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An angiogenesis platform using a cubic artificial eggshell with patterned blood vessels on chicken chorioallantoic membrane.

ABSTRACT: The chorioallantoic membrane (CAM) containing tiny blood vessels is an alternative to large animals for studies involving angiogenesis and tissue engineering. However, there is no technique to design the direction of growing blood vessels on the CAM at the microscale level for tissue engineering experiments. Here, a methodology is provided to direct blood vessel formation on the surface of a three-dimensional egg yolk using a cubic artificial eggshell with six functionalized membranes. A structure on the lateral side of the eggshell containing a straight channel and an interlinked chamber was designed, and the direction and formation area of blood vessels with blood flow was artfully defined by channels with widths of 70-2000 ?m, without sharply reducing embryo viability. The relationship between the size of interlinked chamber and the induction of blood vessels was investigated to establish a theory of design. Role of negative and positive pressure in the induction of CAM with blood vessels was investigated, and air pressure change in the culture chamber was measured to demonstrate the mechanism for blood vessel induction. Histological evaluation showed that components of CAM including chorionic membrane and blood vessels were induced into the channels. Based on our design theory, blood vessels were induced into arrayed channels, and channel-specific injection and screening were realized, which demonstrated proposed applications. The platform with position- and space-controlled blood vessels is therefore a powerful tool for biomedical research, which may afford exciting applications in studies involved in local stimulation of blood vessel networks and those necessary to establish a living system with blood flow from a beating heart.

SUBMITTER: Huang W

PROVIDER: S-EPMC5393577 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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An angiogenesis platform using a cubic artificial eggshell with patterned blood vessels on chicken chorioallantoic membrane.

Huang Wenjing W Itayama Makoto M Arai Fumihito F Furukawa Katsuko S KS Ushida Takashi T Kawahara Tomohiro T

PloS one 20170417 4

The chorioallantoic membrane (CAM) containing tiny blood vessels is an alternative to large animals for studies involving angiogenesis and tissue engineering. However, there is no technique to design the direction of growing blood vessels on the CAM at the microscale level for tissue engineering experiments. Here, a methodology is provided to direct blood vessel formation on the surface of a three-dimensional egg yolk using a cubic artificial eggshell with six functionalized membranes. A structu ...[more]

PMID: 28414752

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Project description:During chicken embryonic development, skeleton calcification mainly relies on the eggshell, whose minerals are progressively solubilized and transported to the embryo via the chorioallantoic membrane (CAM). However, the molecular components involved in this process remain undefined. We assessed eggshell demineralization and calcification of the embryo skeleton after 12 and 16 d of incubation, and analyzed the expression of several candidate genes in the CAM: carbonic anhydrases that are likely involved in secretion of protons for eggshell dissolution (CA2, CA4, CA9), ions transporters and regulators (CALB1, SLC4A1, ATP6V1B2, SGK1, SCGN, PKD2) and vitamin-D binding protein (GC). Our results confirmed that eggshell weight, thickness, and strength decreased during incubation, with a concomitant increase in calcification of embryonic skeletal system. In the CAM, the expression of CA2 increased during incubation while CA4 and CA9 were expressed at similar levels at both stages. SCL4A1 and SCGN were expressed, but not differentially, between the two stages, while the expression of ATP6V1B2 and PKD2 genes decreased. The expression of SGK1 and TRPV6 increased over time, although the expression of the latter gene was barely detectable. In parallel, we analyzed the expression of these candidate genes in the yolk sac (YS), which mediates the transfer of yolk minerals to the embryo during the first half of incubation. In YS, CA2 expression increases during incubation, similar to the CAM, while the expression of the other candidate genes decreases. Moreover, CALB1 and GC genes were found to be expressed during incubation in the YS, in contrast to the CAM where no expression of either was detected. This study demonstrates that the regulation of genes involved in the mobilization of egg minerals during embryonic development is different between the YS and CAM extraembryonic structures. Identification of the full suite of molecular components involved in the transfer of eggshell calcium to the embryo via the CAM should help to better understand the role of this structure in bone mineralization.

Project description:Introduction: Recently, MicroRNAs have gained increasing popularity as a novel nucleic acid-mediated medicine to regulate cancer-related protein expression. MicroRNA-21 (miR-21) is known as an oncogenic microRNA which is overexpressed in almost all cancers, including ovarian carcinoma that causes cisplatin (cis-Pt) resistance and vascular endothelial growth factor (VEGF) upregulation. So, miRNA-based therapy can be regarded as knocking down miR-21 expression, inducing tumor cell apoptosis, and suppressing tumor-associated angiogenesis. Methods: PEG5k-carboxymethylated polyethyleneimine nanohydrogels (PEG5k-CMPEI) were loaded with AntagomiR-21 (As-21) at different ratios of nitrogen to phosphorus (N/P). Particle size and ζ potential were determined for the As-21 loaded nanohydrogels. In the cellular experiments, miR-21 expression, cytotoxicity, and cis-Pt sensitivity were studied on A2780 ovarian cancer cell lines. Finally, tumor cell apoptosis and tumor cell-associated angiogenesis were explored in vitro and in vivo. Results: The nanohydrogels, featuring homogeneous size distribution and redox-responsiveness, were steadily loaded by As-21 at the optimum N/P ratio of 5 without any aggregation as determined by transmission electron microscopy (TEM). As-21-loaded nanohydrogels caused sequence-specific suppression of miR-21 expression and provoked apoptosis through ROS generation and caspase 3 activation. Cisplatin cytotoxicity was remarkably enhanced in A2780R as compared to A2780S following co-incubation with As-21-loaded nanohydrogels. Interestingly, the condition of the medium derived from As-21 nanohydrogel-treated A2780R cells inhibited VEGF suppression in human umbilical vein endothelial cells (HUVECs) and the formation of tubes in Matrigel. Moreover, the condition medium caused angiogenesis inhibition in the chicken chorioallantoic membrane (CAM) model. Conclusion: These results suggest that nanohydrogel-based delivery of As-21 can be a promising neoadjuvant therapy for treating resistant tumors via apoptosis induction and angiogenesis suppression.

Dataset Information

An angiogenesis platform using a cubic artificial eggshell with patterned blood vessels on chicken chorioallantoic membrane.

Publications

An angiogenesis platform using a cubic artificial eggshell with patterned blood vessels on chicken chorioallantoic membrane.

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