title: Low-intensity pulsed ultrasound stimulates cell proliferation and proteoglycan production in rabbit intervertebral disc cells cultured in alginate.
abstract: Intervertebral disc degeneration, one of the major causes of low-back pain, is known to result from alteration in biosynthesis of proteoglycan in the disc. Therefore, upregulating the synthesis of proteoglycan in intervertebral disc cells may be one approach in treating disc degeneration. Based on the finding that low-intensity pulsed ultrasound stimulates proteoglycan synthesis in rat chondrocytes, we investigated whether low-intensity pulsed ultrasound stimulates biological properties of rabbit intervertebral disc cells in vitro. Nucleus pulposus cells and annulus fibrosus cells isolated from rabbits were cultured in alginate beads. Cells were stimulated for 20 min each day for 5-12 days, starting on the third day after seeding. An ultrasound signal consisting of a 200 micros burst sine wave of 0.5 MHz repeating at 1 kHz, with an intensity of 0, 7.5, 15, 30, 60, 120 mW/cm2 spatial and temporal average, was applied. DNA and proteoglycan synthesis were evaluated by measuring [3H]-thymidine and [35S]-sulfate incorporation. DNA and proteoglycan content in beads were measured by Hoechst 33258 dye method and dimethylmethylene blue assay. Results demonstrated positive effects on DNA synthesis and content, following low-intensity pulsed ultrasound stimulation with intensities of 7.5 and 15 mW/cm2. Furthermore, ultrasound stimulation significantly upregulated [35S]-sulfate incorporation and proteoglycan content compared to the control group, following 5 days of stimulation in both nucleus pulposus and annulus fibrosus cells. These findings suggest the possible application of low-intensity pulsed ultrasound in biological repair of intervertebral disc degeneration.
authors.affiliation: Department of Orthopedic Surgery, Surgical Science, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan. tohru@is.icc.u-tokai.ac.jp
authors.forename: Toru
authors.initials: T
authors.lastname: Iwashina
authors.forename: Joji
authors.initials: J
authors.lastname: Mochida
authors.forename: Takeshi
authors.initials: T
authors.lastname: Miyazaki
authors.forename: Takuya
authors.initials: T
authors.lastname: Watanabe
authors.forename: Sadahiro
authors.initials: S
authors.lastname: Iwabuchi
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ando
authors.forename: Tomomitsu
authors.initials: T
authors.lastname: Hotta
authors.forename: Daisuke
authors.initials: D
authors.lastname: Sakai
mesh_terms: D000464:Alginates; D000818:Animals; D001562:Benzimidazoles / Q000737:chemistry; D018929:Cell Culture Techniques / Q000379:methods; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods; D019902:Chondrocytes / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D004247:DNA / Q000032:analysis / Q000096:biosynthesis; D020723:Glucuronic Acid; D006025:Glycosaminoglycans / Q000032:analysis / Q000096:biosynthesis; D006603:Hexuronic Acids; D007403:Intervertebral Disc / Q000166:cytology*; D008751:Methylene Blue / Q000031:analogs & derivatives / Q000737:chemistry; D011509:Proteoglycans / Q000032:analysis / Q000096:biosynthesis*; D011817:Rabbits; D014465:Ultrasonics
chemical_list: D000464:Alginates; D001562:Benzimidazoles; D006025:Glycosaminoglycans; D006603:Hexuronic Acids; D011509:Proteoglycans; C435946:dimethylmethylene blue; C447680:hoechst 32258; C013786:A73025; D020723:Glucuronic Acid; D004247:DNA; D008751:Methylene Blue