Optical waveguide lightmode spectroscopy (OWLS) to monitor cell proliferation quantitatively

The use of microscopic observations used for in situ monitoring of cell proliferation in the production of epidermal autografts is not satisfactory. In particular, the identification of the projected cell area from microscopic pictures by image analysis (IA) depends on intensity edges and level of contrasts and is thus limited to subconfluent cultures. Some of these problems can be solved by using optical waveguide lightmode spectroscopy (OWLS), which measures the effective refractive index of a thin layer above an Si(Ti)O(2) waveguide surface. In this study the use of OWLS to monitor cell adhesion, spreading, and growth was studied. The sensitivity of the method was investigated by using three different cell lines, two fibroblasts and one hepatoma cell line. Cell proliferation of two strains of fibroblasts and hepatoma cells was monitored up to 2 days with the OWLS. In parallel, cell density was determined at different time points microscopically using an additional window in the measuring chamber. The cell density of fully spread cells ( approximately 4 h after attachment) was found to be proportional to the OWLS signal. In long-term cultures the influence of the cell density from single cells to confluent cell cultures upon the OWLS signal was investigated. The exponentially growing number of hepatoma resulted in a linear increase of the sensor signal. Due to this and to the fact that the proliferating cells exhibit contact inhibition, it was concluded that the cell contact area must decrease exponentially. The results show the strength of OWLS for monitoring the adhesion and proliferation of anchorage-dependent cells in applications where an on-line indicator of the total biomass is needed. Additionally, OWLS provides metabolic information through detection of the cell mass in close contact with the waveguide.     

On-line monitoring of adhesion and proliferation of cultured hepatoma cells using optical waveguide lightmode spectroscopy (OWLS)

Monitoring of cell adhesion, cell spreading, and cell proliferation opens attractive perspectives in the on-line control of monolayer cell cultures in toxicity tests, in bioreactors as used for the serial production of skin grafts, or in extracorporeal liver devices. In this study the hepatoma Hep G2 cell adhesion and proliferation was monitored using an integrated optical method, optical waveguide lightmode spectroscopy (OWLS). This method is based upon refractive index measurements within a 100-nm thin layer above a Si(Ti)O(2) surface on which the cells were cultured and exposed to cytotoxic and cytostatic agents. The OWLS signal was proportional to cell density during the spreading period (4 h), and in long-term experiments (46 h) the OWLS signal correlated on a logarithmic scale with cell density. After administration of the protein synthesis inhibitor cycloheximide (4 microg/mL) to fully spread hepatoma cells, cell growth was arrested and change of the OWLS signal became noticeable within 6 h after drug administration. For exposure to increasing concentrations of the anticancer drug cyclophosphamide (2.5-20 mM) a concentration-dependent reduction of the OWLS signal was found. For cycloheximide and cyclophospamide the OWLS signal was also confirmed by cell viability measurements using the neutral red assay, the thiazolylblue tetrazoliumbromide assay, total protein measurements, and cell morphology. It was demonstrated that the OWLS signal detects minor changes in cell adhesion, which serve as indicators of metabolic state and growth behavior. OWLS is thus a quantitative tool to characterize impaired cell growth mediated by culture medium, by extracellular matrix, or after exposure to a toxin.     

Optical waveguide lightmode spectroscopy as a new method to study adhesion of anchorage-dependent cells as an indicator of metabolic state

Optical Waveguide Lightmode Spectroscopy (OWLS) is based on measurements of the effective refractive index of a thin layer above the waveguide. Its potential as a whole-cell biosensor was demonstrated recently monitoring adhesion and spreading of Baby Hamster Kidney (BHK) cells on-line. In this work the OWLS is shown to be a promising tool to study the adhesion, morphology and metabolic state of fibroblasts in real time. A new design of the measuring chamber allowed simultaneous observation by phase-contrast microscopy and made the adsorbed cell density controlable and reproducible. The OWLS signal correlated quantitatively with the contact-area between the fibroblasts and the waveguide. The OWLS signals for adhesion and spreading of three different fibroblast cell lines were in good agreement with their morphology identified by phase-contrast microscopy. The cell adhesion and cell shape changes were examined in three scenarios: (a) serum-induced spreading of the surface attached fibroblasts was followed until it was completed, and the OWLS signal remained constant for over 12 h; (b) the fully spread cells were exposed to the microtubuli-disrupting colchicine and a decrease of the OWLS signal was monitored; (c) in a similar experiment with benzalkonium chloride, a strong skin irritant, a concentration-dependent response of the signal was found. The results show the strength of the OWLS method for monitoring the adhesion behavior of anchorage-dependent cells such as fibroblasts. It has a great potential as a whole-cell biosensor for high throughput screening in toxicology.     

The effect of UV irradiation on uracil thin layer measured by optical waveguide lightmode spectroscopy

The polycrystalline uracil thin-layer dosimeter is a well-established method to monitor the biological effects of the environmental ultraviolet (UV) radiation. It is based on the optical density (OD) decrease of the uracil layer in the UV absorption band due to photodimerization of the crystal caused by UV irradiation. In the present study, we report measurements made with optical waveguide lightmode spectroscopy (OWLS) to characterize the changes in the optogeometrical parameters of the uracil layer caused by an artificial UV source. It is shown that UV irradiation causes a decrease in the refractive index and an increase of the optical anisotropy. The determined kinetic parameters of the UV dose-sensor response curves correlate well with results of OD measurements, but the sensitivity of OWLS is about ten times higher. The results show that OWLS is capable of analyzing the UV response of the uracil layer and opens the way for dosimetrical applications.     

Resonant waveguide grating biosensor for living cell sensing

This article presents theoretical analysis and experimental data for the use of resonant waveguide grating (RWG) biosensors to characterize stimulation-mediated cell responses including signaling. The biosensor is capable of detecting redistribution of cellular contents in both directions that are perpendicular and parallel to the sensor surface. This capability relies on online monitoring cell responses with multiple optical output parameters, including the changes in incident angle and the shape of the resonant peaks. Although the changes in peak shape are mainly contributed to stimulation-modulated inhomogeneous redistribution of cellular contents parallel to the sensor surface, the shift in incident angle primarily reflects the stimulation-triggered dynamic mass redistribution (DMR) perpendicular to the sensor surface. The optical signatures are obtained and used to characterize several cellular processes including cell adhesion and spreading, detachment and signaling by trypsinization, and signaling through either epidermal growth factor receptor or bradykinin B2 receptor. A mathematical model is developed to link the bradykinin-mediated DMR signals to the dynamic relocation of intracellular proteins and the receptor internalization during B2 receptor signaling cycle. This model takes the form of a set of nonlinear, ordinary differential equations that describe the changes in four different states of B2 receptors, diffusion of proteins and receptor-protein complexes, and the DMR responses. Classical analysis shows that the system converges to a unique optical signature, whose dynamics (amplitudes, transition time, and kinetics) is dependent on the bradykinin signal input, and consistent with those observed using the RWG biosensors. This study provides fundamentals for probing living cells with the RWG biosensors, in general, optical biosensors.     

New insights into the molecular mechanisms of biomembrane structural changes and interactions by optical biosensor technology

Biomolecular-membrane interactions play a critical role in the regulation of many important biological processes such as protein trafficking, cellular signalling and ion channel formation. Peptide/protein–membrane interactions can also destabilise and damage the membrane which can lead to cell death. Characterisation of the molecular details of these binding-mediated membrane destabilisation processes is therefore central to understanding cellular events such as antimicrobial action, membrane-mediated amyloid aggregation, and apoptotic protein induced mitochondrial membrane permeabilisation. Optical biosensors have provided a unique approach to characterising membrane interactions allowing quantitation of binding events and new insight into the kinetic mechanism of these interactions. One of the most commonly used optical biosensor technologies is surface plasmon resonance (SPR) and there have been an increasing number of studies reporting the use of this technique for investigating biophysical analysis of membrane-mediated events. More recently, a number of new optical biosensors based on waveguide techniques have been developed, allowing membrane structure changes to be measured simultaneously with mass binding measurements. These techniques include dual polarisation interferometry (DPI), plasmon waveguide resonance spectroscopy (PWR) and optical waveguide light mode spectroscopy (OWLS). These techniques have expanded the application of optical biosensors to allow the analysis of membrane structure changes during peptide and protein binding. This review provides a theoretical and practical overview of the application of biosensor technology with a specific focus on DPI, PWR and OWLS to study biomembrane-mediated events and the mechanism of biomembrane disruption. This article is part of a Special Issue entitled: Lipid–protein interactions.     

Application of the optical waveguide lightmode spectroscopy to monitor lipid bilayer phase transition

An instrument for optical waveguide lightmode spectroscopy (OWLS) was designed and developed for measurements at different and controlled temperatures in a range of 15 degrees C around room temperature. The instrument allows to scan the waveguide modes at different wavelengths on the same optical chip using different lasers. This instrument was used to monitor DMPC lipid bilayer main phase transition around the critical temperature. The main problem in these experiments is that the OWLS measurements do not give enough information about an optically anisotropic system like a lipid bilayer. Experimental OWLS data at two different wavelengths can however approximately solve the problem. The temperature dependence of the thickness and the refractive indices (ordinary and extraordinary) for the lipid bilayer around the phase transition is presented. (A theoretical derivation of the extraordinary refractive index is given in.)     

Optical guiding by photoreceptor cells.

The potential for any morphological part of a photoreceptor cell to function as an optical waveguide is determined 1) by the dimensionless parameter V as shown by Snyder (IEEE Trans., Microwave Theory Tech. 17: 1133, 1969) and by Snyder, Pask and Mitchell (J. Opt. Soc. Am. 63: 59, 1973), and 2) by the nature of the optical excitation for that part of the cell. The effects of these two factors are considered for the myoids and outer segments of vertebrate photoreceptor cells and for the crystalline tracts and rhabdomers of invertebrate photoreceptor cells. Examples of both guiding and nonguiding myoids, outer segments, rhabdomers and tracts are cited and some consequences for vision are analyzed.     

Measurement of film thickness up to several hundreds of nanometers using optical waveguide lightmode spectroscopy

Up to now, most studies based on optical waveguide lightmode spectroscopy (OWLS) were dedicated to thin adlayers, assumed to be isotropic and homogeneous, for which data analysis was based on an approximation of the mode equations valid when the thickness is small with respect to the wavelength of the laser light. The aim of the present paper is to extend the use of OWLS to thicker deposited layers (up to approximately 400 nm). Both the simplified and extended models are compared in terms of optical parameters, i.e. the refractive index nA, the thickness dA, and the optical mass QA, for experimental data obtained with polyelectrolyte multilayer films. The deviation of these parameters can be quite large when derived using the simplified model instead of the extended model. This observation evidences that OWLS is well suited for the study of "thick" films if the appropriate model is applied to the data analysis.     

Effects of temperature, pH, and salt concentration on beta-lactoglobulin deposition kinetics studied by optical waveguide lightmode spectroscopy

Deposition kinetics of beta-lactoglobulin at a solid-liquid interface was studied with optical waveguide lightmode spectroscopy (OWLS) over a range of temperatures between 61 and 83 degrees C. A new temperature-controlled cell for OWLS measurements allows fast, on-line monitoring of the deposit formation at elevated temperatures. Primary protein layers were deposited at 25 degrees C in order to precondition and stabilize the waveguide surface. Sustained deposition lasting from a few minutes (around 80 degrees C) to hours (below 70 degrees C) resulted in multilayer deposits up to several tens of nanometers thick. The measured deposition rates were strongly influenced by temperature, pH, and NaCl concentration. Deposition rates decreased with increasing pH from 5.5. to 7.4, in a trend similar to that for noncovalent aggregation of beta-lactoglobulin in solution. Activation energies for deposition rates decreased with increasing pH, from 340 kJ/mol at pH 5.5 to 230 kJ/mol at pH 7.4 and were similar to the activation energies for denaturation of beta-lactoglobulin in solution.     

Development of immunosensor based on OWLS technique for determining Aflatoxin B1 and Ochratoxin A

Mycotoxins are toxic secondary metabolites produced by a number of different fungi, and can be present in a wide range of food and feed commodities including cereal grains, oil seeds, dried fruits, apple juice, wine and meat products from animals fed contaminated meal. Many mycotoxins are highly resistant, and survive food processing, and therefore enter the food chain and provide a threat to human health. The optical waveguide lightmode spectroscopy (OWLS) technique has been applied to the detection of Aflatoxin and Ochratoxin in both competitive and in direct immunoassays. After immobilizing the antibody or antigen conjugate for the direct or indirect measurement, respectively, the sensor chip was used in flow-injection analyser (FIA) system. When using non-competitive method, sensor responses were obtained first only at analyte concentrations of 5-10 ng ml(-1). In both cases, the responses were very unstable. For competitive sensor investigation with the sensitized chip first the optimal dilution rate of monoclonal antibodies was determined, for the measurement of Ochratoxin A and Aflatoxin B1 the monoclonal antibody stock solution was diluted to 1 microg ml(-1) and to a 1:400 dilution, respectively. During the competitive measurement standard solutions were mixed with monoclonal antibodies at the appropriate concentration, the mixture was incubated for 1 min and injected into the OWLS system. The sensitive detection range of the competitive detection method was between 0.5 and 10 ng ml(-1) in both cases. After the establishment of the indirect method, barley and wheat flour samples were measured, and the results were in good correlation by those measured by enzyme linked immuno-sorbent assay (ELISA). Regression coefficient between the two methods for Ochratoxin and Aflatoxin was determined as 0.96 and 0.89, respectively.     

Identification of two distinct regions of the type III connecting segment of human plasma fibronectin that promote cell type-specific adhesion

The principal region of the human plasma fibronectin molecule mediating the adhesion of melanoma cells appears to be the alternatively spliced type III connecting segment (IIICS (Humphries, M. J., Akiyama, S. K., Komoriya, A., Olden, K., and Yamada, K. M. (1986a) J. Cell Biol., in press]. A series of overlapping synthetic peptides spanning the entire IIICS (CS peptides) were examined for their effects on B16-F10 melanoma cell adhesion to the parent fibronectin molecule. Two nonadjacent CS peptides, designated CS1 and CS5, were inhibitory. In contrast, neither inhibited fibronectin-mediated spreading of fibroblastic baby hamster kidney cells. When N-terminal cysteine derivatives of the CS peptides were conjugated to IgG by covalent cross-linking with N-succinimidyl-3(2-pyridyldithio)propionate, both the CS1 and CS5 conjugates promoted B16-F10 melanoma cell spreading. All conjugates were inactive for spreading of baby hamster kidney cells, confirming the cell type specificity of the IIICS adhesion site. Determination of the amounts of CS peptide required to support melanoma cell adhesion revealed that the activity of CS1 was only 2.4-fold lower than that of the intact fibronectin molecule. CS5 was approximately 320-fold less active than fibronectin, suggesting that the CS1 region may be the major site of interaction with the melanoma cell surface. The adhesion-promoting activities of CS1-IgG and CS5-IgG were additive as were the inhibitory activities of the free peptides for B16-F10 cell spreading on fibronectin. These findings suggest that both regions of the IIICS can function separately or together in mediating the interaction of melanoma cells with fibronectin. Since CS1 and CS5 are each found in separate alternatively spliced regions of the IIICS, it is conceivable that the adhesion-promoting activity of fibronectin for different cell types may be under complex regulation.     

A whole-cell biosensor as in vitro alternative to skin irritation tests

This study presents the time-resolved detection of chemically induced stress upon intracellular signaling cascades by using genetically modified sensor cells based on the human keratinocyte cell line HaCaT. The cells were stably transfected with a HSP72-GFP reporter gene construct to create an optical sensor cell line expressing a stress-inducible reporter protein. The time- and dose-dependent performance of the sensor cells is demonstrated and discussed in comparison to a label-free impedimetric monitoring approach (electric cell-substrate impedance sensing, ECIS). Moreover, a microfluidic platform was established based on μSlidesI(0,4)Luer to allow for a convenient, sterile and incubator-independent time-lapse microscopic observation of the sensor cells. Cell growth was successfully achieved in this microfluidic setup and the cellular response to a cytotoxic substance could be followed in real-time and in a non-invasive, sensitive manner. This study paves the way for the development of micro-total analysis systems that combine optical and impedimetric readouts to enable an overall quantitative characterization of changes in cell metabolism and morphology as a response to toxin exposure. By recording multiple parameters, a detailed discrimination between competing stress- or growth-related mechanisms is possible, thereby presenting an entirely new in vitro alternative to skin irritation tests.     

Modulation of growth factor induced fiber outgrowth in rat pheochromocytoma (PC12) cells by a fibronectin receptor antibody

Rat pheochromocytoma PC12 cells respond to the binding of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) by extending neurites in a manner resembling sympathetic neurons. This response requires cell attachment to an appropriate substratum (Fujii et al., J. Neurosci., 2:1157, 1982); attachment factors which function in this capacity include the adhesive proteins fibronectin and laminin. Incubating PC12 cells with a polyclonal antiserum directed against a putative 140-kDa fibroblast cell surface fibronectin receptor (anti-gp140) perturbed spreading but not attachment of the cells to fibronectin and laminin substrates. However, in the presence of anti-gp 140 or its Fab fragments, NGF-stimulated neurite outgrowth was dramatically reduced. The antibody also caused a retraction of previously extended neurites. SDS-PAGE analysis of immunoprecipitates of PC12 cells surface labeled with 125I identified a prominent 120-140-kDa band, suggesting that the site of anti-gp140 action in PC12 cells is also through a fibronectin receptor.     

Detection of cellular response to titanium dioxide nanoparticle agglomerates by sensor cells using heat shock protein promoter

Nanotechnology is becoming increasingly important for products used in our daily lives, such as the masses of titanium dioxide nanoparticle agglomerates (TiO₂ NPs) used in the pharmaceutical industry, for cosmetic products, or for pigments. Meanwhile, a serious lack of detailed information concerning the interaction between the nanomaterials and cells limits their biological and medical applications. Sensing technology is very important for understanding these interactions. We have shown that TiO₂ NPs induce heat shock protein 70B' (HSP70B') mRNA [Okuda‐Shimazaki et al., 2010. Int J Mol Sci 11:2383–2392]. In the current work, sensor cells for detection of cellular responses to NPs were prepared by transfecting an HSP70B' promoter–reporter plasmid. First, to find suitable cells for detection, five different mammalian cell lines were chosen as potential sensor cells. The results showed TiO₂ NP response in some cell lines, although different sensor cells had different TiO₂ NP response levels, as heat shock response ability is important for the detection. Then, we studied the TiO₂ NP time‐course response and dose response. The results indicated that our sensor cells can detect TiO₂ NP cellular responses. Our work should aid in understanding the interactions between bio‐nanomaterials and cells. Biotechnol. Bioeng. 2012; 109: 3112–3118. © 2012 Wiley Periodicals, Inc.     

Analysis of the prestarvation response in growing cells of Dictyostelium discoideum

We have previously shown that growing cells of Dictyostelium discoideum (strains NC4 and AX3) produce a soluble substance that accumulates in the medium in proportion to cell density; this substance regulates the production of certain proteins previously thought to be induced by starvation [Clarke et al., 1987]. We suggest the name PSF (prestarvation factor) for this substance. During growth, Dictyostelium cells monitor the relative concentrations of PSF and food bacteria. When PSF reaches a sufficiently high level relative to the concentration of bacteria, synthesis of PSF-regulated proteins is induced. We propose the name prestarvation response for this induction, which takes place in exponentially growing cells several generations before the food bacteria are depleted. We have explored the mechanism by which the food bacteria inhibit the response of Dictyostelium cells to PSF. We find that the bacteria do not inactivate PSF or inhibit its production; instead, they affect the ability of NC4 cells to detect PSF, possibly by binding to the same cell surface receptor. In the absence of bacteria, as during axenic growth of AX3 cells, the prestarvation response occurs at much lower cell densities, probably accounting for the presence of certain developmentally regulated mRNAs and proteins in axenic cultures.     

The Rap GTPases regulate integrin-mediated adhesion, cell spreading, actin polymerization, and Pyk2 tyrosine phosphorylation in B lymphocytes

Integrin-mediated adhesion plays an important role in B cell development and activation. Signaling initiated by antigens, chemokines, or phorbol esters can rapidly convert integrins to an activated adhesion-competent state. The binding of integrins to their ligands can then induce actin-dependent cell spreading, which can facilitate cell-cell adhesion or cell migration on extracellular matrices. The signaling pathways involved in integrin activation and post-adhesion events in B cells are not completely understood. We have previously shown that anti-Ig antibodies, the chemokine stromal cell-derived factor-1 (SDF-1; CXCL12), and phorbol esters activate the Rap1 and Rap2 GTPases in B cells and that Rap activation is essential for SDF-1-induced B cell migration (McLeod, S. J., Li, A. H. Y., Lee, R. L., Burgess, A. E., and Gold, M. R. (2002) J. Immunol. 169, 1365-1371; Christian, S. L., Lee, R. L., McLeod, S. J., Burgess, A. E., Li, A. H. Y., Dang-Lawson, M., Lin, K. B. L., and Gold, M. R. (2003) J. Biol. Chem. 278, 41756-41767). We show here that preventing Rap activation by expressing Rap-specific GTPase-activating protein II (RapGAPII) significantly decreased lymphocyte function-associated antigen-1- and alpha(4) integrin-dependent binding of murine B cell lines to purified adhesion molecules and to other cells. Conversely, augmenting Rap activation by expressing a constitutively active form of Rap2 enhanced B cell adhesion, showing for the first time that Rap2 can promote integrin activation. We also show that blocking Rap activation inhibited anti-Ig-induced cell spreading and phorbol ester-induced actin polymerization as well as anti-Ig- and SDF-1-induced phosphorylation of Pyk2, a tyrosine kinase involved in morphological changes and chemokine-induced B cell migration. Thus, the Rap GTPases regulate integrin-mediated B cell adhesion as well as processes that control B cell morphology and migration.     

The cell adhesion domain of type XVII collagen promotes integrin-mediated cell spreading by a novel mechanism

Type XVII collagen (BP180) is a keratinocyte transmembrane protein that exists as the full-length protein in hemidesmosomes and as a 120-kDa shed ectodomain in the extracellular matrix. The largest collagenous domain of type XVII collagen, COL15, has been described previously as a cell adhesion domain (Tasanen, K., Eble, J. A., Aumailley, M., Schumann, H., Baetge, J, Tu, H., Bruckner, P., and Bruckner-Tuderman, L. (2000) J. Biol. Chem. 275, 3093-3099). In the present work, the integrin binding of triple helical, human recombinant COL15 was tested. Solid phase binding assays using recombinant integrin alpha(1)I, alpha(2)I, and alpha(10)I domains and cell spreading assays with alpha(1)beta(1)- and alpha(2)beta(1)-expressing Chinese hamster ovary cells showed that, unlike other collagens, COL15 was not recognized by the collagen receptors. Denaturation of the COL15 domain increased the spreading of human HaCaT keratinocytes, which could migrate on the denatured COL15 domain as effectively as on fibronectin. Spreading of HaCaT cells on the COL15 domain was mediated by alpha(5)beta(1) and alpha(V)beta(1) integrins, and it could be blocked by RGD peptides. The collagen alpha-chains in the COL15 domain do not contain RGD motifs but, instead, contain 12 closely related KGD motifs, four in each of the three alpha-chains. Twenty-two overlapping, synthetic peptides corresponding to the entire COL15 domain were tested; three peptides, all containing the KGD motif, inhibited the spreading of HaCaT cells on denatured COL15 domain. Furthermore, this effect was lost by mutation from D to E (KGE instead of KGD). We suggest that the COL15 domain of type XVII collagen represents a specific collagenous structure, unable to interact with the cellular receptors for other collagens. After being shed from the cell surface, it may support keratinocyte spreading and migration.     

Quantifying the contribution of actin networks to the elastic strength of fibroblasts

The structural models created to understand the cytoskeletal mechanics of cells in suspension are described here. Suspended cells can be deformed by well-defined surface stresses in an Optical Stretcher [Guck, J., Ananthakrishnan, R., Mahmood, H., Moon, T.J., Cunningham, C.C., K?s, J., 2001. The optical stretcher: a novel laser tool to micromanipulate cells. Biophys. J. 81(2), 767-784], a two-beam optical trap designed for the contact-free deformation of cells. Suspended cells have a well-defined cytoskeleton, displaying a radially symmetric actin cortical network underlying the cell membrane with no actin stress fibers, and microtubules and intermediate filaments in the interior. Based on experimental data using suspended fibroblasts, we create two structural models: a thick shell actin cortex model that describes cell deformation for a localized stress distribution on these cells and a three-layered model that considers the entire cytoskeleton when a broad stress distribution is applied. Applying the models to data, we obtain a (actin) cortical shear moduli G of approximately 220 Pa for normal fibroblasts and approximately 185 Pa for malignantly transformed fibroblasts. Additionally, modeling the cortex as a transiently crosslinked isotropic actin network, we show that actin and its crosslinkers must be co-localized into a tight shell to achieve these cortical strengths. The similar moduli values and cortical actin and crosslinker densities but different deformabilities of the normal and cancerous cells suggest that a cell's structural strength is not solely determined by cytoskeletal composition but equally importantly by (actin) cytoskeletal architecture via differing cortical thicknesses. We also find that although the interior structural elements (microtubules, nucleus) contribute to the deformed cell's exact shape via their loose coupling to the cortex, it is the outer actin cortical shell (and its thickness) that mainly determines the cell's structural response.     

Introducing RGD peptides on PHBV films through PEG-containing cross-linkers to improve the biocompatibility

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable polyester, has been a good candidate of biomaterial employed in tissue engineering. However, the PHBV film is hydrophobic and has no recognition sites for cell attachment. In this study, PHBV films are activated by ammonia plasma treatment to produce amino groups on the surface, followed by sequential reactions with a heterobifunctional cross-linker containing a segment of poly(ethylene glycol) (PEG) and further with RGD-containing peptides. XPS analyses of modified surfaces after each reaction step reveal that the RGD-containing peptides have been covalently grafted onto PHBV films. The result of cell viability assay indicates that the RGD-modified PHBV films exhibit a distinctly improved cellular compatibility. Moreover, according to the results of serum adsorption tests by optical waveguide lightmode spectroscopy (OWLS) and fibrinogen adsorption tests by enzyme-linked immunosorbent assay (ELISA) on unmodified and modified PHBV surfaces, the introduced PEG chains can significantly decrease the nonspecific adsorption of proteins from serum and fibrinogen from plasma, thus decreasing the risk of thrombus formation and improving the blood compatibility of implanted materials.