Exploring NF-kB silencing biosafety in mesenchymal stem cells as a possible strategy for osteogenic augmentation

Abstract

Introduction: the decrease in oestrogen levels after menopause increases inflammation, activating the NF-kB pathway in mesenchymal stem cells (MSCs). This pathway has a marked anti-osteogenic effect and plays critical roles in cellular function. Our hypothesis is that silencing key genes in the canonical and non-canonical NF-kB pathways in endogenous MSCs using the Aptamer-Lipid Nanoparticle-Gapmer system previously designed by our group could be a viable strategy for the treatment of osteoporosis. However, it is essential to verify that such silencing does not compromise basic cellular functions, given the multiple roles of NF-kB in regulating immune responses, apoptosis, and cellular homeostasis. Materials and methods: the murine MSC line C3H10T1/2 was used to assess the silencing of key genes (Ikka, Ikkb, Nemo, and Nik) in the canonical and non-canonical NF-kB pathways using GapmeRs. Gene expression levels were measured post-silencing and compared with those achieved using commercial inhibitors (BMS-345541 and MLN120B). Additionally, basic cellular function assays, including proliferation, chemotaxis, cell migration, and viability, were conducted to evaluate the safety of NF-kB silencing. Results: silencing Ikka, Ikkb, Nemo, and Nik resulted in a significant reduction in gene expression in vitro. While NF-kB activation with lipopolysaccharide (LPS) significantly increased the expression of target genes such as IL-6 and NFKB1A, this increase was blocked after gene silencing, reaching levels comparable to those achieved with commercial inhibitors. Cellular function assays showed no significant changes in proliferation, chemotaxis, cell migration, or viability following silencing. Conclusions: NF-kB pathway silencing using GapmeRs does not negatively impact basic cellular functions, suggesting that this approach is safe and efficient. These findings support its potential clinical application in promoting the osteogenic differentiation of MSCs for osteoporosis treatment.