TRPV4 channel activation improves the tensile properties of self-assembled articular cartilage constructs

Abstract

A persistent hurdle in the field of tissue regeneration is to produce tissues with biochemical and biomechanical properties robust enough to meet the aggressive physiological demands of the native milieu. In an effort to improve these properties tissues grown in vitro are often subjected to mechanical stimuli that aim to recapitulate the in vivo physiology. These mechanical stimuli are thought to produce downstream alterations in intracellular ion concentrations, which ultimately give rise to increased biosynthesis. There is mounting evidence that these perturbations in the cellular microenvironment are regulated by the Ca2+-permeable transient receptor potential vanilloid 4 (TRPV4) channel. In this study we examined the effects of targeted TRPV4 activation on self-assembled articular cartilage constructs. The objectives of this study were: (i) to determine whether TRPV4 activation would enhance self-assembled constructs; (ii) to identify an optimal treatment time window for TRPV4 activation; and (iii) to compare TRPV4 activation which Na+/K+ pump inhibition, which has previously been shown to improve the construct tensile properties. This study employed a two phase approach. In Phase I self-assembled constructs were grown for 4weeks and subjected to treatment with the TRPV4 agonist 4α-phorbol-12,13-didecanoate (4α-PDD) during three treatment time windows: t=6–10, t=10–14, and t=14–18days. Treatment for t=10–14days produced an 88% increase in collagen and a 153% increase in tensile stiffness. This treatment window was carried forward to Phase II. In Phase II we performed a head to head comparison between TRPV4 activation using 4α-PDD and Na+/K+ pump inhibition using ouabain. Treatment with 4α-PDD produced improvements on a par with ouabain (91–107% increases in tensile stiffness). The results of this study demonstrate the effectiveness of ion channel modulation as a strategy for improving engineered tissues. To our knowledge this is the first study to examine TRPV4 channel activation in tissue engineering.