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Na/K-ATPase (NKA) is a p-type transmembrane enzyme formed by three different subunits (α, β, and γ gamma). Primarily responsible for transporting sodium and potassium through the cell membrane, it also plays a critical role in intracellular signaling. The activation of diverse intracellular pathways may trigger cell death, survival, or even cell proliferation. Changes in the NKA functions or expression in isoforms subunits impact pathological conditions, such as cancer. The NKA function affects cell adhesion, motility, and migration, which are different in the physiological and pathological states. All enzyme subunits take part in the cell adhesion process, with the β subunit being the most studied. Thus, herein we aim to highlight NKA' central role in cell adhesion, motility, and migration in cancer cells.

Metastasis is a frequent complication of cancer and accounts for more than 60% of patients' mortality. Despite technological advancements, treatment options are still limited. Ion channels participate in the regulation of cell adhesion, whilst the regulation of cell adhesion further controls metastasis formation. However, to develop a new ion channel inhibitor targeting metastasis takes tremendous effort and resources; therefore, drug repurposing is an emerging strategy in oncology. In previous studies, we have developed a metal-based nanoslit surface plasmon resonance (SPR) platform to examine the influence of drugs on the cell adhesion process. In this work, we developed a scanner-based cell adhesion kinetic examination (CAKE) system that is capable of monitoring the cell adhesion process by measuring color changes of SPR biosensors. The system's performance was demonstrated by screening the anti-metastasis ability of compounds from a commercial ion-channel inhibitor library. Out of the 274 compounds from the inhibitor library, zinc pyrithione (ZPT) and terfenadine were demonstrated to influence CL1-5 cell adhesion. The cell responses to the two compounds were then compared with those by traditional cell adhesion assays where similar behavior was observed. Further investigation of the two compounds using wound healing and transwell assays was performed and inhibitions of both cell migration and invasion by the two compounds were also observed. The results indicate that ZPT and terfenadine are potential candidates for anti-metastasis drugs. Our work has demonstrated the label-free drug screening ability of our CAKE system for finding potential drugs for cancer treatment.

For a long time, proteins with enzymatic activity have not been usually considered to carry out other functions different from catalyzing chemical reactions within or outside the cell. Nevertheless, in the last few years several reports have uncovered the participation of numerous enzymes in other processes independently of their catalytic activity, placing them in the category of moonlighting proteins. Some moonlighting enzymes have been shown to participate in complex processes such as cell adhesion. Cell adhesion plays a physiological role in multiple processes: it enables cells to establish close contact with one another, allowing communication; it is a key step during cell migration; it is also involved in tightly binding neighboring cells in tissues, etc. Importantly, cell adhesion is also of great importance in pathophysiological scenarios like migration and metastasis establishment of cancer cells. Cell adhesion is strictly regulated through numerous switches: proteins, glycoproteins and other components of the cell membrane. Recently, several cell membrane enzymes have been reported to participate in distinct steps of the cell adhesion process. Here, we review a variety of examples of membrane bound enzymes participating in adhesion of immune cells.

A strategy for monitoring the dynamics of cell adhesion is reported based on localized surface plasmon resonance (LSPR) sensor, which consists of homogenous plasmonic Al nanopyramid array (NPA). The subtle change of cell-substrate interface could be detected since the LSPR sensor can response to the refractive index changes in the vicinity of its surface. We simulated the extinction spectra of homogenous Al NPA biosensor during cell adhesion, and obtained a linear relationship between the LSPR peak shifts and the changes in cell-substrate contact area. On the basis of this mechanism, we applied an Al NPA sensor to monitor the adhesion process of bone mesenchymal stem cells (BMSCs). We attained a dynamic curve of BMSCs adhesion based on the time-dependent LSPR peak shift. Different stages have been identified by the velocity variation of the peak shift, as attaching, fast-spreading, low-spreading and balancing, respectively. Compared to image analysis based on microscope, this LSPR biosensing strategy based on Al NPA shows higher accuracy in monitoring the initial process of cell adhesion. This LSPR biosensor based on homogeneous Al NPA is promising to achieve real-time, semi-quantitative and full-automatic monitoring of cell adhesion process, and can be applied in multiple fields such as cytopathology, oncology and regenerative medicine.

This paper presents a novel development for a full Eulerian coupling method between a fluid and capsule with hyperelastic membrane to address a cell adhesion process. The capsule motion in the fluid field and their mechanical interactions are expressed in the Eulerian framework, while solving the capsule adhesion process by stochastic events of ligand-receptor bindings in a discrete level, with using Lagrangian markers distributed on the implicit surface of the capsule. In addition to this, numerical improvements of the full Eulerian method for calculating an interface normal vector and dealing with multiple capsules by introducing the temporarily constructed level set function. A numerical validity of the present development is investigated through a comparison with available numerical data in a validation problem, and a capability for the capsule adhesion is shown in a single capsule adhesion and platelet adhesion in a suspension with blood cells.

Advances in tissue engineering require the development of new biomaterials with adequate properties of cell attachment and growth. The properties of biomaterials can be improved by incorporation of bioactive molecules to enhance in vitro and/or in vivo functions. In this work, we study the role of a wheat germin-like protease inhibitor (GLPI), free or immobilized in biocompatible matrices to improve cell-attachment ability on different mammalian cell lines. The phylogenetic relationships and functional diversity of the GLPI were analyzed among diverse genera to get insights into sequence motif conservations. The cytocompatibility effect of free GLPI on C2C12 premyoblastic cells and B16 cells as tumoral model has been tested. GLPI promoted proliferation and metabolic activity of both cell types on in vitro models, not showing cytotoxic effects. Furthermore, GLPI was immobilized in chitin microparticles and in chitosan films; we demonstrated an accelerated cell adhesion process in both biomaterials., This paper was supported by Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, PICT Start up No 0008 and PICT Raíces No 0959), Ministerio de Ciencia y Tecnología de la Nación (MINCyT, Programa Raíces Siembra), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC), Universidad Nacional de Mar del Plata. This work was also supported by the Ministerio de Economía y Competitividad (MINECO) (CONSOLIDER project CSD2009-00088). AYM, RP and CAC belong to CONICET. JRM belong to Comisión de Investigaciones Científicas de la Provincia de Buenos Aires.

We report the preparation of microporous surfaces prepared from immiscible polymer blends using the Breath Figures methodology. The selected polymer blend is formed by two biodegradable polymers i.e. poly(ε-caprolactone) and poly(lactic acid) both known to be biocompatible and presenting two distinct kinetics of biodegradation. These two polymers form immiscible polymer blends upon solvent casting in low relative humidity conditions. However, when quenching the melt using fast cooling processes or by evaporation in a moist atmosphere, micrometer size phase-separated domains have been clearly identified by Raman Confocal. This allowed us to analyze cell adhesion as well as proliferation on both planar (those obtained from melt quenching) and microporous surfaces (those obtained by the breath figures approach) formed from different blend compositions. Using C166-GFP and RAW264.7 cell types as a model we could observe an improved adhesion of the C166-GFP on top of the microporous surfaces evidencing the role of the surface topography on the cell adhesion process. In addition, preliminary results on the RAW264.7 cell adhesion indicated the polarization to M2 phenotypes instead of M1 which is a pre-requisite for the potential use of this material for implantology purposes., We gratefully acknowledge financial support from the Spanish National Science Foundation (CSIC) and the Ministerio de Ciencia, Innovacion ´ y Universidades (MINECO) grant number MAT2016-78437- R, FONDOS FEDER and RTI2018-096328-B-I00. The authors would like to acknowledge the International Atomic Energy Agency (IAEA) for providing the necessary expenses for the fellowship (Code No.: C6/LEB/ 17004), and the Lebanese Atomic Energy Commission of the National Council of Scientific Research in Lebanon.

Biological lasers which utilize Fabry–Pérot (FP) cavities have attracted tremendous interest due to their potential in amplifying subtle biological changes. Transverse laser modes generated from cells serve as distinct fingerprints of individual cells; however, most lasing signals lack the ability to provide key information about the cell due to high complexity of transverse modes. The missing key, therefore, hinders it from practical applications in biomedicine. This study reveals the key mechanism governing the frequency distributions of transverse modes in cellular lasers. Spatial information of cells including curvature can be interpreted through spectral information of transverse modes by means of hyperspectral imaging. Theoretical studies are conducted to explore the correlation between the cross‐sectional morphology of a cell and lasing frequencies of transverse modes. Experimentally, the spectral characteristics of transverse modes are investigated in live and fixed cells with different morphological features. By extracting laser modes in frequency domain, the proposed concept is applied for studying cell adhesion process and cell classification from rat cortices. This study expands a new analytical dimension of cell lasers, opening an avenue for subcellular analysis in biophotonic applications., This study reveals the key mechanism governing the frequency distributions of transverse modes in single cell biological lasers. Morphological information of cells can be interpreted through hyperspectral imaging of laser modes. Applications including cell classification and monitoring of cell adhesion are explored and demonstrated.

Surface modification of the substrate as a component of in vitro cell culture and tissue engineering, using bio-active molecules including extracellular matrix (ECM) proteins or peptides derived ECM proteins can modulate the surface properties and thereby induce the desired signaling pathways in cells. The aim of this study was to evaluate the behavior of human bone marrow mesenchymal stem cells (hBM-MSCs) on glass substrates modified with fibronectin (Fn), collagen (Coll), RGD peptides (RGD) and designed peptide (R-pept) as bio-active molecules. The glass coverslips were coated with fibronectin, collagen, RGD peptide and R-peptide. Bone marrow mesenchymal stem cells were cultured on different substrates and the adhesion behavior in early incubation times was investigated using scanning electron microscopy (SEM) and confocal microscopy. The MTT assay was performed to evaluate the effect of different bio-active molecules on MSCs proliferation rate during 24 and 72 h. Formation of filopodia and focal adhesion (FA) complexes, two steps of cell adhesion process, were observed in MSCs cultured on bio-active molecules modified coverslips, specifically in Fn coated and R-pept coated groups. SEM image showed well adhesion pattern for MSCs cultured on Fn and R-pept after 2 h incubation, while the shape of cells cultured on Coll and RGD substrates indicated that they might experience stress condition in early hours of culture. Investigation of adhesion behavior, as well as proliferation pattern, suggests R-peptide as a promising bio-active molecule to be used for surface modification of substrate in supporting and inducing cell adhesion and proliferation.

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.

Stem cells (SCs) can self-renew or differentiate into different cell types, which makes them an ideal cell source for therapies based on tissue engineering. Despite these characteristics, the employment of SCs in clinics has seen alternating fortunes because of our limited understanding of the signals governing SC functions and fate, which impairs our ability to engineer systems to deliver SCs in vivo and to guide their correct biological processes. However, experimental evidence demonstrated that SCs are able to recognize biochemical and biophysical signals displayed by material surfaces; most importantly, cells integrate these signals to elaborate fate decisions. Although the mechanisms underlying signal recognition and response have not been thoroughly characterized, there is a general consensus that the cell adhesion process plays a central role. Adhesion represents a communication gate between exogenous signals and intracellular signaling cascades involving the cytoskeleton and the nucleus. In this work we present recent findings on materials engineered to control cell functions through adhesion processes. In particular, we emphasize the role of material signals on SC behavior. Finally, we discuss a few sensing and transductive molecular mechanisms in an effort to draw out unifying elements concerning cell recognition of and reaction to biophysical/biochemical material signals aimed at controlling cell fate through cell adhesion. Microneedles and nanomedicine are two out of several active innovative approaches used to enhance the transdermal drug and vaccine delivery. Their application individually or in combination have shown to be very promising and attracted considerable interest by researchers from both industry and academia over the last 2 decades. Combining the two technologies has growing interest and has shown to be promising approach not only in enhancing the transdermal drug and vaccine permeation for even difficult drug molecules, such as hydrophilic and macromolecules, but also impart protection and controlling the drug release rates. In Subchapter 2.2, we aim to highlight the advances, which has been made in using these two technologies as an individual or in combination with drug and vaccine delivery. Metal implants, in the form of screws, plates, or pins, are extensively used in the treatment of fractures and nonunions or as replacements for malfunctioned joints. These bones implants face many challenges for acceptance and survival upon insertion in the human body. These challenges include severe inflammation, bacterial invasion, and poor biointegration with the traumatized tissue. Titania (TiO 2 ) nanotubes (TNTs) arrays engineered on the surface of Ti implants by simple and scalable electrochemical anodization process, which have been widely explored as a new nanoengineering approach to improve the process of osseointegration and at the same time to be used as depots for loading drugs and their controllable release for localized delivery, and therapeutic purpose. Several advanced functions can be introduced into these multifunctional implants, including biopolymers, nanoparticles or external stimulation (e.g., electrical, electromagnetic and ultrasound) to release the loaded therapeutic agents in a desired manner when required. Subchapter 2.3 highlights the developed concepts of drug releasing implants based on TNTs for enhancing osteogenesis at the bone-implant interface, as an alternative approach to systemic delivery of therapeutic agents. The reconstruction of skeletal defects is a continuing challenge. Bone and dentin are mineralized hard tissues. The primary inorganic component of these two tissues is crystalline hydroxyapatite and the primary organic component is type I collagen, and these two components are cell-manufactured materials. Tissue-engineering strategies using novel biomaterials have emerged as a promising potential for the treatment of mineralization defects. Some of the recent bioinspired biomaterials utilize proteins and peptides as nanoscale building blocks. A key functional property is that biomaterials need to be cell-compatible and mimic the dynamic nature of the extracellular matrix. ECM is a complex environment comprising a plethora of macromolecules. Another class of novel biomaterials that are currently being developed are peptide-based hydrogels. Peptide-based engineered scaffolds present several advantages over traditional protein scaffolds as the control over hydrogel properties and can be easily tailored to the requirement of the tissue. Biomaterials generated by the self-assembly process have varied applications as it mimics nature’s method of material synthesis. Therefore, concepts of protein-based self-assembly can be utilized for constructing useful biomaterials.

It is well-known that cell adhesion is important in many biological processes such as cell migration and proliferation. A better understanding of the cell adhesion process will shed insight into these cellular biological responses as well as cell adhesion-related diseases treatment. However, there is little research which has attempted to investigate the process of cell adhesion and its mechanism. Thus, this paper aims to study the time-dependent adhesion properties of single living chondrocytes using an advanced coupled experimental-numerical approach. Atomic Force Microscopy (AFM) tips will be used to apply lateral forces to detach chondrocytes that are seeded for three different periods. An advanced Finite Element Analysis (FEA) model combining porohyperelastic (PHE) constitutive model and cohesive zone formulation is developed to explore the mechanism of adhesion. The results revealed that the cells can resist normal traction better than tangential traction in the beginning of adhesion. This is when the cell adhesion molecules establish early attachment to the substrates. After that when the cells are spreading, stress fiber bundles generate tangential traction on the substrate to form strong adhesion. Both simulation and experimental results agree well with each other, providing a powerful tool to study the cellular adhesion process.

Cancer is the second leading cause of death, preceded only by cardiovascular diseases, and there is epidemiological evidence that demonstrate this tendency is emerging worldwide. Brazil has an extensive vegetal biodiversity with more than 55,000 species listed. Such biodiversity collaborates with the finding of compounds which could be the basis for the design of new anti-tumor drugs, with fewer side effects than the conventional chemotherapy used currently. Cedrelone is a limonoid isolated from Trichilia catigua (Meliaceae) which is a native Brazilian plant. This study demonstrates that cedrelone inhibits proliferation, adhesion, migration and invasion of breast tumor cells from the line MDA-MB-231. The effects of cell migration and invasion on MDA-MB-231 cell may be explained, at least in part, by the ability of cedrelone to inhibit MMP activity. We also demonstrate that cedrelone is able to induce apoptosis in MDA-MB-231 cells. There are only a few works investigating the effect of limonoids in cellular processes closely related to tumor progression such as adhesion, migration and invasion. To the best of our knowledge, this is the first work describing the effects of a limonoid on tumor and non-tumor cell adhesion process.

Protein adsorption and receptor-ligand binding are two key first steps in the cell adhesion process. The ability to predetermine the success or failure of these processes would significantly advance the field of tissue engineering. This study examines fibronectin adsorption on functionalized electrospun polycaprolactone (PCL) scaffolds and determines the affinity of cell receptors for the adsorbed fibronectin onto each scaffold. After determining the affinity values, model plots were developed for each scaffold type based on the amount of fibronectin on the surface of the scaffold. The ability to theoretically predict the level of cell binding to a tissue engineering scaffold would significantly impact the decision of what scaffold type to use for a specific application. Results show that aminated PCL scaffolds adsorb significantly more protein than hydrolyzed PCL scaffolds; however, greater α5β1–fibronectin binding occurs on the hydrolyzed scaffolds. This is attributed to a higher affinity between the receptors on the cell and the fibronectin adsorbed onto hydrolyzed scaffolds compared with aminated scaffolds., POLYM. ENG. SCI., 54:2587–2594, 2014. © 2013 Society of Plastics Engineers

Mussel-inspired poly(dopamine) (PDA) coating is proven to be a simple, versatile, and effective strategy to promote cell adhesion onto various substrates. In this study, the initial adhesive behavior of human umbilical vein endothelial cells (HUVECs) is evaluated on a PDA coating under serum-free conditions. It is found that HUVECs can attach directly to and spread with well-organized cytoskeleton and fibrillar adhesions on the PDA surface, whereas cells adhere poorly to and barely spread on the control polycaprolactone surface. Endogenous fibronectin and α5 β1 integrin are found to be involved in the cell adhesion process. These findings will lead to a better understanding of interactions between cells and PDA coating, paving the way for the further development of PDA.

Arachidonic acid (AA) is a common dietary n-6 cis polyunsaturated fatty acid that under physiological conditions is present in an esterified form in cell membrane phospholipids, and it might be present in the extracellular microenvironment. AA and its metabolites are implicated in FAK activation and cell migration in MDA-MB-231 breast cancer cells, and an epithelial-to-mesenchymal-like transition process in mammary non-tumorigenic epithelial cells MCF10A. During malignant transformation is present an altered expression of glycosiltransferases, which promote changes on the glycosilation of cell-surface proteins. The β-1,4-galactosyltransferase I (GalT I) is an enzyme that participates in a variety of biological functions including cell growth, migration, and spreading. However, the participation of AA in the regulation of GalT I expression and the role of this enzyme in the cell adhesion process in breast cancer cells remains to be investigated. In the present study, we demonstrate that AA induces an increase of GalT I expression through a PLA2α, Src, ERK1/2, and LOXs activities-dependent pathway in MDA-MB-231 breast cancer cells. Moreover, MDA-MB-231 cells adhere to laminin via GalT I expression and pretreatment of cells with AA induces an increase of cell adhesion to laminin. In conclusion, our findings demonstrate, for the first time, that AA promotes an increase of GalT I expression through an AA metabolism, Src and ERK1/2 activities-dependent pathway, and that GalT I plays a pivotal role in cell adhesion to laminin in MDA-MB-231 breast cancer cells.

During transformation of a normal cell to a cell capable of forming a cancerous growth, cellular morphology, the cytoskeleton, and focal contacts undergo significant changes. These changes should be capable of being characterized via real-time monitoring of the dynamic cell adhesion process and viscoelastic properties of cells. Here, we describe use of the quartz crystal microbalance (QCM) to distinguish the dynamic cell adhesion signatures of human normal (HMEC) versus malignant (MCF-7) mammary epithelial cells. The significantly reduced QCM responses (changes in frequency [Δf] and motional resistance ΔR) of MCF-7 cells compared with those of HMECs mirror the cancer cells’ morphological features as observed via optical microscope. We analyzed the initial 2-h cell adhesion kinetics, suggesting cell–cell cooperativity for HMECs and no or weak cell–cell interactions for MCF-7 cells. We propose that changes of the ΔR/Δf ratio, which we term the cell viscoelastic index (CVI), reflect the establishment of cytoskeleton structure and dynamic viscoelastic properties of living cells. The CVI decreases significantly on initiation of cell to surface interactions as cells establish their cytoskeletal structures. During the cell adhesion process, MCF-7 cells were consistently softer, exhibiting up to a 2.5-fold smaller CVI when compared with HMECs.

Objective:Transglutaminase 2 (TG2) is a multifunctional protein with an important role in vascular biology, where it is involved in cell-matrix interaction, cell attachment and cell population expansion. In efforts to elucidate the role of TG2 in endothelial cell biology, in this study, we measured several endothelial cell characteristics in cells where TG2 was specifically knocked down by RNAi.Materials and methods:The effect of small interfering RNA (siRNA)-TG2 on human umbilical vein endothelial cells was studied. Adhesion and cell viability were assessed by chemical reduction of MTT, and cell proliferation was analysed by flow cytometry. Apoptosis was evaluated by annexin V/PI dual staining and protein expression level was assayed by western blotting.Results:We found that siRNA-TG2 reduced endothelial cell number, lead to cell adhesion deficiency, cell cycle arrest in G(1) phase and induction of apoptosis. Our results show that exogenously added TG2 could reverse loss of adhesion but did not overcome the defect in cell proliferation, nor could it inhibit siRNA-TG2-induced apoptosis.Conclusion:We conclude that TG2 loss in endothelial cells causes reduction in cell number as a result of cell cycle arrest, flaws in adhesion and induction of apoptosis. Our results imply that reduction in cell number and increased apoptosis in response to TG2 silencing is independent of the cell adhesion process. Altogether, our findings underline the significance of TG2 in endothelial cell cycle progression and cell survival, in vitro.

Ultraviolet (UV)-photofunctionalization of titanium substantially enhances the strength and quality of osseointegration by promoting osteogenic cellular attachment and proliferation. However, the mechanism underlying the initial interaction between the cells and the surface of the material remains to be elucidated, especially where the influence of surface roughness is excluded as a factor. The effect of UV-photofunctionalization on the adhesive strength and cellular stiffness of a single osteoblast and its association with the extent of cell spread, cytoskeletal development and focal adhesion assembly on a very smooth titanium surface was evaluated. Rat bone marrow-derived osteoblasts were cultured on UV-treated or untreated mirror-polished titanium disks. The mean critical shear force required to initiate detachment of a single osteoblast (n=10) was >2000nN on a UV-treated surface at 3h incubation, which was 17 times greater than that on an untreated surface. The mean total energy required to complete the detachment of osteoblasts (n=10) was consistently >60pJ on a UV-treated titanium surface after 24h culture, which was up to 42 times greater than that on an untreated surface. Cellular shear modulus, which represents cellular stiffness, was consistently greater on a UV-treated surface than on an untreated surface after 24h incubation (n=10). This strengthening of cell adhesion and cellular mechanical properties on UV-treated titanium was accompanied by enhanced cell spread and actin fiber development and increased levels of vinculin expression. These results indicate that UV-photofunctionalization substantially strengthens osteoblast retention on titanium bulk material with no topographical features, and that this is associated with enhancement of intracellular structural development during the cell adhesion process.

Protein-protein interactions and/or signaling activities at focal adhesions, where integrin-mediated adhesion to extracellular matrix occurs, are critical for the regulation of adhesion-dependent cellular functions. Although the phosphorylation and activities of focal adhesion molecules have been intensively studied, the effects of the O-GlcNAc modification of their Ser/Thr residues on cellular functions have been largely unexplored. We investigated the effects of O-GlcNAc modification on actin reorganization and morphology of rat insulinoma INS-1 cells after glucosamine (GlcN) treatment. We found that paxillin, a key adaptor molecule in focal adhesions, could be modified by O-GlcNAc in INS-1 cells treated with GlcN and in pancreatic islets from mice treated with streptozotocin. Ser-84/85 in human paxillin appeared to be modified by O-GlcNAc, which was inversely correlated to Ser-85 phosphorylation (Ser-83 in rat paxillin). Integrin-mediated adhesion signaling inhibited the GlcN treatment-enhanced O-GlcNAc modification of paxillin. Adherent INS-1 cells treated with GlcN showed restricted protrusions, whereas untreated cells showed active protrusions for multiple-elongated morphologies. Upon GlcN treatment, expression of a triple mutation (S83A/S84A/S85A) resulted in no further restriction of protrusions. Together these observations suggest that murine pancreatic β cells may have restricted actin organization upon GlcN treatment by virtue of the O-GlcNAc modification of paxillin, which can be antagonized by a persistent cell adhesion process.

Cell adhesion to scaffolds has remained one of the challenges in tissue engineering. Although protein surface modification has been proven to enhance cell adhesion and retention, its specificity depending on cell and biomaterial types means that the best protein and concentration must be established for each specific application. This review focuses on the improvement of cell adhesion for human mesenchymal stem cells with an osteogenesis approach. A brief outline of the cell adhesion process and extracellular matrix proteins precedes an overview of works focused on the adhesion of mesenchymal stem cells and osteoblasts to biomaterials and this effect in their differentiation into osteoblasts.

A human hepatoma cell line (HepG2) was cultured on positively and negatively charged polyelectrolytes. Cell/surface adhesion and cell shape evolution were followed with quartz microbalance with dissipation (QCM-D) and optical microscopy as a function of time, respectively. In particular, substrates coated with poly(ethyleneimine) (PEI) led to fast cell attachment and fur- ther spreading, with average maximum frequency Df 5 79 Hz and dissipation DD 5 40 3 10 26 .O n the contrary, no cell spreading was observed on poly(sodium-4-styrenesulfonate) (PSS), with Df 5 33 Hz and DD 5 4.5 3 10 26 . Atomic force microscopy (AFM) was used to investigate the influence of cell shape on its mechanical properties. Considering the cells as an homogenous solid material, the corresponding elastic modulus was estimated using the Hertz model. The elastic modulus was calculated at the central part of the cell, and the average values obtained were 191 6 14 Pa and 941 6 58 Pa for cells adsorbed on PSS and PEI, respectively. Thus, different cell-substrate interac- tion implied different cell mechanical properties reflected in a higher elastic modulus for stronger cell/substrate interaction. The combination of QCM-D, AFM, and optical microscopy allowed the online study of the cell adhesion process, and the mechanical properties of the adhered cells. Microsc. Res. Tech. 72:957-964, 2009. V C 2009 Wiley-Liss, Inc.

Background and Objective: In the present study, micro-channel arrays were fabricated on the surface of plastic-based disposable chips. The cell adhesion process and the detection of plaque-forming macrophages were observed. Further, we evaluated cell adhesion in a fluid system in vitro. Material and Methods: Features of the micro-channel (1.4 mm wide and 10 mm long) included twenty micro-pillars (with a projection of 200 μm diameter and 250 μm high) coated in a 50 μm thick silicon rubber layer, which were regularly arranged at the bottom of each channel. The efficiency of cell capture was expected to increase by arrangement of micro-pillars in a micro-channel. Mouse macrophage RAW264.7 cells, stimulated for 24 h with lipopolysaccharide (LPS) derived from periodontopathic bacteria, were circulated continuously for 2 h at room temperature by the pump in a chip. Results: Control cells had not formed plaques on micro-pillars 20 min into the experiment. By contrast, LPS-activated macrophages produced plaques at the side walls of micro-pillars after 20 min. The plaques grew during the flow test, and image shading became clearer with increasing flow time for 120 min. The maximal adhesion rate per unit area appeared at 20% for control cells, whereas the peak was shifted to 30% for LPS-activated macrophages (n = 20). The average adhesion rate was 3.0 ± 2.0% for control cells and 5.0 ± 3.9% for LPS-activated macrophages (n = 100). Conclusion: These findings indicate that LPS-activated macrophages accumulate in micro-channel arrays, and suggest that macrophage plaque formation is a two-step procedure: (1) LPS-activated macrophages adhere physically to the silicon rubber layer on micro-pillars; and (2) consequently, the cells adhere to the activated macrophage layer.

Tumor cell metastasis is a complex, multi-step process that is a major cause of death and morbidity amongst cancer patients. Cell adhesion plays a critical role in the development of metastatic cancer, and it is mediated by interactions between receptors on the cell surface and ligands of the extracellular matrix or other surfaces. Therefore, inhibition of the cell adhesion process appears to be an effective method of preventing metastasis. This work describes a genetically engineered polypeptide with the potential to prevent cell adhesion and inhibit metastasis. We have found that the cell penetrating peptide Tat, fused with elastin-like polypeptide (ELP) inhibited adhesion, spreading, invasion and migration of SKOV-3 ovarian cancer cells in cell culture. Furthermore, we have also confirmed that Tat-ELP has anti-metastatic potential in an experimental ovarian cancer metastasis model in vivo, causing approximately 80% reduction in the tumor burden. Since cell attachment is an important step in tumor cell invasion and metastasis, these results suggest a novel role of Tat-ELP as a therapeutic intervention in cancer metastasis. Electronic supplementary material The online version of this article (doi:10.1007/s10585-009-9237-z) contains supplementary material, which is available to authorized users.

A major turning point in the biomaterials field would be to develop tools that can offer greater insight into cell behaviour on material surfaces. Obtaining this information is very important for the development of long-term implantable materials because it can aid in improving cell adhesion and proliferation properties. The amalgamation of multiple disciplines has already produced many interesting techniques and approaches for the characterisation of cell adhesion processes and force adhesion strength determination on biomaterials. In this review, the authors provide an overview of the recent techniques developed for the noninvasive in situ study of the adhesion process as well as systems that allow the measurement of adhesion force strengths over biomaterials. Techniques based on light internal reflection, electrochemical impedance spectroscopy, and the quartz crystal microbalance (QCM) are discussed for their capabilities in investigating the cell adhesion process. Conversely, techniques such as flow cells, centrifugation, and cytodetachers are presented for the adhesion force measurement. An emphasis on atomic force microscopy (AFM) will demonstrate its ability to probe both the cell adhesion process and cell adhesion force, depending on the approach used. A discussion is followed on the strengths and/or weaknesses of these techniques. Finally, new trends and possible long-term directions for determining both adhesion process and force are highlighted.

The three-dimensional (3-D) arrangement of cells within tissues is integral to their development and function. Advances in stem cell science and regenerative medicine have stimulated interest in the replication of this architecture in vitro. We have developed a versatile method for controlling short-term cell–cell and cell–matrix interactions via a facile cell surface engineering process that enables the rapid formation of specific 3-D interactions for a range of cell types. We demonstrate that chemical modification of cell surfaces and matrix proteins can artificially accelerate the cell adhesion process and confirm the ability to control the formation of multicellular aggregates with defined architectures and heterotypic cell types. Direct comparison with a natural aggregation process seen during differentiation of embryonic stem (ES) cells revealed increased expression of developmental regulatory proteins and a concomitant enhancement of ES cell differentiation. Furthermore, this new methodology has numerous applications in generating layered structures. For example, we demonstrate improved transfer of therapeutic human keratinocytes onto a dermal layer in a skin repair model. Biotechnol. Bioeng. 2007; 97: 1617–1625. © 2007 Wiley Periodicals, Inc.

It is known that Photodynamic Therapy (PDT) induces changes in the cytoskeleton, the cell shape, and the adhesion properties of tumour cells. In addition, these targets have also been demonstrated to be involved in the development of PDT resistance. The reversal of PDT resistance by manipulating the cell adhesion process to substrata has been out of reach. Even though the existence of cell adhesion-mediated PDT resistance has not been reported so far, it cannot be ruled out. In addition to its impact on the apoptotic response to photodamage, the cytoskeleton alterations are thought to be associated with the processes of metastasis and invasion after PDT. In this review, we will address the impact of photodamage on the microfilament and microtubule cytoskeleton components and its regulators on PDT-treated cells as well as on cell adhesion. We will also summarise the impact of PDT on the surviving and resistant cells and their metastatic potential. Possible strategies aimed at taking advantage of the changes induced by PDT on actin, tubulin and cell adhesion proteins by targeting these molecules will also be discussed. Fil: Di Venosa, Gabriela Mariana. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Centro de Investigacion Sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Perotti, Christian. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Centro de Investigacion Sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. University of Calgary. Southern Alberta Research Institute; Canadá Fil: Batlle, Alcira Maria del C.. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Centro de Investigacion Sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Casas, Adriana Gabriela. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Centro de Investigacion Sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina

The cell adhesion process and the molecular interactions that determine its kinetics were investigated using a thickness shear mode (TSM) sensor. The goal of this study was to correlate sensor readings with the progression of cell adhesion. In particular, the specific effects of receptor-mediated adhesion, the glycocalyx, and surface charge on initial cell-surface attachment and steady-state adhesion of endothelial cells were investigated. We found a strong correlation between resistance changes (DeltaR) and the development of cell adhesion strength by comparing the sensor readings with independently assessed cell adhesion. The result showed that integrin binding determines the kinetics of initial cell attachment while heparan sulfate proteoglycan (HSPG) modulates steady-state adhesion strength. Coating the sensor surface with the positively charged poly-d-lysine (PDL) enhanced the initial interaction with substratum. These data confirm our current understanding of the contribution of these three phenomena to the adhesion process. The real-time monitoring capability of this technique with high temporal resolution provides more detailed information on the kinetics of the different stages of the adhesion process. This technique has the potential to facilitate the evaluation of biomaterials and surface treatments used for implants and tissue-engineering scaffolds for their bioactive effects on the cell adhesion process.

The Thickness Shear Mode (TSM) quartz crystal resonator has been extensively used as sensitive sensor in various electrochemical and biological applications. This technique based on the propagation of an ultrasonic shear wave generated by a sinusoidal electric field through a piezoelectric quartz resonator, provides a non-destructive and powerful means to probe changes at solid-solid or solid-liquid interfaces. In this study, TSM was used to characterize cell-polymer interactions developing during the cell adhesion process. TSM sensing was used to monitor the inhibiting properties of bioactive polymers towards fibroblast McCoy adhesion processes. For this purpose, thin films of various bioactive polymers exhibiting either carboxylate or/and sulfonate functional groups were deposited onto the TSM. Measurements of the time variation of the electrical motional resistance in the vicinity of the mechanical sensor resonant frequency were performed as the quartz crystal resonator was either coated with the continuous polymer phase or polymer plus cell suspensions. Cell adhesion processes on these surfaces was investigated by cell counting and the quartz resonator-based technique. Inhibition of fibroblast McCoy adhesion onto thin polymer films of various chemical compositions was analyzed and discussed in the perspective of a possible application of these bioactive polymers to fabricate intraocular lenses able to prevent secondary cataract phenomena.

Cbp, a C-terminal Src kinase (Csk)-binding protein, is a transmembrane phosphoprotein that has been implicated in the regulation of the Src family kinase (SFK) through recruiting Csk, a negative regulator of SFK, to a membrane microdomain of lipid rafts. To examine the contribution of Cbp to cell adhesion signaling mediated by SFK, we investigated the kinase responsible for phosphorylating Cbp and the mode of phosphorylation during the cell adhesion process. The results obtained by using mutant mice or cells that lack Csk and/or a member of SFK, Fyn, reveal that Cbp is phosphorylated predominantly by raft-localized Fyn in vivo . Upon cell adhesion onto fibronectin, Cbp becomes transiently phosphorylated (consistent with SFK activation) and recruits Csk to lipid rafts. These events are completed before the full activation of focal adhesion kinase, indicating that the transient activation and down-regulation of SFK in lipid rafts are earlier events in cell adhesion signaling. In Csk-deficient cells, continuous hyperactivation of SFK leads to continuous hyperphosphorylation of Cbp, accompanied by impaired cell spreading and migration. Silencing of Cbp by RNA interference also induced impaired cell spreading. These findings suggest that Cbp could serve as a sensor of SFK activity in early stages of cell adhesion signaling, and that Csk-mediated down-regulation of SFK is essential to allow dynamic cellular events involved in the regulation of cell spreading and migration.

Specific association of tissue transglutaminase (tTG) with matrix fibronectin (FN) results in the formation of an extracellular complex (tTG-FN) with distinct adhesive and pro-survival characteristics. tTG-FN supports RGD-independent cell adhesion of different cell types and the formation of distinctive RhoA-dependent focal adhesions following inhibition of integrin function by competitive RGD peptides and function blocking anti-integrin antibodies alpha5beta1. Association of tTG with its binding site on the 70-kDa amino-terminal FN fragment does not support this cell adhesion process, which seems to involve the entire FN molecule. RGD-independent cell adhesion to tTG-FN does not require transamidating activity, is mediated by the binding of tTG to cell-surface heparan sulfate chains, is dependent on the function of protein kinase Calpha, and leads to activation of the cell survival focal adhesion kinase. The tTG-FN complex can maintain cell viability of tTG-null mouse dermal fibroblasts when apoptosis is induced by inhibition of RGD-dependent adhesion (anoikis), suggesting an extracellular survival role for tTG. We propose a novel RGD-independent cell adhesion mechanism that promotes cell survival when the anti-apoptotic role mediated by RGD-dependent integrin function is reduced as in tissue injury, which is consistent with the externalization and binding of tTG to fibronectin following cell damage/stress.

The cell adhesion and spreading behavior on surfaces of poly(ethyleneterephtalate) and poly(hydroxymethylsiloxane) micropatterned by focused 15 keV Ga+ beams has been studied. It has been found that while no modification in the cell adhesion process could be observed for unirradiated and irradiated areas on the patterned surfaces, in the case of polyhydroxymethylsiloxane the cell adhesion process is basically confined within the irradiated areas and a clear dependence of the cell ordering on the lateral size of the irradiated areas is observed. The results are discussed in terms of the specific spatially resolved chemical modification induced by Ga+ irradiation onto the two different polymers. Thus, the irradiation-induced modification of composition, functional groups concentration, surface free energy, and nanoscale morphology have been studied by means of x-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectrometry, contact angle, and atomic force microscopy. The cell adhesion and s...

MAP kinase activation by growth factors depends on cell adhesion to the extracellular matrix. Disrupting the cell adhesion process in NIH 3T3 fibroblasts induced an almost complete inhibition of MAP kinase, which was impaired by proteasome inhibitors. In the absence of cell anchorage, c-Raf-1 expression was dramatically decreased after 24 h. This down-regulation was suppressed by proteasome inhibitors, suggesting that a proteasome-dependent degradation of Raf occurred in the absence of cell adhesion. Proteasome inhibitors did not affect Raf-1 levels in adherent cells, indicating that this degradation only occurred in the absence of cell adhesion. Finally, ectopic coexpression of Raf-1 and ubiquitin in HEK-293 and NIH 3T3 cells generated ubiquitylated forms of Raf-1, both in adherent and suspended cells, suggesting a possible ubiquitin-dependent degradation of the protein.

Cell adhesion directly influences cell growth, differentiation and migration as well as morphogenesis, integrity and repair. The extracellular matrix (ECM) elaborated by osteoblast cells constitutes a regulator of the cell adhesion process and then of the related phenomenon. These regulatory effects of ECM are mediated through integrins and some of them are able to bind RGD sequences. The aim of this study was to determine the role of the sequence and the structure of RGD-containing peptides (linear and cyclic) as well as their role in the cell adhesion process. Cell adhesion assays onto ECM proteins coated surfaces were performed using a range of linear and cyclic RGD-containing peptides. We showed a different human osteoprogenitor cell adhesion according to the coating for ECM proteins and for RGD-peptides. Inhibition assays using peptides showed different responses depending on the coated protein. Depending on the amino-acid sequence and the structure of the peptides (cyclic/linear), we observed 100% inhibition of cell adhesion onto vitronectin. These results suggest the importance of sequence, structure and conformation of the peptide, which may play a crucial function in the ligand/receptor interaction and/or in the stability of the interaction.

A quartz crystal microbalance (QCM) technique was used to reveal the effect of alkanethiol self-assembled monolayers (SAMs) on the viscoelastic property and cell adhesion process of cultured fibroblasts (L929), by measuring the change in frequency (Δf ) and resistance (ΔR). Four types of SAMs having different functional groups including hydroxyl (OH), carboxylic acid (COOH), amine (NH2) and methyl (CH3) were used in this study. The QCM measurements during cell adhesion showed that floating cells were rapidly adsorbed on all functionalized surfaces as a soft cell monolayer which their stiffness was increased by cell spreading process. Initially, the Δf and ΔR were both positive shifts for COOH, OH and NH2 surfaces indicated fluid-like behavior of adherent cells. A multi-step decreasing of both Δf and ΔR were observed in case of COOH and OH surfaces represented solid-like behavior of cell spreading. The CH3 surface was used as a control because no cell adhesion occurs. QCM responses and morphological changes during cell adhesion process were resulted from viscoelastic nature of mammalian cells. The changes of cell morphology and viscoelastic property during cell adhesion process strongly depend on the surface functionality which can be observed by QCM technique.

The interaction of LFA-1 and ICAM-1 plays an important role in the cell adhesion process. On the basis of previously reported SAR and structural information on the binding of our p-arylthiocinnamide series to LFA-1, we have identified the cyclic amide (C-ring) as a site for modification. Improvement in potency and, more importantly, in the physical properties and pharmacokinetic profiles of the leading compounds resulted from this modification. One of the best compounds (11f) is also shown to reduce myocardial infarct size in rat.

Maspin is a tumor-suppressor serpin (serine protease inhibitor), which inhibits cell invasion and migration. Here, we analyzed maspin function in cell adhesion in nontransformed mammary epithelial cells and investigated the underlying mechanism involved in this process. We report that maspin acts in the early steps in the cell adhesion process. Addition of recombinant maspin rapidly increased MCF-10A cell adhesion to the endogenously deposited matrix, and conversely both an antimaspin antibody (Ab) and maspin knockdown by RNA interference resulted in decreased cell adhesion. Mutation analyses revealed that a region of 86 amino acids located between aa 139 and aa 225 was responsible for maspin effect on adhesion. In addition, we show that maspin is associated with detergent-insoluble cortical cytoskeleton elements. Collectively, these results suggest that maspin is part of the supramolecular structure of the adhesion plaque and it modulates cell adhesion via a beta1 integrin-dependent mechanism.

Hereditary inclusion body myopathy (GNE myopathy) is a neuromuscular disorder due to mutation in key sialic acid biosynthetic enzyme, GNE. The pathomechanism of the disease is poorly understood as GNE is involved in other cellular functions beside sialic acid synthesis. In the present study, a HEK293 cell-based model system has been established where GNE is either knocked down or over-expressed along with pathologically relevant GNE mutants (D176V and V572L). The subcellular distribution of recombinant GNE and its mutant showed differential localization in the cell. The effect of mutation on GNE function was investigated by studying hyposialylation of cell membrane receptor, β1-integrin. Hyposialylated β1-integrin localized to internal vesicles that was restored upon supplementation with sialic acid. Fibronectin stimulation caused migration of hyposialylated β1-integrin to the cell membrane and co-localization with focal adhesion kinase (FAK) leading to increased focal adhesion formation. This further activated FAK and Src, downstream signaling molecules and led to increased cell adhesion. This is the first report to show that mutation in GNE affects β1-integrin-mediated cell adhesion process in GNE mutant cells.

Nature provides cleverly concerted mechanisms which are able to control specific and non-specific interactions between cells and biological surfaces in a dynamic environment.[1] The successful imitation of this ability is of crucial importance for the understanding of cell adhesion to man-made surfaces,[2] and will aid in making significant impact in the future development and fabrication of new, functional biomaterials,[3] long-term antifouling surfaces[4] and novel in vitro diagnostic tools.[5] Researchers have previously fabricated surfaces, typically using self-assembled monolayers (SAMs)[6] in an attempt to control and understand cell adhesion, via the introduction of specific terminal moieties to the SAM to elicit specific surface properties.[7] However, despite the advancements achieved by investigating the interface as a ‘static’ environment, the mechanism behind the cell adhesion process remain unclear[8] representing one of the biggest challenges for many scientific areas ranging from tissue engineering,[9] medicine,[10] cell biology,[11] immunology[12] and marine biofouling.[13] In particular, researchers have tended to use cells to classify surfaces (i.e., bactericidal, repellent) while the ideal approach would be using the surface to understand cell behaviour. Towards this aim, scientists have discovered the possibility to control surface properties by switching their characteristics in response to particular needs.[1b, 14] Surfaces presenting tuneable moieties have been widely used in the study of specific protein-repellent/adhesion phenomena.[15] However, there are only a few reports on the control of non-specific cell adhesion[16] due to the lack of specific targeting on the cell surface, and the difficulties encountered in the interpretation of the outcome of end-point assays.[17] To date, dynamic control over cell adhesion has been achieved by using “smart” surfaces mostly formed by stimuli-responsive polymers.[18] For instance, the thermo-responsive polymer poly(N-isopropylacrylamide) and the closely related polyacrylamides have been widely investigated for preparing biologically relevant switchable surfaces,[19] as well as polymeric materials that can tune their properties from bacteriostatic to non-fouling and antimicrobial.[20] However, compared to such polymer systems, SAMs will allow higher precision of distribution and faster changes in surface properties.[21] Therefore, utilising such dynamic SAMs, the possibility exists to monitor the first interaction between a cell and a surface in real-time. Additionally, the passage between reversible and non-reversible cell adhesion can be ascertained. SAMs with stimuli-responsive characteristics have been used for the adhesion of smaller analytes such as proteins,[15a-d] and DNA,[22] and control specific interactions with cell surface receptors[23] but there has been no attempt to study non-specific cell adhesion on such smart surfaces.[24] In this context, the development of a reversible and fast electrochemical switchable surface, based on homogeneously distributed two-component SAMs represents a promising tool for studying cell-surface interactions in vitro and in real-time, and is a significant step forward for the fabrication of similar systems in vivo. The use of an applied potential for changing the surface properties will allow the study of bacterial interactions without perturbing their natural environment, and therefore their viability (as it may happen by changing the pH and temperature).

When bioceramics are implanted into a body, proteins in the fluid adsorb and subsequently cells adhere on the surface. To detect the structural characteristics at the interface between the surfaces and cells, a quartz crystal microbalance with dissipation (QCM-D) technique is one of excellent in situ analytical methods. The QCM-D detecting the interfacial phenomena with the protein adsorption and cell adhesion has been recently researched, and hydroxyapatite sensor surfaces fabricated by an electrophoretic method have been also studied for understanding the interface. The interfacial viscoelastic properties (shear viscosity, elastic shear modulus and tan δ ) of the interface were estimated using a Voigt-based viscoelastic model from the measured frequency (Δ f ) and dissipation shift (Δ D ) curves. The different cell adhesion process, interfacial viscoelasticity, and morphology depending on the surfaces were successfully evaluated by the QCM-D technique. This chapter summarized the studies on the QCM-D technique that clarify the interfacial phenomena between bioceramics and cells.

We present a phase shifting interferometry system for the study of the early adhesion process for osteoblast-like cells, through an interference microscope. Optical phase maps from the cells are obtained experimentally as a function of cell adhesion time. The process is carried out on surfaces of metallic materials relevant to the development of bone implants. The surfaces were subjected to various levels of mechanical polishing and their roughness was measured using the same experimental technique mentioned before. Morphological changes of the cell can be measured over their optical phase maps while the cell adhesion process is accomplished. The experimental technique shows a suitable feature as to the observed time scale, and also shows a high stockiness and precision for the determination of the optical phase.

Surface plasmon resonance (SPR) sensor provides a useful means to study the concentration change on the sensor chip. In this study, we developed a living cells adhesion behavior monitoring technology, using SPR biosensor to detect the refractive index change induced by the living C6 cells attachment. Combined with the cell images under microscope, the re-distribution of molecules between the cell-matrix during the cell adhesion process was discussed.

The adhesion process of osteoblast-like cells on hydroxyapatite (HAp) and oxidized polystyrene (PSox) was investigated using a quartz crystal microbalance with dissipation (QCM-D), confocal laser scanning microscope (CLSM), and atomic force microscope (AFM) techniques in order to clarify the interfacial phenomena between the surfaces and cells. The interfacial viscoelastic properties (shear viscosity (η(ad)), elastic shear modulus (μ(ad)), and tan δ) of the preadsorbed protein layer and the interface layer between the surfaces and cells were estimated using a Voigt-based viscoelastic model from the measured frequency (Δf) and dissipation shift (ΔD) curves. In the ΔD-Δf plots, the cell adhesion process on HAp was classified as (1) a mass increase only, (2) increases in both mass and ΔD, and (3) slight decreases in mass and ΔD. On PSox, only ΔD increases were observed, indicating that the adhesion behavior depended on the surface properties. The interfacial μ(ad) value between the material surfaces and cells increased with the number of adherent cells, whereas η(ad) and tanδ decreased slightly, irrespective of the surface. Thus, the interfacial layer changed the elasticity to viscosity with an increase in the number. The tan δ values on HAp were higher than those on PSox and exceeded 1.0. Furthermore, the pseudopod-like structures of the cells on HAp had periodic stripe patterns stained with a type I collagen antibody, whereas those on PSox had cell-membrane-like structures unstained with type I collagen. These results indicate that the interfacial layers on PSox and HAp exhibit elasticity and viscosity, respectively, indicating that the rearrangements of the extracellular matrix and cytoskeleton changes cause different cell-surface interactions. Therefore, the different cell adhesion process, interfacial viscoelasticity, and morphology depending on the surfaces were successfully monitored in situ and evaluated by the QCM-D technique combined with other techniques.

Takeshi T, Keisuke N, Takaaki I, Makoto Y, Tatsuji N. Involvement of adhesion molecule in in vitro plaque-like formation of macrophages stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide. J Periodont Res 2010; 45: 550–556. © 2010 John Wiley & Sons A/S Background and Objective: Inflammatory agents, such as lipopolysaccharide (LPS), in periodontal pockets may promote atherogenesis by activating leukocytes. In our previous study, we developed a microchannel chip to observe the cell adhesion process in a fluid system. The objective of this investigation was to examine the mechanism by which periodontopathic bacterial LPS enhances plaque-like formation on a microchannel chip. Material and Methods: To evaluate the effect of Aggregatibacter actinomycetemcomitans LPS on the expression of adhesion molecules, e.g. intercellular adhesion molecule 1 (ICAM-1), lymphocyte function-associated antigen 1 (LFA-1) and L-selectin, on the surface of murine macrophage RAW264.7 cells, the expression of each adhesion molecule was examined by flow cytometry and western blot analysis. Moreover, a flow test on the microchannel chip involving anti-adhesion molecule antibodies was conducted to clarify which adhesion molecule is related to plaque-like formation of RAW264.7 cells. Results: The expressions of ICAM-1 and LFA-1 on the surface of RAW 264.7 cells increased following 12 h culture with LPS; L-selectin expression was unaffected. An increase in ICAM-1 expression was also confirmed by western blot analysis. The flow test revealed that anti-ICAM-1 antibody inhibited plaque-like formation of LPS-stimulated macrophages on the micropillars of the microchannel chip. Conclusion: These findings indicate that ICAM-1 plays an important role in plaque-like formation of LPS-stimulated macrophages. Our microchannel chip is a suitable tool for the investigation of etiological factors of atherosclerosis, including periodontitis, in vitro.

Cell adhesion is a process which is initiated by the attachment of cells to specific sites in adhesive matrix proteins via cell surface receptors of the integrin family. This is followed by a reorganization of cytoskeletal elements which results in cell spreading and the formation of focal adhesion plaques. We have examined the effects of a class of small galactosaminoglycan-containing proteoglycans on the various stages of cell adhesion to fibronectin-coated substrates. Our results indicate that dermatan sulfate proteoglycans (DSPGs) derived from cartilage, as well as other related small proteoglycans, inhibit the initial attachment of CHO cells and rat embryo fibroblasts to substrates composed of the 105-kD cell-binding fibronectin fragment, but do not affect cell attachment to intact fibronectin. Although this effect involves binding of DSPGs to the substrate via the protein core, the intact proteoglycan is necessary for the observed activity. Isolated core proteins are inactive. The structural composition of the galactosaminoglycan chain does not appear to be functionally significant since both chondroitin sulfate and various dermatan sulfate proteoglycans of this family inhibit cell attachment to the fibronectin fragment. Neither the percentage of cells spread nor the mean area of spread cells adhering to substrates of intact fibronectin was significantly affected by the DSPGs. However, significantly fewer cells formed focal adhesions in the presence of DSPGs as compared with untreated control cells. These results suggest that the binding of small galactosaminoglycan-containing proteoglycans to a fibronectin substrate may affect several stages in the cell adhesion process.

In this paper preliminary results for the detection of the cell adhesion process with shear-horizontal polarized surface acoustic wave devices (SH-SAW) are presented. The murine fibroblast cell line L929 was investigated. Therefore a special measurement setup was developed. It enables us to perform long term measurements at stable ambient conditions. Different amounts of cells where seeded. According to the number of cells different slopes of resonant frequency increase vs. time were found.

Heterotropic association of tissue transglutaminase (TG2) with extracellular matrix-associated fibronectin (FN) can restore the adhesion of fibroblasts when the integrin-mediated direct binding to FN is impaired using RGD-containing peptide. We demonstrate that the compensatory effect of the TG-FN complex in the presence of RGD-containing peptides is mediated by TG2 binding to the heparan sulfate chains of the syndecan-4 cell surface receptor. This binding mediates activation of protein kinase Calpha (PKCalpha) and its subsequent interaction with beta(1) integrin since disruption of PKCalpha binding to beta(1) integrins with a cell-permeant competitive peptide inhibits cell adhesion and the associated actin stress fiber formation. Cell signaling by this process leads to the activation of focal adhesion kinase and ERK1/2 mitogen-activated protein kinases. Fibroblasts deficient in Raf-1 do not respond fully to the TG-FN complex unless either the full-length kinase competent Raf-1 or the kinase-inactive domain of Raf-1 is reintroduced, indicating the involvement of the Raf-1 protein in the signaling mechanism. We propose a model for a novel RGD-independent cell adhesion process that could be important during tissue injury and/or remodeling whereby TG-FN binding to syndecan-4 activates PKCalpha leading to its association with beta(1) integrin, reinforcement of actin-stress fiber organization, and MAPK pathway activation.

The endosperm cells undergo marked structural changes in association with their adhesion to the inner surface of the bean seed coat. An increase in the abundance of organelles is found within the cytoplasm. Plastids become very obvious as their size increases. Rough endoplasmic reticulum cisternae, together with many mitochondria and dictyosomes, are present throughout the cytoplasm. The cytoplasmic features are similar to those of branched parenchyma cells and the inner epidermis of the seed coat. The cell adhesion process involves the formation of an electron-dense substance that associates with the fibrillar wall material. These complexes eventually form a layer between the phenolic surface of the seed coat and the endosperm wall and likely serve as the cementing substance. Besides the contribution of the endosperm cells, it is suggested that the inner epidermis of the seed coat plays an important role in the adhesion process.