Cell Adhesion Strength Research Articles

Regulation of human cerebro-microvascular endothelial baso-lateral adhesion and barrier function by S1P through dual involvement of S1P1 and S1P2 receptors

Herein we show that S1P rapidly and acutely reduces the focal adhesion strength and barrier tightness of brain endothelial cells. xCELLigence biosensor technology was used to measure focal adhesion, which was reduced by S1P acutely and this response was mediated through both S1P1 and S1P2 receptors. S1P increased secretion of several pro-inflammatory mediators from brain endothelial cells. However, the magnitude of this response was small in comparison to that mediated by TNFα or IL-1β. Furthermore, S1P did not significantly increase cell-surface expression of any key cell adhesion molecules involved in leukocyte recruitment, included ICAM-1 and VCAM-1. Finally, we reveal that S1P acutely and dynamically regulates microvascular endothelial barrier tightness in a manner consistent with regulated rapid opening followed by closing and strengthening of the barrier. We hypothesise that the role of the S1P receptors in this process is not to cause barrier dysfunction, but is related to controlled opening of the endothelial junctions. This was revealed using real-time measurement of barrier integrity using ECIS ZΘ TEER technology and endothelial viability using xCELLigence technology. Finally, we show that these responses do not occur simply though the pharmacology of a single S1P receptor but involves coordinated action of S1P1 and S1P2 receptors.

Real-time monitoring of contractile properties of H9C2 cardiomyoblasts by using a quartz crystal microbalance

A QCM was used for the study of the contractility of H9C2 cells and assessment of the inotropic drug–cell interactions based on changes in the cells' viscoelasticity and cell adhesion strength.

An accelerated buoyancy adhesion assay combined with 3-D morphometric analysis for assessing osteoblast adhesion on microgrooved substrata

An accelerated negative buoyancy method has been developed to assess cell adhesion strength. This method has been used in conjunction with 3-D morphometric analysis to understand the effects of surface topology on cell response. Aligned micro-grooved surface topographies (with a range of groove depths) were produced on stainless steel 316L substrates by laser ablation. An investigation was carried out on the effect of the micro-grooved surface topography on cell adhesion strength, cell and nucleus volumes, cell phenotypic expression and attachment patterns. Increased hydrophobicity and anisotropic wettability was observed on surfaces with deeper grooves. A reduction was noted in cell volume, projected areas and adhesion sites for deeper grooves, linked to lower cell proliferation and differentiation rates and also to reduced adhesion strength. The results suggest that the centrifugation assay combined with three-dimensional cell morphometric analysis has considerable potential for obtaining improved understanding of the cell/substrate interface.

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.

Cell adhesion strength from cortical tension - an integration of concepts.

Morphogenetic mechanisms such as cell movement or tissue separation depend on cell attachment and detachment processes, which involve adhesion receptors as well as the cortical cytoskeleton. The interplay between the two components is of stunning complexity. Most strikingly, the binding energy of adhesion molecules is usually too small for substantial cell-cell attachment, pointing to a main deficit in our present understanding of adhesion. In this Opinion article, I integrate recent findings and conceptual advances in the field into a coherent framework for cell adhesion. I argue that active cortical tension is best viewed as an integral part of adhesion, and propose on this basis a non-arbitrary measure of adhesion strength - the tissue surface tension of cell aggregates. This concept of adhesion integrates heterogeneous molecular inputs into a single mechanical property and simplifies the analysis of attachment-detachment processes. It draws attention to the enormous variation of adhesion strengths among tissues, whose origin and function is little understood.

Enhancement of Endothelial Cell Retention on ePTFE Vascular Constructs by siRNA-Mediated SHP-1 or SHP-2 Gene Silencing

Insufficient retention of endothelial cells to the luminal surface of small-diameter tissue engineered vascular grafts has precluded clinical translation. This study utilized a gene silencing strategy to enhance the adhesion strength of vascular endothelial cells to fibronectin-coated expanded polytetrafluoroethylene, a common vascular graft material. SHP-1 and SHP-2 are both phosphatases known to inhibit the formation of focal contacts. By employing siRNA to knockdown the expression of SHP-1 or SHP-2, we observed a significant improvement in cell retention following 6 h of pulsatile fluid shear stress. At an average fluid shear stress of 15 dyn/cm2, cell retention was improved from approximately 30% for control groups to approximately 70 and 85% for cells treated with SHP-1 and SHP-2 specific siRNA, respectively (n = 8 for all groups). We also observed that treatment with SHP-1 or SHP-2 specific siRNA caused a modest increase in focal contact density and did not cause a significant effect on the expression of the endothelial cell markers VEGFR-2, VE-cadherin, and PECAM-1. These findings indicate that molecular modulation may be an effective strategy for improving the retention of endothelial cells within tissue engineered vascular grafts, which may in turn improve their clinical performance.

Binding Forces of Streptococcus mutans P1 Adhesin

Streptococcus mutans is a Gram-positive oral bacterium that is a primary etiological agent associated with human dental caries. In the oral cavity, S. mutans adheres to immobilized salivary agglutinin (SAG) contained within the salivary pellicle on the tooth surface. Binding to SAG is mediated by cell surface P1, a multifunctional adhesin that is also capable of interacting with extracellular matrix proteins. This may be of particular importance outside of the oral cavity as S. mutans has been associated with infective endocarditis and detected in atherosclerotic plaque. Despite the biomedical importance of P1, its binding mechanisms are not completely understood. In this work, we use atomic force microscopy-based single-molecule and single-cell force spectroscopy to quantify the nanoscale forces driving P1-mediated adhesion. Single-molecule experiments show that full-length P1, as well as fragments containing only the P1 globular head or C-terminal region, binds to SAG with relatively weak forces (∼50 pN). In contrast, single-cell analyses reveal that adhesion of a single S. mutans cell to SAG is mediated by strong (∼500 pN) and long-range (up to 6000 nm) forces. This is likely due to the binding of multiple P1 adhesins to self-associated gp340 glycoproteins. Such a cooperative, long-range character of the S. mutans-SAG interaction would therefore dramatically increase the strength and duration of cell adhesion. We also demonstrate, at single-molecule and single-cell levels, the interaction of P1 with fibronectin and collagen, as well as with hydrophobic, but not hydrophilic, substrates. The binding mechanism (strong forces, cooperativity, broad specificity) of P1 provides a molecular basis for its multifunctional adhesion properties. Our methodology represents a valuable approach to probe the binding forces of bacterial adhesins and offers a tractable methodology to assess anti-adhesion therapy.

Measurement systems for cell adhesive forces.

Cell adhesion to the extracellular matrix (ECM) involves integrin receptor-ligand binding and clustering to form focal adhesion (FA) complexes, which mechanically link the cell's cytoskeleton to the ECM and regulate fundamental cell signaling pathways. Although elucidation of the biochemical events in cell-matrix adhesive interactions is rapidly advancing, recent studies show that the forces underlying cell-matrix adhesive interactions are also critical to cell responses. Therefore, multiple measurement systems have been developed to quantify the spatial and temporal dynamics of cell adhesive forces, and these systems have identified how mechanical events influence cell phenotype and FA structure-function relationships under physiological and pathological settings. This review focuses on the development, methodology, and applications of measurement systems for probing (a) cell adhesion strength and (b) 2D and 3D cell traction forces.

Regulation of cell adhesion and cell sorting at embryonic boundaries.

Embryonic boundaries are sharp delimitations that prevent intermingling between different cell populations. They are essential for the development of well-organized structures and ultimately a functional organism. It has been long believed that this process was driven by global differences in cell adhesion strength, or expression of different types of adhesion molecules. The actual picture turns out to be quite different: Boundaries should be viewed as abrupt discontinuities, where cortical contractility is acutely upregulated in response to specific cell surface contact receptors which act as repulsive cues. Cell adhesion is also modulated along the interface, in different ways depending on the type of boundary, but in all cases the process is subordinated to the function of the cortical actomyosin cytoskeleton.

Quantifying Cell Adhesion through Impingement of a Controlled Microjet

The impingement of a submerged, liquid jet onto a cell-covered surface allows assessing cell attachment on surfaces in a straightforward and quantitative manner and in real time, yielding valuable information on cell adhesion. However, this approach is insufficiently characterized for reliable and routine use. In this work, we both model and measure the shear stress exerted by the jet on the impingement surface in the micrometer-domain, and subsequently correlate this to jet-induced cell detachment. The measured and numerically calculated shear stress data are in good agreement with each other, and with previously published values. Real-time monitoring of the cell detachment reveals the creation of a circular cell-free area upon jet impingement, with two successive detachment regimes: 1), a dynamic regime, during which the cell-free area grows as a function of both the maximum shear stress exerted by the jet and the jet diameter; followed by 2), a stationary regime, with no further evolution of the cell-free area. For the latter regime, which is relevant for cell adhesion strength assessment, a relationship between the jet Reynolds number, the cell-free area, and the cell adhesion strength is proposed. To illustrate the capability of the technique, the adhesion strength of HeLa cervical cancer cells is determined ((34 ± 14) N/m2). Real-time visualization of cell detachment in the dynamic regime shows that cells detach either cell-by-cell or by collectively (for which intact parts of the monolayer detach as cell sheets). This process is dictated by the cell monolayer density, with a typical threshold of (1.8 ± 0.2) × 109 cells/m2, above which the collective behavior is mostly observed. The jet impingement method presents great promises for the field of tissue engineering, as the influence of both the shear stress and the surface characteristics on cell adhesion can be systematically studied.

Effect of laser-dimpled titanium surfaces on attachment of epithelial-like cells and fibroblasts.

PURPOSEThe objective of this study was to conduct an in vitro comparative evaluation of polished and laserdimpled titanium (Ti) surfaces to determine whether either surface has an advantage in promoting the attachment of epithelial-like cells and fibroblast to Ti.MATERIALS AND METHODSForty-eight coin-shaped samples of commercially pure, grade 4 Ti plates were used in this study. These discs were cleaned to a surface roughness (Ra: roughness centerline average) of 180 nm by polishing and were divided into three groups: SM (n=16) had no dimples and served as the control, SM15 (n=16) had 5-µm dimples at 10-µm intervals, and SM30 (n=16) had 5-µm dimples at 25-µm intervals in a 2 × 4 mm2 area at the center of the disc. Human gingival squamous cell carcinoma cells (YD-38) and human lung fibroblasts (MRC-5) were cultured and used in cell proliferation assays, adhesion assays, immunofluorescent staining of adhesion proteins, and morphological analysis by SEM. The data were analyzed statistically to determine the significance of differences.RESULTSThe adhesion strength of epithelial cells was higher on Ti surfaces with 5-µm laser dimples than on polished Ti surfaces, while the adhesion of fibroblasts was not significantly changed by laser treatment of implant surfaces. However, epithelial cells and fibroblasts around the laser dimples appeared larger and showed increased expression of adhesion proteins.CONCLUSIONThese findings demonstrate that laser dimpling may contribute to improving the periimplant soft tissue barrier. This study provided helpful information for developing the transmucosal surface of the abutment.

Thermoresponsive nanostructured surfaces generated by the Langmuir-Schaefer method are suitable for cell sheet fabrication.

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Block copolymers composed of polystyrene and PIPAAm (St-IPAAms) having various chain lengths and compositions were synthesized by reversible addition-fragmentation chain transfer radical polymerization. The St-IPAAm Langmuir film at an air-water interface was horizontally transferred onto a hydrophobically modified glass substrate while regulating its density. Atomic force microscopy images clearly visualized nanoscaled sea-island structures on the surface. By adjusting both the composition of St-IPAAms and the density of immobilized PIPAAms, a series of thermoresponsive surfaces was prepared to control the strength, rate, and quality of cell adhesion and detachment through changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was more rapidly recovered on the optimized surfaces than on conventional PIPAAm surfaces. These unique PIPAAm surfaces are suggested to be useful for controlling the strength of cell adhesion and detachment.

Modulation of selectin-mediated adhesion of flowing lymphoma and bone marrow cells by immobilized SDF-1.

The α-chemokine, stromal-derived factor-1 (SDF-1), has been linked to the homing of circulating tumor cells to bone. SDF-1 is expressed by bone microvascular cells and osteoblasts and normally functions to attract blood-borne hematopoietic stem and progenitor cells to marrow. It has been shown that treatment of cancer cells with soluble SDF-1 results in a more aggressive phenotype; however, the relevance of the administration of the soluble protein is unclear. As such, a flow device was functionalized with P-selectin and SDF-1 to mimic the bone marrow microvasculature and the initial steps of cell adhesion. The introduction of SDF-1 onto the adhesive surface was found to significantly enhance the adhesion of lymphoma cells, as well as low-density bone marrow cells (LDBMC), both in terms of the number of adherent cells and the strength of cell adhesion. Thus, SDF-1 has a synergistic effect with P-selectin on cancer cell adhesion and may be sufficient to promote preferential metastasis to bone.

Dual-functioning phage-derived peptides encourage human bone marrow cell-specific attachment to mineralized biomaterials

Cell instructive mineralized biomaterials are a promising alternative to conventional auto-, allo-, and xenograft therapies for the reconstruction of critical sized defects. Extracellular matrix proteins, peptide domains, and functional motifs demonstrating cell and mineral binding activity have been used to improve cell attachment. However, these strategies vary in their tissue regeneration outcomes due to lack of specificity to both regenerative cell populations and the material substrates. In order to mediate cell-specific interactions on apatite surfaces, we identified peptide sequences with high affinity towards apatite (VTKHLNQISQSY, VTK) and clonally derived human bone marrow stromal cells (DPIYALSWSGMA, DPI) using phage display. The primary aims of this study were to measure apatite binding affinity, human bone marrow stromal cell (hBMSC) adhesion strength, and peptide specificity to hBMSCs when the apatite and cell-specific peptides are combined into a dual-functioning peptide. To assess binding affinity to hydroxyapatite (HA), binding isotherms were constructed and peptide binding affinity (K1) determined. HBMSC, MC3T3 and mouse dermal fibroblast (MDF) adhesion strength on biomimetic apatite functionalized with single- and dual-functioning peptide sequences were evaluated using a centrifugation assay. DPI-VTK had the highest binding strength towards hBMSCs (p < 0.01). DPI-VTK, while promoting strong initial attachment to hBMSCs, did not encourage strong adhesions to MC3T3s or fibroblasts (p < 0.01). Taken together, phage display is a promising strategy to identify preferential cell and material binding peptide sequences that can tether specific cell populations onto specific biomaterial chemistries.

Enhanced cell adhesion and alignment on micro-wavy patterned surfaces.

Various micropatterns have been fabricated and used to regulate cell adhesion, morphology and function. Micropatterns created by standard photolithography process are usually rectangular channels with sharp corners (microgrooves) which provide limited control over cells and are not favorable for cell-cell interaction and communication. This paper proposes a new micropattern with smooth wavy surfaces (micro-waves) to control the position and orientation of cells. To characterize cell growth and responses on the micro-patterned substrates, bovine aortic endothelial cells were seeded onto surfaces with micro-grooves and micro-waves for 24 h. As a result, the cells on the micro-wavy pattern appeared to have a lower death rate and better alignment compared to those on the micro-grooved pattern. In addition, flow-induced shear stress was applied to examine the adhesion strength of cells on the micro-wavy pattern. Results showed that cells adhered to the wavy surface displayed both improved alignment and adhesion strength compared to those on the flat surface. The combination of increased alignment, lower death rate and enhanced adhesion strength of cells on the micro-wavy patterns will offer advantages in potential applications for cell phenotype, proliferation and tissue engineering.

Two-dimensional numerical study of flow dynamics of a nucleated cell tethered under shear flow

When blood components (e.g., leukocytes and platelets) adhere to a surface (e.g., blood vessel wall), shear flow causes the elongation of the non-adherent part of the cell membrane forming a long thin cylinder shape (i.e., cell tether). The formation of cell tether is important for regulation of cell adhesion strength and stabilization of cell rolling, and may significantly affect the flow dynamics inside the vessel, as well as the motion of other cells and bioactive molecules. Although significant efforts have been made to reveal mechanisms underlying cell tether formation, the role of nucleus, nucleus/cell volume ratio, nucleus/plasma viscosity ratio and cytoplasm/plasma viscosity ratio remains unknown. As such, we developed a two-dimensional mathematical model, in which leukocytes are regarded as compound viscoelastic capsules with a nucleus. We investigated the effects of several factors on flow dynamic characteristics of tethered cells, including the cell length, the inclination angle, the drag and lift forces acting on the cell. The presence of a nucleus (with nucleus/cell volume ratio of 0.44) led to a decrease of 33.8% in the cell length and an increase of 152%, 113% and 43.6% in the inclination angle, the drag force and lift force respectively compared to those of a cell without nucleus. For a cell with nucleus/cell volume ratio of 0.2, a 10-fold increase in cytoplasm/plasma viscosity ratio resulted in a decrease of 19.3% in the cell length and an increase of 93.9%, 155% and 131% in the inclination angle, the drag force and lift force respectively. These results indicate that nucleus and cytoplasm play a significant role in flow dynamics of nucleated cells tethered under shear flow. The developed mathematical model could be used to further understand the mechanisms of cell-adhesion related bioprocesses and to optimize the conditions for cell manipulation in microfluidics.

MC3T3-E1 osteoblast adhesion to laser induced hydroxyapatite coating on Ti alloy

An in vitro cell study evaluating cell adhesion to hydroxyapatite (HA) coated prosthetic Ti-6Al- 4V alloy via laser treatment is presented in comparison with uncoated alloy. Based on our previous in vitro biocompatibility study, which demonstrated higher cell attachment and proliferation with MC3T3-E1 pre- osteoblast cells, the present investigation aims to reveal the effect of laser coating Ti alloy with HA on the adhesion strength of bone-forming cells against centrifugal forces. Remaining cells on different substrates after centrifugation were visualized using fluorescent staining. Semi-quantifications on the numbers of cells were conducted based on fluorescent images, which demonstrated higher numbers of cells retained on HA laser treated substrates post centrifugation. The results indicate potential increase in the normalized maximum force required to displace cells from HA coated surfaces versus uncoated control surface. The possible mechanisms that govern the enhancing effect were discussed, including surface roughness, chemistry, wettability, and protein adsorption. The improvement in cell adhesion through laser treatment with a biomimetic coating could be useful in reducing tissue damage at the prosthetic to bone junction and minimizing the loosening of prosthetics over time.

Response to "Comment on 'A method to measure cellular adhesion utilizing a polymer micro-cantilever'" [Appl. Phys. Lett. 104, 236103 (2014)

This reply is in response to a Comment posed by Eibl on our recently published Letter1 as mentioned in the title. While the primary focus of this paper was to present a new adhesion measurement tool and the results1 are preliminary in nature as reiterated in the text several times (p. 2, paragraph 1, column 1 and p. 5, last paragraph, column 1), we would be happy to address these concerns. We agree with the author of the Comment that additional work is required after functionalizing the micro-cantilever surface with cellular adhesion receptor proteins to achieve optimal physiological measurements as we also indicated in our manuscript1 (page 5, 1st paragraph, column 2). As we discussed1 (p. 4, paragraph 2, column 1) 3,3′-dithiobis[sulfosuccinimidylpropionate] (DTSSP) coated cantilever surface adheres covalently to the surface proteins of the cell membrane.2–4 We interpreted the differences observed in cells lines as an evidence reflecting their unique cell properties and our studies confirm widely accepted results.5–11 The main point of criticism of author of the Comment is that “such measurements… may not accurately reflect physiological aspects of cell-cell adhesion.” We would like to emphasize the fact that all in vitro studies involving cell culture are always limited in their physiological significance. However, we believe that meaningful results can be inferred by chemical cross-linking as explained below. In the Comment, the author states that we “assume” that “all the covalent bonds between DTSSP and membrane proteins rupture.” We did not make such an assumption. There are four possible rupturing point scenarios (as alluded to also by the author) when the cantilever is retracted from the cell membrane: (a) The linkage between the disulfide group of the DTSSP and the gold surface of the cantilever breaks requiring energy equivalent to (∼45 kcal/mol).12 (b) The bond between DTSSP and the NH2-groups on cell transmembrane proteins breaks (C-N bond: requiring a bond energy of approx. 175 kcal/mol).13 (c) The transmembrane proteins binding within the phospholipid bilayer of the cellular membrane is disrupted. Finally, (d) the binding between the cell membrane with the substratum or reciprocally with either the cell membrane of neighboring cells or the adjacent extracellular matrix (ECM) is compromised. The first two events outlined above are highly unlikely because of the inherent requirement of very strong bond energies. In our experimental setup, we suspect that retraction of the cantilever may have extracted some of the transmembrane proteins, as suggested by outcome (c) above, since they require much less energy than breaking covalent bonds. In relevant studies, cross-linkers14–17 such as DTSSP and Bis[2-(Sulfosuccinimidooxycarbonyloxy)ethyl] (sulfo-BSOCOES) have been used to quantify cell adhesion molecules (CAMs, generally transmembrane proteins) by covalently anchoring the CAM to an external substrate using cross-linkers and then lysing the rest of cells by using surfactants. This way the authors14–17 were able to estimate the protein density on the cell's surface and used that information to determine relative quantity of CAMs and thereby cell adhesion strength. It is also possible that upon cantilever retraction we replicated outcome (d), but in the absence of detailed analysis, it is not possible to speculate further. Significant to this discussion, cross linkers have been used to attach cells to cantilevers for cell-cell adhesion measurements18,19 and the fact that epithelial cells exhibit greater adhesive strength compared to mesenchymal cells.6,20

Contribution of stress wave and cavitation bubble in evaluation of cell–cell adhesion by femtosecond laser-induced impulse

When an intense femtosecond laser is focused in a cell culture medium, shock wave, stress wave, and cavitation bubble are generated at the laser focal point. Cell–cell adhesion can be broken at the cellular level by the impacts of these factors. We have applied this breaking of the adhesion to an estimation of the cell–cell adhesion strength. In this application, it is important to identify which of these factors is the dominant factor that breaks the adhesion. Here we investigated this issue using streptavidin-coated microbeads adhering to a biotin-coated substrate as a mimic of the cell–cell adhesion. The results indicated that the break was induced mainly by the stress wave, not by the impact of the cavitation bubble.

Geometrical Micropillars Combined with Chemical Surface Modifications – Independency of Actin Filament Spatial Distribution in Primary Osteoblasts

Cell-biomaterial interactions are strongly affected by topographical and chemical surface characteristics. We found out earlier that geometric titanium (Ti) pillar structures in the micrometer range induce the cells to rearrange their actin cytoskeleton in short fibers solely on the top of the pillars. As a result, cell physiology was hampered concerning collagen I synthesis and spreading capacity. Furthermore, the position-dependent initial cell adhesion strength was declined near the edges. We asked whether these observed cellular effects can be performed only in combination with Ti or occur independently of chemical surface features. In addition, the specific culture conditions, e.g. serum content or influence of gravity, were of interest. Human primary osteoblasts were cultured in Osteoblast Growth Medium with serum containing SupplementMix on pure silicon pillars (5x5x5 μm) or on samples additionally sputtered with Ti (as reference) or gold. To offer the cells ligands for their adhesion receptors, we coated the pillars with collagen I or alternatively with a plasma polymer layer from allylamine. Different from standard culture conditions, the cells were cultured against gravity as well as without serum. The actin cytoskeleton was stained with phalloidin-TRITC after 24 h and analyzed by confocal laser scanning microscopy. Interestingly, on all modifications tested the cell’s actin cytoskeleton was distinctly organized in short fibers on the top of the pillars. Thus, we were able to exclude the influence of (i) the material chemistry (gold, silicon, physical plasma vs. Ti), (ii) the protein deposition on the pillar top and edges, and (iii) the impression caused by gravity.