It is very useful to evaluate the content and 3D distribution of extracellular matrix non-destructively in tissue engineering. This study evaluated the feasibility of using micro-computed tomography (microCT) with Hexabrix to measure quantitatively sulfated glycosaminoglycans (GAGs) of engineered cartilage. Rabbit chondrocytes at passage 2 were used to produce artificial cartilages in polyglycolic acid scaffolds in vitro. Engineered cartilages were incubated with Hexabrix 320 for 20 min and analyzed via microCT scanning. The number of voxels in the 2D and 3D scanning images were counted to estimate the amount of sulfated GAGs. The optimal threshold value for quantification was determined by regression analysis. The 2D microCT images of an engineered cartilage showed positive correlation with the histological image of Safranin-O staining. Quantitative data obtained with the 3D microCT images of 14 engineered cartilages showed strong correlation with sulfated GAGs contents obtained by biochemical analysis (R(2) = 0.883, p < 0.001). Repeated exposure of engineered cartilages to Hexabrix 320 and microCT scanning did not significantly affect cell viability, total DNA content, or the total content of sulfated GAGs. We conclude that microCT imaging using Hexabrix 320 provides high spatial resolution and sensitivity to assess the content and 3D distribution of sulfated GAGs in engineered cartilages. It is expected to be a valuable tool to evaluate the quality of engineered cartilage for commercial development in the future.
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