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Egg-in-cube: design and fabrication of a novel artificial eggshell with functionalized surface.

Huang W, Arai F, Kawahara T - PLoS ONE (2015)

Bottom Line: To test the effectiveness of the design, the cubic eggshells were used to culture chick embryos and survivability was confirmed when PDMS membranes with adequate oxygen permeability were used.Additionally, the chick embryo tissues could be accessed and manipulated from outside the cubic eggshell, by using mechanical tools without breakage of the eggshell.The proposed "Egg-in-Cube" with functionalized surface has great potential to serve as a promising platform for biomedical research.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Functions Engineering, Kyushu Institute of Technology, Wakamatsu-ku, Kitakyushu, Japan.

ABSTRACT
An eggshell is a porous microstructure that regulates the passage of gases to allow respiration. The chick embryo and its circulatory system enclosed by the eggshell has become an important model for biomedical research such as the control of angiogenesis, cancer therapy, and drug delivery test, because the use of embryo is ethically acceptable and it is inexpensive and small. However, chick embryo and extra-embryonic blood vessels cannot be accessed freely and has poor observability because the eggshell is tough and cannot be seen through, which limits its application. In this study, a novel artificial eggshell with functionalized surface is proposed, which allows the total amount of oxygen to pass into the egg for the chick embryo culturing and has high observability and accessibility for embryo manipulation. First, a 40-mm enclosed cubic-shaped eggshell consisting of a membrane structure and a rigid frame structure is designed, and then the threshold of the membrane thickness suitable for the embryo survival is figured out according to the oxygen-permeability of the membrane structure. The designed artificial eggshell was actually fabricated by using polydimethylsiloxane (PDMS) and polycarbonate (PC) in the current study. Using the fabricated eggshell, chick embryo and extra-embryonic blood vessels can be observed from multiple directions. To test the effectiveness of the design, the cubic eggshells were used to culture chick embryos and survivability was confirmed when PDMS membranes with adequate oxygen permeability were used. Since the surface of the eggshell is transparent, chick embryo tissue development could be observed during the culture period. Additionally, the chick embryo tissues could be accessed and manipulated from outside the cubic eggshell, by using mechanical tools without breakage of the eggshell. The proposed "Egg-in-Cube" with functionalized surface has great potential to serve as a promising platform for biomedical research.

No MeSH data available.


Related in: MedlinePlus

Spatial control of embryo blood vessel formation on the patterned side membranes.The total oxygen volume permeating into each cubic eggshell was calculated to be comparable to that of cubic eggshells fabricated from six 0.3-mm-thick PDMS membranes. (a) Typical images of blood vessel formation in oxygenated channels with widths of 5, 10, and 16 mm, respectively. Polystyrene plates were glued on a 0.1-mm-thick PDMS membrane to make an oxygen non-permeable (NP) area. From day 4 to 7, blood vessels grew selectively in the oxygen-permeable channels. (b) Area ratios of blood vessels on a whole lateral-side membrane (width: 32 mm) made from a 0.3-mm-thick membrane and in the channels with widths of 5, 10, and 16 mm (0.1-mm-thick membrane). From day 4 to 7, the area of blood vessel formation decreased prominently in the narrow channel because of the decrease of oxygenated area on the patterned lateral-side membrane. (c) Height ratios of the area of blood vessel formation from day 4 to 7. On a whole lateral-side membrane or in the oxygenated channel with a width of 16 mm, blood vessels spread rapidly after the transferring of egg contents. On the other hand, blood vessels grew slowly in the oxygenated channel with a width of 5 mm. The error bar is SEM (n = 5). The detailed process of image analysis and definition of Ab and Hb are shown in S4 Fig.
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pone.0118624.g007: Spatial control of embryo blood vessel formation on the patterned side membranes.The total oxygen volume permeating into each cubic eggshell was calculated to be comparable to that of cubic eggshells fabricated from six 0.3-mm-thick PDMS membranes. (a) Typical images of blood vessel formation in oxygenated channels with widths of 5, 10, and 16 mm, respectively. Polystyrene plates were glued on a 0.1-mm-thick PDMS membrane to make an oxygen non-permeable (NP) area. From day 4 to 7, blood vessels grew selectively in the oxygen-permeable channels. (b) Area ratios of blood vessels on a whole lateral-side membrane (width: 32 mm) made from a 0.3-mm-thick membrane and in the channels with widths of 5, 10, and 16 mm (0.1-mm-thick membrane). From day 4 to 7, the area of blood vessel formation decreased prominently in the narrow channel because of the decrease of oxygenated area on the patterned lateral-side membrane. (c) Height ratios of the area of blood vessel formation from day 4 to 7. On a whole lateral-side membrane or in the oxygenated channel with a width of 16 mm, blood vessels spread rapidly after the transferring of egg contents. On the other hand, blood vessels grew slowly in the oxygenated channel with a width of 5 mm. The error bar is SEM (n = 5). The detailed process of image analysis and definition of Ab and Hb are shown in S4 Fig.

Mentions: An advantage of the developed cubic eggshell is that the thickness of the PDMS membrane (oxygen permeability) can be freely changed under the conditions of Equation (1) shown under the Design of the artificial (cubic) eggshell in this article. For example, the permeating oxygen volume can be adjusted to an appropriate level for chick embryo development by changing the thickness of the membranes and oxygen non-permeable plates. In this experiment, in order to figure out the relationship between the oxygen permeable area and the growth of blood vessels on the lateral-side membranes, the lateral-side membranes of the cubic eggshell were fabricated using patterned membranes with oxygen permeable and non-permeable areas of different widths. We attached two polystyrene plates on the left and right sides of the lateral-side membrane to form two oxygen non-permeable areas as shown in Fig. 7(A). The middle area of the side face was a 0.1-mm-thick PDMS membrane with widths of 5, 10, or 16 mm. According to Equation (1) shown under the Design of the artificial (cubic) eggshell in this article, since there was a large decrease in the oxygen-permeating area on the face with a 5- or 10-mm-wide channel, we changed the membrane thickness of another side face from 0.3 mm to 0.1 mm, which ensured that the total oxygen permeating volume was comparable to that permeating in the eggshell fabricated using the 0.3-mm-thick PDMS membrane. In the channel with a width of 16 mm, since the oxygen permeating rate for 0.1 mm is more than twice as large as that for 0.3 mm, oxygen permeation will be relatively larger than that permeating over the whole face made of a 0.3-mm-thick membrane. Therefore, the total permeating oxygen volume of the patterned eggshell is comparable to that of the cubic eggshell fabricated using 0.3-mm-thick PDMS membranes. In order to clarify the changes of blood vessel formation in the channel with widths of 5, 10, and 16 mm, the blood vessel formation on the 0.3-mm-thick lateral-side membrane (width: 32 mm) was used as a control. From Fig. 7(A), we found that blood vessels formed selectively in the oxygen permeable channels made of the 0.1-mm-thick PDMS membranes. Since the lateral-side membrane was transparent and the bottom edge of the spreading blood vessel network could be clearly observed, we calculated the degree of vasculature using the area revealed by the bottom edge. In this study, we selected eggs with almost the same volume, and the spreading area and height of blood vessel network was divided by the area occupied by egg albumen and its depth, respectively. This quotient was defined as the area/height ratio. The detailed process of image analysis is shown in S4 Fig.Fig. 7(B) shows that the area ratio of blood vessel network decreased when the width of the oxygen permeable channel became narrow. In addition, as shown in Fig. 7(C), the growth speed of blood vessel network in the narrow channel was also slower than that in a wider one. These results suggest that, by precisely controlling the oxygen permeation on one surface with a designed pattern, we can create new functions such as regulation of the growth and spacing of new blood vessels. This result demonstrates promising applications of these eggshells for biomedical research, such as cardiovascular simulation.


Egg-in-cube: design and fabrication of a novel artificial eggshell with functionalized surface.

Huang W, Arai F, Kawahara T - PLoS ONE (2015)

Spatial control of embryo blood vessel formation on the patterned side membranes.The total oxygen volume permeating into each cubic eggshell was calculated to be comparable to that of cubic eggshells fabricated from six 0.3-mm-thick PDMS membranes. (a) Typical images of blood vessel formation in oxygenated channels with widths of 5, 10, and 16 mm, respectively. Polystyrene plates were glued on a 0.1-mm-thick PDMS membrane to make an oxygen non-permeable (NP) area. From day 4 to 7, blood vessels grew selectively in the oxygen-permeable channels. (b) Area ratios of blood vessels on a whole lateral-side membrane (width: 32 mm) made from a 0.3-mm-thick membrane and in the channels with widths of 5, 10, and 16 mm (0.1-mm-thick membrane). From day 4 to 7, the area of blood vessel formation decreased prominently in the narrow channel because of the decrease of oxygenated area on the patterned lateral-side membrane. (c) Height ratios of the area of blood vessel formation from day 4 to 7. On a whole lateral-side membrane or in the oxygenated channel with a width of 16 mm, blood vessels spread rapidly after the transferring of egg contents. On the other hand, blood vessels grew slowly in the oxygenated channel with a width of 5 mm. The error bar is SEM (n = 5). The detailed process of image analysis and definition of Ab and Hb are shown in S4 Fig.
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Related In: Results  -  Collection

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pone.0118624.g007: Spatial control of embryo blood vessel formation on the patterned side membranes.The total oxygen volume permeating into each cubic eggshell was calculated to be comparable to that of cubic eggshells fabricated from six 0.3-mm-thick PDMS membranes. (a) Typical images of blood vessel formation in oxygenated channels with widths of 5, 10, and 16 mm, respectively. Polystyrene plates were glued on a 0.1-mm-thick PDMS membrane to make an oxygen non-permeable (NP) area. From day 4 to 7, blood vessels grew selectively in the oxygen-permeable channels. (b) Area ratios of blood vessels on a whole lateral-side membrane (width: 32 mm) made from a 0.3-mm-thick membrane and in the channels with widths of 5, 10, and 16 mm (0.1-mm-thick membrane). From day 4 to 7, the area of blood vessel formation decreased prominently in the narrow channel because of the decrease of oxygenated area on the patterned lateral-side membrane. (c) Height ratios of the area of blood vessel formation from day 4 to 7. On a whole lateral-side membrane or in the oxygenated channel with a width of 16 mm, blood vessels spread rapidly after the transferring of egg contents. On the other hand, blood vessels grew slowly in the oxygenated channel with a width of 5 mm. The error bar is SEM (n = 5). The detailed process of image analysis and definition of Ab and Hb are shown in S4 Fig.
Mentions: An advantage of the developed cubic eggshell is that the thickness of the PDMS membrane (oxygen permeability) can be freely changed under the conditions of Equation (1) shown under the Design of the artificial (cubic) eggshell in this article. For example, the permeating oxygen volume can be adjusted to an appropriate level for chick embryo development by changing the thickness of the membranes and oxygen non-permeable plates. In this experiment, in order to figure out the relationship between the oxygen permeable area and the growth of blood vessels on the lateral-side membranes, the lateral-side membranes of the cubic eggshell were fabricated using patterned membranes with oxygen permeable and non-permeable areas of different widths. We attached two polystyrene plates on the left and right sides of the lateral-side membrane to form two oxygen non-permeable areas as shown in Fig. 7(A). The middle area of the side face was a 0.1-mm-thick PDMS membrane with widths of 5, 10, or 16 mm. According to Equation (1) shown under the Design of the artificial (cubic) eggshell in this article, since there was a large decrease in the oxygen-permeating area on the face with a 5- or 10-mm-wide channel, we changed the membrane thickness of another side face from 0.3 mm to 0.1 mm, which ensured that the total oxygen permeating volume was comparable to that permeating in the eggshell fabricated using the 0.3-mm-thick PDMS membrane. In the channel with a width of 16 mm, since the oxygen permeating rate for 0.1 mm is more than twice as large as that for 0.3 mm, oxygen permeation will be relatively larger than that permeating over the whole face made of a 0.3-mm-thick membrane. Therefore, the total permeating oxygen volume of the patterned eggshell is comparable to that of the cubic eggshell fabricated using 0.3-mm-thick PDMS membranes. In order to clarify the changes of blood vessel formation in the channel with widths of 5, 10, and 16 mm, the blood vessel formation on the 0.3-mm-thick lateral-side membrane (width: 32 mm) was used as a control. From Fig. 7(A), we found that blood vessels formed selectively in the oxygen permeable channels made of the 0.1-mm-thick PDMS membranes. Since the lateral-side membrane was transparent and the bottom edge of the spreading blood vessel network could be clearly observed, we calculated the degree of vasculature using the area revealed by the bottom edge. In this study, we selected eggs with almost the same volume, and the spreading area and height of blood vessel network was divided by the area occupied by egg albumen and its depth, respectively. This quotient was defined as the area/height ratio. The detailed process of image analysis is shown in S4 Fig.Fig. 7(B) shows that the area ratio of blood vessel network decreased when the width of the oxygen permeable channel became narrow. In addition, as shown in Fig. 7(C), the growth speed of blood vessel network in the narrow channel was also slower than that in a wider one. These results suggest that, by precisely controlling the oxygen permeation on one surface with a designed pattern, we can create new functions such as regulation of the growth and spacing of new blood vessels. This result demonstrates promising applications of these eggshells for biomedical research, such as cardiovascular simulation.

Bottom Line: To test the effectiveness of the design, the cubic eggshells were used to culture chick embryos and survivability was confirmed when PDMS membranes with adequate oxygen permeability were used.Additionally, the chick embryo tissues could be accessed and manipulated from outside the cubic eggshell, by using mechanical tools without breakage of the eggshell.The proposed "Egg-in-Cube" with functionalized surface has great potential to serve as a promising platform for biomedical research.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Functions Engineering, Kyushu Institute of Technology, Wakamatsu-ku, Kitakyushu, Japan.

ABSTRACT
An eggshell is a porous microstructure that regulates the passage of gases to allow respiration. The chick embryo and its circulatory system enclosed by the eggshell has become an important model for biomedical research such as the control of angiogenesis, cancer therapy, and drug delivery test, because the use of embryo is ethically acceptable and it is inexpensive and small. However, chick embryo and extra-embryonic blood vessels cannot be accessed freely and has poor observability because the eggshell is tough and cannot be seen through, which limits its application. In this study, a novel artificial eggshell with functionalized surface is proposed, which allows the total amount of oxygen to pass into the egg for the chick embryo culturing and has high observability and accessibility for embryo manipulation. First, a 40-mm enclosed cubic-shaped eggshell consisting of a membrane structure and a rigid frame structure is designed, and then the threshold of the membrane thickness suitable for the embryo survival is figured out according to the oxygen-permeability of the membrane structure. The designed artificial eggshell was actually fabricated by using polydimethylsiloxane (PDMS) and polycarbonate (PC) in the current study. Using the fabricated eggshell, chick embryo and extra-embryonic blood vessels can be observed from multiple directions. To test the effectiveness of the design, the cubic eggshells were used to culture chick embryos and survivability was confirmed when PDMS membranes with adequate oxygen permeability were used. Since the surface of the eggshell is transparent, chick embryo tissue development could be observed during the culture period. Additionally, the chick embryo tissues could be accessed and manipulated from outside the cubic eggshell, by using mechanical tools without breakage of the eggshell. The proposed "Egg-in-Cube" with functionalized surface has great potential to serve as a promising platform for biomedical research.

No MeSH data available.


Related in: MedlinePlus