Regeneration of Damaged Tendon-Bone Junctions (Entheses)-TAK1 as a Potential Node Factor.

Musculoskeletal dysfunctions are highly prevalent due to increasing life expectancy. Consequently, novel solutions to optimize treatment of patients are required. The current major research focus is to develop innovative concepts for single tissues. However, interest is also emerging to generate applications for tissue transitions where highly divergent properties need to work together, as in bone-cartilage or bone-tendon transitions. Finding medical solutions for dysfunctions of such tissue transitions presents an added challenge, both in research and in clinics. This review aims to provide an overview of the anatomical structure of healthy adult entheses and their development during embryogenesis. Subsequently, important scientific progress in restoration of damaged entheses is presented. With respect to enthesis dysfunction, the review further focuses on inflammation. Although molecular, cellular and tissue mechanisms during inflammation are well understood, tissue regeneration in context of inflammation still presents an unmet clinical need and goes along with unresolved biological questions. Furthermore, this review gives particular attention to the potential role of a signaling mediator protein, transforming growth factor beta-activated kinase-1 (TAK1), which is at the node of regenerative and inflammatory signaling and is one example for a less regarded aspect and potential important link between tissue regeneration and inflammation.

Increased Collagen Turnover Impairs Tendon Microstructure and Stability in Integrin α2ß1-Deficient Mice.

Integrins are a family of transmembrane proteins, involved in substrate recognition and cell adhesion in cross-talk with the extra cellular matrix. In this study, we investigated the influence of integrin α2ß1 on tendons, another collagen type I-rich tissue of the musculoskeletal system. Morphological, as well as functional, parameters were analyzed in vivo and in vitro, comparing wild-type against integrin α2ß1 deficiency. Tenocytes lacking integrin α2ß1 produced more collagen in vitro, which is similar to the situation in osseous tissue. Fibril morphology and biomechanical strength proved to be altered, as integrin α2ß1 deficiency led to significantly smaller fibrils as well as changes in dynamic E-modulus in vivo. This discrepancy can be explained by a higher collagen turnover: integrin α2ß1-deficient cells produced more matrix, and tendons contained more residual C-terminal fragments of type I collagen, as well as an increased matrix metalloproteinase-2 activity. A greatly decreased percentage of non-collagenous proteins may be the cause of changes in fibril diameter regulation and increased the proteolytic degradation of collagen in the integrin-deficient tendons. The results reveal a significant impact of integrin α2ß1 on collagen modifications in tendons. Its role in tendon pathologies, like chronic degradation, will be the subject of future investigations.

ERK1/2 drives IL-1ß-induced expression of TGF-ß1 and BMP-2 in torn tendons.

Diseased and injured tendons develop fibrosis, driven by factors including TGF-ß, BMPs and CTGF. IL-1ß and its signal transducer Erk1/2 are known to regulate TGF-ß expression in animal tendons. We utilised tissues and cells isolated from patients with shoulder tendon tears and tendons of healthy volunteers to advance understanding of how inflammation induces fibrosis in diseased human tendons. ERK1/2 expression was reduced in torn (diseased) compared to healthy patient tendon tissues. We next investigated the fibrotic responses of tendon-derived cells isolated from healthy and diseased human tendon tissues in an inflammatory milieu. IL-1ß treatment induced profound ERK1/2 signalling, TGFB1 and BMP2 mRNA expression in diseased compared to healthy tendon-derived cells. In the diseased cells, the ERK1/2 inhibitor (PD98059) completely blocked the IL-1ß-induced TGFB1 and partially reduced BMP2 mRNA expression. Conversely, the same treatment of healthy cells did not modulate IL-1ß-induced TGFB1 or BMP2 mRNA expression. ERK1/2 inhibition did not attenuate IL-1ß-induced CTGF mRNA expression in healthy or diseased tendon cells. These findings highlight differences between ERK1/2 signalling pathway activation and expression of TGF-ß1 and BMP-2 between healthy and diseased tendon tissues and cells, advancing understanding of inflammation induced fibrosis during the development of human tendon disease and subsequent repair.

Embryo movements regulate tendon mechanical property development.

Tendons transmit forces from muscles to bones to enable skeletal motility. During development, tendons begin to bear load at the onset of embryo movements. Using the chick embryo model, this study showed that altered embryo movement frequency led to changes in elastic modulus of calcaneal tendon. In particular, paralysis led to decreased modulus, whereas hypermotility led to increased modulus. Paralysis also led to reductions in activity levels of lysyl oxidase (LOX), an enzyme that we previously showed is required for cross-linking-mediated elaboration of tendon mechanical properties. Additionally, inhibition of LOX activity abrogated hypermotility-induced increases in modulus. Taken together, our findings suggest embryo movements are critical for tendon mechanical property development and implicate LOX in this process. These exciting findings expand current knowledge of how functional tendons form during development and could guide future clinical approaches to treat tendon defects associated with abnormal mechanical loading in uteroThis article is part of the Theo Murphy meeting issue 'Mechanics of development'.

Postnatal tendon growth and remodeling require platelet-derived growth factor receptor signaling.

Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in the fundamental biological activities of many cells that compose musculoskeletal tissues. However, little is known about the role of PDGFR signaling during tendon growth and remodeling in adult animals. Using the hindlimb synergist ablation model of tendon growth, our objectives were to determine the role of PDGFR signaling in the adaptation of tendons subjected to a mechanical growth stimulus, as well as to investigate the biological mechanisms behind this response. We demonstrate that both PDGFRs, PDGFRα and PDGFRß, are expressed in tendon fibroblasts and that the inhibition of PDGFR signaling suppresses the normal growth of tendon tissue in response to mechanical growth cues due to defects in fibroblast proliferation and migration. We also identify membrane type-1 matrix metalloproteinase (MT1-MMP) as an essential proteinase for the migration of tendon fibroblasts through their extracellular matrix. Furthermore, we report that MT1-MMP translation is regulated by phosphoinositide 3-kinase/Akt signaling, while ERK1/2 controls posttranslational trafficking of MT1-MMP to the plasma membrane of tendon fibroblasts. Taken together, these findings demonstrate that PDGFR signaling is necessary for postnatal tendon growth and remodeling and that MT1-MMP is a critical mediator of tendon fibroblast migration and a potential target for the treatment of tendon injuries and diseases.

Leptin accelerates the pathogenesis of heterotopic ossification in rat tendon tissues via mTORC1 signaling.

Leptin, an adipocyte-derived cytokine associated with bone metabolism, is believed to play a critical role in the pathogenesis of heterotopic ossification (HO). The effect and underlying action mechanism of leptin were investigated on osteogenic differentiation of tendon-derived stem cells (TDSCs) in vitro and the HO formation in rat tendons. Isolated rat TDSCs were treated with various concentrations of leptin in the presence or absence of mTORC1 signaling specific inhibitor rapamycin in vitro. A rat model with Achilles tenotomy was employed to evaluate the effect of leptin on HO formation together with or without rapamycin treatment. In vitro studies with TDSCs showed that leptin increased the expression of osteogenic biomarkers (alkaline phosphatase, runt-related transcription factor 2, osterix, osteocalcin) and enhanced mineralization of TDSCs via activating the mTORC1 signal pathway (as indicated by phosphorylation of p70 ribosomal S6 kinase 1 and p70 ribosomal S6). However, mTORC1 signaling blockade with rapamycin treatment suppressed leptin-induced osteogenic differentiation and mineralization. In vivo studies showed that leptin promoted HO formation in the Achilles tendon after tenotomy, and rapamycin treatment blocked leptin-induced HO formation. In conclusion, leptin can promote TDSC osteogenic differentiation and heterotopic bone formation via mTORC1 signaling in both vitro and vivo model, which provides a new potential therapeutic target for HO prevention.

Laminin and Matrix metalloproteinase 11 regulate Fibronectin levels in the zebrafish myotendinous junction.

BACKGROUND: Remodeling of the extracellular matrix (ECM) regulates cell adhesion as well as signaling between cells and their microenvironment. Despite the importance of tightly regulated ECM remodeling for normal muscle development and function, mechanisms underlying ECM remodeling in vivo remain elusive. One excellent paradigm in which to study ECM remodeling in vivo is morphogenesis of the myotendinous junction (MTJ) during zebrafish skeletal muscle development. During MTJ development, there are dramatic shifts in the primary components comprising the MTJ matrix. One such shift involves the replacement of Fibronectin (Fn)-rich matrix, which is essential for both somite and early muscle development, with laminin-rich matrix essential for normal function of the myotome. Here, we investigate the mechanism underlying this transition. RESULTS: We show that laminin polymerization indirectly promotes Fn downregulation at the MTJ, via a matrix metalloproteinase 11 (Mmp11)-dependent mechanism. Laminin deposition and organization is required for localization of Mmp11 to the MTJ, where Mmp11 is both necessary and sufficient for Fn downregulation in vivo. Furthermore, reduction of residual Mmp11 in laminin mutants promotes a Fn-rich MTJ that partially rescues skeletal muscle architecture. CONCLUSIONS: These results identify a mechanism for Fn downregulation at the MTJ, highlight crosstalk between laminin and Fn, and identify a new in vivo function for Mmp11. Taken together, our data demonstrate a novel signaling pathway mediating Fn downregulation. Our data revealing new regulatory mechanisms that guide ECM remodeling during morphogenesis in vivo may inform pathological conditions in which Fn is dysregulated.

bFGF- and CaPP-Loaded Fibrin Clots Enhance the Bioactivity of the Tendon-Bone Interface to Augment Healing.

BACKGROUND: Tendon-to-bone healing is a complex and slow process, and the rate of poor healing remains high. In recent years, several new strategies have been developed that enhance tendon-to-bone healing by increasing the bioactivity. Fibrin clots have been widely used to improve tissue healing and tissue engineering, HYPOTHESIS: Modified fibrin clots can improve the bioactivity of the tendon-bone interface and histological appearance. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 27 male New Zealand White rabbits were used. Of these, 3 were used for cell isolation, and the remaining 24 rabbits were divided into 2 groups (12 per group) for an in vivo partial patellectomy study. The setting time, degradation time, and basic fibroblast growth factor (bFGF) and ceramide-activated protein phosphatase (CaPP) release kinetics of bFGF- and CaPP-loaded fibrin clots were modified appropriately for early tendon-to-bone healing. In an in vitro experiment, the bFGF- and CaPP-loaded fibrin clots were assessed for cell migration and proliferation by microscopy, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, and DAPI (4',6-diamidino-2-phenylindole) assay. Quantitative real-time reverse transcription polymerase chain reaction and a Western blot assay were performed to test for an induction effect of the bFGF- and CaPP-loaded fibrin clots. Finally, for the in vivo experiment, the rabbits were divided into 2 treatment groups: one with bFGF- and CaPP-loaded fibrin clots and one without bFGF- and CaPP-loaded fibrin clots after partial patellectomy in patella-patellar tendon sutured sites. A histological evaluation was performed at 2, 4, and 6 weeks after surgery. RESULTS: The sitting time and degradation time of the bFGF- and CaPP-loaded fibrin clots were set at 15 seconds and more than 2 weeks, respectively, and the porosity was minimized to achieve the highest levels of cell migration and growth. In the bFGF-CaPP group of the in vitro experiment, cell proliferation increased to a greater extent relative to the control group (P < .05); the mRNA expression of osteopontin, alkaline phosphatase, runt-related transcription factor 2, vascular endothelial growth factor, and collagen type I was upregulated (P < .05); and the relative protein expression of these factors was enhanced (P < .05). In vivo, hematoxylin and eosin staining showed that the tendon-to-bone connections were more mature and more arranged when treated with bFGF- and CaPP-loaded fibrin clots than when untreated, and the histological scores were higher. CONCLUSION: bFGF- and CaPP-loaded fibrin clots enhanced cell migration and proliferation and the expression of related genes and proteins, which increased the bioactivity of the tendon-bone interface and resulted in the histological improvement of tendon-to-bone healing. CLINICAL RELEVANCE: As fibrin clots have already been used in clinical practice, bFGF- and CaPP-loaded fibrin clots can be further used to augment healing in the early stages of tendon-to-bone healing.

Peritendinous elastase treatment induces tendon degeneration in rats: A potential model of tendinopathy in vivo.

The purpose of this study was to investigate the role of elastase on tendinopathy, as well as to evaluate the potential for peritendinous injections of elastase into rats to cause tendinopathy. We first investigated the expression of elastase in the tendons of patients with tendinopathy, and then established the effects of elastase injection on the Achilles tendons of rats. Ultrasonographic and incapacitance testing was used to conduct tests for 8 weeks. Tendon tissues were collected for histological observation and protein levels of collagen type I and type III were detected using Western blotting. The percentage of elastase-positive cells increased in human specimens with grades II and III tendinopathy. The rat model demonstrated that the thickness of the tendon increased after elastase injection during Week 2-8. Hypercellularity and focal lesions were detected after Week 2. The expression of elastase was increased and elastin was decreased in Week 8. Collagen type I expression was decreased, but type III was increased in Week 4. These results suggested that elastase may be involved in the development of chronic tendinopathy, and that peritendinous injection of elastase may result in tendinopathy in rats.

Ageing does not result in a decline in cell synthetic activity in an injury prone tendon.

Advancing age is a well-known risk factor for tendon disease. Energy-storing tendons [e.g., human Achilles, equine superficial digital flexor tendon (SDFT)] are particularly vulnerable and it is thought that injury occurs following an accumulation of micro-damage in the extracellular matrix (ECM). Several authors suggest that age-related micro-damage accumulates due to a failure of the aging cell population to maintain the ECM or an imbalance between anabolic and catabolic pathways. We hypothesized that ageing results in a decreased ability of tendon cells to synthesize matrix components and matrix-degrading enzymes, resulting in a reduced turnover of the ECM and a decreased ability to repair micro-damage. The SDFT was collected from horses aged 3-30 years with no signs of tendon injury. Cell synthetic and degradative ability was assessed at the mRNA and protein levels. Telomere length was measured as an additional marker of cell ageing. There was no decrease in cellularity or relative telomere length with increasing age, and no decline in mRNA or protein levels for matrix proteins or degradative enzymes. The results suggest that the mechanism for age-related tendon deterioration is not due to reduced cellularity or a loss of synthetic functionality and that alternative mechanisms should be considered.

Lysyl Oxidase Activity Is Required for Ordered Collagen Fibrillogenesis by Tendon Cells.

Lysyl oxidases (LOXs) are a family of copper-dependent oxido-deaminases that can modify the side chain of lysyl residues in collagen and elastin, thereby leading to the spontaneous formation of non-reducible aldehyde-derived interpolypeptide chain cross-links. The consequences of LOX inhibition in producing lathyrism are well documented, but the consequences on collagen fibril formation are less clear. Here we used ß-aminoproprionitrile (BAPN) to inhibit LOX in tendon-like constructs (prepared from human tenocytes), which are an experimental model of cell-mediated collagen fibril formation. The improvement in structure and strength seen with time in control constructs was absent in constructs treated with BAPN. As expected, BAPN inhibited the formation of aldimine-derived cross-links in collagen, and the constructs were mechanically weak. However, an unexpected finding was that BAPN treatment led to structurally abnormal collagen fibrils with irregular profiles and widely dispersed diameters. Of special interest, the abnormal fibril profiles resembled those seen in some Ehlers-Danlos Syndrome phenotypes. Importantly, the total collagen content developed normally, and there was no difference in COL1A1 gene expression. Collagen type V, decorin, fibromodulin, and tenascin-X proteins were unaffected by the cross-link inhibition, suggesting that LOX regulates fibrillogenesis independently of these molecules. Collectively, the data show the importance of LOX for the mechanical development of early collagenous tissues and that LOX is essential for correct collagen fibril shape formation.

H3K9MTase G9a is essential for the differentiation and growth of tenocytes in vitro.

Cell differentiation is controlled by specific transcription factors. The functions and expression levels of these transcription factors are regulated by epigenetic modifications, such as histone modifications and cytosine methylation of the genome. In tendon tissue, tendon-specific transcription factors have been shown to play functional roles in the regulation of tenocyte differentiation. However, the effects of epigenetic modifications on gene expression and differentiation in tenocytes are unclear. In this study, we investigated the epigenetic regulation of tenocyte differentiation, focusing on the enzymes mediating histone 3 lysine 9 (H3K9) methylation. In primary mouse tenocytes, six H3K9 methyltransferase (H3K9MTase) genes, i.e., G9a, G9a-like protein (GLP), PR domain zinc finger protein 2 (PRDM2), SUV39H1, SUV39H2, and SETDB1/ESET were all expressed, with increased mRNA levels observed during tenocyte differentiation. In mouse embryos, G9a and Prdm2 mRNAs were expressed in tenocyte precursor cells, which were overlapped with or were adjacent to cells expressing a tenocyte-specific marker, tenomodulin. Using tenocytes isolated from G9a-flox/flox mice, we deleted G9a by infecting the cells with Cre-expressing adenoviruses. Proliferation of G9a-null tenocytes was significantly decreased compared with that of control cells infected with GFP-expressing adenoviruses. Moreover, the expression levels of tendon transcription factors gene, i.e., Scleraxis (Scx), Mohawk (Mkx), Egr1, Six1, and Six2 were all suppressed in G9a-null tenocytes. The tendon-related genes Col1a1, tenomodulin, and periostin were also downregulated. Consistent with this, Western blot analysis showed that tenomodulin protein expression was significantly suppressed by G9a deletion. These results suggested that expression of the H3K9MTase G9a was essential for the differentiation and growth of tenocytes and that H3K9MTases may play important roles in tendinogenesis.

Tendon overload results in alterations in cell shape and increased markers of inflammation and matrix degradation.

Tendon injury is thought to involve both damage accumulation within the matrix and an accompanying cell response. While several studies have characterized cell and matrix response in chronically injured tendons, few have assessed the initial response of tendon to overload-induced damage. In this study, we assessed cell response to cyclic loading. Fascicle bundles from the equine superficial digital flexor tendon were exposed to cyclic loading in vitro, designed to mimic a bout of high-intensity exercise. Changes in cell morphology and protein-level alterations in markers of matrix inflammation and degradation were investigated. Loading resulted in matrix damage, which was accompanied by cells becoming rounder. The inflammatory markers cyclooxygenase-2 and interleukin-6 were increased in loaded samples, as were matrix metalloproteinase-13 and the collagen degradation marker C1,2C. These results indicate upregulation of inflammatory and degradative pathways in response to overload-induced in vitro, which may be initiated by alterations in cell strain environment because of localized matrix damage. This provides important information regarding the initiation of tendinopathy, suggesting that inflammation may play an important role in the initial cell response to tendon damage. Full understanding of the early tenocyte response to matrix damage is critical in order to develop effective treatments for tendinopathy.

Role of metalloproteinases in tendon pathophysiology.

Tendons play a crucial role in musculoskeletal functioning because they physically connect bones and muscles making the movement of articular joints possible. The molecular composition of tendons mostly include collagen I fibrils, which aggregate together to form fibers to form a fascicle. A complex network composed of resident cells (i.e., tenocytes) and extracellular matrix macromolecules (glycosaminoglycans, proteoglycans, glycoproteins and other non collagenous proteins) interact and define the structure of tendons and their properties. Development, renewal and remodeling of tendons composition occur at all ages of living organisms so the homeostasis of proteolytic systems is a critical issue. A major role is played by Metalloproteinases, a family of Zn(2+)-dependent endopeptidases involved in the catabolism of several components of the extracellular matrix, such as collagens, proteoglycans, fibronectin and many others. Among these, two main classes are mostly involved in tendon pathophysiology, namely the Matrix Metalloproteinases (MMPs) and a Disintegrin-like and Metalloproteinase domain with Thrombospondin motifs (ADAMTSs). This study analyses the various aspects of the roles played by Metalloproteinases in the physiological and pathological processes of tendons.

Cyclical strain modulates metalloprotease and matrix gene expression in human tenocytes via activation of TGFß.

Tendinopathies are a range of diseases characterised by degeneration and chronic tendon pain and represent a significant cause of morbidity. Relatively little is known about the underlying mechanisms; however onset is often associated with physical activity. A number of molecular changes have been documented in tendinopathy such as a decrease in overall collagen content, increased extracellular matrix turnover and protease activity. Metalloproteinases are involved in the homeostasis of the extracellular matrix and expression is regulated by mechanical strain. The aims of this study were to determine the effects of strain upon matrix turnover by measuring metalloproteinase and matrix gene expression and to elucidate the mechanism of action. Primary Human Achilles tenocytes were seeded in type I rat tail collagen gels in a Flexcell™ tissue train system and subjected to 5% cyclic uniaxial strain at 1Hz for 48h. TGFß1 and TGFßRI inhibitor were added to selected cultures. RNA was measured using qRT-PCR and TGFß protein levels were determined using a cell based luciferase assay. We observed that mechanical strain regulated the mRNA levels of multiple protease and matrix genes anabolically, and this regulation mirrored that seen with TGFß stimulation alone. We have also demonstrated that the inhibition of the TGFß signalling pathway abrogated the strain induced changes in mRNA and that TGFß activation, rather than gene expression, was increased with mechanical strain. We concluded that TGFß activation plays an important role in mechanotransduction. Targeting this pathway may have its place in the treatment of tendinopathy.

MMP inhibition as a potential method to augment the healing of skeletal muscle and tendon extracellular matrix.

The extracellular matrix (ECM) of skeletal muscle and tendon is composed of different types of collagen molecules that play important roles in the transmission of forces throughout the body, and in the repair and regeneration of injured tissues. Fibroblasts are the primary cells in muscle and tendon that maintain, repair, and modify the ECM in response to mechanical loading, injury, and inactivity. Matrix metalloproteinases (MMPs) are enzymes that digest collagen and other structural molecules, which are synthesized and excreted by fibroblasts. MMPs are required for baseline ECM homeostasis, but disruption of MMP regulation due to injury or disease can alter the normal ECM architecture and prevent proper force transmission. Chronic injuries and diseases of muscles and tendons can be severely debilitating, and current therapeutic modalities to enhance healing are quite limited. This review will discuss the mechanobiology of MMPs, and the potential use of MMP inhibitors to improve the treatment of injured and diseased skeletal muscle and tendon tissue.

Early events of overused supraspinatus tendons involve matrix metalloproteinases and EMMPRIN/CD147 in the absence of inflammation.

BACKGROUND: The principal feature of tendon degeneration is structural change of the extracellular matrix (ECM) including collagens. In painful tendons, alterations of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been described; however, the initial molecular mechanism at the origin of these alterations is still poorly understood. A rat model of supraspinatus tendon overuse has been developed, which may be predictive of pathological tendon alterations. PURPOSE: To determine which MMPs are involved in early ECM remodeling during overuse and their relationship with the inflammatory context. STUDY DESIGN: Controlled laboratory study. METHODS: Analyses were performed on rat supraspinatus tendons at 2 and 4 weeks of overuse on a downhill treadmill. Transcript levels of MMPs and TIMPs were assessed by semiquantitative reverse transcription polymerase chain reaction. Western blotting and/or immunolabeling were used for MMP-2, MMP-3, MMP-13, and extracellular MMP inducer (EMMPRIN, also called cluster of differentiation [CD] 147) detection. In situ and/or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gelatin zymography was performed for MMP-2 and MMP-9. TIMP activity was revealed by reverse zymography. Inflammation was assessed by cytokine antibody array and/or immunolabeling. RESULTS: Compared with a control, overused supraspinatus tendons showed a significantly higher gelatinolytic activity at 2 weeks, which slightly decreased at 4 weeks. MMP-9 and MMP-13 were undetectable; MMP-3 was downregulated in overused tendons. Only MMP-2, particularly its active form, and the MMP-2 activator MMP-14 were upregulated at 2 weeks of overuse when an increase in TIMP-2 transcripts was observed. MMP-2 upregulation occurred in the absence of inflammation but was associated with an increase of EMMPRIN/CD147. CONCLUSION: EMMPRIN/CD147-regulated MMP-2 and MMP-14, associated with low MMP-3, appear as the main characteristics of ECM remodeling in early overused tendons. Whether alterations in the pattern of these MMPs are an adaptive response or a repair response that may degenerate into tendinosis, is still uncertain. Moreover, there seems to be no indication for an inflammatory response to overuse, suggesting that the increased metalloproteinase activity is rather a response to a mechanical stress than an inflammatory one. CLINICAL RELEVANCE: Any strategy aimed at preventing full-thickness tears resulting from initial tendon matrix alterations should consider these changes in MMP-3, MMP-2, and MMP-14, or further upstream, EMMPRIN.

Collagen type I and decorin expression in tenocytes depend on the cell isolation method.

BACKGROUND: The treatment of rotator cuff tears is still challenging. Tendon tissue engineering (TTE) might be an alternative in future. Tenocytes seem to be the most suitable cell type as they are easy to obtain and no differentiation in vitro is necessary. The aim of this study was to examine, if the long head of the biceps tendon (LHB) can deliver viable tenocytes for TTE. In this context, different isolation methods, such as enzymatic digestion (ED) and cell migration (CM), are investigated on differences in gene expression and cell morphology. METHODS: Samples of the LHB were obtained from patients, who underwent surgery for primary shoulder arthroplasty. Using ED as isolation method, 0.2% collagenase I solution was used. Using CM as isolation method, small pieces of minced tendon were put into petri-dishes. After cell cultivation, RT-PCR was performed for collagen type I, collagen type III, decorin, tenascin-C, fibronectin, Scleraxis, tenomodulin, osteopontin and agreccan. RESULTS: The total number of isolated cells, in relation to 1 g of native tissue, was 14 times higher using ED. The time interval for cell isolation was about 17 hours using ED and approximately 50 days using CM. Cell morphology in vitro was similar for both isolation techniques. Higher expression of collagen type I could be observed in tenocyte-like cell cultures (TLCC) using ED as isolation method (p < 0.05), however decorin expression was higher in TLCC using CM as isolation method (p < 0.05). Dedifferentiation potential seemed to be similar for both isolation techniques. CONCLUSION: In summary tenocyte-like cells can be obtained with both isolation methods (ED and CM) from the LHB. As no obvious disadvantage could be seen using ED, this method is more suitable for clinical use, as time for cell isolation is shorter and a remarkably higher number of cells can be obtained. However, both isolation methods can further be improved.

RhoA/ROCK, cytoskeletal dynamics, and focal adhesion kinase are required for mechanical stretch-induced tenogenic differentiation of human mesenchymal stem cells.

Human bone marrow mesenchymal stem cells (hMSCs) have the potential to differentiate into tendon/ligament-like lineages when they are subjected to mechanical stretching. However, the means through which mechanical stretch regulates the tenogenic differentiation of hMSCs remains unclear. This study examined the role of RhoA/ROCK, cytoskeletal organization, and focal adhesion kinase (FAK) in mechanical stretch-induced tenogenic differentiation characterized by the up-regulation of tendon-related marker gene expression. Our findings showed that RhoA/ROCK and FAK regulated mechanical stretch-induced realignment of hMSCs by regulating cytoskeletal organization and that RhoA/ROCK and cytoskeletal organization were essential to mechanical stretch-activated FAK phosphorylation at Tyr397. We also demonstrated that this process can be blocked by Y-27632 (a specific inhibitor of RhoA/ROCK), cytochalasin D (an inhibitor of cytoskeletal organization) or PF 573228 (a specific inhibitor of FAK). The results of this study suggest that RhoA/ROCK, cytoskeletal organization, and FAK compose a "signaling network" that senses mechanical stretching and drives mechanical stretch-induced tenogenic differentiation of hMSCs. This work provides novel insights regarding the mechanisms of tenogenesis in a stretch-induced environment and supports the therapeutic potential of hMSCs.

Tendinopatías / Tendinopathies

Objetivo: Realizar una revisión bibliográfica, sobre la etiología, etiopatogenia y tratamiento de las tendinopatías, así como revisar la terminología utilizada. Material y método: Se consultaron diversas fuentes electrónicas y en papel. Se utilizó Pub-med como motor de búsqueda. Resultados: Estructuralmente, la lesión se caracteriza por una alteración de los tenocitos, una desorganización del colágeno, un aumento de la sustancia fundamental y un aumento de los vasos sanguíneos. Junto a todo ello, aparece una alteración de las metaloproteasas y sus inhibidores. Estas sustancias forman parte de la homeostasis normal del tendón, pero determinados factores de riesgo pueden alterar la regulación normal de estas sustancias y podrían contribuir a iniciar y mantener el proceso de forma indefinida. También se ha descrito que el proceso de apoptosis o muerte celular programada iniciada por un citocromo y una enzima podrían estar en su origen patogénico. Conclusiones: Persiste un desconocimiento del proceso que origina y mantiene la lesión. Debido a ello, en la actualidad se han propuesto diversas opciones terapéuticas con más o menos éxito, pero ninguna con una eficacia totalmente satisfactoria. Desde el punto de vista terminológico, el término usado de tendinitis no es adecuado (AU)

Objetive: To extensively review all publicated data regarding etiology, pathogenesis and treatment of this disease and, review the terminology that was used in these processes. Material y Methods: We consulted some electronic and paper knowledge sources and Pubmed was used as a search engine. Results: Morphologically, the basic lesion is a cellular alteration of tenocytes, collagen disorganization, and an increase in matrix content and blood vessels. Moreover, metalloproteases and its inhibitors are disturbed. A programmed apoptosis of cells initiated by a cytocrom has been suggested as the origin of the disease. Conclusions: Due to ignorance of it origin, no real effective treatment has been yet achieved. Some therapeutic interventions have been proposed, with variable degree of success. From the standpoint of terminology, the term most used of tendinitis is not suitable (AU)