scholarly journals Depletion of Embryonic Macrophages Leads to a Reduction in Angiogenesis in the Ex Ovo Chick Chorioallantoic Membrane Assay

Cells ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 5
Author(s):  
Hanna Tay ◽  
Charis Du Cheyne ◽  
Kristel Demeyere ◽  
Jurgen De Craene ◽  
Lobke De Bels ◽  
...  
Keyword(s):  
Chorioallantoic Membrane ◽  
Collagen Type I ◽  
Type I ◽  
Cam Assay ◽  
In Vivo Models ◽  
Chick Chorioallantoic Membrane ◽  
Angiogenesis Assay ◽  
Fold Reduction ◽  
Phosphate Buffered Saline

Macrophages play an important but poorly understood role in angiogenesis. To investigate their role in vessel formation, relevant in vivo models are crucial. Although the chick chorioallantoic membrane (CAM) model has been frequently used as an angiogenesis assay, limited data are available on the involvement of chicken macrophages in this process. Here, we describe a method to deplete macrophages in the ex ovo chick CAM assay by injection of clodronate liposomes and show that this depletion directly affects vascularisation of collagen onplants. Chicken embryos were injected intravenously with either clodronate or phosphate-buffered saline (PBS) liposomes, followed by placement of collagen type I plugs on the CAM to quantify angiogenic ingrowth. Clodronate liposome injection led to a significant 3.4-fold reduction of macrophages compared with control embryos as measured by immunohistochemistry and flow cytometry. Furthermore, analysis of vessel ingrowth into the collagen plugs revealed a significantly lower angiogenic response in macrophage-depleted embryos compared with control embryos, indicating that chicken embryonic macrophages play an essential function in the development of blood vessels. These results demonstrate that the chick CAM assay provides a promising model to investigate the role of macrophages in angiogenesis.

Evaluation of Electro-Spun Tubular Scaffolds to Create an Anastomosis Using the CAM Assay

2013 ◽  
Author(s):  
Adam Orendain ◽  
Jose Carrasco ◽  
Eniko T. Enikov ◽  
Gholam Peyman
Keyword(s):  
Growth Factor ◽  
Vascular Disease ◽  
Vascular Tissue ◽  
Uv Light ◽  
Chorioallantoic Membrane ◽  
Neovascular Glaucoma ◽  
Collagen Type I ◽  
Type I ◽  
Cam Assay ◽  
Chick Chorioallantoic Membrane

Central retinal vein occlusion (CRVO) is a vascular disease characterized by thrombosis of the retinal veins that can eventually lead to ischemia. Ischemic CRVO can then cause macular degeneration and neovascular glaucoma causing partial to full blindness. In this study, we determined the feasibility of electrospinning tubular scaffolds for treating CRVO and vascular disease. Electrospinning was utilized to produce customizable scaffolds from nano-bers using collagen type I. Scaffolds were treated with glutaraldehyde, glycine, ethanol, UV light, and combinations of the treatments for the purpose cross-linking and to study their angiogenic effects. Structural properties of the scaffolds were analyzed with scanning electron micrsoscopy (SEM). Scaffolds were immobilized with human recombinant vascular endothelial growth factor (rhVEGF165) to investigate the drug-delivering abilities of the electrospun materials and as a method to produce vascularization. The chick chorioallantoic membrane (CAM) assay was used to examine the effects of VEGF immobilizations and to evaluate the feasibility of creating an anastomosis to treat CRVO. Collagen onplants (non-electrospun) and electrospun implants were made on day 10 of embryonic development. Findings show collagen loaded with rhVEGF165 had improved vasculature and pro-angiogenic properties. The present study suggests that collagen can immobilize and release growth factor, be electrospun to mimic the ultrastructure of native blood vessels, and holds promise for vascular tissue engineering.

scholarly journals The Chorioallantoic Membrane Assay in Nanotoxicological Research—An Alternative for In Vivo Experimentation

Nanomaterials ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2328
Author(s):  
Christoph R. Buhr ◽  
Nadine Wiesmann ◽  
Rachel C. Tanner ◽  
Jürgen Brieger ◽  
Jonas Eckrich
Keyword(s):  
Imaging Techniques ◽  
Chorioallantoic Membrane ◽  
Drug Carriers ◽  
Human Organism ◽  
Cam Assay ◽  
In Ovo ◽  
In Vivo Models ◽  
Toxicological Profile ◽  
Application Fields

Nanomaterials unveil many applicational possibilities for technical and medical purposes, which range from imaging techniques to the use as drug carriers. Prior to any human application, analysis of undesired effects and characterization of their toxicological profile is mandatory. To address this topic, animal models, and rodent models in particular, are most frequently used. However, as the reproducibility and transferability to the human organism of animal experimental data is increasingly questioned and the awareness of animal welfare in society increases at the same time, methodological alternatives are urgently required. The chorioallantoic membrane (CAM) assay is an increasingly popular in ovo experimental organism suitable for replacement of rodent experimentation. In this review, we outline several application fields for the CAM assay in the field of nanotoxicology. Furthermore, analytical methods applicable with this model were evaluated in detail. We further discuss ethical, financial, and bureaucratic aspects and benchmark the assay with other established in vivo models such as rodents.

Antiangiogenic Iridoids from Stachys ocymastrum and Premna resinosa

Planta Medica ◽  
2019 ◽  
Vol 85 (11/12) ◽  
pp. 1034-1039 ◽  
Author(s):  
Anna Maria Iannuzzi ◽  
César Muñoz Camero ◽  
Massimiliano DʼAmbola ◽  
Valeria DʼAngelo ◽  
Smain Amira ◽  
...  
Keyword(s):  
Chorioallantoic Membrane ◽  
Zebrafish Embryos ◽  
Iridoid Glucoside ◽  
Leaf Extracts ◽  
In Vivo Models ◽  
Antiangiogenic Activity ◽  
Chick Chorioallantoic Membrane ◽  
Vessel Growth

AbstractIn this paper, the isolation of one new iridoid glucoside, 6β-acetoxyipolamiide (1), and thirteen (2 – 14) known congeners from two Lamiaceae species, Stachys ocymastrum and Premna resinosa, leaf extracts is reported. The structural determination of the isolated compounds was performed by mono- and bidimensional NMR spectroscopic analysis as well as MS experiments. The isolates were assayed for their antiangiogenic activity by two in vivo models, zebrafish embryos and chick chorioallantoic membrane assays. The compounds with a significant antiangiogenic activity in both assays were β-hydroxyipolamiide (2), ipolamiide (3), and buddlejoside A5 (8). 6-O-α-l-(3″-O-p-Methoxycinnamoyl-4″-O-acetyl)rhamnopyranosyl catalpol (13) and 6-O-α-l-(2″-trans-caffeoyl)rhamnopyranosyl catalpol (6) showed the best antiangiogenic response on blood vessel growth in zebrafish embryos, whereas saccatoside (10) and 6-O-α-l-(2″-O - p-methoxycinnamoyl-3″-O-acetyl)rhamnopyranosyl catalpol (14) resulted in a strong reduction of capillary formation in the chorioallantoic membrane assay.

scholarly journals Angiogenic Effects of Collagen/Mesoporous Nanoparticle Composite Scaffold Delivering VEGF165

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Joong-Hyun Kim ◽  
Tae-Hyun Kim ◽  
Min Sil Kang ◽  
Hae-Won Kim
Keyword(s):  
Type I Collagen ◽  
Composite Scaffold ◽  
Subcutaneous Tissue ◽  
Chorioallantoic Membrane ◽  
Silica Nanoparticle ◽  
Angiogenic Factor ◽  
Type I ◽  
Chick Chorioallantoic Membrane ◽  
Endothelial Growth Factor

Vascularization is a key issue for the success of tissue engineering to repair damaged tissue. In this study, we report a composite scaffold delivering angiogenic factor for this purpose. Vascular endothelial growth factor (VEGF) was loaded on mesoporous silica nanoparticle (MSN), which was then incorporated within a type I collagen sponge, to produce collagen/MSN/VEGF (CMV) scaffold. The CMV composite scaffold could release VEGF sustainably over the test period of 28 days. The release of VEGF improved the cell proliferation. Moreover, thein vivoangiogenesis of the scaffold, as studied by the chick chorioallantoic membrane (CAM) model, showed that the VEGF-releasing scaffold induced significantly increased number of blood vessel complexes when compared with VEGF-free scaffold. The composite scaffold showed good biocompatibility, as examined in rat subcutaneous tissue. These results demonstrate that the CMV scaffold with VEGF-releasing capacity can be potentially used to stimulate angiogenesis and tissue repair.

scholarly journals Neuroprotection in early stages of Alzheimers Disease is promoted by Transthyretin angiogenic properties

2021 ◽  
Author(s):  
Tiago Gião ◽  
Joana Saavedra ◽  
José Ricardo Vieira ◽  
Marta Teixeira Pinto ◽  
Gemma Arsequell ◽  
...  
Keyword(s):  
Chorioallantoic Membrane ◽  
Tube Formation ◽  
Cam Assay ◽  
Vessel Length ◽  
Chick Chorioallantoic Membrane ◽  
Brain Microvessels ◽  
Formation Conditions ◽  
Therapeutic Avenue ◽  
Important Therapeutic Target

While still controversial, it has been demonstrated that vascular defects can precede the onset of the other AD hallmarks features, making it an important therapeutic target. Given that the protein transthyretin (TTR) has been established as neuroprotective in AD, here we investigated the influence of TTR in the vasculature. AD transgenic mice with TTR genetic reduction, AD/TTR+/-, exhibited a thicker BM in brain microvessels and decreased vessel length than animals with normal TTR levels, AD/TTR+/+. Further in vivo investigation, using the chick chorioallantoic membrane (CAM) assay, revealed that TTR is a pro-angiogenic molecule. Also, TTR increased the expression of key angiogenic molecules, by endothelial cells under tube formation conditions. We showed that TTR reduction leads to a thicker BM in AD mice than in NT animals, strengthening the idea that TTR is a neuroprotective protein. We also studied the effect of TTR tetrameric stabilization on BM thickness, showing that AD mice treated with iododiflunisal (IDIF) displayed a significant reduction of BM thickness and increased vessel length when compared to non-treated littermates. Our in vivo results show the involvement of TTR in angiogenesis, particularly as a modulator of vascular alterations occurring in AD. Since TTR is decreased early in AD, its tetrameric stabilization can represent a therapeutic avenue for the early treatment of AD through the maintenance of the vascular structure.

scholarly journals Microvascular Experimentation in the Chick Chorioallantoic Membrane as a Model for Screening Angiogenic Agents including from Gene-Modified Cells

2021 ◽  
Vol 23 (1) ◽  
pp. 452
Author(s):  
Donna C. Kennedy ◽  
Barbara Coen ◽  
Antony M. Wheatley ◽  
Karl J. A. McCullagh
Keyword(s):  
Low Cost ◽  
Chorioallantoic Membrane ◽  
Visual Assessment ◽  
Cam Assay ◽  
Chick Chorioallantoic Membrane ◽  
Assay Procedure ◽  
Angiogenic Effect ◽  
Cultivation Techniques

The chick chorioallantoic membrane (CAM) assay model of angiogenesis has been highlighted as a relatively quick, low cost and effective model for the study of pro-angiogenic and anti-angiogenic factors. The chick CAM is a highly vascularised extraembryonic membrane which functions for gas exchange, nutrient exchange and waste removal for the growing chick embryo. It is beneficial as it can function as a treatment screening tool, which bridges the gap between cell based in vitro studies and in vivo animal experimentation. In this review, we explore the benefits and drawbacks of the CAM assay to study microcirculation, by the investigation of each distinct stage of the CAM assay procedure, including cultivation techniques, treatment applications and methods of determining an angiogenic response using this assay. We detail the angiogenic effect of treatments, including drugs, metabolites, genes and cells used in conjunction with the CAM assay, while also highlighting the testing of genetically modified cells. We also present a detailed exploration of the advantages and limitations of different CAM analysis techniques, including visual assessment, histological and molecular analysis along with vascular casting methods and live blood flow observations.

scholarly journals Neuroprotection in early stages of Alzheimer’s disease is promoted by transthyretin angiogenic properties

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Tiago Gião ◽  
Joana Saavedra ◽  
José Ricardo Vieira ◽  
Marta Teixeira Pinto ◽  
Gemma Arsequell ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Chorioallantoic Membrane ◽  
Tube Formation ◽  
Cam Assay ◽  
Vessel Length ◽  
Angiogenic Potential ◽  
Chick Chorioallantoic Membrane ◽  
Brain Microvessels

Abstract Background While still controversial, it has been demonstrated that vascular defects can precede the onset of other AD hallmarks features, making it an important therapeutic target. Given that the protein transthyretin (TTR) has been established as neuroprotective in AD, here we investigated the influence of TTR in the vasculature. Methods We evaluated the thickness of the basement membrane and the length of brain microvessels, by immunohistochemistry, in AβPPswe/PS1A246E (AD) transgenic mice and non-transgenic mice (NT) bearing one (TTR+/−) or two (TTR+/+) copies of the TTR gene. The angiogenic potential of TTR was evaluated in vitro using the tube formation assay, and in vivo using the chick chorioallantoic membrane (CAM) assay. Results AD transgenic mice with TTR genetic reduction, AD/TTR+/−, exhibited a thicker BM in brain microvessels and decreased vessel length than animals with normal TTR levels, AD/TTR+/+. Further in vivo investigation, using the CAM assay, revealed that TTR is a pro-angiogenic molecule, and the neovessels formed are functional. Also, TTR increased the expression of key angiogenic molecules such as proteins interleukins 6 and 8, angiopoietin 2, and vascular endothelial growth factor, by endothelial cells, in vitro, under tube formation conditions. We showed that while TTR reduction also leads to a thicker BM in NT mice, this effect is more pronounced in AD mice than in NT animals, strengthening the idea that TTR is a neuroprotective protein. We also studied the effect of TTR tetrameric stabilization on BM thickness, showing that AD mice treated with the TTR tetrameric stabilizer iododiflunisal (IDIF) displayed a significant reduction of BM thickness and increased vessel length, when compared to non-treated littermates. Conclusion Our in vivo results demonstrate the involvement of TTR in angiogenesis, particularly as a modulator of vascular alterations occurring in AD. Since TTR is decreased early in AD, its tetrameric stabilization can represent a therapeutic avenue for the early treatment of AD through the maintenance of the vascular structure.

scholarly journals Collagen-Based Electrospun Materials for Tissue Engineering: A Systematic Review

2021 ◽  
Vol 8 (3) ◽  
pp. 39
Author(s):  
Britani N. Blackstone ◽  
Summer C. Gallentine ◽  
Heather M. Powell
Keyword(s):  
Systematic Review ◽  
Tissue Engineering ◽  
Collagen Type I ◽  
The Body ◽  
Pool Data ◽  
Type I ◽  
High Concentrations ◽  
Increased Strength

Collagen is a key component of the extracellular matrix (ECM) in organs and tissues throughout the body and is used for many tissue engineering applications. Electrospinning of collagen can produce scaffolds in a wide variety of shapes, fiber diameters and porosities to match that of the native ECM. This systematic review aims to pool data from available manuscripts on electrospun collagen and tissue engineering to provide insight into the connection between source material, solvent, crosslinking method and functional outcomes. D-banding was most often observed in electrospun collagen formed using collagen type I isolated from calfskin, often isolated within the laboratory, with short solution solubilization times. All physical and chemical methods of crosslinking utilized imparted resistance to degradation and increased strength. Cytotoxicity was observed at high concentrations of crosslinking agents and when abbreviated rinsing protocols were utilized. Collagen and collagen-based scaffolds were capable of forming engineered tissues in vitro and in vivo with high similarity to the native structures.

scholarly journals Properties of a bovine collagen type I membrane for guided bone regeneration applications

e-Polymers ◽  
2021 ◽  
Vol 21 (1) ◽  
pp. 210-221
Author(s):  
Igor S. Brum ◽  
Carlos N. Elias ◽  
Jorge J. de Carvalho ◽  
Jorge L. S. Pires ◽  
Mario J. S. Pereira ◽  
...  
Keyword(s):  
Bone Regeneration ◽  
Collagen Type ◽  
Mechanical Stability ◽  
Collagen Type I ◽  
Guided Bone Regeneration ◽  
Bone Volume ◽  
Male Rats ◽  
Toxicity Tests ◽  
Type I

Abstract Dental implant treatment requires an available bone volume in the implantation site to ensure the implant’s mechanical stability. When the bone volume is insufficient, one must resort to surgical means such as guided bone regeneration (GBR). In GBR surgery, bone grafts and membranes are used. The objective of this work is to manufacture and characterize the in vitro and in vivo properties of resorbable collagen type I membranes (Green Membrane®) for GBR. Membrane surface morphology was characterized by SEM and roughness was measured using an interferometric noncontact 3D system. In vivo skin sensitization and toxicity tests have been performed on Wistar rats. Bone defects were prepared in 24 adult male rats, filled with biomaterials (Blue Bone® and Bio Oss®) and covered with collagen membranes to maintain the mechanical stability of the site for bone regeneration. The incisions were closed with simple stitches; and 60 days after the surgery, the animals were euthanized. Results showed that the analyzed membrane was homogeneous, with collagen fiber webs and open pores. It had no sign of cytotoxicity and the cells at the insertion site showed no bone morphological changes. There was no tissue reaction and no statistical difference between Blue Bone® and Bio Oss® groups. The proposed membrane has no cytotoxicity and displays a biocompatibility profile that makes it suitable for GBR.

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