Response to acute hyperglycemia and high fructose in cultured tenocytes.

High monosaccharide levels are intimately associated with diabetes and impact tendon cells through inflammation and impairment in metabolic homeostasis. Experiments were designed to understand the responses elicited by cultured tenocytes under monosaccharide stress induced by hyperglycemia and hyperfructosemia. We simulated hyperglycemia and hyperfructosemia in vitro by treating tenocytes with media containing sublethal concentrations of glucose and fructose, respectively. Exposure of tenocytes to high glucose and high fructose altered the levels of IL-1ß, IL-2, IL-6, IL10 and IL-17A. AMPK expression was increased in high-glucose and decreased in high-fructose groups. High fructose increased the level of IRS-1 compared with the control. Increased mitochondrial superoxide levels and compromised mitochondrial membrane integrity were exhibited by both the groups. The findings from the network analysis revealed many altered genes that are related to pathways for enzyme-linked receptor protein signaling, positive regulation of metabolic processes, transmembrane receptor tyrosine kinase pathway, insulin receptor signaling and regulation of cytokine production. Overall, the data suggest that the tenocytes under high monosaccharide levels exhibit survival responses by altering the expression status of cytokines and metabolic mediators that are involved in the underlying pathogenesis of tendinopathy.

The tendon microenvironment: Engineered in vitro models to study cellular crosstalk.

Tendinopathy is a multi-faceted pathology characterized by alterations in tendon microstructure, cellularity and collagen composition. Challenged by the possibility of regenerating pathological or ruptured tendons, the healing mechanisms of this tissue have been widely researched over the past decades. However, so far, most of the cellular players and processes influencing tendon repair remain unknown, which emphasizes the need for developing relevant in vitro models enabling to study the complex multicellular crosstalk occurring in tendon microenvironments. In this review, we critically discuss the insights on the interaction between tenocytes and the other tendon resident cells that have been devised through different types of existing in vitro models. Building on the generated knowledge, we stress the need for advanced models able to mimic the hierarchical architecture, cellularity and physiological signaling of tendon niche under dynamic culture conditions, along with the recreation of the integrated gradients of its tissue interfaces. In a forward-looking vision of the field, we discuss how the convergence of multiple bioengineering technologies can be leveraged as potential platforms to develop the next generation of relevant in vitro models that can contribute for a deeper fundamental knowledge to develop more effective treatments.

Hypoxic Incubation Conditions for Optimized Manufacture of Tenocyte-Based Active Pharmaceutical Ingredients of Homologous Standardized Transplant Products in Tendon Regenerative Medicine.

Human fetal progenitor tenocytes (hFPT) produced in defined cell bank systems have recently been characterized and qualified as potential therapeutic cell sources in tendon regenerative medicine. In view of further developing the manufacture processes of such cell-based active pharmaceutical ingredients (API), the effects of hypoxic in vitro culture expansion on key cellular characteristics or process parameters were evaluated. To this end, multiple aspects were comparatively assessed in normoxic incubation (i.e., 5% CO2 and 21% O2, standard conditions) or in hypoxic incubation (i.e., 5% CO2 and 2% O2, optimized conditions). Experimentally investigated parameters and endpoints included cellular proliferation, cellular morphology and size distribution, cell surface marker panels, cell susceptibility toward adipogenic and osteogenic induction, while relative protein expression levels were analyzed by quantitative mass spectrometry. The results outlined conserved critical cellular characteristics (i.e., cell surface marker panels, cellular phenotype under chemical induction) and modified key cellular parameters (i.e., cell size distribution, endpoint cell yields, matrix protein contents) potentially procuring tangible benefits for next-generation cell manufacturing workflows. Specific proteomic analyses further shed some light on the cellular effects of hypoxia, potentially orienting further hFPT processing for cell-based, cell-free API manufacture. Overall, this study indicated that hypoxic incubation impacts specific hFPT key properties while preserving critical quality attributes (i.e., as compared to normoxic incubation), enabling efficient manufacture of tenocyte-based APIs for homologous standardized transplant products.

Collagen XII mediated cellular and extracellular mechanisms regulate establishment of tendon structure and function.

Tendons have a uniaxially aligned structure with a hierarchical organization of collagen fibrils crucial for tendon function. Collagen XII is expressed in tendons and has been implicated in the regulation of fibrillogenesis. It is a non-fibrillar collagen belonging to the Fibril-Associated Collagens with Interrupted Triple Helices (FACIT) family. Mutations in COL12A1 cause myopathic Ehlers Danlos Syndrome with a clinical phenotype involving both joints and tendons supporting critical role(s) for collagen XII in tendon development and function. Here we demonstrate the molecular function of collagen XII during tendon development using a Col12a1 null mouse model. Col12a1 deficiency altered tenocyte shape, formation of interacting cell processes, and organization resulting in impaired cell-cell communication and disruption of hierarchal structure as well as decreased tissue stiffness. Immuno-localization revealed that collagen XII accumulated on the tenocyte surface and connected adjacent tenocytes by building matrix bridges between the cells, suggesting that collagen XII regulates intercellular communication. In addition, there was a decrease in fibrillar collagen I in collagen XII deficient tenocyte cultures compared with controls suggesting collagen XII signaling specifically alters tenocyte biosynthesis. This suggests that collagen XII provides feedback to tenocytes regulating extracellular collagen I. Together, the data indicate dual roles for collagen XII in determination of tendon structure and function. Through association with fibrils it functions in fibril packing, fiber assembly and stability. In addition, collagen XII influences tenocyte organization required for assembly of higher order structure; intercellular communication necessary to coordinate long range order and feedback on tenocytes influencing collagen synthesis. Integration of both regulatory roles is required for the acquisition of hierarchal structure and mechanical properties.

The Collagen-Based Medical Device MD-Tissue Acts as a Mechanical Scaffold Influencing Morpho-Functional Properties of Cultured Human Tenocytes.

Mechanotransduction is the ability of cells to translate mechanical stimuli into biochemical signals that can ultimately influence gene expression, cell morphology and cell fate. Tenocytes are responsible for tendon mechanical adaptation converting mechanical stimuli imposed during mechanical loading, thus affecting extracellular matrix homeostasis. Since we previously demonstrated that MD-Tissue, an injectable collagen-based medical compound containing swine-derived collagen as the main component, is able to affect tenocyte properties, the aim of this study was to analyze whether the effects triggered by MD-Tissue were based on mechanotransduction-related mechanisms. For this purpose, MD-Tissue was used to coat Petri dishes and cytochalasin B was used to deprive tenocytes of mechanical stimulation mediated by the actin cytoskeleton. Cell morphology, migration, collagen turnover pathways and the expression of key mechanosensors were analyzed by morphological and molecular methods. Our findings confirm that MD-Tissue affects collagen turnover pathways and favors cell migration and show that the MD-Tissue-induced effect represents a mechanical input involving the mechanotransduction machinery. Overall, MD-Tissue, acting as a mechanical scaffold, could represent an effective medical device for a novel therapeutic, regenerative and rehabilitative approach to favor tendon healing in tendinopathies.

Multi-omic single cell analysis resolves novel stromal cell populations in healthy and diseased human tendon.

Tendinopathy accounts for over 30% of primary care consultations and represents a growing healthcare challenge in an active and increasingly ageing population. Recognising critical cells involved in tendinopathy is essential in developing therapeutics to meet this challenge. Tendon cells are heterogenous and sparsely distributed in a dense collagen matrix; limiting previous methods to investigate cell characteristics ex vivo. We applied next generation CITE-sequencing; combining surface proteomics with in-depth, unbiased gene expression analysis of > 6400 single cells ex vivo from 11 chronically tendinopathic and 8 healthy human tendons. Immunohistochemistry validated the single cell findings. For the first time we show that human tendon harbours at least five distinct COL1A1/2 expressing tenocyte populations in addition to endothelial cells, T-cells, and monocytes. These consist of KRT7/SCX+ cells expressing microfibril associated genes, PTX3+ cells co-expressing high levels of pro-inflammatory markers, APOD+ fibro-adipogenic progenitors, TPPP3/PRG4+ chondrogenic cells, and ITGA7+ smooth muscle-mesenchymal cells. Surface proteomic analysis identified markers by which these sub-classes could be isolated and targeted in future. Chronic tendinopathy was associated with increased expression of pro-inflammatory markers PTX3, CXCL1, CXCL6, CXCL8, and PDPN by microfibril associated tenocytes. Diseased endothelium had increased expression of chemokine and alarmin genes including IL33.

The transcription factor scleraxis differentially regulates gene expression in tenocytes isolated at different developmental stages.

The transcription factor scleraxis (SCX) is expressed throughout tendon development and plays a key role in directing tendon wound healing. However, little is known regarding its role in fetal or young postnatal tendons, stages in development that are known for their enhanced regenerative capabilities. Here we used RNA-sequencing to compare the transcriptome of adult and fetal tenocytes following SCX knockdown. SCX knockdown had a larger effect on gene expression in fetal tenocytes, affecting 477 genes in comparison to the 183 genes affected in adult tenocytes, indicating that scleraxis-dependent processes may differ in these two developmental stages. Gene ontology, network and pathway analysis revealed an overrepresentation of extracellular matrix (ECM) remodelling processes within both comparisons. These included several matrix metalloproteinases, proteoglycans and collagens, some of which were also investigated in SCX knockdown tenocytes from young postnatal foals. Using chromatin immunoprecipitation, we also identified novel genes that SCX differentially interacts with in adult and fetal tenocytes. These results indicate a role for SCX in modulating ECM synthesis and breakdown and provide a useful dataset for further study into SCX gene regulation.

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.

Alda-1, an activator of ALDH2, ameliorates Achilles tendinopathy in cellular and mouse models.

Achilles tendinopathy has a high re-injury rate and poor prognosis. Development of effective therapy for Achilles tendinopathy is important. Excessive accumulation of ROS and resulting oxidative stress are believed to cause tendinopathy. Overproduction of hydrogen peroxide (H2O2), the most common ROS, could lead to the tendinopathy by causing oxidative damage, activation of endoplasmic reticulum (ER) stress and apoptotic death of tenocytes. Activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) is expected to alleviate oxidative stress and ER stress. Alda-1 is a selective and potent activator of ALDH2. In this study, we examined the cytoprotective benefit of Alda-1, an activator of ALDH2, on H2O2-induced Achilles tendinopathy in cellular and mouse models. We prepared cellular and mouse models of Achilles tendinopathy by treating cultured Achilles tenocytes and Achilles tendons with oxidative stressor H2O2. Subsequently, we studied the protective benefit of Alda-1 on H2O2-induced Achilles tendinopathy. Alda-1 pretreatment attenuated H2O2-induced cell death of cultured Achilles tenocytes. Treatment of Alda-1 prevented H2O2-induced oxidative stress and depolarization of mitochondrial membrane potential in tenocytes. Application of Alda-1 attenuated H2O2-triggered mitochondria- and ER stress-mediated apoptotic cascades in cultured tenocytes. Alda-1 treatment ameliorated the severity of H2O2-induced Achilles tendinopathy in vivo by preventing H2O2-induced pathological histological features of Achilles tendons, apoptotic death of Achilles tenocytes and upregulated expression of inflammatory cytokines IL-1ß and TNF-α. Our results provide the evidence that ALDH2 activator Alda-1 ameliorates H2O2-induced Achilles tendinopathy. Alda-1 could be used for preventing and treating Achilles tendinopathy.

The Effects of Corticosteroid Injection in the Healthy and Damaged Achilles Tendon Model: Histopathological and Biomechanical Experimental Study in Rats.

OBJECTIVE: To show the effects of corticosteroids on inflammatory reactions in the injured Achilles tendon in rats. MATERIAL AND METHOD: Thirty-two adult Wistar Albino rats were used in the study. The rats were divided into 4 groups. In the first group (Intact Saline), saline solution was injected to the intact Achilles tendon. In the second group (Intact Corticosteroid), corticosteroid was injected to the intact tendon. In the third group (Injured Saline), saline solution was injected to the injured Achilles tendon. In the fourth group (Injured Corticosteroid), corticosteroid was injected to the injured tendon. All groups were sacrificed on day 30 and Achilles tendons were taken and prepared for histological and biomechanical evaluation. RESULTS: According to the biomechanical test; mean load-to-failure of the Intact Saline group was significantly lower than the Intact Corticosteroid (p=0.016), Injured Saline (p=0.001) and Injured Corticosteroid) (p=0.012) groups. According to the histopathological evaluation, tenocyte mean of the Intact Saline group was statistically lower than the Injured Saline and Injured Corticosteroid groups. Tenocyte mean of the Intact Corticosteroid group was statistically significantly lower than the Injured Saline and Injured Corticosteroid groups. The ground substance mean of the Intact Saline group was significantly lower than the Injured Saline and Injured Corticosteroid groups. The ground substance mean of the Intact Corticosteroid group was significantly lower than the Injured Saline and Injured Corticosteroid groups. There was no statistically significant difference between the groups in terms of calcification. CONCLUSION: It has been found that there is biomechanical and histopathological significant benefit of intra-tendon corticosteroid administration in the experimentally generated Achilles tendon injury model.

Effects of Substrate Stiffness on Morphology and MMP-1 Gene Expression in Tenocytes Stimulated With Interleukin-1ß.

Tendon cells, tenocytes, are constantly subjected to mechanical stress in vivo, which maintains a level of cellular tension. When a tendon is subjected to overloading, local rupture of collagen fibers are induced, which deprives tenocytes of mechanical stress, lowers their cellular tension level and upregulates their catabolism. In addition, leukocytes are attracted to the rupture sites and produce interleukin-1ß (IL-1ß), and this exogenous IL-1ß also stimulates tenocyte catabolism. We tested a hypothesis that catabolic tenocytes with low cellular tension at the rupture sites excessively respond to the exogenous IL-1ß and further upregulate matrix metalloproteinase 1 (MMP-1) gene expression. Tenocytes from rabbit Achilles tendon were cultured on the following substrates: glass or polydimethylsiloxane micropillar substrates with a height of 2, 4, or 8 µm. Following a 3-day IL-1ß stimulation at a concentration of 0, 1, 10, or 100 pM, the effects of IL-1ß stimulation on cell morphology and MMP-1 gene expression was analysed with fluorescent microscopy and fluorescence in situ hybridization, respectively. In addition, the effects of IL-1ß stimulation on cell membrane fluidity were examined. It was demonstrated that the cells on 8-µm-height micropillars exhibited a greater response than those on rigid substrates with flat (glass) and topologically the same surface (2-µm-height micropillars) to IL-1ß when supplied at the same concentration. Besides this, membrane fluidity was lower in the cells on micropillars. Therefore, it appears that cellular attachment to softer substrates lowers the cellular actin cortex tension, reducing the membrane fluidity and possibly elevating the sensitivity of IL-1 receptors to ligand binding. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:150-159, 2020.

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.

High glucose alters tendon homeostasis through downregulation of the AMPK/Egr1 pathway.

Diabetes mellitus (DM) is associated with higher risk of tendinopathy, which reduces tolerance to exercise and functional activities and affects lifestyle and glycemic control. Expression of tendon-related genes and matrix metabolism in tenocytes are essential for maintaining physiological functions of tendon. However, the molecular mechanisms involved in diabetic tendinopathy remain unclear. We hypothesized that high glucose (HG) alters the characteristics of tenocyte. Using in vitro 2-week culture of tenocytes, we found that expression of tendon-related genes, including Egr1, Mkx, TGF-ß1, Col1a2, and Bgn, was significantly decreased in HG culture and that higher glucose consumption occurred. Down-regulation of Egr1 by siRNA decreased Scx, Mkx, TGF-ß1, Col1a1, Col1a2, and Bgn expression. Blocking AMPK activation with Compound C reduced the expression of Egr1, Scx, TGF-ß1, Col1a1, Col1a2, and Bgn in the low glucose condition. In addition, histological examination of tendons from diabetic mice displayed larger interfibrillar space and uneven glycoprotein deposition. Thus, we concluded that high glucose alters tendon homeostasis through downregulation of the AMPK/Egr1 pathway and the expression of downstream tendon-related genes in tenocytes. The findings render a molecular basis of the mechanism of diabetic tendinopathy and may help develop preventive and therapeutic strategies for the pathology.

Development of organic solvent-free micro-/nano-porous polymer scaffolds for musculoskeletal regeneration.

The use of biomaterial scaffolds has been an enormous field of research in tissue engineering, where the aim is to use graft materials for assisting the human body in recovering lost functions. Currently, there are many ways biomaterial scaffolds can be fabricated; however, many of these techniques involve the use of toxic organic solvents during the process. As biocompatibility is one of the mandatory requirements in designing a successful scaffold, there is an interest in fabricating scaffolds that are completely organic solvent-free. This paper describes the development and characterization of novel micro-/nano-fibrillar composites (MFC/NFC) that can produce scaffolds which are completely free from organic solvents. In this research, the cytocompatibility of these materials have been tested in vitro using mouse osteoblast-like cells and primary rat tenocytes, where cell numbers increase over the culture period, demonstrating the material viability. Gene expression analysis of primary rat tenocytes on MFC/NFC scaffolds demonstrate tenocytic behavior, and histology studies show an increase in cell formation on NFC scaffolds. This study establishes the potential of using the MFC/NFC technique to produce completely organic solvent-free scaffolds capable of hosting musculoskeletal cells, in the hope of providing a graft material for non-union skeletal fractures and rotator cuff repairs. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1393-1404, 2017.

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.