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Orthopaedic Biomechanics The effects of notochordal cells on matrix production by nucleus pulposus and bone marrow stromal cells I.T.M. Arkesteijn, L.A. Smolders*, S. Spillekom*, E. Potier, B.P. Meij*, K. Ito, M.A. Tryfonidou* *Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University Introduction The cell viability was similar in all culture groups on day 1 and 28. Most cases of back pain can be attributed to intervertebral disc The GAG/DNA amount increased significantly in time in all groups, degeneration, starting in the nucleus pulposus (NP). At birth the NP but NCs had no additive effects on NPCs or MSCs (fig 2). Histology contains two distinct cell types: small nucleus pulposus cells showed that NCs and non-clustered NPCs produced more (NPCs, fig 1A) and large, vacuolated notochordal cells (NCs, fig proteoglycans than MSCs and clustered NPCs. 1C), which disappear with age. Since the onset of degeneration occurs soon after the loss of NCs, it is believed that NCs play a central role in maintaining a healthy NP. As the number of NPCs also decreases during degeneration, it has been proposed to complement the NP cell population with bone marrow stromal cells (MSCs). In this study we hypothesize that NCs can upregulate matrix production by NPCs and MSCs. Figure 2: GAG/DNA assessing the effect of A) NCs on NPCs, B) NCs on MSCs. ACs used as control. Red bars=day 1, blue bars=day 15, green bars=day 28. $=p<0.05 difference in increase in time. Furthermore, in line with the biochemical analysis aggrecan gene Figure 1: H&E staining of canine NPCs on A) day 1 and B) day 28 (insert: clustered cells). Canine NCs on C) day 1 and D) day 28. Scale bar=50 µm expression (fig 3) did not increase in the NC containing co-cultures. Study design • Fresh NCs were isolated from mongrel dogs; NPCs, MSCs and articular chondrocytes (ACs, to control if effects are NC-specific) were isolated from Beagles. Cells were cultured in alginate beads (1.2%) in 400 mOsm hg-DMEM+10% FBS +1% P/S in 5% O2 for 28 days and the following groups were compared: Single cell cultures 3*106 cells/ml alginate Co-cultures 6*106 cells/ml alginate A) NC; NPC; AC A) NPC+NC; NPC+AC B) NC; MSC; AC B) MSC+NC; MSC+AC • Samples of day 15 and 28 were compared to day 1 for : Cell Figure 3: Aggrecan expression assessing the effect of A) NCs on NPCs, B) NCs on MSCs. ACs used as control. Red bars=day 1, blue bars=day 15, green bars=day 28. #=p<0.001 difference in increase in time. $=p<0.05 difference in increase in time. viability (calcein-AM, PI); cell morphology (H&E); DNA content Collagen type II expression increased in MSC+NC co-cultures (Hoechst dye assay); Glycosaminoglycan (GAG) content (DMMB compared to MSCs alone. However, this effect was much smaller assay); Matrix distribution (hematoxylin, safranin-O, fast green); Gene than in the MSC+AC control group. Brachyury expression was only expression (NC markers – brachyury, CK-18; ECM markers - detected in the groups containing NCs, and increased significantly in aggrecan, collagen type I & II; matrix degradation and remodelling – time in the NC and MSC+NC group and decreased for NPC+NC. MMP-13, ADAMTS5, TIMP1) Conclusions Results In this study, NCs were not able to up-regulate extracellular matrix On day 1 the NCs were large, vacuolated cells, but in time the production or disc matrix protein gene expression by NPCs and vacuole size decreased (fig. 1A,B). Like NPCs (fig 1C,D), MSCs and MSCs. This could be due the loss of NC phenotype in time that could ACs developed clusters in time. be attributed to suboptimal NC-culture conditions. Department of Biomedical Engineering This work was supported by AOSpine International through an AOSpine Research Network grant (SRN2011_11)