The role of mechanotransduction in tendon.

Tendons play an important role in the maintenance of motor function by connecting muscles and bones and transmitting forces. Particularly, the role of mechanical stress has primarily focused on the key mechanism of tendon homeostasis, with much research on this topic. With the recent development of molecular biological techniques, the mechanisms of mechanical stress sensing and signal transduction have been gradually elucidated with identification of mechanosensor in tendon cells and the master regulator in tendon development. This review provides a comprehensive overview of the structure and function of tendon tissue, including the role for physical performance and the detailed mechanism of mechanotransduction in its regulation. An important lesson is that the role of mechanotransduction in tendon tissue is only partially clarified, indicating the complexity of the mechanisms of motor function and fueling increasing interest in uncovering these mechanisms.

This review summarizes current knowledge of the mechanobiology of tendons. Appropriate mechanical stress can enhance the anabolic effects of tenocytes via the activation of PIEZO1-MKX/SCX cascade, which can alter the mechanical properties of tendon tissue and may also affect physical performance. However, the elucidation of the mechanisms by which structural changes in tendons affect physical performance appears to be still partial. Further clarification of tendon function is anticipated to lead to applications in enhancing physical performance and the extension of healthy life expectancy.

Comparison of early clinical outcome in carpal tunnel release - mini-open technique with palmar incision vs. endoscopic technique with wrist crease incision.

BACKGROUND: The purpose of this study was to examine two techniques for Carpal Tunnel Syndrome, mini-Open Carpal Tunnel Release (mini-OCTR) and Endoscopic Carpal Tunnel Release (ECTR), to compare their therapeutic efficacy. METHODS: Sixteen patients who underwent mini-OCTR in palmar incision and 17 patients who underwent ECTR in the wrist crease incision were included in the study. All patients presented preoperatively and at 1, 3, and 6 months postoperatively and were assessed with the Visual Analogue Scale (VAS) and the Disabilities of Arm, Shoulder and Hand Score (DASH). We also assessed the pain and cosmetic VAS of the entire affected hand or surgical wound, and the patient's satisfaction with the surgery. RESULTS: In the objective evaluation, both surgical techniques showed improvement at 6 months postoperatively. The DASH score was significantly lower in the ECTR group (average = 3 months: 13.6, 6 months: 11.9) than in the mini-OCTR group (average = 3 months: 27.3, 6 months: 20.6) at 3 and 6 months postoperatively. Also, the pain VAS score was significantly lower in the ECTR group (average = 17.1) than in the mini-OCTR group (average = 36.6) at 3 months postoperatively. The cosmetic VAS was significantly lower in the ECTR group (average = 1 month: 15.3, 3 months: 12.2, 6 months: 5.41) than in the mini-OCTR group (average = 1 month: 33.3, 3 months: 31.2, 6 months: 24.8) at all time points postoperatively. Patient satisfaction scores tended to be higher in the ECTR group (average = 3.3) compared to the mini-OCTR group (average = 2.7). CONCLUSIONS: ECTR in wrist increase incision resulted in better pain and cosmetic recovery in an early postoperative phase compared with mini-OCTR in palmar incision. Our findings suggest that ECTR is an effective technique for patient satisfaction.

The Effectiveness of Rehabilitation after Open Surgical Release for Trigger Finger: A Prospective, Randomized, Controlled Study.

BACKGROUND: It is not clear whether rehabilitation after surgery for trigger finger is effective. The aim of this study was to reveal its effectiveness for trigger finger. METHODS: This study was a randomized, controlled trial that included patients who underwent operations for trigger fingers. The patients in the rehabilitation group had postoperative occupational therapy (OT) for 3 months, while the patients in the control group were not referred for rehabilitation but received advice for a range of motion exercises. We evaluated the severity of trigger finger, Disability of Arm-Shoulder-Hand (DASH) score, pain-visual analogue scale (VAS), grip strength, whether they gained a full range of motion (ROM), and complications before and after surgery. RESULTS: Finally, 29 and 28 patients were included in the control and rehabilitation groups, respectively. At final follow-up, the DASH score, grip strength, and ROM were significantly improved in the rehabilitation group compared to that preoperatively. At final follow-up, pain was significantly improved in both groups from that preoperatively. There were no significant differences in the results, including the DASH score, grip strength, ROM and pain-VAS between the control and rehabilitation groups at the final follow-up. Subgroup analysis showed that there is a significant difference in the DASH score of patients doing housework or light work and those with a duration of symptoms >12 months between the control and rehabilitation groups at the final follow-up.

Fracture Line Distributions of Undisplaced Distal Radius Fractures in Relation to Rupture of the Extensor Pollicis Longus Tendon.

Rupture of the extensor pollicis longus (EPL) tendon is a known complication after undisplaced distal radius fracture (DRF). However, no report has revealed the relationship between EPL tendon rupture and the fracture pattern. Thus, this study aimed to investigate the characteristics of fractures at risk of EPL tendon rupture using fracture line mapping of undisplaced DRFs. This study used computed tomography imaging data of undisplaced DRFs with (n=18) and without EPL tendon rupture (n=52). Fracture lines obtained from 3D reconstruction data were drawn manually after matching with a 2D template wrist model. Fracture maps represented the fracture line distribution by superimposing the fracture lines of all 70 patients. Heat maps showed the relative frequency of the fracture lines as a gradual color change. Fracture lines of cases with EPL tendon rupture were concentrated in the proximal border of Lister's tubercle. By contrast, fracture lines of cases without EPL tendon rupture were relatively dispersed.

Motion-capture Analysis of Mice Using a Video Recorded on an iPhone Camera.

When focusing on quick movements in the analysis of animal behavior, a high-speed camera can be used as a powerful tool. There are many options for high-speed cameras to record movement. In recent years, the quality and sophistication of videos captured on cell phones have evolved so much that the iPhone's slow-motion video system can function as a tool for behavior analysis. Here, we describe a method to analyze the movement of the ankle joint and jump speed during the jumping action of mice, using an iPhone.

The mechanosensitive ion channel PIEZO1 is expressed in tendons and regulates physical performance.

How mechanical stress affects physical performance via tendons is not fully understood. Piezo1 is a mechanosensitive ion channel, and E756del PIEZO1 was recently found as a gain-of-function variant that is common in individuals of African descent. We generated tendon-specific knock-in mice using R2482H Piezo1, a mouse gain-of-function variant, and found that they had higher jumping abilities and faster running speeds than wild-type or muscle-specific knock-in mice. These phenotypes were associated with enhanced tendon anabolism via an increase in tendon-specific transcription factors, Mohawk and Scleraxis, but there was no evidence of changes in muscle. Biomechanical analysis showed that the tendons of R2482H Piezo1 mice were more compliant and stored more elastic energy, consistent with the enhancement of jumping ability. These phenotypes were replicated in mice with tendon-specific R2482H Piezo1 replacement after tendon maturation, indicating that PIEZO1 could be a target for promoting physical performance by enhancing function in mature tendon. The frequency of E756del PIEZO1 was higher in sprinters than in population-matched nonathletic controls in a small Jamaican cohort, suggesting a similar function in humans. Together, this human and mouse genetic and physiological evidence revealed a critical function of tendons in physical performance, which is tightly and robustly regulated by PIEZO1 in tenocytes.

Single Cell RNA Sequencing Reveals Critical Functions of Mkx in Periodontal Ligament Homeostasis.

The periodontal ligament (PDL) comprises a fibrous tissue that connects teeth to alveolar bone and is essential for periodontal function. The transcription factor mohawk homeobox (Mkx) is expressed in the PDL where it plays an important role in the development and maintenance of the PDL. However, the precise and critical functions of Mkx in the cell populations comprising PDL have not yet been elucidated. The present study aimed to clarify the effects of a Mkx deficiency on PDL cellular heterogeneity and differences between gene expression in PDL tissues from wild-type (WT) (Mkx +/+ ) and Mkx knockout (Mkx -/- ) rats using single-cell RNA sequencing. We identified 12 cell clusters comprising mesenchymal cells and macrophages. The expression of Mkx and scleraxis (Scx; another key transcription factor of PDL), was mutually exclusive, and partitioned mesenchymal cell clusters into Mkx and Scx types that dominantly expressed proteoglycans and elastic fibers, and type 1 and 3 collagen, respectively. Ossification-related genes were upregulated in mesenchymal cell and osteoblast clusters with more Mkx -/- than Mkx +/+ PDLs. Increased number of cells and inflammatory mediators were observed in macrophage clusters of Mkx -/- PDL. These results suggested that Mkx plays an important role in maintaining PDL homeostasis by regulating specific cell populations and gene expression.

Generation of a tendon-like tissue from human iPS cells.

Tendons and ligaments are essential connective tissues that connect the muscle and bone. Their recovery from injuries is known to be poor, highlighting the crucial need for an effective therapy. A few reports have described the development of artificial ligaments with sufficient strength from human cells. In this study, we successfully generated a tendon-like tissue (bio-tendon) using human induced pluripotent stem cells (iPSCs). We first differentiated human iPSCs into mesenchymal stem cells (iPSC-MSCs) and transfected them with Mohawk (Mkx) to obtain Mkx-iPSC-MSCs, which were applied to a newly designed chamber with a mechanical stretch incubation system. The embedded Mkx-iPSC-MSCs created bio-tendons and exhibited an aligned extracellular matrix structure. Transplantation of the bio-tendons into a mouse Achilles tendon rupture model showed host-derived cell infiltration with improved histological score and biomechanical properties. Taken together, the bio-tendon generated in this study has potential clinical applications for tendon/ligament-related injuries and diseases.

The effect of mechanical stress on enthesis homeostasis in a rat Achilles enthesis organ culture model.

Tendons and ligaments are jointed to bones via an enthesis that is essential to the proper function of the muscular and skeletal structures. The aim of the study is to investigate the effect of mechanical stress on the enthesis. We used ex vivo models in organ cultures of rat Achilles tendons with calcaneus including the enthesis. The organ was attached to a mechanical stretching apparatus that can conduct cyclic tensile strain. We made the models of 1-mm elongation (0.5 Hz, 3% elongation), 2-mm elongation (0.5 Hz, 5% elongation), and no stress. Histological evaluation by Safranin O staining and Toluidin Blue and Picro Sirius red staining was conducted. Expression of sex-determining region Y-box 9 (Sox9), scleraxis (Scx), Runt-related transcription factor 2 (Runx2), and matrix metalloproteinase 13 (Mmp13) were examined by real-time polymerase chain reaction and immunocytochemistry. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end-labeling and live/dead staining and was conducted for evaluation of the apoptosis and cell viability. The structure of the enthesis was most maintained in the model of 1-mm elongation. The electronic microscope showed that the enthesis of the no stress model had ill-defined borders between fibrocartilage and mineralized fibrocartilage, and that calcification of mineralized fibrocartilage occurred in the model of 2-mm elongation. Sox9 and Scx was upregulated by 1-mm elongation, whereas Runx2 and Mmp13 were upregulated by 2-mm elongation. Apoptosis was inhibited by low stress. The results of this study suggested that 1-mm elongation can maintain the structure of the enthesis, while 2-mm elongation promotes degenerative changes.

Both microRNA-455-5p and -3p repress hypoxia-inducible factor-2α expression and coordinately regulate cartilage homeostasis.

Osteoarthritis (OA), the most common aging-related joint disease, is caused by an imbalance between extracellular matrix synthesis and degradation. Here, we discover that both strands of microRNA-455 (miR-455), -5p and -3p, are up-regulated by Sox9, an essential transcription factor for cartilage differentiation and function. Both miR-455-5p and -3p are highly expressed in human chondrocytes from normal articular cartilage and in mouse primary chondrocytes. We generate miR-455 knockout mice, and find that cartilage degeneration mimicking OA and elevated expression of cartilage degeneration-related genes are observed at 6-months-old. Using a cell-based miRNA target screening system, we identify hypoxia-inducible factor-2α (HIF-2α), a catabolic factor for cartilage homeostasis, as a direct target of both miR-455-5p and -3p. In addition, overexpression of both miR-455-5p and -3p protect cartilage degeneration in a mouse OA model, demonstrating their potential therapeutic value. Furthermore, knockdown of HIF-2α in 6-month-old miR-455 knockout cartilage rescues the elevated expression of cartilage degeneration-related genes. These data demonstrate that both strands of a miRNA target the same gene to regulate articular cartilage homeostasis.

Mkx regulates the orthodontic tooth movement via osteoclast induction.

INTRODUCTION: The periodontal ligament (PDL) plays an important role in orthodontic tooth movement; however, the underlying molecular mechanism remains unclear. We have previously reported that the Mohawk homeobox (Mkx), a tendon-specific transcription factor, is expressed in the PDL and regulates its homeostasis. MATERIALS AND METHODS: In the present study, we examined the role of Mkx in orthodontic tooth movement via bone remodeling induced by mechanical stimulation in Mkx-deficient rats, which are widely used as experimental animals for orthodontic force application. Orthodontic tooth movement of the maxillary first molar was performed in 7-week-old male Mkx-deficient rats (n = 4) and wild-type Wistar rats (n = 4) using coil springs for 14 days. Hematoxylin and eosin (H&E) staining and tartrate-resistant acid phosphatase (TRAP) staining were performed to evaluate morphological changes and osteoclasts. Furthermore, changes in the expression of receptor activator nuclear factor-kappa B ligand (RANKL) were demonstrated using immunostaining. RESULTS: The amount of tooth movement was significantly lower in Mkx-deficient rats than in wild-type rats. The number of TRAP-positive cells was suppressed in Mkx-deficient rats on the compression side. CONCLUSION: Orthodontic tooth movement experiments in Mkx-deficient rats suggested that Mkx is involved in osteoclast induction at the alveolar bone surface on the compression side. This study reveals the possibility that Mkx plays a mechanosensory role in orthodontic tooth movement by inducing RANKL expression and osteoclastogenesis.

MicroRNA Expression Profiling, Target Identification, and Validation in Chondrocytes.

MicroRNAs (miRNAs) are a class of noncoding small RNAs, which play a critical role in various biological processes including musculoskeletal formation and arthritis pathogenesis via regulating target gene expressions, raising the potentially substantial effects on gene expression networks. Over 2000 miRNAs are encoded in the human genome and a single miRNA potentially targets hundreds of genes. To examine the expression and function of miRNAs in chondrocytes and arthritis pathogenesis, we describe the protocols for the current miRNA related experiments including miRNA expression profiling by (1) Next Generation Sequencing and by TaqMan Array system, (2) miRNA target prediction by TargetScan, (3) miRNA target screening by cell-based reporter library assay, and (4) miRNA and its target interaction by HITS-CLIP (high-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation) in cartilage and chondrocyte research.

Regulation of tendon and ligament differentiation.

Tendons transmit power from muscles to bones, and ligaments maintain the stability of joints, thus producing smooth and flexible movements of articular joints. However, tendons have poor self-healing ability upon damage due to injuries, diseases, or aging. To maintain homeostasis or promote regeneration of the tendon/ligament, it is critical to understand the mechanism responsible for the coordination of tendon/ligament-specific gene expression and subsequent cell differentiation. In this review, we have discussed the core molecular mechanisms involved in the development and homeostasis of tendons and ligaments, with particular focus on transcription factors, signaling, and mechanical stress.

In vitro Neo-Genesis of Tendon/Ligament-Like Tissue by Combination of Mohawk and a Three-Dimensional Cyclic Mechanical Stretch Culture System.

Tendons and ligaments are pivotal connective tissues that tightly connect muscle and bone. In this study, we developed a novel approach to generate tendon/ligament-like tissues with a hierarchical structure, by introducing the tendon/ligament-specific transcription factor Mohawk (MKX) into the mesenchymal stem cell (MSC) line C3H10T1/2 cells, and by applying an improved three-dimensional (3D) cyclic mechanical stretch culture system. In our developed protocol, a combination of stable Mkx expression and cyclic mechanical stretch synergistically affects the structural tendon/ligament-like tissue generation and tendon related gene expression. In a histological analysis of these tendon/ligament-like tissues, an organized extracellular matrix (ECM), containing collagen type III and elastin, was observed. Moreover, we confirmed that Mkx expression and cyclic mechanical stretch, induced the alignment of structural collagen fibril bundles that were deposited in a fibripositor-like manner during the generation of our tendon/ligament-like tissues. Our findings provide new insights for the tendon/ligament biomaterial fields.

Transcriptional, epigenetic and microRNA regulation of growth plate.

Endochondral ossification is a critical event in bone formation, particularly in long shaft bones. Many cellular differentiation processes work in concert to facilitate the generation of cartilage primordium to formation of trabecular structures, all of which occur within the growth plate. Previous studies have revealed that the growth plate is tightly regulated by various transcription factors, epigenetic systems, and microRNAs. Hence, understanding these mechanisms that regulate the growth plate is crucial to furthering the current understanding on skeletal diseases, and in formulating effective treatment strategies. In this review, we focus on describing the function and mechanisms of the transcription factors, epigenetic systems, and microRNAs known to regulate the growth plate.

The transcription factors regulating intervertebral disc development.

Damage to the intervertebral discs (IVDs) occurs due to aging or excessive mechanical stress, causing a series of IVD-related degenerative diseases, such as spinal disc herniation and spondylosis. These IVD-related diseases are difficult to cure, partially because the regeneration ability of IVDs is not sufficient. As a novel strategy for treatment of IVD-related diseases, mesenchymal stem cell transplantation to the damaged discs has been reported in animal studies. To further develop and improve this approach, it is necessary to gain a better understanding of the molecular network regulating IVD development by critical transcription factors. Recent findings reveal that during IVD development, nucleus pulposus and annuls fibrosus differentiation is coordinated by a series of transcription factors, such as Mkx, Pax1, 9, Shh, Foxa1, 2, T-Brachyury, and Sox5, 6, 9. The combination of mesenchymal stem cell transplantation with the regulation of these molecules may provide a novel strategy for treatment of degenerative disc diseases.

Wwp2 maintains cartilage homeostasis through regulation of Adamts5.

The WW domain-containing protein 2 (Wwp2) gene, the host gene of miR-140, codes for the Wwp2 protein, which is an HECT-type E3 ubiquitin ligases abundantly expressed in articular cartilage. However, its function remains unclear. Here, we show that mice lacking Wwp2 and mice in which the Wwp2 E3 enzyme is inactivated (Wwp2-C838A) exhibit aggravated spontaneous and surgically induced osteoarthritis (OA). Consistent with this phenotype, WWP2 expression level is downregulated in human OA cartilage. We also identify Runx2 as a Wwp2 substrate and Adamts5 as a target gene, as similar as miR-140. Analysis of Wwp2-C838A mice shows that loss of Wwp2 E3 ligase activity results in upregulation of Runx2-Adamts5 signaling in articular cartilage. Furthermore, in vitro transcribed Wwp2 mRNA injection into mouse joints reduces the severity of experimental OA. We propose that Wwp2 has a role in protecting cartilage from OA by suppressing Runx2-induced Adamts5 via Runx2 poly-ubiquitination and degradation.

[Cartilage/chondrocyte research and osteoarthritis. Current topics and future of ligament regeneration therapy.]

Ligaments of joint have an essential role of proper mobilization and stabilization between bone and bone. Damage to ligaments caused by ageing, injury, and arthritis induce a disability of musculoskeletal system and has a problem to reduce our quality of life. To aim for the regeneration of ligaments, we have researched from the point of view of the developmet, found out that the transcription factor Mohawk has been important for the development and homeostasis of tendons and ligaments, and analyzed its function. Furthermore, we have also attempted to induce stem cells to tendon and ligament cells to produce type Ⅰ collagen fibers. In this article, we outline the mechanism of the development that has been reported including our approaches.

Essential role of Mohawk for tenogenic tissue homeostasis including spinal disc and periodontal ligament.

Tendons and ligaments play essential roles in connecting muscle and bone and stabilizing the connections between bones. The damage to tendons and ligaments caused by aging, injury, and arthritis induces the dysfunction of the musculoskeletal system and reduces the quality of life. Current therapy for damaged tendons and ligaments depends on self-repair; however, it is difficult to reconstruct normal tissue. Regeneration therapy for tendons and ligaments has not been achieved, partly because the mechanism, cell biology, and pathophysiology of tendon and ligament development remain unclear. This review summarizes the role of the transcription factor, Mohawk, which controls tendon and ligament cell differentiation, in the maintenance of cell homeostasis, as well as its function in disease and the possibility of new therapeutic approaches.

The transcription factor mohawk homeobox regulates homeostasis of the periodontal ligament.

The periodontal ligament (PDL), which connects the teeth to the alveolar bone, is essential for periodontal tissue homeostasis. Although the significance of the PDL is recognized, molecular mechanisms underlying PDL function are not well known. We report that mohawk homeobox (Mkx), a tendon-specific transcription factor, regulates PDL homeostasis by preventing its degeneration. Mkx is expressed in the mouse PDL at the age of 10 weeks and expression remained at similar levels at 12 months. In Mkx-/- mice, age-dependent expansion of the PDL at the maxillary first molar (M1) furcation area was observed. Transmission electron microscopy (TEM) revealed that Mkx-/- mice presented collagen fibril degeneration in PDL with age, while the collagen fibril diameter gradually increased in Mkx+/+ mice. PDL cells lost their shape in Mkx-/- mice, suggesting changes in PDL properties. Microarray and quantitative polymerase chain reaction (qPCR) analyses of Mkx-/- PDL revealed an increase in osteogenic gene expression and no change in PDL- and inflammatory-related gene expression. Additionally, COL1A1 and COL1A2 were upregulated in Mkx-overexpressing human PDL fibroblasts, whereas osteogenic genes were downregulated. Our results indicate that Mkx prevents PDL degeneration by regulating osteogenesis.