Specific growth factors during the expansion and redifferentiation of adult human articular chondrocytes enhance chondrogenesis and cartilaginous tissue formation in vitro

TLDR

Adult human articular chondrocytes were expanded in serum‑containing medium with or without mitogens (EGF, PDGFbb, FGF‑2, TGFβ1, or FGF‑2/TGFβ1) and then redifferentiated in 3‑D pellets using serum‑supplemented, serum‑free, or serum‑free medium plus TGFβ1/dexamethasone. All tested mitogens increased proliferation and dedifferentiation; FGF‑2/TGFβ1 yielded the fastest growth and lowest CII/CI and Agg/Ver ratios, while redifferentiation in serum‑free medium was superior for EGF, PDGFbb, and FGF‑2 expanded cells; adding TGFβ1/dexamethasone to serum‑free medium amplified CII/CI 96‑fold in FGF‑2/TGFβ1 expanded cells, producing the greatest collagen II and glycosaminoglycan accumulation and demonstrating that expansion‑phase growth factors modulate both proliferation and subsequent 3‑D redifferentiation capacity.

Abstract

Adult human articular chondrocytes were expanded in a medium with 10% serum (CTR) or further supplemented with different mitogens (i.e., EGF, PDGFbb, FGF-2, TGF beta 1, or FGF-2/TGF beta 1). Cells were then induced to redifferentiate in 3D pellets using serum-supplemented medium (SSM), serum-free medium (SFM), or SFM supplemented with factors inducing differentiation of chondroprogenitor cells (i.e., TGF beta 1 and/or dexamethasone). All factors tested during expansion enhanced chondrocyte proliferation and dedifferentiation, as assessed by the mRNA ratios of collagen type II to type I (CII/CI) and aggrecan to versican (Agg/Ver), using real-time PCR. FGF-2/TGF beta 1-expanded chondrocytes displayed the lowest doubling times, CII/CI and Agg/Ver ratios, averaging, respectively, 50, 0.2 and 15% of CTR-expanded cells. Redifferentiation in pellets was more efficient in SFM than SSM only for EGF-, PDGFbb- or FGF-2-expanded chondrocytes. Upon supplementation of SFM with TGF beta and dexamethasone (SFM TD), CII/CI ratios decreased 4.4-fold for EGF- and PDGFbb-expanded chondrocytes, but increased 96-fold for FGF-2/TGF beta 1-expanded cells. Chondrocytes expanded with FGF-2/TGF beta 1 and redifferentiated in SFM TD expressed the largest mRNA amounts of CII and aggrecan and generated cartilaginous tissues with the highest accumulation of glycosaminoglycans and collagen type II. Our results provide evidence that growth factors during chondrocyte expansion not only influence cell proliferation and differentiation, but also the cell potential to redifferentiate and respond to regulatory molecules upon transfer into a 3D environment.