Progression of chondrocyte signaling responses to mechanical stimulation in 3-D gel culture

Abstract

Mechanical stimulation of 3-D chondrocyte cultures increases extracellular matrix (ECM) production and mechanical stiffness in regenerating cartilage. The goal of this study was to examine the progression of chondrocyte signaling responses to mechanical stimulation in 3-D culture during tissue regeneration. To investigate the role of integrins in chondrocyte mechanotransduction, function-blocking antibodies and small-molecule antagonists were used to disrupt integrin-matrix interactions during dynamic compression of chondrocytes in 3-D agarose culture. At early days in culture, blocking [alpha]v[beta]3 integrin abolished dynamic compression stimulation of proteoglycan synthesis, independent of effects in free-swell culture, while blocking [alpha]5[beta]1 integrins abolished the effect of compression only when blocking in free-swell increased proteoglycan synthesis. This suggests that disrupting [alpha]v[beta]3 and [alpha]5[beta]1 interactions with the ECM influences proteoglycan synthesis in distinct pathways and that [alpha]v[beta]3 more directly influences the mechanical response. To further distinguish individual mechanotransduction pathways, we investigated the temporal gene transcription response of chondrocytes to ramp-and-hold compression on Days 1, 10, and 28 in 3-D agarose culture. Clustered and individual gene expression profiles changed temporally and in magnitude over time in culture. Day 1 cultures differed from Days 10 and 28, reflecting changes in cell microenvironment with development of pericellular and extracellular matrices. Comparisons with the response of intact tissue to compression suggested similar regulatory mechanisms. We further investigated MAPkinase (ERK1/2, p38, JNK) and Akt activation on Days 1 and 28 in agarose culture through phosphorylation state-specific Western blotting.

(cont.) Compression induced transient ERK1/2 phosphorylation on both days, with Day 28 levels similar to intact tissue. Unique from tissue behavior, only slight transient p38 phosphorylation was observed on Day 28, and SEK phosphorylation was undetected. Akt was uniquely regulated in intact cartilage compared to MAPks, with decreased total Akt levels over time under static compression. In contrast, compression transiently decreased pAkt levels in agarose cultures, with no changes in total Akt. Changes in the chondrocyte responses to compression with time in agarose culture suggest that cells sense different forces and respond differently with time; further studies may help optimize mechanical loading for tissue-engineering purposes. These studies provide a basis for further examination of mechanotransduction in cartilage.