Stem cells are widely known in the state of the art of cell-based therapies. Recently, ADSCs are becoming a popular resource of adult stem cells across different fields, and latest publications show its wide application for the treatment of soft tissue injuries like tendon injuries, which represent a high percentage of the consultations in orthopaedic practitioners. Molecular-based therapies and local deliveries are necessary for an effective treatment of chronic tendon injuries. In this study, human ADSCs were selected to investigate its differentiation potential into the tendon phenotype. Customised cell culture media was used as the differentiation factor. In the present study, ADSCs were used in passage 3 to ensure pluripotency in vitro. Using the customised cell culture media, its time, concentration and frequency of refreshment effects were investigated. On the selected time points different techniques were performed: 1,) cells were harvested, and messenger RNA (mRNA) was examined by Real Time Polymerase Chain Reaction (RT-PCR), analysing the expression of common tendon and extracellular matrix (ECM) markers. Protein expression was determined by Western Blotting. 2) Collagen content was analysed by tissue digestion and colorimetric techniques. 3) Deoxyribonucleic Acid (DNA) was stained, and fluorescent imaging was used to characterise nuclear roundness. 4) Metabolic activity of the cultures was assessed using CellTiter 96® Aqueous One Solution (MTS). 5) Cell proliferation was evaluated using CyQuant® Cell Proliferation Assay.Introduction
Materials and Methods
Energy storing tendons such as the equine superficial digital flexor tendon (SDFT) stretch and recoil with each stride and therefore require a high degree of compliance compared to tendons with a purely positional function, such as the equine common digital extensor tendon (CDET). This extra extensibility is provided by a specialised interfascicular matrix (IFM), which provides greater sliding and recoil between adjacent fascicles in energy storing tendons. However, the composition of the IFM remains largely undefined. We hypothesised that the IFM in the SDFT has a distinct composition, with a greater abundance of proteoglycans and elastin which facilitate extension and recoil. Transverse and longitudinal sections were cut from the mid-metacarpal regions of SDFTs and CDETs from 5 horses aged 3–7 years. Sections were stained using Alcian blue/Periodic acid Schiff to detect proteoglycans, elastic Van Giesson's to detect elastin, and immunohistochemistry was performed using antibodies for decorin, biglycan, fibromodulin, lumican and lubricin. Resultant images were graded by blinded observers to assess staining intensity in the IFM and fascicular matrix (FM), and statistical significance determined using ANOVA.Introduction
Materials and Methods
The exact mechanisms leading to tendinopathies and tendon ruptures remain poorly understood while their occurrence is clearly associated with exercise. Overloading is thought to be a major factor contributing to the development of tendon pathologies. However, as animal studies have shown, heavy loading alone won't cause tendinopathies. It has been speculated, that malfunctioning adaptation or healing processes might be involved, triggering tendon tissue degeneration. By analysing the expression of the entirety of degrading enzymes (degradome) in pathological and non-pathological, strained and non-strained tendon tissue, the aim of this study was to identify common or opposite patterns in gene regulation. This approach may generate new targets for future studies. RNA was extracted from different tendon tissues: normal (n=7), tendinopathic (n=4) and ruptured (n=4) Achilles tendon; normal (n=4) and tendinopathic (n=4) posterior tibialis tendon; normal hamstrings tendon with or without subjection to static strain (n=4). The RNA was reverse transcribed, then pooled per group The expression of 538 protease genes was analysed using Taqman low-density array quantitative RT-PCR. To be considered relevant, changes had to be at least 4fold and measurable at a level below 36 Cts.Introduction
Materials and Methods
Tendinopathies are debilitating and painful conditions. They are believed to result from repetitive overuse, which can create micro-damage that accumulates over time, and initiates a catabolic cell response. The aetiology of tendinopathy remains poorly understood, therefore the ideal treatment remains unclear. However, current data support the use of eccentric exercise as an effective treatment. In a previous study, we have shown that eccentric loading generates perturbations in the tendon at 10Hz, which is not present during other less effective loading regimes. Consequently, we hypothesis that 10Hz loading initiates an increased anabolic response in tenocytes, that can promote tendon repair. Human tenocytes from healthy hamstring tendons and tendinopathic Achilles tendons were derived by collagenase digest and outgrowth respectively. Tenocytes were seeded into 3D collagen gels. The gels were fixed in custom-made chambers and placed in an incubator for 24hrs whilst gene expression stabilised. After 24hrs, cyclic uniaxial strain at 1% ± 1% was applied to the cells, at either 1Hz (n=4) or 10Hz (n=4) using a Bose loading system. After 15 minutes of cyclic strain, the samples were maintained in chambers under 1% static strain for 24 hrs after which gene expression was characterised using RT-PCR.Introduction
Materials and Methods
Whilst all tendons connect muscle to bone, energy storing (ES) tendons, such as the equine superficial digital flexor tendon (SDFT) play an additional role, storing energy to improve locomotion efficiency. ES tendons experience significantly higher strains during locomotion than other positional tendons, such as the common digital extensor tendon (CDET). Our previous work has demonstrated that the interfascicular matrix (IFM) is more extensible in ES tendons, allowing ES tendons to stretch further during use. However, ES tendons must also recoil efficiently to perform their energy storing function. It has not been yet established if the IFM is able to recoil and recover after loading. Thus, this project aimed to determine the recoil capacity of the IFM in both the ES and positional tendons from young and old horses. Five young (3–7 years) and five old (17–20 years) SDFTs and CDETs were dissected from the forelimbs of 10 euthanized horses. Groups of 2 intact fascicles (bounded by IFM) were dissected from each tendon. Using a custom-made dissection rig and a polarised light microscope, samples were dissected, and the opposing end of each fascicle was cut transversely, leaving a 10 mm length of IFM. IFM samples were tested in shear, by preconditioning with 10 loading cycles then pulling to failure. The hysteresis and stress relaxation that occurred during preconditioning were calculated.Introduction
Materials and Methods
Fatigue loading has an age-specific effect on tendon fascicle micro-mechanics, with greater fibre sliding in aged samples indicating a decreased mechanical integrity, and a reduced ability to withstand cyclic loading, which may partially explain the age-related risk of tendon injury. The human Achilles and equine superficial digital flexor (SDFT) tendons function as energy stores, experiencing large, repetitive stresses and strains1 and are therefore highly susceptible to injury, particularly in aged individuals. We have previously observed rotation within SDFT fascicles in response to applied strain, which indicates the presence of helical sub-structures within this tendon. Further, we have shown that this rotation decreases with ageing, suggesting alterations to the helix sub-structure and a difference in the extension mechanisms in aged tendons. We therefore hypothesise that cyclic fatigue loading (FL) will result in alterations in fascicle extension mechanisms which are age specific.Summary Statement
Introduction
Tendon micromechanics were investigated using 2 methods. When collagen deformation was measured directly, higher levels of inter-fibre sliding were observed than when tenocyte nuclei were tracked. This suggests that under high strain tenocytes become unattached from the collagen fibres. Fibre extension and inter-fibre sliding have both been reported during tendon extension, but fibre sliding is believed to be the predominant mechanism in normal healthy tendon function. Fatigue damage is known to result in structural changes and reduced mechanical properties, but its influence on micromechanics is unknown. This work aimed: To investigate the effect of fatigue loading on bovine digital extensor fascicle micromechanics, comparing fibre extension and fibre sliding, hypothesising that the relative importance of these may change due to fatigue damage. To compare two techniques for characterising micromechanics: bleaching of a grid to directly measure collagen deformation, and using the cells as fiducial markers of fibre movement.Summary Statement
Introduction
We have shown that integrin mRNA expression is regulated by the application of mechanical load. This indicates that mechanical loading may modify cell sensitivity to perceive further load through increased interaction with the ECM. Tendinopathies are a range of diseases characterised by pain and insidious degeneration. Although poorly understood, onset is often associated with physical activity. We have previously investigated the regulation by mechanical strain of metalloproteinase gene expression in human tenocyte in a 3D collagen matrix. Integrins are important in cellular interaction with the ECM and are reported to mediate mechanotransduction in various non-tendon tissues. We have reported that TGFbeta activation is a key player in the regulation of metalloproteinases in response to mechanical load, which may be mediated by integrins. This project aims to investigate the effect of cyclic loading and TGFbeta stimulation on integrin expression by human tenocytes, in collagen and fibrin matrices.Summary Statement
Introduction
Most cases of tendinopathy are believed to be overuse injuries rather than the result of a chronic event. The investigation of the fatigue properties of tendon is therefore of critical importance. This work considered the cyclic stress-relaxation and creep behaviour of two contrasting bovine tendon types – the largely postional digital extensor and the more energy storing deep digital flexor tendon. Fascicles were cyclically loaded (1Hz), to 1800 cycles of stress relaxation or to failure in creep, stopping some tests at 300, 900 or 1200 cycles to perform quasi-static failure tests or confocal imaging using a highly concentrated Acridine Orange solution. Creep tests were cycled to 60% of the ultimate tensile strength (UTS), while for stress relaxation, cyclic deformation to the strain associated with 60% UTS was used. Flexor tendon fascicles were found to exhibit reduced stress relaxation at all time points compared to the extensor fascicles and also showed an increase in the mean cycles to failure during creep testing. Evidence of fatigue damage was clear in the confocal images with breakdown of the collagen fibre alignment evident from 300 cycles; however it appears that some damage could occur without effect on the UTS of the fascicle. Despite what appears to be superior fatigue resistance in the flexor tendon fascicles, the matrix damage, certainly at early time points, appeared visually to be as severe as that observed with the extensor tendon fascicles.
The purpose of this study was to profile the mRNA expression for the 23 known matrix metalloproteinases (MMPs), 4 tissue inhibitor of metalloproteinases (TIMPs) and 19 ADAMTSs (a disintegrin and metalloproteinase with thrombospontin motif) in Dupuytren's Disease and normal palmar fascia. Dupuytren's Disease (DD) is a fibroproliferative disorder affecting the palmar fascia, leading to contractures. The MMPs and ADAMTSs are related enzymes collectively responsible for turnover of the extracellular matrix. The balance between the proteolytic action of the MMPs and ADAMTSs and their inhibition by the TIMPs underpins many pathological processes. Deviation in favour of proteolysis is seen in e.g. invasive carcinomata, whereas an imbalance towards inhibition causes e.g. fibrosis. A group of patients with end-stage gastric carcinoma was treated with a broad spectrum MMP inhibitor in an attempt to reduce the rate of carcinoma advancement; a proportion developed a ‘musculoskeletal syndrome’ resembling DD. Tissue samples were obtained from patients undergoing surgery to correct contractures caused by DD and from healthy controls undergoing carpal tunnel decompression. The DD tissue was separated macroscopically into cord and nodule. Total RNA was extracted and mRNA expression analysed by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), normalised to 18S rRNA. Comparing across all genes, the DD nodule, DD cord and normal palmar fascia samples each had a distinct mRNA expression profile. Statistically significant (p<0.05) differences in mRNA expression included: higher MMP-2, -7 and ADAMTS-3 levels in both cord and nodule; higher MMP-1, -14, TIMP-1 and ADAMTS-4 and -5 in nodule alone, lower MMP-3 in nodule and cord and lower TIMP-2, -3 and -4 and ADAMTS-1 and -8 levels in nodule alone. The distinct mRNA profile of each group suggests differences in extracellular proteolytic activity which may underlie the process of fascial remodelling in DD.
The matrix metalloproteinases (MMPs) and ADAMTSs (a disintegrin and metalloproteinase with thrombos-pontin motif) are related enzymes collectively responsible for turnover of the extracellular matrix. The balance between the proteolytic action of the MMPs and ADAMTSs, and their inhibition by the tissue inhibitors of metalloproteinases (TIMPs), underpins many pathological processes. Deviation in favour of proteolysis is seen in e.g. invasive carcinoma, whereas an imbalance towards inhibition causes e.g. fibrosis. Dupuytren’s Disease (DD) is a fibroproliferative disorder affecting the palmar fascia, leading to contractures. A group of patients with end-stage gastric carcinoma were treated with a broad spectrum MMP inhibitor in an attempt to reduce the rate of tumour advancement: a proportion developed a ‘musculoskeletal syndrome’ resembling DD. Several groups have looked at subsets of the metalloproteinase family in relation to DD, but to date, a study of the gene expression of all of the members has not been published. We therefore set out to profile the mRNA expression for the 23 known MMPs, 4 TIMPs &
19 ADAMTSs in DD and normal palmar fascia. Tissue samples were obtained from patients undergoing surgery to correct contractures caused by DD and from healthy controls undergoing carpal tunnel decompression. The DD tissue was separated macroscopically into cord and nodule. Total RNA was extracted and mRNA expression analysed by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), normalised to 18S rRNA. Comparing across all genes, the DD nodule, DD cord and normal palmar fascia samples each had a distinct mRNA expression profile. Statistically significant (p<
0.05) differences in mRNA expression included: higher MMP-2, -7 and ADAMTS-3 levels in both cord and nodule; higher MMP-1, -14, TIMP-1 and ADAMTS-4 and -5 in nodule alone, lower MMP-3 in nodule and cord and lower TIMP-2, -3 and -4 and ADAMTS-1 and -8 levels in nodule alone. The distinct mRNA profile of each group suggests differences in extracellular proteolytic activity which may underlie the process of fascial remodelling in DD. Further in vitro experiments are planned based on these observed differences in gene expression.
Only 30% of the normal tendon sections showed any positive staining at all Compared to 36% of ruptured tendon and 43% of the painful tendinopathy sections.
There is a paucity of nerve tissue within these tendons, which may have implications for the neurogenic hypothesis of tendon degeneration There appear be more nerve fibres in vascular areas of the painful tendinopathy biopsies There may be more nerve fibres in the peritendinous tissue
