AbstractMagnetic resonance imaging (MRI) is a promising non-invasive method to assess cartilage regeneration based on the quantitative relationship between MRI features and concentrations of the major components in the extracellular matrix (ECM). To this end,in vitroexperiments are performed to investigate the relationship and reveal the underlying mechanism. A series of collagen (COL) and glycosaminoglycan (GAG) solutions at different concentrations are prepared, andT1andT2relaxation times are measured with or without a contrast agent (Gd-DTPA2−) by MRI. Fourier transform infrared spectrometry is also used to measure the contents of biomacromolecule-bound water and other water, allowing theoretical derivation of the relationship between biomacromolecules and the resultingT2values. It has been revealed that the MRI signal in the biomacromolecule aqueous systems is mainly influenced by the protons in hydrogens of biomacromolecule-bound water, which we divide into inner-bound water and outer-bound water. We have also found that COL results in higher sensitivity of bound water than GAG inT2mapping. Owing to the charge effect, GAG regulates the penetration of the contrast agent during dialysis and has a more significant effect onT1values than COL. Considering that COL and GAG are the most abundant biomacromolecules in the cartilage, this study is particularly useful for the real-time MRI-guided assessment of cartilage regeneration. A clinical case is reported as anin vivodemonstration, which is consistent with ourin vitroresults. The established quantitative relation plays a critical academic role in establishing an international standard ISO/TS24560-1:2022 ‘Clinical evaluation of regenerative knee articular cartilage using delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) andT2mapping’ drafted by us and approved by International Standard Organization.