The degenerative joint disease osteoarthritis (OA) is in urgent need of disease-modifying OA drugs, which targets the structural tissue changes driving the disease. Currently, the most advanced drug in class is Sprifermin (recombinant human fibroblast growth factor 18, rhFGF18) that utilizes an anabolic approach to regenerate the degenerated articular cartilage of OA patients. Understanding the biological effects of Sprifermin on cartilage is key to exploiting the full clinical potential of the drug. We therefore aimed to investigate the mode of action behind the cartilage regenerative effect of Sprifermin, using ex vivo models. Ex vivo cultures of articular cartilage from healthy bovine, healthy human individuals and human OA patients were stimulated with Sprifermin, FGF18, IGF-I (positive control) or placebo formulation (negative control), using various doses and dosing regimens. Pre-culturing with OSM and TNF-α was also used to induce an inflammatory state before treatment. Effects on metabolic activity (AlamarBlue), cell proliferation (PCNA staining), cartilage extracellular matrix (ECM) remodeling (release of biomarkers ProC2, CS846, C2M, AGNx2, acMMP9 and AGNx1) and cartilage quality (expression of chondrocyte phenotype markers COL2A1, ACAN and SOX9, and fibrocartilage marker COL1A1, and Safranin O/Fast green staining) were monitored. The results, regardless of species and pathological stages of the articular cartilage, demonstrated the ability of Sprifermin to activate chondrocytes in their natural matrix ex vivo. The bovine studies (Paper I) confirmed the well-known anabolic effects of Sprifermin (increased cell proliferation, metabolic activity, and type II collagen and aggrecan formation), while revealing a novel catabolic effect (increased aggrecanase activity, but not MMP activity). In parallel, studies showed that inflammatory pre-culturing of the cartilage prior to treatment altered these responses. Both bovine and human OA cartilage studies (Paper I and II) indicated a temporal multiphasic pattern of ECM remodeling during Sprifermin treatment. The human OA cartilage cultures revealed an early phase (day 7-28) of increased aggrecanase-mediated aggrecan degradation and decreased type II collagen formation, and a late phase (day 35-70) of stabilized aggrecanase-mediated aggrecan degradation and increased type II collagen formation. After 70 days, the relative expression of four cartilage markers indicated a sustained cartilage quality (ACAN > COL2A1 > SOX9 > COL1A1), but no difference was observed between treatments. In conclusion, a temporal biphasic process of ECM remodeling was observed in articular cartilage ex vivo during Sprifermin-induced cartilage regeneration. An early catabolic phase of increased aggrecanase activity and decreased type II collagen formation was followed by a late anabolic phase of type II collagen and aggrecan formation. Parallel chondrocyte proliferation and increased metabolic activity were indicative of a growing chondrocyte population. Finally, induction of an inflammatory state before treatment appeared to change the response to Sprifermin.