Identifying the Irreversible Mechanical Behaviour of Individual Mineralised Collagen Fibril Assemblies

Lead Research Organisation: Heriot-Watt University
Department Name: Sch of Engineering and Physical Science

Abstract

Osteoporosis and osteoarthritis affect millions of patients around the world and are eventually characterised by a reduction of bone strength that results in increased rates of fractures. Life expectancy continues to rise but patient specific treatment solutions to optimally manage those patients are sill not available. Such solutions could consist of tailored medication strategies and, at a later stage, tailored implant solutions which require a thorough understanding of the mechancial competence of structural tissue. While the mechanical behaviour of bone is currently well characterised at the upper level of tissue organisation, the underlying nonlinear mechanical properties of mineralised collagen fibre assemblies, however, remain obscured by structural features such as cellular porosity, lamellar organisation, cement lines, cracks and other interfaces. Starting from preliminary pilot experiments, this proposal aims at performing simultaneous uniaxial micropillar strength tests and structural measurements using small-angle X-ray scattering and wide-angle X-ray diffraction on micron-sized volumes of the extra-cellular matrix (ECM) and, thus, on mineralised collagen fibre assemblies only. This project will result in a versatile and powerful experimental framework that will be used to understand the structure-mechanics relation of ECM with an unprecedented spatial resolution of the mechanical experiment. The results of this project will inform the engineering of patient-specific material solutions in silico through all relevant length scales starting from the ECM level. This, in turn, will foster the development and realisation of production technologies for manufacturing patient-specific "implants on demand" which could be offered as a service or embedded in a hospital. The novel experimental techniques may be useful for testing and developing functional thin films such as implant coatings, investigating the impact of pathological changes on the ECM, or even to reduce, refine, and replace animal experiments.

Planned Impact

Society: As an ageing society UK is highly affected since over 11 million (approximately 17 %) inhabitants suffer from osteoporosis or osteoarthritis. Osteoporosis itself is associated with a dramatic increase in mortality and it causes women beyond menopause to spend more days in hospital than due to diabetes, heart attack or breast cancer. The diseases would be highly manageable if diagnostics, treatments, and compliance could be improved. A corner stone could be the development of patient specific diagnoses and treatment strategies. Those strategies necessarily rely on a thorough understanding of structure-mechanics relationships of the affected tissues at the extra-cellular matrix level. The present project makes a major contribution to this end.

Science: Simultaneous uniaxial strength tests and X-ray diffraction measurements on micron-sized volumes of the extra-cellular matrix are a breakthrough to understand biological hard tissues. The proposed experiments are a new technical challenge that could be extended to a much broader class of biological, biomimetic and non-biological composites and nanostructured materials. It will lead directly to novel questions and challenges on different tissue types both healthy and subject to pathological changes.

Specifically targeting bone in such a challenging project will help to understand the mechanical behaviour of the fundamental building blocks of bone and the scale transition of bone mechanical properties beyond the elastic loading regime in general. This is a prerequisite to develop optimal strategies for diagnosis, treatment, and the management of patients with musculoskeletal diseases. The results will have a direct and substantial impact in my field of research and will inform a broad audience of researchers ranging from theoretical and experimental biomechanics, through tissue engineering and synthetic biology, to pre-clinical and clinical orthopaedics.

Technology: The versatile and powerful experimental framework developed at the core of this project will be of use for a wide range of researchers crossing disciplines from geoscience, to biomedical and energy engineering, materials science, to functional coatings and optical materials. This will be realised through the collaborations within this project and then by making the novel experimental framework available for new scientific and industrial collaborations.

Publications

10 25 50
 
Description We successfully implemented novel experimental techniques combining micropillar compression testing with simultaneous X-ray diffraction techniques. This allows to probe micron-sized volumes in a uniaxial fashion and supplement such measurements with deformation measurements on the nanometre length scale. In the course of the project this setup will be used to investigate scale transitions of the strains in mineralised tissues such as bone. Using a uniaxial model system, i.e. mineralised turkey leg tendon, we aim at unravelling the irreversible behaviour of mineralised collagen fibrils. In addition, we developed imaging methods to image tested micropillars in 3D at a spatial resolution of up to 10 nm.

We have developed a novel constitutive model that explains the micro- and nanomechanical behaviour of mineralised collagen fibres. We used both the developed experimental techniques and the model to investigate this behaviour under wet, quasi-phyisologic conditions.
Exploitation Route The whole setup is portable and can be implemented in diffraction beamlines at synchrotrons other than the European Synchrotron Radiation Facility. Furthermore, it is an integral part of our own hierarchical material mechanics laboratory.

Currently, the results form critical data for two larger proposals. One in the environmental sciences and one in engineering. The developed model together with the identified data are freely available and pave the way to formulate a unified model for bone tissue.
Sectors Aerospace, Defence and Marine,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other

 
Description Methods and tools have been used in outreach to wider public in form of images (artistic), photos, and laymen engagement.
First Year Of Impact 2019
Sector Healthcare
Impact Types Societal

 
Description Standard beam time application
Amount € 43,000 (EUR)
Funding ID ME-1472 
Organisation European Synchrotron Radiation Facility 
Sector Charity/Non Profit
Country France
Start 09/2017 
End 09/2017
 
Description Standard beam time application
Amount € 43,000 (EUR)
Funding ID ME-1415 
Organisation European Synchrotron Radiation Facility 
Sector Charity/Non Profit
Country France
Start 08/2016 
End 08/2016
 
Description Standard beam time application
Amount € 43,000 (EUR)
Funding ID ME-1458 
Organisation European Synchrotron Radiation Facility 
Sector Charity/Non Profit
Country France
Start 12/2017 
End 12/2017
 
Title A statistical constitutive model for the micro- and nanomechanical behaviour of mineralised collagen fibres 
Description Computational models could be a cornerstone in digital healthcare to tackle the socio-economic burden associated with bone-related diseases such as osteoporosis, osteoarthritis and bone cancer. This includes improved diagnoses, the manufacturing of custom implants and the monitoring of bone implant systems in personalised medicine. Such models strongly depend on understanding the complex characteristics of bone as a material on its different hierarchical levels. To address this, we need a comprehensive modelling framework that is able to simulate the nonlinear mechanical behaviour of bone tissue from the molecular up to the macroscopic length scale. There is, however, still limited knowledge on the mineralised collagen fibre. This fibre is a fibril-matrix reinforced composite and bone's mechanical building block at the microscale consisting of mineralised collagen fibrils and mineral particles. Here, we report an elasto-plastic statistical constitutive model that bridges the gap between the nano- and micrometre length scale and by quantifying average collagen deformations may connect continuum and molecular levels. By embedding two shear lag models, we can explain the mineralised collagen fibre behaviour and how load is transferred between its main mechanical components. We used experimental results at the fibre level from combining micropillar compression and X-ray scattering. Furthermore, synchrotron radiation X-ray phase-contrast nanometre computed tomography was used to quantify the ultrastructure of the fibril-matrix composite. The model outputs statistical distributions for micro- and nanomechanical responses including strain ratios between the fibre and its components, e.g. deformations of the mineralised collagen fibrils and mineral particles. As such, it directly simulates results from combined mechanical testing and X-ray scattering analyses of composite materials. Heterogeneous fibril deformation helps to explain small strain ratios independently reported for mineralised tissues in such experiments. The model achieves a mean accuracy of 96% compared to micro- and nanoscale experiments. To assess the generality of our model, we validated it against independent results for bone extracellular matrix. Our model explains bone's fundamental microscale mechanical unit and closes a gap between molecular and continuum levels. Our model helps to understand nonlinear bone mechanics and is generally applicable for fibril-reinforced composites that can be used in the design of bio-inspired materials. 
Type Of Material Model of mechanisms or symptoms - human 
Year Produced 2019 
Provided To Others? Yes  
Impact See description above 
 
Title Facilitating micropillar testing under physiological conditions 
Description After successfully combining micropillar testting, a method to probe micron sized volumes of extra-cellular matrix, with small- and wide angle X-ray diffraction, we extended the mehtod to allow for testing under quasi-physiological conditions. This allows to measure strains in the molecular network of a micron sized specimen of extra-cellular matrix closer to the actual properties of the living tissue. 
Type Of Material Improvements to research infrastructure 
Year Produced 2018 
Provided To Others? Yes  
Impact Novel experimental procedure established at beamline ID13 of the European Synchrotron Radiation Facility. As method and devices are portable, this can be used at, e.g., Diamond light source. 
 
Title In situ micropillar compression test in SAXS/WAXD synchrotron beamline 
Description We combined micropillar testting, a method to probe micron sized volumes of extra-cellular matrix, with small- and wide angle X-ray diffraction for the first time. This allows to measure strains in the molecular network of a micron sized specimen of extra-cellular matrix and extends the compression test on the micrometer length scale to deliver answers on the molecular level. 
Type Of Material Improvements to research infrastructure 
Year Produced 2016 
Provided To Others? Yes  
Impact Novel experimental procedure established at beamline ID13 of the European Synchrotron Radiation Facility. 
 
Title Synchrotron nanoCT analyses for mineralised collagen fibrils 
Description Developed novel scanning to resolve individual mineralised collagen fibres (~7 µm diameter 14 µm height) at 20 nm spatial resolution. Developed novel analyses protocols to estimate number of mineralised collagen fibrils in fibre. 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2018 
Provided To Others? No  
Impact novel ultra-high resolution imaging protocol to be used for mineralised tissues is generally accessible for synchrotron users Manuscript in preparation 
 
Description Dry micropillar compression SAXS/WAXD experiments 
Organisation European Synchrotron Radiation Facility
Country France 
Sector Charity/Non Profit 
PI Contribution We performed micropillar compression experiments on micron-sized samples combined with SAXS/WAXD measurements. This was possible
Collaborator Contribution The team of ID13 provided support in writing the beam time application. Do to its successful acceptance full hands on support of the whole team during our experiment was given.
Impact Novel experimental scheme (micropillar testing combined with SAXS/WAXD for the first time) 1st experimental report
Start Year 2015
 
Description SAXS/WAXD evaluation 
Organisation University of Grenoble
Department Laboratory for Interdisciplinary Physics
Country France 
Sector Academic/University 
PI Contribution A team member has been travelling to Grenoble to get training in the evaluation of SAXS/WAXD signals and to start the experimental evaluation. The successful experiment at ID13 of the European Synchrotron Radiation Facility (ESRF) has opened up new routes for testing and will foster additional experiments with this group.
Collaborator Contribution University of Grenoble provides full hands on training in the evaluation of SAXS and WAXD signals acquired in step-wise loading experiments conducted at ID13 (ESRF).
Impact step-wise loading experiment at ID13 of joint evaluation of mechanical, SAXS and WAXD data
Start Year 2016
 
Description Sample preparation 
Organisation University of Bern
Country Switzerland 
Sector Academic/University 
PI Contribution The team from Heriot-Watt travelled to Bern to prepare the necessary test specimens for the grant. This was necessary since the laboratory facilities at Heriot-Watt were under construction when the project started. A team member stayed for four weeks to accomplish the production of 200 specimens for the micromechanical tests.
Collaborator Contribution The team of the ISTB provided access to their preparation facility and stored the raw material. Most importantly their workshop produced 200 high precision sample holders that had a suitable accuracy to be used in synchrotron light nano-computed tomography that could not have been produced otherwise.
Impact 200 high-precision sample holders for SEM, FIB/SEM, and synchrotron light nano-computed tomography 200 pre-specimens for further preparation in SEM
Start Year 2016
 
Description BBC Worldservices interviews 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Recorded an interview on 24/09/2017 giving brief background of the project and its aims.
Live interview 25/09/2017 BBC breakfast radio giving brief background of the project and its aims.
Year(s) Of Engagement Activity 2017