Development of an Ultrasound Stimulated Whole Disc Culture System

Objective: To develop a whole disc culture system with ultrasound stimulation. Summary of background data: Intervertebral disc degeneration is one of the most common spinal disorders. Nevertheless, no effective treatment or preventative strategies are available in the management of degenerative disc disease. Low-intensity pulsed ultrasound (LIPUS), which has been proved to be affective in assisting bone healing and stimulating bone growth, is a safe and non-invasive treatment modality. Bench top studies conducted at the cellular level have shown the evidence of LIPUS stimulation in nucleus pulposus proliferation and extracellular matrix regeneration. Nevertheless, the potential effect of LIPUS on the intervertebral disc at tissue or organ level are yet to be explored. Therefore, the goal of the current project is to develop an ultrasound stimulated disc culture system used for the study of effectiveness of LIPUS on the biomechanical, biological and biochemical properties of the degenerative disc. Methods: (1) Development of the disc culture system: The system includes bioreactor chamber, media circulation system, pressure loading system and ultrasound system. The bioreactor chamber served as a culture environment for disc. Media circulation system supplied disc nutrient cycling with flow rate 200μl/min. The loading system mimics the daily disc loading in human body, which provided the dynamic loading at 0.2-0.8 MPa for 16 hours and static loading at 0.2 MPa for 8 hour. The ultrasound system was able to stimulate the disc at different power, frequency, and duty cycle. (2) Calibration of ultrasound probe: Acoustic power meter and ultrasound machine were used to calibrate the output power of probe. First, the frequency was tuned up to its best driving frequency, then the magnification was adjusted to match the output power and the nominal power in 0.2W, 1W, 2W, 2W. (3) The energy field inside the bioreactor: 1 MHz transducer and thermocouples were used to measure the energy field. The input frequency was 1 MHz, and the power was 3 W. This experiment is divided into two parts. In the first part, we measured ultrasound energy in seven points inside bioreactor. In the second part, we recorded temperature in 15 mins with and without disc, and calculated the temperature rise and heating rate. (4) Organ culture test using porcine discs: Live/Dead assay were used for the assessment of disc viability. To verify the survival rate, discs were assigned to two groups, the intact group and the cultured group. The cell viability rate of the intact group was measured at day 0, and the cultured group was measured after 7 days of incubation. Result: (1) The calibration rate of ultrasound probes are closed to one, the probes are therefore determined as accurate output sources. (2) From the measurement of energy field inside the bioreactor, we knew that output energy did not attenuate in front of ultrasound probe, but it reduced when transducer moved to other two directions. In addition, the results of thermocouple have shown that the energy passed through the disc and was absorbed. (3) Specimens did not survive over 7 days at present; thus, two groups were changed to three groups for better understanding of the cell viability in the culture process, which were intact group, 3 days group, and 6 days group. The cell viability rates of intact group and 3 days group were close, but the 6 days group shown a significant decrease. The potential infection risks may be the source of specimen, such as the endophyte generated inside the disc, human contamination, and liquid leakage from the connections of bioreactor. Conclusion: The construction of whole culture system has been established, but the infection is an issue that needs to be solved. Keywords: whole disc culture system, low-intensity pulsed ultrasound, ultrasound calibration