Engineering the synovial lining–sublining architecture in a human 3D model to study macrophage–fibroblast interactions in arthritis

The synovial membrane is a key regulator of joint homeostasis, inflammation, and tissue remodeling, yet human in vitro models that reproduce its spatial organization while enabling controlled investigation of macrophage-fibroblast-like stromal cell (FLS) interactions remain limited. Here, we developed a xeno-free, spatially organized 3D synovial membrane model that reconstructs the structural relationship between the macrophage lining and fibroblast-rich sublining compartment. The model was generated by establishing an apically organized macrophage-containing lining-like compartment on a transwell membrane, followed by stepwise assembly of FLS laden hydrogel constructs. Hydrogel screening identified a 1:3 VitroGel® RGD formulation as the most suitable condition to support FLS viability, homogeneous cell distribution, and stable tissue organization. Experimentally induced M1-like and M2-like macrophages were incorporated, and the model was stimulated with TNF-α, TGF-β, or OA synovial fluid (SF). Cytotoxicity, proliferation, metabolic activity, gene expression, and cytokine secretion were analyzed. Pharmacological responsiveness was evaluated using adalimumab and 4-octyl itaconate (4-OI). The spatially organized construct enabled compartment-specific analysis of macrophage-FLS communication. M1-like macrophages induced inflammatory FLS responses characterized by elevated IL1B, IL6, TNFA, and MMP1 expression, whereas M2-like macrophages promoted matrix-remodeling and myofibroblast-related programs, including increased TGFB and ACTA2 expression. Exposure to OA-SF and cytokine stimulation induced FLS hyperproliferation, metabolic adaptation, and distinct transcriptional responses. Treatment with adalimumab suppressed TNF-driven inflammatory responses, whereas 4-OI reduced selected inflammatory markers and altered remodeling-associated responses. Together, these findings establish a spatially organized, xeno-free 3D synovial membrane model as a defined and experimentally tractable platform for studying macrophage-FLS communication and synovial activation. By combining physiologically inspired tissue organization with compartment-specific analysis, the model provides a human in vitro platform for investigating OA-associated inflammatory and remodeling processes and evaluating pharmacological modulation under defined experimental conditions.