Rat Achilles Tendon Research Articles

Effect of storage and preconditioning of healing rat Achilles tendon on structural and mechanical properties

Tendon tissue storage and preconditioning are often used in biomechanical experiments and whether this generates alterations in tissue properties is essential to know. The effect of storage and preconditioning on dense connective tissues, like tendons, is fairly understood. However, healing tendons are unlike and contain a loose connective tissue. Therefore, we investigated if storage of healing tendons in the fridge or freezer changed the mechanical properties compared to fresh tendons, using a pull-to-failure or a creep test. Tissue morphology and cell viability were also evaluated. Additionally, two preconditioning levels were tested. Rats underwent Achilles tendon transection and were euthanized 12 days postoperatively. Statistical analyzes were done with one-way ANOVA or Student’s t-test. Tissue force and stress were unaltered by storage and preconditioning compared to fresh samples, while high preconditioning increased the stiffness and modulus (p ≤ 0.007). Furthermore, both storage conditions did not modify the viscoelastic properties of the healing tendon, but altered transverse area, gap length, and water content. Cell viability was reduced after freezing. In conclusion, preconditioning on healing tissues can introduce mechanical data bias when having extensive tissue strength diversity. Storage can be used before biomechanical testing if structural properties are measured on the day of testing.

Histopathological and immunohistochemical investigation of the local and systemic effects of tranexamic acid on the healing of the Achilles tendon in rats.

Objectives This study aims to compare the effects of systemic and local applications of tranexamic acid (TXA) on tendon healing using a rat Achilles tendon injury model.Patients and methods Thirty-six adult male albino Wistar rats (aging 3-4 months; weighing 350 to 400 g) were used in this study conducted between December 2019 and January 2020. The Achilles tendon was performed bilateral tenotomy and surgically repaired. Postoperatively, 1 mL of TXA was administered to each leg locally in the local group, whereas 2 mL of TXA was intraperitoneally administered in the systemic group. The control group was left untreated. Half of the rats were sacrificed on Day 15 and the other half on Day 30. Tendon healing was evaluated with the Bonar and the Movin scoring systems and immunohistochemical methods.Results The systemic group had the highest Bonar and Movin scores on Day 15. All groups exhibited tendon healing on Day 30, with no significant differences among the groups. The tenocyte morphology was found to be more impaired in both TXA groups on Day 30 (p=0.013). Ground substance scores were lower in the systemic group on Day 30 (p=0.028). The fiber structure and arrangement scores were higher in the systemic group on Day 15 (p=0.007 and p=0.032). Immunohistochemical analyses showed that galectin-3 values exhibited a significant difference in all groups on Day 30 (p=0.020). In all groups, it was determined that type I collagen values showed an increasing trend on Day 30, compared to the values on Day 15, whereas type III collagen values showed a decreasing trend.Conclusion Our results demonstrated that local and systemic use of TXA does not impair tendon healing. Although advanced studies are needed, our study suggests that TXA application reduces the development of fibrosis.

Liquid Poly-N-acetyl Glucosamine (sNAG) Improves Achilles Tendon Healing in a Rat Model.

The Achilles tendon, while the strongest and largest tendon in the body, is frequently injured. Even after surgical repair, patients risk re-rupture and long-term deficits in function. Poly-N-acetyl glucosamine (sNAG) polymer has been shown to increase the rate of healing of venous leg ulcers, and use of this material improved tendon-to-bone healing in a rat model of rotator cuff injury. Therefore, the purpose of this study was to investigate the healing properties of liquid sNAG polymer suspension in a rat partial Achilles tear model. We hypothesized that repeated sNAG injections throughout healing would improve Achilles tendon healing as measured by improved mechanical properties and cellular morphology compared to controls. Results demonstrate that sNAG has a positive effect on rat Achilles tendon healing at three weeks after a full thickness, partial width injury. sNAG treatment led to increased quasistatic tendon stiffness, and increased tangent and secant stiffness throughout fatigue cycling protocols. Increased dynamic modulus also suggests improved viscoelastic properties with sNAG treatment. No differences were identified in histological properties. Importantly, use of this material did not have any negative effects on any measured parameter. These results support further study of this material as a minimally invasive treatment modality for tendon healing.

Micro-Groove Patterned PCL Patches with DOPA for Rat Achilles Tendon Regeneration

Achilles tendon regeneration using current tissue engineering techniques requires morphometrically mimetic tendon biomaterials to attain the required microstructural tendon tissue and to achieve the necessary mechanical properties. In this study, we used nano- and micro-groove patterned polycaprolactone patches (PCL-400, PCL-5000), which offer adequate elastic modulus and biocompatibility. The PCL patches were fabricated via capillary force lithography and were surface modified with DOPA for increased hydrophilicity. We hypothesized that the resulting biologically surface-modified elastic groove patterned patches would enhance the cell behaviors in vitro and the rat Achilles tendon regeneration in vivo. The micro-groove patterned PCL patches with DOPA coating (DOPA-PCL-5000) exhibited excellent cell elongation, tenogenic differentiation and YAP expression of the mesenchymal stem cells in our in vitro study and these results were further confirmed by application to the rat Achilles tendon rupture model in our in vivo study. DOPA-PCL-5000 induced dramatic regeneration of rat Achilles tendon compared to the other groups, which was further confirmed by the Achilles functional index and histological analysis at 8 weeks. In conclusion, DOPA-PCL-5000 can be used not only for damaged Achilles tendon, but also for various functional tendon regeneration techniques such as rotator cuff, patellar tendon, and flexor tendon.

Histological evaluation of the effect of low-frequency electric stimulation on healing Achilles tendons in rats.

ABSTRACTPurpose:Histologically evaluate the effects of low frequency electrical stimulation in the treatment of Achilles tendon injuries in rats.Methods:Thirty-four rats underwent Achilles tendon tenotomy and tenorrhaphy. They were randomly allocated in two groups. Half of the sample constituted the experiment group, whose lesions were stimulated with 2 Hz, nonpolarized current and 1 mA, for 14 days. The other animals formed the control group. They were evaluated at 2, 4 and 6 weeks. The histological study was carried out, the collagen density and the wound maturity index were measured.Results:The healing score was higher in the group stimulated at the 6th week (p = 0.018). The density collagen 1 was higher in the group treated at the three times (p = 0.004) and that collagen 3 was higher in the group treated at 6 weeks (p = 0.004). Together, collagen 1 and 3 were higher in the group stimulated at 4 and 6 weeks (p = 0.009, p = 0.004). The maturity index was higher in this group at the three moments (p = 0.017 p = 0.004 and p = 0.009).Conclusion:Low frequency electric stimulation improved healing and increased the quantity of collagen.

Tenogenic effects of silymarin following experimental Achilles tendon transection in rats.

Tendon healing is prolonged due to the small number of cells, poor circulation, and low metabolism. The optimal tendon healing and its complete functional recovery have always been a challenge for researchers. Silymarin possesses anti-inflammatory, anti-oxidant, analgesic, and regenerative properties. The present study aimed to investigate the effects of silymarin on healing the Achilles tendon in rats. Twenty-four male Wistar rats were divided into two groups of control and treatment. After surgical preparation, a complete transverse incision was made in the middle part of the Achilles tendon, and then a modified Kessler suture was placed. The control group received 1.00 mL normal saline for five consecutive days, and the treatment group received 50.00 mg kg-1 of silymarin suspended in 1.00 mL normal saline for five days, orally. During the experimental period, Achilles functional index (AFI) was recorded. Six weeks after surgery, sampling was done. Histopathologically, a significant increase in the density of collagen fibers and reduction in neovascularization and inflammatory cells infiltration were observed in the treatment group. The biomechanical evaluation showed a significant increase in tensile strength of the tendon in the treatment group compared to the control group. The AFI results were concomitant with the results stated above, indicating an improvement in the AFI of rats in the treatment group. The present study results showed that oral administration of silymarin improved tissue healing indices, biomechanical properties, and functional index, leading to optimal healing of experimental Achilles tendon injury in the rat.

Collagen and chondroitin sulfate functionalized bioinspired fibers for tendon tissue engineering application

Functional tendon tissue engineering depends on harnessing the biochemical and biophysical cues of the native tendon extracellular matrix. In this study, we fabricated highly-aligned poly(L-lactic acid) (PLLA) fibers with surfaces decorated by two of the crucial tendon ECM components, type 1 collagen (COL1) and chondroitin sulfate (CS), through a coaxial stable jet electrospinning approach. Effects of the biomimetic COL1-CS (shell)/PLLA (core) fibers on the tenogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro were investigated. Higher rates of cell spreading and proliferation are observed on the aligned COL1-CS/PLLA fibers compared to that on the plain PLLA fibers. Expression of the tendon-associated genes scleraxis (SCX) and COL1 as well as protein tenomodulin (TNMD) are significantly increased. Introduction of mechanical stimulation gives rise to synergistic effect on tenogenic differentiation of hMSCs. Higher expression of TGF-β2, TGFβR-II, and Smad3 by the cells on the COL1-CS/PLLA fiber substrates are observed, which indicates that COL1-CS/PLLA ultrafine fibers dictate the hMSC tenogenic differentiation through activating the TGF-β signaling pathway. Animal study in rat Achilles tendon repair model corroborated the promoting role of COL1-CS/PLLA in regenerating a tendon-like tissue. Thus, our highly aligned biomimicking fibers may serve as an efficient scaffolding system for functional tendon regeneration.

Diminishing effects of mechanical loading over time during rat Achilles tendon healing.

Mechanical loading affects tendon healing and recovery. However, our understanding about how physical loading affects recovery of viscoelastic functions, collagen production and tissue organisation is limited. The objective of this study was to investigate how different magnitudes of loading affects biomechanical and collagen properties of healing Achilles tendons over time. Achilles tendon from female Sprague Dawley rats were cut transversely and divided into two groups; normal loading (control) and reduced loading by Botox (unloading). The rats were sacrificed at 1, 2- and 4-weeks post-injury and mechanical testing (creep test and load to failure), small angle x-ray scattering (SAXS) and histological analysis were performed. The effect of unloading was primarily seen at the early time points, with inferior mechanical and collagen properties (SAXS), and reduced histological maturation of the tissue in unloaded compared to loaded tendons. However, by 4 weeks no differences remained. SAXS and histology revealed heterogeneous tissue maturation with more mature tissue at the peripheral region compared to the center of the callus. Thus, mechanical loading advances Achilles tendon biomechanical and collagen properties earlier compared to unloaded tendons, and the spatial variation in tissue maturation and collagen organization across the callus suggests important regional (mechano-) biological activities that require more investigation.

Bionic Silk Fibroin Film Induces Morphological Changes and Differentiation of Tendon Stem/Progenitor Cells

Purpose Tendon injuries are common musculoskeletal system disorders, but the ability for tendon regeneration is limited. Silk fibroin (SF) film may be suitable for tendon regeneration due to its excellent biocompatibility and physical properties. This study is aimed at evaluating the application value of bionic SF film in tendon regeneration. Methods Tendon stem/progenitor cells (TSPCs) were isolated from rat Achilles tendon and characterized based on their surface marker expression and multilineage differentiation potential. SF films with smooth or bionic microstructure surfaces (5, 10, 15, 20 μm) were prepared. The morphology and mechanical properties of natural tendons and SF films were characterized. TSPCs were used as the seed cells, and the cell viability and cell adhesion morphology were analyzed. The tendongenesis-related gene expression of TSPCs was also evaluated using quantitative polymerase chain reaction. Results Compared to the native tendon, only the 10, 15, and 20 μm SF film groups had comparable maximum loading and ultimate stress, with the exception of the breaking elongation rate. The 10 μm SF film group had the highest percentage of oriented cells and the most significant changes in cell morphology. The most significant upregulations in the expression of COL1A1, TNC, TNMD, and SCX were also observed in the 10 μm SF film group. Conclusion SF film with a bionic microstructure can serve as a tissue engineering scaffold and provide biophysical cues for the use of TSPCs to achieve proper cellular adherence arrangement and morphology as well as promote the tenogenic differentiation of TSPCs, making it a valuable customizable biomaterial for future applications in tendon repair.

Galectin-3 Enhances Osteogenic Differentiation of Precursor Cells From Patients With Diffuse Idiopathic Skeletal Hyperostosis via Wnt/β-Catenin Signaling

Diffuse idiopathic skeletal hyperostosis (DISH) is a noninflammatory skeletal disease characterized by the progressive ectopic ossification and calcification of ligaments and enthuses. However, specific pathogenesis remains unknown. Bone marrow mesenchymal stem cells (BMSCs) are a major source of osteoblasts and play vital roles in bone metabolism and ectopic osteogenesis. However, it is unclear whether BMSCs are involved in ectopic calcification and ossification in DISH. The current study aimed to explore the osteogenic differentiation abilities of BMSCs from DISH patients (DISH-BMSCs). Our results showed that DISH-BMSCs exhibited stronger osteogenic differentiation abilities than normal control (NC)-BMSCs. Human cytokine array kit analysis showed significantly increased secretion of Galectin-3 in DISH-BMSCs. Furthermore, Galectin-3 downregulation inhibited the increased osteogenic differentiation ability of DISH-BMSCs, whereas exogenous Galectin-3 significantly enhanced the osteogenic differentiation ability of NC-BMSCs. Notably, the increased Galectin-3 in DISH-BMSCs enhanced the expression of β-catenin as well as TCF-4, whereas attenuation of Wnt/β-catenin signaling partially alleviated Galectin-3-induced osteogenic differentiation and activity in DISH-BMSCs. In addition, our results noted that Galectin-3 interacted with β-catenin and enhanced its nuclear accumulation. Further in vivo studies showed that exogenous Galectin-3 enhanced ectopic bone formation in the Achilles tendon in trauma-induced rats by activating Wnt/β-catenin signaling. The current study indicated that enhanced osteogenic differentiation of DISH-BMSCs was mainly attributed to the increased secretion of Galectin-3 by DISH-BMSCs, which enhanced β-catenin expression and its nuclear accumulation. Our study helps illuminate the mechanisms of pathological osteogenesis and sheds light on the possible development of potential therapeutic strategies for DISH treatment. © 2022 American Society for Bone and Mineral Research (ASBMR).

Dry Needling of a Healthy Rat Achilles Tendon Increases Its Gene Expressions: A Pilot Study.

Tendon dry needling is a potential treatment for tendinopathies. Several hypotheses have been proposed to explain its underlying mechanisms. No studies (to the best of our knowledge) have investigated changes in gene expression. To investigate histological and gene expression changes after the application of dry needling to the healthy Achilles tendons of rats. Six Sprague-Dawley male rats were randomly divided into two groups: no intervention or dry needling. Dry needling consisted of three sessions (once per week) to the Achilles tendon. Molecular expression of several genes involved in tendon repair and remodeling (e.g., Cox2, Mmp2, Mmp9, Col1a1, Col3a1, Vefg, and Scx) was assessed 7days after the last needling session (day 28) or 28days after for the no-intervention group. Histological tissue changes were determined with hematoxylin-eosin analyses. The hematoxylin-eosin-stained images revealed no substantial differences in collagen structure or the presence of inflammatory cells between the dry needling and no-intervention groups. A significant increase in the molecular expression of Cox2, Mmp2, Col3a1, and Scx genes was observed in Achilles tendons treated with dry needling when compared with the no-intervention group. This animal pilot study found that the application of dry needling to the healthy Achilles tendons of rats is able to increase the expression of genes associated with collagen regeneration and tissue remodeling of the extracellular matrix with no further histological damage to the tendon.

Modulation of vascular response after injury in the rat Achilles tendon alters healing capacity.

Tendons are relatively hypovascular but become hypervascular during both injury and degeneration. This is due to the angiogenic response, or the formation of new blood vessels, to tissue injury. The objective of this study was to evaluate the effect of vascular modulation in the rat Achilles tendons during healing. Fischer rats received a bilateral Achilles incisional injury followed by local injections of vascular endothelial growth factor (VEGF), anti-VEGF antibody (B20.4-1-1), or salineeither early or late during the healing process. Vascular modulation and healing were evaluated using multiple in vivo ultrasound imaging modalities, in vivo functional assessment, and ex vivo measures of tendon compositional and mechanical properties. The late delivery of anti-VEGF antibody, B20, caused a temporary reduction in healing capacity during a time point where vascularity was also decreased, and then an improvement during a later time point where vascularity was increased relative to control. However, VEGF delivery had a minimal impact on healing and vascular changes in both early and late delivery times. This study was the first to evaluate vascular changes using both in vivo imaging methods and ex vivo histological methods, as well as functional and mechanical outcomes associated with these vascular changes. Clinical significance: this study demonstrates that the alteration of vascular response through the delivery of angiogenic growth factors has the ability to alter tendon healing properties.

Higher BMP Expression in Tendon Stem/Progenitor Cells Contributes to the Increased Heterotopic Ossification in Achilles Tendon With Aging.

Although the mineralization in tendon tissue has been reported in a series of aging and disease models, the underlying mechanisms remain unknown. This study aimed to describe the appearance of heterotopic ossification in rat Achilles tendon and further verify whether this tissue metaplasia is related to the enhanced osteogenic differentiation of tendon stem/progenitor cells (TSPCs) owing to the higher expression of bone morphogenetic proteins (BMP-2/4/7) with aging. The male SD rats, aged 4, 8, and 20 months (M), were used. The analyses of ossification and BMP expression in tendon were tested by radiological view (X-ray and CT), histological staining [hematoxylin and eosin (HE), Alcian blue, and Alizarin red], immunohistochemistry, and Western blot. The osteogenic differentiation potential and BMP expression of TSPCs were examined by Alizarin red S staining and real-time PCR. TSPCs were treated with BMP-2 or noggin, and the osteogenic differentiation potential was also examined. X-ray and CT showed the appearance of heterotopic ossification in tendon, and the volume and density of ossification was increased with aging. Histological staining showed the appearance of calcified region surrounded by chondrocyte-like cells and the increased osteogenesis-related gene and BMP expression in ossified tendon with aging. Moreover, the osteogenic differentiation potential and BMP expression in TSPCs isolated from ossified tendon were increased with aging. Additionally, BMP-2 increased the calcium nodule formation and osteogenesis-related gene expression in TSPCs. The addition of noggin inhibited BMP-induced enhancement of osteogenic differentiation. Thus, these findings suggested that the enhanced osteogenic differentiation of TSPCs contributes to the increased heterotopic ossification in aged tendon, which might be induced by the higher expression of BMPs with aging.

Tendon stem cell-derived exosomes regulate inflammation and promote the high-quality healing of injured tendon

BackgroundTendon stem cells (TSCs) have been reported to hold promises for tendon repair and regeneration. However, less is known about the effects of exosomes derived from TSCs. Therefore, we aimed to clarify the healing effects of TSC-derived exosomes (TSC-Exos) on tendon injury.MethodsThe Achilles tendons of Sprague-Dawley male rats were used for primary culture of TSCs and tenocytes, and exosomes were isolated from TSCs. The proliferation of tenocytes induced by TSC-Exos was analyzed using an EdU assay; cell migration was measured by cell scratch and transwell assays. We used western blot to analyze the role of the PI3K/AKT and MAPK/ERK1/2 signaling pathways. In vivo, Achilles tendon injury models were created in Sprague-Dawley rats. Rats (n = 54) were then randomly assigned to three groups: the TSC-Exos group, the GelMA group, and the control group. We used immunofluorescence to detect changes in the expression of inflammatory and apoptotic markers at 1 week after surgery. Histology and changes in expression of extracellular matrix (ECM)-related indices were assessed by hematoxylin-eosin (H&E) staining and immunohistochemistry at 2 and 8 weeks. The collagen fiber diameter of the healing tendon was analyzed at 8 weeks by transmission electron microscopy (TEM).ResultsTSC-Exos were taken up by tenocytes, which promoted the proliferation and migration of cells in a dose-dependent manner; this process may depend on the activation of the PI3K/AKT and MAPK/ERK1/2 signaling pathways. At 1 week after surgery, we found that inflammation and apoptosis were significantly suppressed by TSC-Exos. At 2 and 8 weeks, tendons treated with TSC-Exos showed more continuous and regular arrangement in contrast to disorganized tendons in the GelMA and control groups, and TSC-Exos may help regulate ECM balance and inhibited scar formation. Further, at 8 weeks, the TSC-Exos group had a larger diameter of collagen compared to the control group.ConclusionsOur data suggest that TSC-Exos could promote high-quality healing of injured tendon, which may be a promising therapeutic approach for tendon injury.

Improving the regenerative microenvironment during tendon healing by using nanostructured fibrin/agarose-based hydrogels in a rat Achilles tendon injury model.

Achilles tendon injuries are a frequent problem in orthopaedic surgery due to their limited healing capacity and the controversy surrounding surgical treatment. In recent years, tissue engineering research has focused on the development of biomaterials to improve this healing process. The aim of this study was to analyze the effect of tendon augmentation with a nanostructured fibrin-agarose hydrogel (NFAH) or genipin cross-linked nanostructured fibrin-agarose hydrogel (GP-NFAH), on the healing process of the Achilles tendon in rats. NFAH, GP-NFAH, and MatriDerm (control) scaffolds were generated (five in each group). A biomechanical and cell-biomaterial-interaction characterization of these biomaterials was then performed: Live/Dead Cell Viability Assay, water-soluble tetrazolium salt-1 (WST-1) assay, and DNA-released after 48 hours. Additionally, a complete section of the left Achilles tendon was made in 24 Wistar rats. Animals were separated into four treatment groups (six in each group): direct repair (Control), tendon repair with MatriDerm, or NFAH, or GP-NFAH. Animals were euthanized for further histological analyses after four or eight weeks post-surgery. The Achilles tendons were harvested and a histopathological analysis was performed. Tensile test revealed that NFAH and GP-NFAH had significantly higher overall biomechanical properties compared with MatriDerm. Moreover, biological studies confirmed a high cell viability in all biomaterials, especially in NFAH. In addition, in vivo evaluation of repaired tendons using biomaterials (NFAH, GP-NFAH, and MatriDerm) resulted in better organization of the collagen fibres and cell alignment without clinical complications than direct repair, with a better histological score in GP-NFAH. In this animal model we demonstrated that NFAH and GP-NFAH had the potential to improve tendon healing following a surgical repair. However, future studies are needed to determine the clinical usefulness of these engineered strategies. Cite this article: Bone Joint J 2020;102-B(8):1095-1106.

Short- and Long-Term Exercise Results in a Differential Achilles Tendon Mechanical Response.

The study was conducted to define the biomechanical response of rat Achilles tendon after a single bout of exercise and a short or long duration of daily exercise. We hypothesized that a single bout or a short duration of exercise would cause a transient decrease in Achilles tendon mechanical properties and a long duration of daily exercise would improve these properties. One hundred and thirty-six Sprague-Dawley rats were divided into cage activity (CA) or exercise (EX) groups for a single bout, short-term, or long-term exercise. Animals in single bout EX groups were euthanized, 3, 12, 24, or 48 h upon completion of a single bout of exercise (10 m/min, 1 h) on a flat treadmill. Animals in short-term EX groups ran on a flat treadmill for 3 days, 1, or 2 weeks while animals in long-term EX groups ran for 8 weeks. Tendon quasi-static and viscoelastic response was evaluated for all Achilles tendons. A single bout of exercise increased tendon stiffness after 48 h of recovery. Short-term exercise up to 1 week decreased cross-sectional area, stiffness, modulus, and dynamic modulus of the Achilles tendon. In contrast, 8 weeks of daily exercise increased stiffness, modulus, and dynamic modulus of the tendon. This study highlights the response of Achilles tendons to single and sustained bouts of exercise. Adequate time intervals are important to allow for tendon adaptations when initiating a new training regimen and overall beneficial effects to the Achilles tendon.

Bimodal Whole-Mount Imaging of Tendon Using Confocal Microscopy and X-ray Micro-Computed Tomography

BackgroundThree-dimensional imaging modalities for optically dense connective tissues such as tendons are limited and typically have a single imaging methodological endpoint. Here, we have developed a bimodal procedure utilising fluorescence-based confocal microscopy and x-ray micro-computed tomography for the imaging of adult tendons to visualise and analyse extracellular sub-structure and cellular composition in small and large animal species.ResultsUsing fluorescent immunolabelling and optical clearing, we visualised the expression of the novel cross-species marker of tendon basement membrane, laminin-α4 in 3D throughout whole rat Achilles tendons and equine superficial digital flexor tendon 5 mm segments. This revealed a complex network of laminin-α4 within the tendon core that predominantly localises to the interfascicular matrix compartment. Furthermore, we implemented a chemical drying process capable of creating contrast densities enabling visualisation and quantification of both fascicular and interfascicular matrix volume and thickness by x-ray micro-computed tomography. We also demonstrated that both modalities can be combined using reverse clarification of fluorescently labelled tissues prior to chemical drying to enable bimodal imaging of a single sample.ConclusionsWhole-mount imaging of tendon allowed us to identify the presence of an extensive network of laminin-α4 within tendon, the complexity of which cannot be appreciated using traditional 2D imaging techniques. Creating contrast for x-ray micro-computed tomography imaging of tendon using chemical drying is not only simple and rapid, but also markedly improves on previously published methods. Combining these methods provides the ability to gain spatio-temporal information and quantify tendon substructures to elucidate the relationship between morphology and function.

MicroRNA-21-3p Engineered Umbilical Cord Stem Cell-Derived Exosomes Inhibit Tendon Adhesion.

PurposeAs a common complication of tendon injury, tendon adhesion is an unresolved problem in clinical work. The aim of this study was to investigate whether human umbilical cord mesenchymal stem cell-derived exosomes (HUMSC-Exos), one of the most promising new-generation cell-free therapeutic agents, can improve tendon adhesion and explore potential-related mechanisms.MethodsThe rat Achilles tendon injury adhesion model was constructed in vivo, and the localization of HUMSC-Exos was used to evaluate the tendon adhesion. Rat fibroblast cell lines were treated with transforming growth factor β1 (TGF-β1) and/or HUMSC-Exos in vitro, and cell proliferation, apoptosis and gene expression were measured. MicroRNA (miRNA) sequencing and quantitative PCR (qPCR) analysis confirmed differential miRNAs. A specific miRNA antagonist (antagomir-21a-5p) was used to transform HUMSC-Exos and obtain modified exosomes to verify its efficacy and related mechanism of action.ResultsIn this study, we found HUMSC-Exos reduced rat fibroblast proliferation and inhibited the expression of fibrosis genes: collagen III (COL III) and α-smooth muscle actin (α-SMA) in vitro. In the rat tendon adhesion model, topical application of HUMSC-Exos contributed to relief of tendon adhesion. Specifically, the fibrosis and inflammation-related genes were simultaneously inhibited by HUMSC-Exos. Further, miRNA sequencing of HUMSCs and HUMSC-Exos showed that miR-21a-3p was expressed at low abundance in HUMSC-Exos. The antagonist targeting miR-21a-3p was recruited for treatment of HUMSCs, and harvested HUMSC-Exos, which expressed low levels of miR-21a-3p, and expanded the inhibition of tendon adhesion in subsequent in vitro experiments.ConclusionOur results indicate that HUMSC-Exos may manipulate p65 activity by delivering low-abundance miR-21a-3p, ultimately inhibiting tendon adhesion. The findings may be promising for dealing with tendon adhesion.

Microcurrent and adipose-derived stem cells modulate genes expression involved in the structural recovery of transected tendon of rats.

Tendon injuries are common and have a high incidence of re-rupture that can cause loss of functionality. Therapies with adipose-derived stem cells (ASC) and the microcurrent (low-intensity electrical stimulation) application present promising effects on the tissue repair. We analyzed the expression of genes and the participation of some molecules potentially involved in the structural recovery of the Achilles tendon of rats, in response to the application of both therapies, isolated and combined. The tendons were distributed in five groups: normal (N), transected (T), transected and ASC (C) or microcurrent (M) or with ASC, and microcurrent (MC). Microcurrent therapy was beneficial for tendon repair, as it was observed a statistically significant increase in the organization of the collagen fibers, with involvement of the TNC, CTGF, FN, FMDO, and COL3A1 genes as well as PCNA, IL-10, and TNF-α. ASC therapy significantly increased the TNC and FMDO genes expression with no changes in the molecular organization of collagen. With the association of therapies, a significant greater collagen fibers organization was observed with involvement of the FMOD gene. The therapies did not affect the expression of COL1A1, SMAD2, SMAD3, MKX, and EGR1 genes, nor did they influence the amount of collagen I and III, caspase-3, tenomodulin (Tnmd), and hydroxyproline. In conclusion, the application of the microcurrent isolated or associated with ASC increased the organization of the collagen fibers, which can result in a greater biomechanical resistance in relation to the tendons treated only with ASC. Future studies will be needed to demonstrate the biological effects of these therapies on the functional recovery of injured tendons.

Heterogeneity of proteome dynamics between connective tissue phases of adult tendon.

Maintenance of connective tissue integrity is fundamental to sustain function, requiring protein turnover to repair damaged tissue. However, connective tissue proteome dynamics remain largely undefined, as do differences in turnover rates of individual proteins in the collagen and glycoprotein phases of connective tissue extracellular matrix (ECM). Here, we investigate proteome dynamics in the collagen and glycoprotein phases of connective tissues by exploiting the spatially distinct fascicular (collagen-rich) and interfascicular (glycoprotein-rich) ECM phases of tendon. Using isotope labelling, mass spectrometry and bioinformatics, we calculate turnover rates of individual proteins within rat Achilles tendon and its ECM phases. Our results demonstrate complex proteome dynamics in tendon, with ~1000 fold differences in protein turnover rates, and overall faster protein turnover within the glycoprotein-rich interfascicular matrix compared to the collagen-rich fascicular matrix. These data provide insights into the complexity of proteome dynamics in tendon, likely required to maintain tissue homeostasis.