Cell Adhesion Force Research Articles

KCa3.1 channel inhibition leads to an ICAM-1 dependent increase of cell-cell adhesion between A549 lung cancer and HMEC-1 endothelial cells.

Early metastasis leads to poor prognosis of lung cancer patients, whose 5-year survival rate is only 15%. We could recently show that the Ca2+ sensitive K+ channel KCa3.1 promotes aggressive behavior of non-small cell lung cancer (NSCLC) cells and that it can serve as a prognostic marker in NSCLC. Since NSCLC patients die of metastases, we investigated whether KCa3.1 channels contribute to poor patient prognosis by regulating distinct steps of the metastatic cascade. We investigated the extravasation of NSCLC cells and focused on their adhesion to endothelial cells and on transendothelial migration. We quantified the adhesion forces between NSCLC cells and endothelial cells by applying single cell force spectroscopy, and we monitored transendothelial migration using live-cell imaging. Inhibition of KCa3.1 channels with senicapoc or KCa3.1 silencing increases the adhesion force of A549 lung cancer cells to human microvascular endothelial cells (HMEC-1). Western blotting, immunofluorescence staining and biotinylation assays indicate that the elevated adhesion force is due to increased expression of ICAM-1 in both cell lines when KCa3.1 channels are downregulated. Consistent with this interpretation, an anti-ICAM-1 blocking antibody abolishes the KCa3.1-dependent increase in adhesion. Senicapoc inhibits transendothelial migration of A549 cells by 50%. Selectively silencing KCa3.1 channels in either NSCLC or endothelial cells reveals that transendothelial migration depends predominantly on endothelial KCa3.1 channels.In conclusion, our findings disclose a novel function of KCa3.1 channels in cancer. KCa3.1 channels regulate ICAM-1 dependent cell-cell adhesion between endothelial and cancer cells that affects the transmigration step of the metastatic cascade.

In Vitro Evaluation of PCL and P(3HB) as Coating Materials for Selective Laser Melted Porous Titanium Implants.

Titanium is widely used as a bone implant material due to its biocompatibility and high resilience. Since its Young’s modulus differs from bone tissue, the resulting “stress shielding” could lead to scaffold loosening. However, by using a scaffold-shaped geometry, the Young’s modulus can be adjusted. Also, a porous geometry enables vascularisation and bone ingrowth inside the implant itself. Additionally, growth factors can improve these effects. In order to create a deposit and release system for these factors, the titanium scaffolds could be coated with degradable polymers. Therefore, in the present study, synthetic poly-ε-caprolactone (PCL) and the biopolymer poly(3-hydroxybutyrate) (P(3HB)) were tested for coating efficiency, cell adhesion, and biocompatibility to find a suitable coating material. The underlying scaffold was created from titanium by Selective Laser Melting (SLM) and coated with PCL or P(3HB) via dip coating. To test the biocompatibility, Live Cell Imaging (LCI) as well as vitality and proliferation assays were performed. In addition, cell adhesion forces were detected via Single Cell Force Spectroscopy, while the coating efficiency was observed using environmental scanning electron microscopy (ESEM) and energy-dispersive X-ray (EDX) analyses. Regarding the coating efficiency, PCL showed higher values in comparison to P(3HB). Vitality assays revealed decent vitality values for both polymers, while values for PCL were significantly lower than those for blank titanium. No significant differences could be observed between PCL and P(3HB) in proliferation and cell adhesion studies. Although LCI observations revealed decreasing values in cell number and populated area over time on both polymer-coated scaffolds, these outcomes could be explained by the possibility of coating diluent residues accumulating in the culture medium. Overall, both polymers fulfill the requirements regarding biocompatibility. Nonetheless, since only PCL coating ensured the maintenance of the porous implant structure, it is preferable to be used as a coating material for creating a deposit and release system for growth factors.

Abstract 429: The Effects of Fluvastatin and Methyl-β-Cyclodextrin-Mediated Cholesterol Depletion on the Biomechanics of Vascular Smooth Muscle Cells in Atherosclerosis

Atherosclerosis is a leading cause of death worldwide. Phenotypic shifting, alteration in cell adhesion, and migration toward inflammatory site of blood vessel wall are all critical contributions of vascular smooth muscle cells (VSMC) to the progression of atherosclerosis. Knowing that cholesterol is a main participant of fatty deposition in atherosclerotic lesions, cholesterol’s role in orchestrating cell migration, mechanics and spreading has grown more apparent. Growing evidences suggests that cholesterol loaded into VSMC in atherosclerosis is much larger than previously known, and about 40% of the total foam cells in the atherosclerotic plaque were VSMC-derived. Emerging studies have increasingly categorized cholesterol as major player in regulating VSMC stiffness and N-Cadherin mediated cell-cell adhesions. Modulating their activity or expression may block VSMC migration during the progression of atherosclerosis. In this study, the effects of a 3-hydroxy-3-methylglutaryl-coenzyme-A reductase inhibitor, fluvastatin and Methyl-β-Cyclodextrin-Mediated (MβCD) cholesterol depletion on VSMCs N-cadherin adhesion and cellular stiffness were addressed. VSMCs cholesterol quantification and lactate dehydrogenase assays demonstrated significant reduction in total cellular cholesterol content by approx. 67% for fluvastatin and 40% for MβCD. The atomic force microscope (AFM) was used to investigate real time adhesion and stiffness of living VSMCs. A proprietary software package written in Matlab was used to estimate Young’s modulus of the cell cortex. Cell adhesion was measured for 50-70% confluent cells with N-Cadherin coated stylus AFM probes on an AFM mounted on an inverted microscope. Our results suggested that fluvastatin and MβCD mediated cholesterol depletion increased N-cadherin mediated cell adhesion force by approx. 27% and 17% respectively. In addition, fluvastatin and MβCD respectively reduced VSMCs stiffness by approx. 24% and 29 % compared to control VSMCs. Altogether, the knowledge that we obtained in this project may lead to a novel therapeutic strategy that could potentially control and block VSMC migration and prevent atherosclerotic plaque formation by deciphering and regulating N-cadherin mediated adhesion

Evaluation of initial cell adhesion on poly (2-methoxyethyl acrylate) (PMEA) analogous polymers

Cell adhesion is a major concern in biomaterial development. Generally, cells adhere to polymeric substrates via the interaction between integrins and proteins adsorbed on the substrates. Previously, it was reported that poly (2-methoxyethyl acrylate) (PMEA) and its analogous polymers can alter the integrin dependency for cell adhesion. In particular, integrin-independent adhesion was observed on PMEA. However, initial adhesion mechanisms, including integrin-independent adhesion mechanisms, on PMEA are not well characterized. In this study, initial cell adhesion within 10 min was characterized on PMEA analogous polymers. Protein adsorption was suppressed on PMEA compared with tissue culture polystyrene, but the cell adhesion site in adsorbed fibronectin was exposed to the cells similarly. HT-1080 cells adhered on PMEA in a serum medium even in the presence of EDTA, suggesting that the cells adhered via both integrin-dependent and integrin-independent mechanisms. Finally, the cell adhesion force was measured by single-cell force spectroscopy. The cell adhesion force was not changed on PMEA in serum and serum-free media, suggesting that the cells adhered on PMEA directly. In conclusion, the control of protein adsorption is useful for regulating integrin dependency for cell adhesion and following the expression of cell functions regulated by integrins.

High aspect ratio silicon nanowires control fibroblast adhesion and cytoskeleton organization

Cell–cell and cell–matrix interactions are essential to the survival and proliferation of most cells, and are responsible for triggering a wide range of biochemical pathways. More recently, the biomechanical role of those interactions was highlighted, showing, for instance, that adhesion forces are essential for cytoskeleton organization. Silicon nanowires (Si NWs) with their small size, high aspect ratio and anisotropic mechanical response represent a useful model to investigate the forces involved in the adhesion processes and their role in cellular development. In this work we explored and quantified, by single cell force spectroscopy (SCFS), the interaction of mouse embryonic fibroblasts with a flexible forest of Si NWs. We observed that the cell adhesion forces are comparable to those found on collagen and bare glass coverslip, analogously the membrane tether extraction forces are similar to that on collagen but stronger than that on bare flat glass. Cell survival did not depend significantly on the substrate, although a reduced proliferation after 36 h was observed. On the contrary both cell morphology and cytoskeleton organization revealed striking differences. The cell morphology on Si-NW was characterized by a large number of filopodia and a significant decrease of the cell mobility. The cytoskeleton organization was characterized by the absence of actin fibers, which were instead dominant on collagen and flat glass support. Such findings suggest that the mechanical properties of disordered Si NWs, and in particular their strong asymmetry, play a major role in the adhesion, morphology and cytoskeleton organization processes. Indeed, while adhesion measurements by SCFS provide out-of-plane forces values consistent with those measured on conventional substrates, weaker in-plane forces hinder proper cytoskeleton organization and migration processes.

The selective cytotoxicity of DSF-Cu attributes to the biomechanical properties and cytoskeleton rearrangements in the normal and cancerous nasopharyngeal epithelial cells

Cancer initiation and progression follow complex changes of cellular architecture and biomechanical property. Cancer cells with more submissive (or “softer”) than their healthy counterparts attributed to the reorganization of the complex cytoskeleton structure, may be considered as a potential anti-tumor therapeutic target. In this study, atomic force microscopy (AFM) was carried out to detect the topographical and biophysical changes of nasopharyngeal carcinoma CNE-2Z cells and normal nasopharyngeal epithelial cells NP69-SV40T by treating the Disulfiram chelated with Cu2+ (DSF-Cu). DSF-Cu induced the apoptotic population, ROS production and decreased the NF-κB-p65 expression of CNE-2Z cells, which was much higher than those of NP69-SV40T cells. DSF-Cu caused the obvious changes of cell morphology and membrane ultrastructure in CNE-2Z cells. The roughness decreased and stiffness increased significantly in CNE-2Z cells, which correlated with the rearrangement of intracellular F-actin, FLNa and α-tubulin structures in CNE-2Z cells. And the adhesion force of CNE-2Z cells was also increased accompanied with the increased E-cadherin expression. However, these results could not be observed in the NP69-SV40T cells even the concentration of DSF reached up to 400nM. Finally, the detection of cell wound scratch assay confirmed DSF-Cu could inhibit the migration of CNE-2Z cells, but no effect on NP69-SV40T cells. These findings demonstrated the selective cytotoxicity of DSF-Cu in CNE-2Z cells may attribute to the different mechanical properties and cytoskeleton rearrangement from the normal nasopharyngeal epithelial cells.

Cellular Shear Adhesion Force Measurement and Simultaneous Imaging by Atomic Force Microscope

This paper presents a sensitive and fast cellular shear adhesion force measurement method that uses atomic force microscopy (AFM). AFM is used both as a tool for imaging cells on the nano-scale and as a force sensor for the measurement of the shear adhesion force between a cell and the substrate. After cell imaging, the measurement of cellular shear adhesion force is made based on the different positions of the cell on the nano-scale. Moreover, various pushing speeds of probe and various locations of cells were used in experiments to study their influences. It was found that the measurement of cell adhesion in the upper portion of the cell is different from that in the lower portion. This may reveal that cancer cells have high metastasis tendency after culture for 16–20 h, which is significant for preventing metastasis in patients diagnosed with early cancer lesions. Furthermore, the cellular shear adhesion forces of two types of living cancer cell were obtained based on the measurements of AFM cantilever deflections in the torsional and vertical directions. The results demonstrate that the shear adhesion force of cancer cells is twice as much as that of the same type of cancer cells with tumor-necrosis-factor-related apoptosis-inducing ligand. The proposed method can also be used to measure the cellular shear adhesion force between a cell and the substrate, and for the simultaneous exploration of cells using AFM imaging and manipulation.

Essential arterial hypertension patients present higher cell adhesion forces, contributing to fibrinogen-dependent cardiovascular risk.

The increase of erythrocyte aggregation by high fibrinogen levels may be an indicator of cardiovascular risk. γ' fibrinogen variant has been considered as a possible player in enhancing aggregation. Here, we assessed, at the single-cell level, the influence of fibrinogen on erythrocyte aggregation in essential arterial hypertension. We also aimed at understanding how γ' fibrinogen is altered in this disease. Using atomic force microscopy (AFM), we show that the work and force necessary for erythrocyte-erythrocyte detachment is higher for patients than for healthy donors, with these parameters further increasing in both groups when higher fibrinogen concentrations are present. This can be associated with changes in blood flow, due to transient bridging of two erythrocytes by fibrinogen, representing an important cardiovascular risk factor. γ' fibrinogen can influence the increased risk in essential arterial hypertension, as we demonstrate that its levels are significantly increased in these patients' blood. Nevertheless, this cannot be the only cause for the changes observed in the AFM data.

The focal adhesion targeting domain of p130Cas confers a mechanosensing function.

Members of the Cas family of focal adhesion proteins contain a highly conserved C-terminal focal adhesion targeting (FAT) domain. To determine the role of the FAT domain in these proteins, we compared wild-type exogenous NEDD9 with a hybrid construct in which the NEDD9 FAT domain had been exchanged for the p130Cas (also known as BCAR1) FAT domain. Fluorescence recovery after photobleaching (FRAP) revealed significantly slowed exchange of the fusion protein at focal adhesions and significantly slower two-dimensional migration. No differences were detected in cell stiffness as measured using atomic force microscopy (AFM) and in cell adhesion forces measured with a magnetic tweezer device. Thus, the slowed migration was not due to changes in cell stiffness or adhesion strength. Analysis of cell migration on surfaces of increasing rigidity revealed a striking reduction of cell motility in cells expressing the p130Cas FAT domain. The p130Cas FAT domain induced rigidity-dependent phosphorylation of tyrosine residues within NEDD9. This in turn reduced post-translational cleavage of NEDD9, which we show inhibits NEDD9-induced migration. Collectively, our data therefore suggest that the p130Cas FAT domain uniquely confers a mechanosensing function.

The relationship between cell adhesion force activation on nano/micro-topographical surfaces and temporal dependence of cell morphology.

Interaction between adherent cells and extracellular matrix/scaffold surface features performs a crucial role in inducing physiological functions via signal transduction. Topographical design of scaffold surfaces, therefore, has the potential to promote physiological functions such as cell proliferation and differentiation. This study utilizes quantitative evaluation of cell-material interaction to identify how temporal dependence of cell morphology impacts cell adhesion force activation on nano/micro-ordered topographical surfaces. Nano-rough and micro-dot/line-patterned poly-lactic acid substrates were prepared to enable: (i) examination of the morphology of lamellipodia/filopodia, focal adhesion coupled with vinculin accumulations, and actin-filaments of osteoblast-like cells; and (ii) assay of the cell detachment force by single cell force spectroscopy. The quantitative evaluation results evidenced that in the initial period (cell adhesion time after initial attachment on any location, ta < 1 h), while nano-topographical surface enhanced detachment force of "spherical" cells, micro-topographical surfaces did not have this effect. Significantly, the identical micro-topographical surfaces were able to enhance detachment force of "spreading" cells in intermediate (1 < ta < 12 h) and long-term periods (ta > 24 h). These findings could be utilized in the design of scaffold surfaces to promote cell-material interaction (e.g. strengthening of the cell-substrate adhesion force), in tissue engineering.

Interactions of the extremely acidophilic archaeon Ferroplasma acidiphilum with acidophilic bacteria during pyrite bioleaching

Bioleaching has been applied as a successful technique for metal recovery from various mineral sources like low-grade ores, waste materials and tailings. Mixed cultures of bioleaching microorganisms have a high performance in mineral dissolution. Thus far, microbial interactions in bioleaching communities are poorly understood. In this paper, the acidophilic archaeon Ferroplasma acidiphilum and the bacteria Leptospirillum ferriphilum and Sulfobacillus thermosulfidooxidans were chosen to study their interactions during pyrite leaching. The initial attachment to pyrite and pyrite leaching efficiency of pure and mixed populations were investigated. The data indicate: (i) attachment and bioleaching efficiency of L. ferriphilum was reduced in the presence of F. acidiphilum. However, the combination of F. acidiphilum and S. thermosulfidooxidans showed increased leaching, although the initial attachment rate was reduced, when compared to pure cultures. Thus, synergistic or antagonistic interactions may exist between F. acidiphilum and S. thermosulfidooxidans or F. acidiphilum and L. ferriphilum, respectively; (ii) pre-established biofilms of L. ferriphilum inhibited initial attachment to pyrite by cells of F. acidiphilum and did not promote pyrite leaching by F. acidiphilum. In contrast, inactivated biofilm cells of S. thermosulfidooxidans enhanced pyrite bioleaching by F. acidiphilum; (iii) adhesion forces of cells to an AFM tip (Si3N4) seemed to be not correlated to attachment and bioleaching capacity; and (iv) lectins were applied to show and distinguish single species in mixed biofilm populations. Physical contact between cells of S. thermosulfidooxidans and F. acidiphilum was visible.

Influence of csgD and ompR on Nanomechanics, Adhesion Forces, and Curli Properties of E. coli.

Curli are bacterial appendages involved in the adhesion of cells to surfaces; their synthesis is regulated by many genes such as csgD and ompR. The expression of the two curli subunits (CsgA and CsgB) in Escherichia coli (E. coli) is regulated by CsgD; at the same time, csgD transcription is under the control of OmpR. Therefore, both genes are involved in the control of curli production. In this work, we elucidated the role of these genes in the nanomechanical and adhesive properties of E. coli MG1655 (a laboratory strain not expressing significant amount of curli) and its curli-producing mutants overexpressing OmpR and CsgD, employing atomic force microscopy (AFM). Nanomechanical analysis revealed that the expression of these genes gave origin to cells with a lower Young's modulus (E) and turgidity (P0), whereas the adhesion forces were unaffected when genes involved in curli formation were expressed. AFM was also employed to study the primary structure of the curli expressed through the freely jointed chain (FJC) model for polymers. CsgD increased the number of curli on the surface more than OmpR did, and the overexpression of both genes did not result in a greater number of curli. Neither of the genes had an impact on the structure (total length of the polymer and number and length of Kuhn segments) of the curli. Our results further suggest that, despite the widely assumed role of curli in cell adhesion, cell adhesion force is also dictated by surface properties because no relation between the number of curli expressed on the surface and cell adhesion was found.

Nanonet Force Microscopy for Measuring Cell Forces

The influence of physical forces exerted by or felt by cells on cell shape, migration, and cytoskeleton arrangement is now widely acknowledged and hypothesized to occur due to modulation of cellular inside-out forces in response to changes in the external fibrous environment (outside-in). Our previous work using the non-electrospinning Spinneret-based Tunable Engineered Parameters’ suspended fibers has revealed that cells are able to sense and respond to changes in fiber curvature and structural stiffness as evidenced by alterations to focal adhesion cluster lengths. Here, we present the development and application of a suspended nanonet platform for measuring C2C12 mouse myoblast forces attached to fibers of three diameters (250, 400, and 800 nm) representing a wide range of structural stiffness (3–50 nN/μm). The nanonet force microscopy platform measures cell adhesion forces in response to symmetric and asymmetric external perturbation in single and cyclic modes. We find that contractility-based, inside-out forces are evenly distributed at the edges of the cell, and that forces are dependent on fiber structural stiffness. Additionally, external perturbation in symmetric and asymmetric modes biases cell-fiber failure location without affecting the outside-in forces of cell-fiber adhesion. We then extend the platform to measure forces of (1) cell-cell junctions, (2) single cells undergoing cyclic perturbation in the presence of drugs, and (3) cancerous single-cells transitioning from a blebbing to a pseudopodial morphology.

Investigating cell-substrate and cell-cell interactions by means of single-cell-probe force spectroscopy.

Cell adhesion forces are typically a mixture of specific and nonspecific cell-substrate and cell-cell interactions. In order to resolve these phenomena, Atomic Force Microscopy appears as a powerful device which can measure cell parameters by means of manipulation of single cells. This method, commonly known as cell-probe force spectroscopy, allows us to control the force applied, the area of interest, the approach/retracting speed, the force rate, and the time of interaction. Here, we developed a novel approach for in situ cantilever cell capturing and measurement of specific cell interactions. In particular, we present a new setup consisting of two different half-surfaces coated either with recrystallized SbpA bacterial cell surface layer proteins (S-layers) or integrin binding Fibronectin, on which MCF-7 breast cancer cells are incubated. The presence of a clear physical boundary between both surfaces benefits for a quick detection of the region under analysis. Thus, quantitative results about SbpA-cell and Fibronectin-cell adhesion forces as a function of the contact time are described. Additionally, the importance of the cell spreading in cell-cell interactions has been studied for surfaces coated with two different Fibronectin concentrations: 20 μg/mL (FN20) and 100 μg/mL (FN100), which impact the number of substrate receptors. Microsc. Res. Tech. 80:124-130, 2017. © 2016 Wiley Periodicals, Inc.

A Review on Quantitative Measurement of Cell Adhesion Strength.

There is an explosion of interest in characterizing cell adhesion process and the mechanochemical players that is critical to a wide range of physiological and pathological consequences. Much, however, still remains uncertain and controversial about the quantitative measurement of cell adhesion strength (i.e., cell-substratum adhesion force, cell-cell adhesion force, etc.). The main reason for this deficit is a lack of well-established and comprehensive experimental methods that quantitatively measure cell adhesion strength at cellular and even molecular levels. This leads to the need for conducting a literature review on the methods for characterizing the adhesion force of anchorage-dependent cells. In this review article, we discern and critique available techniques for measuring cell adhesion strength that is required for an adherent cell (or cells) of interest to detach from the neighboring cell or the substratum. This review focuses on recent advances in the technique for measuring cell adhesion strength to weigh up the strengths and weaknesses of each technique, together with a brief description for the biological aspects of cell adhesion process. The summary of this article might help us both to develop a standard method for measuring cell adhesion strength and to broaden and deepen our understanding of cell adhesion process.

Tension-compression asymmetry in the binding affinity of membrane-anchored receptors and ligands.

Cell adhesion plays a crucial role in many biological processes of cells, e.g., immune responses, tissue morphogenesis, and stem cell differentiation. An essential problem in the molecular mechanism of cell adhesion is to characterize the binding affinity of membrane-anchored receptors and ligands under different physiological conditions. In this paper, a theoretical model is presented to study the binding affinity between a large number of anchored receptors and ligands under both tensile and compressive stresses, and corroborated by demonstrating excellent agreement with Monte Carlo simulations. It is shown that the binding affinity becomes lower as the magnitude of the applied stress increases, and drops to zero at a critical tensile or compressive stress. Interestingly, the critical compressive stress is found to be substantially smaller than the critical tensile stress for relatively long and flexible receptor-ligand complexes. This counterintuitive finding is explained by using the Euler instability theory of slender columns under compression. The tension-compression asymmetry in the binding affinity of anchored receptors and ligands depends subtly on the competition between the breaking and instability of their complexes. This study helps in understanding the role of mechanical forces in cell adhesion mediated by specific binding molecules.

The Micromechanics of Cellularized Extracellular Matrix

Collagen gels are widely used in experiments on cell mechanics because they mimic the extracellular matrix in physiological conditions. Collagen gels are often characterized by their bulk rheology; however, variations in the gel microstructure and cell adhesion forces cause the mechanical properties to be inhomogeneous at the cellular scale. We study the mechanics of type I collagen on the scale of tens to hundreds of microns by using holographic optical tweezers (HOT) to apply pN forces to microparticles embedded in the collagen network. We find that in response to optical forces particle displacements are inhomogeneous, anisotropic and asymmetric. Gels prepared at 21C and 37C show qualitative difference in their micromechanical characteristics. We also demonstrate that contracting cells remodel the micromechanics of their surrounding extracellular matrix in a strain- and distance- dependent manner.

Molecular Tension Sensors for Probing the Mechanical and Chemical Roles of Distinct Integrin Classes

Integrins are heterodimeric transmembrane proteins that mediate cell attachment to the extracellular matrix (ECM). Mammalian cells express numerous α/β-integrin heterodimers, each with a unique combination of affinities for individual ECM components and distinct roles in intracellular signal transduction. Notably, αvβ3, αvβ5, and α5β1 integrins recognize structurally similar RGD-based binding motifs, but play distinct, and central, roles in cancer metastasis (αvβ3 integrin), septic shock (αvβ5 integrin), and angiogenic sprouting (α5β1 integrin). In this study, we used Forster resonance energy transfer (FRET)-based molecular tension sensors (MTSs) that report on the mechanical forces exerted by individual integrin heterodimers to probe the roles of αvβ3, αvβ5, and α5β1 integrins in cell adhesion and traction force generation. We developed MTS variants that incorporated a minimal RGD sequence derived from fibronectin (MTSRGD), an RGD sequence derived from vitronectin (MTSVN), or the complete ninth and tenth type-III domains from fibronectin (MTSFN). Using primary dermal fibroblasts, we found that both MTSRGD and MTSVN recruited αvβ3 and αvβ5 integrins to large, force-generating adhesions at the cell periphery. In contrast, MTSFN supported the recruitment of α5β1 integrin to force-producing adhesions. However, measurements at the single-molecule level revealed that the forces experienced by individual MTSRGD molecules were on average larger than those experienced by MTSFN. These and other data suggest that force production at the level of individual integrins is regulated by the interaction of the integrin with its extracellular ligand. More broadly, our results imply that αv-containing and α5β1 integrins play distinct roles within adhesions, and that ligand affinity and traction force generation are not necessarily coupled at the level of individual integrins.

Using cell monolayer rheology to probe average single cell mechanical properties.

The cell monolayer rheology technique consists of a commercial rotational rheometer that probes the mechanical properties of a monolayer of isolated cells. So far we have described properties of an entire monolayer. In this short communication, we show that we can deduce average single cell properties. Results are in very good agreement with earlier work on single cell mechanics. Our approach provides a mean of 105-106 adherent cells within a single experiment. This makes the results very reproducible. We extend our work on cell adhesion strength and deduce cell adhesion forces of fibroblast cells on fibronectin coated glass substrates.

Enhanced Fibroblast Cellular Ligamentization Process to Polyethylene Terepthalate Artificial Ligament by Silk Fibroin Coating.

Artificial ligaments utilized in reconstruction of anterior cruciate ligament (ACL) are usually made of polyethylene terepthalate (PET) because of its good mechanical properties in vivo. However, it was found that the deficiencies in hydrophilicity and biocompatibility of PET hindered the process of ligamentization. Therefore, surface modification of the PET is deemed as a solution in resolving such problem. Silk fibroin (SF), which is characterized by good biocompatibility and low immunogenicity in clinical applications, was utilized to prepare a coating on the PET ligament (PET+SF) in this work. At first, decrease of hydrophobicity and appearance of amino groups were found on the surface of artificial PET ligament after coating with SF. Second, mouse fibroblasts were cultured on the two different kinds of ligament in order to clarify the possible effect of SF coating. It was proved that mouse fibroblasts display better adhesion and proliferation on PET+SF than PET ligament according to the results of several technical methods including SEM observation, cell adhesive force and spread area test, and mRNA analysis. Meanwhile, methylthiazolyldiphenyl-tetrazolium bromide and DNA content tests showed that biocompatibility of PET+SF is better than PET ligament. In addition, collagen deposition tests also indicated that the quantity of collagen in PET+SF is higher than PET ligament. Based on these results, it can be concluded that SF coating is suggested to be an effective approach to modify the surface of PET ligament and enhance the "ligamentization" process in vivo accordingly.