Diosmin-Capped Silver Nanoparticles Promote Osteogenic Differentiation of Human Periodontal Ligament Stem Cells: Box–Behnken Optimization and in vitro Evaluation

Abstract

Aim: Diosmin is a bioactive flavonoid with anti-inflammatory, anti-oxidant, and osteogenic properties; however, its poor water solubility limits its therapeutic use. To overcome this, the study developed Diosmin-loaded silver nanoparticles (AgNPs), leveraging the bone-regenerative potential of silver, as a unique strategy to improve the osteogenic differentiation of periodontal ligament stem cells (PDLSCs). Methods: The nanoparticles loaded with the extracted Diosmin were prepared using a scalable green reduction method and optimized using the Box–Behnken design (BBD) and response surface methodology. Key factors (silver nitrate concentration, Diosmin concentration, and reaction temperature) were assessed for their effects on the particle size, zeta potential, and polydispersity index. This study also evaluated the effects of Diosmin-loaded AgNPs on the viability and osteogenic differentiation of human PDLSCs. Alizarin Red staining and alkaline phosphatase activity were used to assess osteogenic differentiation. Moreover, real-time quantitative polymerase chain reaction (RT-qPCR) was used to track the expression of osteogenic-associated markers, osteoprotegerin (OPG) and runt-related transcription factor 2 (RUNX2), as well as proinflammatory markers, TNF-α and IL-1β. Results: The optimized Diosmin-loaded AgNPs identified by the BBD showed high desirability, with particle size of 181.0± 8.45 nm using dynamic light scattering technique (DLS) and a negative surface charge of − 21.45± 1.27 mV, confirmed by transmission electron microscopy (TEM) photomicrographs. Moreover, energy-dispersive X-ray (EDX) analysis showed the preparation of AgNPs through showing their elemental peaks. Stability studies demonstrated that formulations remained stable at 4.0± 2.0°C for up to 3 months. The results showed that, in contrast to silver nitrate (AgNO 3 ), Diosmin-loaded AgNPs promoted osteogenic differentiation and modulated the expression of inflammatory markers, indicating a potential role in the regulation of the local inflammatory microenvironment associated with osteogenesis. Conclusion: Green AgNPs have proven their efficacy as a potential approach for osteogenic differentiation. Keywords: diosmin, silver nanoparticles, green synthesis, box-behnken design, osteogenic differentiation, periodontal ligament stem cells