Novel multimodal MRI and MicroCT imaging approach to quantify angiogenesis and 3D vascular architecture of biomaterials

Abstract

Quantitative assessment of functional perfusion capacity and vessel architecture is critical when validating biomaterials for regenerative medicine purposes and requires high-tech analytical methods. Here, combining two clinically relevant imaging techniques, (magnetic resonance imaging; MRI and microcomputed tomography; MicroCT) and using the chorioallantoic membrane (CAM) assay, we present and validate a novel functional and morphological three-dimensional (3D) analysis strategy to study neovascularization in biomaterials relevant for bone regeneration. Using our new pump-assisted approach, the two scaffolds, Optimaix (laminar structure mimicking entities of the diaphysis) and DegraPol (highly porous resembling spongy bone), were shown to directly affect the architecture of the ingrowing neovasculature. Perfusion capacity (MRI) and total vessel volume (MicroCT) strongly correlated for both biomaterials, suggesting that our approach allows for a comprehensive evaluation of the vascularization pattern and efficiency of biomaterials. Being compliant with the 3R-principles (replacement, reduction and refinement), the well-established and easy-to-handle CAM model offers many advantages such as low costs, immune-incompetence and short experimental times with high-grade read-outs when compared to conventional animal models. Therefore, combined with our imaging-guided approach it represents a powerful tool to study angiogenesis in biomaterials.

Evolving applications of the egg: chorioallantoic membrane assay and ex vivo organotypic culture of materials for bone tissue engineering

“…µCT is a sensitive method to assess bone formation or remodelling and has been used to calculate the overall bone gain/loss of human femoral bone cores 50,51 (Figure 3), chick femurs [145][146][147][148] (Figure 7) and could be used to assess bone formation within scaffolds on the CAM. 149 Magnetic resonance imaging (MRI) to measure vascular perfusion of scaffolds or materials on the CAM has also been described. 116,130,149 Alternatively, a marker that targets hydroxyapatite was used to highlight mineralised deposits on scaffolds.…”

“…151 The injection of contrast agent in this study also necessitated the use of a surgical microscope, which may not be accessible for all researchers. Woloszyk et al 149 combined MRI in ovo using gadolinium-based contrast agent and µCT post-perfusion with 'Microfil' to provide an overall 3D assessment of functional blood flow and quantification of vascularisation. Using a material like cortical bone (Optimaix ™ ), and another similar to cancellous bone (DegraPol ® ), the method was validated, and the functional blood flow calculated by MRI correlated to total vessel volume determined by µCT.…”

“…µCT is a sensitive method to assess bone formation or remodelling and has been used to calculate the overall bone gain/loss of human femoral bone cores 50 , 51 ( Figure 3 ), chick femurs 145 – 148 ( Figure 7 ) and could be used to assess bone formation within scaffolds on the CAM. 149 …”

“…Magnetic resonance imaging (MRI) to measure vascular perfusion of scaffolds or materials on the CAM has also been described. 116 , 130 , 149 Alternatively, a marker that targets hydroxyapatite was used to highlight mineralised deposits on scaffolds. 150 An advantage of MRI would be the ability to non-invasively assess functional vasculature formed at the surface, middle or tissue interface of the scaffold; 116 , 149 however, concerns with this method include the availability of an MRI machine and the reported use of ketamine to sedate the embryo during imaging.…”

“… 116 , 130 , 149 Alternatively, a marker that targets hydroxyapatite was used to highlight mineralised deposits on scaffolds. 150 An advantage of MRI would be the ability to non-invasively assess functional vasculature formed at the surface, middle or tissue interface of the scaffold; 116 , 149 however, concerns with this method include the availability of an MRI machine and the reported use of ketamine to sedate the embryo during imaging. At the time of writing, no studies could be found reporting the change, if any, in vascular resistance to topical ketamine on the CAM; however, the change in vascular resistance should be equal in all test subjects if a consistent method is used, allowing comparisons between materials on the CAM to be made.…”