Short Duration ◽  
Young's Modulus ◽  
Vascular Grafts ◽  
Ultimate Strain ◽  
Burst Pressure ◽  
Chemical Treatments ◽  
Retention Strength

In vivo tissue-engineered vascular grafts constructed in the subcutaneous spaces of graft recipients have functioned well clinically. Because the formation of vascular graft tissues depends on several recipient conditions, chemical pretreatments, such as dehydration by ethanol (ET) or crosslinking by glutaraldehyde (GA), have been attempted to improve the initial mechanical durability of the tissues. Here, we compared the effects of short-duration (10 min) chemical treatments on the mechanical properties of tissues. Tubular tissues (internal diameter, 5 mm) constructed in the subcutaneous tissues of beagle dogs (4 weeks, n = 3), were classified into three groups: raw tissue without any treatment (RAW), tissue dehydrated with 70% ET (ET), and tissue crosslinked with 0.6% GA (GA). Five mechanical parameters were measured: burst pressure, suture retention strength, ultimate tensile strength (UTS), ultimate strain (%), and Young’s modulus. The tissues were also autologously re-embedded into the subcutaneous spaces of the same dogs for 4 weeks (n = 2) for the evaluation of histological responses. The burst pressure of the RAW group (1275.9 ± 254.0 mm Hg) was significantly lower than those of ET (2115.1 ± 262.2 mm Hg, p = 0.0298) and GA (2570.5 ± 282.6 mm Hg, p = 0.0017) groups. Suture retention strength, UTS or the ultimate strain did not differ significantly among the groups. Young’s modulus of the ET group was the highest (RAW: 5.41 ± 1.16 MPa, ET: 12.28 ± 2.55 MPa, GA: 7.65 ± 1.18 MPa, p = 0.0185). No significant inflammatory tissue response or evidence of residual chemical toxicity was observed in samples implanted subcutaneously for four weeks. Therefore, short-duration ET and GA treatment might improve surgical handling and the mechanical properties of in vivo tissue-engineered vascular tissues to produce ideal grafts in terms of mechanical properties without interfering with histological responses.

Abstract 580: The Production of a Bio-Engineered Endothelial Intima From Cultured Cells Using Whole Cardiac Cadaveric Extracellular Matrix.

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Stefan Kren ◽  
Gabriel Caron ◽  
Doris A Taylor
Keyword(s):  
Endothelial Cells ◽  
Rat Heart ◽  
Cultured Cells ◽  
Fluorescent Microscopy ◽  
Nuclear Staining ◽  
Endothelial Lining ◽  
Arterial Tree ◽  
The Matrix

Background; Bioengineered solutions to failing cardiac tissue have been difficult to achieve due partially to adverse interactions between circulating blood and the engineered surface. The aim of this study was to determine if by using naturally-derived ECM and cultured endothelial cells, a bioengineered whole-heart vascular intima could be generated. The matrix substrate for organ culture was produced by a perfusion-based detergent decelluarization of cadaveric rat heart. This process maintained ECM protein integrity as indicated by a glycosaminoglycan assay, with ~ equivalent amounts present relative to cadaveric rat heart. Its acellular nature was confirmed by loss of > 96% DNA (p = 0.001) compared to normal rat heart. In vitro infusion of aqueous dye or Mercox resin suggested a complete arterial tree, with structural preservation of vascular conduits. In vivo perfusability of the ECM was demonstrated by heterotopic transplantation with anticoagulation (n=4) into RNU rats for 7 days. Recellularization of the vascular tree was attempted by In Vitro Langendorff perfusion of 2 x 107 rat aortal endothelial cells (ECs) followed by a 7d incubation with escalating pulsatile flow in a 3D bioreactor. CellTracker Green assessed EC viability and permitted visualization of engrafted cells by fluorescent microscopy. Vessels of different diameters contained “patches” of confluent endothelium with complete circumferential lining of many of the matrix conduits. ECs lining both chamber walls and trabeculae were also observed. Nuclear staining showed 537.8 +/− 67.6 ECs / mm2 on endocardial surfaces, as well as 311.7 +/− 61.8 ECs / mm2 in vessels. To enhance the delivery of cells into the ventricular walls, a microcanulization of the brachiocephalic artery with sustained aortal perfusion was undertaken. This technique diverted more cells to the vasculature and more broadly distributed the cells in each area resulting in a lower cell density; 199.8 +/− 25.0 ECs / mm2 in vessels vs 125.8 +/− 43.4 ECs / mm2 on endocardial surfaces. In conclusion, these data suggest that by using detergent prepared acellular ECM of a whole organ, generation of a complete endothelial lining of vascular structures may be possible.

Development Of 3D Models For Studying Megakaryopoiesis In Vitro

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3696-3696
Author(s):  
Lindsay Wray ◽  
Christian A Di Buduo ◽  
David L. Kaplan ◽  
Alessandra Balduini
Keyword(s):  
Mechanical Properties ◽  
Bone Marrow ◽  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Peripheral Blood ◽  
Platelet Production ◽  
Vascular Niche ◽  
Proplatelet Formation ◽  
Bone Marrow Vascular Niche

Abstract Introduction Silk fibroin, derived from Bombyx mori silkworm cocoons, is used extensively in biomaterials and regenerative medicine. The useful characteristics of this protein include self-assembly, robust mechanical properties, biocompatibility and biodegradability. Moreover, silk can be enhanced through a variety of chemical modifications that affect cell attachment, growth and differentiation. Thrombocytopenia occurs when a patient suffers from an abnormally low platelet count in the peripheral blood; usually a result of disease, trauma, or cancer treatment. To treat these patients, it is estimated that two million platelet transfusions are performed in the U.S. each year. This high demand for platelets has created a clinical demand for studying the causes of thrombocytopenia and alternative routes for treatment. Platelets are anuclear cells that are released into the bloodstream in the bone marrow by megakaryocytes via the extension of long filaments called proplatelets. It is hypothesized that platelet production from megakaryocytes is regulated by environmental factors at the site of bone marrow vascular niche. Studies of megakaryopoiesis are typically performed on extracellular matrix protein-coated culture plates and transwell membranes. While these initial studies have provided invaluable insight into the process of megakaryopoiesis, the goal of the present project was to create a bone marrow model that mimics the vascular niche for functional in vitro platelet production. We hypothesized that a silk-based in vitro tissue model would allow the effects of substrate surface properties and endothelial co-culture on megakaryopoiesis to be studied in a holistic manner, thereby enabling further elucidation of the mechanisms involved in the process of platelet production. Results In order to more closely mimic the bone marrow vascular niche structure, a porous silk sponge was assembled around the silk vessel-like tubes. Megakarycytes seeded in the porous silk sponge migrated toward the silk tube and released platelets into the tube lumen. The perfusion bioreactor moved the platelets into the platelet collecting bags. After perfusion the platelets were collected and analyzed by flow cytometry. The bioreactor platelets exhibited similar morphology, CD41 positive staining, and activation compared to peripheral blood platelet controls. Megakaryocyte attachment and proplatelet formation through the silk vascular wall were improved by altering the silk properties. Silk functionalized by entrapping extracellular matrix proteins within the tube membrane resulted in increased megakaryocyte attachment and proplatelet compared to unfunctionalized silk tube controls. Silk surface roughness improved megakaryocyte attachment compared to the control but did not affect proplatelets. Decreasing the silk stiffness improved proplatelets, but did not significantly affect megakaryocyte attachment. Co-culture with endothelial cells improved megakaryocyte attachment while maintaining a high level of proplatelet formation. Additionally, megakaryocyte and endothelial cell co-culture on the silk vessel model resulted in an icreased platelet production compared to megakaryocytes cultured alone. Conclusions The goal of this project was to develop an in vitro model of megakaryopoiesis using a tissue engineering approach. Using human megakaryocytes and endothelial cells, we demonstrate the following advanced features of the silk-based model: (1) immobilization of extracellular matrix components within the membrane, (2) tunable surface topography, (3) tunable mechanical properties, (4) physiologically relevant thickness for appropriate proplatelet extension, and (5) controlled localization of a vascular endothelium. Thus, by functionalizing silk, we can control megakaryocyte function on silk. The broader impact of this work offers a versatile new tool for studying megakaryocyte development and platelet production in vitro. Disclosures: No relevant conflicts of interest to declare.

A newly designed tympanostomy stent with TiO2 coating to reduce Pseudomonas aeruginosa biofilm formation

2018 ◽  
Vol 33 (4) ◽  
pp. 599-605 ◽  
Author(s):  
Hosung Joe ◽  
Young Joon Seo
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Surface Area ◽  
Biofilm Formation ◽  
Fluorescent Protein ◽  
Otitis Media With Effusion ◽  
Fluorescent Microscopy ◽  
Confocal Laser ◽  
Tympanostomy Tube ◽  
Definitive Evidence

Biofilm formation has been implicated as a cause of post-tympanostomy tube otorrhea in patients suffering from otitis media with effusion, and biofilms have been found to adhere to all available types of tympanostomy tubes (TT) made from silicone. In this study, we present a novel stent designed with a reduced surface area and a titanium dioxide (TiO2) coating to prevent biofilm formation. Using a radio frequency power supply, tympanostomy stents (TS) made from Nitinol (Nikel-titanium) were coated with TiO2 to form an oxide layer on the metallic target. We successfully reproduced biofilms with carbenicillin-resistant Pseudomonas aeruginosa strain, PAO1-GFP (green fluorescent protein) on the tubes in vitro. We then compared the levels of biofilm formation by this strain on the two types of implants using several methods, including bacterial quantification, electron microscopy, and confocal laser fluorescent microscopy. Our results provide definitive evidence that the combination of the TiO2 coating and minimized surface area of the Nitinol stent inhibited the P. aeruginosa biofilm formation. The ability of the TS to prevent viable bacteria colonization (over 10 folds, compared to silicone TT) was verified by anti-biofilm test. Future studies will reveal more useful in reducing otorrhea and plugging complications as a novel tympanostomy tube.

Organ-specific change inDolichos biflorus lectin binding by myocardial endothelial cells during in vitro cultivation

1993 ◽  
Vol 29 (1) ◽  
pp. 25-31 ◽  
Author(s):  
Johanna Plendl ◽  
Laura Hartwell ◽  
Robert Auerbach
Keyword(s):  
Endothelial Cells ◽  
Lectin Binding ◽  
In Vitro Cultivation ◽  
Specific Change ◽  
Organ Specific

scholarly journals An Electromagnetic System for Inducing a Localized Force Gradient in an ECM and Its Influence on HMVEC Sprouting

2017 ◽  
Vol 23 (1) ◽  
pp. 70-82 ◽  
Author(s):  
Hian Hian See ◽  
Sahan C. B. Herath ◽  
Rerngchai Arayanarakool ◽  
Yue Du ◽  
Evan Tan ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Experimental Results ◽  
Microvascular Endothelial Cells ◽  
Electromagnetic System ◽  
Magnetic Forces ◽  
Subsequent Response ◽  
Microvascular Endothelial

Mechanical properties of the extracellular matrix (ECM) have been observed to influence the behavior of cells. Investigations on such an influence commonly rely on using soluble cues to alter the global intrinsic ECM properties in order to study the subsequent response of cells. This article presents an electromagnetic system for inducing a localized force gradient in an ECM, and reports the experimentally observed effect of such a force gradient on in vitro angiogenic sprouting of human microvascular endothelial cells (HMVECs). This force gradient is realized through the induction of magnetic forces on the superparamagnetic microparticle–embedded ECM ( sECM). Both analytical and statistically meaningful experimental results demonstrate the effectiveness of this approach in influencing the behavior of a targeted HMVEC sprout without affecting that of other sprouts nearby. These results suggest the possibility of selectively controlling the in vitro behavior of cells by the induction of a localized force gradient in the ECM.

scholarly journals Replication-Competent ΔNS1 Influenza A Viruses Expressing Reporter Genes

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 698
Author(s):  
Aitor Nogales ◽  
Michael Schotsaert ◽  
Raveen Rathnasinghe ◽  
Marta L. DeDiego ◽  
Adolfo García-Sastre ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Influenza A ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Fluorescent Microscopy ◽  
Reporter Genes ◽  
In Vivo Studies ◽  
Structural Protein ◽  
Infected Cells

The influenza A virus (IAV) is able to infect multiple mammalian and avian species, and in humans IAV is responsible for annual seasonal epidemics and occasional pandemics of respiratory disease with significant health and economic impacts. Studying IAV involves laborious secondary methodologies to identify infected cells. Therefore, to circumvent this requirement, in recent years, multiple replication-competent infectious IAV expressing traceable reporter genes have been developed. These IAVs have been very useful for in vitro and/or in vivo studies of viral replication, identification of neutralizing antibodies or antivirals, and in studies to evaluate vaccine efficacy, among others. In this report, we describe, for the first time, the generation and characterization of two replication-competent influenza A/Puerto Rico/8/1934 H1N1 (PR8) viruses where the viral non-structural protein 1 (NS1) was substituted by the monomeric (m)Cherry fluorescent or the NanoLuc luciferase (Nluc) proteins. The ΔNS1 mCherry was able to replicate in cultured cells and in Signal Transducer and Activator of Transcription 1 (STAT1) deficient mice, although at a lower extent than a wild-type (WT) PR8 virus expressing the same mCherry fluorescent protein (WT mCherry). Notably, expression of either reporter gene (mCherry or Nluc) was detected in infected cells by fluorescent microscopy or luciferase plate readers, respectively. ΔNS1 IAV expressing reporter genes provide a novel approach to better understand the biology and pathogenesis of IAV, and represent an excellent tool to develop new therapeutic approaches against IAV infections.

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