• Hormoz Sheikh, Karen Zakharian, Ramiro Perez De La Torre, Christopher Facek, Adrian Vasquez, G Rasul Chaudhry, David Svinarich, and Mick J Perez-Cruet.
• University of Uppsala, Sweden.
• J Neurosurg Spine. 2009 Mar 1; 10 (3): 265-72.

ObjectThere is currently no biologic therapy to repair or restore a degenerated intervertebral disc. A potential solution may rest with embryonic stem cells (ESCs), which have a potential to grow indefinitely and differentiate into a variety of cell types in vitro. Prior studies have shown that ESCs can be encouraged to differentiate toward specific cell lineages by culture in selective media and specific growth environment. Among these lineages, there are cells capable of potentially producing nucleus pulposus (NP) in vivo. In this investigation, the authors studied ESCderived chondroprogenitors implanted into a degenerated disc in a rabbit. For this purpose, a rabbit model of disc degeneration was developed.MethodsA percutaneous animal model of disc degeneration was developed by needle puncture of healthy intact discs in 16 New Zealand white rabbits. Series of spine MR imaging studies were obtained before disc puncture and after 2, 6, and 8 weeks. Prior to implantation, murine ESCs were cultured with cis-retinoic acid, transforming growth factor beta, ascorbic acid, and insulin-like growth factor to induce differentiation toward a chondrocyte lineage. After confirmation by MR imaging, degenerated disc levels were injected with chondrogenic derivatives of ESCs expressing green fluorescent protein. At 8 weeks post-ESC implantation, the animals were killed and the intervertebral discs were harvested and analyzed using H & E staining, confocal fluorescent microscopy, and immunohistochemical analysis. Three intervertebral disc groups were analyzed in 16 rabbits, as follows: 1) Group A, control: naïve, nonpunctured discs (32 discs, levels L4-5 and L5-6); 2) Group B, experimental control: punctured disc (16 discs, level L2-3); and 3) Group C, experimental: punctured disc followed by implantation of chondroprogenitor cells (16 discs, level L3-4).ResultsThe MR imaging studies confirmed intervertebral disc degeneration at needle-punctured segments starting at approximately 2 weeks. Postmortem H & E histological analysis of Group A discs showed mature chondrocytes and no notochordal cells. Group B discs displayed an intact anulus fibrosus and generalized disorganization within fibrous tissue of NP. Group C discs showed islands of notochordal cell growth. Immunofluorescent staining for notochordal cells was negative for Groups A and B but revealed viable notochordal-type cells within experimental Group C discs, which had been implanted with ESC derivatives. Notably, no inflammatory response was noted in Group C discs.ConclusionsThis study illustrates a reproducible percutaneous model for studying disc degeneration. New notochordal cell populations were seen in degenerated discs injected with ESCs. The lack of immune response to a xenograft of mouse cells in an immunocompetent rabbit model may suggest an as yet unrecognized immunoprivileged site within the intervertebral disc space.

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