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

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.

Osteoarthritis and Cartilage Regeneration: Focus on Pathophysiology and Molecular Mechanisms

“…Timur et al attempted to dissect the mechanism underlying the chondrogenic effect of physiological osmolarity on in vitro cultured human articular cartilage chondrocytes [21]. While normal expansion culture medium with plasma osmolarity (280 mOsm) induces chondrocyte dedifferentiation, higher levels of oxygenation mimicking physiological osmolarity of the healthy cartilage (in a range of 380-450 mOsm) favors the elevated expression of the chondrogenic marker gene COL2A1.…”

Bioreactor-Controlled Physoxia Regulates TGF-β Signaling to Alter Extracellular Matrix Synthesis by Human Chondrocytes

“…To assess the effect of physoxia on the chondrocytic phenotype, we screened the expression of ECM markers associated with a differentiated (i.e., wanted) and de-differentiated (i.e., unwanted, fibroblastic) phenotype of articular chondrocytes. We used the expression of the core protein of aggrecan ( ACAN ), an important proteoglycan (PG) of the ECM of articular chondrocytes, and that of the structural key marker protein collagen type II ( COL2A1 ) [37], as indicators of a proper phenotype (Figure 2). Expression of both genes showed an inverse pO 2 -dependency, up-regulation of mRNA abundances with down-regulated pO 2 levels.…”

“…Ordering details and instructions for using the TGF-β2-specific ELISA assay can be found elsewhere [37]. Briefly, medium was collected and centrifuged at 1200 rpm for 8 min to remove cell debris.…”

Aggrecan and COMP Improve Periosteal Chondrogenesis by Delaying Chondrocyte Hypertrophic Maturation

“…GAG-bound TGF-β is able to stimulate neocartilage formation ( Park et al, 2008 ) and it was recently shown that under cartilage physiological osmolarity TGF-β signaling was increased. This was associated with an improved chondrocyte phenotype ( Tan Timur et al, 2019 ). Indeed, also in our studies we observed increased (but not significant) TGF-β3 expression in the Aggrecan-supplemented conditions.…”