Combination of bone morphogenetic protein 9 and transforming growth factor BETA-1 improves cartilaginous matrix formation by equine chondrocytes in three-dimensional culture

Abstract

Purpose: Osteoarthritis is not only common in humans, but also highly prevalent in horses. In vitro, chondrocytes are used to study disease processes under controlled conditions and the effects of (new) drugs at the cellular level. Bone morphogenic protein 9 (BMP9) has been shown to be a potent modulator of cartilage development by bovine articular chondrocytes. The hypertrophic effect of BMP9 could be antagonized by a low dose of TGFb-1. We compared the effects of BMP9 and TGFb-1 on cartilaginous matrix deposition by equine chondrocytes in pellet culture and hypothesized that BMP9 would synergize with a low dose of TGFb-1 to preserve the healthy chondrocyte phenotype. Methods: Pellets from healthy equine chondrocytes (P1; n ¼ 2 Shetland Pony donors) were cultured for 28 days with chondrogenic medium alone or supplemented with the following: TGFb-1-low (0.1 ng/ml), TGFb-1-standard (10 ng/ml), BMP9 (50ng/ml), combination-low (50 ng/ ml BMP9 þ 0.1 ng/ml TGFb-1), combination-standard: (50 ng/ml BMP9 þ 10 ng/ml TGFb-1), or combination-low for one week, followed by TGFb-1-standard (i.e. Switch group). Bern scoring and collagen type-2 (COL2) staining of histology sections and glycosaminoglycan (GAG) and DNA analsyis were performed at days 7, 21, and 28. Gene expression of chondrogenic markers (aggrecan, collagen type-2, TGFb-1) and hyper-trophic or fibrosis markers (ADAMTS5, collagen type-1, collagen type-3, MMP3, MMP13) were measured at day 7 and 28. Statistical analysis was conducted between TGb-1-standard and the other groups. Results: Analysis of Safranin-O/Fast-green stained sections demonstrated higher Bern scores for the BMP9-treated groups at all time points (fig.1). At day 7, COL2 staining was stronger for groups that received BMP9. GAG content normalized for DNA was significantly higher for the BMP9 and combination-low group at days 21 and 28. At both time points, aggrecan expression was significantly higher than standard in all groups that received BMP9 during the entire culture period. However, COL2 expression was significantly higher for combination groups only, at day 7. MMP3 was significantly upregulated in the BMP9 and combination-low groups at day 28, whereas MMP13 expression was significantly downregulated in these two groups at the same time point. Conclusions: BMP9 improved matrix deposition by equine chon-drocytes based on pellet size, GAG/DNA, and intensity of Safranin-O and COL2 staining. Although the combination of BMP9 with low doses of TGFb-1 upregulated MMP3 expression, it downregulated the expression of the late hypertrophy marker MMP13 while upregulating aggrecan and COL2 expression. Further analysis of hypertrophy markers and underlying signalling pathways should be considered for a deeper understanding of the observed synergistic effect. Altogether, a combination of BMP9 with a low dose of TGFb-1 significantly improved chondrogenesis of equine chondrocytes. Purpose: Sprifermin is a truncated form of fibroblast growth factor (FGF) 18 known to induce chondrocyte proliferation and type II collagen formation [1,2]. Data from preclinical investigations show that cartilage formation happens in different phases after therapy with sprifermin, starting with a phase of cartilage degradation during the induction of proliferation of chondrocytes followed by a phase of cartilage for-mation/production of extracellular matrix. The aim was to investigate the effect of intra-articular (IA) administrated sprifermin on cartilage turnover as compared to placebo by measurement of markers in longitudinal synovial fluid samples of patients participating in the FORWARD study. Methods: Synovial fluids (SF) from participants with baseline and at least one follow-up sample (baseline (BL) at three consecutive weeks in six month intervals through to week (wk) 80, fig.A available from the phase II clinical trial evaluating the efficacy and safety of intraarticularly delivered sprifermin [3] were selected for the investigations. Biochemical markers were measured in available SF samples of the placebo (saline IA, n¼38) and the highest sprifermin dose group (100 mg/IAx4, n¼59). Each included patient had baseline and at least one FU sample available. Samples were pretreated with ultrasound and centrifugation to decrease viscosity. Markers measured were PRO-C2 (type II collagen formation), huARGS (aggrecan degradation), and FBN-C (fibronectin). Markers were technically validated for synovial fluid measurement. Data were normalized to baseline to investigate the median change over time. Results: Baseline mean (SD) levels of the markers in SF at BL were: PRO-C2, 21.4 (13.6) ng/mL, huARGS, 1117 (516) pM and FBN-C, 2556 (1959) ng/mL. PRO-C2 was initially decreased (from BL to wk 2) after injection with sprifermin; however, the level was increased at the beginning of a new injection cycle followed by a decrease after injection of sprifermin (Fig.B). Overall PRO-C2 levels increased over time in therapy with sprifermin, while no change was observed for the placebo arm. huARGS showed a similar pattern as PRO-C2-there was an overall increase in ARGS over time in the sprifermin group (fig.C). Interestingly ARGS continuously decreased over time in the placebo group. FBN-C is continuously increased after injection's cycles, whereas no effect was seen in the placebo group (fig.D). Conclusions: Confirmatory of the preclinical investigations we saw a biphasic response on cartilage turnover after injection with sprifermin. Cartilage formation and chondrocyte proliferation was only modulated by sprifermin, and cartilage degradation (ARGS) was temporal induced and reduced by sprifermin and saline injections, respectively.1. Gigout A, Guehring H, Froemel D, Meurer A, Ladel C, Reker D, et al. Sprifermin (rhFGF18) enables proliferation of chondrocytes producing a hyaline cartilage matrix. Osteoarthr Cartil. 2017;25. 2. Reker D, Kjelgaard-Petersen CF, Siebuhr AS, Michaelis M, Gigout A, Karsdal MA, et al. Sprifermin (rhFGF18) modulates extracellular matrix turnover in cartilage explants ex vivo. J Abstracts / Osteoarthritis and Cartilage 28 (2020) S86eS527 S496