Rabbit tendon cells produce MMP-3 in response to fluid flow without significant calcium transients
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.
Section snippets
Materials and methods
Cell populations were isolated from tendon according to the method described by Banes and co-workers (Banes et al., 1988). Cells from the Achilles tendon of male New Zealand White rabbits (age 5–7 months) were used in this study. The tendons were removed aseptically from rabbits sacrificed in unrelated experiments. The paratenon was separated from the Achilles tendon, incubated at 37°C in 0.5% collagenase for 5 min, then in 0.25% trypsin for 15 min. These enzymatic treatments released fibroblasts
Results
The 6 h flow protocol at 1 dyn/cm2 resulted in marked changes in gene expression (Fig. 2). Immediately post-flow, COX-2 was induced in experimental cells. After the 9 h incubation, COX-2 expression was still elevated, and MMP-1, MMP-3 and IL-1β were also expressed in the experimental cells; note that these genes were barely, or not detectable in control cells (C0 and C9 in Fig. 2). To substantiate the changes in gene expression, we also measured the levels of MMP-3 protein released into the
Discussion
Shear stress is one type of mechanical stimulus to which tendon cells are likely subjected in vivo. The objective of this research was to evaluate if rabbit tendon cells respond to fluid flow. This study is the first to report that tendon cells can alter gene expression in response to fluid flow. To our knowledge, this is also the first report of the sensitivity of the MMP-1 and MMP-3 genes to fluid flow. This induction is not a complete surprise, however, as AP-1 binding has been shown to
Acknowledgements
This work was supported by the Alberta Heritage Foundation of Medical Research, NIH-AR38121 and the Hunt Foundation. The authors wish to thank Marco Lotano for assistance with the cell culture and Joanne Bruno for help in staining the cells to visualize the actin cytoskeleton.
References (29)
- et al.
Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cellsa hypothesis for calcium-dependent and calcium-independent events activated by flow
Journal of Biomechanics
(1995) - et al.
Effects of shear stress on eicosanoid gene expression and metabolite production in vascular endothelium as studied in a novel biomechanical perfusion model
Biochemical and Biophysical Research Communications
(2000) - et al.
Analysis of cumulative strain in tendons and tendon sheaths
Journal of Biomechanics
(1987) - et al.
The effect of shear stress on fibroblasts derived from Dupuytren's tissue and normal palmar fascia
Journal of Hand Surgery [American]
(1998) - et al.
Pregnancy induces complex changes in the pattern of mRNA expression in knee ligaments of the adolescent rabbit
Matrix Biology
(1998) - et al.
Intracellular calcium response of ACL and MCL ligament fibroblasts to fluid-induced shear stress
Cell Signalling
(1997) - et al.
Mitogen-activated protein kinase signaling in bovine articular chondrocytes in response to fluid flow does not require calcium mobilization
Journal of Biomechanics
(2000) - et al.
Stimulation of transcription factors NF kappa B and AP1 in endothelial cells subjected to shear stress
Biochemical and Biophysical Research Communications
(1994) - et al.
Control of endothelial cell gene expression by flow
Journal of Biomechanics
(1995) - et al.
Fluid shear stress stimulates prostaglandin and nitric oxide release in bone marrow-derived preosteoclast-like cells
Biochemical and Biophysical Research Communications
(2000)
Investigation of low-force high-frequency activities on the development of carpal-tunnel syndrome
Journal of Clinical Biomechanics
Response of rabbit Achilles tendon to chronic repetitive loading
Connective Tissue Research
Mechanoreception at the cellular levelthe detection, interpretation, and diversity of responses to mechanical signals
Biochemistry and Cell Biology
Cell populations of tendon: a simplified method for isolation of synovial cells and internal fibroblastsconfirmation of origin and biologic properties
Journal of Orthopaedic Research
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