Culturing disc endplates
Citation for published version (APA):Potier, E., Wang, P. H., & Ito, K. (2009). Culturing disc endplates. Poster session presented at Mate Poster Award 2009 : 14th Annual Poster Contest.
Document status and date: Published: 01/01/2009
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DNA, GAG, hydroxyproline content
DNA content remained stable up to 21 days (Fig. 3).
GAG and hydroxyproline stayed constant up to day 7, but decreased at day 21 (Fig. 3).
Tissue morphology
Proteoglycan staining was less intense at day 21 than at day 1 and 7, with EP layers thinning observed at day 7 and 21 (Fig. 4).
Discussion
Culture conditions were appropriate for EP viability, but not sufficient to maintain EP phenotype (GAG loss, EP thinning).
Will investigate the capacity of high osmotic pressure (similar to native nucleus pulposus environment) to impede proteoglycan loss in EP explant culture.
Introduction
Intervertebral discs (IVDs) consist of a firm, flexible outer layer (annulus fibrosus) surrounding a gelatinous, compressible core (nucleus pulposus). Endplates (EPs) are cartilaginous layers that lie between the IVD and the vertebral body (Fig. 1). As nucleus pulposus is avascular, its nutrient and oxygen supply relies on diffusion from the vertebral body blood vessels through the EPs [1].
Due to aging or some diseases, EPs can calcify, impairing nutrient diffusion to the nucleus pulposus [2] and potentially leading to disc degeneration, a major cause of low back pain.
Overall objective: To investigate the calcification process
of the disc EPs, and a way to reverse it.
Study objective:To establish an explant culture system for disc EPs.
Methods
Cartilaginous EPs were harvested from bovine tails (24-30 month old; n=3, each donor in duplicate) and consisted of cores (Ø 10mm) surrounded by thin layers of bone on one side and of nucleus pulposus on the other.
EPs were cultured for 3 weeks in hg-DMEM, 10% FBS, 25 µg/ml ascorbic acid, 0.1 mM NEAA, 0.4 mM L-proline. At day 0, 7, and 21 cell viability was evaluated by calcein/propidium iodide staining; tissue phenotype was evaluated by Hoechst dye, DMMB, and chloramin-T assays for DNA, glycoaminoglycan (GAG), and hydroxyproline (representative of collagen) contents respectively; and tissue morphology was assessed with safranin-O/Fast Green staining for proteoglycans and collagens.
Results
Cell viability
No significant differences were observed between day 0, 7, and 21 (Fig. 2).
Culturing disc endplates
E. Potier, P.H. Wang, K. Ito
/ Department of Biomedical Engineering
Figure 2. Cell Viability. Calcein/propidium iodide staining. Representative of 6 explants/group. Scale bar: 100 µm.
References
[1] Moore, 2000, Eur Spine J 9 [2] Roberts, 1996, Spine 21 Figure 1. IVD and endplate structure.
Endplate Nucleus Pulposus
Annulus Fibrosus
Vascular bud Vertebral body
Figure 4. Tissue morphology. Safranin-O/Fast Green staining, Hematoxylin counter-staining. Representative of 6
explants/group. Scale bar: 250 µm.
Figure 3. DNA, GAG, hydroxyproline contents. Per mg of dry weight. Values are means +/- St Dev. N=6/group.
Day 0 Day 7 Day 21
DNA GAG Hydroxyproline
µµµµ g D N A /m g d ry w e ig h t µµµµ g G A G /m g d ry w e ig h t 0 0.5 1 1.5 2 0 7 21 Days in culture D N A /d ry w e ig h t (u g /m g ) 0 3 6 9 12 0 7 21 Days in culture G A G /d ry w e ig h t (u g /m g ) 0 50 100 150 200 250 0 7 21 Days in culture G A G /d ry w e ig h t (u g /m g )
Day 1 Day 7 Day 21
µµµµ g D N A /m g d ry w e ig h t µµµµ g G A G /m g d ry w e ig h t µµµµ g H Y P /m g d ry w e ig h t
