Cyclic Mechanical Strain Research Articles

Effects of RSC96 Schwann Cell-Derived Exosomes on Proliferation, Senescence, and Apoptosis of Dorsal Root Ganglion Cells In Vitro.

BackgroundStress urinary incontinence is a common condition in women and can be associated with peripheral nerve injury. Exosomes. derived from Schwann cells, can enhance the regeneration of axons of the peripheral nervous system. This study aimed to investigate the effects of RSC96 Schwann cell-derived exosomes in a novel in vitro model of dorsal root ganglion (DRG) cell injury induced by cyclic mechanical strain (CMS).Material/MethodsRSC96 Schwann cells and DRG cells were cultured in vitro. CMS in DRG cells involved mechanical stretch trauma with 5333 μ strain. ExoQuick-TC polymer was used to precipitate exosomes from RSC96 Schwann cell culture medium and identified by nanoparticle tracking analysis, electron microscopy, and Western blot for detection of CD9 and tumor susceptibility gene 101 (Tsg101) protein. Cultured DRG cells were treated with RSC96 Schwann cell-derived exosomes, followed by measurement of cell viability, proliferation, senescence, and apoptosis using the cell counting kit-8 (CCK-8) assay, senescence-associated beta-galactosidase (SA-β-gal) staining, and Hoechst 33258 (blue) fluorescence nucleic acid staining using flow cytometry.ResultsMechanical stretch with 5333 μ strain for 8 hours at 1 Hz decreased the activity of cultured DRG cells. RSC96 Schwann cell-derived exosomes promoted cell proliferation and significantly inhibited apoptosis and senescence of DRG cells following injury induced by CMS.ConclusionsAn in vitro model of DRG cell injury induced by CMS, showed that RSC96 Schwann cell-derived exosomes had a protective effect. The effects of Schwann cell-derived exosomes on peripheral nerve injury, including in stress urinary incontinence, require future in vivo studies.

Novel roles for scleraxis in regulating adult tenocyte function

BackgroundTendinopathies are common and difficult to resolve due to the formation of scar tissue that reduces the mechanical integrity of the tissue, leading to frequent reinjury. Tenocytes respond to both excessive loading and unloading by producing pro-inflammatory mediators, suggesting that these cells are actively involved in the development of tendon degeneration. The transcription factor scleraxis (Scx) is required for the development of force-transmitting tendon during development and for mechanically stimulated tenogenesis of stem cells, but its function in adult tenocytes is less well-defined. The aim of this study was to further define the role of Scx in mediating the adult tenocyte mechanoresponse.ResultsEquine tenocytes exposed to siRNA targeting Scx or a control siRNA were maintained under cyclic mechanical strain before being submitted for RNA-seq analysis. Focal adhesions and extracellular matrix-receptor interaction were among the top gene networks downregulated in Scx knockdown tenocytes. Correspondingly, tenocytes exposed to Scx siRNA were significantly softer, with longer vinculin-containing focal adhesions, and an impaired ability to migrate on soft surfaces. Other pathways affected by Scx knockdown included increased oxidative phosphorylation and diseases caused by endoplasmic reticular stress, pointing to a larger role for Scx in maintaining tenocyte homeostasis.ConclusionsOur study identifies several novel roles for Scx in adult tenocytes, which suggest that Scx facilitates mechanosensing by regulating the expression of several mechanosensitive focal adhesion proteins. Furthermore, we identified a number of other pathways and targets affected by Scx knockdown that have the potential to elucidate the role that tenocytes may play in the development of degenerative tendinopathy.

Mechanical loading induces primary cilia disassembly in tendon cells via TGF\u03b2 and HDAC6

This study used isolated human tenocytes to test the hypothesis that cyclic mechanical strain directly stimulates primary cilia disassembly, and to elucidate the mechanisms involved. Cells were seeded onto flexible membranes and strained at 0–3%; 1 Hz, for up to 24 hours. Cilia length and prevalence progressively reduced with increasing strain duration but showed full recovery within 2 hours of strain removal. The response to loading was not influenced by actin organisation as seen in other cell types. However, the loading response could be recreated by treatment with TGFβ. Furthermore, treatment with the HDAC6 inhibitor Tubacin, or a TGFβ receptor inhibitor both prevented strain induced cilia disassembly. These data are the first to describe primary cilia expression in isolated tenocytes, showing that mechanical strain regulates cilia expression independent of changes in tendon extracellular matrix. Furthermore, we show that cilia disassembly is mediated by the activation of TGFβ receptors leading to activation of HDAC6. Previous studies have shown that cilia are required for TGFβ signalling and that tendon mechanosignalling is mediated by TGFβ. The present study therefore suggests a novel feedback mechanism whereby cilia disassembly inhibits prolonged TGFβ activation in response to continuous cyclic loading.

High amplitude and low frequency cyclic mechanical strain promotes degeneration of human nucleus pulposus cells via the NF-κB p65 pathway.

Disc degeneration alters the structure and function of intervertebral discs and is the basis of spinal degenerative diseases. To establish the molecular mechanism of intervertebral disc degeneration caused by mechanical strain, this study examined the effects of different amplitude (3%, 9%, 19%) cyclic mechanical strain (CMS) at a low frequency (0.01 Hz) on the secretion of cartilage extracellular matrix, expression of inflammatory cytokines and catabolic proteases, and activation of NF-κB signaling pathway in human nucleus pulposus cells. We also investigated effects of low frequency and high amplitude (19%) CMS on degeneration of human nucleus pulposus cells in the presence or absence of p65 inhibitor, p65 silencing shRNA, or p65 overexpression. While 3% CMS did not significantly decrease aggrecan or type II collagen expression, or increase TNF-α, IL-1β, IL-6 expression, 9% and 19% CMS showed the significant effects. Low frequency and high amplitude (19%) CMS was found to promote p65 activation in human nucleus pulposus cells, and IL-1β was found to promote p65 nuclear translocation though IκB kinase phosphorylation. Furthermore, degeneration process of nucleus pulposus cells was found attenuated in the presence of p65 inhibitor or p65 silencing shRNA, but promoted with p65 overexpression. These data suggest that high amplitude and low frequency CMS could promote degeneration of human nucleus pulposus cells significantly via the NF-κB p65 pathway. Our findings have uncovered the effect of CMS on human nucleus pulposus cell degeneration and have identified a previously unknown intrinsic underlying mechanism.

Protective role of Nrf2 against mechanical-stretch-induced apoptosis in mouse fibroblasts: a potential therapeutic target of mechanical-trauma-induced stress urinary incontinence.

We investigated the protective effect and underlying molecular mechanism of nuclear factor-E2-related factor 2 (Nrf2) against mechanical-stretch-induced apoptosis in mouse fibroblasts. Normal cells, Nrf2 silencing cells, and Nrf2 overexpressing cells were respectively divided into two groups-nonintervention and cyclic mechanical strain (CMS)-subjected to CMS of 5333μ (1.0Hz for 4h), six groups in total (control, CMS, shNfe212, shNfe212+CMS, LV-shNfe212, and LV-shNfe212+CMS). After treatment, cell apoptosis; cell-cycle distribution; expressions of Nrf2, Bax, Bcl-2, Cyt-C, caspase-3, caspase-9, cleaved-caspase-3, and cleaved-caspase-9; mitochondrial membrane potential (ΔΨm); reactive oxygen species (ROS); and malondialdehyde (MDA) levels were measured. Thirty virgin female C57BL/6 mice were divided into two groups: control (without intervention) and vaginal distension (VD) groups, which underwent VD for 1 h with an 8-mm dilator (0.3ml saline). Leak-point pressure (LPP) was tested on day 7 after VD; Nrf2 expression, apoptosis, and MDA levels were then measured in urethra and anterior vaginal wall. Mechanical stretch decreased Nrf2 messenger RNA (mRNA) and protein expressions. Overexpression of Nrf2 alleviated mechanical-stretch-induced cell apoptosis; S-phase arrest of cell cycle; up-regulation of Bax, cytochrome C (Cyt-C), ROS, MDA, ratio of cleaved-caspase-3/caspase-3 and cleaved-caspase-9/caspase-9; and exacerbated the decrease of Bcl2 and ΔΨm in L929 cells. On the contrary, silencing of Nrf2 showed opposite effects. Besides, VD reduced LPP levels and Nrf2 expression and increased cell apoptosis and MDA generation in the urethra and anterior vaginal wall. Nrf2 exhibits a protective role against mechanical-stretch -induced apoptosis on mouse fibroblasts, which might indicate a potential therapeutic target of mechanical-trauma-induced stress urinary incontinence (SUI).

Microimpedance tomography system to monitor cell activity and membrane movements in a breathing lung-on-chip

Abstract We report about a new microimpedance tomography (MITO) system integrated in a lung-on-chip that recapitulates the thin alveolar barrier including the cyclic mechanical strain of the breathing movements. The system enables to detect changes that take place in the lung alveolar barrier located at 1 mm from the detection system. This leaves space for the three-dimensional deflection of the lung alveolar barrier. The aim of the MITO is to monitor both the electrochemical and the mechanical changes occurring in the lung alveolar barrier using impedimetric coplanar electrodes. Distant and real-time monitoring of changes in the resistivity of a human lung epithelial cell monolayer challenged with Triton X-100 could easily be detected. An exponential drop of 7% in impedance magnitude was recorded following the permeabilization of the monolayer. While the membrane is deflected to mimic the respiratory movements, the impedance readout can be correlated to the mechanical strain in the alveolar barrier. Small variations of the mechanical strain due to the density of the cell population can be detected. A mechanical strain difference of 0.4% was monitored between epithelial cells just seeded on the alveolar membrane and the resulting confluent layer 24 h later. The system is produced using a flexible printed circuit board (PCB) bonded to the lung-on-chip device made of polydimethylsiloxane (PDMS). It brings impedance-based cell and organ function monitoring onto organs-on-chips on a single, cost-efficient and integrable layer that does not interfere with the biomimetic capabilities of the chip.

Adaptive responses of murine osteoblasts subjected to coupled mechanical stimuli.

Restitution of the natural organization and orientation of cells is imperative for the construction of functional tissue scaffolds. While numerous studies have exploited mechanical methods to engineer tissues with the desired cellular architecture, fundamental knowledge is still lacking in understanding the manner in which morphological features can be modulated through coupled mechanical cues. To address this knowledge gap, the adhesion and alignment response of murine osteoblast cells under the synergistic effects of matrix rigidity and cyclic mechanical loading was investigated. This was accomplished by applying cyclic mechanical strain (1% at 0.05Hz) to MC3T3-E1 cells seeded on PDMS substrates of different elastic moduli (1.22, 1.70 and 2.04MPa). Results demonstrate that the overall cell density and expression of inactive vinculin increased on substrates subjected to cyclic stimulus in comparison to substrates under static loading. Conversely, in terms of the adhesion response, osteoblasts exhibited an increased growth of focal adhesion complexes under static substrates. Interestingly, results also elucidate that substrates of a stiffer matrix exposed to cyclic stimulus, had a significantly higher percentage of osteoblasts aligned parallel to the direction of the applied strain, as well as a higher degree of internal order with respect to the strain axis, in comparison to both cells seeded on substrates of lower stiffness under cyclic loading or under static conditions. These findings suggest the role of cyclic mechanical strain coupled with matrix rigidity in eliciting mechanosensitive adaptations in cell functions that allow for the reconstitution of the spatial and orientational assembly of cells in vivo for tissue engineering.

Stretchable Piezoelectric Substrate Providing Pulsatile Mechanoelectric Cues for Cardiomyogenic Differentiation of Mesenchymal Stem Cells.

Ex vivo induction of cardiomyogenic differentiation of mesenchymal stem cells (MSCs) before implantation would potentiate therapeutic efficacy of stem cell therapies for ischemic heart diseases because MSCs rarely undergo cardiomyogenic differentiation following implantation. In cardiac microenvironments, electric pulse and cyclic mechanical strain are sequentially produced. However, no study has applied the pulsatile mechanoelectric cues (PMEC) to stimulate cardiomyogenic differentiation of MSCs ex vivo. In this study, we developed a stretchable piezoelectric substrate (SPS) that can provide PMEC to human MSCs (hMSCs) for cardiomyogenic differentiation ex vivo. Our data showed that hMSCs subjected to PMEC by SPS underwent promoted cardiac phenotype development: cell alignment and the expression of cardiac markers (i.e., cardiac transcription factors, structural proteins, ion channel proteins, and gap junction proteins). The enhanced cardiac phenotype development was mediated by the upregulation of cardiomyogenic differentiation-related autocrine factor expression, focal adhesion kinase, and extracellular signal-regulated kinases signaling pathways. Thus, SPS providing electrical and mechanical regulation of stem cells may be utilized to potentiate hMSC therapies for myocardial infarction and provide a tool for the study of stem cell biology.

Mechanical Testing for Stretchable Electronics

Stretchable electronics have been a subject of increased research over the past decade (Lacour, S., et al., 2006, “Mechanisms of Reversible Stretchability of Thin Metal Films on Elastomeric Substrates,” Appl. Phys. Lett., 88(20), p. 204103; Lacour, S., et al., 2004, “Design and Performance of Thin Metal Film Interconnects for Skin-Like Electronic Circuits,” IEEE Electron Device Lett., 25(4), pp. 179–181; and Maghribi, M., et al., 2005, “Stretchable Micro-Electrode Array,” International IEEE-EMBS Conference on Microtechnologies in Medicine and Biology, pp. 80–83.). Although stretchable electronic devices are a relatively new area for the semiconductor/electronics industries, recent market research indicates that the market could be worth more than $900 million by 2023 (PR Newswire, 2015, “Stretchable Electronics Market Worth $911.37 Million by 2023,” PR Newswire, Albuquerque, NM.). This paper investigates mechanical testing methods designed to test the stretching capabilities of potential products across the electronics industry to help quantify and understand the mechanical integrity, response, and the reliability of these devices. Typically, the devices consist of stiff modules connected by stretchable traces (Loher, T., et al., 2006, “Stretchable Electronic Systems,” Electronics Packaging Technology Conference (EPTC '06), pp. 271–276.). They require electrical and mechanical connectivity between the modules to function. In some cases, these devices will be subject to biaxial and/or cyclic mechanical strain, especially for wearable applications. The ability to replicate these mechanical strains and understand their effect on the function of the devices is critical to meet performance, process, and reliability requirements. In this paper, methods for simulating biaxial and out-of-plane strains similar to what may occur in a wearable device on the human body are proposed. Electrical and/or optical monitoring (among other methods) can be used to determine cycles to failure depending on expected failure modes. Failure modes can include trace damage in stretchable regions, trace damage in functional component regions, or bulk stretchable material damage, among others. Three different methods of applying mechanical strain are described, including a stretchable air bladder method, membrane test method, and lateral expansion method.

Human cardiac fibroblasts adaptive responses to controlled combined mechanical strain and oxygen changes in vitro.

Upon cardiac pathological conditions such as ischemia, microenvironmental changes instruct a series of cellular responses that trigger cardiac fibroblasts-mediated tissue adaptation and inflammation. A comprehensive model of how early environmental changes may induce cardiac fibroblasts (CF) pathological responses is far from being elucidated, partly due to the lack of approaches involving complex and simultaneous environmental stimulation. Here, we provide a first analysis of human primary CF behavior by means of a multi-stimulus microdevice for combined application of cyclic mechanical strain and controlled oxygen tension. Our findings elucidate differential human CFs responses to different combinations of the above stimuli. Individual stimuli cause proliferative effects (PHH3+ mitotic cells, YAP translocation, PDGF secretion) or increase collagen presence. Interestingly, only the combination of hypoxia and a simulated loss of contractility (2% strain) is able to additionally induce increased CF release of inflammatory and pro-fibrotic cytokines and matrix metalloproteinases.

Lamina cribrosa in glaucoma.

This article presents, summarizes, and interprets the most recent advances in the study and understanding of the lamina cribrosa in glaucoma, in the context of previous work. The lamina is an active living structure that responds to strain by changing morphology at the micro-scale and macro-scale in glaucoma. Changes in lamina cribrosa morphology in glaucoma include posteriorization of the laminar insertion into the sclera, increased cupping or depth of the lamina cribrosa, and the development of focal lamina cribrosa defects. These lamina cribrosa changes are associated with disk hemorrhages and visual field damage, and are detectable with clinical imaging techniques such as optical coherence tomography. Glaucomatous changes in the lamina cribrosa are thought to be driven by cellular processes mediated by focal cyclical mechanical strain. Strain is eye specific and mediated by intraocular pressure, cerebrospinal fluid pressure, scleral and lamina cribrosa morphology, and structural stiffness; deleterious lamina cribrosa strains can occur at all levels of mean intraocular pressure. Laminar morphology is ever changing in health and disease, and recent studies have identified several promising morphological changes that are indicative of glaucoma susceptibility, onset, and progression.

Abstract A38: Biophysical Regulation of Breast Cancer Metastasis

Abstract Introduction: Despite many advances in our understanding of breast cancer biology over the last decades, there are no therapies that can effectively prevent or treat metastatic cancer. During the growth of the primary tumor mass and its dissemination through the body during metastasis, cancer cells are exposed to a host of mechanical environments generated by the local matrix compliance, pressure and tension from tumor mass expansion, and fluidic shear stresses from interstitial and vascular fluid flow. These biophysical forces are emerging as powerful regulators of cancer growth, quiescence and metastasis; however, our understanding of the mechanisms of the biomechanical regulation of tumor biology remains very limited. The overall goal was to identify the role of mechanical forces in regulating key steps in tumor metastasis and progression. Results: We recently developed a mesofluidic system that allows the high throughput study of tumor cell interactions under flow. This device allows us to simulate the adhesion of cancer cells to endothelial cells under physiological flow conditions to model this step of the metastatic cascade and to apply shear forces to cells in a 96-well format. In addition, we have designed a system to apply mechanical stretch to cells in a high throughput format (576 wells simultaneously). Using these two systems, we examined the interplay between mechanical cues and the propensity of breast cancer cells to metastasize and undergo epithelial-to-mesenchymal transition (EMT). Mechanical strain increases circulating tumor cell adhesion to endothelial cells and extracellular matrix (ECM). We applied cyclic mechanical strain (5% maximal strain) to MDA-MB-231 and MCF-7 cancer cells for 24 hours and then performed adhesion assays of the cancer cells to activated and non-activated endothelial cells (ECS) and purified ECM proteins. We found that cyclic strain increased cancer cell adhesion to activated ECs in comparison to non-strained cells. In addition cancer cells exposed to mechanical strain adhered more to collagen I, laminin, and vitronectin, while they adhered less to collagen II and fibronectin in comparison to non-strained control cells. To determine which integrins were involved in the strain-induced change in adhesion, we treated the cells with a library of integrin inhibitors while applying strain for 24 hours. Cilengitide, P11, ATN-161, Bio 1211, and RGDS peptides reduced the adhesion of cancer cells back to the level of non-strained cancer cells, indicating the role of αvβ3, αvβ5, α5β1, and α4β1 integrins in the biophysical regulation of circulating tumor cell adhesion. Cyclic strain alters the ability of breast cancer cells to invade through an endothelial monolayer. We next used the high throughput system to apply multiple levels of strain to the cancer cell lines (0, 2.5, 5, 7.5, 10, 12.5, 15 and 17.5% strain). We then trypsinized the cells and measured their invasion in a Transwell assay with a confluent layer of endothelial cells cultured on a porous membrane. We found that the mid-level strains ranging from 7.5-15% strain decreased the invasiveness of the MDA-MB-231 cells compared to the non-strained control cells. For the MCF-7 cells, mechanical strain of 5% or higher led to decreased invasion through the endothelial layer. Mechanical forces alter signaling through the TGF-β and Yap/Taz pathways as well the expression of markers of epithelial-to-mesenchymal transition (EMT). We applied cyclic strain at multiple levels (2.5-17.5% strain) to breast cancer cells for 24 hours then immunostained for various signaling proteins. In MDA-MB-231 cells, we saw a significant decrease in the intensity of Smad2/3 and nuclear phospho-Smad2/3 at mid-range strains (5-12.5% strain) as well as an increase in the nuclear localization of Yap/Taz using fluorescent immunostaining. At high levels of strain (15 and 17.5% strain), there was increased nuclear p-Smad2/3 intensity. Consistent with these findings, we found increased Smad activity in the cells using a luciferase reporter assay. An analysis of EMT markers using PCR, western blotting and immunostaining showed a reduction in mesenchymal markers including α−SMA, Slug, ZEB, fibronectin and vimentin with mid-range mechanical strain levels. Conclusion: Together, our studies demonstrate that mechanical forces can profoundly alter the propensity of cancer cells to adhere and invade during metastasis. Moreover, the response to mechanical strain is dependent on the magnitude of the strain and cancer cell type. These effects are mediated, in part, through integrin interactions and the differential regulation of the TGF-β and Hippo pathways by biophysical forces. Citation Format: Adrianne Spencer, Jason Lee, Katerina Lee, Darshil Choksi, Jerry Wang, Christopher Spruell, Aaron Baker. Biophysical Regulation of Breast Cancer Metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A38.

Mechanical Signaling in NF1 Osteoblast Cells

Neurofibromatosis Type I (NF1) syndrome is characterized by neurofibromas andneural tumors but is also associated with skeletal abnormalities. The cellularpathophysiology of skeletal abnormalities in NF1 is not understood. Theseabnormalities result from constitutive active RAS and its downstream effectors, RASERKpathway, due to mutation of NF1 gene which converts active RAS-GTP intoinactive RAS-GDP. In osteoblast cells, RAS-ERK pathway is involved in cellproliferation and differentiation and is also involved in mechanical signals transduction. In this study, we propose that Nf1 mutation in osteoblast cells will affect the responseto mechanical stimulation through the RAS pathway. The Flexcell tension system wasused to mechanically stimulate calvarial osteoblast precursor from conditional knockoutmice, Nf1(ob-/-), and wild type calvarial osteoblast precursor cells, (WT. Theprotocol of cyclic mechanical strain was 2% to 4% elongation at 0.16 Hz (10 cycles perminute) for 24h. Mechanically stimulated cells showed lower expression levels of theosteoblast marker gene, RUNX2, measured at 4h and 8h post-stretch. Mineralizedmatrix deposition, assessed by Alizarin red staining, was decreased in Nf1(ob-/-)compared to (WT) cells following mechanical stimulation. the Nf1(ob-/-) and WTosteoblast precursor cells were then treated with RAS inhibitor (FTI-277), for 4h and8h. RUNX2 expression level was increased in Nf1(ob-/-) cells compared to non-treatedcells. However, the opposite result was seen in (WT) cells. The FTI-277 treatmentresulted in lower RUNX2 expression level and lower mineralized matrix deposition. This response of (WT) cells was normal. However, the Nf1(ob-/-) response showedthat these cells although they have hyper-active RAS, but when it is exposed to stress,it loses its ability to express osteoblast markers or lay down mineralized matrix. Ourresults indicate that, the hyper-active RAS in NF1 mutant osteoblast will result in cellsbeing stuck in proliferative state and unable to differentiate.

Cyclic Strain and Hypertension Increase Osteopontin Expression in the Aorta.

Hypertension has a direct impact on vascular hypertrophy and is a known risk factor for the development of atherosclerosis. Osteopontin (OPN) has emerged as an important protein mediator of inflammation and remodeling of large arteries. However, its role and mechanism of regulation in the setting of hypertension is still unknown. Our objectives for this study were therefore to investigate the role of OPN in hypertension-induced vascular remodeling and inflammation. OPN Knockout (KO) and wild type (WT) mice were made hypertensive with angiotensin II (Ang II) infusion for seven days. We observed that OPN KO aortas were protected against Ang II-induced medial hypertrophy and inflammation, despite comparable increases in systolic blood pressure (SBP) in both groups. OPN expression was increased in WT aortas from hypertensive mice (induced by either Ang II or norepinephrine). OPN expression was increased in aortic smooth muscle cells (SMCs) subjected to cyclic mechanical strain suggesting that mechanical deformation of the aortic wall is responsible in part for the increased OPN expression induced by hypertension. Finally, we utilized hypertensive transgenic smooth muscle cell-specific catalase overexpressing (TgSMC-Cat) mice to determine the role of H2O2 in mediating hypertension-induced increases in OPN expression. We also found that the hypertension-induced increase in OPN expression was inhibited in transgenic smooth muscle cell-specific catalase overexpressing (TgSMC-Cat) mice, suggesting that H2O2, plays a vital role in mediating the hypertension-induced increase in OPN expression. Taken together, these results define a potentially important role for OPN in the pathophysiology of hypertension.

Early responses of human periodontal ligament fibroblasts to cyclic and static mechanical stretching.

To compare the mechanotransduction caused by cyclic and static mechanical strains in human periodontal ligament fibroblasts (hPDLFs) cultured under identical conditions. hPDLFs, originating from the same donors, were exposed either to cyclic or to static tensile strain using specially designed devices and under identical culture conditions. Activation of all members of mitogen-activated protein kinases (MAPKs) was monitored by western immunoblot analysis. Expression levels of immediate/early genes c-fos and c-jun were assessed with quantitative real-time polymerase chain reaction. Time course experiments revealed that both types of stresses activate the three members of MAPK, that is ERK, p38, and JNK, with cyclic stress exhibiting a slightly more extended activation. Further downstream, both stresses upregulate the immediate/early genes c-fos and c-jun, encoding components of the activator protein-1 (AP-1), a key transcription factor in osteoblastic differentiation; again cyclic strain provokes a more intense upregulation. Six hours after the application of both strains, MAPK activation and gene expression return to basal levels. Finally, cells exposed to cyclic stress for longer periods are distributed approximately perpendicular to the axis of the applied strain, whereas cells exposed to static loading remain in a random orientation in culture. The findings of the present study indicate similar, although not identical, immediate/early responses of hPDLs to cyclic and static stretching, with cyclic strain provoking a more intense adaptive response of these cells to mechanical deformation.

The driving force governing room temperature grain coarsening in thin gold films

Strong room-temperature grain coarsening in gold films on polyimide induced by cyclic uniaxial mechanical strain is demonstrated. Detailed electron backscatter diffraction analysis revealed that, in contrast to the predictions of shear-coupled grain boundary migration model, the grain coarsening is isotropic and coarsened grains do not exhibit any specific crystallographic orientations or misorientations to the neighboring grains. It is shown that a thermodynamic model where the driving force appears due to the difference in yield stresses between the grains with different sizes provides an adequate explanation of the experimental data.

Cyclic tensile strain promotes the osteogenic differentiation of a bone marrow stromal cell and vascular endothelial cell co-culture system

Mechanical stimuli and neovascularization are closely coupled to osteogenic differentiation and new bone formation. The purpose of present study was to detect the effect of cyclic mechanical strain on a co-culture system of bone marrow stromal cells (BMSCs) and vascular endothelial cells (VECs) and to clarify the related mechanisms. Primary BMSCs and VECs were isolated from Sprague-Dawley rats and co-cultured at various ratios (1:0, 1:2, 1:4, 4:1, 2:1, 1:1, and 0:1). To determine optimized loading conditions, the cells were then subjected to various cyclic tensile strains (0%, 3%, 6% and 9%) using a Flexcell 5000 mechanical loading system. A protocol of 6% strain on the co-cultured cells at a 1:1 ratio was selected as the optimized culture conditions based on the best osteogenic effects, which included increased ALP activity, matrix mineralization and the expressions of VEGF, Runx-2 and Col-1. The VEGF-R inhibitor tivozanib was used to analyze the paracrine role of VEGF, and the osteogenesis-promoting effects of 6% tensile strain were abrogated in the co-cultured cells treated with tivozanib. These results demonstrate that cyclic tensile strain promotes osteogenic differentiation in BMSC/VEC co-culture systems, possibly via a VEC-mediated paracrine effect of VEGF on BMSCs.

Combined effects of proinflammatory cytokines and intermittent cyclic mechanical strain in inhibiting osteogenicity in human periodontal ligament cells.

Mechanical strain plays an important role in bone formation and resorption during orthodontic tooth movement. The mechanism has not been fully studied, and the process becomes complex with increased amounts of periodontal patients seeking orthodontic care. Our aims were to elucidate the combined effects of proinflammatory cytokines and intermittent cyclic strain (ICS) on the osteogenic capacity of human periodontal ligament cells. Cultured human periodontal ligament cells were exposed to proinflammatory cytokines (interleukin-1β 5 ng/mL and tumor necrosis factor-α 10 ng/mL) for 1 and 5 days, and ICS (0.5 Hz, 12% elongation) was applied for 4 h per day. The autocrine of inflammatory cytokines was measured by enzyme-linked immunosorbent assay. The expression of osteoblast markers runt-related transcription factor 2 and rabbit collagen type I was determined using real-time polymerase chain reaction and Western blot. The osteogenic capacity was also detected by alkaline phosphatase (ALP) staining, ALP activity, and alizarin red staining. We demonstrated that ICS impaired the osteogenic capacity of human periodontal ligament cells when incubated with proinflammatory cytokines, as evidenced by the low expression of ALP staining, low ALP activity, reduced alizarin red staining, and reduced osteoblast markers. These data, for the first time, suggest that ICS has a negative effect on the inductive inhibition of osteogenicity in human PDL cells mediated by proinflammatory cytokines.

Time-dependent combinatory effects of active mechanical loading and passive topographical cues on cell orientation.

Mechanical stretching and topographical cues are both effective mechanical stimulations for regulating cell morphology, orientation, and behaviors. The competition of these two mechanical stimulations remains largely underexplored. Previous studies have suggested that a small cyclic mechanical strain is not able to reorient cells that have been pre-aligned by relatively large linear microstructures, but can reorient those pre-aligned by small linear micro/nanostructures if the characteristic dimension of these structures is below a certain threshold. Likewise, for micro/nanostructures with a given characteristic dimension, the strain must exceed a certain magnitude to overrule the topographic cues. There are however no in-depth investigations of such "thresholds" due to the lack of close examination of dynamic cell orientation during and shortly after the mechanical loading. In this study, the time-dependent combinatory effects of active and passive mechanical stimulations on cell orientation are investigated by developing a micromechanical stimulator. The results show that the cells pre-aligned by linear micro/nanostructures can be altered by cyclic in-plane strain, regardless of the structure size. During the loading, the micro/nanostructures can resist the reorientation effects by cyclic in-plane strain while the resistive capability (measured by the mean orientation angle change and the reorientation speed) increases with the increasing characteristic dimension. The micro/nanostructures also can recover the cell orientation after the cessation of cyclic in-plane strain, while the recovering capability increases with the characteristic dimension. The previously observed thresholds are largely dependent on the observation time points. In order to accurately evaluate the combinatory effects of the two mechanical stimulations, observations during the active loading with a short time interval or endpoint observations shortly after the loading are preferred. This study provides a microengineering solution to investigate the time-dependent combinatory effects of the active and passive mechanical stimulations and is expected to enhance our understanding of cell responses to complex mechanical environments. Biotechnol. Bioeng. 2016;113: 2191-2201. © 2016 Wiley Periodicals, Inc.

Cyclic Strain of Precision Cut Lung Slices (PCLS) Induces Pro-Inflammatory and Pro-Proliferative Signalling

RATIONALE:Cyclic strain of the lung contributes to its development and growth, but can also precipitate pulmonary and systemic injury, known as ventilator-associated lung injury. In vitro cyclic mechanical strain (CMS) models of alveolar epithelial cells are limited by their inability to replicate the complex environment of the lung. Precision cut lung slices (PCLS) offer a novel and accessible model of CMS in human lung with preserved cell-cell and cell-matrix interactions. We hypothesized that CMS would induce pro-inflammatory signalling in PCLS.METHODS:Murine and human lung samples were agarose-inflated and sliced to 400-500μm. PCLS were adhered to a collagen-coated flexible membrane using peripheral application of cyanoacrylate, and CMS was applied at either static, low, or high magnitude CMS conditions. Supernatant was collected for analysis of IL-8 generation and cells were fixed and labelled for markers of mitosis and ERK phosphorylation.RESULTS:CMS increased IL-8 generation and cell proliferation in both murine and human PCLS when compared to static controls. Increasing duration and magnitude of CMS led to increased IL-8 release. Proliferating cells were identified spatially as alveolar epithelial type II cells.CONCLUSION:CMS increased cell proliferation and injury in murine and human PCLS when compared with static controls in a time and CMS-dependent manner. Proliferating cells correlated spatially with alveolar epithelial type II cells. Ongoing work aims to characterise the cells in which pro-inflammatory and proliferative signalling is induced, and also the mechanotransduction pathways in terms of MAPK and mitochondrial ROS signalling, and cytoskeletal reorganisation. These data demonstrate that PCLS provide a viable and translational model to study the effects of CMS in human lung and are the first description, to our knowledge, of CMS-induced cell proliferation and injury in human PCLS.