Silver nanoparticles induce stress-associated upregulation of VEGF and FGF in fibroblasts as a compensatory response.

Background: Silver nanoparticles (AgNPs) are widely incorporated into wound dressings and tissue-engineering scaffolds due to their antimicrobial properties; however, their direct effects on fibroblast biology and angiogenic signaling remain incompletely understood. Fibroblasts play a key role in tissue repair through secretion of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). Elucidating how AgNPs influence fibroblast stress response and growth factor expression is critical for the safe design of regenerative biomaterials. Methods: Commercially standardized AgNPs were characterized by transmission electron microscopy (TEM) to determine particle size and dispersion. Mouse NIH/3T3 fibroblasts were exposed to various concentrations of AgNPs. Intracellular AgNP localization was visualized by TEM. Reactive oxygen species (ROS) generation was evaluated at 6 h using a dichloro-dihydro-fluorescein diacetate (DCFH-DA)-based assay, with additional cell-free controls to assess nanoparticle-probe interactions, including potential fluorescence quenching effects. Cell viability and migration were evaluated at 24 h using PrestoBlue and wound healing assays, respectively. VEGF and FGF protein expression were quantified by Western blot after 24 h of exposure. Results: AgNPs with an average diameter of 27.35 ± 11.92 nm were effectively internalized and accumulated in nuclei, perinuclear regions, mitochondria, and cytoplasmic vacuoles. At 6 h, ROS levels showed an increasing trend with AgNP exposure, although not statistically significant. Cell-free experiments demonstrated a dose-dependent reduction in DCF fluorescence in the presence of AgNPs, confirming nanoparticle-induced quenching and suggesting that intracellular ROS levels may be underestimated. At 24 h, AgNPs significantly reduced cell viability at 100 µg/mL, while migration exhibited a non-significant increase at 50 µg/mL. Despite these stress responses, VEGF and FGF levels increased significantly in a concentration-dependent manner, reaching statistical significance at the highest concentration. Conclusion: AgNP exposure induces intracellular accumulation and early oxidative stress, followed by upregulation of VEGF and FGF in fibroblasts. However, the absence of corresponding functional enhancement in cell migration, together with cytotoxic effects at higher concentrations, indicates that this response reflects a compensatory stress-associated mechanism rather than functional angiogenesis. These findings highlight the importance of validating nanoparticle-assay interactions and emphasize the need for careful dose optimization in the development of AgNP-based regenerative biomaterials.