Advanced glycation end products impair the repair of injured tendon: a study in rats.

BACKGROUND: The AGEs levels in tissues of diabetics and elderly tend to be higher than in normal individuals. This study aims to determine the effects of AGEs on Achilles tendon repair. MATERIALS AND METHODS: Thirty-six male eight-week-old Sprague Dawley rats were selected in this study. The rats were randomly divided into two experimental groups and a control group after the transection of the Achilles tendon. During the tendon repair, the experimental groups were injected around the Achilles tendon with 350mmol/L (low dose group) and 1000mmol/L (high dose group) D-ribose 0.2 ml respectively to increase the AGEs level, while in the control group were given the same amount of PBS. The injections were given twice a week for six weeks. Collagen-I, TNF-α, and IL-6 expression in the healed Achilles tendon was assessed. Additionally, macroscopic, pathological, and biomechanical evaluations of Achilles tendon repair were conducted. RESULTS: The repaired Achilles tendons in the high dose group showed severe swelling and distinctive adhesions. The histological score went up with the increase of the AGEs in the Achilles tendon (p<0.001). TNF- α and IL-6 in the Achilles tendon increased (p<0.001, p<0.001), and the production of collagen-I decreased with the accumulation of AGEs in the repaired Achilles tendon (p<0.001). The tensile strength of Achilles tendon in the high dose group was impaired significantly. CONCLUSION: In current study, the compromised tendon repair model induced by AGEs was successfully established in rat. The study demonstrated that AGEs significantly impair Achilles tendon repair.

Evaluation of suitable reference genes for qPCR normalisation of gene expression in a Achilles tendon injury model.

Tendons are one of the major load-bearing tissues in the body; subjected to enormous peak stresses, and thus vulnerable to injury. Cellular responses to tendon injury are complex, involving inflammatory and repair components, with the latter employing both resident and recruited exogenous cell populations. Gene expression analyses are valuable tools for investigating tendon injury, allowing assessment of repair processes and pathological responses such as fibrosis, and permitting evaluation of therapeutic pharmacological interventions. Quantitative polymerase chain reaction (qPCR) is a commonly used approach for such studies, but data obtained by this method must be normalised to reference genes: genes known to be stably expressed between the experimental conditions investigated. Establishing suitable tendon injury reference genes is thus essential. Accordingly we investigated mRNA expression stability in a rat model of tendon injury, comparing both injured and uninjured tendons, and the effects of rapamycin treatment, at 1 and 3 weeks post injury. We used 11 candidate genes (18S, ACTB, AP3D1, B2M, CSNK2A2, GAPDH, HPRT1, PAK1IP1, RPL13a, SDHA, UBC) and assessed stability via four complementary algorithms (Bestkeeper, deltaCt, geNorm, Normfinder). Our results suggests that ACTB, CSNK2A2, HPRT1 and PAK1IP1 are all stably expressed in tendon, regardless of injury or drug treatment: any three of these would serve as universally suitable reference gene panel for normalizing qPCR expression data in the rat tendon injury model. We also reveal 18S, UBC, GAPDH, and SDHA as consistently poor scoring candidates (with the latter two exhibiting rapamycin- and injury-associated changes, respectively): these genes should be avoided.

The Efficacy of Intratissue Percutaneous Electrolysis (EPI®) and Nutritional Factors for the Treatment of Induced Tendinopathy in Wistar Rats: Hepatic Intermediary Metabolism Effects.

Achilles tendinopathy (TP) is characterized as the third most common disease of the musculoskeletal system, and occurs in three phases. There is currently no evidence of effective treatment for this medical condition. In this study, the modulatory effects of the minimally invasive technique intratissue percutaneous electrolysis (EPI) and combinations of EPI with four nutritional factors included in the diet, hydroxytyrosol (HT), maslinic acid (MA), glycine, and aspartate (AA), on hepatic intermediary metabolism was examined in Wistar rats with induced tendinopathy at various stages of TP. Results obtained showed that induced tendinopathy produced alterations in the liver intermediary metabolisms of the rats. Regarding carbohydrate metabolism, a reduction in the activity of pro-inflammatory enzymes in the later stages of TP was observed following treatment with EPI alone. Among the combined treatments using nutritional factors with EPI, HT+EPI and AA+EPI had the greatest effect on reducing inflammation in the late stages of TP. In terms of lipid metabolism, the HT+EPI and AA+EPI groups showed a decrease in lipogenesis. In protein metabolism, the HT+EPI group more effectively reduced the inflammatory effects of induced TP. Treatment with EPI combined with nutritional factors might help regulate intermediary metabolism in TP disease and reduce the inflammation process.

Dexamethasone treatment influences tendon healing through altered resolution and a direct effect on tendon cells.

Inflammation, corticosteroids, and loading all affect tendon healing, with an interaction between them. However, underlying mechanisms behind the effect of corticosteroids and the interaction with loading remain unclear. The aim of this study was to investigate the role of dexamethasone during tendon healing, including specific effects on tendon cells. Rats (n = 36) were randomized to heavy loading or mild loading, the Achilles tendon was transected, and animals were treated with dexamethasone or saline. Gene and protein analyses of the healing tendon were performed for extracellular matrix-, inflammation-, and tendon cell markers. We further tested specific effects of dexamethasone on tendon cells in vitro. Dexamethasone increased mRNA levels of S100A4 and decreased levels of ACTA2/α-SMA, irrespective of load level. Heavy loading + dexamethasone reduced mRNA levels of FN1 and TenC (p < 0.05), while resolution-related genes were unaltered (p > 0.05). In contrast, mild loading + dexamethasone increased mRNA levels of resolution-related genes ANXA1, MRC1, PDPN, and PTGES (p < 0.03). Altered protein levels were confirmed in tendons with mild loading. Dexamethasone treatment in vitro prevented tendon construct formation, increased mRNA levels of S100A4 and decreased levels of SCX and collagens. Dexamethasone during tendon healing appears to act through immunomodulation by promoting resolution, but also through an effect on tendon cells.

Pharmacological modulation of gp130 signalling enhances Achilles tendon repair by regulating tenocyte migration and collagen synthesis via SHP2-mediated crosstalk of the ERK/AKT pathway.

Tendon injuries typically display limited reparative capacity, often resulting in suboptimal outcomes and an elevated risk of recurrence or rupture. While cytokines of the IL-6 family are primarily recognised for their inflammatory properties, they also have multifaceted roles in tissue regeneration and repair. Despite this, studies examining the association between IL-6 family cytokines and tendon repair remained scarce. gp130, a type of glycoprotein, functions as a co-receptor for all cytokines in the IL-6 family. Its role is to assist in the transmission of signals following the binding of ligands to receptors. RCGD423 is a gp130 modulator. Phosphorylation of residue Y759 of gp130 recruits SHP2 and SOCS3 and inhibits activation of the STAT3 pathway. In our study, RCGD423 stimulated the formation of homologous dimers of gp130 and the phosphorylation of Y759 residues without the involvement of IL-6 and IL-6R. Subsequently, the phosphorylated residues recruited SHP2, activating the downstream ERK and AKT pathways. These mechanisms ultimately promoted the migration ability of tenocytes and matrix synthesis, especially collagen I. Moreover, RCGD423 also demonstrated significant improvements in collagen content, alignment of collagen fibres, and biological and biomechanical function in a rat Achilles tendon injury model. In summary, we demonstrated a promising gp130 modulator (RCGD423) that could potentially enhance tendon injury repair by redirecting downstream signalling of IL-6, suggesting its potential therapeutic application for tendon injuries.

Unveiling Metabolic Similarities of Entheses in Patients with Psoriasis and Psoriatic Arthritis Using Noninvasive In Vivo Molecular Imaging: Results From a Cross-sectional Exploratory Study.

OBJECTIVE: We assessed and compared molecular tissue changes at the entheses in patients with psoriasis (PsO) and psoriatic arthritis (PsA) and in healthy controls (HCs) in vivo using multispectral optoacoustic tomography (MSOT) and described their relationship with clinical and ultrasound findings of enthesitis. METHODS: A cross-sectional study (MSOT and Arthrosonography in PsA) in biologic disease-modifying antirheumatic drug-naïve patients with PsA and PsO and HCs was performed. Participants underwent clinical, ultrasonographic, and MSOT examination of six entheses (lateral humeral epicondyle, distal patellar tendon attachment, and Achilles tendon attachment). MSOT-measured hemoglobin (Hb), oxygen saturation (SO2), collagen, and lipid levels were quantified, and mean differences between groups were calculated using linear mixed effects models. MSOT-measured analytes were compared between entheses with and without clinical and ultrasound anomalies. RESULTS: Ninety participants were included (30 PsO, 30 PsA, and 30 HCs), 540 entheses were clinically assessed, and 540 ultrasound and 830 MSOT scans were obtained. Patients with PsA and PsO showed increased oxygenated Hb (PsA: P = 0.003; PsO: P = 0.054) and SO2 (PsA: P < 0.001; PsO: P = 0.001) levels and decreased collagen signals (PsA: P < 0.001; PsO: P < 0.001) compared with HCs, with more pronounced changes in PsA. Significantly lower collagen levels (P = 0.01) and increased lipids (P = 0.03) were recorded in tender entheses compared with nontender ones. Erosions and enthesophytes on ultrasound were associated with significant differences in SO2 (P = 0.014) and lipid signals (P = 0.020), respectively. CONCLUSION: Patients with PsA and PsO exhibit an analogous metabolic pattern at the entheses that is exacerbated in the presence of inflammation. These findings support the notion of a psoriatic disease spectrum characterized by common immunometabolic tissue changes.

Malvidin attenuates trauma-induced heterotopic ossification of tendon in rats by targeting Rheb for degradation via the ubiquitin-proteasome pathway.

The pathogenesis of trauma-induced heterotopic ossification (HO) in the tendon remains unclear, posing a challenging hurdle in treatment. Recognizing inflammation as the root cause of HO, anti-inflammatory agents hold promise for its management. Malvidin (MA), possessing anti-inflammatory properties, emerges as a potential agent to impede HO progression. This study aimed to investigate the effect of MA in treating trauma-induced HO and unravel its underlying mechanisms. Herein, the effectiveness of MA in preventing HO formation was assessed through local injection in a rat model. The potential mechanism underlying MA's treatment was investigated in the tendon-resident progenitor cells of tendon-derived stem cells (TDSCs), exploring its pathway in HO formation. The findings demonstrated that MA effectively hindered the osteogenic differentiation of TDSCs by inhibiting the mTORC1 signalling pathway, consequently impeding the progression of trauma-induced HO of Achilles tendon in rats. Specifically, MA facilitated the degradation of Rheb through the K48-linked ubiquitination-proteasome pathway by modulating USP4 and intercepted the interaction between Rheb and the mTORC1 complex, thus inhibiting the mTORC1 signalling pathway. Hence, MA presents itself as a promising candidate for treating trauma-induced HO in the Achilles tendon, acting by targeting Rheb for degradation through the ubiquitin-proteasome pathway.

Elevated fluid and glycosaminoglycan content in the Achilles tendon contribute to higher intratendinous pressures: Implications for Achilles tendinopathy.

BACKGROUND: Tendinopathy alters the compositional properties of the Achilles tendon by increasing fluid and glycosaminoglycan content. It has been speculated that these changes may affect intratendinous pressure, but the extent of this relationship remains unclear. Therefore, we aimed to investigate the impact of elevated fluid and glycosaminoglycan content on Achilles tendon intratendinous pressure and to determine whether hyaluronidase (HYAL) therapy can intervene in this potential relationship. METHODS: Twenty paired fresh-frozen cadaveric Achilles tendons were mounted in a tensile-testing machine and loaded up to 5% strain. Intratendinous resting (at 0% strain) and dynamic pressure (at 5% strain) were assessed using the microcapillary infusion technique. First, intratendinous pressure was measured under native conditions before and after infusion of 2 mL physiological saline. Next, 80 mg of glycosaminoglycans were administered bilaterally to the paired tendons. The right tendons were additionally treated with 1500 units of HYAL. Finally, both groups were retested, and the glycosaminoglycan content was analyzed. RESULTS: It was found that both elevated fluid and glycosaminoglycan content resulted in higher intratendinous resting and dynamic pressures (p < 0.001). HYAL treatment induced a 2.3-fold reduction in glycosaminoglycan content (p = 0.002) and restored intratendinous pressures. CONCLUSION: The results of this study demonstrated that elevated fluid and glycosaminoglycan content in Achilles tendinopathy contribute to increased intratendinous resting and dynamic pressures, which can be explained by the associated increased volume and reduced permeability of the tendon matrix, respectively. HYAL degrades glycosaminoglycans sufficiently to lower intratendinous pressures and may, therefore, serve as a promising treatment.

Roles of Plasminogen Activator Inhibitor-1 in Heterotopic Ossification Induced by Achilles Tenotomy in Thermal Injured Mice.

Heterotopic ossification (HO) is the process by which ectopic bone forms at an extraskeletal site. Inflammatory conditions induce plasminogen activator inhibitor 1 (PAI-1), an inhibitor of fibrinolysis, which regulates osteogenesis. In the present study, we investigated the roles of PAI-1 in the pathophysiology of HO induced by trauma/burn treatment using PAI-1-deficient mice. PAI-1 deficiency significantly promoted HO and increased the number of alkaline phosphatase (ALP)-positive cells in Achilles tendons after trauma/burn treatment. The mRNA levels of inflammation markers were elevated in Achilles tendons of both wild-type and PAI-1-deficient mice after trauma/burn treatment and PAI-1 mRNA levels were elevated in Achilles tendons of wild-type mice. PAI-1 deficiency significantly up-regulated the expression of Runx2, Osterix, and type 1 collagen in Achilles tendons 9 weeks after trauma/burn treatment in mice. In in vitro experiments, PAI-1 deficiency significantly increased ALP activity and mineralization in mouse osteoblasts. Moreover, PAI-1 deficiency significantly increased ALP activity and up-regulated osteocalcin expression during osteoblastic differentiation from mouse adipose-tissue-derived stem cells, but suppressed the chondrogenic differentiation of these cells. In conclusion, the present study showed that PAI-1 deficiency promoted HO in Achilles tendons after trauma/burn treatment partly by enhancing osteoblast differentiation and ALP activity in mice. Endogenous PAI-1 may play protective roles against HO after injury and inflammation.

Early delivery of human umbilical cord mesenchymal stem cells improves healing in a rat model of Achilles tendinopathy.

Objective: This study aimed to explore the efficacy and optimal delivery time of human umbilical cord mesenchymal stem cells (hUC-MSCs) in treating collagenase-induced Achilles tendinopathy. Methods: Achilles tendinopathy in rats at early or advanced stages was induced by injecting collagenase I into bilateral Achilles tendons. A total of 28 injured rats were injected with a hUC-MSC solution or normal saline into bilateral tendons twice and sampled after 4 weeks for histological staining, gene expression analysis, transmission electron microscope assay and biomechanical testing analysis. Results: The results revealed better histological performance and a larger collagen fiber diameter in the MSC group. mRNA expression of TNF-α, IL-1ß and MMP-3 was lower after MSC transplantation. Early MSC delivery promoted collagen I and TIMP-3 synthesis, and strengthened tendon toughness. Conclusion: hUC-MSCs demonstrated a therapeutic effect in treating collagenase-induced Achilles tendinopathy, particularly in the early stage of tendinopathy.

Anti-inflammatory effects of sericin and swimming exercise in treating experimental Achilles tendinopathy in rat.

The aim of this study was to assess the effectiveness of combining sericin with swimming exercise as a treatment for type-I collagenase-induced Achilles tendinopathy (AT) in rats, with a focus on inflammatory cytokines. An experimental AT model was established using type-I collagenase in male Sprague-Dawley rats, categorized into five groups: Group 1 (Control + Saline), Group 2 (AT), Group 3 (AT + exercise), Group 4 (AT + sericin), and Group 5 (AT + sericin + exercise). Intratendinous sericin administration (0.8 g/kg/mL) took place from days 3 to 6, coupled with 30 min daily swimming exercise sessions (5 days/week, 4 weeks). Serum samples were analyzed using ELISA for tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), interleukin-10 (IL-10), and total antioxidant-oxidant status (TAS-TOS), alongside histopathological and immunohistochemical assessments of Achilles tendon samples. Elevated TNF-α and IL-1ß and decreased IL-10 levels were evident in Group 2; Of these, TNF-α and IL-1ß were effectively reduced and IL-10 increased across all treatment groups, particularly groups 4 and 5. Serum TAS was notably lower in Group 2 and significantly increased in Group 5 compared to Group 2. Histopathologically, Group 2 displayed severe degeneration, irregular fibers, and round cell nuclei, while Group 5 exhibited decreased degeneration and spindle-shaped fibers. The Bonar score increased in Group 2 and decreased in groups 4 and 5. Collagen type-I alpha-1 (Col1A1) expression was notably lower in Group 2 (P = 0.001) and significantly increased in groups 4 and 5 compared to Group 2 (P = 0.011 and 0.028, respectively). This study underscores the potential of sericin and swimming exercises in mitigating inflammation and oxidative stress linked to AT pathogenesis, presenting a promising combined therapeutic strategy.

Novel application of in vivo microdialysis in a rat Achilles tendon acute injury model.

Tendon injury and healing involve intricate changes to tissue metabolism, biology, and inflammation. Current techniques often require animal euthanasia or tissue destruction, limiting assessment of dynamic changes in tendon, including treatment response, disease development, rupture risk, and healing progression. Microdialysis, a minimally invasive technique, offers potential for longitudinal assessment, yet it has not been applied to rat tendon models. Therefore, the objective of this study is to adapt a novel application of an in vivo assay, microdialysis, using acute injury as a model for extreme disruption of the tendon homeostasis. We hypothesize that microdialysis will be able to detect measurable differences in the healing responses of acute injury with high specificity and sensitivity. Overall results suggest that microdialysis is a promising in vivo technique for longitudinal assessment for this system with strong correlations between extracellular fluid (ECF) and dialysate concentrations and reasonable recovery rates considering the limitations of this model. Strong positive correlations were found between dialysate and extracellular fluid (ECF) concentration for each target molecule of interest including metabolites, inflammatory mediators, and collagen synthesis and degradation byproducts. These results suggest that microdialysis is capable of detecting changes in tendon healing following acute tendon injury with high specificity and sensitivity. In summary, this is the first study to apply microdialysis to a rat tendon model and assess its efficacy as a direct measurement of tendon metabolism, biology, and inflammation.NEW & NOTEWORTHY This study adapts a novel application of microdialysis to rat tendon models, offering a minimally invasive avenue for longitudinal tendon assessment. Successfully detecting changes in tendon healing after acute injury, it showcases strong correlations between extracellular fluid and dialysate concentrations. The results highlight the potential of microdialysis as a direct measure of tendon metabolism, biology, and inflammation, bypassing the need for animal euthanasia and tissue destruction.

Chronological Changes in the Expression and Localization of Sox9 between Achilles Tendon Injury and Functional Recovery in Mice.

Tendons help transmit forces from the skeletal muscles and bones. However, tendons have inferior regenerative ability compared to muscles. Despite studies on the regeneration of muscles and bone tissue, only a few have focused on tendinous tissue regeneration, especially tendon regeneration. Sex-determining region Y-box transcription factor 9 (Sox9) is an SRY-related transcription factor with a DNA-binding domain and is an important control factor for cartilage formation. Sox9 is critical to the early-to-middle stages of tendon development. However, how Sox9 participates in the healing process after tendon injury is unclear. We hypothesized that Sox9 is expressed in damaged tendons and is crucially involved in restoring tendon functions. We constructed a mouse model of an Achilles tendon injury by performing a 0.3 mm wide partial excision in the Achilles tendon of mice, and chronologically evaluated the function restoration and localization of the Sox9 expressed in the damaged sites. The results reveal that Sox9 was expressed simultaneously with the formation of the pre-structure of the epitenon, an essential part of the tendinous tissue, indicating that its expression is linked to the functional restoration of tendons. Lineage tracing for Sox9 expressed during tendon restoration revealed the tendon restoration involvement of cells that switched into Sox9-expressing cells after tendon injury. The stem cells involved in tendon regeneration may begin to express Sox9 after injury.

Bioactive and Elastic Emulsion Electrospun DegraPol Tubes Delivering IGF-1 for Tendon Rupture Repair.

Tendon injuries can result in two major drawbacks. Adhesions to the surrounding tissue may limit the range of motion, while fibrovascular scar formation can lead to poor biomechanical outcomes. Prosthetic devices may help to mitigate those problems. Emulsion electrospinning was used to develop a novel three-layer tube based on the polymer DegraPol (DP), with incorporated insulin-like growth factor-1 (IGF-1) in the middle layer. Scanning electron microscopy was utilized to assess the fiber diameter in IGF-1 containing pure DP meshes. Further characterization was performed with Fourier Transformed Infrared Spectroscopy, Differential Scanning Calorimetry, and water contact angle, as well as through the assessment of mechanical properties and release kinetics from ELISA, and the bioactivity of IGF-1 by qPCR of collagen I, ki67, and tenomodulin in rabbit Achilles tenocytes. The IGF-1-containing tubes exhibited a sustained release of the growth factor up to 4 days and showed bioactivity by significantly upregulated ki67 and tenomodulin gene expression. Moreover, they proved to be mechanically superior to pure DP tubes (significantly higher fracture strain, failure stress, and elastic modulus). The novel three-layer tubes intended to be applied over conventionally sutured tendons after a rupture may help accelerate the healing process. The release of IGF-1 stimulates proliferation and matrix synthesis of cells at the repair site. In addition, adhesion formation to surrounding tissue can be reduced due to the physical barrier.

Optogenetic-induced muscle loading leads to mechanical adaptation of the Achilles tendon enthesis in mice.

Skeletal shape depends on the transmission of contractile muscle forces from tendon to bone across the enthesis. Loss of muscle loading impairs enthesis development, yet little is known if and how the postnatal enthesis adapts to increased loading. Here, we studied adaptations in enthesis structure and function in response to increased loading, using optogenetically induced muscle contraction in young (i.e., growth) and adult (i.e., mature) mice. Daily bouts of unilateral optogenetic loading in young mice led to radial calcaneal expansion and warping. This also led to a weaker enthesis with increased collagen damage in young tendon and enthisis, with little change in adult mice. We then used RNA sequencing to identify the pathways associated with increased mechanical loading during growth. In tendon, we found enrichment of glycolysis, focal adhesion, and cell-matrix interactions. In bone, we found enrichment of inflammation and cell cycle. Together, we demonstrate the utility of optogenetic-induced muscle contraction to elicit in vivo adaptation of the enthesis.

Pro-inflammatory activity of long noncoding RNA FOXD2-AS1 in Achilles tendinopathy.

Achilles tendinopathy is a prevalent clinical problem that plagues athletes and general populations. Achilles tendon healing is a complex process, and so far, there is no successful long-term solution to Achilles tendinopathy in the field of microsurgery due to its poor natural regeneration ability. Limitations in understanding the pathogenesis of Achilles tendon development and Achilles tendon injury hinder clinical treatment developments. There is an increasing demand for innovative conservative treatments that can improve Achilles tendon injury. In this study, a Sprague-Dawley rat model of Achilles tendinopathy was established. Lentiviral vectors that interfere with the expression of FOXD2-AS1, miR-21-3p, or PTEN were injected every 3 days. Rats were euthanized after 3 weeks, and the effect of FOXD2-AS1, miR-21-3p, or PTEN on Achilles tendon healing was analyzed by histological observation, biomechanical test, and examinations of inflammatory factors and tendon markers. As measured, downregulating FOXD2-AS1 or upregulating miR-21-3p improved histological structure, suppressed inflammation, promoted the expression of tendon markers, and optimized the biomechanical properties of Achilles tendon. Upregulating PTEN was capable of reversing the promoting effect of inhibition of FOXD2-AS1 on Achilles tendon healing. As concluded, deficiency of FOXD2-AS1 accelerates the healing of Achilles tendon injury and improves tendon degeneration by regulating the miR-21-3p/PTEN axis and promoting the activation of the PI3K/AKT signaling pathway.

CCN1 expression is regulated by mechanical stimuli in tendons.

Tendon overuse injuries are common, but the processes that govern tendon response to mechanical load are not fully understood. A series of experiments of in vitro and in vivo experiments was devised to study to the relationship between mechanical stimuli and the matricellular protein Cellular Communication Network Factor 1 (CCN1) in tenocytes and tendons. First, human and murine tenocytes were subjected to cyclic uniaxial loading in order to evaluate changes in CCN1 gene expression as a response to mechanical stimuli. Then, baseline Ccn1 gene expression in different murine tendons (Achilles, patellar, forearm, and tail) was examined. Finally, changes in Ccn1 expression after in vivo unloading experiments were examined. It was found that CCN1 expression significantly increased in both human and murine tenocytes at 5 and 10% cyclical uniaxial strain, while 2.5% strain did not have any effect on CCN1 expression. At baseline, the Achilles, patellar, and forearm tendons had higher expression levels of Ccn1 as compared to tail tendons. Twenty-four hours of immobilization of the hind-limb resulted in a significant decrease in Ccn1 expression in both the Achilles and patellar tendons. In summary, CCN1 expression is up-regulated in tenocytes subjected to mechanical load and down-regulated by loss of mechanical load in tendons. These results show that CCN1 expression in tendons is at least partially regulated by mechanical stimuli.

eEF2 improves dense connective tissue repair and healing outcome by regulating cellular death, autophagy, apoptosis, proliferation and migration.

Outcomes following human dense connective tissue (DCT) repair are often variable and suboptimal, resulting in compromised function and development of chronic painful degenerative diseases. Moreover, biomarkers and mechanisms that guide good clinical outcomes after DCT injuries are mostly unknown. Here, we characterize the proteomic landscape of DCT repair following human Achilles tendon rupture and its association with long-term patient-reported outcomes. Moreover, the potential regulatory mechanisms of relevant biomarkers were assessed partly by gene silencing experiments. A mass-spectrometry based proteomic approach quantified a large number (769) of proteins, including 51 differentially expressed proteins among 20 good versus 20 poor outcome patients. A novel biomarker, elongation factor-2 (eEF2) was identified as being strongly prognostic of the 1-year clinical outcome. Further bioinformatic and experimental investigation revealed that eEF2 positively regulated autophagy, cell proliferation and migration, as well as reduced cell death and apoptosis, leading to improved DCT repair and outcomes. Findings of eEF2 as novel prognostic biomarker could pave the way for new targeted treatments to improve healing outcomes after DCT injuries.Trial registration: NCT02318472 registered 17 December 2014 and NCT01317160 registered 17 March 2011, with URL http://clinicaltrials.gov/ct2/show/NCT02318472 and http://clinicaltrials.gov/ct2/show/study/NCT01317160 .

Fast, non-eccentrically loaded exercise worsens tendinopathic healing responses in a murine model.

OBJECTIVE: To advance the understanding of how alterations in exercise speed and grade (flat vs 17° incline or decline) affect the quality of tendon healing, and to determine if a biomarker relationship exists between serum levels of a ColX breakdown product (CXM) and animals exposed to treadmill running protocols. ANIMALS: 35 male mice (C57BL/6J), 8 weeks of age. PROCEDURES: Mice were preconditioned on a treadmill for 14 days. Tendinopathy was then induced by 2 intra-tendinous TGFß1 injections followed by randomization into 7 exercise groups. Exercise capacity and objective gait analysis were measured weekly. Mice were euthanized and histopathologic analysis and evaluation of serum CXM levels were performed. Statistics were conducted using a 2-way ANOVA (exercise capacity), Mixed Effects Model (gait analysis, effect of preconditioning), and 1-way ANOVA (gait analysis, the effect of injury, and rehabilitation normalized to baseline; CXM serum analysis), all with Tukey post hoc tests and significance set to P < .05. RESULTS: Exercise at a fast-flat speed demonstrated inferior tendinopathic healing at the cellular level and impaired stance braking abilities, which were compensated for by increased propulsion. Mice exposed to exercise (at any speed or grade) demonstrated higher systemic levels of CXM than those that were cage rested. However, no ColX immunostaining was observed in the Achilles tendon or calcaneal insertion. CLINICAL RELEVANCE: Exercise at a fast speed and in absence of eccentric loading components (incline or decline) demonstrated inferior tendinopathic healing at the cellular level and impaired braking abilities that were compensated for by increased propulsion.

Pdgfrß+ lineage cells transiently increase at the site of Achilles tendon healing.

The purpose of this study was to track platelet-derived growth factor receptor-ß (Pdgfr-ß) lineage cells at the site of Achilles tendon injury over time. Pdgfr-ß-CreERT2 :Ai9 mice were generated to track Pdgfr-ß lineage cells in adult mice. A surgical Achilles transection injury model was employed to examine the presence of Pdgfr-ß lineage cells in the healing tendon over time, with five mice per time point at 3, 7, 14, 28, and 56 days postoperatively. Histology and immunohistochemistry for tdTomato (Pdgfr-ß lineage cells), PCNA (proliferating cell nuclear antigen, cell proliferation), and α-SMA (α-smooth muscle actin, myofibroblasts) were performed. The percentage of cells at the healing tendon site staining positive for tdTomato and PCNA were quantified. Over 75% of cells at the injury site were Pdgfr-ß lineage cells at Days 3, 7, and 14, and this percentage decreased significantly by Days 28 and 56 postinjury. Cell proliferation at the injury site peaked on Day 7 and decreased thereafter. Immunohistochemistry for α-SMA demonstrated minimal colocalization of myofibroblasts with Pdgfr-ß lineage cells. This study demonstrates that in a mouse model of Achilles tendon injury, Pdgfr-ß lineage cells' presence at the injury site is transient. Thus, we conclude that they are unlikely to be the cells that differentiate into myofibroblasts and directly contribute to tendon fibrous scar formation. Clinical Significance: This study provides some insight into the presence of Pdgfr-ß lineage cells (including pericytes) following Achilles injury, furthering our understanding of tendon healing.