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Three-dimensional (3D) quantitative localization of multiple biomolecular components within their native cellular context holds immense promise across various areas of biomedicine. While confocal Raman spectral imaging allows label-free detection of biomolecules within the 3D biological samples, achieving quantitative 3D mapping of multiple biomolecules has proven challenging. To address this limitation, we present an integrated quantitative bio-analytical methodology designed to elevate semi-quantitative volumetric Raman imaging analysis to a fully quantitative level, enabling precise visualization and assessment of the 3D distribution of multiple key biomolecular components in biological samples. We herein showcased the utility of quantitative Raman analysis in chemometric phenotyping of 3D human engineered cartilage, including the compositional changes during the maturation process of chondrocytes in hydrogels which mimics the development of native human cartilage. The structural changes of cartilage from week 3 to week 15 were analysed to investigate the changes in the localisation of different kinds of extracellular matrices. By employing spectral-unmixing techniques, we performed simultaneous analysis and comparison of six biological components within the human engineered cartilage, using the native human cartilage sample as the positive control. Our results demonstrate a progressive maturation of the engineered cartilage towards native human cartilage, characterized by significant differences in the levels of elastin and glycogen. These findings hold crucial implications for the viability and survival of tissue-engineered cartilage following implantation into human patients. This innovative approach opens new avenues for in-depth investigations of tissue development, disease progression, and therapeutic interventions.
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