Tendon Functional Extracellular Matrix Screen, Hazel R. C.; Berk, David E.; Kadler, Karl E. ...
Journal of orthopaedic research,
June 2015, Volume:
33, Issue:
6
Journal Article
Histological examination of pathological tendon generally does not reveal signs of inflammation. However, the inflammatory cytokine IL-6 has been shown to be expressed in ruptured rotator cuff ...tendon. The aim of this study was to investigate the expression of IL-6 family members in painful posterior tibialis tendon (PTT) and in painful and ruptured Achilles tendon (AT) compared with normal tendon.
AT samples were obtained from cadavers (normal) or from patients undergoing surgical procedures to treat chronic painful tendinopathy or ruptured tendon. PTT samples were obtained from patients undergoing surgery for other reasons (normal) and from patients with PTT dysfunction (painful). Total RNA was extracted and mRNA expression was analysed by quantitative real-time PCR.
Collagen type I α-chain I (COL1A1) expression was increased in both painful PTT and AT compared with normal. Ciliary neurotrophic factor levels were increased in painful PTT only. In the painful AT, cyclooxygenase-2 (COX2) and IL-6 expression increased compared with normal. In the ruptured AT, levels of VEGF A, COX2, oncostatin-M, leukaemia inhibitory factor and IL-6 expression were higher compared with both normal and painful AT. IL-6R expression decreased in both painful and ruptured AT compared with normal.
Painful AT and PTT show different expression patterns, indicating a substantial difference between those two tendinopathies. Inflammatory markers are up-regulated in painful and particularly in ruptured AT, pointing towards a role of inflammation not only in rupture healing, but also in Achilles tendinopathy.
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Structure-function relationships in tendons are directly influenced by the arrangement of collagen fibres. However, the details of such arrangements in functionally distinct tendons ...remain obscure. This study demonstrates the use of quantitative polarised light microscopy (qPLM) to identify structural differences in two major tendon compartments at the mesoscale: fascicles and interfascicular matrix (IFM). It contrasts functionally distinct positional and energy storing tendons, and considers changes with age. Of particular note, the technique facilitates the analysis of crimp parameters, in which cutting direction artefact can be accounted for and eliminated, enabling the first detailed analysis of crimp parameters across functionally distinct tendons.
IFM shows lower birefringence (0.0013 ± 0.0001 −), as compared to fascicles (0.0044 ± 0.0005 −), indicating that the volume fraction of fibres must be substantially lower in the IFM. Interestingly, no evidence of distinct fibre directional dispersions between equine energy storing superficial digital flexor tendons (SDFTs) and positional common digital extensor tendons (CDETs) were noted, suggesting either more subtle structural differences between tendon types or changes focused in the non-collagenous components.
By contrast, collagen crimp characteristics are strongly tendon type specific, indicating crimp specialisation is crucial in the respective mechanical function. SDFTs showed much finer crimp (21.1 ± 5.5 µm) than positional CDETs (135.4 ± 20.1 µm). Further, tendon crimp was finer in injured tendon, as compared to its healthy equivalents. Crimp angle differed strongly between tendon types as well, with average of 6.5 ± 1.4° in SDFTs and 13.1 ± 2.0° in CDETs, highlighting a substantially tighter crimp in the SDFT, likely contributing to its effective recoil capacity.
This is the first study to quantify birefringence in fascicles and interfascicular matrix of functionally distinct energy storing and positional tendons. It adopts a novel method – quantitative polarised light microscopy (qPLM) to measure collagen crimp angle, avoiding artefacts related to the direction of histological sectioning, and provides the first direct comparison of crimp characteristics of functionally distinct tendons of various ages.
A comparison of matched picrosirius red stained and unstained tendons sections identified non-homogenous staining effects, and leads us to recommend that only unstained sections are analysed in the quantitative manner.
qPLM is successfully used to assess birefringence in soft tissue sections, offering a promising tool for investigating the structural arrangements of fibres in (soft) tissues and other composite materials.
Tendon Structure and Composition Thorpe, Chavaunne T; Screen, Hazel R C
Advances in experimental medicine and biology,
2016, Volume:
920
Journal Article
Peer reviewed
Tendons are soft, fibrous tissues that connect muscle to bone. Their main function is to transfer muscle generated force to the bony skeleton, facilitating movement around a joint, and as such they ...are relatively passive, inelastic structures, able to resist high forces. Tendons are predominantly composed of collagen, which is arranged in a hierarchical manner parallel to the long axis of the tendon, resulting in high tensile strength. Tendon also contains a range of non-collagenous proteins, present in low amounts, which nevertheless have important functional roles. In this chapter, we describe general tendon composition and structure, and discuss how variations in composition and structure at different levels of the tendon hierarchy confer specific mechanical properties, which are related to tendon function.
(1978) into six levels: 'tropocollagen', 'microfibril', 'subfibril', 'fibril', 'fascicle' and 'tendon'. Since this early publication, however, such terminology has only been partially adopted, with ...subsequent authors introducing their own, and at times contradictory, naming conventions. ...we feel that as tendon research advances, it is increasingly important for researchers to use common terminology that is defined precisely.
Repetitive overload is a primary factor in tendon injury, causing progressive accumulation of matrix damage concurrent with a cellular response. However, it remains unclear how these events occur at ...the initial stages of the disease, making it difficult to identify appropriate treatment approaches. Here, we describe the development of a new model to cyclically load the Achilles tendon (AT) of rats in vivo and investigate the initial structural and cellular responses. The model utilizes controlled dorsiflexion of the ankle joint applied near maximal dorsiflexion, for 10,000 cycles at 3 Hz. Animals were subjected to a single bout of in vivo loading under anaesthesia, and either culled immediately (without recovery from anaesthesia), or 48 h or 4-weeks post-loading. Macro strains were assessed in cadavers, whilst tendon specific microdamage was assessed through collagen-hybridizing peptide (CHP) immunohistochemistry which highlighted a significant rise in CHP staining in loaded ATs compared to contralateral controls, indicating an accumulation of overload-induced damage. Staining for pro-inflammatory mediators (IL-6 and COX-2) and matrix degradation markers (MMP-3 and -13) also suggests an initial cellular response to overload. Model validation confirmed our approach was able to explore early overload-induced damage within the AT, with microdamage present and no evidence of broader musculoskeletal damage. The new model may be implemented to map the progression of tendinopathy in the AT, and thus study potential therapeutic interventions.
Hypercholesterolemia is associated with tendon pathology, but the reasons underpinning this relationship are not well understood. Cholesterol can accumulate in the tendon non-collagenous matrix which ...may affect both global and local tissue mechanics. Changes to the local strain environment within tendon may have significant implications for mechanosensitive tenocytes. Here, we investigated the association between elevated blood cholesterol and presence of tendon lipids in the Achilles tendon. We expected lipids to be localised in the proteoglycan-rich inter-sub-tendon matrix (ISTM), therefore we also sought to examine the impact of this on the biomechanical and viscoelastic properties of the ISTM.
The Achilles tendons of 32 young wild-type (SD) and 32 apolipoprotein E knock-out rats (ApoE
) were harvested at 15.6 ± 2.3 weeks of age. 32 specimens underwent histological examination to assess the distribution of lipids throughout sub-tendons and ISTM. The remaining specimens were prepared for biomechanical testing, where the ISTM between the gastrocnemius and soleus sub-tendons was subjected to shear load mechanical testing. A sub-set of tests were video recorded to enable a strain analysis.
ApoE
serum cholesterol was double that of SD rats (mean 2.25 vs. 1.10 mg/ml, p < 0.001) indicating a relatively mild hypercholesterolemia phenotype. Nonetheless, we found histological evidence of esterified lipids in the ISTM and unesterified lipids in the sub-tendons, although the location or intensity of staining was not appreciably different between rat strains. Despite a lack of observable histological differences in lipid content between groups, there were significant differences in the mechanical and viscoelastic behaviour of the Achilles sub-tendon matrix.
Even slightly elevated cholesterol may result in subtle changes to tendon biomechanical properties and hence injury risk. The young age of our cohort and the mild phenotype of our ApoE
rats are likely to have limited our findings and so we also conclude that the ApoE
rat model is not well suited for investigating the biomechanical impact of tendon xanthomas on Achilles sub-tendon function.
Summary
Tendon consists of highly ordered type I collagen molecules that are grouped together to form subunits of increasing diameter. At each hierarchical level, the type I collagen is interspersed ...with a predominantly non‐collagenous matrix (NCM) (Connect. Tissue Res., 6, 1978, 11). Whilst many studies have investigated the structure, organization and function of the collagenous matrix within tendon, relatively few have studied the non‐collagenous components. However, there is a growing body of research suggesting the NCM plays an important role within tendon; adaptations to this matrix may confer the specific properties required by tendons with different functions. Furthermore, age‐related alterations to non‐collagenous proteins have been identified, which may affect tendon resistance to injury. This review focuses on the NCM within the tensional region of developing and mature tendon, discussing the current knowledge and identifying areas that require further study to fully understand structure–function relationships within tendon. This information will aid in the development of appropriate techniques for tendon injury prevention and treatment.
The tendon interfascicular matrix (IFM) binds tendon fascicles together. As a result of its low stiffness behaviour under small loads, it enables non-uniform loading and increased overall ...extensibility of tendon by facilitating fascicle sliding. This function is particularly important in energy storing tendons, with previous studies demonstrating enhanced extensibility, recovery and fatigue resistance in the IFM of energy storing compared to positional tendons. However, the compositional specialisations within the IFM that confer this behaviour remain to be elucidated. It is well established that the IFM is rich in elastin, therefore we sought to test the hypothesis that elastin depletion (following elastase treatment) will significantly impact IFM, but not fascicle, mechanical properties, reducing IFM resilience in all samples, but to a greater extent in younger tendons, which have a higher elastin content. Using a combination of quasi-static and fatigue testing, and optical imaging, we confirmed our hypothesis, demonstrating that elastin depletion resulted in significant decreases in IFM viscoelasticity, fatigue resistance and recoverability compared to untreated samples, with no significant changes to fascicle mechanics. Ageing had little effect on fascicle or IFM response to elastase treatment.
This study offers a first insight into the functional importance of elastin in regional specific tendon mechanics. It highlights the important contribution of elastin to IFM mechanical properties, demonstrating that maintenance of a functional elastin network within the IFM is essential to maintain IFM and thus tendon integrity.
Developing effective treatments or preventative measures for musculoskeletal tissue injuries necessitates the understanding of healthy tissue function and mechanics. By establishing the contribution of specific proteins to tissue mechanical behaviour, key targets for therapeutics can be identified. Tendon injury is increasingly prevalent and chronically debilitating, with no effective treatments available.
Here, we investigate how elastin modulates tendon mechanical behaviour, using enzymatic digestion combined with local mechanical characterisation, and demonstrate for the first time that removing elastin from tendon affects the mechanical properties of the interfascicular matrix specifically, resulting in decreased recoverability and fatigue resistance. These findings provide a new level of insight into tendon hierarchical mechanics, important for directing development of novel therapeutics for tendon injury.
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Organ-on-chip systems are capable of replicating complex tissue structures and physiological phenomena. The fine control of biochemical and biomechanical cues within these microphysiological systems ...provides opportunities for cancer researchers to build complex models of the tumour microenvironment. Interest in applying organ chips to investigate mechanisms such as metastatsis and to test therapeutics has grown rapidly, and this review draws together the published research using these microfluidic platforms to study cancer. We focus on both in-house systems and commercial platforms being used in the UK for fundamental discovery science and therapeutics testing. We cover the wide variety of cancers being investigated, ranging from common carcinomas to rare sarcomas, as well as secondary cancers. We also cover the broad sweep of different matrix microenvironments, physiological mechanical stimuli and immunological effects being replicated in these models. We examine microfluidic models specifically, rather than organoids or complex tissue or cell co-cultures, which have been reviewed elsewhere. However, there is increasing interest in incorporating organoids, spheroids and other tissue cultures into microfluidic organ chips and this overlap is included. Our review includes a commentary on cancer organ-chip models being developed and used in the UK, including work conducted by members of the UK Organ-on-a-Chip Technologies Network. We conclude with a reflection on the likely future of this rapidly expanding field of oncological research.
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