Sprifermin (rhFGF18) modulates extracellular matrix turnover in cartilage explants ex vivo

Abstract

Background: Sprifermin (recombinant human fibroblast growth factor 18) is in clinical development as a potential disease-modifying osteoarthritis drug (DMOAD). In vitro studies have shown that cartilage regenerative properties of sprifermin involve chondrocyte proliferation and extracellular matrix (ECM) production. To gain further insight into the process of sprifermin in the cartilage tissue, this study aimed at investigating the ECM turnover of articular cartilage explants in a longitudinal manner.

Methods: Bovine full-depth articular cartilage explants were stimulated with sprifermin or placebo at weekly intervals, similar to the dosing regimen used in clinical trials. Pre-culturing with oncostatin M and tumour necrosis factor-α, was also used to induce an inflammatory state before treatment. Metabolic activity was measured using AlamarBlue, and chondrocyte proliferation was visualized by immuno-histochemical detection of proliferating cell nuclear antigen. ECM turnover was quantified by biomarker ELISAs; ProC2 reflecting type II collagen formation, CS846 reflecting aggrecan formation, active MMP9, C2M and AGNx2 reflecting matrix metalloproteinase activity, and AGNx1 reflecting aggrecanase activity.

Results: Sprifermin was able to reach the chondrocytes through the extracellular matrix, as it increased cell proliferation and metabolic activity of explants. ProC2 and CS846 was dose-dependently increased (P < 0.05) by sprifermin compared to placebo, while C2M and AGNx2 were unaffected, active MMP9 was slightly decreased, and AGNx1 was slightly increased. Over the course of treatment, the temporal order of ECM turnover responses was AGNx1, then ProC2, followed by CS846 and MMP9. Pro-inflammatory activation of the explants diminished the ECM turnover responses otherwise observed under non-inflammatory conditions.

Conclusions: The data suggest that sprifermin has chondrogenic effects on articular cartilage ex vivo, exerted through a sequential process of ECM turnover; aggrecan degradation seems to occur first, while type II collagen and aggrecan production increased at a later time point. In addition, it was observed that these chondrogenic effects are dependent on the inflammatory status of the cartilage prior to treatment.

Keywords: Chondrocyte and cartilage biology; Osteoarthritis; Other therapeutics.

Fig. 1

Cell proliferation, metabolic activity and type II collagen formation. Bovine cartilage explants were cultured for 3 weeks with weekly administration (72 h duration) of indicated compounds. a Cell proliferation was visualized by staining for PCNA in FFPE tissue slides from three replicate explants for each time point and treatment. One representative explant is shown. Scale bar applies to all pictures. b Metabolic activity was measured using AlamarBlue after 0, 1, 2 and 3 weeks of compound-culturing. Values were baseline-corrected and data presented as mean ± SEM of six replicate explants. c ProC2 (Nordic Bioscience) was measured in conditioned media collected at 0, 1, 2 and 3 weeks of compound-culturing. Values were placebo-corrected and data presented as mean ± SEM of six replicate explants. All results are from one study (Study 1). Unpaired t-test was used to compare the treatment group to the placebo group at each time point. Significance levels are indicated by asterisks; *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Metabolic activity and ECM turnover in bovine cartilage explants. Bovine cartilage explants were pre-cultured for 1 week, and then cultured for further 5 weeks with weekly administration (48 h duration) of indicated compounds. Metabolic activity (a) was measured using AlamarBlue after 5 weeks of compound-culturing. ProC2 (b), C2M (c), AGNx2 (d) and AGNx1 (e) (Nordic Bioscience) was measured in conditioned media collected at 0, 1, 2, 3, 4 and 5 weeks of compound-culturing. Values of biomarkers were placebo-corrected and all data presented as mean ± SEM of six replicate explants. One-way ANOVA was used for multiple comparisons to the placebo group at each time point. Significance levels are indicated by asterisks; *P < 0.05, **P < 0.01, ***P < 0.001. Results from two studies are shown in separate graphs (Left, Study 2; Right, Study 3)

Fig. 3

Metabolic activity and ECM turnover in pro-inflammatory bovine cartilage explants. Bovine cartilage explants were pre-cultured for 1 week with OSM + TNF-α (5/10 ng/mL), and then cultured for further 5 weeks with weekly administration (48 h duration) of indicated compounds. Metabolic activity (a) was measured using AlamarBlue after 5 weeks of compound-culturing. ProC2 (b), C2M (c), AGNx2 (d) and AGNx1 (e) (Nordic Bioscience) was measured in conditioned media collected at 0, 1, 2, 3, 4 and 5 weeks of compound-culturing, and values were placebo-corrected. All values are mean ± SEM of six replicate explants. One-way ANOVA was used for multiple comparisons to the placebo group at each time point. To compare the biomarker responses following pro-inflammatory and non-inflammatory (Fig. 2) pre-culturing, the iAUC of similar doses under each condition were calculated, and presented as mean ± SEM of six replicate explants (graph to the right). Unpaired t-tests were used to compare the two conditions. Significance levels are indicated by asterisks; *P < 0.05, **P < 0.01, ***P < 0.001. All results are from one study (Study 2)

Fig. 4

Sequential process of cartilage regeneration. Cartilage regeneration illustrated as a coordinated sequential process of chondrocyte proliferation and matrix turnover. The process may be initiated by sprifermin signaling to the chondrocytes to start proliferating. However, the chondrocytes are caged by the surrounding matrix, which preclude unlimited expansion. This may force the chondrocytes to initiate matrix degradation processes to expand the lacunae (get rid of their cage), and eventually we may see a larger population of chondrocytes that starts producing new matrix. Note that this figure illustrates a working hypothesis (and is hypothetical) as we do not know whether matrix degradation is required for regeneration of cartilage by pro-proliferative compounds like sprifermin