Three-dimensional characterization of tissue-engineered constructs by contrast-enhanced nanofocus computed...

Area of Science:

• Biomaterials Science
• Tissue Engineering
• Medical Imaging

Background:

• Clinical implementation of tissue-engineered (TE) constructs requires robust quality control (QC) tools for consistent release specifications.
• Advanced, noninvasive methods for monitoring TE construct properties, particularly extracellular matrix (ECM) development, are needed.
• Current QC methods like staining and weight measurements offer limited insights into the 3D structure and spatial distribution of ECM.

Purpose of the Study:

• To demonstrate the proof of concept for contrast-enhanced nanofocus computed tomography (CE-nano-CT) as a whole-construct imaging technique for TE constructs.
• To enable noninvasive 3D visualization and quantification of in vitro engineered ECM within TE constructs.
• To assess the potential of CE-nano-CT in evaluating ECM quantity, homogeneity, and culture-specific structural differences.

Main Methods:

• Engineered ECM in 3D TE scaffolds was cultured under static and perfusion bioreactor conditions.
• Contrast-enhanced nanofocus computed tomography (CE-nano-CT) was employed using two contrast agents: Hexabrix® and phosphotungstic acid (PTA).
• CE-nano-CT results were compared with standard techniques: Live/Dead viability assay, Picrosirius Red staining, and net dry weight measurements.

Main Results:

• Hexabrix® as a contrast agent showed a linear correlation between ECM volume and net dry ECM weight, indicating comprehensive ECM staining.
• Phosphotungstic acid (PTA) stained only a portion of the ECM, attributed to its specific binding properties.
• PTA-stained CE-nano-CT data revealed distinct differences between static and perfusion culture conditions, highlighting culture-specific ECM structures.

Conclusions:

• CE-nano-CT is a promising noninvasive imaging technique for the 3D assessment of engineered ECM in TE constructs.
• The choice of contrast agent (Hexabrix® vs. PTA) influences the extent and specificity of ECM visualization.
• CE-nano-CT provides novel insights into ECM quantity and homogeneity, aiding in the optimization of bioreactor conditions and the development of well-characterized TE products for clinical use.