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 TGFβ-1. We compared the effects of BMP9 and TGFβ-1 on cartilaginous matrix deposition by equine chondrocytes in pellet culture and hypothesized that BMP9 would synergize with a low dose of TGFβ-1 to preserve the healthy chondrocyte phenotype. Methods: Pellets from healthy equine chondrocytes (P1; n = 2 Shetland Pony donors) were cultured for 28 days with chondrogenic medium alone or supplemented with the following: TGFβ-1-low (0.1 ng/ml), TGFβ-1-standard (10 ng/ml), BMP9 (50ng/ml), combination-low (50 ng/ml BMP9 + 0.1 ng/ml TGFβ-1), combination-standard: (50 ng/ml BMP9 + 10 ng/ml TGFβ-1), or combination-low for one week, followed by TGFβ-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, TGFβ-1) and hypertrophic or fibrosis markers (ADAMTS5, collagen type-1, collagen type-3, MMP3, MMP13) were measured at day 7 and 28. Statistical analysis was conducted between TGβ-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 chondrocytes based on pellet size, GAG/DNA, and intensity of Safranin-O and COL2 staining. Although the combination of BMP9 with low doses of TGFβ-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 TGFβ-1 significantly improved chondrogenesis of equine chondrocytes.