gms | German Medical Science

Objectives: During standard expansion culture (i.e. plasma osmolarity, 280 mOsm) chondrocytes inevitably lose their specific phenotype and de-differentiate, which makes them inappropriate for autologous chondrocyte implantation. However, in vitro expansion of human articular chondrocytes (HACs) is still required for cell-based therapies to treat cartilage pathologies. Physiological osmolarity (i.e. 380 mOsm) has been shown to increase collagen type II (COL2) expression in vitro, but the underlying reason is unknown. Transforming growth factor beta (TGF-beta) is an accepted key regulator of chondrocyte differentiation and known to stimulate COL2 production. In this study we therefore aimed to elucidate the role of TGF-beta signaling as a potential molecular mechanism driving the COL2 expression under physiological culture conditions.

Methods: HACs were cultured in cytokine-free medium of 280 or 380 mOsm, respectively, under standard 2D in vitro conditions, with or without lentiviral TGF-beta 2 knockdown (RNAi). Expression of TGF-beta isoforms, TGF-beta superfamily receptors, BMPs and chondrocyte marker genes was evaluated by QPCR. TGF-beta 2 protein secretion (ELISA) and TGF-beta bioactivity, using an established reporter cell line, was determined. Statistical significance (P<0.05) was determined by unpaired two tailed t-tests.

Results and Conclusion: Physiological osmolarity differentially altered TGF-beta isoform expression in a time- and passage-dependent manner. Specifically, TGF-beta 2 expression was increased significantly 2-fold and 1.5-fold at Passage 1 and Passage 2 respectively. TGF-beta 2 protein level was 2.7-fold higher in 380 mOsm versus 280 mOsm cultures. Physiological osmolarity increased TGF-beta activity 2.3-times. Upon TGF-beta 2 isoform-specific knockdown, COL2 expression significantly increased by 4.4-fold and 12-fold at 280 mOsm and 380 mOsm, respectively, as compared to controls. TGF-beta 2 knockdown significantly increased TGF-beta superfamily target gene ID-1 expression levels by 2.8-fold and 4.3-fold at 280 mOsm and 380 mOsm respectively compared to controls. The expression of another TGF-beta superfamily target gene, CTGF, was significantly increased by 5.0-fold and 8.3-fold at 280 mOsm and 380 mOsm respectively after TGF-beta 2 knockdown. Physiological osmolarity and TGF-beta 2 RNAi also induced several BMPs and ALK5.

We showed that TGF-beta 2 knockdown increases COL2 expression in human osteoarthritic chondrocytes in vitro, most likely through a regulatory feedback loop involving BMP induction. This is the first study indicating that TGF-beta signaling is involved in osmolarity-induced chondrocyte marker gene expression and our findings hold potential to influence future cell-based cartilage repair strategies.