The mechanics of the collagen fibrillar network in ageing cartilage

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Type II collagen fibrils in cartilage tissue play a critical but experimentally less understood role in joint biomechanics. Using time-resolved synchrotron X-ray nanomechanics as a novel probe of cartilage fibrillar mechanics, our preliminary data has uncovered hitherto unknown aspects of fibrillar deformation under loading, including transient loss of pre-strain, intrafibrillar disordering indicative of water movement, alteration of collagen pre-strain levels under proteoglycan digestion and in-phase changes of fibril strain, orientation and disorder under cyclic loading. We hypothesize that age-related changes in cartilage, including crosslinking and hydration, will critically alter these fibrillar deformation dynamics, which will have significant effects on joint biomechanical deterioration in ageing.

We will use synchrotron X-ray nanomechanics combined with proteomic characterization of ageing human tissues to test this hypothesis. Further, using crosslinking and hydration as mechanistic variables, we will test their effect under controlled conditions in bovine cartilage. We will characterise age-related alteration in collagen fibrillar dynamics under loading and possible fatigue-related changes. By linking fibrillar response to localized loading, we will clarify how focal damage to cartilage can spread to overall joint degradation.

We will obtain a comprehensive understanding of the structural and mechanical role of the collagen fibrillar network in cartilage, and quantify the mechanical homeostatic changes in ageing. By integrating molecular-level information through microscale mapping, we will be able to link small scale alterations to whole joint deterioration in ageing and musculoskeletal degeneration. This will in turn enable potential clinical impact (understanding the influence of drugs on the mechanics of cartilage at the nanoscale) or in investigating the downstream effect of genetic knockout models of disease.

The project will deliver in three areas: public engagement, academic engagement and industrial/application impact. A significant part of the academic impact activity is integrated into the "Academic Beneficiaries" section above.

In terms of industrial/application impact, by the development of software methods for rapid, high throughput extraction and visualization of nanoscale structural parameters in collagenous fibrous biocomposites like cartilage, we will facilitate the application of high brilliance synchrotron X-ray microprobe scattering SAXS by the wider bioengineering and biomechanics community. Taking advantage of the structural and compositional commonalities across hydrated collagenous tissues like cartilage, tendon, skin and intervertebral disc, we will develop software scripts which will perform reduction, fitting and display of nanostructural parameters arising from the fibrillar collagen SAXS pattern. These parameters include but are not limited to: fibril D-period, fibril orientation, degree of orientation, fibrillar radial distribution, gap/overlap ratio and lateral intermolecular spacing. By working with the software team developing the general X-ray diffraction analysis program DAWN (www.dawnsci.org) we will embed these scripts into the pipelines for data processing present in this software.

As a result, non-specialist users (like biologists, bioengineers or clinicians) would be able to drop in a series of acquired experimental synchrotron X-ray diffraction patterns acquired from a spatial map or time series of a collagenous tissue into DAWN, and with minimal user intervention, acquire a 2D map or time-plot of collagen fibrillar orientation, pre-strain or any other parameter extractable from the SAXS pattern. By combining high throughput data analysis, advanced and attractive data graphical representations and full automatization, such software scripts will significantly lower the technical barrier surrounding certain synchrotron X-ray imaging methods in the eyes of non-specialists, and facilitate their application to a wide range of biological and bioengineering questions. To enable this, Diamond Light Source has committed a significant portion of an expert software PDRA's time to work with our team to develop and implement these routines into DAWN over the course of the Research Objectives.

The second area of impact is a planned series of public engagement activities at the Royal Society Summer Exhibition, the Big Bang Science Fair and at the Centre of the Cell at the Blizzard Institute, Whitechapel (London). These activities will engage and enlighten the public on the excitement of a relatively overlooked aspect of biology - the role of the extracellular matrix - and to convey the spirit of multidisciplinary research in STEM subjects. The stand will consist of two main parts. The first will enable users to generate fibril-matrix architectures of the types found in the body (cartilage, skin etc.) using 3D printing, combined with interactive visualization of the X-ray diffraction patterns produced by the structures generated and supported by movies of acquired experimental data and tissue-microscopy. The second will emphasize the role of the fibre-matrix interactions by combining interactive modification of relative material properties of fibre and matrix, along with their orientations, with simulations of observed mechanical behaviours. These activities are chosen to cover different audiences: while the Royal Society and the Big Bang Science Fair will attract visitors across the UK, the Centre of the Cell exhibitions in East London have a special focus on widening participation and science outreach in the community.

Lastly, this project will impact academic research via application of novel combinations of methods - like the synchrotron X-ray and proteomics approaches - which will impact bioengineering, biophysics, cartilage development, mechanobiology and development of X-ray methods.