Abstract
Intervertebral disc degeneration (IVDD) is linked to low back pain. Microstructural changes during degeneration have previously been imaged using 2D sectioning techniques and 3D methods which are limited to small specimens and prone to inducing artefacts from sample preparation. This study explores micro computed X-ray tomography (microCT) methods with the aim of resolving IVD 3D microstructure whilst minimising sample preparation artefacts. Low X-ray absorption contrast in non-mineralised tissue can be enhanced using staining and phase contrast techniques. A step-wise approach, including comparing three stains, was used to develop microCT for bovine tail IVD using laboratory and synchrotron sources. Staining successfully contrasted collagenous structures; however not all regions were stained and the procedure induced macroscopic structural changes. Phase contrast microCT of chemically fixed yet unstained samples resolved the nucleus pulposus, annulus fibrosus and constituent lamellae, and finer structures including collagen bundles and cross-bridges. Using the same imaging methods native tissue scans were of slightly lower contrast but free from sample processing artefacts. In the future these methods may be used to characterise structural remodelling in soft (non-calcified) tissues and to conduct in situ studies of native loaded tissues and constructs to characterise their 3D mechanical properties.
Highlights
Low back pain (LBP) affects up to 84% of the population at some point during their lives[1]
I2KI fully penetrated the segment whilst phosphomolybdic acid (PMA) failed to penetrate the whole annulus fibrosus (AF) and phosphotungstic acid (PTA) staining was largely confined to the segment edges
The quarter segments scanned on the Phoenix XMT laboratory system after two weeks of incubation showed I2KI fully stained the intervertebral disc (IVD) tissue but PMA and PTA stains had not completely penetrated the samples
Summary
Low back pain (LBP) affects up to 84% of the population at some point during their lives[1]. IVD micro-structure has been characterised by 2D imaging of either tissue sections or the surface of sequentially peeled AF lamellae[4,5,16,17,18,19] These approaches are: (i) destructive and prone to inducing artefacts, (ii) disruptive of the large residual strains which characterise the unloaded IVD state[20], (iii) reliant on chemical fixation or partial dehydration of the tissues which alters their structure and mechanical properties[21,22] and (iv) confined to imaging relatively small regions of the disc. X-ray micro-computed tomography (microCT) has the potential to circumvent these issues by imaging native (non-chemically fixed) tissues in 3D at microscopic resolutions. These methods may be used to characterise age-related structural remodelling in cartilaginous tissues and to map the 3D mechanical properties of tissues and tissue-engineered constructs
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