Rabbit tendon cells produce MMP-3 in response to fluid flow without significant calcium transients

Abstract

Forces applied to tendon during movement cause cellular deformation, as well as fluid movement. The goal of this study was to test the hypothesis that rabbit tendon fibroblasts detect and respond to fluid-induced shear stress. Cells were isolated from the paratenon of the rabbit Achilles tendon and then subjected to fluid flow at 1 dyn/cm² for 6 h in a specially designed multi-slide flow device. The application of fluid flow led to an increased expression of the collagenase-1 (MMP-1), stromelysin-1 (MMP-3), cyclooxygenase II (COX-2) and interleukin-1β (IL-1β) genes. The release of proMMP-3 into the medium exhibited a dose-response with the level of fluid shear stress. However, not all cells aligned in the direction of flow. In other experiments, the same cells were incubated with the calcium-reactive dye FURA-2 AM, then subjected to laminar fluid flow in a parallel plate flow chamber. The cells did not significantly increase intracellular calcium concentration when exposed to fluid shear stress levels of up to 25 dyn/cm². These results show that gene expression in rabbit tendon cells is sensitive to fluid flow, but that signal transduction is not dependent on intracellular calcium transients. The upregulation of the MMP-1, MMP-3 and COX-2 genes shows that fluid flow could be an important mechanical stimulus for tendon remodelling or injury.

Introduction

Cells regulate the biosynthetic activity of musculoskeletal tissues (Frank and Hart, 1990). It is generally accepted that this activity is, at least in part, regulated by the mechanical stress–strain state of the cell, and that the cellular stress–strain state depends directly on mechanical loading. For example, when a muscle contracts, the corresponding tendinous tissue may experience a variety of loading conditions: tension in the free running tendon, compression where the tendon contacts a bony prominence, or shear stress when individual fascicles move past each other or the tendon moves within its sheath (Benjamin and Ralphs, 1997). Therefore, cells on the tendon surface (epitenon and paratenon) are likely subjected to fluid-induced shear stress as interstitial fluid is circulated through tendon matrix past cells. We believe that tendon cells are subjected to fluid-induced shear stress as a consequence of reciprocal displacement, surface contact with extratendinous structures and interstitial fluid movement. Unfortunately, the levels of fluid-induced shear stress in tendons have not been measured or modelled, but numerous investigators have reported that friction occurs between tendons and sheaths (Goldstein et al., 1987; Uchiyama et al., 1995; Smutz et al., 1994).

One of the most rapid cellular responses to a mechanical stimulus is an increase in intracellular calcium (Banes et al., 1995). Elevated calcium levels initiate the transcription of many immediate early genes, which in turn affect the expression of late response genes (van Haasteren et al., 1999). Cells from bone, ligament and cartilage mobilize intracellular calcium in response to fluid flow (Hung et al (1995), Hung et al (1997); Yellowley et al., 1997). Osteoblasts, chondrocytes and endothelial cells exposed to fluid flow also alter gene expression over time (Malek and Izumo, 1995; Pavalko et al., 1998; Hung et al., 2000), but this response has yet to be evaluated with tendon cells.

We have previously used the rabbit Achilles tendon as a model to study how tendons respond to mechanical loading in vivo, and what factors are involved in the development of overuse injuries (Archambault et al., 2001). Since the relative displacement of a tendon in a sheath has been proposed to be a mechanism for tendon injury (Moore et al., 1991), we wanted to evaluate if tendon cells responded to the fluid flow that might occur as a result of this displacement. We hypothesized that rabbit tendon cells would respond to fluid-induced shear stress by increasing intracellular calcium, realigning in the direction of flow and altering gene expression. Particularly, we were interested in the expression of IL-1β, COX-2, MMP-1, and MMP-3, factors that might be involved in tendon injury and matrix degeneration.