Relaxin‐2 as a modifiable hormonal pathway in knee osteoarthritis: Dose dependent effects on chondrocyte phenotype

BACKGROUND: Postmenopausal female individuals are disproportionately affected by knee osteoarthritis (KOA), experiencing earlier onset and more severe pathology compared to their male counterparts. Despite this clinical disparity, the molecular mechanisms underlying female-specific vulnerability remain poorly defined. OBJECTIVE: To evaluate the mechanistic role of relaxin-2 in postmenopausal KOA. DESIGN: This was a translational research study that evaluated relaxin-2 using an in vitro postmenopausal human KOA chondrocyte culture model and in silico network medicine simulation. SETTING: Research laboratory. SPECIMENS: KOA chondrocytes isolated from a 67-year-old postmenopausal female human donor. INTERVENTIONS: Female KOA chondrocytes were treated with varying relaxin-2 concentrations (control: 0 pg/mL, low: 0.496 pg/mL, medium: 49.6 pg/mL, and high: 4960 pg/mL; n = 5-7/group), with doses based on previously reported physiologic serum levels for pre- and postmenopausal female individuals. MAIN OUTCOME MEASURE(S): Effects were evaluated by immunofluorescence analysis of chondrogenicity markers (type II collagen [Col2], aggrecan [ACAN]), fibrotic markers (type I collagen [Col1], type III collagen [Col3]), extracellular matrix degradation markers (matrix metalloproteinase-13 [MMP-13], A Disintegrin And Metalloproteinase with Thrombospondin Motifs 4 [ADAMTS4]), and mitochondrial integrity and function markers (translocase of the outer mitochondrial membrane 20 [TOMM20], Succinate Dehydrogenase Subunit A [SDHA], Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha [PGC-1α]). RESULTS: Relaxin-2 increased Col2 at low and medium concentrations (p < .05), with the Col2/Col1 ratio highest at the low dose relaxin-2. MMP-13 expression was greatest for the medium compared to the control (p = .73), low (p = .002), and high relaxin-2 concentrations (p = .005), whereas ACAN and ADAMTS4 showed no differences across concentrations (p > .05). In silico analysis revealed that postmenopausal female individuals exhibit a fibrotic transcriptomic signature and that mitochondrial function is regulated by relaxin-2 in fibrotic chondrocytes. Computational analyses were validated experimentally, with TOMM20 and SDHA elevated at all relaxin-2 concentrations (p < .05), whereas PGC-1α was reduced at high concentration (p < .05). CONCLUSIONS: These findings demonstrate that relaxin-2 reprograms fibrotic osteoarthritic chondrocytes toward a healthier, less fibrotic state by restoring mitochondrial function.