Mechanical force regulates Sox9 expression at the developing enthesis.

Entheses transmit force from tendons and ligaments to the skeleton. Regional organization of enthesis extracellular matrix (ECM) generates differences in stiffness required for force transmission. Two key transcription factors co-expressed in entheseal tenocytes, scleraxis (Scx) and Sox9, directly control production of enthesis ECM components. Formation of embryonic craniofacial entheses in zebrafish coincides with onset of jaw movements, possibly in response to the force of muscle contraction. We show dynamic changes in scxa and sox9a mRNA levels in subsets of entheseal tenocytes that correlate with their roles in force transmission. We also show that transcription of a direct target of Scxa, Col1a, in enthesis ECM is regulated by the ratio of scxa to sox9a expression. Eliminating muscle contraction by paralyzing embryos during early stages of musculoskeletal differentiation alters relative levels of scxa and sox9a in entheses, primarily owing to increased sox9a expression. Force-dependent TGF-ß (TGFß) signaling is required to maintain this balance of scxa and sox9a expression. Thus, force from muscle contraction helps establish a balance of transcription factor expression that controls specialized ECM organization at the tendon enthesis and its ability to transmit force.

Development and maintenance of tendons and ligaments.

Tendons and ligaments are fibrous connective tissues vital to the transmission of force and stabilization of the musculoskeletal system. Arising in precise regions of the embryo, tendons and ligaments share many properties and little is known about the molecular differences that differentiate them. Recent studies have revealed heterogeneity and plasticity within tendon and ligament cells, raising questions regarding the developmental mechanisms regulating tendon and ligament identity. Here, we discuss recent findings that contribute to our understanding of the mechanisms that establish and maintain tendon progenitors and their differentiated progeny in the head, trunk and limb. We also review the extent to which these findings are specific to certain anatomical regions and model organisms, and indicate which findings similarly apply to ligaments. Finally, we address current research regarding the cellular lineages that contribute to tendon and ligament repair, and to what extent their regulation is conserved within tendon and ligament development.

Secretome from myoblasts statically loaded at low intensity promotes tenocyte proliferation via the IGF-1 receptor pathway.

Exercise is widely recognized as beneficial for tendon healing. Recently, it has been described that muscle-derived molecules secreted in response to static exercise influence tendon healing. In this study, the optimal static loading intensity for tendon healing and the composition of secretome released by myoblasts in response to different intensities of static strain were investigated. In an in vitro coculture model, myoblasts were mechanically loaded using a Flexcell Tension System. Tenocytes were seeded on transwell inserts that allowed communication between the tenocytes and myoblasts without direct contact. Proliferation and migration assays, together with RNA sequencing, were used to determine potential cellular signaling pathways. The secretome from myoblasts exposed to 2% static loading increased the proliferation and migration of the cocultured tenocytes. RNA-seq analysis revealed that this loading condition upregulated the expression of numerous genes encoding secretory proteins, including insulin-like growth factor-1 (IGF-1). Confirmation of IGF-1 expression and secretion was carried out using qPCR and enzyme-linked immunosorbt assay (ELISA), revealing a statistically significant upregulation in response to 2% static loading in comparison to both control conditions and higher loading intensities of 5% and 10%. Addition of an inhibitor of the IGF-1 receptor (PQ401) to the tenocytes significantly reduced myoblast secretome-induced tenocyte proliferation. In conclusion, IGF-1 may be an important molecule in the statically loaded myoblast secretome, which is responsible for influencing tenocytes during exercise-induced healing.

Exosomes derived from mir-337-3p over-expressing tendon stem cells protect against apoptosis of tenocytes via targeting caspase3.

BACKGROUND: Tendons are important dense fibrous structures connecting muscle to bone, and tendon stem cells (TDSCs) affect their repair and regeneration. The role of TDSC-derived exosomes (TDSC-Exos) is still being unexplored; therefore, this study aimed to investigate the protective effect of TDSC-Exos on tenocytes. METHODS: The TDSCs and tenocytes were all derived from Sprague Dawley (SD) rats. The expression of positive and negative markers of TDSCs were detected by flow cytometry, and the multi-differentiation ability was also detected to identify TDSCs. Exos were derived from TDSCs using ultracentrifugation; furthermore, Exos enriched with microRNA(miR)-377-3p were generated from TDSCs stably overexpressing miR-377-3p after transfection, identified with transmission electron microscopy (TEM), western blot and PKH26 staining assay. Moreover, the cell functions of tenocytes were evaluated by MTT, EdU, transwell, and flow cytometry. Dual luciferase reporter and RNA pull-down assays were used to verify the binding sites of miR-337-3p and caspase3 (CASP3) predicted by Targetscan. RESULTS: Exos (miR-337-3p) were taken up by tenocytes, and promoted the proliferation, migration, and invasion and suppressed the apoptosis of tenocytes in a dose-dependent manner. Bioinformatics analysis showed that CASP3 was a target of miR-377-3p, which was further verified by luciferase and RNA pull-down assays. Moreover, over-expressed CASP3 reversed the effects of Exos (miR-337-3p) on cell functions of tenocytes. CONCLUSIONS: Our findings suggest that Exos derived from miR-337-3p over-expressing TDSCs could potentially protect against tenocyte apoptosis by regulating CASP3. This novel therapeutic approach holds promise for the treatment of tendon injury, offering a glimmer of hope for improved patient outcomes.

Basic calcium phosphate crystals induce the expression of extracellular matrix remodelling enzymes in tenocytes.

OBJECTIVES: Basic calcium phosphate (BCP) crystals contribute to several syndromes associated with tendon disease, including acute calcific tendinitis and Milwaukee shoulder syndrome. Interactions between BCP crystals and tenocytes (tendon cells) may contribute to these clinical syndromes. This study aimed to determine the direct effects of BCP crystals on tenocyte function and viability. METHODS: In vitro assays were used to assess changes in human tenocytes cultured with BCP crystals. Real-time PCR was used to determine changes in the expression of tendon-related genes and extracellular matrix remodelling enzymes (MMPs; a disintegrin and metalloproteases, ADAMTS; and tissue inhibitor of metalloproteinases, TIMPs). ELISA was used to measure protein concentrations in tenocyte supernatants. MTT and alamarBlue™ assays were used to determine changes in cell viability. RESULTS: BCP crystals upregulated tenocyte gene expression of MMP-1, MMP-3, ADAMTS-4 and TIMP-1 after 24 h. Time-course experiments showed expression peaked at 8 h for TIMP-1 and 48 h for MMP-1 and ADAMTS-4. Cyclooxygenase (COX)-1 gene expression was upregulated after 48 h. Tenocytes did not alter expression of scleraxis and tendon collagens, and expression of pro-inflammatory cytokines was not induced with BCP crystals. BCP crystals increased tenocyte release of prostaglandin E2 (PGE2) and MMP-1 protein after 24 h. However, neither COX-1 inhibition nor COX-2 inhibition led to consistent change in BCP crystal-induced tenocyte gene expression of extracellular matrix remodelling enzymes. BCP crystals had no effect on tenocyte viability. CONCLUSION: BCP crystals induce extracellular matrix remodelling enzymes, but not inflammatory cytokines, in tenocytes.

Mechanical stretch facilitates tenomodulin expression to induce tenocyte migration via MAPK signaling pathway.

Tenomodulin (Tnmd) is a type II transmembrane glycoprotein that regulates tendon development and maturation. Our previous study indicated that mechanical stretch could induce Tnmd expression to promote tenocyte migration, associated with reinforcement of fibrous actin (F-actin) stress fibers and chromatin decondensation. However, the detailed molecular mechanisms of this processes are far from clear. Activation of mitogen-activated protein kinase (MAPK) signaling occurs in response to various extracellular stimuli and controls a large number of fundamental cellular processes. The present study we investigated the influence of MAPK signaling on mechanical stretch-induced Tnmd expression and its action way. Expression and activities of extracellular signal-related kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 MAPK (p38) were determined by Western blot. Cell migration was detected by Transwell assay. Immunofluorescence staining was used to detect F-actin stress fibers. Nuclear chromatin decondensation was detected by in situ DNaseI sensitivity assay. It was found that mechanical stretch promoted Tnmd expression by activating ERK1/2, JNK and p38 signaling. The inhibition of the ERK1/2, JNK or p38 repressed mechanical stretch-promoted tenocyte migration and mechanical stretch-induced reinforcement of F-actin stress fibers. However, only ERK1/2 and p38 inhibitor could repress mechanical stretch-induced chromatin decondensation, and the JNK inhibitor had no significant effect. Moreover, latrunculin (Lat A), the most widely used reagent to depolymerize actin filaments, could inhibit the stretch-induced chromatin decondensation. Taken together, our findings elucidated a molecular pathway by which a mechanical signal is transduced via activation of MAPK signaling to influence reinforcement of F-actin stress fibers and chromatin decondensation, which could further lead Tnmd expression to promote tenocyte migration.

Mechanical stretch promotes tenocyte migration via chromatin remodelling-mediated nuclear morphology changes.

Wound healing and function recovery of injured tendons are still a big challenge for orthopaedic surgery. Evidence in clinic shows that early controlled motion has significant favourable effects on tendon healing; however, the mechanisms involved in are not fully understood. In the present study, it was shown that an appropriate mechanical stretch (10% strain, 0.5 Hz for 1 h) evidently promotes rat tenocyte migration and nuclear morphology changes. The farther research discovered that mechanical stretch had no effect on Lamin A/C expression, but it could promote chromatin decondensation. Moreover, the histone modification plays an important role in mechanical stretch-mediated chromatin decondensation. Inhibition histone modification could inhibit mechanical stretch-promoted nuclear morphology changes and tenocyte migration. These results indicating that mechanical stretch may promote tenocyte migration via chromatin remodelling-mediated nuclear morphology changes, which contribute to a better understanding of the role of mechanical stretch on tenocyte migration and repair of injured tendon.

Quantifying the magnitude of local tendon injury from electrosurgical transection.

BACKGROUND: Electrocautery is a common surgical technique and is often used during shoulder arthroplasty to elevate or transect the subscapularis tendon. The relative amount of tissue damage caused by cautery as opposed to sharp transection is not currently known. The purpose of this study was to examine local tissue damage resulting from electrocautery vs. sharp transection with a scalpel. We hypothesized that the electrosurgical unit would cause higher collateral tissue damage and cell death compared with sharp transection. METHODS: Twelve cadaveric ovine shoulders were randomized to either the electrosurgical or sharp transection group. The infraspinatus tendon was isolated, and a partial-thickness transection was made using either a monopolar electrosurgical device (Bovie) or No. 10 scalpel blade. Tendon explants were then visualized with confocal microscopy to evaluate tissue architecture. A live/dead assay was performed using microscopy imaging analysis software. Comparisons between Bovie and scalpel transection were made using the Mann-Whitney U test, and the cell death percentage at standardized distances from the transection site was compared between groups using a mixed-model analysis. Significance was defined at P < .05. RESULTS: The cellular and tendon fibril architecture was well maintained beyond the scalpel transection site, whereas Bovie transection disrupted the architecture beyond its transection path. The percentage of dead cells in the Bovie group (74.9% ± 31.2%) was significantly higher than that in the scalpel group (27.6% ± 29.9%, P = .0004). Compared with the transection site, the cell death percentage after Bovie transection significantly declined at 2.5 mm whereas that after scalpel transection significantly declined at 1 mm from the transection site. CONCLUSION: There was a significantly higher dead cell percentage in the Bovie transection group, indicating extensive damage beyond the local incision site, compared with sharp transection. Electrosurgical transection of the ovine infraspinatus tendon ex vivo caused higher cell death and greater tissue architecture disruption compared with sharp scalpel transection.

Three-dimensional ultrastructure reconstruction of tendinous components at the bifurcation of the bovine superficial digital flexor tendon using array and STEM tomographies.

Tendons transmit force from muscle to bone for joint movement. Tenocytes are a specialized type of fibroblast that produces collagen fibrils in tendons. Their cytoplasmic processes form a network surrounding collagen fibrils to define a collagen fibre. Glycosaminoglycan (GAG) chains link collagen fibrils and adhere at the D-band of the collagen fibril. In this study, we used array and scanning transmission electron microscope (STEM) tomographies to reconstruct the three-dimensional ultrastructure of tenocytes, collagen fibres, collagen fibrils and GAG chains at the bifurcation of the bovine hindlimb superficial digital flexor tendon (SDFT). Collagen fibrils comprising a collagen fibre were not aligned uniformly and had at least two running directions. Spindle-shaped tenocytes were arranged along the long axis of a plurality of collagen fibres, where two groups of collagen fibrils with oblique directions to each other exhibited an oblique overlap of the two collagen fibril layers. Collagen fibrils with different running directions were observed in separating layers of about 300 nm in thickness and had diameters of 0-200 nm. About 40% of all collagen fibrils had a peak in the range of 20-40 nm. STEM analysis of the same site where the crossing of collagen fibres was observed by transmission electron microscopy demonstrated the outline of collagen fibrils with a clear D-banding pattern at a regular interval. Collagen fibrils were reconstructed three-dimensionally using continuous images acquired by STEM tomography, which confirmed that the collagen fibrils at the crossing sites did not orientate in layers, but were woven one by one. Higher magnification observation of GAG chains attached between the crossing collagen fibrils revealed numerous GAG chains arranged either vertically or obliquely on collagen fibrils. Furthermore, GAG chains at the cross of collagen fibrils connected the closest D-bands. GAG chains are thought to be universally present between collagen fibrils of the tendon. These observations by array and STEM tomographies increase our knowledge of the anatomy in the bifurcation of the bovine hindlimb SDFT and demonstrate the utility of these new imaging technologies.

Development of pluripotent stem cell-based human tenocytes.

Human pluripotent stem cells (PSCs) are used as a platform for therapeutic purposes such as cell transplantation therapy and drug discovery. Another motivation for studying PSCs is to understand human embryogenesis and development. All cell types that make up the body tissues develop through defined trajectories during embryogenesis. For example, paraxial mesoderm is considered to differentiate into several cell types including skeletal muscle cells, chondrocytes, osteocytes, dermal fibroblasts, and tenocytes. Tenocytes are fibroblast cells that constitute the tendon. The step-wise narrowing fate decisions of paraxial mesoderm in the embryo have been modeled in vitro using PSCs; however, deriving tenocytes from human-induced PSCs and their application in cell therapy have long been challenging. PSC-derived tenocytes can be used for a source of cell transplantation to treat a damaged or ruptured tendon due to injury, disorder, or aging. In this review, we discuss the latest research findings on the use of PSCs for studying the biology of tenocyte development and their application in therapeutic settings.

Stretch-Induced Tenomodulin Expression Promotes Tenocyte Migration via F-Actin and Chromatin Remodeling.

The mechanosensitive gene tenomodulin (Tnmd) is implicated in tendon maturation and repair. However, the mechanism by which mechanical loading regulates Tnmd's expression and its role in tenocyte migration is yet to be defined. Here, we show that Tnmd and migration were upregulated in uniaxial cyclic stress-stimulated tenocytes. The knockdown of Tnmd reduced cell migration in the presence and absence of mechanical loading, suggesting that Tnmd is involved in tenocyte migration. Moreover, the treatment of stress-stimulated tenocytes with the actin inhibitor latrunculin (Lat A), histone acetyltransferase inhibitor anacardic acid (ANA), or histone demethylases inhibitor GSK-J4 suppressed Tnmd expression and tenocyte migration. These results show that actin stress fiber formation and chromatin decondensation regulates Tnmd expression, which might then regulate tenocyte migration. Thus, this study proposes the involvement of the actin and chromatin mechanotransduction pathway in the regulation of Tnmd and reveals a novel role of Tnmd in tenocyte migration. The identification of Tnmd function in tenocyte migration provides insight into the molecular mechanisms involved in Tnmd-mediated tendon repair.

Secretome from In Vitro Mechanically Loaded Myoblasts Induces Tenocyte Migration, Transition to a Fibroblastic Phenotype and Suppression of Collagen Production.

It is known that mechanical loading of muscles increases the strength of healing tendon tissue, but the mechanism involved remains elusive. We hypothesized that the secretome from myoblasts in co-culture with tenocytes affects tenocyte migration, cell phenotype, and collagen (Col) production and that the effect is dependent on different types of mechanical loading of myoblasts. To test this, we used an in vitro indirect transwell co-culture system. Myoblasts were mechanically loaded using the FlexCell® Tension system. Tenocyte cell migration, proliferation, apoptosis, collagen production, and several tenocyte markers were measured. The secretome from myoblasts decreased the Col I/III ratio and increased the expression of tenocyte specific markers as compared with tenocytes cultured alone. The secretome from statically loaded myoblasts significantly enhanced tenocyte migration and Col I/III ratio as compared with dynamic loading and controls. In addition, the secretome from statically loaded myoblasts induced tenocytes towards a myofibroblast-like phenotype. Taken together, these results demonstrate that the secretome from statically loaded myoblasts has a profound influence on tenocytes, affecting parameters that are related to the tendon healing process.

Single-cell transcriptomic analysis identifies extensive heterogeneity in the cellular composition of mouse Achilles tendons.

Tendon is a dense connective tissue that stores and transmits forces between muscles and bones. Cellular heterogeneity is increasingly recognized as an important factor in the biological basis of tissue homeostasis and disease, yet little is known about the diversity of cell types that populate tendon. To address this, we determined the heterogeneity of cell populations within mouse Achilles tendons using single-cell RNA sequencing. In assembling a transcriptomic atlas of Achilles tendons, we identified 11 distinct types of cells, including three previously undescribed populations of tendon fibroblasts. Prior studies have indicated that pericytes, which are found in the vasculature of tendons, could serve as a potential source of progenitor cells for adult tendon fibroblasts. Using trajectory inference analysis, we provide additional support for the notion that pericytes are likely to be at least one of the progenitor cell populations for the fibroblasts that compose adult tendons. We also modeled cell-cell interactions and identified previously undescribed ligand-receptor signaling interactions involved in tendon homeostasis. Our novel and interactive tendon atlas highlights previously underappreciated heterogeneity between and within tendon cell populations. The atlas also serves as a resource to further the understanding of tendon extracellular matrix assembly and maintenance and in the design of therapies for tendinopathies.

Widespread diversity in the transcriptomes of functionally divergent limb tendons.

KEY POINTS: Tendon is a hypocellular, matrix-rich tissue that has been excluded from comparative transcriptional atlases. These atlases have provided important knowledge about biological heterogeneity between tissues, and our study addresses this important gap. We performed measures on four of the most studied tendons, the Achilles, forepaw flexor, patellar and supraspinatus tendons of both mice and rats. These tendons are functionally distinct and are also among the most commonly injured, and therefore of important translational interest. Approximately one-third of the filtered transcriptome was differentially regulated between Achilles, forepaw flexor, patellar and supraspinatus tendons within either mice or rats. Nearly two-thirds of the transcripts that are expressed in anatomically similar tendons were different between mice and rats. The overall findings from this study identified that although tendons across the body share a common anatomical definition based on their physical location between skeletal muscle and bone, tendon is a surprisingly genetically heterogeneous tissue. ABSTRACT: Tendon is a functionally important connective tissue that transmits force between skeletal muscle and bone. Previous studies have evaluated the architectural designs and mechanical properties of different tendons throughout the body. However, less is known about the underlying transcriptional differences between tendons that may dictate their designs and properties. Therefore, our objective was to develop a comprehensive atlas of the transcriptome of limb tendons in adult mice and rats using systems biology techniques. We selected the Achilles, forepaw digit flexor, patellar, and supraspinatus tendons due to their divergent functions and high rates of injury and tendinopathies in patients. Using RNA sequencing data, we generated the Comparative Tendon Transcriptional Database (CTTDb) that identified substantial diversity in the transcriptomes of tendons both within and across species. Approximately 30% of filtered transcripts were differentially regulated between tendons of a given species, and nearly 60% of the filtered transcripts present in anatomically similar tendons were different between species. Many of the genes that differed between tendons and across species are important in tissue specification and limb morphogenesis, tendon cell biology and tenogenesis, growth factor signalling, and production and maintenance of the extracellular matrix. This study indicates that tendon is a surprisingly heterogenous tissue with substantial genetic variation based on anatomical location and species.

Scleraxis lineage cells contribute to organized bridging tissue during tendon healing and identify a subpopulation of resident tendon cells.

During tendon healing, it is postulated that tendon cells drive tissue regeneration, whereas extrinsic cells drive pathologic scar formation. Tendon cells are frequently described as a homogenous, fibroblast population that is positive for the marker Scleraxis (Scx). It is controversial whether tendon cells localize within the forming scar tissue during adult tendon healing. We have previously demonstrated that S100 calcium-binding protein A4 (S100a4) is a driver of tendon scar formation and marks a subset of tendon cells. The relationship between Scx and S100a4 has not been explored. In this study, we assessed the localization of Scx lineage cells (ScxLin) following adult murine flexor tendon repair and established the relationship between Scx and S100a4 throughout both homeostasis and healing. We showed that adult ScxLin localize within the scar tissue and organize into a cellular bridge during tendon healing. Additionally, we demonstrate that markers Scx and S100a4 label distinct populations in tendon during homeostasis and healing, with Scx found in the organized bridging tissue and S100a4 localized throughout the entire scar region. These studies define a heterogeneous tendon cell environment and demonstrate discrete contributions of subpopulations during healing. These data enhance our understanding and ability to target the cellular environment of the tendon.-Best, K. T., Loiselle, A. E. Scleraxis lineage cells contribute to organized bridging tissue during tendon healing and identify a subpopulation of resident tendon cells.

Age-associated changes in the response of tendon explants to stress deprivation is sex-dependent.

Purpose of the Study: The incidence of tendon injuries increases dramatically with age, which presents a major clinical burden. While previous studies have sought to identify age-related changes in extracellular matrix structure and function, few have been able to explain fully why aged tissues are more prone to degeneration and injury. In addition, recent studies have also demonstrated that age-related processes in humans may be sex-dependent, which could be responsible for muddled conclusions in changes with age. In this study, we investigate short-term responses through an ex vivo explant culture model of stress deprivation that specifically questions how age and sex differentially affect the ability of tendons to respond to altered mechanical stimulus.Materials and Methods: We subjected murine flexor explants from young (4 months of age) and aged (22-24 months of age) male and female mice to stress-deprived culture conditions for up to 1 week and investigated changes in viability, cell metabolism and proliferation, matrix biosynthesis and composition, gene expression, and inflammatory responses throughout the culture period.Results and Conclusions: We found that aging did have a significant influence on the response to stress deprivation, demonstrating that aged explants have a less robust response overall with reduced metabolic activity, viability, proliferation, and biosynthesis. However, age-related changes appeared to be sex-dependent. Together, this work demonstrates that the aging process and the subsequent effect of age on the ability of tendons to respond to stress-deprivation are inherently different based on sex, where male explants favor increased activity, apoptosis, and matrix remodeling while female explants favor reduced activity and tissue preservation.

Exogenous micro-RNA and antagomir modulate osteogenic gene expression in tenocytes.

Tendinopathy is a common and disabling condition that is difficult to treat. The pathomolecular events behind tendinopathy remain uncertain. Micro-RNAs (miRNAs, miRs) are short non-coding RNAs that regulate gene expression and may play a role in tendinopathy development. Tenocytes were obtained from human patellar tendons in patients undergoing anterior cruciate ligament (ACL) reconstruction. Micro-RNA mimics and antagomirs for miR-30d, 26a, and 29a were separately transfected into tenocyte culture. Gene expression for scleraxis, collagen 1 alpha 1 (COL1A1), collagen 3 alpha 1 (COL3A1), interleukin-1-beta (IL-1ß), interleukin-6 (IL-6), bone morphogenic protein 2 (BMP2), bone morphogenic protein 12 (BMP12), and osteocalcin was determined for each miRNA mimic and antagomir transfection using real-time quantitative PCR (qPCR). The results showed that exogenous miR-29a downregulated BMP2 and BMP12, while miR-26a and miR-30d did not have a significant effect on tenocyte gene expression. These findings suggest miR-29a contributes to tendon homeostasis and can serve as a potential therapeutic target in treating tendinopathy.

Different Frequency of Cyclic Tensile Strain Relates to Anabolic/Catabolic Conditions Consistent with Immunohistochemical Staining Intensity in Tenocytes.

Tenocytes are mechanosensitive cells intimately adapting their expression profile and hence, their phenotype to their respective mechanomilieu. The immunolocalization and expression intensity of tenogenic, anabolic and catabolic markers in tenocytes in response to in vitro mechanical loading have not been monitored by immunohistochemical staining (IHC). Thus, we investigated the association between IHC intensities, different stimulation frequencies, and tenogenic metabolism using a versatile mechanical stretcher. Primary tenocytes obtained from murine Achilles tendons were transferred to poly(dimethylsiloxane) (PDMS) elastomeric chamber. Chambers were cyclically stretched by 5% in uniaxial direction at a variation of tensile frequency (1 or 2 Hz) for 3 h. After stretching, cell physiology, IHC intensities of tendon-related markers, and protein level of the angiogenesis marker vascular endothelial growth factor (VEGF) were evaluated. Cell proliferation in tenocytes stimulated with 1 Hz stretch was significantly higher than with 2 Hz or without stretch, while 2 Hz stretch induced significantly reduced cell viability and proliferation with microscopically detectable apoptotic cell changes. The amount of scleraxis translocated into the nuclei and tenomodulin immunoreactivity of tenocytes treated with stretch were significantly higher than of non-stretched cells. The collagen type-1 expression level in tenocytes stretched at 1 Hz was significantly higher than in those cultivated with 2 Hz or without stretching, whereas the matrix metalloproteinase (MMP)-1 and MMP-13 immunoreactivities of cells stretched at 2 Hz were significantly higher than in those stimulated with 1 Hz or without stretching. The secreted VEGF-protein level of tenocytes stretched at 2 Hz was significantly higher than without stretching. Our IHC findings consistent with cell physiology suggest that appropriate stretching can reproduce in vitro short-term tenogenic anabolic/catabolic conditions and allow us to identify an anabolic stretching profile.

Sustained Exposure of Substance P Causes Tendinopathy.

Recently, neuromediators such as substance P (SP) have been found to be important factors in tendon homeostasis. Some studies have found SP to be the cause of inflammation and tendinopathy, whereas others have determined it to be a critical component of tendon healing. As demonstrated by these conflicting findings, the effects of SP on tendinopathy remain unclear. In this study, we hypothesized that the duration of SP exposure determines its effect on the tendons, with repetitive long-term exposure leading to the development of tendinopathy. First, we verified the changes in gene and protein expression using in vitro tenocytes with 10-day exposure to SP. SP and SP + Run groups were injected with SP in their Achilles tendon every other day for 14 days. Achilles tendons were then harvested for biomechanical testing and histological processing. Notably, tendinopathic changes with decreased tensile strength, as observed in the Positive Control, were observed in the Achilles in the SP group compared to the Negative Control. Subsequent histological analysis, including Alcian blue staining, also revealed alterations in the Achilles tendon, which were generally consistent with the findings of tendinopathy in SP and SP + Run groups. Immunohistochemical analysis revealed increased expression of SP in the SP group, similar to the Positive Control. In general, the SP + Run group showed worse tendinopathic changes. These results suggest that sustained exposure to SP may be involved in the development of tendinopathy. Future research on inhibiting SP is warranted to target SP in the treatment of tendinopathy and may be beneficial to patients with tendinopathy.

Possible association of oestrogen and Cryba4 with masticatory muscle tendon-aponeurosis hyperplasia.

OBJECTIVE: Masticatory muscle tendon-aponeurosis hyperplasia, which is associated with limited mouth opening, progresses very slowly from adolescence. The prevalence rates of this disease are higher among women than among men, suggesting oestrogen involvement. As parafunctional habits are frequently observed, mechanical stress is likely involved in the pathogenesis and advancement of this disease. To elucidate the pathological condition, we examined the effect of oestrogen on tenocyte function and the relationship between mechanical stress and crystallin beta A4 (Cryba4), using murine TT-D6 tenocytes. MATERIALS AND METHODS: Cell proliferation assays, RT-PCR, real-time RT-PCR, Western blot analysis and mechanical loading experiments were performed. RESULTS: The physiological dose of oestrogen increased the levels of scleraxis and tenomodulin in TT-D6 tenocytes. In contrast, forced expression of Cryba4 inhibited scleraxis expression in these cells. Surprisingly, oestrogen significantly promoted cell differentiation in the Cryba4-overexpressing TT-D6 tenocytes. Moreover, tensile force induced Cryba4 expression in these tendon cells. CONCLUSION: Oestrogen and Cryba4 may be associated with the progression of masticatory muscle tendon-aponeurosis hyperplasia.