Moderate Exercise Mitigates the Detrimental Effects of Aging on Tendon Stem Cells

Aging is known to cause tendon degeneration whereas moderate exercise imparts beneficial effects on tendons. Since stem cells play a vital role in maintaining tissue integrity, in this study we aimed to define the effects of aging and moderate exercise on tendon stem/progenitor cells (TSCs) using in vitro and in vivo models. TSCs derived from aging mice (9 and 24 months) proliferated significantly slower than TSCs obtained from young mice (2.5 and 5 months). In addition, expression of the stem cell markers Oct-4, nucleostemin (NS), Sca-1 and SSEA-1 in TSCs decreased in an age-dependent manner. Interestingly, moderate mechanical stretching (4%) of aging TSCs in vitro significantly increased the expression of the stem cell marker, NS, but 8% stretching decreased NS expression. Similarly, 4% mechanical stretching increased the expression of Nanog, another stem cell marker, and the tenocyte-related genes, collagen I and tenomodulin. However, 8% stretching increased expression of the non-tenocyte-related genes, LPL, Sox-9 and Runx-2, while 4% stretching had minimal effects on the expression of these genes. In the in vivo study, moderate treadmill running (MTR) of aging mice (9 months) resulted in the increased proliferation rate of aging TSCs in culture, decreased lipid deposition, proteoglycan accumulation and calcification, and increased the expression of NS in the patellar tendons. These findings indicate that while aging impairs the proliferative ability of TSCs and reduces their stemness, moderate exercise can mitigate the deleterious effects of aging on TSCs and therefore may be responsible for decreased aging-induced tendon degeneration.     

Extracorporeal Shock Wave Treatment (ESWT) Improves In Vitro Functional Activities of Ruptured Human Tendon-Derived Tenocytes

In vitro models of human tenocytes derived from healthy as well as from ruptured tendons were established, characterized and used at very early passage (P1) to evaluate the effects of Extracorporeal Shock Wave Treatment (ESWT). The molecular analysis of traditional tenocytic markers, including Scleraxis (Scx), Tenomodulin (Tnm), Tenascin-C (Tn-C) and Type I and III Collagens (Col I and Col III), permitted us to detect in our samples the simultaneous expression of all these genes and allowed us to compare their levels of expression in relationship to the source of the cells and treatments. In untreated conditions, higher molecular levels of Scx and Col I in tenocytes from pathological compared to healthy samples have been detected, suggesting – in the cells from injured tendon – the natural trigger of an early differentiation and repairing program, which depends by Scx and requires an increase in collagen expression. When ESWT (at the dose of 0.14 mJ/mm²) was applied to cultured tenocytes explanted from injured source, Scx and Col I were significantly diminished compared to healthy counterpart, indicating that such natural trigger maybe delayed by the treatment, in order to promote cellular repair. Herein, we show for the first time that ESWT enhances in vitro functional activities of ruptured tendon-derived tenocytes, such as proliferation and migration, which could probably contributes to tendon healing in vivo.     

Substance P is a mechanoresponsive, autocrine regulator of human tenocyte proliferation

It has been hypothesised that substance P (SP) may be produced by primary fibroblastic tendon cells (tenocytes), and that this production, together with the widespread distribution of the neurokinin-1 receptor (NK-1 R) in tendon tissue, could play an important role in the development of tendinopathy, a condition of chronic tendon pain and thickening. The aim of this study was to examine the possibility of endogenous SP production and the expression of NK-1 R by human tenocytes. Because tendinopathy is related to overload, and because the predominant tissue pathology (tendinosis) underlying early tendinopathy is characterized by tenocyte hypercellularity, the production of SP in response to loading/strain and the effects of exogenously administered SP on tenocyte proliferation were also studied. A cell culture model of primary human tendon cells was used. The vast majority of tendon cells were immunopositive for the tenocyte/fibroblast markers tenomodulin and vimentin, and immunocytochemical counterstaining revealed that positive immunoreactions for SP and NK-1 R were seen in a majority of these cells. Gene expression analyses showed that mechanical loading (strain) of tendon cell cultures using the FlexCell© technique significantly increased the mRNA levels of SP, whereas the expression of NK-1 R mRNA decreased in loaded as compared to unloaded tendon cells. Reduced NK-1 R protein was also observed, using Western blot, after exogenously administered SP at a concentration of 10⁻⁷ M. SP exposure furthermore resulted in increased cell metabolism, increased cell viability, and increased cell proliferation, all of which were found to be specifically mediated via the NK-1 R; this in turn involving a common mitogenic cell signalling pathway, namely phosphorylation of ERK1/2. This study indicates that SP, produced by tenocytes in response to mechanical loading, may regulate proliferation through an autocrine loop involving the NK-1 R.     

Efficient expansion of mouse primary tenocytes using a novel collagen gel culture method

Development of regenerative therapies for damaged tendons remains a great challenge, largely because of lack of information regarding the mechanisms responsible for differentiation of tenocytes. Mouse tenocytes have not been fully characterized owing to the absence of efficient and reproducible methods for their in vitro expansion without losing phenotypic features. The objective of the study was to establish an improved and reliable method for stable primary culture of mouse tenocytes by using collagen gel. Achilles and tail tendon tissues were harvested and embedded in collagen gel. After 10 days of continuous culture, the gel was digested and cells were passaged on tissue culture-treated plastic dishes. Mouse tenocytes cultured in collagen gel exhibited significantly shorter doubling time and higher numbers of proliferation when maintained on the plastic dishes compared with those cultured without using gel. Transmission electron microscopic analyses showed that cultured tenocytes retained some morphological features of tenocytes in tendon tissues, such as cell–cell junctional complex formation, well-developed rough endoplasmic reticulum, and mitochondria in their cytoplasm. mRNA expression of tenocyte markers (tenomodulin, type I collagen, periostin, and scleraxis) was higher in cells cultured in collagen gel than in those cultured in the absence of gel. Our results show that tenocytes cultured using the collagen gel method express typical lineage markers and exhibit improved growth characteristics, thus providing a stable platform for studying molecular mechanisms that control their differentiation.     

Effect of Young Extrinsic Environment Stimulated by Hypoxia on the Function of Aged Tendon Stem Cell

Tendon stem cells (TSCs), recently identified as tendon cells, play an important role in maintaining the homeostasis of tendon tissue. Age-related decrease in the function of TSCs has been reported. Recent reports demonstrated that hypoxic condition is advantageous for efficient expansion of TSCs. Moreover, the impaired function of aged stem cells could be modulated by exposing them to a young environment. Therefore, we investigated the effects of hypoxic-conditioned culture medium (HCCM) from young TSCs on the proliferation, migration, senescence, and tenocyte phenotype of aged TSCs. TSCs were isolated, and the conditioned medium was collected. There were 4 groups: young TSCs, aged TSCs, aged TSCs + aged HCCM, and aged TSCs + young HCCM. The proliferative capacity, migration, β-galactosidase activity, and tenogenic differentiation potential of TSCs were assessed. Our results showed that HCCM enhanced the proliferation and migration potential of aged TSCs. Moreover, the senescence-associated β-galactosidase activity of aged TSCs was decreased by young HCCM. After being cultured in the young HCCM, the expressions of tenocyte-related genes in aged TSCs were significantly enhanced. Together, results of this study indicate that HCCM from young TSCs may represent an effective strategy to improve the impaired function of aged TSCs.     

Gap junction permeability between tenocytes within tendon fascicles is suppressed by tensile loading

Gap junction communication is an essential component in the mechanosensitive response of tenocytes. However, little is known about direct mechanoregulation of gap junction turnover and permeability. The present study tests the hypothesis that mechanical loading alters gap junction communication between tenocyte within tendon fascicles. Viable tenocytes within rat tail tendon fasicles were labelled with calcein-AM and subjected to a fluorescent loss induced by photobleaching (FLIP) protocol. A designated target cell within a row of tenocytes was continuously photobleached at 100% laser power whilst recording the fluorescent intensity of neighbouring cells. A mathematical compartment model was developed to estimate the intercellular communication between tenocytes based upon the experimental FLIP data. This produced a permeability parameter, k, which quantifies the degree of functioning gap functions between cells as confirmed by the complete inhibition of FLIP by the inhibitor 18α-glycyrrhentic acid. The application of 1N static tensile load for 10?min had no effect on gap junction communication. However, when loading was increased to 1?h, there was a statistically significant reduction in gap junction permeability. This coincided with suppression of connexin 43 protein expression in loaded samples as determined by confocal immunofluorescence. However, there was an upregulation of connexin 43 mRNA. These findings demonstrate that tenocytes remodel their gap junctions in response to alterations in mechanical loading with a complex mechanosensitive mechanism of breakdown and remodelling. This is therefore the first study to show that tenocyte gap junctions are not only important in transmitting mechanically activated signals but that mechanical loading directly regulates gap junction permeability.     

Creating an Animal Model of Tendinopathy by Inducing Chondrogenic Differentiation with Kartogenin

Previous animal studies have shown that long term rat treadmill running induces over-use tendinopathy, which manifests as proteoglycan accumulation and chondrocytes-like cells within the affected tendons. Creating this animal model of tendinopathy by long term treadmill running is however time-consuming, costly and may vary among animals. In this study, we used a new approach to develop an animal model of tendinopathy using kartogenin (KGN), a bio-compound that can stimulate endogenous stem/progenitor cells to differentiate into chondrocytes. KGN-beads were fabricated and implanted into rat Achilles tendons. Five weeks after implantation, chondrocytes and proteoglycan accumulation were found at the KGN implanted site. Vascularity as well as disorganization in collagen fibers were also present in the same site along with increased expression of the chondrocyte specific marker, collagen type II (Col. II). In vitro studies confirmed that KGN was released continuously from KGN-alginate in vivo beads and induced chondrogenic differentiation of tendon stem/progenitor cells (TSCs) suggesting that chondrogenesis after KGN-bead implantation into the rat tendons is likely due to the aberrant differentiation of TSCs into chondrocytes. Taken together, our results showed that KGN-alginate beads can be used to create a rat model of tendinopathy, which, at least in part, reproduces the features of over-use tendinopathy model created by long term treadmill running. This model is mechanistic (stem cell differentiation), highly reproducible and precise in creating localized tendinopathic lesions. It is expected that this model will be useful to evaluate the effects of various topical treatments such as NSAIDs and platelet-rich plasma (PRP) for the treatment of tendinopathy.     

Characterization of type I,III and V collagens in high-density cultured tenocytes by triple-immunofluorescence technique

The purpose of this study is to examine the intracellular distribution of collagen types I, III and V in tenocytes using triple-label immunofluorescence staining technique in high-density tenocyte culture on Filter Well Inserts (FWI). The tenocytes were incubated for 4 weeks under monolayer conditions and for 3 weeks on FWI. At the end of the third week of high-density culture, we observed tenocyte aggregation followed by macromass cluster formation. Immunofluorescence labeling with anti-collagen type I antibody revealed that the presence of collagen type I was mostly around the nucleus. Type III collagen was more diffused in the cytoplasm. Type V collagen was detected in fibrillar and vesicular forms in the cytoplasm. We conclude that, the high-density culture on FWI is an appropriate method for the production of tenocytes without loosing specialized processes such as the synthesis of different collagen molecules. We consider that the high-density culture system is suitable for in vitro applications which affect tendon biology and will improve our understanding of the biological behavior of tenocytes in view of adequate matrix structure synthesis. Such high-density cultures may serve as a model system to provide sufficient quantities of tenocytes to prepare tenocyte-polymer constructs for tissue engineering applications in tendon repair.     

Exosomes from tendon stem cells promote injury tendon healing through balancing synthesis and degradation of the tendon extracellular matrix

Tendon injuries are common musculoskeletal system disorders in clinical, but the regeneration ability of tendon is limited. Tendon stem cells (TSCs) have shown promising effect on tissue engineering and been used for the treatment of tendon injury. Exosomes that serve as genetic information carriers have been implicated in many diseases and physiological processes, but effect of exosomes from TSCs on tendon injury repair is unclear. The aim of this study is to make clear that the effect of exosomes from TSCs on tendon injury healing. Exosomes were harvested from conditioned culture media of TSCs by a sequential centrifugation process. Rat Achilles tendon tendinopathy model was established by collagenase‐I injection. This was followed by intra‐Achilles‐tendon injection with TSCs or exosomes. Tendon healing and matrix degradation were evaluated by histology analysis and biomechanical test at the post‐injury 5 weeks. In vitro, TSCs treated with interleukin 1 beta were added by conditioned medium including exosomes or not, or by exosomes or not. Tendon matrix related markers and tenogenesis related markers were measured by immunostaining and western blot. We found that TSCs injection and exosomes injection significantly decreased matrix metalloproteinases (MMP)‐3 expression, increased expression of tissue inhibitor of metalloproteinase‐3 (TIMP‐3) and Col‐1a1, and increased biomechanical properties of the ultimate stress and maximum loading. In vitro, conditioned medium with exosomes and exosomes also significantly decreased MMP‐3, and increased expression of tenomodulin, Col‐1a1 and TIMP‐3. Exosomes from TSCs could be an ideal therapeutic strategy in tendon injury healing for its balancing tendon extracellular matrix and promoting the tenogenesis of TSCs.     

Mechanical force modulates scleraxis expression in bioartificial tendons

Following tendon injury, cartilage, bone and fat metaplasia are often observed, making the optimization of tenocyte differentiation an important clinical goal. In this study we examined the effect of static and cyclic mechanical loading on the expression of genes which play a role in tenocyte differentiation and function, namely scleraxis (Scx) and Type I collagen (Col1a1), and determined the effect of varying mechanical parameters including (1) static vs dynamic load, (2) increasing strain magnitude, (3) inclusion of 10 s rest periods, and (4) increasing cycle number. Cyclic loading resulted in a greater increase of tenocyte gene expression than static loading over 3 weeks in culture. Increasing strain levels potentiated the induction of tenocyte genes. The insertion of a 10 s rest periods further enhanced tenocyte gene expression, as did increasing repetition numbers. These results suggest that mechanical signaling exerts an important influence on the expression of genes which play a role in determining the tendon phenotype. Further work is required to confirm and extend these findings in primary cells such as resident tendon progenitor/stem cells, in order to provide an improved understanding of biology from which optimized rehabilitation programs can be developed.     

Differential expression and cellular localization of novel isoforms of the tendon biomarker tenomodulin

Tenomodulin (Tnmd, also called Tendin) is classified as a type II transmembrane glycoprotein and is highly expressed in developing as well as in mature tendons. Along with scleraxis (scx), Tnmd is a candidate marker gene for tenocytes. Its function is unknown, but it has been reported to have anti-angiogenic properties. Results in a knockout mouse model did not substantiate that claim. It has homology to chondromodulin-I. Single nucleotide polymorphisms of TNMD have been associated with obesity, macular degeneration, and Alzheimer's disease in patients. In the present study, three Tnmd isoforms with deduced molecular weights of 20.3 (isoform II), 25.4 (isoform III), and 37.1 (isoform I) kDa were proposed and verified by Western blot from cells with green fluorescent protein-linked, overexpressed constructs, tissue, and by qPCR of isoforms from human tissues and cultured cells. Overexpression of each Tnmd isoform followed by immunofluorescence imaging showed that isoforms I and II had perinuclear localization while isoform III was cytoplasmic. Results of qPCR demonstrated differential expression of each Tnmd isoform in patient's specimens taken from flexor carpi radialis, biceps brachii, and flexor digitorum profundus tendons. Knockdown of Tnmd increased the expression of both scleraxis (scx) and myostatin, indicating a potential negative feedback loop between Tnmd and its regulators. Knockdown of all Tnmd isoforms simultaneously also reduced tenocyte proliferation. I-TASSER protein three-dimensional conformation modeling predictions indicated each Tnmd isoform had different structures and potential functions: isoform 1, modeled as a cytosine methyltransferase; isoform 2, a SUMO-1-like SENP-1 protease; and isoform 3, an α-syntrophin, plextrin homology domain scaffolding protein. Further functional studies with each Tnmd isoform may help us to better understand regulation of tenocyte proliferation, tendon development, response to injury and strain, as well as mechanisms in tendinoses. These results may indicate novel therapeutic targets in specific tenomodulin isoforms as well as treatments for tendon diseases.     

Shear Elastic Modulus on Patellar Tendon Captured from Supersonic Shear Imaging: Correlation with Tangent Traction Modulus Computed from Material Testing System and Test–Retest Reliability

Characterization of the elastic properties of a tendon could enhance the diagnosis and treatment of tendon injuries. The purpose of this study was to examine the correlation between the shear elastic modulus on the patellar tendon captured from a Supersonic Shear Imaging (SSI) and the tangent traction modulus computed from a Material testing system (MTS) on 8 fresh patellar pig tendons (Experiment I). Test–retest reliability of the shear elastic modulus captured from the SSI was established in Experiment II on 22 patellar tendons of 11 healthy human subjects using the SSI. Spearman Correlation coefficients for the shear elastic modulus and tangent traction modulus ranged from 0.82 to 1.00 (all p<0.05) on the 8 tendons. The intra and inter-operator reliabilities were 0.98 (95% CI: 0.93–0.99) and 0.97 (95% CI: 0.93–0.98) respectively. The results from this study demonstrate that the shear elastic modulus of the patellar tendon measured by the SSI is related to the tangent traction modulus quantified by the MTS. The SSI shows good intra and inter-operator repeatability. Therefore, the present study shows that SSI can be used to assess elastic properties of a tendon.     

Effect of indomethacin and lactoferrin on human tenocyte proliferation and collagen formation in vitro

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in patients with injuries and inflammation of tendon and ligament, and as post-surgical analgesics. The aim of this study is to investigate the effect of indomethacin, a classic NSAID and its combinational effect with an anabolic agent of skeletal tissue, lactoferrin, on the proliferation and collagen formation of human tenocytes in vitro. A factorial experimental design was employed to study the dose-dependent effect of the combination of indomethacin and lactoferrin. The results showed that indomethacin at high concentration (100 μM) inhibited human tenocyte proliferation in culture medium with 1–10% fetal bovine serum (FBS) in vitro. Also, high dose of indomethacin inhibited the collagen formation of human tenocytes in 1% FBS culture medium. Lactoferrin at 50–100 μg/ml promoted human tenocyte survival in serum-free culture medium and enhanced proliferation and collagen synthesis of human tenocytes in 1% FBS culture medium. When 50–100 μg/ml lactoferrin was used in combination with 100–200 μM indomethacin, it partially rescued the inhibitory effects of indomethacin on human tenocyte proliferation, viability and collagen formation. To our knowledge, it is the first evidence that lactoferrin is anabolic to human tenocytes in vitro and reverses potential inhibitory effects of NSAIDs on human tenocytes.     

Predicting tenocyte expression profiles and average molecular concentrations in Achilles tendon ECM from tissue strain and fiber damage

In this study, we propose a method for quantitative prediction of changes in concentrations of a number of key signaling, structural and effector molecules within the extracellular matrix of tendon. To achieve this, we introduce the notion of elementary cell responses (ECRs). An ECR defines a normal reference secretion profile of a molecule by a tenocyte in response to the tenocyte’s local strain. ECRs are then coupled with a model for mechanical damage of tendon collagen fibers at different straining conditions of tendon and then scaled up to the tendon tissue level for comparison with experimental observations. Specifically, our model predicts relative changes in ECM concentrations of transforming growth factor beta, interleukin 1 beta, collagen type I, glycosaminoglycan, matrix metalloproteinase 1 and a disintegrin and metalloproteinase with thrombospondin motifs 5, with respect to tendon straining conditions that are consistent with the observations in the literature. In good agreement with a number of in vivo and in vitro observations, the model provides a logical and parsimonious explanation for how excessive mechanical loading of tendon can lead to under-stimulation of tenocytes and a degenerative tissue profile, which may well have bearing on a better understanding of tendon homeostasis and the origin of some tendinopathies.     

Torsion and Antero-Posterior Bending in the In Vivo Human Tibia Loading Regimes during Walking and Running

Bending, in addition to compression, is recognized to be a common loading pattern in long bones in animals. However, due to the technical difficulty of measuring bone deformation in humans, our current understanding of bone loading patterns in humans is very limited. In the present study, we hypothesized that bending and torsion are important loading regimes in the human tibia. In vivo tibia segment deformation in humans was assessed during walking and running utilizing a novel optical approach. Results suggest that the proximal tibia primarily bends to the posterior (bending angle: 0.15°–1.30°) and medial aspect (bending angle: 0.38°–0.90°) and that it twists externally (torsion angle: 0.67°–1.66°) in relation to the distal tibia during the stance phase of overground walking at a speed between 2.5 and 6.1 km/h. Peak posterior bending and peak torsion occurred during the first and second half of stance phase, respectively. The peak-to-peak antero-posterior (AP) bending angles increased linearly with vertical ground reaction force and speed. Similarly, peak-to-peak torsion angles increased with the vertical free moment in four of the five test subjects and with the speed in three of the test subjects. There was no correlation between peak-to-peak medio-lateral (ML) bending angles and ground reaction force or speed. On the treadmill, peak-to-peak AP bending angles increased with walking and running speed, but peak-to-peak torsion angles and peak-to-peak ML bending angles remained constant during walking. Peak-to-peak AP bending angle during treadmill running was speed-dependent and larger than that observed during walking. In contrast, peak-to-peak tibia torsion angle was smaller during treadmill running than during walking. To conclude, bending and torsion of substantial magnitude were observed in the human tibia during walking and running. A systematic distribution of peak amplitude was found during the first and second parts of the stance phase.     

Influence of a single loading episode on gene expression in healing rat Achilles tendons

Mechanical loading stimulates tendon healing via mechanisms that are largely unknown. Genes will be differently regulated in loaded healing tendons, compared with unloaded, just because of the fact that healing processes have been changed. To avoid such secondary effects and study the effect of loading per se, we therefore studied the gene expression response shortly after a single loading episode in otherwise unloaded healing tendons. The Achilles tendon was transected in 30 tail-suspended rats. The animals were let down from the suspension to load their tendons on a treadmill for 30 min once, 5 days after tendon transection. Gene expression was studied by Affymetrix microarray before and 3, 12, 24, and 48 h after loading. The strongest response in gene expression was seen 3 h after loading, when 150 genes were up- or downregulated (fold change ≥2, P ≤ 0.05). Twelve hours after loading, only three genes were upregulated, whereas 38 were downregulated. Fewer than seven genes were regulated after 24 and 48 h. Genes involved in the inflammatory response were strongly regulated at 3 and 12 h after loading; this included upregulation of iNOS, PGE synthase, and IL-1β. Also genes involved in wound healing/coagulation, angiogenesis, and production of reactive oxygen species were strongly regulated by loading. Microarray results were confirmed for 16 selected genes in a repeat experiment (N = 30 rats) using real-time PCR. It was also confirmed that a single loading episode on day 5 increased the strength of the healing tendon on day 12. In conclusion, the fact that there were hardly any regulated genes 24 h after loading suggests that optimal stimulation of healing requires a mechanical loading stimulus every day.     

Uphill running improves rat Achilles tendon tissue mechanical properties and alters gene expression without inducing pathological changes

Overuse Achilles tendinopathy is a common and challenging problem in sports medicine. Little is known about the etiology of this disorder, and the development of a good animal model for overuse tendinopathy is essential for advancing insight into the disease mechanisms. Our aim was to test a previously proposed rat model for Achilles tendon overuse. Ten adult male Sprague-Dawley rats ran on a treadmill with 10° incline, 1 h/day, 5 days/wk (17-20 m/min) for 12 wk and were compared with 12 control rats. Histological, mechanical, and gene-expression changes were measured on the Achilles tendons after the intervention, and local tendon glucose-uptake was measured before and after the intervention with positron emission tomography. No differences were detected between runners and controls in tissue histology or in glucose uptake, indicating that tendon pathology was not induced. Greater tendon tissue modulus (P < 0.005) and failure stress/body weight (P < 0.02) in runners compared with controls further supported that tendons successfully adapted to uphill running. Several genes of interest were regulated after 12 wk of running. Expression of collagen III and insulin-like growth factor I was increased, while collagen I was unchanged, and decreases were seen in noncollagen matrix components (fibromodulin and biglycan), matrix degrading enzymes, transforming growth factor-β1, and connective tissue growth factor. In conclusion, the tested model could not be validated as a model for Achilles tendinopathy, as the rats were able to adapt to 12 wk of uphill running without any signs of tendinopathy. Improved mechanical properties were observed, as well as changes in gene-expression that were distinctly different from what is seen in tendinopathy and in response to short-term tendon loading.     

Maturational alterations in gap junction expression and associated collagen synthesis in response to tendon function

Energy-storing tendons including the equine superficial digital flexor tendon (SDFT) contribute to energetic efficiency of locomotion at high-speed gaits, but consequently operate close to their physiological strain limits. Significant evidence of exercise-induced microdamage has been found in the SDFT which appears not to exhibit functional adaptation; the degenerative changes have not been repaired by the tendon fibroblasts (tenocytes), and are proposed to accumulate and predispose the tendon to rupture during normal athletic activity. The anatomically opposing common digital extensor tendon (CDET) functions only to position the digit, experiencing significantly lower levels of strain and is rarely damaged by exercise. A number of studies have indicated that tenocytes in the adult SDFT are less active in collagen synthesis and turnover than those in the immature SDFT or the CDET. Gap junction intercellular communication (GJIC) is known to be necessary for strain-induced collagen synthesis by tenocytes. We postulate therefore that expression of GJ proteins connexin 43 and 32 (Cx43; Cx32), GJIC and associated collagen expression levels are high in the SDFT and CDET of immature horses, when the SDFT in particular grows significantly in cross-sectional area, but reduce significantly during maturation in the energy-storing tendon only. The hypothesis was tested using tissue from the SDFT and CDET of foetuses, foals, and young adult Thoroughbred horses. Cellularity and the total area of both Cx43 and Cx32 plaques/mm² of tissue reduced significantly with maturation in each tendon. However, the total Cx43 plaque area per tenocyte significantly increased in the adult CDET. Evidence of recent collagen synthesis in the form of levels of neutral salt-soluble collagen, and collagen type I mRNA was significantly less in the adult compared with the immature SDFT; procollagen type I amino-propeptide (PINP) and procollagen type III amino-propeptide (PIIINP) levels per mm² of tissue and PINP expression per tenocyte also decreased with maturation in the SDFT. In the CDET PINP and PIIINP expression per tenocyte increased in the adult, and exceeded those in the adult SDFT. The level of PINP per mm² was greater in the adult CDET than in the SDFT despite the higher cellularity of the latter tendon. In the adult SDFT, levels of PIIINP were greater than those of PINP, suggesting relatively greater synthesis of a weaker form of collagen previously associated with microdamage. Tenocytes in monolayers showed differences in Cx43 and Cx32 expression compared with those in tissue, however there were age- and tendon-specific phenotypic differences, with a longer time for 50% recovery of fluorescence after photobleaching in adult SDFT cells compared with those from the CDET and immature SDFT. As cellularity reduces following growth in the SDFT, a failure of the remaining tenocytes to show a compensatory increase in GJ expression and collagen synthesis may explain why cell populations are not able to respond to exercise and to repair microdamage in some adult athletes. Enhancing GJIC in mature energy-storing tendons could provide a strategy to increase the cellular synthetic and reparative capacity.     

A synthetic mechano-growth factor E peptide promotes rat tenocyte migration by lessening cell stiffness and increasing F-actin formation via the FAK-ERK1/2 signaling pathway

Tendon injuries are common in sports and are frequent reasons for orthopedic consultations. The management of damaged tendons is one of the most challenging problems in orthopedics. Mechano-growth factor (MGF), a recently discovered growth repair factor, plays positive roles in tissue repair through the improvement of cell proliferation and migration and the protection of cells against injury-induced apoptosis. However, it remains unclear whether MGF has the potential to accelerate tendon repair. We used a scratch wound assay in this study to demonstrate that MGF-C25E (a synthetic mechano-growth factor E peptide) promotes the migration of rat tenocytes and that this promotion is accompanied by an elevation in the expression of the following signaling molecules: focal adhesion kinase (FAK) and extracellular signal regulated kinase1/2 (ERK1/2). Inhibitors of the FAK and ERK1/2 pathways inhibited the MGF-C25E-induced tenocyte migration, indicating that MGF-C25E promotes tenocyte migration through the FAK-ERK1/2 signaling pathway. The analysis of the mechanical properties showed that the Young's modulus of tenocytes was decreased through treatment of MGF-C25E, and an obvious formation of pseudopodia and F-actin was observed in MGF-C25E-treated tenocytes. The inhibition of the FAK or ERK1/2 signals restored the decrease in Young's modulus and inhibited the formation of pseudopodia and F-actin. Overall, our study demonstrated that MGF-C25E promotes rat tenocyte migration by lessening cell stiffness and increasing pseudopodia formation via the FAK-ERK1/2 signaling pathway.