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Background: Annulus fibrosus (AF) regeneration is critical to intervertebral disc repair, yet remains challenging due to the remarkable heterogeneity of AF tissue.Since the stiffness of substrate determines stem cell lineage commitment (Cell 2006, 126:677-89), this study aimed to explore whether the differentiation of AF-derived stem cells (AFSCs) (PLoS One 2014, accepted), a newly identified cell source for AF tissue engineering, could be regulated by substrate stiffness.