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Bovine explant model of degeneration of the intervertebral disc.

Roberts S, Menage J, Sivan S, Urban JP - BMC Musculoskelet Disord (2008)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Spinal Studies, Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, Shropshire SY10 7AG, UK. sally.roberts@rjah.nhs.uk

ABSTRACT

Background: 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.

Methods: 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.

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(a) H&E stained sections at 3 weeks post-incubation with 20 mg trypsin, 20 mg papain or no enzyme (control). (b) Toluidine blue stained sections demonstrating severe loss of metachromasia from all of the disc in enzyme treated discs compared to non-enzymatically digested, where loss is diminished and only in the outer annulus.
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Figure 3: (a) H&E stained sections at 3 weeks post-incubation with 20 mg trypsin, 20 mg papain or no enzyme (control). (b) Toluidine blue stained sections demonstrating severe loss of metachromasia from all of the disc in enzyme treated discs compared to non-enzymatically digested, where loss is diminished and only in the outer annulus.

Mentions: These changes were also seen microscopically with central fissures seen clearly on all enzyme treated discs (Figure 3a). There were obviously no cells within the nuclear region which was digested, but in the surrounding matrix the cells were generally healthy in appearance (though no cell viability measurements were made) and occurring at a similar density as in the controls. Toluidine blue stained sections of untreated control discs demonstrated some loss of metachromasia, particularly in the outer annulus. However, sections of enzyme treated discs, both those treated with papain and trypsin, showed severe and much greater loss of metachromasia at all time points (Figure 3b). Control discs, incubated for 3 weeks had little metachromasia in the outer annulus; this pattern is similar to that of normal, freshly obtained discs, reflecting the lower GAG content found in this region. Blood vessels were seen in the outer annulus fibrosus of both enzyme treated and untreated discs. The morphology of these, the cells within them and the disc cells themselves appeared normal, even after 3 weeks of incubation (Figure 4).


Bovine explant model of degeneration of the intervertebral disc.

Roberts S, Menage J, Sivan S, Urban JP - BMC Musculoskelet Disord (2008)

(a) H&E stained sections at 3 weeks post-incubation with 20 mg trypsin, 20 mg papain or no enzyme (control). (b) Toluidine blue stained sections demonstrating severe loss of metachromasia from all of the disc in enzyme treated discs compared to non-enzymatically digested, where loss is diminished and only in the outer annulus.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2266744&req=5

Figure 3: (a) H&E stained sections at 3 weeks post-incubation with 20 mg trypsin, 20 mg papain or no enzyme (control). (b) Toluidine blue stained sections demonstrating severe loss of metachromasia from all of the disc in enzyme treated discs compared to non-enzymatically digested, where loss is diminished and only in the outer annulus.
Mentions: These changes were also seen microscopically with central fissures seen clearly on all enzyme treated discs (Figure 3a). There were obviously no cells within the nuclear region which was digested, but in the surrounding matrix the cells were generally healthy in appearance (though no cell viability measurements were made) and occurring at a similar density as in the controls. Toluidine blue stained sections of untreated control discs demonstrated some loss of metachromasia, particularly in the outer annulus. However, sections of enzyme treated discs, both those treated with papain and trypsin, showed severe and much greater loss of metachromasia at all time points (Figure 3b). Control discs, incubated for 3 weeks had little metachromasia in the outer annulus; this pattern is similar to that of normal, freshly obtained discs, reflecting the lower GAG content found in this region. Blood vessels were seen in the outer annulus fibrosus of both enzyme treated and untreated discs. The morphology of these, the cells within them and the disc cells themselves appeared normal, even after 3 weeks of incubation (Figure 4).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Spinal Studies, Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, Shropshire SY10 7AG, UK. sally.roberts@rjah.nhs.uk

ABSTRACT

Background: 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.

Methods: 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.

Show MeSH
Related in: MedlinePlus