Dynamic Compression of Chondrocyte-Agarose Constructs Reveals New Candidate Mechanosensitive Genes

Abstract

Articular cartilage is physiologically exposed to repeated loads. The mechanical properties of cartilage are due to its extracellular matrix, and homeostasis is maintained by the sole cell type found in cartilage, the chondrocyte. Although mechanical forces clearly control the functions of articular chondrocytes, the biochemical pathways that mediate cellular responses to mechanical stress have not been fully characterised. The aim of our study was to examine early molecular events triggered by dynamic compression in chondrocytes. We used an experimental system consisting of primary mouse chondrocytes embedded within an agarose hydrogel; embedded cells were pre-cultured for one week and subjected to short-term compression experiments. Using Western blots, we demonstrated that chondrocytes maintain a differentiated phenotype in this model system and reproduce typical chondrocyte-cartilage matrix interactions. We investigated the impact of dynamic compression on the phosphorylation state of signalling molecules and genome-wide gene expression. After 15 min of dynamic compression, we observed transient activation of ERK1/2 and p38 (members of the mitogen-activated protein kinase (MAPK) pathways) and Smad2/3 (members of the canonical transforming growth factor (TGF)-β pathways). A microarray analysis performed on chondrocytes compressed for 30 min revealed that only 20 transcripts were modulated more than 2-fold. A less conservative list of 325 modulated genes included genes related to the MAPK and TGF-β pathways and/or known to be mechanosensitive in other biological contexts. Of these candidate mechanosensitive genes, 85% were down-regulated. Down-regulation may therefore represent a general control mechanism for a rapid response to dynamic compression. Furthermore, modulation of transcripts corresponding to different aspects of cellular physiology was observed, such as non-coding RNAs or primary cilium. This study provides new insight into how chondrocytes respond to mechanical forces.

The Effect of Mechanical Loading on Articular Cartilage

“…The main function explored is the mechanotransduction, the molecular mechanism by which cells respond to mechanical stimuli. The development of AC in vivo [38], indeed, is influenced by mechanical stimuli, since they have both anabolic and catabolic effects on chondrocytes [39,40]. Understanding the mechanically-induced chondrogenesis of mesenchymal stem cells (MSCs) is of great interest for cartilage tissue engineering research, with the aim to produce cartilage with functionally competent mechanical properties.…”

Mechanotransduction in primary human osteoarthritic chondrocytes is mediated by metabolism of energy, lipids, and amino acids

“…MMP-13) which results in the breakdown of ECM molecules. Dynamic loading has been shown to promote these anabolic responses in chondrocytes; whereas, static loading has been shown to inhibit them [9, 10]. Dynamic compression has been shown to alter signal transduction including activation of GTPase signaling via the Rho-A and ROCK pathways, Erk-1 and -2, MAPK and SEK, and Smad2 [9, 11-13].…”

Digoxin and Adenosine Triphosphate Enhance the Functional Properties of Tissue-Engineered Cartilage

“…Interestingly, these cellular changes are also known to occur in chondrocytes during mechanical compression of cartilage. [62][63][64] Suramin, on the other hand, a reported inhibitor of the P2Y receptor, abolished ATPinduced changes in the Young's modulus, but did not affect ATP-induced changes in the collagen content. These results indicate that ATP can act by pathways independent of the P2Y receptor.…”