Project

Discipline

bioreactor; uni-axial compression; torsion; relative gene expression; live-dead assay

Lead
Lay summary
Aim of the StudyThis biomechanical project aims to demonstrate the importance of combined mechanical force and motion to the cell activity and metabolism of intervertebral disc (IVD) cells. We have developed an in vitro organ culture system of intact IVDs that allows to control parameters associated with disc degeneration. Further, combined axial torsion and cyclic compression have never been investigated at the disc cell level in situ in the intervertebral disc. Microscopic changes to the extracellular matrix and gene expression of disc cells will be monitored under physiological and "hyperphysiological" mechanical loading.Background and ImpactLow back pain is one of the major causes of high socioeconomic costs world-wide in the modern societies. About 80% of Swiss People suffer at least once in their lifetime from severe low back pain. Correlative evidence points towards a link between low back pain and degenerative disc disease (DDD). Genetics but also epi-genetic factors, like endplate calcification, mechanical overloading or endplate fractures, play a critical role in the risk for the development of degenerative disc disease. The degeneration of the IVD is accompanied by increased cell death, activation of catabolic enzymes, and finally also biochemical changes of the extracellular matrix. The project aims to demonstrate the importance of combined mechanical force and motion to the cell activity and metabolism of intervertebral disc cells. The results gained from these experiments may provide insights into the mechanisms leading to disc degeneration and may help in finding therapy strategies to prevent or to delay DDD.MethodologyWe have established an in vitro culture system of intact bovine or rabbit caudal IVDs with intact endplates, where cell viability can be maintained for up to 21 days under diurnal uni-axial compression (= control group). A novel loading scheme with 2D of freedom will be applied with two periods of cyclic compression and/or axial torsion. Excised spine motion segments either from the adult cow tail or the adult rabbit will be cultured in vitro with the endplates attached under controlled physiological conditions. From each disc, cell viability, gene expression of disc cells, cell activity and apoptosis will be monitored. For this project a novel design of 2D of freedom bioreactor is being developed, that handles 4 disc units at a time, which can be controlled independently either force- or way-driven.

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Last update: 21.02.2013

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Background. Low back pain is one of the major causes of high socioeconomic costs world-wide in the modern societies. About 80% of Swiss People suffer at least once in their lifetime from severe low back pain. Correlative evidence points towards a link between low back pain and degenerative disc disease (DDD). Genetics but also epi-genetic factors, like endplate calcification, mechanical overloading or endplate fractures, play a critical role in the risk for the development of degenerative disc disease. The degeneration of the IVD is accompanied by increased cell death, activation of catabolic enzymes, and finally also biochemical changes of the extracellular matrix. Previous in vivo and in vitro studies indicate relatively moderate cellular responses to uniaxial compressive loading, possibly due to the lack of shear and other complex forces. Here, we propose to increase the complexity of the mechanical loading regime by the addition of axial torsion during organ culture. It is known that pure torsion of ~2° will increase disc height, and decrease intra-discal pressure. The project aims to demonstrate the importance of combined mechanical force and motion to the cell activity and metabolism of intervertebral disc cells.Hypothesis 1 The two different regions of the IVD, the nucleus pulposus and the annulus fibrosus, will respond differently to the two mechanical stimuli, i.e. cyclic compression and torsion. Torsion can be “sensed” by the annulus fibrosus cells and will activate matrix production of large macro-molecules, i.e. collagen type 1, and to some extent collagen type 2. Intermediate or no changes are expected for the nucleus pulposus under torsion.Aim 1 To demonstrate regional-specific differences with respect to gene expression and cell activity between the nucleus pulposus and the annulus fibrosus in response to different applied forces / motions and to develop more realistic loading regimes.Hypothesis 2 “Hyperphysiological torsion” (2 x 4h ± 5°) will cause an increase of cell death in the the annulus fibrosus, whereas “hyperphysiological compression” (2 x 4h ± 5Hz) will cause an increase of cell death in the nucleus pulposus. Finally, 2D hyperphysiological loading, i.e. combination of hyperphysiological torsion with high frequency compressive loading further increases degeneration.Aim 2 To induce in vitro disc degeneration, i.e. increased cell death, by either excessive torsion, high frequency cyclic compression or a combination of both.Hypothesis 3 There is an optimal combination of torsion and cyclic compression (frequency and amplitude) that may be beneficial for disc cells.Aim 3 To induce in vitro disc degeneration by mechanical overload or physiological stress and to identify an optimal loading regime using cyclic compression and torsion, which activates anabolic genes, cell activity and GAG production over a mid-term culture period. Experimental Design We have established an in vitro culture system of intact bovine or rabbit caudal IVDs with intact endplates, where cell viability can be maintained for up to 21 days under diurnal (16 h at 0.8 MPa and 8 h at 0.2 MPa) uni-axial compression (= control group). A novel loading scheme with 2D of freedom will be applied with two periods of cyclic compression and/or axial torsion. Excised spine motion segments either from the adult cow tail or the adult rabbit will be cultured in vitro with the endplates attached under controlled physiological conditions. From each disc, cell viability, gene expression of disc cells, cell activity and apoptosis will be monitored.Significance of Research This in vitro organ culture system of intact IVDs allows control of parameters associated with disc degeneration. Combined axial torsion and cyclic compression have never been investigated at the disc cell level in situ in the intervertebral disc. The results gained from these experiments may provide insights into the mechanisms leading to disc degeneration and may help in finding therapy strategies to prevent or to delay DDD.