Abstract
Introduction
Physical disruption of the extracellular matrix influences the mechanical and chemical environment of intervertebral disc cells. We hypothesise that this can explain degenerative changes such as focal proteoglycan loss, impaired cell-matrix binding, cell clustering, and increased activity of matrix-degrading enzymes.
Methods
Disc tissue samples were removed surgically from 11 patients (aged 34–75 yrs) who had a painful but non-herniated disc. Each sample was divided into a pair of specimens (approximately 5mm3), which were cultured at 37°C under 5% CO2. One of each pair was allowed to swell, while the other was restrained by a perspex ring. Live-cell imaging was performed with a wide field microscope for 36 hrs. Specimens were then sectioned at 5 and 30 μm for histology and immunofluorescence using a confocal microscope. Antibodies were used to recognise free integrin receptor α5β1, matrix metalloprotease MMP-1, and denatured collagen types I-III. Proteoglycan content of the medium, analysed using the colorimetric DMMB assay, was used to assess tissue swelling and GAG loss. Constrained/unconstrained results were compared using matched-pair t-tests.
Results
Time-lapse cinematography revealed small cell movements in unconstrained specimens, for up to 12 hrs. By 36 hrs, unconstrained (free swelling) samples showed greater: loss of GAG's (p<0.003), loss of integrin binding (p<0.02), synthesis of MMP-1 (p<0.03), and collagen denaturation (p<0.009). Cell clustering was evident in all tissues after 36 hrs.
Conclusion
Swelling of disrupted disc tissue disturbs cell-matrix binding, increases matrix degradation, and allows increased proteoglycan loss. This sequence of events could follow disc injury or herniation in-vivo.
