Establishment of an intervertebral disc whole-organ culture model with intact endplates

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Journal o f Biomechanics 2006, Vol. 39 (Suppl 1)

explicit relationship has been established between the elastic solid matrix properties, and a triphasic medium at an equilibrium state. Using these theoretical programs, indentation creep data of seventeen bovine specimens were analyzed. The intrinsic mechanical properties of the tissue were obtained together with the FCD values. Moreover, the cartilage sample exactly at the indented site can be harvested for biochemical GAG assay for sitespecific, biomechanics-biochemical correlations. The FCD values calculated by the triphasic theory are verified by standard biochemical assays with a correlation coefficient close to one. This accuracy may also be due to the site-specific harvesting of specimens at the indentation site. The intrinsic mechanical properties calculated from analysis of our triphasic indentation data are also consistent with those reported in literature. In conclusion, our triphasic indentation algorithm provides an excellent method to determine the relationship between the mechanical properties and proteoglycan content of articular cartilage with a hitherto unmatched degree of specificity of the materials tested at the indentation site. 4862 Mo, 14:45-15:00 (P10) Effect o f glycosaminoglycan (GAG) depletion on cartilage friction under various tribological conditions J. Katta, Z. Jin, E. Ingham, J. Fisher. IMBE, School ef Mechanical Engineering, University of Leeds, Leeds, UK GAGs have been shown to play an important role in the deformation and interstitial water pressurization of articular cartilage and in turn its load bearing mechanisms and friction characteristics. A pin-on-plate machine was utilized in this study to test the hypothesis that depleting GAGs from cartilage will increase its friction coefficient under different tribological conditions. Tests were divided into three models (n=6 each) based on the sliding conditions - Dynamic4 (4 mm/s sliding velocity; 4 mm stroke length), Dynamic2 (2 mm/s sliding velocity; 2 mm stroke length), and Static model (4 mm/s startup velocity) to provide different loading and unloading conditions on articular cartilage. For each pair of bovine cartilage pin (9mm Dia) and plate (20 15mm 2) in any model, the baseline coefficient of friction was determined with PBS as the lubricant under 25 N load. The samples were then treated with either chondroitinase ABC (CABC) or buffer (control) for 24 hours. Friction tests were then repeated to calculate the percentage change in the coefficient of friction for each pair. The study is the first to report the effect of GAG depletion on cartilage friction properties over a range of loading conditions in a cartilage sliding against cartilage configuration. CABC treatment rendered the cartilage tissue soft due to the loss of compressive stiffness and histological staining confirmed the loss of GAGs from the cartilage samples. CABC treated samples showed a statistically significant increase (>50%) in friction coefficients in the Dynamic4 model and this was believed to be due to the loss of biphasic lubrication operating in that model. In the case of the Dynamic2 and Static models where boundary lubrication played a major role, there was no significant difference between the friction levels between control tests and CABC treatment tests, consistent with our previous findings [1]. It remains to be established though, whether any boundary lubricant other than GAGs, such as lubricin/SZP or phospholipids that remain unaffected due to CABC treatment control the friction properties of cartilage under predominantly static loading conditions. References [1] Pickard J., et al. Proc. Inst. Mech. Eng. [H] 1998; 212(3): 177-82.

2.2. Disc Mechanics 4846 Mo, 16:00-16:15 (P12) Perfusion block results in decreased diffusion into ovine lumbar intervertebral discs M.J. van der Werf 1, E Lambers 2, P. Lezuo 1, O. Maissen 1, K. Ito 1,2. 1AO Research Institute, Daves, Switzerland, 2Dept. of Biemed. Eng., Eindhoven Univ. of Tech., The Netherlands Introduction: Cells inside the disc rely on diffusion through the endplate for nutrition and removal of waste products. Although correlation has been found between occlusion of endplate vascular openings and disc degeneration [1], causality has never been demonstrated. Therefore, an in vivo model for nutrient insufficiency induced disc degeneration is being developed. In this study, we determine if a method for disrupted endplate perfusion results in inhibited disc diffusion. Methods: Under general anaesthesia, cp-Ti-foils were inserted into slots in sheep lumbar vertebrae, parallel and adjacent to the endplates overlying the nuclear region of L2-L3 and L4-L5 discs. Inhalation gas mixture was changed from 98% 02 to 69/29% N20/O 2. In the next 35 minutes, intranuclear concentrations of 02 and N20 were measured amperometrically. Post-mortem, the vertebral vasculature was infused with Procion red and sagittal sections examined to quantify perfusion. Mechanical tests on cadaver motion segments were done to ensure that the procedure did not affect intradiscal pressure.

Oral Presentations Results: After change in gas mixture, the discs received less 0 2 and the drop over time was clearly seen, slower in the control discs than in blocked discs. The N20 diffusion was clearly inhibited by the block. After 35 minutes the N20 had increased more than 350% in the control discs, but in the blocked discs the increase was only 25%. In the control discs there were 1.5±0.99 perfused capillary buds per mm of endplate, whereas in the blocked discs this was 0.79±0.58 (p<0.01). No significant effect on the intradiscal pressure could be measured. Discussion: In this study N20 was used as a tracer for nutrient diffusion into the disc. It was demonstrated that a partial block of diffusion of N20 into the disc is achieved. This model will be used in future longer term studies. References [1] Benneker et al. Spine 2005.

6589 Mo, 16:15-16:30 (P12) Establishment o f an intervertebral disc whole-organ culture model with intact endplates D. Haschtmann, J.V. Stoyanov, L. Ettinger, S.J. Ferguson. MEM Research Center, University of Bern, Bern, Switzerland Introduction: Investigating intervertebral disc and endplate degeneration with current in-vive animal models often remains difficult due to the complexity of the involved metabolic and signaling cascades. In contrast, more controlled but simplified in-vitro systems with use of isolated cells or disc fragments are often afflicted with reduced cell viability or cell dedifferentiation in consequence of the unconstrained culture conditions. Therefore, there is a demand for a controlled culture model with preserved metabolic properties that offers the possibility to investigate disc and endplate pathologies in a structurally intact organ. Methods: Naturally constrained intervertebral disc/endplate units were isolated from female 6-months-old New Zealand White rabbits and cultured for up to 7 weeks in cell culture media (DMEM/F12, 10% FCS, 25~tg/ml ascorbate, 50~tg/ml gentamicin). Cell viability, metabolic activity, extracellular matrix (ECM) composition and ECM gene expression profile were monitored using the Live/Dead Cell Viability Test®, tetrazolium salt reduction (WST-8), proteoglycan- and DNA quantification assays and quantitative PCR. Results: Cell viability initially after disc isolation was 81±7%, which did not change significantly up to 49 days (p =0.12). While proteoglycan (GAG) and DNA content decreased slightly [GAG: non-significant decrease of 32±25%, DNA: decrease of 39±14% (p=0.0495)], matrix genes exhibited a degenerative profile with an up-regulation of type I collagen [Annulus (A): 14.5±2.9 fold, Nucleus (N): 162±11.2 fold] and suppression of collagen type II (A: >98%, N: 89±14%) and aggrecan genes (A: 88±8%, N: 84±4%). Additionally, cell metabolic activity was reduced to 33±12% of the initial value. Discussion: The established whole organ disc/endplate system exhibits major aspects of physiological disc properties with a degenerative pattern and therefore allows studying various aspects of disc metabolism and degeneration. Preservation of the bony endplates ensures structural integrity. Acknowledgements: AO Foundation, Switzerland.

5883 Mo, 16:30-16:45 (P12) Effect of limited nutrition on intervertebral disc health: an in vitro investigation on w h o l e organ disc explants with endplates B. Gantenbein 1, S. JLinger 1, T. GrL)nhagen 2, C.R. Lee 1, C.C. van Donkelaar2, M. Alini 1, K. Ito 1,2. 1AO Research Institute, Davos, Switzerland, 2Dept. of Biomed. Eng., Eindhoven Univ. of Tech., Eindhoven, Netherlands Introduction: In addition to genetic factors and ageing, limited nutrition and loading conditions are associated with an increased risk for disc degeneration. To investigate these latter effects and their interactions, we have recently developed an in vitro system for culturing whole intervertebral disc (IVD) explants. In vitro culture system: After systemic anti-coagulation and post-mortem vascular evacuation, caudal discs and adjacent endplates were excised from sheep tails. Freshly prepared discs were compared to those cultured under diurnal loading with media bathing all surfaces for 7 days. There was a small but non-significant decrease in mean cell viability in both annulus and nucleus tissues. GAG synthesis rates did not change significantly. Although gene expression in both tissue types revealed a non-significant down-regulation of anabolic genes and a significant "switching on" of catabolic genes, there were no obvious signs of matrix degradation. To our knowledge, this is the first report showing in vitro maintenance of a moderate size healthy disc with intact endplates. Limited nutrition and loading: Glucose concentration diffusing into the disc has been shown to play a key role for cell survival of disc cell populations, in particular for nucleus pulposus cells. To determine such effects in situ, glucose concentration in the DMEM media was reduced (22.5mM versus 10-15mM glucose) and adjusted with ribose to maintain the same osmolality. Discs were kept under physiological diurnal uni-axial loading (0.2/0.6MPa, 8/16h) with