Mutations in COMP cause familial carpal tunnel syndrome.

Carpal tunnel syndrome (CTS) is the most common peripheral nerve entrapment syndrome, affecting a large proportion of the general population. Genetic susceptibility has been implicated in CTS, but the causative genes remain elusive. Here, we report the identification of two mutations in cartilage oligomeric matrix protein (COMP) that segregate with CTS in two large families with or without multiple epiphyseal dysplasia (MED). Both mutations impair the secretion of COMP by tenocytes, but the mutation associated with MED also perturbs its secretion in chondrocytes. Further functional characterization of the CTS-specific mutation reveals similar histological and molecular changes of tendons/ligaments in patients' biopsies and the mouse models. The mutant COMP fails to oligomerize properly and is trapped in the ER, resulting in ER stress-induced unfolded protein response and cell death, leading to inflammation, progressive fibrosis and cell composition change in tendons/ligaments. The extracellular matrix (ECM) organization is also altered. Our studies uncover a previously unrecognized mechanism in CTS pathogenesis.

Tendinosis develops from age- and oxygen tension-dependent modulation of Rac1 activity.

Age-related tendon degeneration (tendinosis) is characterized by a phenotypic change in which tenocytes display characteristics of fibrochondrocytes and mineralized fibrochondrocytes. As tendon degeneration has been noted in vivo in areas of decreased tendon vascularity, we hypothesized that hypoxia is responsible for the development of the tendinosis phenotype, and that these effects are more pronounced in aged tenocytes. Hypoxic (1% O2 ) culture of aged, tendinotic, and young human tenocytes resulted in a mineralized fibrochondrocyte phenotype in aged tenocytes, and a fibrochondrocyte phenotype in young and tendinotic tenocytes. Investigation of the molecular mechanism responsible for this phenotype change revealed that the fibrochondrocyte phenotype in aged tenocytes occurs with decreased Rac1 activity in response to hypoxia. In young hypoxic tenocytes, however, the fibrochondrocyte phenotype occurs with concomitant decreased Rac1 activity coupled with increased RhoA activity. Using pharmacologic and adenoviral manipulation, we confirmed that these hypoxic effects on the tenocyte phenotype are linked directly to the activity of RhoA/Rac1 GTPase in in vitro human cell culture and tendon explants. These results demonstrate that hypoxia drives tenocyte phenotypic changes, and provide a molecular insight into the development of human tendinosis that occurs with aging.

Amplification of Mitochondrial Activity in the Healing Response Following Rotator Cuff Tendon Injury.

Mitochondrial function following rotator cuff tendon injury (RCI) influences the tendon healing. We examined the mitochondrial morphology and function under hypoxia in the shoulder tendon tissue from surgically-induced tenotomy-RCI rat model and cultured swine tenocytes. The tendon tissue was collected post-injury on 3-5 (Group-A), 10-12 (Group-B), and 22-24 (Group-C), days and the corresponding contralateral tendons were used as control for each group. There was higher protein expression of citrate synthase (P < 0.0001) [10.22 MFI (mean fluorescent intensity)] and complex-1 (P = 0.0008) (7.86 MFI) in Group-A and Group-B that decreased in Group-C [(P = 0.0201) (5.78 MFI and (P = 0.7915) (2.32 MFI), respectively] compared to control tendons. The ratio of BAX:Bcl2 (Bcl2 associated x protein:B cell lymphoma 2) in RCI tendons increased by 50.5% (Group-A) and 68.4% (Group-B) and decreased by 25.8% (Group-C) compared to normoxic controls. Hypoxia increased ß-tubulin expression (P = 0067) and reduced PGC1-α (P = 0412) expression in the isolated swine tenocytes with no effect on the protein expression of Complex-1 (P = 7409) and citrate synthase (P = 0.3290). Also, the hypoxic tenocytes exhibited about 4-fold increase in mitochondrial superoxide (P < 0.0001), altered morphology and mitochondrial pore integrity, and increase in mitochondrial density compared to normoxic controls. These findings suggest the critical role of mitochondria in the RCI healing response.

Elevated TGF ß2 serum levels in Emery-Dreifuss Muscular Dystrophy: Implications for myocyte and tenocyte differentiation and fibrogenic processes.

Among rare diseases caused by mutations in LMNA gene, Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B are characterized by muscle weakness and wasting, joint contractures, cardiomyopathy with conduction system disorders. Circulating biomarkers for these pathologies have not been identified. Here, we analyzed the secretome of a cohort of patients affected by these muscular laminopathies in the attempt to identify a common signature. Multiplex cytokine assay showed that transforming growth factor beta 2 (TGF ß2) and interleukin 17 serum levels are consistently elevated in the vast majority of examined patients, while interleukin 6 and basic fibroblast growth factor are altered in subgroups of patients. Levels of TGF ß2 are also increased in fibroblast and myoblast cultures established from patient biopsies as well as in serum from mice bearing the H222P Lmna mutation causing Emery-Dreifuss Muscular Dystrophy in humans. Both patient serum and fibroblast conditioned media activated a TGF ß2-dependent fibrogenic program in normal human myoblasts and tenocytes and inhibited myoblast differentiation. Consistent with these results, a TGF ß2 neutralizing antibody avoided fibrogenic marker activation and myogenesis impairment. Cell intrinsic TGF ß2-dependent mechanisms were also determined in laminopathic cells, where TGF ß2 activated AKT/mTOR phosphorylation. These data show that TGF ß2 contributes to the pathogenesis of Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B and can be considered a potential biomarker of those diseases. Further, the evidence of TGF ß2 pathogenetic effects in tenocytes provides the first mechanistic insight into occurrence of joint contractures in muscular laminopathies.

Local application of tranexamic acid affects tendon healing negatively in the late period.

OBJECTIVES: This study aims to evaluate the potential adverse effects of tranexamic acid (TA) on tendon healing. MATERIALS AND METHODS: Twelve male Wistar-Albino rats (weighing 300 g to 350 g) were used in the study. Rats were divided into two groups. Right legs of the rats were determined as the TA group and left legs as the serum physiologic (SP) group. Bilateral Achilles tenotomy was performed and surgically repaired. For the right side, 1 mL of TA and for the left side, 1 mL of SP were applied. Half of the rats were sacrificed at the third week and the other half at the sixth week and tendon samples were collected from the extremities. Histological analyses were performed according to the tendon scoring system (Bonar classification). RESULTS: Tenocyte cell morphology was better in the third week in TA group than in SP group. In terms of colloidal organization, SP groups gave superior results in all weeks. An analysis of total tendon healing scores revealed that the results of the third week TA groups were superior to the results of the sixth week TA groups. Tenocyte morphology and total tendon healing scores of rats in the sixth week TA group were statistically significantly lower compared to the third week TA group (tenocyte morphology p=0.009, total score p=0.041). CONCLUSION: In this study, we detected that locally administered TA has an adverse effect on tendon healing in late period. However, further immunohistochemical and biomechanical studies are needed to support these results.

Therapeutic potential of hAECs for early Achilles tendon defect repair through regeneration.

Cell-based therapy holds great promise for tendon disorders, a widespread debilitating musculoskeletal condition. Even if the cell line remains to be defined, preliminary evidences have proven that amniotic-derived cells possess in vitro and in vivo a great tenogenic potential. This study investigated the efficacy of transplanted human amniotic epithelial cells (hAECs) by testing their early regenerative properties and mechanisms involved on a validated ovine Achilles tendon partial defect performed on 29 animals. The injured tendons treated with hAECs recovered rapidly, in 28 days, structural and biomechanical properties undertaking a programmed tissue regeneration, differently from the spontaneous healing tissues. hAECs remained viable within the host tendons establishing with the endogenous progenitor cells an active dialogue. Through the secretion of modulatory factors, hAECs inhibited the inflammatory cells infiltration, activated the M2 macrophage subpopulation early recruitment, and accelerated blood vessel as well as extracellular matrix remodelling. In parallel, some in situ differentiated hAECs displayed a tenocytelike phenotype. Both paracrine and direct hAECs stimulatory effects were confirmed analysing their genome profile before and after transplantation. The 49 human up-regulated transcripts recorded in transplanted hAECs belonged to tendon lineage differentiation (epithelial-mesenchymal transition, connective specific matrix components, and skeleton or muscle system development-related transcripts), as well as the in situ activation of paracrine signalling involved in inflammatory and immunomodulatory response. Altogether, these evidences support the hypothesis that hAECs are a practicable and efficient strategy for the acute treatment of tendinopathy, reinforcing the idea of a concrete use of amniotic epithelial cells towards the clinical practice.

Hyperglycemia Augments the Adipogenic Transdifferentiation Potential of Tenocytes and Is Alleviated by Cyclic Mechanical Stretch.

Diabetes mellitus is associated with damage to tendons, which may result from cellular dysfunction in response to a hyperglycemic environment. Tenocytes express diminished levels of tendon-associated genes under hyperglycemic conditions. In contrast, mechanical stretch enhances tenogenic differentiation. However, whether hyperglycemia increases the non-tenogenic differentiation potential of tenocytes and whether this can be mitigated by mechanical stretch remains elusive. We explored the in vitro effects of high glucose and mechanical stretch on rat primary tenocytes. Specifically, non-tenogenic gene expression, adipogenic potential, cell migration rate, filamentous actin expression, and the activation of signaling pathways were analyzed in tenocytes treated with high glucose, followed by the presence or absence of mechanical stretch. We analyzed tenocyte phenotype in vivo by immunohistochemistry using an STZ (streptozotocin)-induced long-term diabetic mouse model. High glucose-treated tenocytes expressed higher levels of the adipogenic transcription factors PPARγ and C/EBPs. PPARγ was also highly expressed in diabetic tendons. In addition, increased adipogenic differentiation and decreased cell migration induced by high glucose implicated a fibroblast-to-adipocyte phenotypic change. By applying mechanical stretch to tenocytes in high-glucose conditions, adipogenic differentiation was repressed, while cell motility was enhanced, and fibroblastic morphology and gene expression profiles were strengthened. In part, these effects resulted from a stretch-induced activation of ERK (extracellular signal-regulated kinases) and a concomitant inactivation of Akt. Our results show that mechanical stretch alleviates the augmented adipogenic transdifferentiation potential of high glucose-treated tenocytes and helps maintain their fibroblastic characteristics. The alterations induced by high glucose highlight possible pathological mechanisms for diabetic tendinopathy. Furthermore, the beneficial effects of mechanical stretch on tenocytes suggest that an appropriate physical load possesses therapeutic potential for diabetic tendinopathy.

Cell Toxicity in Fibroblasts, Tenocytes, and Human Mesenchymal Stem Cells-A Comparison of Necrosis and Apoptosis-Inducing Ability in Ropivacaine, Bupivacaine, and Triamcinolone.

PURPOSE: To analyze the ability of ropivacaine, bupivacaine, and triamcinolone to induce apoptosis and necrosis in fibroblasts, tenocytes, and human mesenchymal stem cells. METHODS: Human dermal fibroblasts, adipose-derived human mesenchymal stem cells (hMSCs), and tenocytes gained from the rotator cuff tendon were seeded with a cell density of 0.5 × 104/cm2. One specimen of ropivacaine, bupivacaine, and triamcinolone was tested separately on the cells with separate concentrations of 0.5%, 0.25%, and 0.125% for each specimen. The negative control received no agent, only a change of medium. The incubation period for each agent was 30 minutes. After a change of medium and 1 hour, 24 hours, and 7 days of incubation, 104 cells were harvested and analyzed via fluorescence-activated cell sorting with double-staining with annexin V and propidium iodide. Statistical analysis to determine significant difference (P < .05) between the groups with SPSS statistics 23 through one-way analysis of variance with a univariate general linear model was performed. RESULTS: Bupivacaine showed necrosis-inducing effects on fibroblasts and tenocytes, with the necrotic effect peaking at 0.5% and 0.25%. Ropivacaine and triamcinolone caused no significant necrosis. Compared with fibroblasts and tenocytes, hMSCs did not show significant necrotic or apoptotic effects after exposure to bupivacaine. Overall, no significant differences in apoptosis were detected between different cell lines, varying concentrations, or time measurements. CONCLUSIONS: Bupivacaine 0.5% and 0.25% have the most necrosis-inducing effects on fibroblasts and tenocytes. Ropivacaine caused less necrosis than bupivaine. Compared with fibroblasts and tenocytes, hMSCs were not affected by necrosis using any of the tested agents. A significant apoptosis-inducing effect could not be detected for the different cell lines. CLINICAL RELEVANCE: Possible cell toxicity raises questions of concern for intra-articular injections using local anesthetics and corticosteroids. The present study demonstrates the necrotic and apoptotic effects of ropivacaine, bupivacaine, and triamcinolone and may give recommendations for intra-articular use of local anesthetics and corticosteroids.

Effects of boric acid on the healing of Achilles tendons of rats.

PURPOSE: Tendinous lesions are among the most frequent pathologies encountered in sportsmen. The objectives of new treatments are to improve the healing process and reduce the recovery time. Boron plays an important role in the wound repair process by increasing components of extracellular matrix and angiogenesis. This animal study aimed to investigate the effect of boric acid on healing of the Achilles tendon. METHODS: The right Achilles tendons of 40 rats were completely sectioned, and the rats were randomly divided into five groups. Each group consisted of eight rats. Groups 1 and 2 were oral boric acid groups with the doses of 4 and 8 mg/kg/day boric acid, respectively. Group 3 was the local boric acid group (8 mg/kg boric acid intratendinous injection). Group 4 was administered both oral and local boric acid (8 mg/kg/day orally and 8 mg/kg boric acid intratendinous injection), and group 5 was the control group with no boric acid application. At the end of the fourth week, all the rats were killed and histopathological examination of the Achilles tendon repair site was made. RESULTS: Histopathological examination of the tissue sections revealed more properly oriented collagen fibres, more normal cellular distribution of tenocytes and more properly organized vascular bundles in group 1 and group 2, which were the groups administered oral boric acid. Pathological sum scores of groups 1 and 2 were less than those of the other groups, and the differences between the oral boric acid groups (group 1 and group 2) and the other three groups (groups 3, 4 and 5) were statistically significant (p = 0.001). CONCLUSION: As boric acid is safe and toxicity even after very high doses is unusual, oral boric acid may be used as an agent to improve the healing process of tendon injuries. However, biomechanical tests should also be performed to show the effect of boric acid on strength and endurance of the tendon before it can be used in clinical practice.