This article has Open Peer Review reports available.
Bovine explant model of degeneration of the intervertebral disc
© Roberts et al; licensee BioMed Central Ltd. 2008
Received: 16 October 2007
Accepted: 25 February 2008
Published: 25 February 2008
Many new treatments for degeneration of the intervertebral disc are being developed which can be delivered through a needle. These require testing in model systems before being used in human patients. Unfortunately, because of differences in anatomy, there are no ideal animal models of disc degeneration. Bovine explant model systems have many advantages but it is not possible to inject any significant volume into an intact disc. Therefore we have attempted to mimic disc degeneration in an explant bovine model via enzymatic digestion.
Bovine coccygeal discs were incubated with different concentrations of the proteolytic enzymes, trypsin and papain, and maintained in culture for up to 3 weeks. A radio-opaque solution was injected to visualise cavities generated. Degenerative features were monitored histologically and biochemically (water and glycosaminoglycan content, via dimethylmethylene blue).
Results and Conclusion
The central region of both papain and trypsin treated discs was macro- and microscopically fragmented, with severe loss of metachromasia. The integrity of the surrounding tissue was mostly in tact with cells in the outer annulus appearing viable. Biochemical analysis demonstrated greatly reduced glycosaminoglycan content in these compared to untreated discs. We have shown that bovine coccygeal discs, treated with proteolytic enzymes can provide a useful in vitro model system for developing and testing potential new treatments of disc degeneration, such as injectable implants or biological therapies.
There is currently a great deal of interest in developing new therapies to address degeneration of the intervertebral disc, due to the association of disc degeneration with low back pain. These therapies range from injectable synthetic nucleus replacements to biological approaches such as cell therapy [1, 2]. Before being used in humans, any potential new therapies should be tested in model systems, either in vitro or in vivo. Animal models of disc degeneration are limited, with no ideal model . Small animals such as rodents or rabbits have a different cell type and anatomy from that found in humans and large animals are expensive, do not develop the same pattern of degeneration as in humans, and some of these also have a different cell type.
Bovine caudal discs have been developed as a suitable model for many in vitro studies of the intervertebral disc, having similar physico-chemical properties to the human disc . However, these bovine discs have such a high swelling-pressure that it is virtually impossible to inject any significant volume into them, thus they cannot be used for testing nuclear replacements unless they are modified. In this paper we describe a technique for developing an explant model of disc degeneration in the bovine caudal disc that would be suitable for testing injectable nucleus pulposus replacements. We have characterised the properties of the disc following enzymatic digestion which results in some similar changes as are seen in degeneration of the human intervertebral disc. This highlights its potential uses and limitations for testing novel therapeutic developments for degenerative intervertebral discs.
Bovine tails were obtained from animals aged 18–30 months at a local abattoir within 1 hour of death. Before dissection tails were washed in 500 parts per million of chlorine to minimise risk of infection. Skin was removed and motion segments, 1.5 to 2 cm in diameter, were dissected from the tail, under sterile conditions. They were cleaned of soft tissues, including muscles and ligaments. The segments were washed thoroughly with sterile phosphate buffered saline (PBS, Invitrogen, Paisley) before washing for 10 minutes with standard culture medium (Dulbecco's modified Eagles medium (DMEM:F12, Invitrogen) supplemented with 500 μg/ml ascorbic acid (Sigma), 10% foetal bovine serum (PAA Laboratories, Yeovil), 0.05% Gentamicin (Invitrogen) and 0.005% Fungizone (Invitrogen)). Thirty eight motion segments (1.5–2.3 cm in diameter, mean 1.8 cm ± 0.3) were obtained from the central region of tails from approximately 12 animals.
(ii) Enzyme Digestion
For induction of degeneration of the nucleus pulposus, motion segments were injected with i) 20 mg/ml of papain (18 U/mg protein, Sigma) in buffer (0.01 M cysteine hydrochloride, 0.5 mM EDTA, 0.2 M sodium acetate, pH 6), ii) trypsin, used at 1, 5, 10 or 20 mg/ml trypsin in PBS (12,400 U/mg protein Sigma), or iii) untreated, with nothing injected (as 'controls'). It was difficult to be accurate in recording the exact volume injected, due to subsequent back pressure but it was estimated that 70–100 μl was used per treatment. Motion segments were cultured in standard culture medium at 37°C in a 5%CO2 humidified atmosphere. At day 1 the action of the trypsin was stopped by injection with FBS; the medium was changed on all the motion segments at day 1 and then every 3 or 4 days. Three samples (randomised from different animals) for each treatment were harvested at 1, 2 and 3 weeks at which time they were X-rayed, cut in half sagittally and photographed. One half was processed for histology and the other was used for biochemical assessment. In addition 3 untreated discs were excised and analysed similarly to provide control baseline values ('normals').
The disc dimensions (circumference, diameter and height) were measured in a separate sample of 8 motion segments before (V0) and after (V1) digestion with papain for one week. These measurements were used to make an estimate of disc volume. Disc volume used to give an indication of the degree of swelling which might occur during the incubation.
(iii) Radiography and histology
Intact motion segments were injected with a radio-opaque solution (Omnipaque, Amersham, Buckinghamshire) before X-raying (32 kV for 80 seconds exposure) and then dissected using a band saw. The volume injected was recorded. One half of the motion segment was fixed in formol saline for 24 hours before transferring to decalcifying solution (10% formic acid in formalin) for 1–2 weeks until decalcification was complete (as assessed with radiographs). Samples were then embedded in paraffin wax and 4 μ thick sections were cut and stained with either haematoxylin and eosin (H&E) or toluidine blue . Morphology was assessed by standard light microscopy using both bright and polarised light.
(iv) Biochemical assessment
Bone was removed from the cranial and caudal aspects of the remaining half motion segment. The central half of the disc was cut into 2 columns, which were then cut into approximately 1 mm thick slices from the outer annulus, through to the mid nucleus to provide a profile of glycosaminoglycan (GAG) and water contents. The pre-weighed slices of disc were dried to a constant weight at 60°C and the water content calculated as percentage of total wet weight.
The dried disc slices were then digested with 1 ml papain solution (2.5 mg of papain in 20 ml of 0.01 M cysteine hydrochloride, 0.5 mM EDTA, 0.2 M sodium acetate, pH 6) at 60°C for 4 hours. The GAG content was measured using a dimethylmethylene blue (DMB (Serva)) assay , modified to a 96 well plate method.
The dimensions of the motion segments changed little during the enzymatic digestion and incubation. The original estimated volume of the disc (V0) was 1.10 ± 0.17 cm whereas 1 week later (V1) it was 1.11 ± 0.16 cm (n = 8). The volume which could be injected was 136 ± 44 μl (n = 8), ie 12% of the volume of the disc.
Any new therapy developed for the treatment of intervertebral disc degeneration to alleviate or delay the onset of associated back pain must go through the inevitable series of testing prior to human trials. Models of disc degeneration have severe limitations  with none providing the ideal test environment. Certain properties, for example cytotoxicity, can be carried out on cell cultures. However, disc cells isolated from their extracellular matrix have a completely different morphology and size than when retained within it , such that explant models are more likely to reflect the in vivo situation than isolated cell models. Whilst the use of explant models will not preclude the complete use of whole animal in vivo models, they may be able to answer many of the questions which have to be asked and minimise the number and hence reduce the investment in cost and ethics otherwise required to take a new product to clinical trials and the marketplace.
There are already several in vitro models described in the literature which, although they are characterised by additional techniques (eg cell number and viability, immunohistochemistry, qRT-PCR) to those used in this study, many involve small species, for example mouse , rat [9, 10] or rabbit . These species have inherent limitations of size and notochordal cells within the nucleus pulposus region. More recently larger discs from bigger animals such as sheep and cattle have been cultured as explant models with the possibility of studying mechanisms of disc degeneration [12, 13]. Likewise several models have utilised enzyme digestion of the extracellular matrix (summarised in reviews [4, 14]. Many of these were developed to mimic and understand better the physiological response to the treatment of chemonucleolysis. More recently there is renewed interest in this as a clinical treatment, with chondroitinase ABC as an alternative enzyme. Imai et al  use this in a rabbit model of chemonucleolysis, whilst it has also been suggested to be useful for a model of degeneration in mice. Antoniou et al  have used different enzymes to examine the effect of degradation of individual matrix components (for example, collagen or proteoglycans) on magnetic resonance imaging parameters. Little has been detailed, however, on the GAG profile, which is probably the most important biochemical parameter relating to disc degeneration.
There are real physical limitations of injecting any significant volume into a healthy disc (though small volumes such as might be used for testing growth factor or even cell therapy can obviously be introduced). In contrast, in the clinical setting where it is envisaged that newly designed implants might be injected into a degenerate human lumbar intervertebral disc, it can often be easy to inject volumes of at least 2 ml of a substance (i.e. approximately 10% of its volume). Hence there is a requirement for a model system of a degenerate disc which is larger than the animal discs most commonly used in such tests and which has properties more similar to those of the human disc. The bovine coccygeal disc provides such samples, with similar properties in terms of composition, swelling pressure and metabolism to those seen in human discs . We have shown in this study that breakdown and loss of GAGs can be induced, sufficient to inject a potential nuclear replacement for subsequent testing. (Several nuclear replacements are being investigated by other groups, for example, NuCore® Injectable Nucleus (Spine Wave Inc, Shelton, CT, USA) and the BioDisc™ (Cryolife Inc, Kennesaw, GA, USA)).
There have been some studies examining explant culture with and without the vertebral endplates attached, indicating that bovine discs without endplates and ovine with endplates could maintain viability for up to 1 week [12, 13]. The vertebral bone (and ligamentous tissue) certainly seemed to be an advantage in the present study in that swelling was severely restricted, with no significant or obvious swelling of the whole motion segment, although it is likely that the similar water content seen in the treated and control discs was due to lack of restraint against swelling via musculature, ligaments etc such as would occur in vivo. In addition, the enzymes used to treat the motion segments did not appear to have affected the histology of the remaining surrounding tissue greatly or any differently from that seen in the untreated controls. Both trypsin and papain created a 'space' allowing material to be injected, but trypsin may be preferable on a cost-basis and is certainly much cheaper than other enzymes such as chymopapain or collagenase. Likewise, there was little difference between time-points studied, particularly for papain, hence this would be recommended to use for the shortest interval.
This model could have several uses for testing newly developed products. These could include application systems, mechanical properties of implants or their adhesive and interaction properties. Indeed, preliminary data has been obtained with this model, whereby an injectable gel has been inserted and tested under either hydrostatic load (approximately 4 kg for 2 hrs) or cyclic loading (4 kg, 600 cycles at a frequency of 1 Hz; Dr S Sarit, personal communication). In addition to testing implants, they could also be used to assess biological approaches and, for example, monitor injected cells. When developing new therapies, the choice of the ideal test system depends very much on the questions being asked. In this study we have shown that the use of a general proteolytic enzyme together with a bovine coccygeal explant system can provide a means of producing a cheap, reproducible system suitable for testing some of the new generation of injectable implants which are being developed in the spine field.
Several injectable novel therapies are being developed worldwide for treating degeneration of the intervertebral disc associated with back pain. As with all such developments these require testing and evaluation in the laboratory prior to taking to the clinic. Unfortunately 'normal' discs have a high swelling pressure and resist injecting any volume of substance into. Hence we have developed an explant model which mimics some aspects of disc degeneration and would be suitable for testing the new generation of injectable therapies for treating degenerate discs in humans.
We are grateful to the Engineering and Physical Sciences Research Council and Biotechnology and Biological Sciences Research Council for funding of this project and Annie Kerr for help in manuscript preparation.
- Thomas J, Lowman A, Marcolongo M: Novel associated hydrogels for nucleus pulposus replacement. J Biomed Mater Res. 2003, 67: 1329-1337.View ArticleGoogle Scholar
- Meisel HJ, Siodla V, Ganey T, Minkus Y, Hutton WC, Alasevic O: Clinical experience in cell-based therapeutics: Disc chondrocyte transplantation – A treatment for degenerated or damaged intervertebral disc. Biomolecular Engineering. 2006, 24: 5-21.View ArticlePubMedGoogle Scholar
- Alini M, Eisenstein SM, Ito K, Little CB, Kettler A, Masuda K, Melrose J, Ralphs JR, Stokes IAF, Wilke H-J: Are animal models useful for studying human disc disorders/degeneration?. Eur Spine J. 2008, 17: 2-19.View ArticlePubMedGoogle Scholar
- Oshima H, Ishihara H, Urban JPG, Tsuji H: The use of coccygeal discs to study intervertebral disc metabolism. J Orthop Res. 1993, 11: 332-338.View ArticlePubMedGoogle Scholar
- Roberts S, Menage J: Microscopic Methods for the Analysis of Engineered Tissues. Methods in Molecular Biology: Biopolymer Methods in Tissue Engineering. Edited by: Hollander AP, Hatton PV. 2004, Totowa: Humana Press Inc, 238: 171-195.View ArticleGoogle Scholar
- Farndale RW, Buttle DJ, Barrett AJ: Improved Quantitation and Discrimination of Sulphated Glycosaminoglycans by use of Dimethylmethylene Blue. Biochim Biophys Acta. 1986, 883: 173-177.View ArticlePubMedGoogle Scholar
- Roberts S, Menage J, Duance VC, Wotton S, Ayad S: Collagen Types Around the Cells of the Intervertebral Disc and Cartilage End Plate: An Immunolocalization Study. Spine. 1991, 16: 1030-1038.View ArticlePubMedGoogle Scholar
- Ariga K, Yonenobu K, Nakase T, Hosono N, Okuda S, Meng W, Tamura Y, Yoshikawa H: Mechanical stress-induced apoptosis of endplate chondrocytes in organ-cultured mouse intervertebral discs. Spine. 2003, 28: 1528-1533.PubMedGoogle Scholar
- Risbud MV, Di Martino A, Guttapalli A, Seghatoleslami R, Denaro V, Vaccaro A, Albert T, Shapiro I: Toward an Optimum System for Intervertebral Disc Organ Culture. Spine. 2006, 31: 884-890.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: 2652-2659.View ArticlePubMedGoogle Scholar
- Chiba K, Andersson GB, Masuda K, Momohara S, Williams JM, Thonar JM: A new Culture System to Study the Metabolism of the Intervertebral Disc in vitro. Spine. 1998, 23: 1821-1828.View ArticlePubMedGoogle Scholar
- Lee CR, Iatridis JC, Poveda L, Alini M: In vitro organ culture of the bovine intervertebral disc-effects of vertebral endplate and potential for mechanobiology. Spine. 2006, 31: 515-522.View ArticlePubMedGoogle Scholar
- Gantenbein B, Grunhagen T, Lee CR, van Donkelaar CC, Alini M, Ito K: An in vitro organ culturing system for intervertebral disc explants with vertebral endplates: a feasibilty study with ovine caudal discs. Spine. 2006, 31: 2665-2673.View ArticlePubMedGoogle Scholar
- Lotz JC: Animal models of intervertebral disc degeneration. Spine. 2004, 29: 2742-2750.View ArticlePubMedGoogle Scholar
- Imai Y, Okuma M, An HS, Nakagawa K, Yamada M, Muehleman C, Thonar E, Masuda K: Restoration of disc height loss by recombinant human osteogenic protein-1 injection into intervertebral discs undergoing degeneration induced by an intradiscal injection of chondroitinase ABC. Spine. 2007, 32: 1197-1205.View ArticlePubMedGoogle Scholar
- Norcross JP, Lester GE, Weinhold P, Dahners LE: An in vivo model of degenerative disc disease. J Orthop Res. 2003, 21: 183-185.View ArticlePubMedGoogle Scholar
- Antoniou J, Mwale F, Demers CN, Beaudoin G, Goswami T, Aebi M, Alini M: Quantitative magnetic resonance imaging of enzymatically induced degradation of the nucleus pulposus of intervertebral discs. Spine. 2006, 31: 1547-1554.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/9/24/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.