Preparation and in vitro characterization of luteolin-loaded liposome/GelMA composite microspheres and associated macrophage and osteogenic responses

Abstract Background The chronic inflammatory microenvironment of periodontitis impairs periodontal tissue repair. Biomaterials that combine local delivery with immunomodulatory and osteogenic functions may support periodontal regeneration. This in vitro study developed a luteolin-loaded liposome/GelMA composite microsphere platform and evaluated its material properties and cell responses. Methods Luteolin-loaded nanoliposomes (Lipo@Lut) were prepared by thin-film dispersion and immobilized in photocrosslinked gelatin methacryloyl (GelMA) microspheres to form GMs/Lipo@Lut. Physicochemical characteristics, in vitro release, cytocompatibility, LPS-stimulated RAW264.7 responses, and osteogenic responses of mouse periodontal ligament stem cells (mPDLSCs) in direct culture and a RAW264.7–mPDLSC Transwell co-culture model were evaluated in vitro. Results The composite microspheres had a porous architecture and released luteolin more slowly than Lipo@Lut during the 28-day in vitro study. Under LPS-stimulated conditions, GMs/Lipo@Lut was associated with lower TNF-α and iNOS mRNA expression, higher Arg-1 and IL-10 mRNA expression, and stronger Arg-1 immunofluorescence in RAW264.7 cells. Together, these findings demonstrated macrophage-associated inflammatory marker changes under the tested in vitro conditions. Representative ALP and Alizarin Red S staining suggested enhanced osteogenic differentiation of mPDLSCs in direct culture and in the Transwell model. Image-based semiquantification showed a significantly higher Alizarin Red S-positive area fraction in the GMs/Lipo@Lut group in both models, while ALP staining remained qualitative. OCN and RUNX2 protein expression also increased in direct culture. Conclusions In vitro, GMs/Lipo@Lut showed a slower luteolin-release profile than Lipo@Lut and was associated with immunomodulatory and osteogenic responses in the tested cell models. These findings support further evaluation of this platform for periodontal regeneration; however, efficacy, safety, retention, and degradation behavior require validation in appropriate in vivo periodontitis models.