Midportion Achilles tendinopathy (tendinosis) is a troublesome painful condition, often characterised by pain, local swelling, tenderness and functional disability. Despite extensive research, the pathogenesis is poorly understood and treatment remains challenging. Features related to the peritendinous connective tissue can be of importance. Recently it has been suggested that the plantaris tendon might be involved in this condition. Furthermore, it has been hypothesised that tendon pain and the tendinosis-related tissue changes in tendinopathy might be mediated by signalling substances such as glutamate and acetylcholine. A clinical observation, not scientifically evaluated, has been that unilateral treatment for bilateral Achilles tendinosis can lead to an effect on the contralateral side.      The aim of this work was to examine the morphology and innervation patterns in the plantaris tendon and the peritendinous connective tissue in between the Achillles and plantaris tendons in midportion Achilles tendinopathy, and to evaluate if plantaris tendon removal has an effect on Achilles tendon structure. Another aim was to determine if unilateral treatment for Achilles tendinopathy targeting the peritendinous connective tissue can result in bilateral recovery. Furthermore the presence of non-neuronal cholinergic and glutamate systems was examined.      Sections of plantaris tendons with adjacent peritendinous connective tissue from patients with midportion Achilles tendinopathy were stained for morphology (H&E), and innervation patterns were evaluated using antibodies against general nerve marker (PGP9.5), sensory (CGRP) and sympathetic (TH) nerve fibres and Schwann cells (S-100β). Furthermore immunostainings against non-neuronal aceylcholine (ChAT) and glutamate signalling components (glutamate, VGluT2, NMDAR1) were performed. Plantaris tendon cells were cultured and also stained for glutamate signalling components, and were stimulated with glutamate and glutamate receptor agonist NMDA. Furthermore, Ultrasound Tissue Characterisation (UTC) was used to monitor the integrity of the Achilles tendon collagen structure after plantaris tendon removal.      Plantaris tendons exhibited tendinosis-like tissue patterns such as hypercellularity, collagen disorganisation and large numbers of blood vessels. The peritendinous connective tissue between the plantaris and Achilles tendons contained large numbers of fibroblasts and blood vessels and to some extent macrophages and mast cells. A marked innervation was found in the peritendinous connective tissue and there were also nerve fibres in the loose connective tissue spaces within the tendon tissue proper. Most nerve fibres were identified as sensory fibres. Some nerve fascicles in the peritendinous connective tissue showed absence of axons but homogenous reactions for Schwann cell marker. Tenocytes and cells in the peritendinous connective tissue expressed ChAT, glutamate, VGluT2 and NMDAR1. Tendon cells in vitro expressed VGluT2, NMDAR1 and glutamate. UTC showed significant improvement of Achilles tendon integrity 6 months after surgical plantaris tendon removal and scraping procedure. Eleven out of thirteen patients reported of a bilateral recovery after unilateral surgical treatment.      The results of this work show that plantaris tendons exhibit tendinosis-like tissue changes, internal innervation and features that suggest occurrence of glutamate and acetylcholine production and signalling. Plantaris removal improves Achilles tendon structure suggesting possible compressive/shearing interference between the Achilles and plantaris tendons in tendinopathy. The peritendinous connective tissue shows marked innervation, which thus might transmit pain when being compressed. The partial absence of axons indicates a possible nerve degeneration. On the whole, the study gives new evidence favouring that the plantaris tendon and the peritendinous connective tissue might be of importance for pain and the tendinopathy process in midportion Achilles tendinopathy.

by Pau Hon-Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 146-179). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.i / ABBREVIATIONS --- p.iii / ABSTRACT (English) --- p.v / ABSTRACT (Chinese) --- p.ix / FLOW CHART --- p.xi / INDEX OF FIGURES --- p.xxi / INDEX OF TABLES --- p.xxiv / PUBLICATION LIST --- p.xxv / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Basic Structure of Tendons --- p.3 / Chapter 1.2 --- Anatomy of Patellar Tendon --- p.9 / Chapter 1.3 --- Cellular Characteristics of Fibroblasts --- p.11 / Chapter 1.4 --- Healing Process in Tendons --- p.13 / Chapter 1.5 --- Chronic Tendon Disorder --- p.19 / Chapter 1.6 --- Aims and Objectives --- p.25 / Chapter 1.7 --- Study Plan --- p.26 / Chapter CHAPRER 2 --- MATERIALS AND METHODOLOGY --- p.35 / Chapter 2.1 --- Collection of human Tissues Samples --- p.38 / Chapter 2.2 --- Tissue Culture --- p.40 / Chapter 2.3 --- Preparation of Conditioned Medium --- p.46 / Chapter 2.4 --- Proliferation Response of Tendon Fibroblasts --- p.48 / Chapter 2.5 --- Invasion Assay --- p.53 / Chapter 2.6 --- Chick Chorioallantonic Membrane Model --- p.56 / Chapter 2.7 --- SDS-PAGE --- p.58 / Chapter 2.8 --- Enzyme Linked Immunosorbent Assay --- p.63 / Chapter 2.9 --- Immunocytochemical Staining --- p.68 / Chapter 2.10 --- Statistical Analysis --- p.69 / Chapter CHAPTER 3 --- RESULTS --- p.74 / Chapter 3.1 --- Patient's Information --- p.76 / Chapter 3.2 --- Primary Explant Cell Culture --- p.77 / Chapter 3.3 --- Proliferation Response of Tendon Fibroblasts --- p.79 / Chapter 3.4 --- Invasion Assay --- p.82 / Chapter 3.5 --- Chick Chorioallantonic Membrane Model --- p.83 / Chapter 3.6 --- SDS-PAGE --- p.85 / Chapter 3.7 --- Enzyme Linked Immunosorbent Assay --- p.86 / Chapter 3.8 --- Immunocytochemical Staining --- p.88 / Chapter CHAPTER 4 --- DISCUSSION --- p.116 / Chapter 4.1 --- Higher Proliferation Capacity of Tendinosis Fibroblasts --- p.118 / Chapter 4.2 --- Tendinosis Fibroblasts Secrete Angiogenesis Factors to Enhance Angiogenesis --- p.123 / Chapter 4.3 --- Tendinosis Fibroblasts Secrete More Proteolytic Enzyme --- p.126 / Chapter 4.4 --- Proinflammatory Characteristics in Tendinosis Fibroblasts --- p.129 / Chapter 4.5 --- Comparison of in vitro and Histological Study --- p.132 / Chapter 4.6 --- Tendinosis Fibroblasts Seems to be Transformed --- p.134 / Chapter 4.7 --- Limitation of Study --- p.136 / Chapter CHAPTER 5 --- FUTURE RESEARCH DIRECTION --- p.140 / Chapter 5.1 --- Further Exploration of the Factors Secreted in Tendinosis Fibroblasts --- p.141 / Chapter 5.2 --- Proteoglycan Synthesis in Tendinosis Fibroblasts --- p.142 / Chapter 5.3 --- Tendinosis Animal Model and Clinical Study --- p.143 / Chapter CHAPTER 6 --- CONCLUSION --- p.144 / BIBLIOGRAPHY --- p.146 / APPENDIX --- p.180

Thèse (M.Sc.)--Université Laval, 2004. / Bibliogr. Publié aussi en version électronique.

La mise en place du système musculo-(exo)squelettique de la drosophile est un modèle d’organisation particulièrement propice à l’étude des interactions tissulaires au cours du développement.Notre étude vise à, d’une part, comprendre la myogenèse appendiculaire à travers l’étude des interactions précoces entre les précurseurs de tendon et les myoblastes, et d’autre part, étudier les mécanismes de différenciation des précurseurs de tendons associés au disque de patte. Dans ce contexte nous avons adapté la méthode GRASP (GFP Reconstitution Across Synaptic Partners) ainsi que l’imagerie en temps réel à notre modèle pour démontrer l’existence des interactions cellulaires entre les précurseurs de tendons et les myoblastes, nous avons aussi mis au point une approche cellule-spécifique afin de trier les précurseurs de tendons et les myoblastes associés au disque de patte, ce qui nous a permis d’obtenir dans un premier temps les données transcriptomiques des précurseurs de tendons. J’ai également étudié l’impact de l’altération des précurseurs de tendon sur le comportement des myoblastes associés et inversement. Nos résultats montrent que l’altération du développement des tendons entraîne une désorganisation spatiale des myoblastes environnants. Dans la seconde partie de mon projet, je me suis intéressée à l’implication de la voie Notch et des gènes de la famille odd-skipped dans la différenciation et la morphogenèse des précurseurs de tendon. J’ai ainsi démontré que Notch est nécessaire et localement suffisant pour induire l’expression de stripe et que les gènes odd-skipped et stripe coopèrent en aval cette voie pour permettre l’invagination et l’élongation sous forme de tube des longs tendons internes de la patte. / The formation of the musculo-(exo)skeletal system in drosophila is a remarkable example of tissue patterning making it a suitable model for studying multiple tissue interactions during development.The aim of our study is to better understand appendicular myogenesis through the identification of early interactions between tendon and muscle precursors, and by investigating the mechanisms governing the specification of tendon cell precursors of the leg disc. In order to characterize the interaction between these two tissues, we adapted the GRASP method (GFP Reconstitution Across Synaptic Partners) and set up live imaging experiments to reveal cellular interactions between tendon precursors and myoblasts. We have also conducted a genome wide cell-specific analysis using Fluorescence-activated cell sorting (FACS) on imaginal discs which allowed us to perform a tendon cell specific transcriptional analysis.To test whether reciprocal muscle-tendon interactions are necessary for correct muscle-tendon development, I performed experiments to specifically interfere with the development of tendon or muscle precursors. By altering tendon precursors formation during the early steps of leg development, we affect the spatial localization of the associated myoblasts. These findings provide the first evidence of the developmental impact of early interactions between muscle and tendon precursors in the leg disc.In the second part of my project, I investigated the role of Notch pathway and odd-skipped genes in the differentiation and morphogenesis of tendon precursors. Thus, I have demonstrated that Notch signalling pathway is necessary and locally sufficient for the initiation of stripe expression, and that both odd-skipped genes and stripe are required downstream of Notch to promote morphological changes associated with formation of long tubular tendons.

Master of Science / Department of Clinical Sciences / Walter C. Renberg / Objective: To measure strain in vivo in the calcanean tendon during trotting in canines, and to compare to strain present after tibiotarsal immobilization. Animals: 6 canines Procedures: A Differential Variable Reluctance Transducer[superscript]TM (DVRT®) strain gauge was surgically implanted on the common gastrocnemius tendon. Surface EMG, % strain, and ground reaction forces were measured prior to intervention and after immobilization. Peak vertical force (Fz), vertical impulse, initial, maximum and final strain, and peak-to-peak EMG amplitude were recorded. Data was analyzed using repeated measures analysis of variance and paired t-tests (p[equal to or less than]0.05). Results: Timing of strain data correlated closely to the hind limb footstrike and EMG activity in all dogs. Maximum tendon strain occurred simultaneous with peak Fz. Continued muscle contraction was evident after immobilization. There was no statistical difference in maximum strain after immobilization compared to normal motion. Minimum strain, both at the beginning and end of the strain curve, was significantly decreased with the immobilized state compared to non-immobilized joints. Conclusions and Clinical Relevance: Tibiotarsal immobilization did not eliminate calcaneal tendon strain during weight bearing. Decreased isometric muscle contraction during swing phase of the gait would account for smaller minimum strain in immobilized joints. Immobilization is frequently applied after Achilles tendon rupture to alleviate strain and force on the sutured repair, with possible complications due to the immobilization method. Direct correlation of strain with tendon force was not made in this study. This would be an important factor before adjusting current treatment recommendations.

The motivation for this PhD thesis was to understand the molecular basis of how cells regulate the formation of an organised and mechanically strong extracellular matrix (ECM). In tendon this process begins during embryogenesis with the appearance of bundles of narrow-diameter (~30 nm) collagen fibrils that are parallel to the tendon long axis. At the onset of collagen fibrillogenesis, the cells elongate, the fibrils are constrained within plasma membrane channels with their ends contained in tension-sensitive actin-stabilised plasma membrane protrusions. The mechanism by which actin is reorganised during cell elongation and the formation of tension-sensitive plasma membrane protrusions is poorly understood. The small GTPase RhoA is the major regulator of actin reorganisation into stress fibres, which have been implicated in mechanotransduction, ECM assembly and remodelling. The hypothesis tested by this PhD thesis was that the organisation and tensioning of extracellular collagen fibrils is generated on a blueprint of tensioned actin filaments within the cell. Rho activity is regulated specifically by Rho GTPase activating proteins (RhoGAPs). By comparing the global gene expression of tendon tissues at different developmental stages, Arhgap28, a novel RhoGAP, which is expressed during tendon development but not during postnatal maturation, was identified.Arhgap28 belongs to a large family of RhoGAPs containing the closely related members, Arhgap6 and Arhgap18, which have previously been shown to regulate RhoA and stress fibre formation. Arhgap28 expression was upregulated in embryonic fibroblasts cultured in a 3D, tensioned embryonic tendon-like construct compared to monolayer culture. Arhgap28 expression was further enhanced during the development of mechanical strength and stiffness of the tendon constructs, but downregulated when the tension in tendon constructs was released. Overexpression of a C-terminal V5-tagged Arhgap28 protein caused a reduction in RhoA activation and disruption of stress fibre assembly. Modulation of Rho signalling using lysophosphatidic acid and Y27632 showed that collagen remodelling by cells in collagen gels and tendon constructs is regulated by RhoA signalling. A tissue-wide qPCR analysis identified Arhgap28 in several tissues including tendon, bone, and skin. An Arhgap28 reporter mouse (Arhgap28gt) and an Arhgap28 knockout mouse (Arhgap28del) were also studied to investigate the role of Arhgap28 in tissue organisation in vivo. Arhgap28gt mice showed Arhgap28 expression in bones at E18.5. Homozygous Arhgap28del mice were viable, appeared normal but expressed a truncated Arhgap28 transcript, which if translated, would produce a protein lacking the RhoGAP domain. Therefore, it was hypothesised that knockout mice were normal due to compensation from another RhoGAP. Overexpression of Arhgap6 in Arhgap28-null bone tissues was confirmed. Upregulation in RhoA expression was also detected, further suggesting that Arhgap28 regulates RhoA. Interestingly, a microarray comparison of bone tissues from wild type and Arhgap28-null mice showed that genes linked to bone dysplasia are downregulated in Arhgap28-null bone. Together, these results suggest that formation of a strong and organised collagen ECM is mediated by RhoA-generated cellular tension and that Arhgap28 and Arhgap6 might be co-regulators of this process.

<p>  </p> <p>The disruption of homeostasis in tendon extracellular matrix and altered biomechanical properties lead to poor tendon healing, creating a significant clinical challenge for millions of diabetics. Furthermore, improving blood glucose levels doesn't normalize tendon properties in diabetics. Diabetes-related tendon complications are often associated with advanced glycation end products (AGEs) crosslinking with collagen. However, recent studies have found no evidence of higher collagen crosslinking in diabetics and no correlation between tendon AGE content and tensile strength. The interaction between serum AGEs and AGE receptors (RAGE) is a less explored mechanism of AGE-mediated effects. People with diabetes are more likely to accumulate AGEs in their serum as a result of hyperglycemia, the consumption of AGE-rich foods, and diminished kidney clearance of AGEs. In previous studies, advanced glycation end-products (AGEs) have been shown to inhibit cell proliferation and migration, both of which are critical to tendon healing. We hypothesized that serum AGEs and activation of RAGE represent a mechanism underlying impaired tendon properties with diabetes. The increasing serum AGE levels would impair tendon biomechanical properties and tendon healing, while inhibition of RAGE [Azeliragon (AZ)] would improve tendon mechanics.</p> <p>Db/db mice with naturally elevated serum AGEs and impaired tendon function were treated daily with a RAGE inhibitor [Azeliragon (AZ), n=9] or vehicle (n=10) for three weeks. Patellar tendon stiffness and modulus were greater (p<0.05) in mice receiving AZ (stiffness: 9.6±1.2 N/mm, modulus: 78.2±8.2 MPa) compared to vehicle (5.8±0.9 N/mm, modulus: 49.0±8.3 MPa). Maximum strain (vehicle: 0.9±0.1, AZ: 0.8±0.05) and toughness (vehicle: 6.1±1.4, AZ: 6.5±1.2 J·m−3) were not different between groups (p>0.05). Maximum stress tended to be greater in the AZ group (vehicle: 14.6±2.4, AZ: 23.3±2.9 N/mm2, p=0.156).</p> <p>Ten-week-old non-diabetic mice were assigned to receive daily injections of bovine serum albumin (BSA-only, n=6), BSA and AZ (BSA-AZ, n=5), 200 mg/ml glycated BSA (AGE-BSA, n=4), and AGE with AZ (AGE-AZ, n=6). A full-thickness, partial-width defect was created in both patellar tendons. Treatments were started one week before surgery and continued for three weeks after surgery. Three Tendon stiffness was lower in mice treated with AGEs (p<0.05, 10.8±1.4 N/mm) compared to BSA-only (17.6±1.3 N/mm). Further, tendon stiffness in AGE-treated mice given AZ was not different from AGE-BSA (p<0.05, 12.7±1.8 N/mm). Tendon modulus was lower in mice treated with AGEs (p<0.05, 28.0±7.0 MPa) compared to BSA-only (63.5±9.0 MPa). Additionally, modulus in AGE-treated mice given AZ was not different from AGE-BSA (p>0.05, 47.6±10.4 N/mm). </p> <p>We demonstrate that administering a RAGE inhibitor improves tendon properties in an established mouse model for type 2 diabetes. In healthy mice, serum AGE levels inhibit the recovery of tendon biomechanical properties after injury; RAGE inhibitors did not have an effect on mice given AGEs. Based on these data, we suggest elevated serum AGEs, as seen with diabetes, are associated with poor mechanical properties and delayed tendon healing.</p>

No description available.

Tendon injury is painful and often debilitating, and is a one of the most prevalent soft tissue injuries encountered in the clinic. While common, the underlying molecular and genetic processes of tendon damage and repair remain poorly understood. The work described herein used genome-wide expression analyses to investigate tendon injury and healing from three perspectives. The first identified novel gene expression in tendon fibroblasts following their stimulation with nitric oxide (NO). Of particular relevance to tendon healing was the observation that stimulated fibroblasts express a number of extracellular matrix (ECM) genes in response to NO in a dose-dependent manner, and that NO significantly affects cellular adhesion, a critical process during tendon repair. These findings will be of use when optimising dosages of NO delivery in future work investigating NO as potential treatment for tendon injuries. The second study examined gene expression in an acute tendon injury model in the rat at 1, 7, and 21 days post injury, roughly representing the inflammation, proliferation, and remodelling phase of wound repair. Several novel genes and pathways were found to be differentially expressed at each stage of healing. Of particular interest were the presence of a significant number of genes related to glutamate signaling, a method of cellular communication that has not previously been shown to exist in tendon. Also upregulated were a number of genes which have previously only been associated with embryonic development. Finally, gene expression in a supraspinatus tendinopathy model in the rat was investigated. Several genetic pathways were identified in tendinopathic tendons which have not previously been associated with the disease, and, analogous to the acute injury model study, glutamate signaling gene overexpression was also prevalent. Further in vitro studies showed that the expression of these genes in tendon fibroblasts were stimulated by glutamate treatment, which in turn upregulated pro-apoptotic pathways causing cell death. This may prove to be an important causative factor in the tendon degeneration seen in tendinopathy, in which apoptosis has been identified as playing a significant role. The results of these studies contribute to a better understanding of the aetiology of several extremely common pathologies of this soft tissue, and may help to develop more targeted therapies for increasing the efficacy of tendon healing in future.

ABSTRACT Background: On behalf of the Orthopedic clinic, Västerås, a study has been conducted with focus on comparing the results after conservative and surgical treatment due to complete achillestendon rupture. Aim: To compare results when testing the active and passive range of motion, calf muscle endurance, estimation of pain related to the achillestendon and self-efficacy to be physically active for individuals who have undergone conservative or surgical treatment after complete achillestendon rupture. Method: 14 individuals afflicted by complete achillestendon rupture in 2010 were recruited as a purposive sample. Examination were conducted of: Ankle range of motion with a goniometer, calf muscle endurance through a toe-raise test, estimation of pain intensity related to the achillestendon by VAS and self-efficacy to be physically active through “Exercise self-efficacy scale”. P-value and the median were calculated. Result: The results of ankle range of motion and calf muscle endurance were based on the difference between the injured and the healthy side. When testing active plantarflexion the conservative group had a median of 4 ̊ and the surgical group had a median of 10 ̊. At the toe-raise test the conservative group had a median of 11 toe-raises and the surgical group a median of 7. Through estimation of “Exercise self-efficacy scale” the conservative group had a median of 115 point and the surgical group a median of 94. When testing active dorsiflexion, passive plantarflexion, passive dorsiflexion and estimation of pain related to the achillestendon the median value were 0 for both groups. Conclusion: There was no statistical significance between the groups.   Key words: Achilles tendon, operative, rupture, self efficacy, treatment outcome. / SAMMANFATTNING Bakgrund: På uppdrag av Ortoped kliniken Västerås, har en studie genomförts med fokus på att jämföra resultat efter konservativ respektive operativ behandling i samband med total hälseneruptur. Syfte: Att jämföra resultaten vid test av aktiv och passiv fotledsrörlighet, vadmuskeluthållighet, skattning av smärta relaterat till hälsenan samt self-efficacy för att vara fysiskt aktiv för individer som genomgått konservativ respektive operativ behandling efter total hälseneruptur. Metod: 14 individer som drabbats av total hälseneruptur under 2010 rekryterades enligt ett ändamålsenligt urval. Undersökningar gjordes av fotledsrörlighet med hjälp av goniometer, vadmuskeluthållighet via ett tåhävningstest, skattning av smärtintensitet relaterat till hälsenan via VAS och self-efficacy för att vara fysiskt aktiv via ”Exercise self-efficacy scale”. P-värde och median beräknades. Resultat: Resultaten för fotledsrörligheten och vadmuskeluthålligheten baserade sig på skillnaden mellan frisk och skadad sida. Vid test av aktiv plantarflexion hade den konservativa gruppen en median på 4° och den operativa gruppen en median på 10°. Vid tåhävningstestet hade den konservativa gruppen en median på 11 stycken tåhävningar och den operativa gruppen en median på 7. Vid skattning via ”Exercise self-efficacy scale” hade den konservativa gruppen en median på 115 poäng och den operativa gruppen en median på 94. Vid test av aktiv dorsalflexion, passiv plantarflexion, passiv dorsalflexion samt vid skattning av smärta blev medianvärdet 0 för båda grupperna. Slutsats: Det förelåg ingen statistisk signifikant skillnad mellan grupperna.   Nyckelord: Behandlingsresultat, egen förmåga, hälsena, operationer, ruptur.