An organ culture system to model early degenerative changes of the intervertebral disc
- Ravi K Ponnappan†1, 2,
- Dessislava Z Markova†1,
- Paul JD Antonio3,
- Hallie B Murray3,
- Alexander R Vaccaro1, 2,
- Irving M Shapiro1,
- D Greg Anderson1, 2,
- Todd J Albert1, 2 and
- Makarand V Risbud1Email author
© Ponnappan et al.; licensee BioMed Central Ltd. 2011
Received: 3 February 2011
Accepted: 21 October 2011
Published: 21 October 2011
Back pain, a significant source of morbidity in our society, is related to the degenerative changes of the intervertebral disc. At present, the treatment of disc disease consists of therapies that are aimed at symptomatic relief. This shortcoming stems in large part from our lack of understanding of the biochemical and molecular events that drive the disease process. The goal of this study is to develop a model of early disc degeneration using an organ culture. This approach is based on our previous studies that indicate that organ culture closely models molecular events that occur in vivo in an ex vivo setting.
To mimic a degenerative insult, discs were cultured under low oxygen tension in the presence of TNF-α, IL-1β and serum limiting conditions.
Treatment resulted in compromised cell survival and changes in cellular morphology reminiscent of degeneration. There was strong suppression in the expression of matrix proteins including collagen types 1, 2, 6 and 9, proteoglycans, aggrecan and fibromodulin. Moreover, a strong induction in expression of catabolic matrix metalloproteinases (MMP) 3, 9 and 13 with a concomitant increase in aggrecan degradation was seen. An inductive effect on NGF expression was also noticed. Although similar, nucleus pulposus and annulus fibrosus tissues showed some differences in their response to the treatment.
Results of this study show that perturbations in microenvironmental factors result in anatomical and gene expression change within the intervertebral disc that may ultimately compromise cell function and induce pathological deficits. This system would be a valuable screening tool to investigate interventional strategies aimed at restoring disc cell function.
Intervertebral disc (IVD) degeneration is believed to be one of the major underlying causes of spinal pathology. Low back conditions are ubiquitous, accounting for approximately $100 billion in direct health care-related costs in the US in 2008 [1, 2]. Published reports estimate a higher prevalence of degenerative discs as age increases, and nearly 90% of people over 60 years have at least one degenerative disc on a magnetic resonance imaging . Clinical manifestations stemming from IVD degeneration vary based on the grade of degeneration and associated spinal pathology.
IVD degeneration typically occurs through a progressive and stepwise cascade of events which results from the alteration of cellular and extracellular matrix composition of the nucleus pulposus (NP) and annulus fibrosus (AF) [4, 5]. Early changes in human disc degeneration often occur at the cellular level without an appreciable disruption of the native disc architecture. As the degenerative cascade progresses, structural abnormalities appear within the disc, resulting in macroscopic changes and compensatory healing responses. Higher-grade degenerative processes often lead to clinically evident conditions that may require medical or surgical management. Current research attempts at investigating disc degeneration have focused on models thought to represent the late-stage degenerative changes [6–16]. Inevitably, induction of the degenerative cascade in these models requires the mechanical disruption of the AF and creating structural and morphological changes that mimic late degeneration [15–17]. This naturally results in the inability to focus on early cellular and molecular changes. Other models have described the use of supra-physiological mechanical loading of functional spinal units with external apparatus . While this type of approach does not disrupt the annulus, achievement of controlled and consistent degeneration may rely on uniformity of mechanical loading, require longer induction periods, and bring higher costs often associated with in vivo studies.
As an alternative to some of the in vivo models, other studies have described disc organ culture systems to investigate disc degeneration [17, 19–25]. Some of these reports are feasibility studies designed to describe and characterize techniques to maintain a viable disc organ culture [17, 19, 21], whereas other studies use enzymatic digestion of the proteoglycan matrix or needle puncture injury to replicate the cellular and architectural changes that occur during disc degeneration without intrinsic action of proinflammatory and catabolic mediators (as postulated in human degenerative disc disease, or DDD) [25–27]. To the best of our knowledge, our study is the first attempt to report an in vitro model that induces physiological disc degeneration and allows the investigation of early disc degeneration without annulus disruption.
Materials and methods
Disc harvesting and organ culture
Animal experiments were approved by the Institutional Animal Care and Use Committee. Wistar rats (250 to 300 g; Charles River Laboratories International, Inc., Wilmington, MA, USA) were euthanized with CO2 and the spinal columns were removed en bloc under aseptic conditions. Lumbar IVDs comprising the vertebral endplates, AF and NP were harvested (six discs per animal). Soft tissues and posterior elements were removed, and discs were rinsed in saline solution before being placed in 48-well culture plates. All experimental specimens were cultured in an hypoxia workstation (Invivo2; Ruskinn Technology Ltd, Pencoed, UK) in 1% O2, 5% CO2, and 93% N2 in Dulbecco's modified Eagle's medium (DMEM) containing 1% fetal bovine serum (FBS), tumor necrosis factor-alpha (TNF-α) 100 ng/mL, interleukin-1beta (IL-1β) 10 ng/mL (R&D Systems, Inc., Minneapolis, MN, USA), 50 μg/mL L-ascorbate, 40 mM NaCl, antibiotics, and antimycotics (Cellgro; Mediatech, Inc., Manassas, VA, USA). Concentration of cytokines was based on the literature [28, 29]. Importantly, slightly higher doses of cytokines were chosen to compensate for the lack of systemic feedback present from immune and neuronal cells during degeneration. Control groups were cultured in DMEM containing 20% FBS, 50 μg/mL L-ascorbate, 40 mM NaCl, and antibiotics. The discs were maintained for 3 and 10 days. The complete medium was replaced every 2 days for all groups.
Assessment of molecular diffusion
Assessment of apoptosis
For in situ identification of apoptotic cells, a TACS 2 TdT-Fluor In Situ Apoptosis Detection Kit was used on histological sections in accordance with the instructions of the manufacturer (Trevigen Inc., Gaithersburg, MD, USA). In brief, 8-μm tissue sections from paraffin-embedded discs were deparaffinized in xylene, rehydrated, and pretreated with proteinase K solution for 15 minutes at 37°C. The slides were rinsed twice in deionized water and incubated with 50 μL of labeling reaction mixture for 1 hour at 37°C in a humidified chamber. After washing with phosphate-buffered saline (PBS), 50 μL of Strep-Fluor solution was applied, and the slides were incubated for 20 minutes. The slides were washed three times in PBS and mounted under glass coverslips using fluorescence mounting media (Vector Laboratories, Burlingame, CA, USA) and analyzed under a fluorescence microscope using a 495-nm filter (Nikon eclipse TE 2000-U; Nikon, Tokyo, Japan).
Histology and immunohistochemistry
Whole disc units, including the endplates, were fixed in 10% formaldehyde in PBS for routine histological and immunohistochemical evaluation. After fixation, disc units were serially dehydrated in alcohol. Decalcification was accomplished with 12.5% ethylenediaminetetraacetic acid (EDTA) (pH 7.4) followed by paraffin embedding. The tissue blocks were sectioned parallel to the endplate (6 to 8 μm in thickness), and sections were deparaffinized in xylene and rehydrated through graded ethanol and stained with Alcian blue, eosin, and hematoxylin. For localizing matrix metalloproteinase 3 (MMP-3), sections were incubated with rabbit monoclonal MMP-3 antibody (EP1186Y; Abcam, Cambridge, MA, USA) in 2% bovine serum albumin in PBS at a dilution of 1:250 at 4°C overnight. After washing of the sections, the bound primary antibody was incubated with Alexa fluor-488 conjugated anti-rabbit secondary antibody (Invitrogen Corporation, Carlsbad, CA, USA) at a dilution of 1:100 for 1 hour at room temperature. Sections were mounted in mounting media containing DAPI (4',6' diamino-2-phenylindole) and visualized with a fluorescence microscope (Olympus, Tokyo, Japan).
RNA extraction and quantitative real-time polymerase chain reaction
Primers used for real-time reverse transcription-polymerase chain reaction
Sequence (5' to 3')
Size, base pairs
Collagen type 1
Collagen type 2
Collagen type 6
Collagen type 9
Expression of aggrecan degradation products, MMP-3, MMP-9, and MMP-13 were evaluated by means of Western blot analysis. AF and NP tissue was washed with cold PBS and was extracted with lysis buffer (Cell Signaling Technologies, Danvers, MA, USA) at 4°C for 24 hours. The lysis buffer contains the following: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA (ethylene glycol tetraacetic acid), 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium vanadate (Na3VO4), 1 μg/mL leupeptin, 1 mM PMSF (phenylmethylsulfonyl fluoride), and 1X complete protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN, USA). The tissue lysate was centrifuged for 10 minutes at 14,000g to collect the clear tissue extract. Protein concentration in the extract was determined by using a Pierce BCA protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA). To detect aggrecan core protein fragments, lysates were treated with chondroitinase ABC (Sigma-Aldrich, St. Louis, MO, USA) 0.1 U/mL in 50 mM Tris-acetate EDTA buffer at 37°C for 1 hour. Protein extracts (10 μg) were centrifuged at 4°C for 2 minutes at 14,000g and resolved on NuPAGE 4-12% Bis-Tris Gels (Invitrogen Corporation). Proteins were transferred onto Immobilon-P Membrane (Millipore Corporation, Billerica, MA, USA). The membrane was blocked with 5% nonfat dry milk in PBS with 0.1% Tween-20 and incubated overnight at 4°C in 3% nonfat dry milk in PBS with 0.1% Tween-20 with appropriate antibody (mouse monoclonal [BC-3] to aggrecan ARGxx, rabbit monoclonal [EP1186Y] to MMP-3, rabbit monoclonal [EP1254] to MMP-9, and rabbit polyclonal to MMP-13; all from Abcam). The binding of the secondary antibody was detected by enhanced chemiluminescence (ECL-Plus; GE Healthcare, Little Chalfont, Buckinghamshire, UK). Results were normalized to the content of tubulin detected with monoclonal antibody beta-tubulin (E7; Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, IA, USA).
Data were represented as mean ± standard error from three independent experiments. Statistical analysis was performed with Sigma Plot 11.2 statistical software (Systat Software, Inc., San Jose, CA, USA). Student t test or, where appropriate, Mann-Whitney test was used to measure statistical significance (P <0.05).
Characterization of molecular transport in organ culture
To characterize the diffusion of proteins of different molecular-weight proteins in the organ culture disc, we incubated discs with Firefly (61 kDa) and Renilla (38 kDa) luciferase (Figure 1A). After 24 hours of incubation, the NP was dissected and analyzed for the luciferase activity. Both the Firefly and Renilla luciferase show an increase in luminescence in a concentration-dependent manner within the NP (Figure 1B,C).
Assessment of cellular apoptosis and morphological changes
To ensure that our model reflects cellular and histological changes often seen with early disc degeneration, an assessment of cell viability was performed with the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. Figure 1D shows that TNF-α and IL-1β treatment for 3 days results in an increase in the number of apoptotic cells (Figure 1D-d) both in the NP and AF in comparison with the untreated control discs (Figure 1D-b). We performed a histological analysis of the organ-cultured discs to assess changes in cellular morphology and tissue organization (Figure 1E). Control untreated discs showed lamellar organization of the AF with well-circumscribed NP containing abundant proteoglycan-rich Alcian blue-stained extracellular matrix. NP cells showed well-defined eosinophilic cytosol with prominent cell nuclei. After 3 days of treatment with TNF-α and IL-1β, the discs demonstrated a notable loss of eosinophilic staining of the cytoplasm of the central NP cells. Although tissue architecture was preserved, a decrease in the Alcian blue staining of the extracellular matrix was noted with signs of cell loss and a concomitant increase in vacuole formation. At 10 days after treatment, additional changes of the cellular architecture were observed; the most obvious was a loss of Alcian blue staining of the extracellular matrix. Additionally, the eosinophilic staining of the NP cytosol was nearly gone and lacunae previously occupied by cells were empty. AF architecture, however, was preserved throughout the 10 days of treatment (Figure 1E, bottom row).
Changes in gene expression
Downregulation of critical matrix genes
Upregulation in catabolic gene expression
Changes in expression of genes that are associated with pain and inflammation
The ubiquitous presence of symptomatic DDD is an increasing clinical problem with few reliable treatments. The etiology of DDD is likely multifactorial and remains elusive. Current surgical treatment options for DDD are based on the end-stage changes of the degenerative disc complex with or without concomitant neural compression or instability or both. Medical (non-surgical) management of DDD (specifically discogenic low back pain) centers around symptom relief with supportive measures, whereas surgical management focuses on ablation and reconstruction of the pathological disc. Currently, no specific cure exists to halt or reverse the pathological processes that result in disc degeneration. Therefore, to understand the degenerative process at the molecular level for devising a biologic solution, a reliable model to investigate the cellular changes that initiate disc degeneration is needed. Such a model will assist in evaluating treatments that may be able to halt or reverse the early degenerative process.
Several animal-based models of DDD have been described in the literature and employ various techniques to induce degeneration. Currently, the most commonly used strategies involve in vivo techniques with mechanical disruption of the AF [6, 9, 12, 15–17, 30], mechanical stress of the functional spine unit , or chemical degradation of the NP [8, 31]. Other models capitalize on the natural occurrence of advanced DDD in that species . Regardless, the existing models are geared primarily toward mimicking the advanced degenerative changes resembling grade 4 or 5 human discs on the Pfirrmann scale. Although a few in vitro organ culture models that use injury or chemicals to replicate cellular and molecular changes seen during disc degeneration have been reported [24–26], a true atraumatic model that attempts to characterize the early stages of DDD (Pfirrmann grade 1 to 3) has not been available. As described in this study, an atraumatic in vitro model of disc degeneration uses perturbations in several important microenviromental factors that are known to occur during degenerative cascade. The model allows an analysis of reproducible and quantifiable effects at the cellular level by using gene expression, protein analysis, and histology. Another advantage is that this model offers differential analysis of the AF and the NP.
Our organ culture DDD model allows analysis of cellular changes that occur in degenerative discs prior to injury or disruption of the AF. Using chemical proinflammatory cytokines and conditions that mimic the normal internal disc milieu (hypoxia, hyperosmolarity, and decreased nutrients), our model shows alterations in gene expression, protein expression, and histological disc architecture resembling human degenerative discs in grade 1 or 2. Gene expression analysis shows downregulation of important extracellular matrix components, including collagen types 1, 2, 6, and 9 and fibromodulin. A similar decline is seen in the expression of the major proteoglycan aggrecan, indicating a slowdown of the anabolic or matrix maintenance phase as seen in human DDD [33, 34]. Furthermore, significant induction in expression of MMP-3, -9, and -13, enzymes that are closely associated with disc degeneration in humans, shows promotion of a catabolic state [35–37].
Importantly, Western blot analysis confirmed the presence and upregulation of degradative enzymes (aggrecanase and MMPs), indicating upregulation not only at the mRNA level but also at the protein level. These results strongly support the notion that early phases of disc degeneration are characterized not only by a decrease in synthetic activity of cells but also by a concomitant increase in production of the mediators that promote a catabolic environment within the disc.
The most compelling piece of data that substantiates this model is the histology of the discs at 3 and 10 days after treatment. The progressive loss of the proteoglycan-rich extracellular matrix and changes in cellular morphology and architecture closely resemble DDD in humans. As expected, this phenomenon progressively worsens with treatment duration. Although the disc architecture resembles a grade 3 disc in humans at 10 days after treatment, eosinophilic cytoplasm continues to be seen within the NP, indicating the presence of viable cells. Regardless of treatment duration, significant annular disruption at the structural level was not noted in any of the specimens, indicating a state reminiscent of the early stage of disease in humans. It should be noted that the rat organ culture described here, despite several similarities with the human pathological state, lacks true mechanical loading, although medium osmolarity is raised to mimic loading conditions and to counteract swelling. Therefore, while extrapolating the results from this rat organ culture system to the humans, it would be prudent to consider the inherent differences in tissue size, diffusional distances, and cellular morphology and composition between the species.
This study describes an atraumatic in vitro model to investigate cellular and molecular events associated with IVD degeneration by using a rat disc in organ culture. This model may prove useful to those seeking an atraumatic model to use for investigations of early DDD mechanisms and treatment.
degenerative disc disease
Dulbecco's modified Eagle's medium
fetal bovine serum
nerve growth factor
polymerase chain reaction
reverse transcription-polymerase chain reaction
tumor necrosis factor-alpha.
This work was supported by a research grant from Biomet, Inc. The authors would like to thank Rhonda Walter for histological analysis.
- Hoy D, March L, Brooks P, Woolf A, Blyth F, Vos T, Buchbinder R: Measuring the global burden of low back pain. Best Pract Res Clin Rheumatol. 2010, 24: 155-165. 10.1016/j.berh.2009.11.002.View ArticlePubMedGoogle Scholar
- Dagenais S, Tricco AC, Haldeman S: Synthesis of recommendations for the assessment and management of low back pain from recent clinical practice guidelines. Spine J. 2010, 10: 514-529. 10.1016/j.spinee.2010.03.032.View ArticlePubMedGoogle Scholar
- Boden SD, Davis DO, Dina TS, Patronas NJ, Wiesel SW: Abnormal magnetic-resonance scans of the lumbar spine in asymptomatic subjects. A prospective investigation. J Bone Joint Surg Am. 1990, 72: 403-408.PubMedGoogle Scholar
- Pfirrmann CW, Metzdorf A, Zanetti M, Hodler J, Boos N: Magnetic resonance classification of lumbar intervertebral disc degeneration. Spine. 2001, 26: 1873-1878. 10.1097/00007632-200109010-00011.View ArticlePubMedGoogle Scholar
- Thompson JP, Pearce RH, Schechter MT, Adams ME, Tsang IK, Bishop PB: Preliminary evaluation of a scheme for grading the gross morphology of the human intervertebral disc. Spine. 1990, 15: 411-415. 10.1097/00007632-199005000-00012.View ArticlePubMedGoogle Scholar
- Han B, Zhu K, Li FC, Xiao YX, Feng J, Shi ZL, Lin M, Wang J, Chen QX: A simple disc degeneration model induced by percutaneous needle puncture in the rat tail. Spine. 2008, 33: 1925-1934. 10.1097/BRS.0b013e31817c64a9.View ArticlePubMedGoogle Scholar
- Hoogendoorn RJ, Helder MN, Kroeze RJ, Bank RA, Smit TH, Wuisman PI: Reproducible long-term disc degeneration in a large animal model. Spine. 2008, 33: 949-954. 10.1097/BRS.0b013e31816c90f0.View ArticlePubMedGoogle Scholar
- Masuda K, Aota Y, Muehleman C, Imai Y, Okuma M, Thonar EJ, Andersson GB, An HS: A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration. Spine. 2005, 30: 5-14.PubMedGoogle Scholar
- Moskowitz RW, Ziv I, Denko CW, Boja B, Jones PK, Adler JH: Spondylosis in sand rats: a model of intervertebral disc degeneration and hyperostosis. J Orthop Res. 1990, 8: 401-411. 10.1002/jor.1100080312.View ArticlePubMedGoogle Scholar
- Nuckley DJ, Kramer PA, Del Rosario A, Fabro N, Baran S, Ching RP: Intervertebral disc degeneration in a naturally occurring primate model: radiographic and biomechanical evidence. J Orthop Res. 2008, 26: 1283-1288. 10.1002/jor.20526.View ArticlePubMedGoogle Scholar
- Omlor GW, Nerlich AG, Wilke HJ, Pfeiffer M, Lorenz H, Schaaf-Keim M, Bertram H, Richter W, Carstens C, Guehring T: A new porcine in vivo animal model of disc degeneration: response of anulus fibrosus cells, chondrocyte-like nucleus pulposus cells, and notochordal nucleus pulposus cells to partial nucleotomy. Spine. 2009, 34: 2730-2739. 10.1097/BRS.0b013e3181b723c9.View ArticlePubMedGoogle Scholar
- Reid JE, Meakin JR, Robins SP, Skakle JM, Hukins DW: Sheep lumbar intervertebral discs as models for human discs. Clin Biomech (Bristol, Avon). 2002, 17: 312-314. 10.1016/S0268-0033(02)00009-8.View ArticleGoogle Scholar
- Singh K, Masuda K, An HS: Animal models for human disc degeneration. Spine J. 2005, 5: 267S-279S. 10.1016/j.spinee.2005.02.016.View ArticlePubMedGoogle Scholar
- Yoon SH, Miyazaki M, Hong SW, Tow B, Morishita Y, Hu M, Ahn SJ, Wang JC: A porcine model of intervertebral disc degeneration induced by annular injury characterized with magnetic resonance imaging and histopathological findings. Laboratory investigation. J Neurosurg Spine. 2008, 8: 450-457. 10.3171/SPI/2008/8/5/450.View ArticlePubMedGoogle Scholar
- Zhang H, La Marca F, Hollister SJ, Goldstein SA, Lin CY: Developing consistently reproducible intervertebral disc degeneration at rat caudal spine by using needle puncture. J Neurosurg Spine. 2009, 10: 522-530. 10.3171/2009.2.SPINE08925.View ArticlePubMedGoogle Scholar
- Zhou H, Hou S, Shang W, Wu W, Cheng Y, Mei F, Peng B: A new in vivo animal model to create intervertebral disc degeneration characterized by MRI, radiography, CT/discogram, biochemistry, and histology. Spine. 2007, 32: 864-872. 10.1097/01.brs.0000259835.31062.3d.View ArticlePubMedGoogle Scholar
- Haschtmann D, Stoyanov JV, Ettinger L, Nolte LP, Ferguson SJ: Establishment of a novel intervertebral disc/endplate culture model: analysis of an ex vivo in vitro whole-organ rabbit culture system. Spine. 2006, 31: 2918-2925. 10.1097/01.brs.0000247954.69438.ae.View ArticlePubMedGoogle Scholar
- Kroeber MW, Unglaub F, Wang H, Schmid C, Thomsen M, Nerlich A, Richter W: New in vivo animal model to create intervertebral disc degeneration and to investigate the effects of therapeutic strategies to stimulate disc regeneration. Spine. 2002, 27: 2684-2690. 10.1097/00007632-200212010-00007.View ArticlePubMedGoogle Scholar
- Risbud MV, Izzo MW, Adams CS, Arnold WW, Hillibrand AS, Vresilovic EJ, Vaccaro AR, Albert TJ, Shapiro IM: An organ culture system for the study of the nucleus pulposus: description of the system and evaluation of the cells. Spine. 2003, 28: 2652-2658. 10.1097/01.BRS.0000099384.58981.C6.View ArticlePubMedGoogle Scholar
- Risbud MV, Di Martino A, Guttapalli A, Seghatoleslami R, Denaro V, Vaccaro AR, Albert TJ, Shapiro IM: Toward an optimum system for intervertebral disc organ culture: TGF-beta 3 enhances nucleus pulposus and anulus fibrosus survival and function through modulation of TGF-beta-R expression and ERK signaling. Spine. 2006, 31: 884-890. 10.1097/01.brs.0000209335.57767.b5.View ArticlePubMedGoogle Scholar
- Gawri R, Mwale F, Ouellet J, Roughley PJ, Steffen T, Antoniou J, Haglund L: Development of an organ culture system for long term survival of the intact human intervertebral disc. Spine. 2011, 36: 1835-1842. 10.1097/BRS.0b013e3181f81314.View ArticlePubMedGoogle Scholar
- Chan SC, Gantenbein-Ritter B, Leung VY, Chan D, Cheung KM, Ito K: Cryopreserved intervertebral disc with injected bone marrow-derived stromal cells: a feasibility study using organ culture. Spine J. 2010, 10: 486-496. 10.1016/j.spinee.2009.12.019.View ArticlePubMedGoogle Scholar
- Jünger S, Gantenbein-Ritter B, Lezuo P, Alini M, Ferguson SJ, Ito K: Effect of limited nutrition on in situ intervertebral disc cells under simulated-physiological loading. Spine. 2009, 34: 1264-1271. 10.1097/BRS.0b013e3181a0193d.View ArticlePubMedGoogle Scholar
- Korecki CL, MacLean JJ, Iatridis JC: Dynamic compression effects on intervertebral disc mechanics and biology. Spine. 2008, 33: 1403-1409. 10.1097/BRS.0b013e318175cae7.PubMed CentralView ArticlePubMedGoogle Scholar
- Roberts S, Menage J, Sivan S, Urban JP: Bovine explant model of degeneration of the intervertebral disc. BMC Musculoskelet Disord. 2008, 9: 24-10.1186/1471-2474-9-24.PubMed CentralView ArticlePubMedGoogle Scholar
- Illien-Jünger S, Gantenbein-Ritter B, Grad S, Lezuo P, Ferguson SJ, Alini M, Ito K: The combined effects of limited nutrition and high-frequency loading on intervertebral discs with endplates. Spine. 2010, 35: 1744-1752. 10.1097/BRS.0b013e3181c48019.View ArticlePubMedGoogle Scholar
- Korecki CL, Costi JJ, Iatridis JC: Needle puncture injury affects intervertebral disc mechanics and biology in an organ culture model. Spine. 2008, 33: 235-241. 10.1097/BRS.0b013e3181624504.PubMed CentralView ArticlePubMedGoogle Scholar
- Séguin CA, Pilliar RM, Roughley PJ, Kandel RA: Tumor necrosis factor-alpha modulates matrix production and catabolism in nucleus pulposus tissue. Spine. 2005, 30: 1940-1948. 10.1097/01.brs.0000176188.40263.f9.View ArticlePubMedGoogle Scholar
- Purmessur D, Freemont AJ, Hoyland JA: Expression and regulation of neurotrophins in the nondegenerate and degenerate human intervertebral disc. Arthritis Res Ther. 2008, 10: R99-10.1186/ar2487.PubMed CentralView ArticlePubMedGoogle Scholar
- Greg Anderson D, Li X, Tannoury T, Beck G, Balian G: A fibronectin fragment stimulates intervertebral disc degeneration in vivo. Spine. 2003, 28: 2338-2345. 10.1097/01.BRS.0000096943.27853.BC.View ArticlePubMedGoogle Scholar
- Boxberger JI, Auerbach JD, Sen S, Elliott DM: An in vivo model of reduced nucleus pulposus glycosaminoglycan content in the rat lumbar intervertebral disc. Spine. 2008, 33: 146-154. 10.1097/BRS.0b013e31816054f8.PubMed CentralView ArticlePubMedGoogle Scholar
- Gruber HE, Gordon B, Norton HJ, Kilburn J, Williams C, Zinchenko N, Heath J, Ingram J, Hanley EN: Analysis of cell death and vertebral end plate bone mineral density in the annulus of the aging sand rat. Spine J. 2008, 8: 475-481. 10.1016/j.spinee.2006.11.008.View ArticlePubMedGoogle Scholar
- Singh K, Masuda K, Thonar EJ, An HS, Cs-Szabo G: Age-related changes in the extracellular matrix of nucleus pulposus and anulus fibrosus of human intervertebral disc. Spine. 2009, 34: 10-16. 10.1097/BRS.0b013e31818e5ddd.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhao CQ, Wang LM, Jiang LS, Dai LY: The cell biology of intervertebral disc aging and degeneration. Ageing Res Rev. 2007, 6: 247-261. 10.1016/j.arr.2007.08.001.View ArticlePubMedGoogle Scholar
- Podichetty VK: The aging spine: the role of inflammatory mediators in intervertebral disc degeneration. Cell Mol Biol. 2007, 53: 4-18.PubMedGoogle Scholar
- Millward-Sadler SJ, Costello PW, Freemont AJ, Hoyland JA: Regulation of catabolic gene expression in normal and degenerate human intervertebral disc cells: implications for the pathogenesis of intervertebral disc degeneration. Arthritis Res Ther. 2009, 11: R65-10.1186/ar2693.PubMed CentralView ArticlePubMedGoogle Scholar
- Bachmeier BE, Nerlich A, Mittermaier N, Weiler C, Lumenta C, Wuertz K, Boos N: Matrix metalloproteinase expression levels suggest distinct enzyme roles during lumbar disc herniation and degeneration. Eur Spine J. 2009, 18: 1573-1586. 10.1007/s00586-009-1031-8.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd.