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Orthopaedic Biomechanics Using notochordal cells to regenerate the degenerated intervertebral disc S.A.H. de Vries, I.T.M. Arkesteijn, E. Potier, M.A. Tryfonidou, K. Ito Introduction Disc degeneration is one of the main causes of low back pain and is characterized by the inability of the resident cells to maintain a healthy tissue, due to a decreasing number and/or a change of phenotype. Current treatments are only symptomatic and new approaches addressing the underlying cause of disc degeneration are needed, like cellbased regenerative treatments. In fact, mesenchymal stem cells (MSCs) have already been shown to stop but not reverse this degenerative process [1]. Innovative approaches are therefore needed to further promote disc matrix production by resident and/or regenerative cells. In human adults, the core of the disc is mainly populated by chondrocyte-like nucleus pulposus cells (NPCs). During growth, when the largest amount of matrix is produced, large vacuolated notochordal cells (NCs) are, however, also present. As the disappearance of these cells coincides with the onset of disc degeneration, they are believed to be involved in disc homeostasis. We thus hypothesize that NCs can increase the synthesis of functional matrix by resident cells, the NPCs, or by regenerative cells, the MSCs. and GAG deposition location; and gene expression (qPCR) of osteogenic- and chondrogenic differentiation markers and matrix and remodeling proteins. Results At day 1, the characteristic vacuoles of the NCs were clearly visible, as were the surrounding MSCs and NPCs in the co-culture groups (figure 1). A B C Figure 1: 40x confocal images of A: NCs alone, B: NCs + MSCs, C: NCs + NPCs. Arrows indicate NCs, white arrowheads indicate MSCs and red arrowheads indicate NPCs. Methods As certain dog breeds (chondrodystrophoid) develop disc degeneration similar to the human condition [2], a canine model will be used in this study. Tissue material will be sampled from healthy dogs used in unrelated experiments. Canine NPCs and MSCs will be retrieved from chondrodystrophoid beagles, and NCs from mixed breed dogs. The following groups of cells will be cocultured up to 28 days in 1.2% alginate beads: 1. NCs 5. NCs + MSCs 2. MSCs 6. NCs + NPCs 3. MSCs + TGFb 7. MSCs + NPCs 4. NPCs The cell density is 3 millions cells/ml for the single cultures and 6 millions cells/ml for the co-cultures using a ratio of 1:1. The cells will be cultured in 5% O2 at 37oC, and culture medium consists of DMEM supplemented with 5% ITS. At day 1, 14 and 28 beads will be analyzed: using confocal imaging for cell viability and morphology (Calcein AM, Propidium Iodide); GAG (DMMB assay) and DNA (Hoechst assay) content; histology (Alcian Blue, HE) for morphology / Department of Biomedical Engineering Culture (n=5) is ongoing and samples will be analysed at day 14 and 28. Future Research If co-culture with NCs up-regulates matrix production, the most successful strategy, stimulating either resident cells (NPCs) or regenerative cells (MSCs), will be tested for its therapeutic potential on NP tissue explants [3]. Furthermore, the mechanism behind NC’s up-regulation of matrix production by NPCs/MSCs (i.e. cell-cell contact or molecular interactions) will be deduced, and in case of molecular interactions, the factor(s) responsible for the NC’s up-regulating effect will be identified. Ultimately, this study will provide not only knowledge on the role of NCs in disc regeneration, but also an innovative treatment for patients suffering from disc degeneration. [1] F. Yang V.Y.L. Leung, K.D.K Luk, Mesenchymal stem cells arrest intervertebral disc degeneration through chondrocytic differentiation and stimulation of endogenous cells., Mol Ther, 2009 [2] J. Lotz, Animal models of intervertebral disc degeneration: lessons learned, Spine, 2004 [3] B.G.M. van Dijk, E. Potier, K. Ito. Culturing bovine nucleus pulposus explants by balancing medium osmolarity, Tissue Eng Part C, 2011. This work was supported by an AOSpine International Hans Joerg Wyss Foundation Research Award grant N HJW2011-SU12.