Project

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Evolution of in vitro Intervertebral Disc Culture Systems: Two Degrees of Freedom Loading (Axial Compression and Torsion) to Study Region-Specific and Synergistic Degenerative Processes

Applicant Gantenbein Benjamin
Number 127586
Funding scheme Project funding
Research institution Institut für chirurgische Technologien und Biomechanik (ISTB) Universität Bern
Institution of higher education University of Berne - BE
Main discipline Biomedical Engineering
Start/End 01.01.2010 - 31.12.2012
Approved amount 252'082.00
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All Disciplines (3)

Discipline
Biomedical Engineering
Molecular Biology
Cellular Biology, Cytology

Keywords (5)

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

Lay Summary (English)

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.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Region Specific Response of Intervertebral Disc Cells to Complex Dynamic Loading: An Organ Culture Study Using a Dynamic Torsion-Compression Bioreactor
Chan Samantha C. W., Walser Jochen, Käppeli Patrick, Shamsollahi Mohammad Javad, Ferguson Stephen J., Gantenbein-Ritter Benjamin (2013), Region Specific Response of Intervertebral Disc Cells to Complex Dynamic Loading: An Organ Culture Study Using a Dynamic Torsion-Compression Bioreactor, in PLoS ONE, 8(8), 72489-72489.
Papain-induced in vitro disc degeneration model for the study of injectable nucleus pulposus therapy
Chan Samantha, Bürki Alexander, Bonél Harald, Benneker Lorin, Gantenbein-Ritter Benjamin (2013), Papain-induced in vitro disc degeneration model for the study of injectable nucleus pulposus therapy, in The Spine Journal, epub ahead of print, 0-0.
A papain-induced disc degeneration model for the assessment of thermo-reversible hydrogel-cells therapeutic approach
Malonzo Cherry, Chan SCW, Kabiri A, Eglin David, Grad Sibylle, Bonél Harald, Benneker Lorin, Gantenbein-Ritter Benjamin (2013), A papain-induced disc degeneration model for the assessment of thermo-reversible hydrogel-cells therapeutic approach, in Journal of Tissue Engineering and Regenerative Medicine, 0(0), 0-0.
Intervertebral disc regeneration or repair with biomaterials and stem cell therapy - feasible or fiction?
Chan Samantha, Gantenbein-Ritter Benjamin (2012), Intervertebral disc regeneration or repair with biomaterials and stem cell therapy - feasible or fiction?, in Swiss Weekly Medical, 142(0), 0-0.
Biological response of the intervertebral disc to repetitive short term cyclic torsion.
Chan Samantha C. W., Ferguson Stephen J., Wuertz Karin, Gantenbein-Ritter Benjamin (2011), Biological response of the intervertebral disc to repetitive short term cyclic torsion., in Spine (Phila Pa 1976), 36(24), 2021-30.
Erratum to: The effects of dynamic loading on the intervertebral disc.
Chan Samantha C. W., Ferguson Stephen J., Gantenbein-Ritter Benjamin (2011), Erratum to: The effects of dynamic loading on the intervertebral disc., in Eur Spine J, 20, 1813-1813.
Harvesting technique to prepare intact bovine tail tntervertebral discs for organ culture including the endplates and partial vertebrae
Chan Samantha CW, Gantenbein-Ritter Benjamin (2011), Harvesting technique to prepare intact bovine tail tntervertebral discs for organ culture including the endplates and partial vertebrae, in Journal of Vizualized Experiments, 60, e3490-3490.
The effects of dynamic loading on the intervertebral disc.
Chan Samantha C. W., Ferguson Stephen J., Gantenbein-Ritter Benjamin (2011), The effects of dynamic loading on the intervertebral disc., in Eur Spine J, 20, 1796-1812.
The evolutionary importance of cell ratio between notochordal and nucleus pulposus cells: an experimental 3-D co-culture study.
Gantenbein-Ritter Benjamin, Chan Samantha C. W. (2011), The evolutionary importance of cell ratio between notochordal and nucleus pulposus cells: an experimental 3-D co-culture study., in Eur Spine J, epub ahead of print, NA-NA.

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
World Forum of Spine Research 18.06.2012 Helsinki
Spineweek 2012 28.05.2012 Amsterdam
World Forum of Spine Research 05.07.2010 Montréal, Canada


Communication with the public

Communication Title Media Place Year
Media relations: print media, online media Mit Biorekatoren dem Schmerz auf der Spur Solothurner Zeitung German-speaking Switzerland 09.05.2011

Awards

Title Year
1st poster Prize of Deutsche Wirbelsäulengesellschaft, Stuttgart, DE 2012
Best Poster Award: Effect of short term torsion to the intervertebral disc: An organ culture study 2010

Associated projects

Number Title Start Funding scheme
145003 A new Zeiss LSM 710 laser scanning microscope for the DCR LCI Core Facility 01.12.2012 R'EQUIP
153411 Exploring the mechanism of torsion-compression induced intervertebral disc degeneration and herniation in organ culture 01.05.2014 Project funding
109722 Effect of limited nutrition and cyclic compression on intervertebral disc degeneration 01.01.2006 Project funding
109722 Effect of limited nutrition and cyclic compression on intervertebral disc degeneration 01.01.2006 Project funding

Abstract

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.
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