Organ Culture Stability of the Intervertebral Disc: Rat Versus Rabbit Dongrim Seol, 1 Hyeonghun Choe, 1,2 Prem S. Ramakrishnan, 1 Keewoong Jang, 1,2 Gail L. Kurriger, 1 Hongjun Zheng, 1 Tae-Hong Lim, 2 James A. Martin 1 1 Departments of Orthopedics and Rehabilitation, University of Iowa, Iowa City, Iowa 52242, 2 Biomedical Engineering, University of Iowa, Iowa City, Iowa 52242 Received 2 March 2012; accepted 5 November 2012 Published online 1 March 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22285 ABSTRACT: There is a need to develop mechanically active culture systems to better understand the role of mechanical stresses in intervertebral disc (IVD) degeneration. Motion segment cultures that preserve the native IVD structure and adjacent vertebral bodies are preferred as model systems, but rapid ex vivo tissue degeneration limits their usefulness. The stability of rat and rabbit IVDs is of particular interest, as their small size makes them otherwise suitable for motion segment culture. The goal of this study was to deter- mine if there are substantial differences in the susceptibility of rat and rabbit IVDs to culture-induced degeneration. Lumbar IVD motion segments were harvested from young adult male Sprague–Dawley rats and New Zealand White rabbits and cultured under standard conditions for 14 days. Biochemical assays and safranin-O histology showed that while glycosaminoglycan (GAG) loss was minimal in rabbit IVDs, it was progressive and severe in rat IVDs. In the rat IVD, GAG loss was concomitant with the loss of notochordal cells and the migration of endplate (EP) cells into the nucleus pulposus (NP). None of these changes were evident in the rabbit IVDs. Compared to rabbit IVDs, rat IVDs also showed increased matrix metalloproteinase-3 (MMP-3) and sharply decreased collagen type I and II collagen expression. Together these data indicated that the rabbit IVD was dramatically more stable than the rat IVD, which showed culture-related degenerative changes. Based on these findings we conclude that the rabbit motion segments are a superior model for mechanobiologic studies. ß 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:838–846, 2013 Keywords: lumbar intervertebral disc; animal model; organ culture; disc degeneration In the United States low back pain (LBP) is the second most common medical symptom requiring a physician visit. Costs associated with hospitalization and utiliza- tion of other health care services for LBP amount to nearly $90 billion/year. 1 Discogenic pain associated with degenerative disc disease (DDD) is thought to be a major source of LBP and DDD is considered a prima- ry target of therapies to alleviate back pain. 2,3 The eti- ology of DDD itself is complex, but there is strong evidence to suggest that abnormal mechanical loading plays a role in its pathogenesis. 4 The IVD provides me- chanical support and mobility to the spine and me- chanical stimulation is critical for IVD development and homeostasis. However, inappropriate loads lead- ing to excessive shear and tensile stresses that can lead to disc degeneration. 4 The effects loading on IVD cell viability and matrix integrity are of particular in- terest, as cell death and metabolic disturbances are known to promote disc degeneration. Better knowledge of the cellular mechano-transduction pathway(s) that regulate these responses is needed to identify opportu- nities for therapeutic intervention. Unfortunately, this effort has been hampered by the lack of suitable model systems for studying loading responses. In vivo studies of mechano-transduction in the IVD are complicated by the inability to fully control joint loading parameters. On the other hand, cell culture studies enable full control over mechanical conditions, but only after isolation of cells from their natural ex- tracellular matrix. In contrast, organ culture systems maintain disc cells in their natural context and enable fine loading control. IVD organ cultures derived from small animals are preferable, as their high surface-to- volume ratio facilitates diffusion of nutrients and waste products. 5 Culture models using IVDs from rats or rabbits have been used to test the effects of axial or hydrostatic compression; however, these experiments did not reproduce the tensile and shear loads experi- enced by the IVD in vivo, which are more likely to damage IVD structures than compressive loads. 6–8 Tensile and shear loading requires rigid fixation of IVDs in mechanically active culture systems, which necessitates retaining the vertebral bodies flanking the IVD. Our previous study showed that rat lumbar IVDs with intact endplates and adjacent vertebral bodies could be cultured as intact motion segments for a peri- od of 2 weeks without significant loss of cell viability. 9 Unfortunately, we also observed dramatic glycosami- noglycan (GAG) loss after 1–2 weeks in culture. As GAG preservation is a mandatory precondition for evaluating any effect of mechanical loads on disc de- generation, the culture-related changes rendered the model too unstable for our studies. 10–12 These findings led us to investigate rabbit motion segments as an al- ternative model system. Moreover, there are some inherent biochemical differences between rodent (rat) and mammalian (rabbit) species. We hypothesized that mammalian (rabbit) IVD culture would be more Grant sponsor: Department of Orthopaedic Surgery and Rehabilitation. Correspondence to: James A. Martin (T: þ1-319-335-5810; F: þ1-319-335-5631; E-mail: james-martin@uiowa.edu) ß 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. 838 JOURNAL OF ORTHOPAEDIC RESEARCH JUNE 2013