Extracellular matrix changes in response to sprifermin studied in ex vivo cultures of articular cartilage

Purpose: Osteoarthritis (OA) is a degenerative disease with high prevalence, creating an unmet medical need for drugs to protect and regenerate cartilage. Sprifermin, a truncated form of fibroblast growth factor 18 (rhFGF18), is being investigated as a potential disease-modifying OA drug (DMOAD). Sprifermin has been shown to increase cartilage volume in the knees of OA patients. The few studies published about the mode of action behind its anabolic effects, indicate that full-length (fl) FGF18 induces cartilage formation by increasing chondrocyte proliferation, resulting in increased overall extracellular matrix (ECM) production by chondrocytes. Our hypothesis is that matrix degradation is initially needed during this process in order to expand the lacunae and make room in the matrix for the new chondrocytes. The aim of this study was to characterize the ECM changes occurring in response to direct stimulation with Sprifermin, using biomarkers of cartilage formation (Pro-C2), cartilage degradation with impaired repair capacity (C2M, AGNx2) and reversible form cartilage degradation (AGNx1). Methods: Full depth cartilage explants (FDCex), punched from bovine articular cartilage, were cultured for 45 days in four separate culture plates (P1-4). Initially, the FDCex were pre-incubated either with (P1, P2) or without (P3, P4) oncostatin M (OSM)/TNF-α (5/10 ng/mL) for 7 days, to investigate how catabolic induction influences the response to subsequent anabolic treatment. All FDCex plates then received the same treatments but with either single (P1, P3) or weekly (P2, P4) administration. Treatments were Sprifermin (1, 10, 50, 100 or 500 ng/mL), full-length (fl) FGF18 (100 ng/mL), IGF-I (100 ng/mL) as positive controls for cartilage formation, or placebo as negative control, in six replicates each. Cell viability was measured using AlamarBlue at day 45. To evaluate ECM changes, four biomarkers of cartilage turnover were quantified in supernatants from days 14, 21, 28, 35 and 42 using well-described ELISAs; Pro-C2 reflecting formation of type II collagen, C2M and AGNx2 reflecting matrix metalloproteinase (MMP)-mediated degradation of type II collagen and aggrecan, respectively, and AGNx1 reflecting aggrecanase-mediated degradation of aggrecan. Mean values and standard error of the mean (SEM) were reported. For each timepoint, significance levels from the placebo were found using one-way ANOVA assuming normal distribution. These are indicated by asterisks; *P < 0.05, **P < 0.01. Results: Only weekly administration had an effect on the cartilage turnover biomarkers. When comparing to placebo, the anabolic biomarker Pro-C2 was dose-dependently increased by Sprifermin, a response that reached statistical significance for the 100 and 500 ng/mL at several time points (P < 0.05) compared to the vehicle control (fig. 1A). Pre-incubation of the FDCex with OSM/TNF-α led to a decreased and delayed response in Pro-C2 (fig. 1B), which was only statistically significant for the 500 ng/mL dose (P < 0.05). Pro-C2 was increased by IGF-I (pos. control) at all time points. The catabolic biomarkers C2M and AGNx2 were not affected by any of the treatment doses. On the contrary, AGNx1 was slightly increased by Sprifermin however, it did not reach statistical significance. Cell viability was increased by all treatments in a dose-dependent manner. Generally, the effect of flFGF18 was similar to Sprifermin. Conclusions: The data presented here indicate that Sprifermin induced increased formation of articular cartilage type II collagen ex vivo. In addition, responsiveness of the articular cartilage to Sprifermin was influenced by the catabolic status of the tissue prior to treatment. Generation of MMP-mediated cleavage fragments of type II collagen and aggrecan were unchanged by Sprifermin, but generation of the aggrecanase-mediated cleavage fragment of aggrecan was slightly but not statistically increased. Although additional research is needed to confirm this observation, the slight increase in AGNX1 might suggest that matrix degradation is needed for expansion of lacunae, which may be needed to initiate the process of cartilage formation.