Integrating modelling and experimental approaches to investigate adventitious age-related collagen crosslinking in skeletal tissues

Lead Research Organisation: University College London
Department Name: Institute of Orthopaedics


As we age our tendons, ligaments and bones begin to function less well, resulting in stiffness, reduced mobility and increased frailty. In some individuals this occurs to such an extent that they suffer considerable pain and are unable to undertake daily activities, let alone exercise to maintain good health. Tendons - the soft connective tissues that connect muscles to bone - are particularly vulnerable to age related changes. It is well known that proteins, such as collagen that forms the main force-resisting component of tendon, are susceptible to modification by sugars in the blood. The addition of sugars to collagen results in cross-links forming between neighbouring collagen molecules. Tendon, ligament and bone are composed of the same type of collagen and therefore it is likely that the same sugar mediated crosslinks will form. Although these cross-links are recognised to have a detrimental effect on collagen properties, they have been notoriously difficult to study. Recent advances in mass spectrometry of proteins, computer modelling studies and nano-scale mechanics have made it possible to study these age-related modifications to proteins in much more detail. In our study we will quantify and locate levels of important sugar-mediated cross-links in tendons from individuals of different ages and link this to how the collagen structures perform mechanically and renew themselves within the tendon tissue. In addition, we will investigate ligament and bone tissue to determine whether a similar pattern and level of age related crosslinking is present suggesting commonality in ageing of different skeletal tissues. Glycation mediated cross-links are likely modified by diet, exercise and pharmacological agents and this may provide an opportune way of intervening to reduce the impact of normal ageing and risk of frailty and poor health. The findings of this study have great potential to improve the quality of life and wellbeing into old age in a large proportion of the population.

Technical Summary

This multi-disciplinary project brings together expertise in molecular simulation studies, extracellular matrix biochemistry and nano-scale mechanics to address the high burden of musculoskeletal disease in the ageing population. Our main focus is on the soft connective tissues of the skeleton, specifically tendons. Our study is designed to test the hypothesis that ageing results in advanced glycation end-product (AGE) cross-link formation; specifically glucosepane, within the tendon collagenous matrix, rendering it more resistant to proteolytic degradation and thereby allowing matrix damage to accumulate and mechanical properties to deteriorate. This hypothesis is based on our recent work, which has shown that the collagenous framework in an energy storing tendon is renewed less frequently and accumulates partially cleaved collagen with advancing age, whereas tendon cell activity does not decline. It is well known that irreversible modifications of long-lived proteins accumulate during ageing and that there is an association between AGE formation and changes in properties, although little is known about the mechanisms. We will investigate energy storing and positional human and equine tendons from donors with a wide age range. We will quantify and locate glucosepane cross-links using tandem mass spectrometry and molecular modelling. Levels will be correlated with susceptibility to proteolytic degradation, thermal stability using differential scanning calorimetry and nano-scale mechanics using atomic force microscopy. We will use a range of complementary computational techniques to provide atomic-level information on structure and strength of glucosepane cross-links, preferential location in potential 'hotspots' and their effect on mechanical properties of collagen. Understanding adventitious age-related modifications to proteins holds considerable scope for translation into better healthcare as levels may be modified by diet, exercise and pharmacological agents.

Planned Impact

We anticipate that the benefits of this research will be wide ranging. First, we expect an impact on quality of life, health and wellbeing in the general population. Understanding the causes of deterioration in skeletal tissue mechanical function addresses one of the most challenging aspects of ageing and health decline. The impact of this proposal would be high in terms of revealing the cause of skeletal tissue functional decline with advancing age. Lifestyles changes such as diet and exercise may modify the mechanisms identified in this study which provides a real opportunity for impact. The proposed work will also contribute to understanding of other connective tissues and age related dysfunction, such as osteoarthritis; secondary to ligament degeneration and joint instability; and the connective tissue aspects of diabetes and therefore will benefit a broad range of individuals.
Second, we see this study as having an impact in policy and healthcare provision. The 2010 Government white paper outlining strategy for public health in England has identified a clear need to reduce demand on the National Health Service and policy aims to achieve this by promoting healthy living and preventing illness (Department of Health, 2010). Plans include launching physical activity initiatives so that active ageing becomes the norm. However 30% of the population have a long standing illness and musculoskeletal conditions form the majority of these illnesses, accounting for 70% along with circulatory and mental health disorders.The proposed work has great potential to contribute to screening for early diagnosis and intervention and to directing lifestyle choices to enable healthy and active ageing in line with the Department of Health initiatives. There is a clear need to reduce demand on the National Health Service and policy aims to achieve this by promoting healthy living and preventing illness. The proposed study will contribute to this by providing benefit to Healthcare service providers.
In addition, society will benefit economically from healthcare improvements. The project will contribute to reducing incapacitation of the workforce and improve independent healthy ageing. The study will also have a wider impact on the nation's wealth as 17% of people claiming incapacity benefit have a musculoskeletal condition and it has been estimated that reducing working age ill health could save the UK up to £100 billion a year.
We envisage that the findings of this study may be of commercial interest to those engineering artificial tissues, as the mechanisms which contribute to tissue ageing may be exploited to enhance the mechanical properties of engineered tissues. This may result in attracting research and development investment into the UK or result in spin out companies.
The work also enhances cross disciplinary interactions building on existing synergistic interactions, thus benefiting other scientific disciplines as well as the Healthcare related industries.
The project provides an excellent opportunity for developing the careers of the post-doctoral research associates as they will benefit from working with cutting edge technology under expert guidance and from the interdisciplinary nature of the project. These learned skills could in the future be applied in a non-academic environment.

Department of Health, (2010). CM7985. Stationery office, London .


10 25 50

publication icon
Birch HL (2018) Extracellular Matrix and Ageing. in Sub-cellular biochemistry

publication icon
Birch HL (2016) Influence of Ageing on Tendon Homeostasis. in Advances in experimental medicine and biology

publication icon
Collier TA (2015) Preferential sites for intramolecular glucosepane cross-link formation in type I collagen: A thermodynamic study. in Matrix biology : journal of the International Society for Matrix Biology

publication icon
Lopez-Clavijo A. F. (2015) Glucosepane: investigating formation and structure in tendon collagen in INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY

Description We have made exciting and highly significant advances in this project towards understanding ageing changes in musculoskeletal tissues. We have shown that advanced glycation end-product crosslinks, specifically glucosepane, increase dramatically with increasing age in tendon collagen and we have developed specialised techniques to isolate and quantify glucosepane and to assess the impact of AGE crosslinking on tendon properties.
Glucosepane forms in vivo by a non-enzymatic pathway in long-lived proteins over many years, making it difficult to study. We have developed a novel method for synthesising glucosepane and its 13C labelled analogue from precursors in vitro within 17 hours. Our synthesised glucosepane provides a standard preparation for further characterisation of the crosslink and for quantification of glucosepane levels in tendon tissue. We have used computational studies and density functional theory to thermodynamically characterize the reaction pathway and intermediate products. Our studies have shown a thermodynamically viable process, although many of the transition states reside on a flat potential energy surface and this may explain their very slow formation over years in the body.
We have developed methodology to separate and purify synthesised glucosepane using high-pressure liquid chromatography with UV detection and a second chromatographic method coupled to mass spectrometry for the identification and quantitation of glucosepane. We have extracted and identified glucosepane in tendon tissue and shown that relative levels increase dramatically with increasing donor age. Absolute levels suggest multiple crosslinks per collagen molecule in human Achilles tendon tissue from an old group of donors between 65 and 81 years old.
The exact location of these crosslinks within the collagen molecule is important with regard to impact on the tissue properties. Our computational studies (published in Matrix Biology) have identified 24 positions based on distance criteria where lysine and arginine residues are close enough to form a crosslink between collagen polypeptide chains. A fully atomistic molecular dynamics simulation showed that six of these sites were energetically favourable (exothermic binding enthalpies) for glucosepane formation compared to unbound glucose. Using the same molecular dynamics simulation, we found that six sites were also favourable for formation of an alterative AGE crosslink DOGDIC and only one of these sites overlapped with glucosepane favourable locations (published in Biophysical Chemistry). The presence of glucosepane at these favourable positions has significant implications for the biological function of collagen, particularly as these sites occur where key collagen-biomolecule and collagen-cell interactions occur and is therefore highly likely to affect function.
Our studies using density functional theory and Møller-Plesset perturbation electronic structure calculations suggest that glucosepane is very hydrophilic and may increase hydration around the collagen molecule (published in Journal of Biomolecular Structure & Dynamics). A change in hydration within the collagen molecule or fibril is likely to influence the physical properties of the collagen framework. We have explored the thermodynamic behavior of collagen within native tendon tissue using differential scanning calorimetry. The results show a significant increase in denaturation temperature with increasing age, in line with that expected by additional crosslinking and an increase in the energetic barrier for helix uncoiling. Our results also showed a significant decrease in the enthalpy of denaturation, suggesting a less dense collagen fibril in keeping with the finding that glucosepane is highly hydrophilic and likely to attract water into the collagen fibril. Furthermore, our experimental studies demonstrate significantly higher water content in aged human Achilles tendon tissue samples.
One of the sites identified for glucosepane formation lies close to the site where collagenases cleave the collagen molecule. We have carried out a molecular dynamics study on the structural properties of a full MMP-1-collagen crystallographic structure, whilst exploring the thermodynamic properties of opening the active site entrance in the absence or presence of collagen (published in Journal of Biomolecular Structure & Dynamics). Our results indicate a rigid active site structure, supporting the idea of a local uncoiling of collagen and insertion mechanism. These results suggest the presence of a crosslink might hinder the uncoiling process. Correspondingly, our experimental work confirms previous findings, demonstrating that the ability of collagenase to digest collagen in old Achilles tendon tissue decreases by 64%. The identification of a potential site for glucosepane formation close to the collagenase cleavage site is important, as the ability of the tendon to turnover the matrix is key to maintaining healthy function.
The presence of glucosepane within the collagen molecule is likely to impact on mechanical properties directly or through water binding properties. Our computational studies at a molecular level suggest that the presence of glucosepane increases the stiffness of the collagen molecule in the initial loading phase. Furthermore, computational free energy calculations of intra-molecular glucosepane in short collagen peptides have shown it to significantly decrease free energy of collagen self-association, suggesting that glucosepane stabilises collagen molecule interactions and may reduce sliding between collagen molecules within the fibril when a mechanical load is applied. At the collagen fibril level, using atomic force microscopy techniques, we have found a significant decrease in the compressive modulus of collagen fibrils and a decrease in molecular contour pulling length in the old tendon specimens as may be expected in a more hydrated collagen fibril.
In further experimental work (published in Arthritis Research & Therapy) in collaboration with Professor Peter Clegg at the University of Liverpool, we have used Next Generation Sequencing to characterise the cell response to ageing in human Achilles tendon and to identify molecular signatures and that might contribute to age-related degeneration. This work demonstrated dynamic alterations in RNA with age at numerous genomic levels, indicating changes in the regulation of transcriptional networks. However, the results suggested that ageing is not primarily associated with loss of ability to synthesise matrix proteins and matrix degrading enzymes providing further support for the importance of AGE crosslinking in age related decline in tendon function.
Overall, the results of this study demonstrate a clear relationship between AGE crosslinking and decline in properties important for functional competence and highlight the importance of further work necessary to show cause and effect and ultimately strategies to reduce AGE formation and improve musculoskeletal health into old age.
Exploitation Route Knowledge of the pathway for formation of glucosepane may open up opportunities to intervene in the process to prevent the build up of AGE crosslinks in tissues such as tendon and other tissues where glucosepane is likely to be a problem. The studies on energetically favourable locations for DOGDIC formation suggest that DOGDIC is as favourable as glucosepane, suggesting that the difference in their abundance may be due to availability of the initiating substrate in the tissue, providing another avenue for an interventional approach.
Our findings will be of interest to academics; particularly those modelling protein-to-protein interactions and working to understand irreversible glycation modifications of long-lived proteins and their accumulation during ageing. Although our study focuses on skeletal tissue, specifically tendon, this chemical process is relevant in other tissues and other age related diseases. In addition bioengineers and those working with biomaterials will be interested in our findings as replacement tissues are often collagen based and involve in vitro crosslinking techniques.
The medical profession and other allied healthcare professionals will be able to use our findings to inform treatments of tendinopathies, particularly in older individuals. The findings will also be of interest to those in the medical profession involved in the management of diabetes.
Sectors Education,Healthcare,Leisure Activities, including Sports, Recreation and Tourism,Pharmaceuticals and Medical Biotechnology

Description We have provided educational impact at post-graduate level by taking on 2 PhD students to work adjacent to the PDRAs employed on this project. At the under-graduate level we have run several projects for 3rd year intercalating medical students at UCL. Our findings on this project have been disseminated to Orthopaedic clinicians at the Royal National Orthopaedic Hospital research day and to other clinicians and allied healthcare professionals through invited lectures to Orthopaedic consultants, Osteopaths, Physiotherapists and to those in the Veterinary profession.
First Year Of Impact 2014
Sector Education
Impact Types Societal

Title RNA-Seq of coding RNA 
Description RNA-seq of achilles tendon from young and old donors to study age-related changes in tendon deposited in ArrayExpress 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact Not known 
Description Collagen crosslinks 
Organisation University of Zurich
Country Switzerland 
Sector Academic/University 
PI Contribution Analysis of mature collagen crosslinks in mice tendons
Collaborator Contribution Breeding of mice and measurement of tendon mechanical properties
Impact Publication in preparation
Start Year 2019
Description Glucosepane synthetic standard preparation 
Organisation Yale University
Department Gilder Lehrman Center for the Study of Slavery, Resistance, and Abolition
Country United States 
Sector Academic/University 
PI Contribution Comparison of synthetic glucosepane standard preparation with native tendon tissue glucosepane
Collaborator Contribution Supply of synthetic glucosepane standard preparation
Impact None yet
Start Year 2016
Description Next Generation Sequencing on human tendon 
Organisation University of Liverpool
Department Institute of Ageing and Chronic Disease
Country United Kingdom 
Sector Academic/University 
PI Contribution Collection of human tendon samples and preparation for RNA work. Data interpretation and writing of manuscript.
Collaborator Contribution RNA-Seq practical work and initial analysis of data.
Impact Publication of a paper
Start Year 2014
Title ForceGen 
Description This Java executable jar derives second order bond force constants for bond stretch and bond angle from quantum mechanical Gaussian calculations. The calculations are compatible with the Amber force field family or any force field derived from the second order tensor of the Hessian from molecular fragments. The output has been made compatible with the Gromacs topology format. 
Type Of Technology Software 
Year Produced 2017 
Open Source License? Yes  
Impact The software has had frequent downloads. 
Description Equine Nutrition and Training Conference invited keynote speaker 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Discussion about the impact of exercise on tendons and the nutritional aspects of tendon health

New links with veterinary health professionals
Year(s) Of Engagement Activity 2013
Description Royal National Orthopedic Hospital Research Day invited speaker 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Health professionals
Results and Impact Discussion about the relationship with and impact of findings on other human diseases

Increased awareness of research expertise and potential for translational projects
Year(s) Of Engagement Activity 2014
Description Stanmore Campus Open Day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Approximately 200 people attended an open day organised by the University and partner hospital to inform members of the public about the research, teaching and clinical work carried out on the Campus.
Year(s) Of Engagement Activity 2016
Description Suffolk Osteopaths conference invited keynote speaker 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Research work was presented to a group of Osteopaths. The talk generated much interest and resulted in an invitation to return to provide an update of findings.

None yet
Year(s) Of Engagement Activity 2014,2016
Description Talk to Imperial College London Musculoskeletal Lab 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Research presented to group of academics, post-graduate and under-graduate students interested in musculoskeletal tissues and disease followed by group discussion surrounding the research topic.
Year(s) Of Engagement Activity 2017