Limits...
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

Vessels distributions inside four different samples implanted for 4 weeks in the mice. The size-bar corresponds to 30 μm. The first two samples, A and B, were both prepared with MICROFILL® but, while the sample A was not pre-seeded with BMSCs before implantation, the sample B was pre-seeded with BMSCs. The sample C was also pre-seeded with BMSCs, but after the recovery of the scaffold from the animal it was stained with PTA. The sample D was a BMSC seeded not stained sample. (A) The vessels in the sample A are rendered in red, the scaffold in blue, and the soft tissue in yellow and green. The inset shows the main vessel partially filled with MICROFIL®. (B) The 3D volume of the sample B was reported. The inset shows a very intricate collagen matrix (rendered in yellow) coexists with the vessels (light green). The newly formed bone is rendered in light blue. (C) The 3D volume of sample C is reported. The segmentation renders the vessels in red and the scaffold in blue. The soft tissues were computationally removed from the 3D rendering to highlight the vessels distribution inside the scaffold. The inset shows in red the numerous vessels crossing the soft tissue (segmented in green). (D) The sample D was BMSC seeded but not stained. The vessels are rendered in red, the soft tissue in yellow and green. The inset shows one of the ramified vessels in red.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4561513&req=5

Figure 2: Vessels distributions inside four different samples implanted for 4 weeks in the mice. The size-bar corresponds to 30 μm. The first two samples, A and B, were both prepared with MICROFILL® but, while the sample A was not pre-seeded with BMSCs before implantation, the sample B was pre-seeded with BMSCs. The sample C was also pre-seeded with BMSCs, but after the recovery of the scaffold from the animal it was stained with PTA. The sample D was a BMSC seeded not stained sample. (A) The vessels in the sample A are rendered in red, the scaffold in blue, and the soft tissue in yellow and green. The inset shows the main vessel partially filled with MICROFIL®. (B) The 3D volume of the sample B was reported. The inset shows a very intricate collagen matrix (rendered in yellow) coexists with the vessels (light green). The newly formed bone is rendered in light blue. (C) The 3D volume of sample C is reported. The segmentation renders the vessels in red and the scaffold in blue. The soft tissues were computationally removed from the 3D rendering to highlight the vessels distribution inside the scaffold. The inset shows in red the numerous vessels crossing the soft tissue (segmented in green). (D) The sample D was BMSC seeded but not stained. The vessels are rendered in red, the soft tissue in yellow and green. The inset shows one of the ramified vessels in red.

Mentions: In order to investigate by XRPCμT the 3D micro-vessels distribution inside the scaffold, a high spatial resolution is required. For this reason, we used the in-line propagation setup of the TOMCAT beamline at SLS, sketched in Figure 1, able to achieve a spatial resolution of 0.64 μm. In the table shown in Figure 1B, we reported the characteristics of the different investigated sample groups. Figure 2 shows details of the vessels distribution inside representative samples from the four different groups implanted for 4 weeks in the mice. The first two samples (A and B) were both stained with MICROFIL®, a compound that fills and enhances the opacity of the micro-vascular network. In sample A, no BMSC were seeded on the scaffold, whereas the scaffold of sample B was seeded with BMSC. Sample C was also seeded with BMSC, but, after its recovery from the animal, it was stained with PTA. PTA enhances the soft tissue (ST) signal and thus it was expected to improve the visualization of both the vessels entering the scaffold pores and the collagenous matrix (Campi et al., 2013). Figure 2D displays the tomographic image of the sample D, which was seeded with BMSC (like B and C samples), but neither it was prepared with MICROFIL®, nor it underwent staining treatment.


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)

Vessels distributions inside four different samples implanted for 4 weeks in the mice. The size-bar corresponds to 30 μm. The first two samples, A and B, were both prepared with MICROFILL® but, while the sample A was not pre-seeded with BMSCs before implantation, the sample B was pre-seeded with BMSCs. The sample C was also pre-seeded with BMSCs, but after the recovery of the scaffold from the animal it was stained with PTA. The sample D was a BMSC seeded not stained sample. (A) The vessels in the sample A are rendered in red, the scaffold in blue, and the soft tissue in yellow and green. The inset shows the main vessel partially filled with MICROFIL®. (B) The 3D volume of the sample B was reported. The inset shows a very intricate collagen matrix (rendered in yellow) coexists with the vessels (light green). The newly formed bone is rendered in light blue. (C) The 3D volume of sample C is reported. The segmentation renders the vessels in red and the scaffold in blue. The soft tissues were computationally removed from the 3D rendering to highlight the vessels distribution inside the scaffold. The inset shows in red the numerous vessels crossing the soft tissue (segmented in green). (D) The sample D was BMSC seeded but not stained. The vessels are rendered in red, the soft tissue in yellow and green. The inset shows one of the ramified vessels in red.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561513&req=5

Figure 2: Vessels distributions inside four different samples implanted for 4 weeks in the mice. The size-bar corresponds to 30 μm. The first two samples, A and B, were both prepared with MICROFILL® but, while the sample A was not pre-seeded with BMSCs before implantation, the sample B was pre-seeded with BMSCs. The sample C was also pre-seeded with BMSCs, but after the recovery of the scaffold from the animal it was stained with PTA. The sample D was a BMSC seeded not stained sample. (A) The vessels in the sample A are rendered in red, the scaffold in blue, and the soft tissue in yellow and green. The inset shows the main vessel partially filled with MICROFIL®. (B) The 3D volume of the sample B was reported. The inset shows a very intricate collagen matrix (rendered in yellow) coexists with the vessels (light green). The newly formed bone is rendered in light blue. (C) The 3D volume of sample C is reported. The segmentation renders the vessels in red and the scaffold in blue. The soft tissues were computationally removed from the 3D rendering to highlight the vessels distribution inside the scaffold. The inset shows in red the numerous vessels crossing the soft tissue (segmented in green). (D) The sample D was BMSC seeded but not stained. The vessels are rendered in red, the soft tissue in yellow and green. The inset shows one of the ramified vessels in red.
Mentions: In order to investigate by XRPCμT the 3D micro-vessels distribution inside the scaffold, a high spatial resolution is required. For this reason, we used the in-line propagation setup of the TOMCAT beamline at SLS, sketched in Figure 1, able to achieve a spatial resolution of 0.64 μm. In the table shown in Figure 1B, we reported the characteristics of the different investigated sample groups. Figure 2 shows details of the vessels distribution inside representative samples from the four different groups implanted for 4 weeks in the mice. The first two samples (A and B) were both stained with MICROFIL®, a compound that fills and enhances the opacity of the micro-vascular network. In sample A, no BMSC were seeded on the scaffold, whereas the scaffold of sample B was seeded with BMSC. Sample C was also seeded with BMSC, but, after its recovery from the animal, it was stained with PTA. PTA enhances the soft tissue (ST) signal and thus it was expected to improve the visualization of both the vessels entering the scaffold pores and the collagenous matrix (Campi et al., 2013). Figure 2D displays the tomographic image of the sample D, which was seeded with BMSC (like B and C samples), but neither it was prepared with MICROFIL®, nor it underwent staining treatment.

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