共查询到20条相似文献,搜索用时 15 毫秒
1.
Jasmin Imsirovic Tyler J. Wellman Jarred R. Mondo?edo Elizabeth Bartolák-Suki Béla Suki 《PloS one》2015,10(10)
Cells in the body experience various mechanical stimuli that are often essential to proper cell function. In order to study the effects of mechanical stretch on cell function, several devices have been built to deliver cyclic stretch to cells; however, they are generally not practical for live cell imaging. We introduce a novel device that allows for live cell imaging, using either an upright or inverted microscope, during the delivery of cyclic stretch, which can vary in amplitude and frequency. The device delivers equi-biaxial strain to cells seeded on an elastic membrane via indentation of the membrane. Membrane area strain was calibrated to indenter depth and the device showed repeatable and accurate delivery of strain at the scale of individual cells. At the whole cell level, changes in intracellular calcium were measured at different membrane area strains, and showed an amplitude-dependent response. At the subcellular level, the mitochondrial network was imaged at increasing membrane area strains to demonstrate that stretch can lead to mitochondrial fission in lung fibroblasts. The device is a useful tool for studying transient as well as long-term mechanotransduction as it allows for simultaneous stretching and imaging of live cells in the presence of various chemical stimuli. 相似文献
2.
Here we demonstrate the microfabrication of deep (> 25 microns) polymeric microstructures created by replica-molding polydimethylsiloxane (PDMS) from microfabricated Si substrates. The use of PDMS structures in microfluidics and biological applications is discussed. We investigated the feasibility of two methods for the microfabrication of the Si molds: deep plasma etch of silicon-on-insulator (SOI) wafers and photolithographic patterning of a spin-coated photoplastic layer. Although the SOI wafers can be patterned at higher resolution, we found that the inexpensive photoplastic yields similar replication fidelity. The latter is mostly limited by the mechanical stability of the replicated PDMS structures. As an example, we demonstrate the selective delivery of different cell suspensions to specific locations of a tissue culture substrate resulting in micropatterns of attached cells. 相似文献
3.
WJ Richardson DD van der Voort E Wilson JE Moore Jr. 《Molecular & cellular biomechanics : MCB》2013,10(3):245-265
Non-uniform stress and strain fields are prevalent in many tissues in vivo, and often exacerbated by disease or injury. These mechanical gradients potentially play a role in contributing to pathological conditions, presenting a need for experimental tools to allow investigation of cell behavior within non-uniformly stimulated environments. Herein, we employ two in vitro cell-stretching devices (one previously published; one newly presented) capable of subjecting cells to cyclic, non-uniform stretches upon the surface of either a circular elastomeric membrane or a cylindrical PDMS tube. After 24 hours of cyclic stretch, 10T1/2 cells on both devices showed marked changes in long-axis orientation, with tendencies to align parallel to the direction of minimal deformation. The degree of this response varied depending on location within the stretch gradients. These results demonstrated the feasibility of conducting cell mechanobiology investigations with the two novel devices, while also highlighting the experimental capabilities of non-uniform mechanical environments for these types of studies. Such capabilities include robust data collection for developing mechanobiological dose-response curves, signal threshold identification, and potential spatial targeting for drug delivery. 相似文献
4.
The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated phenomena such as bone remodeling, muscular hypertrophy, and smooth muscle cell plasticity. However, the forces involved are often extremely complex and difficult to monitor and control in vivo. To better investigate the effects of mechanical forces on cells, we have developed an in vitro method for applying uniaxial cyclic tensile strain to adherent cells cultured on elastic membranes. This method utilizes a custom-designed bioreactor with a motorized cam-rotor system to apply the desired force. Here we present a step-by-step video protocol demonstrating how to assemble the various components of each "stretch chamber", including, in this case, a silicone membrane with micropatterned topography to orient the cells with the direction of the strain. We also describe procedures for sterilizing the chambers, seeding cells onto the membrane, latching the chamber into the bioreactor, and adjusting the mechanical parameters (i.e. magnitude and rate of strain). The procedures outlined in this particular protocol are specific for seeding human mesenchymal stem cells onto silicone membranes with 10 microm wide channels oriented parallel to the direction of strain. However, the methods and materials presented in this system are flexible enough to accommodate a number of variations on this theme: strain rate, magnitude, duration, cell type, membrane topography, membrane coating, etc. can all be tailored to the desired application or outcome. This is a robust method for investigating the effects of uniaxial tensile strain applied to cells in vitro. 相似文献
5.
Masuda T Takahashi I Anada T Arai F Fukuda T Takano-Yamamoto T Suzuki O 《Journal of biotechnology》2008,133(2):231-238
Mechanical stimulation is considered to be one of the major epigenetic factors regulating the metabolism, proliferation, survival and differentiation of cells in the skeletal tissues. It is generally accepted that the cytoskeleton can undergo remodeling in response to mechanical stimuli such as tensile strain or fluid flow. Mechanically induced cell deformation is one of the possible mechanotransduction pathways by which chondrocytes sense and respond to changes in their mechanical environment. Mechanical strain has a variety of effects on the structure and function of their cells in the skeletal tissues, such as chondrocytes, osteoblasts and fibroblasts. However, little is known about the effect of the quality and quantity of mechanical strain and the timing of mechanical loading on the differentiation of these cells. The present study was designed to investigate the effect of the deformation of chondrogenic cells, and cyclic compression using a newly developed culture device, by analyzing mechanobiological response to the differentiating chondrocytes. Cyclic compression between 0 and 22% strains, at 23 microHz was loaded on chondrogenic cell line ATDC5 by seeding in a mass mode on PDMS membrane, assuming direct transfer of cyclic deformation from the membrane to the cells at the same frequency. The compressive strain, induced within the membrane, was characterized based on the analysis of the finite element modeling (FEM). The results showed that the tensile strain inhibits the chondrogenic differentiation of ATDC5 cells, whereas the compressive strain enhances the chondrogenic differentiation, suggesting that the differentiation of the chondrogenic cells could be controlled by the amount and the mode of strain. In conclusion, we have developed a unique strain loading culture system to analyze the effect of various types of mechanical stimulation on various cellular activities. 相似文献
6.
Effective tissue engineering requires appropriate selection of cells and scaffold, where the latter serves as a mechanical and biological support for cell growth and functionality. The optimal combination of cell source and scaffold properties can vary for each desired application. Such preconditions necessitate enhanced understanding of the interactions between cells and scaffold within engineered tissue. Several studies have examined the deforming effects cells induce in scaffolds via exertion of contractile forces. In contrast, other studies focus on the scaffold's biochemical and mechanical properties and their effects on cell behavior.This review summarizes the mechanical interplay between cells and scaffold within engineered tissue. We present evidence for contractile forces exerted by cells on three-dimensional (3D) scaffolds and discuss existing methods for their quantification. In addition, we address some theories related to the effects of scaffold stiffness and mechanical stimulation on cell behavior. Further understanding of the reciprocal effects between cells and scaffold will provide both enhanced knowledge regarding the expected properties of engineered tissue and more competent tissue regeneration techniques. 相似文献
7.
Metzler SA Digesu CS Howard JI Filip To SD Warnock JN 《Biomechanics and modeling in mechanobiology》2012,11(3-4):355-361
Soft tissues, such as tendons, skin, arteries, or lung, are constantly subject to mechanical stresses in vivo. None more so than the aortic heart valve that experiences an array of forces including shear stress, cyclic pressure, strain, and flexion. Anisotropic biaxial cyclic stretch maintains valve homeostasis; however, abnormal forces are implicated in disease progression. The response of the valve endothelium to deviations from physiological levels has not been fully characterized. Here, we show the design and validation of a novel stretch apparatus capable of applying biaxial stretch to viable heart valve tissue, while simultaneously allowing for live en face endothelial cell imaging via confocal laser scanning microscopy (CLSM). Real-time imaging of tissue is possible while undergoing highly characterized mechanical conditions and maintaining the native extracellular matrix. Thus, it provides significant advantages over traditional cell culture or in vivo animal models. Planar biaxial tissue stretching with simultaneous live cell imaging could prove useful in studying the mechanobiology of any soft tissue. 相似文献
8.
Tissue models reconstituted from cells and extracellular matrix (ECM) simulate natural tissues. Cytoskeletal and matrix proteins govern the force exerted by a tissue and its stiffness. Cells regulate cytoskeletal structure and remodel ECM to produce mechanical changes during tissue development and wound healing. Characterization and control of mechanical properties of reconstituted tissues are essential for tissue engineering applications. We have quantitatively characterized mechanical properties of connective tissue models, fibroblast-populated matrices (FPMs), via uniaxial stretch measurements. FPMs resemble natural tissues in their exponential dependence of stress on strain and linear dependence of stiffness on force at a given strain. Activating cellular contractile forces by calf serum and disrupting F-actin by cytochalasin D yield "active" and "passive" components, which respectively emphasize cellular and matrix mechanical contributions. The strain-dependent stress and elastic modulus of the active component were independent of cell density above a threshold density. The same quantities for the passive component increased with cell number due to compression and reorganization of the matrix by the cells. 相似文献
9.
C M Waters M R Glucksberg E P Lautenschlager C W Lee R M Van Matre R J Warp U Savla K E Healy B Moran D G Castner J P Bearinger 《Journal of applied physiology》2001,91(4):1600-1610
There is presently significant interest in cellular responses to physical forces, and numerous devices have been developed to apply stretch to cultured cells. Many of the early devices were limited by the heterogeneity of deformation of cells in different locations and by the high degree of anisotropy at a particular location. We have therefore developed a system to impose cyclic, large-strain, homogeneous stretch on a multiwell surface-treated silicone elastomer substrate plated with pulmonary epithelial cells. The pneumatically driven mechanism consists of four plates each with a clamp to fix one edge of the cruciform elastomer substrate. Four linear bearings set at predetermined angles between the plates ensure a constant ratio of principal strains throughout the stretch cycle. We present the design of the device and membrane shape, the surface modifications of the membrane to promote cell adhesion, predicted and experimental measurements of the strain field, and new data using cultured airway epithelial cells. We present for the first time the relationship between the magnitude of cyclic mechanical strain and the extent of wound closure and cell spreading. 相似文献
10.
Collinsworth AM Torgan CE Nagda SN Rajalingam RJ Kraus WE Truskey GA 《Cell and tissue research》2000,302(2):243-251
Effects of mechanical forces exerted on mammalian skeletal muscle cells during development were studied using an in vitro model to unidirectionally stretch cultured C2C12 cells grown on silastic membrane. Previous models to date have not studied these responses of the mammalian system specifically. The silastic membrane upon which these cells were grown exhibited linear strain behavior over the range of 3.6-14.6% strain, with a Poisson's ratio of approximately 0.5. To mimic murine in utero long bone growth, cell substrates were stretched at an average strain rate of 2.36%/day for 4 days or 1.77%/day for 6 days with an overall membrane strain of 9.5% and 10.6%, respectively. Both control and stretched fibers stained positively for the contractile protein, alpha-actinin, demonstrating muscle fiber development. An effect of stretch on orientation and length of myofibers was observed. At both strain rates, stretched fibers aligned at a smaller angle relative to the direction of stretch and were significantly longer compared to randomly oriented control fibers. There was no effect of duration of stretch on orientation or length, suggesting the cellular responses are independent of strain rate for the range tested. These results demonstrate that, under conditions simulating mammalian long bone growth, cultured myocytes respond to mechanical forces by lengthening and orienting along the direction of stretch. 相似文献
11.
Richardson WJ Metz RP Moreno MR Wilson E Moore JE 《Journal of biomechanical engineering》2011,133(10):101008
Mechanical forces are key regulators of cell function with varying loads capable of modulating behaviors such as alignment, migration, phenotype modulation, and others. Historically, cell-stretching experiments have employed mechanically simple environments (e.g., uniform uniaxial or equibiaxial stretches). However, stretch distributions in vivo can be highly non-uniform, particularly in cases of disease or subsequent to interventional treatments. Herein, we present a cell-stretching device capable of subjecting cells to controllable gradients in biaxial stretch via radial deformation of circular elastomeric membranes. By including either a defect or a rigid fixation at the center of the membrane, various gradients are generated. Capabilities of the device were quantified by tracking marked positions of the membrane while applying various loads, and experimental feasibility was assessed by conducting preliminary experiments with 3T3 fibroblasts and 10T1/2 cells subjected to 24 h of cyclic stretch. Quantitative real-time PCR was used to measure changes in mRNA expression of a profile of genes representing the major smooth muscle phenotypes. Genes associated with the contractile state were both upregulated (e.g., calponin) and downregulated (e.g., α-2-actin), and genes associated with the synthetic state were likewise both upregulated (e.g., SKI-like oncogene) and downregulated (e.g., collagen III). In addition, cells aligned with an orientation perpendicular to the maximal stretch direction. We have developed an in vitro cell culture device that can produce non-uniform stretch environments similar to in vivo mechanics. Cells stretched with this device showed alignment and altered mRNA expression indicative of phenotype modulation. Understanding these processes as they relate to in vivo pathologies could enable a more accurately targeted treatment to heal or inhibit disease, either through implantable device design or pharmaceutical approaches. 相似文献
12.
Dynamic fibroblast cytoskeletal response to subcutaneous tissue stretch ex vivo and in vivo 总被引:7,自引:0,他引:7
Langevin HM Bouffard NA Badger GJ Iatridis JC Howe AK 《American journal of physiology. Cell physiology》2005,288(3):C747-C756
Cytoskeleton-dependent changes in cell shape are well-established factors regulating a wide range of cellular functions including signal transduction, gene expression, and matrix adhesion. Although the importance of mechanical forces on cell shape and function is well established in cultured cells, very little is known about these effects in whole tissues or in vivo. In this study we used ex vivo and in vivo models to investigate the effect of tissue stretch on mouse subcutaneous tissue fibroblast morphology. Tissue stretch ex vivo (average 25% tissue elongation from 10 min to 2 h) caused a significant time-dependent increase in fibroblast cell body perimeter and cross-sectional area (ANOVA, P < 0.01). At 2 h, mean fibroblast cell body cross-sectional area was 201% greater in stretched than in unstretched tissue. Fibroblasts in stretched tissue had larger, "sheetlike" cell bodies with shorter processes. In contrast, fibroblasts in unstretched tissue had a "dendritic" morphology with smaller, more globular cell bodies and longer processes. Tissue stretch in vivo for 30 min had effects that paralleled those ex vivo. Stretch-induced cell body expansion ex vivo was inhibited by colchicine and cytochalasin D. The dynamic, cytoskeleton-dependent responses of fibroblasts to changes in tissue length demonstrated in this study have important implications for our understanding of normal movement and posture, as well as therapies using mechanical stimulation of connective tissue including physical therapy, massage, and acupuncture. mechanotransduction; connective tissue; tensegrity; musculoskeletal manipulations; acupuncture 相似文献
13.
Truschel ST Wang E Ruiz WG Leung SM Rojas R Lavelle J Zeidel M Stoffer D Apodaca G 《Molecular biology of the cell》2002,13(3):830-846
The epithelium of the urinary bladder must maintain a highly impermeable barrier despite large variations in urine volume during bladder filling and voiding. To study how the epithelium accommodates these volume changes, we mounted bladder tissue in modified Ussing chambers and subjected the tissue to mechanical stretch. Stretching the tissue for 5 h resulted in a 50% increase in lumenal surface area (from approximately 2900 to 4300 microm(2)), exocytosis of a population of discoidal vesicles located in the apical cytoplasm of the superficial umbrella cells, and release of secretory proteins. Surprisingly, stretch also induced endocytosis of apical membrane and 100% of biotin-labeled membrane was internalized within 5 min after stretch. The endocytosed membrane was delivered to lysosomes and degraded by a leupeptin-sensitive pathway. Last, we show that the exocytic events were mediated, in part, by a cyclic adenosine monophosphate, protein kinase A-dependent process. Our results indicate that stretch modulates mucosal surface area by coordinating both exocytosis and endocytosis at the apical membrane of umbrella cells and provide insight into the mechanism of how mechanical forces regulate membrane traffic in non-excitable cells. 相似文献
14.
Liujing Chen Tu Huang Yini Qiao Fulin Jiang Jingxiang Lan Yimei Zhou Cai Yang Shanyu Yan Kaihui Luo Liping Su Juan Li 《Journal of cellular biochemistry》2019,120(6):8884-8890
Stem cells are promising candidates for cell-based therapies in diverse conditions including regenerating damaged tissues, treating inflammation in virtue of sepsis, acute renal failure, and cardiovascular disease. Advancement of these therapies relies on the ability to guide stem cells to migrate directly and differentiate towards specific cell phenotypes. During the past decade, many researchers have demonstrated that exogenous applied forces could significantly affect the migration and lineage differentiation of stem cells. Besides, recent advances have highlighted the critical role of internal forces due to cell-matrix interaction in the function of stem cells. Stem cells can generate contractile forces to sense the mechanical properties of cell-generated force microenvironment, and thereby perceive mechanical information that directs broad aspects of stem cell functions, including migration and lineage commitment. In the review, we recount the cell-generated force microenvironment of stem cells and discuss the interactions between cell-generated forces with migration and differentiation of stem cells. We also summarize key experimental evidence of a tight linkage between migration and lineage differentiation of stem cells and pose important unanswered questions in this field. 相似文献
15.
Dominique Tremblay Charles M. Cuerrier Lukasz Andrzejewski Edward R. O'Brien Andrew E. Pelling 《Journal of visualized experiments : JoVE》2014,(88)
Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels. 相似文献
16.
Estimates of force generation or shortening obtained from smooth muscle tissues are valid for individual cells only if each cell is contracting homogeneously and if cells anatomically arranged in series are mechanically coupled. These two assumptions were tested and shown to be valid for the pig carotid media under certain conditions. Homogeneity of cellular responses in carotid strips was estimated from the motion of markers on the tissue during K+ -induced isometric contractions. When tissues were stretched to L0 (the optimum length for force generation), there was little marker movement on stimulation. However, considerable marker movement was observed on stimulation at shorter muscle lengths, reflecting localized shortening or stretching. The mechanical coupling of the very small cells in the media was determined by measuring the dependence of cell length on tissue length. Tissues were fixed with glutaraldehyde during isometric contractions at various tissue lengths (0.4--1.1 x L0). The fixed tissues were macerated with acid and the lengths of the dispersed cells were measured. Cell lengths were broadly distributed at all muscle lengths. However, the direct proportionality between mean cell length and muscle length (as a fraction of L0) indicated that cells which are anatomically in series are coupled force-transmitting structures. We conclude that valid estimates of cellular mechanical function in this preparation can be obtained from tissue measurements at lengths greater than about 0.9L0. 相似文献
17.
Mechanosensitive ion channels are expected to play important roles in transducing mechanical stimuli into intracellular signals during the development and morphogenesis of higher plants. We have identified a novel mechanosensitive anion channel in the protoplast of Arabidopsis thaliana mesophyll cells by using the patch-clamp technique. The channel in the outside-out patches could be activated by positive pressure in the pipette while negative pressure had no effect. The amphipathic membrane crenator trinitrophenol, which is supposed to preferentially insert in the outer leaflet of the lipid bilayer of the plasma membrane, synergized with mechanical membrane stretch to activate the channel. These results suggest that the channel activation is mediated by a convex curvature of the plasma membrane. Therefore, activation of this channel may play an important role when cell volume is increasing during cell growth or hypo-osmotic challenge, which is accompanied by membrane stretch with increasingly convex curvature. 相似文献
18.
F. S. Tabatabaei M. Jazayeri P. Ghahari N. Haghighipour 《Molecular & cellular biomechanics : MCB》2014,11(3):209-220
During orthodontic treatments, applied mechanical forces create strain and result in tooth movement through the alveolar bone. This response to mechanical strain is a fundamental biological reaction. The present study evaluated the effect of equiaxial strain within the range of orthodontic forces on the osteogenic differentiation of human dental pulp stem cells (hDPSCs). Following isolation and culture of hDPSCs, 3rd passage cells were transferred on a silicone membrane covered with collagen. Cell adhesion to the membrane was evaluated under scanning electron microscope (SEM). Cells were divided into three groups: the first group was placed in a conventional culture medium, transferred to an equiaxial stretching device (3% strain for 2 weeks). The positive control was placed in an osteogenic medium with no mechanical strain. The negative control group was placed in the conventional culture medium with no mechanical strain either. Study groups were evaluated for expression of osteogenic markers (Alkaline phosphatase and Osteopontin) with immunofluorescence and real time PCR. SEM images revealed optimal adhesion of cells to the silicone membrane. Immunofluorescence study demonstrated that osteocalcin expression occurred after 2 weeks in the two groups under mechanical and chemical signals. After application of equiaxial strain, level of expression of osteogenic markers was significantly higher than in the negative and positive control groups. Based on the study results, static equiaxial strain which mimics the types of orthodontic forces can result in differentiation of hDPSCs to osteoblasts. The results obtained may be used in cell therapy and tissue engineering. 相似文献
19.
Fisher JL Margulies SS 《American journal of physiology. Lung cellular and molecular physiology》2007,292(1):L40-L53
While a number of whole cell mechanical models have been proposed, few, if any, have focused on the relationship among plasma membrane tension, plasma membrane unfolding, and plasma membrane expansion and relaxation via lipid insertion. The goal of this communication is to develop such a model to better understand how plasma membrane tension, which we propose stimulates Na(+)-K(+)-ATPase activity but possibly also causes cell injury, may be generated in alveolar epithelial cells during mechanical ventilation. Assuming basic relationships between plasma membrane unfolding and tension and lipid insertion as the result of tension, we have captured plasma membrane mechanical responses observed in alveolar epithelial cells: fast deformation during fast cyclic stretch, slower, time-dependent deformation via lipid insertion during tonic stretch, and cell recovery after release from stretch. The model estimates plasma membrane tension and predicts Na(+)-K(+)-ATPase activation for a specified cell deformation time course. Model parameters were fit to plasma membrane tension, whole cell capacitance, and plasma membrane area data collected from the literature for osmotically swollen and shrunken cells. Predictions of membrane tension and stretch-stimulated Na(+)-K(+)-ATPase activity were validated with measurements from previous studies. As a proof of concept, we demonstrate experimentally that tonic stretch and consequent plasma membrane recruitment can be exploited to condition cells against subsequent cyclic stretch and hence mitigate stretch-induced responses, including stretch-induced cell death and stretch-induced modulation of Na(+)-K(+)-ATPase activity. Finally, the model was exercised to evaluate plasma membrane tension and potential Na(+)-K(+)-ATPase stimulation for an assortment of traditional and novel ventilation techniques. 相似文献
20.
Nazaré Pereira-Rodrigues Paul-Emile Poleni Denis Guimard Yasuhiko Arakawa Yasuyuki Sakai Teruo Fujii 《PloS one》2010,5(3)