Abstract: Decreased estrogen levels following menopause result in heightened inflammation. One primary pathway activated in mesenchymal stem cells (MSCs) in response to this inflammation is the NF-kB pathway, which exerts an anti-osteogenic effect. We hypothesized that silencing key genes in both canonical and non-canonical NF-kB pathways in MSCs could enhance their osteogenic potential. We silenced Ikka, Ikkb, Nemo, and Nik genes in murine MSCs using specific GapmeRs. Basic cell function assays indicated no significant changes in proliferation, migration, chemotaxis, or viability upon gene silencing. After optimizing culture conditions for pathway activation with LPS and inhibition with commercial inhibitors BMS-345541 and MLN120B, we confirmed efficient pathway inhibition upon gene silencing. Osteogenic differentiation of MSCs following silencing of the various target genes revealed that key osteogenic markers, IBSP and ALPL, notably increased in Ikkb and Nemo-silenced cells. Additionally, Alizarin Red staining showed a significant 1.5-fold increase in mineralization in Ikkb-silenced cells compared to controls. These findings suggest that targeting Ikkb at the level of endogenous MSCs could be a valid approach for enhancing their osteogenic potential, thereby promoting bone regeneration without compromising basic cell functions.

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