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High-Resolution X-Ray Techniques as New Tool to Investigate the 3D Vascularization of Engineered-Bone Tissue.

Bukreeva I, Fratini M, Campi G, Pelliccia D, Spanò R, Tromba G, Brun F, Burghammer M, Grilli M, Cancedda R, Cedola A, Mastrogiacomo M - Front Bioeng Biotechnol (2015)

Bottom Line: We compared samples seeded and not seeded with BMSC, as well as samples differently stained or unstained.Thanks to the high quality of the images, we investigated the 3D distribution of both vessels and collagen matrix and we obtained quantitative information for all different samples.We propose our approach as a tool for quantitative studies of angiogenesis in TE and for any pre-clinical investigation where a quantitative analysis of the vascular network is required.

View Article: PubMed Central - PubMed

Affiliation: Consiglio Nazionale delle Ricerche - Istituto NANOTEC, c/o Dipartimento di Fisica, Università Sapienza , Rome , Italy.

ABSTRACT
The understanding of structure-function relationships in normal and pathologic mammalian tissues is at the basis of a tissue engineering (TE) approach for the development of biological substitutes to restore or improve tissue function. In this framework, it is interesting to investigate engineered bone tissue, formed when porous ceramic constructs are loaded with bone marrow stromal cells (BMSC) and implanted in vivo. To monitor the relation between bone formation and vascularization, it is important to achieve a detailed imaging and a quantitative description of the complete three-dimensional vascular network in such constructs. Here, we used synchrotron X-ray phase-contrast micro-tomography to visualize and analyze the three-dimensional micro-vascular networks in bone-engineered constructs, in an ectopic bone formation mouse-model. We compared samples seeded and not seeded with BMSC, as well as samples differently stained or unstained. Thanks to the high quality of the images, we investigated the 3D distribution of both vessels and collagen matrix and we obtained quantitative information for all different samples. We propose our approach as a tool for quantitative studies of angiogenesis in TE and for any pre-clinical investigation where a quantitative analysis of the vascular network is required.

No MeSH data available.


Related in: MedlinePlus

(A) Typical diffraction radial profile, I(q), measured for ST (red circles), ST/B interface (green circles), and in the scaffold (black squares). The continuous lines represent the best fitting curves using a Gaussian line shape added to a power law behavior as background. (B) Orientation degree of the collagen molecules, given by the area under the peaks of the azimuthal profiles I(Φ), measured in ST (red circles) and at the ST/B interface (green circles). The continuous lines are the Gaussian curve fits above a constant background. (C) XRPCμT detail of the ST/B interface. (D) Spatial distribution of collagen molecular amount and alignment given by the color intensity and by the arrows vectors, respectively. Size-bar = 15 μm.
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Figure 5: (A) Typical diffraction radial profile, I(q), measured for ST (red circles), ST/B interface (green circles), and in the scaffold (black squares). The continuous lines represent the best fitting curves using a Gaussian line shape added to a power law behavior as background. (B) Orientation degree of the collagen molecules, given by the area under the peaks of the azimuthal profiles I(Φ), measured in ST (red circles) and at the ST/B interface (green circles). The continuous lines are the Gaussian curve fits above a constant background. (C) XRPCμT detail of the ST/B interface. (D) Spatial distribution of collagen molecular amount and alignment given by the color intensity and by the arrows vectors, respectively. Size-bar = 15 μm.

Mentions: Despite the high resolution of the tomographic images, some ambiguity may still be present, like in the insets of Figure 2C where the “hairy” region near the bone might be due to capillaries or to collagenous bundles. To discriminate in the intricate network of “hairy” structures (in green Figure 2) between a capillary network and collagenous oriented fibers, XRμD scanning was already used to explore the time evolution of collagen matrix and structure during the bone mineralization process (Cedola et al., 2006). Here, we used the same technique to confirm that the intricate network was due to collagen fibers matrix. Collagen diffraction gives a broad peak around q = 5.6 nm−1, due to the molecular lateral packing. In Figure 5A, we show the typical diffraction radial profiles, I(q), measured in the ST, at the ST/Bone (B) interface and in the scaffold. We could clearly observe how the collagen peak becomes more pronounced as the mineralization arises, i.e., at the ST/B interface. The orientation degree of the collagen molecules is given by the area under the peaks of the azimuthal profiles I(Φ). Typical azimuthal profiles measured in ST and at the ST/B interface, are reported in Figure 5B. Figure 5C shows a typical XRPCμCT image of the ST/B interface, while the spatial distribution of collagen amount and alignment at this interface is reported in Figure 5D. Specifically, the collagen amount, corresponding to the collagen peak area on each measured point, is given by the map intensity; at the same time, the collagen molecules orientation angle and orientation degree are represented by the directions and amplitudes of the black arrows, as in a vector plot. These scanning XRμD results identify the packing and alignment of the collagen molecules close to the newly formed bone far from the scaffold, well distinguished from the (micro)vessels network in the XRPCμT imaging.


High-Resolution X-Ray Techniques as New Tool to Investigate the 3D Vascularization of Engineered-Bone Tissue.

Bukreeva I, Fratini M, Campi G, Pelliccia D, Spanò R, Tromba G, Brun F, Burghammer M, Grilli M, Cancedda R, Cedola A, Mastrogiacomo M - Front Bioeng Biotechnol (2015)

(A) Typical diffraction radial profile, I(q), measured for ST (red circles), ST/B interface (green circles), and in the scaffold (black squares). The continuous lines represent the best fitting curves using a Gaussian line shape added to a power law behavior as background. (B) Orientation degree of the collagen molecules, given by the area under the peaks of the azimuthal profiles I(Φ), measured in ST (red circles) and at the ST/B interface (green circles). The continuous lines are the Gaussian curve fits above a constant background. (C) XRPCμT detail of the ST/B interface. (D) Spatial distribution of collagen molecular amount and alignment given by the color intensity and by the arrows vectors, respectively. Size-bar = 15 μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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Figure 5: (A) Typical diffraction radial profile, I(q), measured for ST (red circles), ST/B interface (green circles), and in the scaffold (black squares). The continuous lines represent the best fitting curves using a Gaussian line shape added to a power law behavior as background. (B) Orientation degree of the collagen molecules, given by the area under the peaks of the azimuthal profiles I(Φ), measured in ST (red circles) and at the ST/B interface (green circles). The continuous lines are the Gaussian curve fits above a constant background. (C) XRPCμT detail of the ST/B interface. (D) Spatial distribution of collagen molecular amount and alignment given by the color intensity and by the arrows vectors, respectively. Size-bar = 15 μm.
Mentions: Despite the high resolution of the tomographic images, some ambiguity may still be present, like in the insets of Figure 2C where the “hairy” region near the bone might be due to capillaries or to collagenous bundles. To discriminate in the intricate network of “hairy” structures (in green Figure 2) between a capillary network and collagenous oriented fibers, XRμD scanning was already used to explore the time evolution of collagen matrix and structure during the bone mineralization process (Cedola et al., 2006). Here, we used the same technique to confirm that the intricate network was due to collagen fibers matrix. Collagen diffraction gives a broad peak around q = 5.6 nm−1, due to the molecular lateral packing. In Figure 5A, we show the typical diffraction radial profiles, I(q), measured in the ST, at the ST/Bone (B) interface and in the scaffold. We could clearly observe how the collagen peak becomes more pronounced as the mineralization arises, i.e., at the ST/B interface. The orientation degree of the collagen molecules is given by the area under the peaks of the azimuthal profiles I(Φ). Typical azimuthal profiles measured in ST and at the ST/B interface, are reported in Figure 5B. Figure 5C shows a typical XRPCμCT image of the ST/B interface, while the spatial distribution of collagen amount and alignment at this interface is reported in Figure 5D. Specifically, the collagen amount, corresponding to the collagen peak area on each measured point, is given by the map intensity; at the same time, the collagen molecules orientation angle and orientation degree are represented by the directions and amplitudes of the black arrows, as in a vector plot. These scanning XRμD results identify the packing and alignment of the collagen molecules close to the newly formed bone far from the scaffold, well distinguished from the (micro)vessels network in the XRPCμT imaging.

Bottom Line: We compared samples seeded and not seeded with BMSC, as well as samples differently stained or unstained.Thanks to the high quality of the images, we investigated the 3D distribution of both vessels and collagen matrix and we obtained quantitative information for all different samples.We propose our approach as a tool for quantitative studies of angiogenesis in TE and for any pre-clinical investigation where a quantitative analysis of the vascular network is required.

View Article: PubMed Central - PubMed

Affiliation: Consiglio Nazionale delle Ricerche - Istituto NANOTEC, c/o Dipartimento di Fisica, Università Sapienza , Rome , Italy.

ABSTRACT
The understanding of structure-function relationships in normal and pathologic mammalian tissues is at the basis of a tissue engineering (TE) approach for the development of biological substitutes to restore or improve tissue function. In this framework, it is interesting to investigate engineered bone tissue, formed when porous ceramic constructs are loaded with bone marrow stromal cells (BMSC) and implanted in vivo. To monitor the relation between bone formation and vascularization, it is important to achieve a detailed imaging and a quantitative description of the complete three-dimensional vascular network in such constructs. Here, we used synchrotron X-ray phase-contrast micro-tomography to visualize and analyze the three-dimensional micro-vascular networks in bone-engineered constructs, in an ectopic bone formation mouse-model. We compared samples seeded and not seeded with BMSC, as well as samples differently stained or unstained. Thanks to the high quality of the images, we investigated the 3D distribution of both vessels and collagen matrix and we obtained quantitative information for all different samples. We propose our approach as a tool for quantitative studies of angiogenesis in TE and for any pre-clinical investigation where a quantitative analysis of the vascular network is required.

No MeSH data available.


Related in: MedlinePlus