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Exposure to Pulsed Low Intensity Ultrasound Stimulates Extracellular Matrix Metabolism of Bovine Intervertebral Disc Cells Cultured in Alginate Beads

Miyamoto, Kei, MD, PhD*; An, Howard S., MD*; Sah, Robert L., MD, ScD§; Akeda, Koji, MD*; Okuma, Masahiko, MD, PhD*; Otten, Lori, MS*; Thonar, Eugene J.M.A., PhD*†‡; Masuda, Koichi, MD*†

doi: 10.1097/01.brs.0000184558.44874.c0
Basic Science
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Study Design. In vitro study on the effects of pulsed low intensity ultrasound on the cellular metabolism of bovine intervertebral disc cells.

Objective. To determine whether pulsed low intensity ultrasound has effects on cell proliferation and extracellular matrix metabolism by bovine intervertebral disc cells.

Summary of Background Data. The application of pulsed low intensity ultrasound is known to be effective in stimulating fracture and cartilage repair. However, the effects of pulsed low intensity ultrasound on intervertebral disc cells are not known.

Methods. Cells of the nucleus pulposus and inner and outer anulus fibrosus were enzymatically isolated from bovine coccygeal tissue and precultured in alginate beads for 14 days. In the ultrasound group, pulsed low intensity ultrasound was administered to the culture for 20 minutes daily for an additional 20 days. The control group was cultured in the same way but without administration of ultrasound. Cell viability, DNA content, proteoglycan and collagen synthesis, and proteoglycan content at days 10 and 20 after the initiation of treatment were evaluated. Characterization of newly synthesized collagen and proteoglycan was performed.

Results. No significant differences in cell viability and DNA content were observed between the two groups. On day 20, proteoglycan synthesis was increased by the application of pulsed low intensity ultrasound in nucleus pulposus and inner and outer anulus fibrosus cells (24%–26% increase, P < 0.001). The application of pulsed low intensity ultrasound increased proteoglycan content in alginate beads containing inner and outer anulus fibrosus cells (P < 0.05). Collagen synthesis by cells isolated from all three zones of the intervertebral disc was increased by the application of pulsed low intensity ultrasound (16%–19% increase, P < 0.05–0.0001).

Conclusions. The application of pulsed low intensity ultrasound stimulated extracellular matrix metabolism in intervertebral disc cells. Pulsed low intensity ultrasound may prove useful for the physical stimulation of cell metabolism for tissue engineering of intervertebral disc tissue.

Pulsed low intensity ultrasound treatment for 20 days significantly increased proteoglycan and collagen synthesis by intervertebral disc cells in alginate beads. The proteoglycan content was significantly increased in inner and outer anulus fibrosus cell-containing alginate beads. Pulsed low intensity ultrasound may be useful in tissue engineering of the intervertebral disc.

From the Departments of *Orthopedic Surgery, †Biochemistry, and ‡Internal Medicine, Rush Medical College at Rush University Medical Center, Chicago, IL; and §Department of Bioengineering & Whitaker Institute of Biomedical Engineering, University of California-San Diego, La Jolla, CA.

Acknowledgment date: August 16, 2004. First revision date: November 17, 2004. Acceptance date: November 24, 2004.

Supported by NIH Grant Nos. P50 AR-39239 and P01-AR48152.

The legal regulatory status of the device(s)/drug(s) that is/are the subject of this manuscript is not applicable in my country.

Institutional funds were received in support of this work. No benefits in any form have been or will be received from a commercial party related directly or indirectly to the subject of this manuscript.

Address correspondence and reprint requests to Koichi Masuda, MD, 1735 W. Harrison St., Cohn 720, Chicago, IL 60612; E-mail: kmasuda@rush.edu

© 2005 Lippincott Williams & Wilkins, Inc.