Introduction

Intervertebral disc degeneration is a common reason for low back pain and is attributed in part to abnormal mechanical loading experienced by the spine.  Our prior work (1) has demonstrated that prolonged excessive mechanical compression increased catabolic and inflammatory gene expression in a rabbit disc cell culture model.  To study the mechanobiological processes involved in disc degeneration, ex-vivo small animal functional spinal units (FSU) are tested under different mechanical loading conditions to observe changes in the biological outcomes.  In this project we developed a compression testing device that can mechanically load and sustain physiological conditions for rodent FSUs and allow for a higher throughput by testing four samples in parallel simultaneously.

Methods

The testing device that was developed uses a standard 12 well cell culture plate, with the sample specimens placed in the 4 corner wells.  The FSUs were isolated from Sprague Dawley rats and consisted of lumbar intervertebral discs and the superior and inferior vertebral bodies.  The samples are supported in each well by placing the inferior vertebra in the center hole of a custom thermoplastic washer.  The samples are cultured in 1ml F12 media.

The samples are loaded through four independent linear actuators (Physik Instrumente M-229.26S).   Each test assembly has a load cell (Sentran PC3-50-000) in series between the actuator and the sample to provide force feedback to the control loop.  The four stepper motors of the actuators are controlled through a custom Matlab program. The control program can apply load to the samples by either ramping up to and holding a prescribed load for a given time, or by cycling the load between prescribed values at a rate up to 1 Hz.  The control program also allows for a period of preconditioning at the beginning of the test.

Additionally, the samples must be maintained at physiological conditions of 37℃ and 5% CO2/O2. The entire bioreactor assembly was designed to fit within a Heracell 150i incubator.   A picture of the completed unit is shown in Figure 1.

Results

The results of a test run are shown in Figure 2.  The target force was calculated to be 7.9 N from the cross-sectional area of the superior vertebra and a target pressure of 1MPa.  The loading profile consists of a period cyclical loading for preconditioning, a rest period, and a ramp up to and hold at the target load. The mean and standard deviation of the load of each actuator during the hold portion of the test was found to be 7.89±0.06 N, 7.90±0.09, 7.92±0.15 N, and 7.92±0.14 N respectively.

Discussion

The design of the bioreactor satisfied all of the design constraints. The controller and actuator place loads on the specimen within the required parameters. In the future we will do biological tests to assess cell function (RT-PCR for expression of genes regulating matrix homeostasis) and cell viability (MTT assays,  TUNEL assays) on nucleus pulposus cells or annulus fibrosus cells of the intervertebral disc post loading in our bioreactor system.