Therapeutic Targets and Signaling Mechanisms in 2D and 3D In Vitro Models of Osteoarthritis.

Osteoarthritis (OA) is a progressive degenerative joint disorder characterized by cartilage breakdown, inflammation, and subchondral bone remodeling. As research increasingly relies on physiologically relevant systems, advanced in vitro models have become essential for exploring OA mechanisms and identifying therapeutic targets. This review synthesizes current developments in two-dimensional (2D) and three-dimensional (3D) in vitro models used to investigate OA-related signaling pathways and therapeutic strategies. This review aims to critically evaluate and compare two-dimensional (2D) and three-dimensional (3D) in vitro OA models, with particular emphasis on their structural characteristics, signaling mechanisms, and translational relevance. Relevant literature published between January 2009 and January 2025 was examined across major scientific databases, including PubMed, Scopus, and Web of Science. Studies employing 2D or 3D in vitro OA models to explore signaling mechanisms or therapeutic interventions were reviewed and integrated into a qualitative narrative synthesis. Across the analyzed literature, 3D models-such as bioprinted constructs, organoids, microfluidic systems, and stem-cell-derived platforms-were consistently reported to more closely replicate OA-related structural, biochemical, and biomechanical processes than traditional 2D systems. These models facilitated deeper insights into key pathways, including NF-κB, Wnt/β-catenin, and TGF-β signaling. Emerging technologies such as joint-on-a-chip systems and patient-specific induced pluripotent stem cell (iPSC) platforms further enhance translational potential by enabling personalized disease modeling. Key signaling pathways identified across the included studies included NF-κB, Wnt/β-catenin, and TGF-β signaling, which were consistently associated with inflammatory responses, chondrocyte hypertrophy, and cartilage remodeling processes. Narrative synthesis of current evidence highlights the substantial progress made in 3D in vitro OA modeling. By incorporating stem cells, bioprinting approaches, physiologically relevant matrix environments, and inflammatory stimuli, these platforms offer improved fidelity in mimicking joint physiology and OA progression. Collectively, they represent an important step toward more accurate drug testing and the development of personalized therapeutic strategies.