Cytoskeletal changes and the system of regulation of alkaline phosphatase activity in human periodontal ligament cells induced by mechanical stress

The periodontal ligament (PDL) is a specialized, mechanically responsive tissue that adapts via cellular responses to equilibrate the effects of mechanical stress on teeth. However, the mechanism of remodelling by which individual cells in periodontal tissue detect and respond to mechanical stress is not well understood. To identify the cellular mechanisms induced by mechanical stress in the periodontal ligament, we examined the effects of cyclic stretching on periodontal ligament fibroblast-like cells (PDL cells). Furthermore, we investigated the effects of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and interaction with peripheral blood mononuclear cells (PBMCs) on mechanically-simulated PDL cells. PDL cells were cultured on type I collagen-coated silicon membranes with 10% FBS alpha-MEM, and then subjected to cyclic mechanical stimulation (1 s stretching/1 s relaxation, 15% maximum elongation). Alkaline phosphatase activity was monitored by cytochemical and spectrophotometric methods. Morphologically, the cells assumed a spindle shape, and the cytoskeletal components, including microtubules and F-actin filaments, were aligned perpendicular to the strain force vector. Cyclic stretching decreased ALPase activity in PDL cells. The anabolic systemic hormone 1,25(OH)(2)D(3) increased ALPase activity, but this effect was suppressed by cyclic stretching. ALPase activities were reduced by co-culture with PBMCs, including lymphocytes and monocytes. This PBMC-induced ALPase reduction was synergistically reduced by cyclic stretching. ALPase activity was decreased by co-culture with PBMCs, and ALPase activity was reduced synergistically by treatment with PBMCs and cyclic stretching. We conclude that PDL cells changed their shape and alignment in response to cyclic stretching. Furthermore, local factors, such as mechanical stress and PBMCs, showed synergistic suppressive effects on ALPase activity.     

Theoretical study of intracellular stress fiber orientation under cyclic deformation

We studied stress fiber orientation under a wide range of uniaxial cyclic deformations. We devised and validated a hypothesis consisting of two parts, as follows: (1) a stress fiber aligns to avoid a mechanical stimulus in the fiber direction under cyclic deformation. This means that, among all allowable directions, a stress fiber aligns in the direction which minimizes the stimulus, i. e., the summation of the changes in length of the stress fiber over one stretch cycle; and (2) there is a limit in the sensitivity of the cellular response to the mechanical stimulus. Due to this sensing limit, the orientation angle in stress fibers is distributed around the angle corresponding to the minimum stimulus. To validate this hypothesis, we approximated an anisotropic deformation of the membrane on which cells were to be cultured. We then obtained the relationships between the stretch range and the fiber angle in the undeformed state which minimize the mechanical stimuli, assuming that the membrane on which stress fibers and cells adhered was homogeneous and incompressible. Numerical simulation results showed that the proposed hypothesis described our previous experimental results well and was consistent with the experimental results in the literature. The simulation results, taking account of the second part of the hypothesis with a small value for the limit in sensitivity to the mechanical stimulus, could explain why cell orientation is distributed so widely with cyclic stretch ranges of <10%. The proposed hypothesis can be applied to various types of deformation because the mechanical stimulus is always sensed and accumulates under cyclic deformation without the necessity of a reference state to measure the stimulus.     

A new experimental system for the extended application of cyclic hydrostatic pressure to cell culture

Mechanical forces have been shown to be important stimuli for the determination and maintenance of cellular phenotype and function. Many cells are constantly exposed in vivo to cyclic pressure, shear stress, and/or strain. Therefore, the ability to study the effects of these stimuli in vitro is important for understanding how they contribute to both normal and pathologic states. While there exist commercial as well as custom-built devices for the extended application of cyclic strain and shear stress, very few cyclic pressure systems have been reported to apply stimulation longer than 48 h. However, pertinent responses of cells to mechanical stimulation may occur later than this. To address this limitation, we have designed a new cyclic hydrostatic pressure system based upon the following design variables: minimal size, stability of pressure and humidity, maximal accessibility, and versatility. Computational fluid dynamics (CFD) was utilized to predict the pressure and potential shear stress within the chamber during the first half of a 1.0 Hz duty cycle. To biologically validate our system, we tested the response of bone marrow progenitor cells (BMPCs) from Sprague Dawley rats to a cyclic pressure stimulation of 120/80 mm Hg, 1.0 Hz for 7 days. Cellular morphology was measured using Scion Image, and cellular proliferation was measured by counting nuclei in ten fields of view. CFD results showed a constant pressure across the length of the chamber and no shear stress developed at the base of the chamber where the cells are cultured. BMPCs from Sprague Dawley rats demonstrated a significant change in morphology versus controls by reducing their size and adopting a more rounded morphology. Furthermore, these cells increased their proliferation under cyclic hydrostatic pressure. We have demonstrated that our system imparts a single mechanical stimulus of cyclic hydrostatic pressure and is capable of at least 7 days of continuous operation without affecting cellular viability. Furthermore, we have shown for the first time that BMPCs respond to cyclic hydrostatic pressure by alterations in morphology and increased proliferation.     

Mechanical force mobilizes zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement

Organs and tissues adapt to acute or chronic mechanical stress by remodeling their actin cytoskeletons. Cells that are stimulated by cyclic stretch or shear stress in vitro undergo bimodal cytoskeletal responses that include rapid reinforcement and gradual reorientation of actin stress fibers; however, the mechanism by which cells respond to mechanical cues has been obscure. We report that the application of either unidirectional cyclic stretch or shear stress to cells results in robust mobilization of zyxin from focal adhesions to actin filaments, whereas many other focal adhesion proteins and zyxin family members remain at focal adhesions. Mechanical stress also induces the rapid zyxin-dependent mobilization of vasodilator-stimulated phosphoprotein from focal adhesions to actin filaments. Thickening of actin stress fibers reflects a cellular adaptation to mechanical stress; this cytoskeletal reinforcement coincides with zyxin mobilization and is abrogated in zyxin-null cells. Our findings identify zyxin as a mechanosensitive protein and provide mechanistic insight into how cells respond to mechanical cues.     

Regulation of stretch-induced JNK activation by stress fiber orientation

Cyclic mechanical stretch associated with pulsatile blood pressure can modulate cytoskeletal remodeling and intracellular signaling in vascular endothelial cells. The aim of this study was to evaluate the role of stretch-induced actin stress fiber orientation in intracellular signaling involving the activation of c-jun N-terminal kinase (JNK) in bovine aortic endothelial cells. A stretch device was designed with the capability of applying cyclic uniaxial and equibiaxial stretches to cultured endothelial cells, as well as changing the direction of cyclic uniaxial stretch. In response to 10% cyclic equibiaxial stretch, which did not result in stress fiber orientation, JNK activation was elevated for up to 6 h. In response to 10% cyclic uniaxial stretch, JNK activity was only transiently elevated, followed by a return to basal level as the actin stress fibers became oriented perpendicular to the direction of stretch. After the stress fibers had aligned perpendicularly and the JNK activity had subsided, a 90-degree change in the direction of cyclic uniaxial stretch reactivated JNK, and this activation again subsided as stress fibers became re-oriented perpendicular to the new direction of stretch. Disrupting actin filaments with cytochalasin D blocked the stress fiber orientation in response to cyclic uniaxial stretch and it also caused the uniaxial stretch-induced JNK activation to become sustained. These results suggest that stress fiber orientation perpendicular to the direction of stretch provides a mechanism for both structural and biochemical adaptation to cyclic mechanical stretch.     

Cyclic Bending Contributes to High Stress in a Human Coronary Atherosclerotic Plaque and Rupture Risk: In Vitro Experimental Modeling and Ex Vivo MRI-Based Computational Modeling Approach

Many acute cardiovascular syndromes such as heart attack and stroke are caused by atherosclerotic plaque ruptures which often happen without warning. MRI-based models with fluid-structure interactions (FSI) have been introduced to perform flow and stress/strain analysis for atherosclerotic plaques and identify possible mechanical and morphological indices for accurate plaque vulnerability assessment. In this paper, cyclic bending was added to 3D FSI coronary plaque models for more accurate mechanical predictions. Curvature variation was prescribed using the data of a human left anterior descending (LAD) coronary artery. Five computational models were constructed based on ex vivo MRI human coronary plaque data to assess the effects of cyclic bending, pulsating pressure, plaque structure, and axial stretch on plaque stress/strain distributions. In vitro experiments using a hydrogel stenosis model with cyclical bending were performed to observe effect of cyclical bending on flow conditions. Our results indicate that cyclical bending may cause more than 100% or even up to more than 1000% increase in maximum principal stress values at locations where the plaque is bent most. Stress increase is higher when bending is coupled with axial stretch, non-smooth plaque structure, or resonant pressure conditions (zero phase angle shift). Effects of cyclic bending on flow behaviors are more modest (21.6% decrease in maximum velocity, 10.8% decrease in flow rate, maximum flow shear stress changes were < 5%). Computational FSI models including cyclic bending, plaque components and structure, axial stretch, accurate in vivo measurements of pressure, curvature, and material properties should lead to significant improvement on stress-based plaque mechanical analysis and more accurate coronary plaque vulnerability assessment.     

Cyclic-stretch induces the apoptosis of myoblast by activation of Caspase-3 protease in a magnitude-dependent manner

Although many studies have been performed investigating the effects of mechanical stress on the generation and differentiation of myoblasts, little is known about the effects of different magnitudes of mechanical stretch on apoptosis in these cells. The aim of this study was to investigate the effects of different magnitudes of cyclic stretch on apoptosis levels in myoblasts and to investigate the possible mechanisms involved. Myoblasts were cultured on flexible membranes and subjected to cyclic strain stress in a magnitude-dependent manner (6%, 12% or 20% surface elongation). Apoptosis rates were evaluated using flow cytometry, transmission electron microscopy, and caspase assays. Fas/FasL expression was determined by Western blot. The application of different magnitudes of cyclic-stretch-induced a magnitude-dependent increase in apoptosis and caspase-3 activity in cultured myoblasts. Furthermore, inhibition of caspase-3 prevented stretch-induced apoptosis in myoblasts but did not change Fas and FasL protein levels. These data indicate that cyclic stretch induces a magnitude- and caspase-3-dependent increase of apoptosis in cultured myoblasts in vitro. Mechanical forces induced activation of caspase-3 through signalling pathways independent of the Fas/FasL system. These results suggest the existence of a novel mechanism for the regulation of myoblast apoptosis by cyclic stretch.     

NF-kappaB responds to mechanical strains in osteoblast-like cells, and lighter strains create an NF-kappaB response more readily

This study was to examine the early responses of nuclear factor kappa B (NF-kappaB) to mechanical strains in MG-63. MG-63 cells were subjected to cyclic uniaxial compressive or tensile strain, produced by a four-point bending system, at 1000 microstrain or 4000 microstrain for 5 min, 15 min, 30 min and 1h, respectively. Control cells received the same treatment with no mechanical stress loading. Expression of NF-kappaB (p60) was measured by Western blotting. NF-kappaB responded rapidly to mechanical stimuli in MG-63 cells. NF-kappaB was activated by cyclic uniaxial stretch at 1000 microstrain while it was restrained under a compressive strain environment at 1000 microstrain (P<0.001). The effects reversed for tension and compression at 4000 microstrain (P<0.001). Furthermore, strains at 1000 microstrain affected NF-kappaB expression much easier than those at 4000 microstrain. This indicates that there may be different responding mechanisms or mechanotransduction pathways for different mechanical stimuli.     

Early responses of osteoblast-like cells to different mechanical signals through various signaling pathways

This study was to examine the effects of mechanical stimuli alone and coupled with some inhibitors of related signaling pathways on early cellular responses. MG-63 cells were subjected to cyclic uniaxial compressive or tensile strain at 4000 microstrain, produced by four-point bending system. The effects of mechanical strains alone and coupled with inhibitors of microfilament and receptor tyrosine kinase (RTK) on activation of extracellular signal-regulated kinase (ERK), c-fos mRNA, and c-Fos protein were examined. ERK could be activated by mechanical stimuli in 5 min and so could be c-fos mRNA and c-Fos protein in 30 min. Tensile stress had a more obvious effect than compressive one. Early cellular responses were connected with cytoskeleton and RTK pathways during the transduction of mechanical signals. The property of strains was an influential factor for the activation effects.     

Effects of the frequency and duration of cyclic stress on the mechanical properties of cultured collagen fascicles from the rabbit patellar tendon

The effects of frequency or duration of cyclic stress on the mechanical properties of collagen fascicles were studied by means of in vitro tissue culture experiments. Collagen fascicles of approximately 300 microm in diameter were obtained from rabbit patellar tendons. During culture, cyclic stress having the peak stress of approximately 2 MPa was applied to the fascicles at 1 Hz for 1 hour/day (1 Hz-1 h group), at 1 Hz for 4 hours/day (1 Hz-4 h group), or at 4 Hz for 1 hour/day (4 Hz-1 h group). The frequency of 4 Hz and the duration of 1 hour/day are considered to be similar to those of the in vivo stress applied to fascicles in the intact rabbit patellar tendon. After culture for 1 or 2 weeks, the mechanical properties of the fascicles were determined using a microtensile tester, and were compared to the properties of non-cultured, fresh fascicles (control group) and the fascicles cultured under no load condition (non-loaded group). The tangent modulus and tensile strength of fascicles in the 4 Hz-1 h group were similar to those in the control group; however, the fascicles of the 1 Hz-1 h and 1 Hz-4 h groups had significantly lower values than those of the control group. There was no significant difference in the tensile strength between the 1 Hz-1 h and non-loaded groups, although the strength in the 1 Hz-4 h group was significantly higher than that of the non-loaded group. It was concluded that the frequency and duration of cyclic stress significantly affect the mechanical properties of cultured collagen fascicles. If we apply cyclic stress having the frequency and duration which are experienced in vivo, the biomechanical properties are maintained at control, normal level. Lower frequencies or less cycles of applied force induce adverse effects.     

Ultrasound echo is related to stress and strain in tendon

The mechanical behavior of tendons has been well studied in vitro. A noninvasive method to acquire mechanical data would be highly beneficial. Elastography has been a promising method of gathering in vivo tissue mechanical behavior, but it has inherent limitations. This study presents acoustoelasticity as an alternative ultrasound-based method of measuring tendon stress and strain by reporting a relationship between ultrasonic echo intensity (B-mode ultrasound image brightness) and mechanical behavior of tendon in vitro. Porcine digital flexor tendons were cyclically loaded in a mechanical testing system while an ultrasonic echo response was recorded. We report that echo intensity closely follows the applied cyclic strain pattern in time with higher strain protocols resulting in larger echo intensity changes. We also report that echo intensity is related nonlinearly to stress and nearly linearly to strain. This indicates that ultrasonic echo intensity is related to the mechanical behavior in a loaded tissue by an acoustoelastic response, as previously described in homogeneous, nearly incompressible materials. Acoustoelasticity is therefore able to relate strain-dependent stiffness and stress to the reflected echo, even in the processed B-mode signals reflected from viscoelastic and inhomogeneous material such as tendon, and is a promising metric to acquire in vivo mechanical data noninvasively.     

Reduced troponin I phosphorylation and increased Ca2+-dependent ATP-consumption in triton X-skinned fiber preparations from Gαq overexpressor mice

Mechanical stress leads to satellite cell activation, which is an important event in the development, growth, and remodeling of postnatal skeletal muscle. Although there is a considerable knowledge on the events involved in skeletal muscle regeneration and development, the precise role of mechanical stress on activation of satellite cells remains unclear. Previously, satellite cells were isolated from adult bovine muscle and it was shown that the cells are multipotent, i.e., capable of proliferating and to differentiating into both myoblasts and adipocytes. This study investigated the cellular mechanisms by which cyclic mechanical stretching modulates the proliferation and differentiation of adult bovine satellite cells. The application of cyclic stretch induced the proliferation of satellite cells and inhibited their differentiation into myotubes. This response is believed to be closely related to the stretch-mediated changes in the expression of myogenic and cell cycle regulatory factors. Cyclic stretching increased the level of extracellular signal-regulated kinase (ERK) phosphorylation, whereas a specific ERK inhibitor (PD98058) blocked the stretch-mediated inhibition of myogenesis in a dose-dependent manner. Overall, this study demonstrates for the first time that cyclic mechanical stretch induces the proliferation of bovine satellite cells and suppresses their myogenic differentiation through the activation of ERK.     

Differential effects of static and cyclic stretching during elastase digestion on the mechanical properties of extracellular matrices.

Enzyme activity plays an essential role in many physiological processes and diseases such as pulmonary emphysema. While the lung is constantly exposed to cyclic stretching, the effects of stretch on the mechanical properties of the extracellular matrix (ECM) during digestion have not been determined. We measured the mechanical and failure properties of elastin-rich ECM sheets loaded with static or cyclic uniaxial stretch (40% peak strain) during elastase digestion. Quasistatic stress-strain measurements were taken during 30 min of digestion. The incremental stiffness of the sheets decreased exponentially with time during digestion. However, digestion in the presence of static stretch resulted in an accelerated stiffness decrease, with a time constant that was nearly 3 x smaller (7.1 min) than during digestion alone (18.4 min). These results were supported by simulations that used a nonlinear spring network model. The reduction in stiffness was larger during static than cyclic stretch, and the latter also depended on the frequency. Stretching at 20 cycles/min decreased stiffness less than stretching at 5 cycles/min, suggesting a rate-dependent coupling between mechanical forces and enzyme activity. Furthermore, pure digestion reduced the failure stress of the sheets from 88 +/- 21 kPa in control to 29 +/- 15 kPa (P < 0.05), while static and cyclic stretch resulted in a failure stress of 7 +/- 5 kPa (P < 0.05). We conclude that not only the presence but the dynamic nature of mechanical forces have a significant impact on enzyme activity, hence the deterioration of the functional properties of the ECM during exposure to enzymes.     

Disc mechanics with trans-endplate partial nucleotomy are not fully restored following cyclic compressive loading and unloaded recovery

Mechanical function of the intervertebral disc is maintained through the interaction between the hydrated nucleus pulposus, the surrounding annulus fibrosus, and the superior and inferior endplates. In disc degeneration the normal transfer of load between disc substructures is compromised. The objective of this study was to explore the mechanical role of the nucleus pulposus in support of axial compressive loads over time. This was achieved by measuring the elastic slow ramp and viscoelastic stress-relaxation mechanical behaviors of cadaveric sheep motion segments before and after partial nucleotomy through the endplate (keeping the annulus fibrosus intact). Mechanics were evaluated at five conditions: Intact, intact after 10,000 cycles of compression, acutely after nucleotomy, following nucleotomy and 10,000 cycles of compression, and following unloaded recovery. Radiographs and magnetic resonance images were obtained to examine structure. Only the short time constant of the stress relaxation was altered due to nucleotomy. In contrast, cyclic loading resulted in significant and large changes to both the stiffness and stress relaxation behaviors. Moreover, the nucleotomy had little to no effect on the disc mechanics after cyclic loading, as there were no significant differences comparing mechanics after cyclic loading with or without the nucleotomy. Following unloaded recovery the mechanical changes that had occurred as a consequence of cyclic loading were restored, leaving only a sustained change in the short time constant due to the trans-endplate nucleotomy. Thus the swelling and redistribution of the remaining nucleus pulposus was not able to fully restore mechanical behaviors. This study reveals insights into the role of the nucleus pulposus in disc function, and provides new information toward the potential role of altered nucleus pulpous function in the degenerative cascade.     

A kinematic model of stretch-induced stress fiber turnover and reorientation

A kinetic model based on constrained mixture theory was developed to describe the reorganization of actin stress fibers in adherent cells in response to diverse patterns of mechanical stretch. The model was based on reports that stress fibers are pre-extended at a “homeostatic” level under normal, non-perturbed conditions, and that perturbations in stress fiber length destabilize stress fibers. In response to a step change in matrix stretch, the model predicts that stress fibers are initially stretched in registry with the matrix, but that these overly stretched fibers are gradually replaced by new fibers assembled with the homeostatic level of stretch in the new configuration of the matrix. In contrast, average fiber stretch is chronically perturbed from the homeostatic level when the cells are subjected to cyclic equibiaxial stretch. The model was able to describe experimentally measured time courses of stress fiber reorientation perpendicular to the direction of cyclic uniaxial stretch, as well as the lack of alignment in response to equibiaxial stretch. The model also accurately described the relationship between stretch magnitude and the extent of stress fiber alignment in endothelial cells subjected to cyclic uniaxial stretch. Further, in the case of cyclic simple elongation with transverse matrix contraction, stress fibers orient in the direction of least perturbation in stretch. In summary, the model predicts that the rate of stretch-induced stress fiber disassembly determines the rate of alignment, and that stress fibers tend to orient toward the direction of minimum matrix stretch where the rate of stress fiber turnover is a minimum.     

Mechanical stretch upregulates IGFBP-1 secretion from decidualized endometrial stromal cells

Decidualization is an essential process of endometrial differentiation for embryo implantation and maintenance of pregnancy. Recently, uterine movement-induced mechanical stress was noticed to have possible effects on endometrial functions. In this study, we addressed the possible effect of mechanical stress on the process of decidualization of endometrial stromal cells (ESC). ESC were cultured on flexible-bottomed culture plates. After decidualization was achieved with estradiol and progesterone for 12 days, cultures were continued for 24 h with or without cyclic stretch (25% elongation) in serum-free conditions at a rate of 2 cycles/min using a computer-operated cell tension system. Concentrations of insulin-like growth factor-binding protein-1 (IGFBP-1), a marker of decidualization, in the conditioned medium were measured by specific ELISA, and IGFBP-1 mRNA expression in the ESC was measured by RT-PCR. Cyclic stretch remarkably increased IGFBP-1 secretion from decidualized ESC. It also increased IGFBP-1 mRNA in decidualized ESC. The increase in IGFBP-1 secretion was inhibited by actinomycin D but not by indomethacin, PD-98059, or H-89. Conditioned medium of decidualized ESC cultured with cyclic stretch increased IGFBP-1 secretion from decidualized ESC cultured under stationary conditions. These findings imply that uterine movement modulates decidualization of the endometrium and has a regulatory effect on reproduction.     

Strain-induced damage reduces echo intensity changes in tendon during loading

Tendon functionality is related to its mechanical properties. Tendon damage leads to a reduction in mechanical strength and altered biomechanical behavior, and therefore leads to compromised ability to carry out normal functions such as joint movement and stabilization. Damage can also accumulate in the tissue and lead to failure. A noninvasive method with which to measure such damage potentially could quantify structural compromise from tendon injury and track improvement over time. In this study, tendon mechanics are measured before and after damage is induced by "overstretch" (strain exceeding the elastic limit of the tissue) using a traditional mechanical test system while ultrasonic echo intensity (average gray scale brightness in a B-mode image) is recorded using clinical ultrasound. The diffuse damage caused by overstretch lowered the stress at a given strain in the tissue and decreased viscoelastic response. Overstretch also lowered echo intensity changes during stress relaxation and cyclic testing. As the input strain during overstretch increased, stress levels and echo intensity changes decreased. Also, viscoelastic parameters and time-dependent echo intensity changes were reduced.