IntroductionBecause current treatments for symptomatic intervertebral disk degeneration focus on pain rather than its cause, with only modest success in the long term, there is a need for new therapies. Since the earliest signs of intervertebral disk degeneration have been observed in the nucleus pulposus (NP), cell therapies focusing on NP regeneration are of great interest. Very early in life, the NP is populated with two cell types: NP cells and notochordal cells (NCs). Incidence and onset of disk degeneration is less and later in certain species that retain their NCs throughout adulthood vs. those that lose their NCs before adolescence.2 Consequently, it has been proposed that NCs might be involved in the maintenance of a healthy NP, and both direct3 and indirect coculture4 of NCs and NP cells have shown increased proteoglycan synthesis by NP cells in the presence of NCs. However, translation of such effects on isolated NP cells to NP tissue may not be straightforward. Thus, in this study, we investigated the in situ effects of NC therapy using an NP explant culture model developed previously in our group.5 Similar to cell cultures, we hypothesized that an injection of NCs in a NP explant would increase the extracellular matrix protein synthesis over 6 weeks in culture.Materials and MethodsPorcine lumbar and thoracic NPs (n = 4 donors,<8 weeks old) were harvested and digested to isolate clusters of NCs. NC clusters were then stained with cell tracker CFSE, to be traceable for the full duration of the experiment. Bovine caudal NPs (n = 12 donors, 24 months old) were harvested and injected with 10 µL of: (a) PBS (sham), (b) PBS + 250,000 NCs in clusters, or (c) PBS + 10 ng TGF-β3. The NPs (n = 6/group) were put in dialysis tubing (15 kD MWCO) and cultured for 42 days at 5% O2 in medium (lgDMEM) containing 13.3% polyethylene glycol (to prevent NP swelling)5, 10% fetal bovine serum, 1% penicillin/streptomycin, 100 mg/L ascorbic acid, and 3.7 g/L bicarbonate. At day 0 and 42, water (wet and dry weight), DNA (Hoechst 33529), gylcosaminoglycans (DMMB), and collagen (Chloramin-T) contents were measured. Furthermore, cell viability (Calcein blue-AM/propidium iodide), extra-cellular matrix distribution (safranin-O/fast green/hematoxylin staining), and gene expression (RT-qPCR for HPRT1, aggrecan, collagen-I & II & X, cytokeratin 8 & 19, vimentin, brachyury) were also determined.ResultsAt the end of the culture, the amounts of viable NCs and NP cells were similar to those before culture. The prestained NCs were viable, but no clear vacuolar structures, as observed at day 0, could be found. In all groups, day 42 water, DNA, proteoglycan, and collagen contents were similar to the values on day 0 (graph 1). Gene expression and histology are currently being analyzed.ConclusionInjection of NCs or TGF-β3 into NP did not result in a substantial anabolic tissue response. As previous studies have shown that both NCs3,4 and TGF-β36 stimulate extracellular matrix synthesis by isolated NP cells embedded in a 3D hydrogel, we tested if the lack of increased synthesis could be attributed to the NP culture system. Instead of limiting NP swelling in an osmotic manner (PEG), the NP was physically constrained using an artificial annulus fibrosus (with 100kD MWCO membrane) and only the sham and TGF-β3 groups were tested. Both groups showed results similar to those in the present study indicating that the NP culture system was not the cause of a diminished response. Hence, the discrepancy between the previously reported results3,4 and the present results might be explained by the following: (1) Change of NC phenotype during 6 weeks of culture: in coculture studies3,4 only the short-term (4 and 14 day) effect of NCs on NP cells was examined. As NCs may only be able to maintain their phenotype for a couple of days outside of their original matrix, their stimulating effects may be diminished in a 6-week study.(2) Different extracellular matrix synthesis by NP cells, when they are removed from their original matrix. NP cells may possibly have higher synthesis rates when removed from the tissue, to recreate a new stable environment. Also under such conditions, smaller differences in matrix synthesis, would be obvious but may still be insignificant when compared to matrix contents of the native NP.In conclusion, the stimulating effect of NCs on NP cells, found in isolated cell studies, was not observed when NCs were injected into NP tissue containing NP cells.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declaredLivshits G, et al. Annals of Rheumatic Disease 2011;70(10):1740–1745Hunter CJ, et al. Tissue Engeneering 2003;9(4):667–677Aguiar DJ, et al. Experimental Cell Research 1999;246(1):129–137Gantenbein-Ritter B, et al. European Spine Journal 2011;epubvan Dijk BGM, et al. Tissue Engeneering Part C Methods 2011;17(11):1089–1096Nishida k, et al. Spine 1999;24(23):2419–2425
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Aug 2, 2023
Kenichiro Kakutani + 4
