Three-Dimensional Perfusion Imaging of Microcirculatory Networks Within Axially Vascularized Artificial Tissue

Area of Science:

• Biomedical Engineering
• Regenerative Medicine
• Vascular Biology

Background:

• Assessing 3D vascular architecture in engineered tissues is challenging due to limitations of 2D histology and microimaging.
• A reproducible method for complete microcirculatory system visualization is needed for axial vascularization techniques.

Purpose of the Study:

• To present an integrated workflow for high-resolution 3D visualization of neovascularization in engineered tissue constructs.
• To overcome limitations of existing techniques for assessing complex vascular networks.

Main Methods:

• Developed an integrated workflow for 3D visualization of neovascularization in rat arteriovenous (AV) loop tissue constructs.
• Utilized intravascular perfusion with a fluorescent dye for vasculature labeling.
• Employed an ethyl cinnamate-based clearing protocol for optical transparency.
• Imaged samples using confocal and light-sheet fluorescence microscopy at 7 and 28 days postimplantation.

Main Results:

• Achieved high-contrast, 3D visualization of the microvascular network in whole-mount and segmental analyses.
• Observed initial AV loop axis visualization at day 7, progressing to a dense capillary plexus by day 28.
• Demonstrated downstream processing compatibility via rehydration and 3D nuclear counterstaining.

Conclusions:

• The presented workflow provides a powerful and reproducible method for detailed structural assessment of microvascular networks in large engineered constructs.
• This technique overcomes key limitations of conventional methods, enabling comprehensive analysis of neovascularization.
• Facilitates advanced structural assessment of microvascular networks in engineered tissues.