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.