Low-intensity pulsed ultrasound stimulates cell proliferation and proteoglycan production in rabbit intervertebral disc cells cultured in alginate

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 μs 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.