Dynamic stiffness drives BMSC chondrogenesis via <i>Ihh</i> -regulated H3K18 lactylation/ <i>Rcan1</i> axis and mitochondrial fission remodeling

Cartilage defects pose significant clinical challenges due to limited regenerative capacity. Dynamic matrix stiffness, mimicking the physiological mechanical microenvironment, shows promise in directing stem cell chondrogenesis, but its molecular mechanisms remain unclear. Bone marrow mesenchymal stem cells (BMSCs) were cultured on engineered hydrogels with static soft (0.033 kPa), dynamic (0.031-0.126 kPa, time-dependent stiffening), and static stiff (0.126 kPa) conditions. We performed small interfering RNA-mediated<i>Ihh</i>knockdown and<i>Rcan1</i>overexpression, with chondrogenic differentiation assessed via COL2/SOX9 immunofluorescence. For molecular analyses, we conducted qPCR, CUT&Tag-PCR, Western blot, RNA-seq, H3K18la-targeted CUT&Tag sequencing, and transmission electron microscopy (TEM) for mitochondrial morphology assessment. Dynamic stiffness significantly enhanced chondrogenic differentiation, as evidenced by immunofluorescence detection of elevated COL2 and SOX9 expression.<i>Ihh</i>mRNA expression levels were upregulated by dynamic stiffness. Transcriptome profiling analysis revealed that<i>Ihh</i>knockdown disrupted the expression of genes involved in the glycolytic pathway, while Western blot results showed that<i>Ihh</i>knockdown inhibited histone H3 lysine 18 lactylation (H3K18la). CUT&Tag sequencing revealed<i>Ihh</i>-dependent H3K18la enrichment at regulatory regions of mitochondria-associated genes, notably<i>Rcan1. Ihh</i>deficiency promoted mitochondrial fission, as evidenced by increased<i>Drp1</i>and<i>Fis1</i>mRNA expression levels and direct observation of enhanced mitochondrial fission via TEM. Crucially,<i>Rcan1</i>overexpression rescued mitochondrial fusion, downregulated fission markers, and reinstated chondrogenic marker expression. Consistently, the LDHA inhibitor FX11 reduced lactate levels, diminished H3K18la, and downregulated<i>Rcan1</i>, confirming the metabolic dependence of this axis. RNA-seq analysis further established that<i>Rcan1</i>overexpression reprogrammed signaling pathways critical for cell differentiation, including ECM-receptor interaction. Dynamic stiffness promotes BMSC chondrogenesis via the<i>Ihh</i>-H3K18la-<i>Rcan1</i>axis, linking mechanical cues to epigenetic regulation of mitochondrial remodeling and providing a novel target for cartilage repair.