MiR-223-3p-loaded hydroxyapatite nanoparticles enhance odontogenesis and immunomodulation in human dental pulp cells in vitro

This study aimed to examine the effects of miR-223-3p delivered through 3-aminopropyltriethoxysilane-functionalized hydroxyapatite nanoparticles (NPs) on the immunomodulatory behavior and odontogenic differentiation of human dental pulp cells. NPs were synthesized and surface-functionalized with 3-aminopropyltriethoxysilane (APTES) to conjugate miR-223-3p (NPs-miR-223-3p). Primary human dental pulp cells (hDPCs) were characterized by flow cytometry, and NPs uptake was visualized by confocal microscopy using fluorescein amidite (FAM)-labeled miR-223-3p. Cell viability and proliferation following NPs treatment were evaluated using the MTT assay. Intracellular miR-223-3p and odontogenic marker gene expression, including ALP, DMP1, and DSPP, were demonstrated by RT-qPCR, and then confirmed by alkaline phosphatase activity and mineral deposition assays using Alizarin Red S and von Kossa staining. Inflammatory responses in hDPCs were induced with lipopolysaccharide and adenosine triphosphate. Immunomodulatory effects were analyzed by RT-qPCR analysis of IL-1β, IL-18, and NLRP3 mRNA levels, together with ELISA quantification of IL-1β and IL-18 protein secretion. NPs exhibited good biocompatibility in hDPCs, without compromising cellular viability or proliferation, and were efficiently internalized at 24 h. Sustained upregulation of miR-223-3p was observed from 24 h up to 5 days post-treatment, confirming the utility of the NPs as an effective delivery platform. Functionally, NPs-mediated miR-223-3p delivery enhanced odontogenic differentiation, concomitant with decreased SMAD3 expression from Day 1 to Day 5, increased expression of ALP, DSPP, and DMP1, and significantly promoted mineralized matrix formation ( P < 0.05). Moreover, under inflammatory stimulation with LPS and ATP, NPs-miR-223-3p treatment was associated with reduced NLRP3 inflammasome expression and IL-1β and IL-18 expression at both transcript and protein levels ( P < 0.05), indicating an anti-inflammatory effect in the context of ATP/LPS stimulation. miR-223-3p delivered by NPs was associated with enhanced odontogenic differentiation and reduced NLRP3-related inflammatory responses in ATP/LPS-stimulated hDPCs, highlighting its potential for application in vital pulp therapy. Not applicable.