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

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.

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.

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.

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