Intervertebral discs (IVDs) have a hierarchical fibrous composite structure, meaning the discs are fibrous on multiple length scales which can give strength and elasticity. The mechanical properties of these composites offer flexibility as collagen fibres reorganise and align at low loads, and high stiffness as fibres are strained at high loads. However, these fibrous tissues are also susceptible to failure, causing different problems in different regions. IVD degeneration is the leading cause of low back pain, incurring significant healthcare costs. However, despite a wealth of clinical evidence, we do not yet know whether there is a regional distinction in collagen fibre architecture and whether deformation under mechanical load is region-specific. In work recently published in Acta Biomaterialia, researchers used synchrotron computed tomography (sCT) to investigate collagen fibre bundles in 3D throughout an intact native rat lumbar IVD under increasing compressive load. For the first time, their results demonstrate that highly-localised changes in curvature, strain and fibre orientation indicate differences in regional strain transfer and mechanical function. The methods they have developed can be used to identify fibre structures more susceptible to injury and degeneration in other soft tissue types.
Sequence of 3D renders of a rat spine segment being compressed from the lower endplate. Image reused from DOI: 10.1016/j.actbio.2019.05.021 under the CC BY 2.0 license.
In the intervertebral disc (IVD), a central gel-like nucleus pulposus (NP) is surrounded by annulus fibrosus (AF) that attaches to the vertebral endplates. The AF has a hierarchical lamellar structure of layered, dense bundles of aligned type I collagen, in which fibre orientation alternates in adjacent lamellae.
Tissue mechanical properties, residual strain (permanent stiffening of tissue) and AF fibre orientation vary across different IVD regions. Clinical findings show a higher incidence of AF damage/herniation in the posterior regions. Although this is associated with disrupted collagen fibre organisation and loss of residual strain, the regional architectural differences in fibre orientation, curvature and organisation into lamellae are not fully understood. It is also unclear what impact these architectural differences have on regional mechanical behaviours.
Previous studies have made strain measurements on dissected IVD segments. However, these typically allow study of a restricted number of fibres on the sample surface. Preparing the samples for these studies involves detaching endplates, cutting through AF and removing the swelling pressure from the NP. This technique does not preserve the residual strain and complex boundary conditions necessary for normal function.
Equally, although recent advances in ultrahigh field MRI imaging now allow 100 μm resolution, it is not yet possible to resolve the complete disc microstructure at the necessary resolution for complete regional comparisons. Seeing the complete picture requires investigating tissue architecture at fibre-level (micrometre) resolution in 3D within an intact, entire IVD under increasing load.
To get the complete picture, the team brought an intact rat IVD to Diamond to perform high-resolution phase-contrast X-ray synchrotron computed tomography (PC-sCT) on the Diamond-Manchester Imaging Branchline I13-2. This beamline is a partially-coherent X-ray source that uses phase-contrast imaging to resolve soft tissue structures without the need for staining. The high signal-to-noise in-line phase contrast imaging offered by a synchrotron source meant scans could be completed in 12 minutes, compared with the hours needed for ‘laboratory’ μCT (tomography without a synchrotron).
X-ray micro-tomography is a technique commonly used for resolving microstructure in intact tissue samples. Soft tissues generally have low X-ray absorption contrast and can deform during imaging, which gives them poor tomographic imaging characteristics. However, prior work demonstrated that in-line PC-sCT can resolve AF fibre architecture in native, unfixed, unstained IVD.
The team's previous approach also allowed them to track micro-scale tissue displacements in 3D by digital volume correlation (DVC) and create regional maps of strain patterns in the AF of intact IVD segments under compression. However, it did not provide information on changes in fibre architecture or fibre strain upon loading.
In this new work, they used DVC to achieve high precision tracking of over ten thousand individual collagen fibres per region across the full AF width within specific lamellae. They could then relate highly-localised measurements of fibre-level orientation, curvature and strain to tissue function. The results offered powerful comparisons across length scales in IVD regions.
Being able to perform detailed characterisation of numerous individual collagen fibres from in situ imaging of intact tissues and structures will enhance our understanding of tissue biomechanics. This technique will allow us to discover the structures that cause some tissue regions to be more susceptible to degeneration.
Lead author Dr Catherine Disney worked on this project during her PhD at the University of Manchester. She said,
We have achieved a considerable improvement in image quality since we first began developing this technique, which has allowed us to resolve the whole IVD structure, compare the structure in different regions, and relate those structural differences to mechanical properties. This technique is applicable to any material with enough fibre structure.
Co-author and Beamline Scientist Dr Andrew Bodey commented,
Catherine and her colleagues have done excellent work with us at Diamond. 3D imaging of soft tissues under compression presents major problems relating to sample deformation during data collection – leading to blurring and resolution loss. Catherine demonstrated that the unusual mechanical properties of IVDs allow data collection without notable deformations, even for samples under compression. This enabled the collection of beautiful 3D imagery that could be used for precise analyses – providing good insights into a medical problem that affects hundreds of millions of people.
To find out more about the I13 beamline or discuss potential applications, please contact Principal Beamline Scientist Christoph Rau: christoph.rau@diamond.ac.uk.
Disney CM et al. Regional variations in discrete collagen fibre mechanics within intact intervertebral disc resolved using synchrotron computed tomography and digital volume correlation. Acta Biomaterialia (2021). DOI:10.1016/j.actbio.2021.10.012.
Disney CM et al. Synchrotron tomography of intervertebral disc deformation quantified by digital volume correlation reveals microstructural influence on strain patterns. Acta Biomaterialia 92: 290-304 (2019): DOI:10.1016/j.actbio.2019.05.021.
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