Original contribution
Effects of Shock Waves on Tenocyte Proliferation and Extracellular Matrix Metabolism

https://doi.org/10.1016/j.ultrasmedbio.2007.11.002Get rights and content

Abstract

The shock wave is an effective noninvasive modality for resolving various tendon pathologies. However, scientific rationale and mechanism of shock wave therapy remains limited. This study aims to investigate the effects of shock waves and their biochemical mechanisms on tenocyte proliferation and collagen synthesis. Tenocytes harvested from Achilles tendons of Sprague-Dawley rats were used in this study. Cell viability was assayed by trypan blue exclusion methods. The colorimetric assay was determined to evaluate the mitochondria activity of the tenocytes after shock wave exposure. Synthesis of collagen, nitric oxide (NO) and transforming growth factor-β1 (TGF-β1) were determined and their gene expression was also studied. The results showed that there was a dose-dependent impairment of cell viability observed in 0.36 mJ/mm2 and 0.68 mJ/mm2 stimulation. In the proliferation assay, low energy level with low impulses (0.36 mJ/mm2 with 50 and 100 impulses) showed positive stimulatory effects, whereas the high energy level with high impulses (0.68 mJ/mm2 with 250 and 500 impulses) had significant inhibitory effects. At 0.36 mJ/mm2, 100 impulse shock waves treatment, up-regulation of proliferating cell nuclear antigen (PCNA) (at 6 and 24 h) and collagen type I, collagen type III and TGF-β1 gene expression (at 24 h) were observed; these were followed by the increases in NO production (at 24 h), TGF-β1 release (at 48 and 96 h) and collagen synthesis (at the 7th day). This study revealed that shock waves can stimulate tenocyte proliferation and collagen synthesis. The associated tenocyte proliferation is mediated by early up-regulation of PCNA and TGF-β1 gene expression, endogenous NO release and synthesis and TGF-β1 protein and then collagen synthesis. (E-mail: [email protected])

Introduction

The shock wave used in extracorporeal shock wave treatment (ESWT) is a focused, high-energy acoustic pressure disturbance created by the translation of energy via an electrohydraulic, electromagnetic or piezoelectric device; the wave is transmitted to the patient through either water or a coupling gel (Sems et al. 2006). Because the initial therapeutic introduction of shock waves to the human body is to noninvasively treat kidney stones (lithotripsy), this technology has evolved to be considered the procedure of primary choice for urolithiasis (Lotan and Pearle 2007). Despite more than 20 years of experience with extracorporeal shock wave lithotripsy (Bihl and Meyers 2001), its use in orthopaedics and traumatology is still considered. Many clinical studies on ESWT have been done to treat orthopaedic disorders, such as epicondylitis, painful heel syndrome, calcific tendonitis of the shoulder, chronic plantar fasciitis, nonunions, pseudarthrosis and femoral head necrosis in adults (Speed 2004). Despite the efficacy in clinical application, scientific evidence of shock wave therapeutic effects and biochemical mechanisms on tenocytes remain limited, and much remains to be learned about the etiology, pathophysiology and management of these tendinopathies.

The shock wave entering the tissue may be reflected or dissipated, depending on the properties of the tissue. The energy of the shock wave may act through mechanical forces generated directly or indirectly via cavitation (Ogden et al. 2001b). The mechanisms of the action of shock waves on soft-tissue conditions are unknown, but possibly include direct stimulation of the “healing” processes, neovascularization, disintegration of calcium and neural effects. These may involve alterations in the permeability of cell membranes, preventing the development of potentials to transmit painful stimuli, as well as direct suppressive effects on nociceptors and a hyperstimulation mechanism that blocks the gate control mechanism (Wang et al 2002a, Rompe et al 1998, Schelling et al 1994). However, these possibilities remain speculative.

ESWT may be delivered in various energy flux densities, measured in mJ/mm2. Although the terms “high” and “low” energy are commonly used in literature, there is no clear consensus on the energy flux densities involved in relation to these terms. Lower-energy flux application (<0.10 to 0.12 mJ/mm2) is tolerated generally with mild to moderate discomfort; high-energy flux applications (>0.12 mJ/mm2) require local or regional anesthesia (Rompe et al. 2001). Individual treatments are usually described according to the number of shocks administered, the generator frequency and the energy level setting. The total amount of energy delivered per session is determined by multiplying the total flux density by the number of shock waves delivered. The multiple combinations of energy flux densities and numbers of shock waves delivered result in differing amounts of total energy delivered to the tissue being treated. The frequency of shock wave delivery is another variable in ESWT (Sems et al. 2006).

This observation emphasizes the importance of the experimental condition and the consequent difficulties in extrapolating results of a cell suspension study to clinical practice. Following these previous studies and considering the wide application of extracorporeal shock wave therapy in the musculoskeletal field, we evaluated the biologic effects of shock waves on tenocytes. The relationship among the energy density of the shock waves, the number of shock waves applied on the different parameters of cell viability and the biologic effects of this kind of therapy also was also investigated. Tenocytes were treated with ESWT with a shock waves generator selecting two different energy levels (0.36, 0.68 mJ/mm2) and four different shock waves numbers (50, 100, 250 and 500 impulses). At the end of the treatment, cell viability was evaluated together with biochemical activity and gene expression of cells at 24, 48 and 96 h. The study aims to elucidate the effects of shock waves on cultured tenocytes and to further investigate the molecular and biochemical mechanisms by which shock waves promote tenocyte proliferation and regulates collagen synthesis in vitro.

Section snippets

Primary culture of rat tenocytes

The Achilles tendons from 16 Sprague-Dawley rats (6 weeks old, weighing 200–250g, from the animal laboratory of National Yang-Ming University) were excised and washed twice in phosphate-buffered saline (PBS). Each tendon was then cut into small pieces (<1 mm in diameter; approximately 1.5–2.0 mm3) and these pieces were individually placed in six-well culture plates. One milliliter of Dulbecco's Modified Eagle Medium (DMEM; Hyclone, Rontegenstraat, Holland), with 10% fetal bovine serum (FBS,

Effects of shock wave on cell viability

Cells in test tubes were exposed to two levels of energy flux density (0.36 and 0.68 mJ/mm2) and four different shock waves numbers (50, 100, 250 and 500 shock waves). The control group was treated under the same preparation without shock wave stimulation. After the shock waves exposure, cell viability assay was done immediately. The results showed 0.36 mJ/mm2 (50, 100 and 250 impulses), which revealed normal viability, whereas 0.36 mJ/mm2 (500 impulses) and 0.68 mJ/mm2 (50, 100, 250 and 500

Discussion

ESWT has been widely used for lithotripsy treatment of different organs, such as the kidneys, biliary tract, urethral tract and salivary glands, and is now used frequently in orthopaedics and traumatology to avoid surgery and alleviate pain (Ogden et al 2001a, Odgen et al 2001b, Thiel 2001). ESWT may be delivered in various energy flux densities, measured in mJ/mm2. Lower-energy flux application (<0.10 to 0.12 mJ/mm2) is generally tolerated, with mild to moderate discomfort; high-energy flux

Acknowledgments

We thank Taipei City Hospital Yang-Ming Branch and Meden International Inc. for their technical support of shock wave device in this research. We also thank Miss Margaret, Man-Ger SUN for her help in the editing and preparation of this manuscript.

References (38)

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