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Int. J. Mol. Sci. 2019, 20(4), 795; https://doi.org/10.3390/ijms20040795

Increased TGF-β and BMP Levels and Improved Chondrocyte-Specific Marker Expression In Vitro under Cartilage-Specific Physiological Osmolarity

1
Laboratory for Experimental Orthopedics, Department of Orthopaedic Surgery, Maastricht University Medical Centre+, 6229 HX Maastricht, The Netherlands
2
Institute of Anatomy and Cell Biology, RWTH Aachen University, 52074 Aachen, Germany.
3
Department of Orthopaedics, Erasmus MC, University Medical Center, 3000 CA Rotterdam, The Netherlands
4
Department of Internal Medicine, Erasmus MC, University Medical Center, 3000 CA Rotterdam, The Netherlands
5
Department of Orthopaedics, University Medical Center Utrecht, 3508 GA Utrecht, The Netherlands
6
Department of Rheumatology & Clinical Immunology, University Medical Center Utrecht, 3508 GA Utrecht, The Netherlands
7
Department of Biomechanical Engineering, Delft University of Technology, 2628 CD Delft, The Netherlands
*
Author to whom correspondence should be addressed.
Received: 30 December 2018 / Revised: 31 January 2019 / Accepted: 7 February 2019 / Published: 13 February 2019
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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. View Full-Text
Keywords: chondrocyte; osmolarity; TGF-β superfamily; signalling; collagen type II; bone morphogenetic proteins chondrocyte; osmolarity; TGF-β superfamily; signalling; collagen type II; bone morphogenetic proteins
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Tan Timur, U.; Caron, M.; van den Akker, G.; van der Windt, A.; Visser, J.; van Rhijn, L.; Weinans, H.; Welting, T.; Emans, P.; Jahr, H. Increased TGF-β and BMP Levels and Improved Chondrocyte-Specific Marker Expression In Vitro under Cartilage-Specific Physiological Osmolarity. Int. J. Mol. Sci. 2019, 20, 795.

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