The Interfascicular Matrix of Energy Storing Tendons Houses Heterogenous Cell Populations Disproportionately Affected by Aging.

Energy storing tendons such as the human Achilles and equine superficial digital flexor tendon (SDFT) are prone to injury, with incidence increasing with aging, peaking in the 5th decade of life in the human Achilles tendon. The interfascicular matrix (IFM), which binds tendon fascicles, plays a key role in energy storing tendon mechanics, and aging alterations to the IFM negatively impact tendon function. While the mechanical role of the IFM in tendon function is well-established, the biological role of IFM-resident cell populations remains to be elucidated. Therefore, the aim of this study was to identify IFM-resident cell populations and establish how these populations are affected by aging. Cells from young and old SDFTs were subjected to single cell RNA-sequencing, and immunolabelling for markers of each resulting population used to localise cell clusters. Eleven cell clusters were identified, including tenocytes, endothelial cells, mural cells, and immune cells. One tenocyte cluster localised to the fascicular matrix, whereas nine clusters localised to the IFM. Interfascicular tenocytes and mural cells were preferentially affected by aging, with differential expression of genes related to senescence, dysregulated proteostasis and inflammation. This is the first study to establish heterogeneity in IFM cell populations, and to identify age-related alterations specific to IFM-localised cells.

Chemical Markers of Human Tendon Health Identified Using Raman Spectroscopy: Potential for In Vivo Assessment.

The purpose of this study is to determine whether age-related changes to tendon matrix molecules can be detected using Raman spectroscopy. Raman spectra were collected from human Achilles (n = 8) and tibialis anterior (n = 8) tendon tissue excised from young (17 ± 3 years) and old (72 ± 7 years) age groups. Normalised Raman spectra underwent principal component analysis (PCA), to objectively identify differences between age groups and tendon types. Certain Raman band intensities were correlated with levels of advanced glycation end-product (AGE) collagen crosslinks, quantified using conventional destructive biochemistry techniques. Achilles and tibialis anterior tendons in the old age group demonstrated significantly higher overall Raman intensities and fluorescence levels compared to young tendons. PCA was able to distinguish young and old age groups and different tendon types. Raman intensities differed significantly for several bands, including those previously associated with AGE crosslinks, where a significant positive correlation with biochemical measures was demonstrated. Differences in Raman spectra between old and young tendon tissue and correlation with AGE crosslinks provides the basis for quantifying age-related chemical modifications to tendon matrix molecules in intact tissue. Our results suggest that Raman spectroscopy may provide a powerful tool to assess tendon health and vitality in the future.

Effects of booster sessions on self-management interventions for chronic musculoskeletal pain: a systematic review and meta-analysis of randomised controlled trials.

ABSTRACT: Our objective was to investigate the effectiveness of booster sessions after self-management interventions as a means of maintaining self-management behaviours in the treatment of chronic musculoskeletal pain. We searched MEDLINE, EMBASE, Science Citation Index, Cochrane Central Register of Controlled Trials, and PsychINFO. Two authors independently identified eligible trials and collected data. We calculated the odds ratio for the analyses of dichotomous data and standardised mean differences (SMDs) with 95% confidence interval (CI) for continuous variables. Our search identified 14 studies with a total of 1695 patients. All studies were at high risk of bias and provided very low quality evidence. For the primary outcomes, booster sessions had no evidence of an effect on improving patient-reported outcomes on physical function (SMD -0.13, 95% CI -0.32 to -0.06; P = 0.18), pain-related disability (SMD -0.16, 95% CI -0.36 to 0.03; P = 0.11), and pain self-efficacy (SMD 0.15, 95% CI -0.07 to 0.36; P = 0.18). For the secondary outcomes, booster sessions caused a significant reduction in patient-reported pain catastrophising (SMD -0.42, 95% CI -0.64 to -0.19; P = 0.0004) and no evidence of an effect on patient-reported pain intensity, depression, coping, or treatment adherence. There is currently little evidence that booster sessions are an effective way to prolong positive treatment effects or improve symptoms of long-term musculoskeletal conditions after self-management interventions. However, the studies were few with high heterogeneity, high risk of bias, and overall low quality of evidence. Our review argues against including booster sessions routinely to self-management interventions for the purpose of behaviour maintenance.

Structure-function specialisation of the interfascicular matrix in the human achilles tendon.

Tendon consists of highly aligned collagen-rich fascicles surrounded by interfascicular matrix (IFM). Some tendons act as energy stores to improve locomotion efficiency, but such tendons commonly obtain debilitating injuries. In equine tendons, energy storing is achieved primarily through specialisation of the IFM. However, no studies have investigated IFM structure-function specialisation in human tendons. Here, we compare the human positional anterior tibial tendon and energy storing Achilles tendons, testing the hypothesis that the Achilles tendon IFM has specialised composition and mechanical properties, which are lost with ageing. Data demonstrate IFM specialisation in the energy storing Achilles, with greater elasticity and fatigue resistance than in the positional anterior tibial tendon. With ageing, alterations occur predominantly to the proteome of the Achilles IFM, which are likely responsible for the observed trends towards decreased fatigue resistance. Knowledge of these key energy storing specialisations and their changes with ageing offers crucial insight towards developing treatments for tendinopathy. STATEMENT OF SIGNIFICANCE: Developing effective therapeutics or preventative measures for tendon injury necessitates the understanding of healthy tendon function and mechanics. By establishing structure-function relationships in human tendon and determining how these are affected by ageing, potential targets for therapeutics can be identified. In this study, we have used a combination of mechanical testing, immunolabelling and proteomics analysis to study structure-function specialisations in human tendon. We demonstrate that the interfascicular matrix is specialised for energy storing in the Achilles tendon, and that its proteome is altered with ageing, which is likely responsible for the observed trends towards decreased fatigue resistance. Knowledge of these key energy storing specialisations and their changes with ageing offers crucial insight towards developing treatments and preventative approaches for tendinopathy.

An equine tendon model for studying intra-tendinous shear in tendons that have more than one muscle contribution.

Human Achilles tendon is composed of three smaller sub-tendons and exhibits non-uniform internal displacements, which decline with age and after injury, suggesting a potential role in the development of tendinopathies. Studying internal sliding behaviour is therefore important but difficult in human Achilles tendon. Here we propose the equine deep digital flexor tendon (DDFT) and its accessory ligament (AL) as a model to understand the sliding mechanism. The AL-DDFT has a comparable sub-bundle structure, is subjected to high and frequent asymmetric loads and is a natural site of injury similar to human Achilles tendons. Equine AL-DDFT were collected and underwent whole tendon level (n=7) and fascicle level (n=7) quasi-static mechanical testing. Whole tendon level testing was performed by sequentially loading through the proximal AL and subsequently through the proximal DDFT and recording regional strain in the free structures and joined DDFT and AL. Fascicle level testing was performed with focus on the inter-sub-bundle matrix between the two structures at the junction. Our results demonstrate a significant difference in the regional strain between the joined DDFT and AL and a greater transmission of force from the AL to the DDFT than vice versa. These results can be partially explained by the mechanical properties and geometry of the two structures and by differences in the properties of the interfascicular matrices. In conclusion, this tendon model successfully demonstrates that high displacement discrepancy occurs between the two structures and can be used as an easy-access model for studying intra-tendinous shear mechanics at the sub-tendon level. STATEMENT OF SIGNIFICANCE: Our study provides a naturally occurring and easily accessible equine model to study the complex behaviour of sub-tendons within the human Achilles tendon, which is likely to play a critical role in the pathogenesis of tendon disease. Our results demonstrate that the difference in material stiffness between the equine AL and DDFT stems largely from differences in the inter-fascicular matrix and furthermore that differences in strain are maintained in distal parts of the tightly joined structure. Furthermore, our results suggest that distribution of load between sub-structures is highly dependent on the morphological relationship between them; a finding that has important implications for understanding Achilles tendon mechanical behaviour, injury mechanisms and rehabilitation.

Individual variation in Achilles tendon morphology and geometry changes susceptibility to injury.

The unique structure of the Achilles tendon, combining three smaller sub-tendons, enhances movement efficiency by allowing individual control from connected muscles. This requires compliant interfaces between sub-tendons, but compliance decreases with age and may account for increased injury frequency. Current understanding of sub-tendon sliding and its role in the whole Achilles tendon function is limited. Here we show changing the degree of sliding greatly affects the tendon mechanical behaviour. Our in vitro testing discovered distinct sub-tendon mechanical properties in keeping with their mechanical demands. In silico study based on measured properties, subject-specific tendon geometry, and modified sliding capacity demonstrated age-related displacement reduction similar to our in vivo ultrasonography measurements. Peak stress magnitude and distribution within the whole Achilles tendon are affected by individual tendon geometries, the sliding capacity between sub-tendons, and different muscle loading conditions. These results suggest clinical possibilities to identify patients at risk and design personalised rehabilitation protocols.

Lysine-arginine advanced glycation end-product cross-links and the effect on collagen structure: A molecular dynamics study.

The accumulation of advanced glycation end-products is a fundamental process that is central to age-related decline in musculoskeletal tissues and locomotor system function and other collagen-rich tissues. However, although computational studies of advanced glycation end-product cross-links could be immensely valuable, this area remains largely unexplored given the limited availability of structural parameters for the derivation of force fields for Molecular Dynamics simulations. In this article, we present the bonded force constants, atomic partial charges and geometry of the arginine-lysine cross-links DOGDIC, GODIC, and MODIC. We have performed in vacuo Molecular Dynamics simulations to validate their implementation against quantum mechanical frequency calculations. A DOGDIC advanced glycation end-product cross-link was then inserted into a model collagen fibril to explore structural changes of collagen and dynamics in interstitial water. Unlike our previous studies of glucosepane, our findings suggest that intra-collagen DOGDIC cross-links furthers intra-collagen peptide hydrogen-bonding and does not promote the diffusion of water through the collagen triple helices.

Postnatal mechanical loading drives adaptation of tissues primarily through modulation of the non-collagenous matrix.

Mature connective tissues demonstrate highly specialised properties, remarkably adapted to meet their functional requirements. Tissue adaptation to environmental cues can occur throughout life and poor adaptation commonly results in injury. However, the temporal nature and drivers of functional adaptation remain undefined. Here, we explore functional adaptation and specialisation of mechanically loaded tissues using tendon; a simple aligned biological composite, in which the collagen (fascicle) and surrounding predominantly non-collagenous matrix (interfascicular matrix) can be interrogated independently. Using an equine model of late development, we report the first phase-specific analysis of biomechanical, structural, and compositional changes seen in functional adaptation, demonstrating adaptation occurs postnatally, following mechanical loading, and is almost exclusively localised to the non-collagenous interfascicular matrix. These novel data redefine adaptation in connective tissue, highlighting the fundamental importance of non-collagenous matrix and suggesting that regenerative medicine strategies should change focus from the fibrous to the non-collagenous matrix of tissue.

Detection of Age-Related Changes in Tendon Molecular Composition by Raman Spectroscopy-Potential for Rapid, Non-Invasive Assessment of Susceptibility to Injury.

The lack of clinical detection tools at the molecular level hinders our progression in preventing age-related tendon pathologies. Raman spectroscopy can rapidly and non-invasively detect tissue molecular compositions and has great potential for in vivo applications. In biological tissues, a highly fluorescent background masks the Raman spectral features and is usually removed during data processing, but including this background could help age differentiation since fluorescence level in tendons increases with age. Therefore, we conducted a stepwise analysis of fluorescence and Raman combined spectra for better understanding of the chemical differences between young and old tendons. Spectra were collected from random locations of vacuum-dried young and old equine tendon samples (superficial digital flexor tendon (SDFT) and deep digital flexor tendon (DDFT), total n = 15) under identical instrumental settings. The fluorescence-Raman spectra showed an increase in old tendons as expected. Normalising the fluorescence-Raman spectra further indicated a potential change in intra-tendinous fluorophores as tendon ages. After fluorescence removal, the pure Raman spectra demonstrated between-group differences in CH2 bending (1450 cm-1) and various ring-structure and carbohydrate-associated bands (1000-1100 cm-1), possibly relating to a decline in cellular numbers and an accumulation of advanced glycation end products in old tendons. These results demonstrated that Raman spectroscopy can successfully detect age-related tendon molecular differences.

Relative orientation of collagen molecules within a fibril: a homology model for homo sapiens type I collagen.

Type I collagen is an essential extracellular protein that plays an important structural role in tissues that require high tensile strength. However, owing to the molecule's size, to date no experimental structural data are available for the Homo sapiens species. Therefore, there is a real need to develop a reliable homology model and a method to study the packing of the collagen molecules within the fibril. Through the use of the homology model and implementation of a novel simulation technique, we have ascertained the orientations of the collagen molecules within a fibril, which is currently below the resolution limit of experimental techniques. The longitudinal orientation of collagen molecules within a fibril has a significant effect on the mechanical and biological properties of the fibril, owing to the different amino acid side chains available at the interface between the molecules.

Glucosepane is associated with changes to structural and physical properties of collagen fibrils.

Collagen glycation, and in particular the formation of advanced glycation end-product (AGE) crosslinks, plays a central role in the ageing process and in many of the long-term complications of diabetes. Glucosepane, the most abundant and relevant AGE crosslink, has been suggested to increase the stiffness of tissue and reduce its solubility, although no evidence is available concerning the mechanisms. We have used a combination of computational and experimental techniques to study a collagen-rich tissue with a relatively simple organisation to further our understanding of the impact of glucosepane on the structural and physical properties of collagen fibrils. Our work shows that glucosepane levels increase dramatically in aged tendon tissue and are associated with the reduced density of collagen packing and increased porosity to water molecules. Our studies provide the basis to understand many of the tissue dysfunctions associated with ageing and diabetes across a range of different tissues types.

AftD functions as an α1 → 5 arabinofuranosyltransferase involved in the biosynthesis of the mycobacterial cell wall core.

Arabinogalactan (AG) is an essential structural macromolecule present in the cell wall of Mycobacterium tuberculosis, serving to connect peptidoglycan with the outer mycolic acid layer. The D-arabinan segment is a highly branched component of AG and is assembled in a step-wise fashion by a variety of arabinofuranosyltransferases (AraT). We have previously used Corynebacterium glutamicum as a model organism to study these complex processes which are otherwise essential in mycobacteria. In order to further our understanding of the molecular basis of AG assembly, we investigated the role of a fourth AraT, now termed AftD by generating single (ΔaftD) and double deletion (ΔaftB ΔaftD) mutants of C. glutamicum. We demonstrate that AftD functions as an α(1 → 5) AraT and reveal the point at which it exerts its activity in the AG biosynthetic pathway.

Extracellular Matrix and Ageing.

The extracellular matrix (ECM) provides the environment for many cells types within the body and, in addition to the well recognised role as a structural support, influences many important cell process within the body. As a result, age-related changes to the proteins of the ECM have far reaching consequences with the potential to disrupt many different aspects of homeostasis and healthy function. The proteins collagen and elastin are the most abundant in the ECM and their ability to function as a structural support and provide mechanical stability results from the formation of supra-molecular structures. Collagen and elastin have a long half-life, as required by their structural role, which leaves them vulnerable to a range of post-translational modifications. In this chapter the role of the ECM is discussed and the component proteins introduced. Major age-related modifications including glycation, carbamylation and fragmentation and the impact these have on ECM function are reviewed.

ForceGen: atomic covalent bond value derivation for Gromacs.

A large number of crystallographic protein structures include ligands, small molecules and post-translational modifications. Atomic bond force values for computational atomistic models of post-translational or non-standard amino acids, metal binding active sites, small molecules and drug molecules are not readily available in most simulation software packages. We present ForceGen, a Java tool that extracts the bond stretch and bond angle force values and equilibrium values from the Hessian of a Gaussian vibrational frequency analysis. The parameters are compatible with force fields derived using the second order tensor of the Hessian. The output is formatted with the Gromacs topology in mind. This study further demonstrates the use of ForceGen over the quantum mechanically derived structures of a small organic solvent, a naturally occurring protein crosslink derived from two amino acids following post-translational modification and the amino acid ligands of a zinc ion. We then derive Laplacian bond orders to understand how the resulting force values relate back to the quantum mechanical model. The parameterisation of the organic solvent, toluene, was verified using Molecular Mechanics simulations. The structural data from the simulation compared well with the quantum mechanical structure and the system density compared well with experimental values.

Combining nano-physical and computational investigations to understand the nature of "aging" in dermal collagen.

The extracellular matrix of the dermis is a complex, dynamic system with the various dermal components undergoing individual physiologic changes as we age. Age-related changes in the physical properties of collagen were investigated in particular by measuring the effect of aging, most likely due to the accumulation of advanced glycation end product (AGE) cross-links, on the nanomechanical properties of the collagen fibril using atomic force microscope nano-indentation. An age-related decrease in the Young's modulus of the transverse fibril was observed (from 8.11 to 4.19 GPa in young to old volunteers, respectively, P<0.001). It is proposed that this is due to a change in the fibril density caused by age-related differences in water retention within the fibrils. The new collagen-water interaction mechanism was verified by electronic structure calculations, showing it to be energetically feasible.

Fascicles and the interfascicular matrix show decreased fatigue life with ageing in energy storing tendons.

Tendon is composed of rope-like fascicles bound together by interfascicular matrix (IFM). The IFM is critical for the function of energy storing tendons, facilitating sliding between fascicles to allow these tendons to cyclically stretch and recoil. This capacity is required to a lesser degree in positional tendons. We have previously demonstrated that both fascicles and IFM in energy storing tendons have superior fatigue resistance compared with positional tendons, but the effect of ageing on the fatigue properties of these different tendon subunits has not been determined. Energy storing tendons become more injury-prone with ageing, indicating reduced fatigue resistance, hence we tested the hypothesis that the decline in fatigue life with ageing in energy storing tendons would be more pronounced in the IFM than in fascicles. We further hypothesised that tendon subunit fatigue resistance would not alter with ageing in positional tendons. Fascicles and IFM from young and old energy storing and positional tendons were subjected to cyclic fatigue testing until failure, and mechanical properties were calculated. The results show that both IFM and fascicles from the SDFT exhibit a similar magnitude of reduced fatigue life with ageing. By contrast, the fatigue life of positional tendon subunits was unaffected by ageing. The age-related decline in fatigue life of tendon subunits in energy storing tendons is likely to contribute to the increased risk of injury in aged tendons. Full understanding of the mechanisms resulting in this reduced fatigue life will aid in the development of treatments and interventions to prevent age-related tendinopathy. STATEMENT OF SIGNIFICANCE: Understanding the effect of ageing on tendon-structure function relationships is crucial for the development of effective preventative measures and treatments for age-related tendon injury. In this study, we demonstrate for the first time that the fatigue resistance of the interfascicular matrix decreases with ageing in energy storing tendons. This is likely to contribute to the increased risk of injury in aged tendons. Full understanding of the mechanisms that result in this reduced fatigue resistance will aid in the development of treatments and interventions to prevent age-related tendinopathy.

Mapping intermolecular interactions and active site conformations: from human MMP-1 crystal structure to molecular dynamics free energy calculations.

The zinc-dependent Matrix Metalloproteinases (MMPs) found within the extracellular matrix (ECM) of vertebrates are linked to pathological processes such as arthritis, skin ulceration and cancer. Although a general backbone proteolytic mechanism is understood, crystallographic data continue to suggest an active site that is too narrow to encompass the respective substrate. We present a fully parameterised Molecular Dynamics (MD) study of the structural properties of an MMP-1-collagen crystallographic structure (Protein Data Bank - 4AUO), followed by an exploration of the free energy surface of a collagen polypeptide chain entering the active site, using a combined meta-dynamics and umbrella sampling (MDUS) approach. We conclude that the interactions between MMP-1 and the collagen substrate are in good agreement with a number of experimental studies. As such, our unrestrained MD simulations and our MDUS results, which indicate an energetic barrier for a local uncoiling and insertion event, can inform future investigations of the collagen-peptide non-bonded association steps with the active site prior to proteolytic mechanisms. The elucidation of such free energy barriers provides a better understanding of the role of the enzyme in the ECM and is important in the design of future MMP inhibitors.

Computational study of glucosepane-water and hydrogen bond formation: an electron topology and orbital analysis.

The collagen protein provides tensile strength to the extracellular matrix in addition to localising cells, proteins and protein cofactors. Collagen is susceptible to a build up of glycation modifications as a result of an exceptionally long half-life. Glucosepane is a collagen cross-linking advanced glycation end product; the structural and mechanical effects of glucosepane are still the subjects of much debate. With the prospect of an ageing population, the management and treatment of age-related diseases is becoming a pressing concern. One area of interest is the isolation of hydrated glucosepane, which has yet to be reported at an atomistic level. This study presents a series of glucosepane-water complexes within an implicit aqueous environment. Electronic structure calculations were performed using density functional theory and a high level basis set. Hydrogen bonds between glucosepane and explicit water were identified by monitoring changes to covalent bonds, calculating levels of electron donation from Natural Bonding Orbital analysis and the detection of bond critical points. Hydrogen bond strength was calculated using second-order perturbation calculations. The combined results suggest that glucosepane is very hydrophilic, with the imidazole feature being energetically more attractive to water than either hydroxyl group, although all hydrogen bonds, regardless of bond strength, were electrostatic in nature. Our results are in growing support of an earlier hypothesis that cross-links may result in an increase in interstitial water retention, which would permit the collagen fibril to swell, thereby potentially affecting the tensile and compression properties and biological function of connective tissues.

Intra-molecular lysine-arginine derived advanced glycation end-product cross-linking in Type I collagen: A molecular dynamics simulation study.

Covalently cross-linked advanced glycation end products (AGE) are among the major post-translational modifications to proteins as a result of non-enzymatic glycation. The formation of AGEs has been shown to have adverse effects on the properties of the collagenous tissue; they are even linked to a number of age related disorders. Little is known about the sites at which these AGEs form or why certain sites within the collagen are energetically more favourable than others. In this study we have used a proven fully atomistic molecular dynamics approach to identify six sites where the formation of the intra-molecular 3-deoxyglucosone-derived imidazolium cross-link (DOGDIC) is energetically favourable. We have also conducted a comparison of these positions with those of the more abundant glucosepane cross-link, to determine any site preference. We show that when we consider both lysine and arginine AGEs, they exhibit a prevalence to form within the gap region of the collagen fibril.

Influence of Ageing on Tendon Homeostasis.

Tendon functional competence and structural integrity rely on homeostasis of tendon cell metabolism and extracellular matrix macromolecules. The clear link between tendinopathies and increasing age suggests a slow change to tendon homeostasis, which increases susceptibility to damage. Despite this well evidenced association between increasing age and tendon damage, changes to tendon mechanical properties with ageing are not clear with different studies reporting conflicting results. More recent research suggests that age-related changes occur at specific sub-structure locations and may be overlooked by measuring properties of the whole tendon. In this chapter we review changes to tendon mechanical properties, structure and composition. Mechanisms speculated to contribute to tendon change with age such as cellular senescence, ageing stem cell population, reactive oxygen species and formation of advanced glycation end-product crosslinks are discussed. Understanding age-related changes to tendon homeostasis are key to understanding increased incidence of tendon injuries in the ageing population.