3R-Project 83-02

The development of an in vitro intervertebral disc organ culture system

Cynthia Lee and Mauro Alini
Biochemistry and Cell Biology Unit; AO Research Institute, 7270 Davos, Switzerland
mauro.alini@aofoundation.org

Keywords: bovine; intervertebral disc; mechanobiology; explants; reduction; replacement

Duration: 2 years Project Completion: 2005

Background and Aim
Lower back pain is the most common health problem in individuals between the ages of 20 and 50 (1) with an estimated annual cost as high as $100 billion per year in the US alone. Despite the prevalence of low back pain, its etiology is largely unknown. There is mounting evidence, however, of a link between degenerated intervertebral discs (IVD) and clinical symptoms.
Several animals models are currently used to evaluate the effects of provoked disc injuries (mechanical and surgical) on disc composition and disc cell metabolism. These include: a) bipedal rats, in which rats forelimbs are removed, forcing the animals to walk on their hind legs and thus increasing mechanical stresses on their IVD (2); b) surgical treatments, such as anulus tears and external fixators (i.e. Ilizarov-type devices fixed to vertebrae and spanning one or more IVD) on sheep, dogs, pigs, rats, or mice to change the normal spine geometry and induce high stresses on certain areas (nucleus or anulus regions) of the instrumented (with fixator) or injured disc (for example: refs 3-8). To our knowledge, there is no suitable in vitro system to study the intervertebral disc.
The aim of this project is to develop a method for culturing intact intervertebral discs in vitro. In this method, discs will be explanted from bovine tails obtained from the local slaughterhouse, such that no animals are sacrificed specifically to fulfill the research aims.

Method and Results
Bovine coccygeal discs will be harvested from the tails of young cattle (6-8mos old) obtained from the local abattoir. It has already been established that such discs are a suitable model of the human lumbar disc (9). The discs will be cultured in a custom-built chamber similar to that used by Oshima, et al. (10). The chambers allow the discs to be submerged in culture medium with additional medium flowing over the top and bottom surfaces of the disc, and allow for loading of the discs by placing calibrated weights on the top of the chamber. To prevent swelling of the disc, the in situ swelling pressure of the disc will be balanced by weights resting on top of the chamber (mechanical compression).
In the first phase of this project, the appropriate culture conditions (magnitude of mechanical compression, media perfusion rate, length of culture) will be evaluated by assessing cell viability, metabolic activity, and matrix composition of the discs. Cell viability will be evaluated qualitatively using the calcein/ethidium live-dead cell assay (LIVE/DEAD^R Viability/Cytotoxicity Kit #L-3224, Molecular Probes) of fresh tissue samples. Metabolic activity will be assessed by ³⁵S-sulfate incorporation (into sulfated proteoglycans) and real-time PCR analyses of genes coding for matrix proteins and proteinases. Matrix composition will be evaluated in terms of water content (hydration) and proteoglycan content.
Once the appropriate baseline culture conditions are established, the utility of this system for mechanobiology studies will be assessed by measuring the cellular response to changing mechanical loads. Real-time PCR analysis will be used to evaluate the time-dependent response of the cells to changes in load magnitude.

Conclusions and Relevance for 3R
The goal for this project is to develop a system that maintains viable, metabolically active intervertebral discs in vitro. Specifically, our aim is to establish a system suitable for studying intervertebral disc mechanobiology. By conducting preliminary experimentsin vitro, we will be able to focus animal studies around the most relevant questions. In the future, this system may also be useful for investigations into potential treatments of disc disease (e.g. pharmaceutical, physiotherapeutic, and tissue engineering treatments).

References
1. Waddell G: The back pain revolution, Edinburgh, Churchill Livingstone, 1998

2. Cassidy JD, Yong-Hing K, Kirkaldy-Willis WH, Wilkinson AA, A study of the effects of bipedism and upright posture on the lumbosacral spine and paravertebral muscles of the Wistar rat, Spine 1988 Mar;13(3):301-8

3. Melrose J, Ghosh P, Taylor TK, Hall A, Osti OL, Vernon-Roberts B, Fraser RD. A longitudinal study of the matrix changes induced in the intervertebral disc by surgical damage to the annulus fibrosus. J Orthop Res. 1992 Sep;10(5):665-76

4. Cole TC, Burkhardt D, Ghosh P, Ryan M, Taylor T, Effects of spinal fusion on the proteoglycans of the canine intervertebral disc, J Orthop Res 3:277-291, 1985.

5. Hutton WC, Toribatake Y, Elmer WA, Ganey TM, Tomita K, Whitesides TE, The effect of compressive force applied to the intervertebral disc in vivo. A study of proteoglycans and collagen, Spine 23:2524-2537, 1998.

6. Kaigle A, Ekstrom L, Holm S, Rostedt M, Hansson T. In vivo dynamic stiffness of the porcine lumbar spine exposed to cyclic loading: influence of load and degeneration. J Spinal Disord. 1998 Feb;11(1):65-70.

7. Iatridis JC, Mente PL, Stokes IA, Aronsson DD, Alini M, Compression-induced changes in intervertebral disc properties in a rat tail model, Spine 24:996-1002, 1999.

8. Lotz, JC, Colliou OK, Chin JR, Duncan NA, Liebenberg E, Compression-induced degeneration of the intervertebral disc: an in vivo mouse model and finite-element study, Spine 23:2493-2506, 1998.

9. Oshima H, Ishihara H, Urban JP, Tsuji H, The use of coccygeal discs to study intervertebral disc metabolism, J Orthop Res 11:332-338, 1993.

10. Oshima H, Urban JP, Bergel DH, Effect of static load on matrix synthesis rates in the intervertebral disc measured in vitro by a new perfusion technique, J Orthop Res 13:22-29, 1995.

11. Lee CR, Iatridis JC, Alini M, An in vitro culture system for mechanoniology studies of he intervertebral disc. Spine (in press), 2006.

12. Lee CR, Poveda L, Iatridis JC, Alini M, Potential and limitations of an in vitro intervertebral dis organ culture system: Application for mechanobiology. Trans ORS, 2004.

13. Gantenbein B, Grünhagen T, Lee CR, von Donkelaar CC, Alinin M, Ito K An in vitro organ culturing system for intervertebral disc explants with vertebral endplates: a feasibility study with ovine caudal discs, Spine (in press), 2006.

14. Lee CR, Grad S, Mac LeanJJ, et al. Effects of mechanical loading on expression of common endogeneous controlm genes by articular chondrocytes and intervertebral disc cells, Anal. Biochem (in press), 2006.

15. Lee CR, Poveda L, Iatridis JC, Alini M, Effects of vertebral endplate conditions on bovine intervertebral disc organ culture: Potential and limitations for mechanobiological studies, Spine (in press), 2006.

16. Lee CR, Iatridis JC, Alini M, Poveda L, In vitro organ culture of the bovine intervertebral dis: Effects of vertebral endplate and potential for mechanobiological studies, Spine 315:, 2006.

Figures

Figure 1: Nucleus cell viability staining of (a) fresh disc and discs cultured for one (b,c) or seven (d-o) days with (b, d-i) or without (c, j-o) vertebral endplate (VEP). Live cells fluoresce green, dead cells fluoresce red. Images represent cells in a 50mm slice of the tissue, starting at a minimum of 50mm from the surface.