Print Email Facebook Twitter Increased TGF-β and BMP Levels and Improved Chondrocyte-Specific Marker Expression In Vitro under Cartilage-Specific Physiological Osmolarity Title Increased TGF-β and BMP Levels and Improved Chondrocyte-Specific Marker Expression In Vitro under Cartilage-Specific Physiological Osmolarity Author Tan Timur, Ufuk (Universiteit Maastricht; Rheinisch-Westfälische Technische Hogeschool) Caron, Marjolein (Universiteit Maastricht) van den Akker, Guus (Universiteit Maastricht) van der Windt, Anna (Erasmus MC) Visser, Jenny (Erasmus MC) van Rhijn, Lodewijk (Universiteit Maastricht) Weinans, Harrie (TU Delft Biomaterials & Tissue Biomechanics; University Medical Center Utrecht) Welting, Tim (Universiteit Maastricht) Emans, Pieter (Universiteit Maastricht) Jahr, H. (Universiteit Maastricht; Rheinisch-Westfälische Technische Hogeschool) Date 2019 Abstract During standard expansion culture (i.e., plasma osmolarity, 280 mOsm) human articular chondrocytes dedifferentiate, making them inappropriate for autologous chondrocyte implantation to treat cartilage defects. Increasing the osmolarity of culture media to physiological osmolarity levels of cartilage (i.e., 380 mOsm), increases collagen type II (COL2A1) expression of human articular chondrocytes in vitro, but the underlying molecular mechanism is not fully understood. We hypothesized that TGF-β superfamily signaling may drive expression of COL2A1 under physiological osmolarity culture conditions. Human articular chondrocytes were cultured in cytokine-free medium of 280 or 380 mOsm with or without siRNA mediated TGF-β2 knockdown (RNAi). Expression of TGF-β isoforms, and collagen type II was evaluated by RT-qPCR and immunoblotting. TGF-β2 protein secretion was evaluated using ELISA and TGF-β bioactivity was determined using an established reporter assay. Involvement of BMP signaling was investigated by culturing human articular chondrocytes in the presence or absence of BMP inhibitor dorsomorphin and BMP bioactivity was determined using an established reporter assay. Physiological cartilage osmolarity (i.e., physosmolarity) most prominently increased TGF-β2 mRNA expression and protein secretion as well as TGF-β bioactivity. Upon TGF-β2 isoform-specific knockdown, gene expression of chondrocyte marker COL2A1 was induced. TGF-β2 RNAi under physosmolarity enhanced TGF-β bioactivity. BMP bioactivity increased upon physosmotic treatment, but was not related to TGF-β2 RNAi. In contrast, dorsomorphin inhibited COL2A1 mRNA expression in human articular chondrocytes independent of the osmotic condition. Our data suggest a role for TGF-β superfamily member signaling in physosmolarity-induced mRNA expression of collagen type II. As physosmotic conditions favor the expression of COL2A1 independent of our manipulations, contribution of other metabolic, post-transcriptional or epigenetic factors cannot be excluded in the underlying complex and interdependent regulation of marker gene expression. Dissecting these molecular mechanisms holds potential to further improve future cell-based chondral repair strategies. Subject bone morphogenetic proteinschondrocytecollagen type IIosmolaritysignallingTGF-β superfamily To reference this document use: http://resolver.tudelft.nl/uuid:358c996f-80e4-42f5-8779-1104b0219e93 DOI https://doi.org/10.3390/ijms20040795 ISSN 1422-0067 Source International Journal of Molecular Sciences (Online), 20 (4) Part of collection Institutional Repository Document type journal article Rights © 2019 Ufuk Tan Timur, Marjolein Caron, Guus van den Akker, Anna van der Windt, Jenny Visser, Lodewijk van Rhijn, Harrie Weinans, Tim Welting, Pieter Emans, H. Jahr Files PDF ijms_20_00795.pdf 1.33 MB Close viewer /islandora/object/uuid:358c996f-80e4-42f5-8779-1104b0219e93/datastream/OBJ/view