Cyclic mechanical stretch regulates the AMPK/Egr1 pathway in tenocytes via Ca2+-mediated mechanosensing.

PURPOSE: Mechanical stimuli are essential for the maintenance of tendon tissue homeostasis. The study aims to elucidate the mechanobiological mechanisms underlying the maintenance of tenocyte homeostasis by cyclic mechanical stretch under high-glucose (HG) condition. MATERIALS AND METHODS: Primary tenocytes were isolated from rat Achilles tendon and 2D-cultured under HG condition. The in vitro effects of a single bout, 2-h cyclic biaxial stretch session (1 Hz, 8%) on primary rat tenocytes were explored through Flexcell system. Cell viability, tenogenic gene expression, intracellular calcium concentration, focal adhesion kinase (FAK) expression, and signaling pathway activation were analyzed in tenocytes with or without mechanical stretch. RESULTS: Mechanical stretch increased tenocyte proliferation and upregulated early growth response protein 1 (Egr1) expression. An increase in intracellular calcium was observed after 30 min of stretching. Mechanical stretch phosphorylated FAK, calmodulin-dependent protein kinase kinase 2 (CaMKK2), and 5' adenosine monophosphate-activated protein kinase (AMPK) in a time-dependent manner, and these effects were abrogated after blocking intracellular calcium. Inhibition of FAK, CaMKK2, and AMPK downregulated the expression of Egr1. In addition, mechanical stretch reinforced cytoskeletal organization via calcium (Ca2+)/FAK signaling. CONCLUSIONS: Our study demonstrated that mechanical stretch-induced calcium influx activated CaMKK2/AMPK signaling and FAK-cytoskeleton reorganization, thereby promoting the expression of Egr1, which may help maintain tendon cell characteristics and homeostasis in the context of diabetic tendinopathy.

Effects of Conscious Control of Scapular Orientation in Oral Cancer Survivors With Scapular Dyskinesis: A Randomized Controlled Trial.

OBJECTIVES: Spinal accessory nerve dysfunction is one of the complications of neck dissection in patients with oral cancer. This study aimed to explore the effects of long-term scapular-focused exercises and conscious control of scapular orientation on scapular movement and quality of life (QoL). METHODS: This study was a randomized controlled trial with concealed allocation, assessor blinding, and intention-to-treat analysis. Thirty-six patients with oral cancer were randomly allocated to the motor-control group (scapular-focused exercise + conscious control of scapular orientation) or the regular-exercise group (scapular-focused exercises only). Both groups received conventional physical therapy after neck dissection for 3 months. Shoulder pain intensity, active range of motion (AROM) of shoulder abduction, scapular muscle strength and activity under maximal voluntary isometric contraction (MVIC), scapular muscle activity when performing scapular movements, and QoL were measured at baseline, 1 month after the start of the intervention, and the end of the intervention. RESULTS: Both groups showed significant improvement in all outcomes except shoulder pain intensity. After the 3-month intervention, the motor-control group had more significant improvement in AROM of shoulder abduction with a 19° difference (95% CI: 10-29, P < .001), muscle strength of upper trapezius with an 11 N difference (95% CI: 2-20; P = .021), and QoL than the regular-exercise group. When performing shoulder horizontal adduction and flexion, the relative value (%MVIC) of serratus anterior was smaller in the motor-control group with a 106%MVIC difference (95% CI: 7-205, P = .037). CONCLUSIONS: Scapular-focused exercises have promising effects on spinal accessory nerve dysfunction. Combining scapular-focused exercises with conscious control of scapular orientation has more remarkable benefits on AROM of shoulder abduction, UT muscle strength, and muscle activation pattern than the scapular-focused exercises alone. Conscious control of scapular orientation should be considered to integrate into scapular-focused exercises in patients with oral cancer and scapular dyskinesis.Trial registry name and URL, and registration number: ClinicalTrials.gov (URL: https://clinicaltrials.gov; Approval No: NCT03545100).

Functional outcomes and quality of life after a 6-month early intervention program for oral cancer survivors: a single-arm clinical trial.

BACKGROUND: Advanced treatment of oral cancer increases survival rates; however, it also increases the risk of developing shoulder dysfunction, dysphagia, oral dysfunction, donor site morbidity and psychological issues. This single-arm preliminary pilot study aims to explore the effects of a six-month early intervention program following reconstructive surgery in oral cancer survivors. METHODS: A total of 65 participants were analyzed following reconstructive surgery. Outcome measurements were taken during the first visit, and at one, three and six months after reconstructive surgery. RESULTS: Scapular muscle strength and shoulder range of motion progressively improved during the 6-month follow-up. The mean Disability of the Arms, Shoulder and Hand (DASH) score showed significant improvement at 1 month (p < .001). Health related QoL showed significant differences between baseline and 6-months post-surgery scores on global health and on most of the function and symptom scales. The predicted return-to-work rate was 80% at one year after the operation. Return-to-work rate differs in different vocational types, with a higher rate of return in the skilled or semi-skilled (87.5%) and self-employed (86.7%). CONCLUSIONS: We suggest that early integrated intervention program with a follow-up of at least six months following reconstructive surgery may help develop and identify intervention guidelines and goals in the initial six months of treatment following neck dissection in oral cancer survivors.

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

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

Tissue transglutaminase is involved in mechanical load-induced osteogenic differentiation of human ligamentum flavum cells.

Mechanical load-induced osteogenic differentiation might be the key cellular event in the calcification and ossification of ligamentum flavum. The aim of this study was to investigate the influence of tissue transglutaminase (TGM2) on mechanical load-induced osteogenesis of ligamentum flavum cells. Human ligamentum flavum cells were obtained from 12 patients undergoing lumbar spine surgery. Osteogenic phenotypes of ligamentum flavum cells, such as alkaline phosphatase (ALP), Alizarin red-S stain, and gene expression of osteogenic makers were evaluated following the administration of mechanical load and BMP-2 treatment. The expression of TGM2 was evaluated by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay (ELISA) analysis. Our results showed that mechanical load in combination with BMP-2 enhanced calcium deposition and ALP activity. Mechanical load significantly increased ALP and OC gene expression on day 3, whereas BMP-2 significantly increased ALP, OPN, and Runx2 on day 7. Mechanical load significantly induced TGM2 gene expression and enzyme activity in human ligamentum flavum cells. Exogenous TGM2 increased ALP and OC gene expression; while, inhibited TG activity significantly attenuated mechanical load-induced and TGM2-induced ALP activity. In summary, mechanical load-induced TGM2 expression and enzyme activity is involved in the progression of the calcification of ligamentum flavum.

Second messengers mediating the proliferation and collagen synthesis of tenocytes induced by low-level laser irradiation.

For decades, low-level laser therapy (LLLT) has widespread applications in tendon-related injuries. Although the therapeutic effect of LLLT could be explained by photostimulation of target tissue and cells, how tenocytes sense photonic energy and convert them into cascades of cellular and molecular events is still not well understood. This study was designed to elucidate the effects of LLLT on cell proliferation and collagen synthesis by examining the associated second messengers including ATP, Ca(2+), and nitric oxide using rat Achilles tenocytes. Moreover, proliferating cell nuclear antigen (PCNA) and transforming growth factor-ß1 (TGF-ß1) related to cell proliferation and matrix metabolism were also studied. The results showed that 904 nm GaAs laser of 1 J/cm(2) could significantly increase the MTT activity and collagen synthesis of tenocytes. Second messengers including ATP and intracellular Ca2+ were increased after laser treatment. Quantitative PCR analysis of tenocytes treated with laser revealed up-regulated expression of PCNA, type I collagen, and TGF-ß1. Besides, laser-induced TGF-ß1 expression was significantly inhibited by extracellular signal-regulated kinase (ERK) specific inhibitor (PD98059). The findings suggested that LLLT stimulated ATP production and increased intracellular calcium concentration. Directly or indirectly via production of TGF-ß1, these second messengers mediated the proliferation of tenocytes and synthesis of collagen.

Centrifugal force induces human ligamentum flavum fibroblasts inflammation through activation of JNK and p38 pathways.

Inflammation has been proposed to be an important causative factor in ligamentum flavum hypertrophy. However, the mechanisms of mechanical load on inflammation of ligamentum flavum remain unclear. In this study, we used an in vitro model of human ligamentum flavum fibroblasts subjected to centrifugal force to elucidate the effects of mechanical load on cultured human ligamentum flavum fibroblasts; we further studied its molecular and biochemical mechanisms. Human ligamentum flavum fibroblasts were obtained from six patients undergoing lumbar spine surgery. Monolayer cultures of human ligamentum flavum fibroblasts were subjected to different magnitudes of centrifugal forces. Cell viability, cell death, biochemical response, and molecular response to centrifugal forces were analyzed. It was found that centrifugal stress significantly suppressed cell viability without inducing cell death. Centrifugal force at 67.1 g/cm(2) for 60 min significantly increases the production of prostaglandin E2 and nitric oxide as well as gene expression of proinflammatory cytokines, including interleukin (IL)-1α, IL-1ß and IL-6, showed that centrifugal force-dependent induction of cyclooxygense-2 and inducible NO synthase required JNK and p38 mitogen-activated protein kinase, but not ERK 1/2 activities. This study suggested that centrifugal force does induce inflammatory responses in human ligamentum flavum fibroblasts. The activation of both JNK and p38 mitogen-activated protein kinase mechanotransduction cascades is a crucial intracellular mechanism that mediates cyclooxygense-2/prostaglandin E2 and inducible NO synthase/nitric oxide production.

Elastin-derived peptides induce inflammatory responses through the activation of NF-κB in human ligamentum flavum cells.

The formation of fibrotic tissue in the ligamentum flavum (LF) is usually preceded by breakdown of elastic fibers. Elastin-derived peptides (EDPs) from breakdown of elastic fibers display a wide range of biological activities in a variety of cells, but there is minimal information regarding the involvement in the processes of LF hypertrophy. The aim of this study is to elucidate the effects of EDPs on cultured human LF cells and to investigate their molecular and biochemical mechanisms. Human LF cells were obtained from 18 patients who underwent lumbar spine surgery. After treatment with different concentrations of EDPs with or without specific inhibitors in culture medium, the viability and proliferation of LF cells, genes expression, and the signaling pathways were evaluated and analyzed. It was found that 50 µg/ml EDPs significantly increased cell proliferation and synthesis of prostaglandin E(2). The gene expression and protein production of proinflammatory cytokines, including interleukin-1α (IL-1α), IL-1ß, and IL-6, were also upregulated. The levels of p-ERK (extracellular signal-regulated kinase) and NF-κB increased immediately following EDP treatment and sustained up to 90 min. It was also found that NF-κB inhibitor, but not ERK1/2 inhibitor, attenuated EDP-dependent induction of IL-1α, IL-1ß, and IL-6 expression, indicating that NF-κB pathways are required for EDP-induced IL-1α, IL-1ß, and IL-6 gene expression in human LF cells. The results of this in vitro experiment suggest that EDPs do induce inflammatory responses in human LF cells and plays the key role in the development of LF hypertrophy.

Effects of low intensity pulsed ultrasound on rat Schwann cells metabolism.

The effects of low intensity pulsed ultrasound to tenocytes and osteocytes are well understood and applied clinically. However, its effects on cultured Schwann cells are still not well elucidated. This study was designed to elucidate the effects of low intensity pulsed ultrasound on cultured Schwann cells and their possible molecular mechanism. Schwann cells were harvested from sciatic nerves of 3-day-old Sprague-Dawley rats. Low intensity pulsed ultrasound stimulator (frequency: 1 MHz, duration: 2 min, duty cycle: 20%, total treatment time: 3 min) was applied to three different culture conditions: regular culture medium containing 0, 5, or 10% fetal bovine serum. The viability, damage, and differentiation of Schwann cells were examined; gene expression was also analyzed. In the presence of 0.3 W/cm(2) pulsed ultrasound stimulation, increases in cell viability and decreases in cell apoptosis were observed in the serum deprivation group; in this culture condition, interleukin-1, tumor necrosis factor-alpha, and protein zero genes expression were downregulated and Desert Hedgehog transcripts gene expression was upregulated. We concluded that intervention with low intensity pulsed ultrasound could promote Schwann cell proliferation, prevent cell death, and keep adequate phenotype presentation for peripheral nerve recovery. The low intensity pulsed ultrasound stimulation to an injured nerve site could be applied as early as possible especially when the microenvironment is almost serum-free to obtain the most benefit.

The cross-talk between transforming growth factor-beta1 and ultrasound stimulation during mechanotransduction of rat tenocytes.

Ultrasound is an effective noninvasive treatment for various tendinopathies. However, how tenocytes convert ultrasound stimulation into cascades of cellular and molecular events is not well understood. The purpose of this study is to elucidate the signaling pathways of tenocytes during ultrasound stimulation. Primary cultures of tenocytes were harvested from Achilles tendons of Sprague-Dawley rats. The viability and proliferation of tenocytes, their genes expression, and the signaling pathways after ultrasound treatment with or without specific inhibitors were evaluated and analyzed. The results showed that ultrasound treatment (100 mW/cm(2) for 20 min) significantly enhanced matrix metalloproteinase 13 (MMP-13), c-Fos, and c-Jun gene expression, increased JNK and p38, but not extracellular signal-regulated kinase-1/2 (ERK1/2), phosphorylation at 5 min, and sustained up to 60 min. JNK inhibitor and p38 inhibitor, but not ERK1/2 inhibitor, attenuated ultrasound-dependent induction of MMP-13 expression, indicating that the JNK and p38 pathways are required for ultrasound-induced MMP-13 expression in tenocytes. We also found that SB431542 (transforming growth factor-beta (TGF-ß) receptor kinases inhibitor) suppressed ultrasound-induced MMP?13 and c-Fos gene expression, and p38 phosphorylation. This study revealed that ultrasound treatment stimulates tenocytes proliferation and regulates their matrix metabolism through the cross-talk between TGF-ß and ultrasound-induced mitogen-activated protein kinases (MAPKs) signaling pathways.