3D models for oral inflammation using gingival fibroblasts, lipopolysaccharides and hypoxia.

Three-dimensional (3D) in vitro models that more faithfully recapitulate human tissue architecture are essential to advance mechanistic insight into inflammatory diseases. We developed and characterized a 3D human gingival fibroblast spheroid-system to dissect the combined effects of hypoxia and inflammation, two central features of host-pathogen interactions in periodontal disease and other chronic inflammatory conditions. Using controlled oxygen environments and stimulation with E. coli lipopolysaccharide (LPS) and IL-1β, we show that hypoxia alone markedly alters spheroid morphology, producing smaller aggregates with thinner rims, while the combination of hypoxia and inflammation yields the most prominent structural disruption. Cross-section analyses reveal condition-dependent remodeling of extracellular matrix and cytoskeletal organization, with coordinated reductions in fibronectin, collagen I/III, integrin β1, and filamentous actin. Gene expression profiling confirms suppression of extracellular matrix (ECM)-related and adhesion genes under hypoxia and combined stress, whereas secretome analysis demonstrates robust upregulation of pro-inflammatory cytokines and chemokines (IL-6, IL-8, MCP-1, TNFα) and angiogenic VEGFα. Despite these marked structural and immunological shifts, cell viability remained largely preserved. This 3D system captures key features of fibroblast responses to inflammatory and hypoxic cues, providing a human-relevant platform for studies of tissue remodeling, cytokine signaling, and microenvironmental stress relevant to host-pathogen-driven disease.