Cell Spreading Area Research Articles

Effect of Functional Groups of Self-Assembled Monolayers on Protein Adsorption and Initial Cell Adhesion.

Surface modification plays a vital role in regulating protein adsorption and subsequently cell adhesion. In the present work, we prepared nanoscaled modified surfaces using silanization and characterized them using Fourier-transform infrared spectroscopy (FTIR), water contact angle (WCA), and atomic force microscopy (AFM). Five different (amine, octyl, mixed, hybrid, and COOH) surfaces were prepared based on their functionality and varying wettability and their effect on protein adsorption and initial cell adhesion was investigated. AFM analysis revealed nanoscale roughness on all modified surfaces. Fetal bovine serum (FBS) was used for protein adsorption experiment and effect of FBS was analyzed on initial cell adhesion kinetics (up to 6 h) under three different experimental conditions: (a) with FBS in media, (b) with preadsorbed FBS on surfaces, and (c) incomplete media, i.e., without FBS. Various cell features such as cell morphology/circularity, cell area and nuclei size were also studied for the above stated conditions at different time intervals. The cell adhesion rate as well as cell spread area were highest in the case of surfaces with preadsorbed FBS. We observed higher surface coverage rate by adhering cells on hybrid (rate, 0.073 h-1) and amine (0.072 h-1) surfaces followed by COOH (0.062 h-1) and other surfaces under preadsorbed FBS condition. Surface treated with cells in incomplete media exhibited least adhesion rate, poor cell spreading and improper morphology. Furthermore, we found that initial cell adhesion rate and Δadhered cells (%) linearly increased with the change in α-helix content of adsorbed FBS on surfaces. Among all the modified surfaces and under all three experimental conditions, hybrid surface exhibited excellent properties for supporting cell adhesion and growth and hence can be potentially used as surface modifiers in biomedical applications to design biocompatible surfaces.

Interpenetrating polymer network hydrogels composed of chitosan and photocrosslinkable gelatin with enhanced mechanical properties for tissue engineering

Gelatin and chitosan (CS) are widely used natural biomaterials for tissue engineering scaffolds, but the poor mechanical properties of pure gelatin or CS hydrogels become a big obstacle that limits their use as scaffolds, especially in load-bearing tissues. This study provided a novel mechanism of forming interpenetrating network (IPN) of gelatin methacryloyl (GelMA) and CS hydrogels by covalent bonds and hydrophobic interactions through photocrosslinking and basification, respectively. By characterization of the compressive and tensile moduli, ultimate tensile stress and strain, it was found that semi-IPN and IPN structure can greatly enhance the mechanical properties of GelMA-CS hydrogels compared to the single network CS or GelMA. Moreover, the increase of either GelMA or CS concentration can strengthen the hydrogel network. Then, the swelling, enzymatic degradation, and morphology of GelMA-CS hydrogels were also systematically investigated. The excellent biocompatibility of GelMA-CS hydrogels was demonstrated by large spreading area of bone mesenchymal stem cells on hydrogel surfaces when CS concentration was <2% (w/v). According to this study, the multiple requirements of properties can be fulfilled by carefully selecting the GelMA and CS compositions for IPN hydrogels.

Poly(amidoamine)-alginate hydrogels: directing the behavior of mesenchymal stem cells with charged hydrogel surfaces

The surface charge of a biomaterial represents a promising tool to direct cellular behavior, which is crucial for therapeutic approaches in regenerative medicine. To expand the understanding of how the material surface charge affects protein adsorption and mesenchymal stem cell behavior, differently charged surfaces with zeta potentials spanning from −25 mV to +15 mV were fabricated by the conjugation of poly(amidoamine) to alginate-based hydrogels. We showed that the increase of the biomaterials surface charge resulted in enhanced quantities of biologically available, surface-attached proteins. Since different surface charges were equalized after protein adsorption, mesenchymal stem cells interacted rather with diverse protein compositions instead of different surface features. Besides an enhanced cell attachment to increasingly positively charged surfaces, the cell spreading area and the expression of adhesion-related genes integrin α5 and tensin 1 were found to be increased after adhesion. Moreover, first results indicate a potential impact of the surface charge on mesenchymal stem cell differentiation towards bone and fat cells. The improved understanding of surface charge-related cell behavior has significant impact on the design of biomedical devices and artificial organs.

Inducement of a spontaneously wrinkled polydimethylsiloxane surface and its potential as a cell culture substrate.

Spontaneous wrinkling of a polydimethylsiloxane (PDMS) surface was induced by repeated thermal shrinkage of liquid PDMS coated onto a cured PDMS layer. We investigated and evaluated the potential of the resulting surface as a cell culture substrate by monitoring the viability, spreading area, and proliferation rate of MG-63 cells cultured on native, wrinkled, and poly-L-lysine (PLL)-coated PDMS surfaces. Cells seeded on the wrinkled and PLL-coated PDMS surfaces spread and adhered better than those on native surfaces. The numbers of attached cells growing on wrinkled and PLL-coated PDMS surfaces were higher than those of cells on a native PDMS surface. The spreading area of cells on the wrinkled surface was similar to that of cells on the PLL-coated surface, and was much larger than that on native PDMS. The proliferation rate of cells on the wrinkled surface was more than double that of cells on native PDMS. Reverse-transcription polymerase chain reaction (RT-PCR) analysis of integrin mRNA expression showed that cells on the wrinkled surface were more tightly attached due to higher expression of the protein than exhibited in cells on native PDMS. Thus, the novel findings of this study are that the induction of a wrinkled PDMS surface through a simple curing process produces a suitable cell culture substrate without need of surface modification, and that its effectiveness is comparable to that of a PLL-coated PDMS surface.

Regulatory actions of 3',5'-cyclic adenosine monophosphate on osteoclast function: possible roles of Epac-mediated signaling.

Alterations in cellular levels of the second messenger 3',5'-cyclic adenosine monophosphate ([cAMP]i ) regulate a wide range of physiologically important cellular signaling processes in numerous cell types. Osteoclasts are terminally differentiated, multinucleated cells specialized for bone resorption. Their systemic regulator, calcitonin, triggers morphometrically and pharmacologically distinct retraction (R) and quiescence (Q) effects on cell-spread area and protrusion-retraction motility, respectively, paralleling its inhibition of bone resorption. Q effects were reproduced by cholera toxin-mediated Gs -protein activation known to increase [cAMP]i , unaccompanied by the [Ca2+ ]i changes contrastingly associated with R effects. We explore a hypothesis implicating cAMP signaling involving guanine nucleotide-exchange activation of the small GTPase Ras-proximate-1 (Rap1) by exchange proteins directly activated by cAMP (Epac). Rap1 activates integrin clustering, cell adhesion to bone matrix, associated cytoskeletal modifications and signaling processes, and transmembrane transduction functions. Epac activation enhanced, whereas Epac inhibition or shRNA-mediated knockdown compromised, the appearance of markers for osteoclast differentiation and motility following stimulation by receptor activator of nuclear factor kappa-Β ligand (RANKL). Deficiencies in talin and Rap1 compromised in vivo bone resorption, producing osteopetrotic phenotypes in genetically modified murine models. Translational implications of an Epac-Rap1 signaling hypothesis in relationship to N-bisphosphonate actions on prenylation and membrane localization of small GTPases are discussed.

Attachment and spatial organisation of human mesenchymal stem cells on poly(ethylene glycol) hydrogels.

Strategies that enable hydrogel substrates to support cell attachment typically incorporate either entire extracellular matrix proteins or synthetic peptide fragments such as the RGD (arginine-glycine-aspartic acid) motif. Previous studies have carefully analysed how material characteristics can affect single cell morphologies. However, the influence of substrate stiffness and ligand presentation on the spatial organisation of human mesenchymal stem cells (hMSCs) have not yet been examined. In this study, we assessed how hMSCs organise themselves on soft (E = 7.4-11.2 kPa) and stiff (E = 27.3-36.8 kPa) poly(ethylene glycol) (PEG) hydrogels with varying concentrations of RGD (0.05-2.5 mM). Our results indicate that hMSCs seeded on soft hydrogels clustered with reduced cell attachment and spreading area, irrespective of RGD concentration and isoform. On stiff hydrogels, in contrast, cells spread with high spatial coverage for RGD concentrations of 0.5 mM or higher. In conclusion, we identified that an interplay of hydrogel stiffness and the availability of cell attachment motifs are important factors in regulating hMSC organisation on PEG hydrogels. Understanding how cells initially interact and colonise the surface of this material is a fundamental prerequisite for the design of controlled platforms for tissue engineering and mechanobiology studies.

MicroRNA-21 drives the switch to a synthetic phenotype in human saphenous vein smooth muscle cells.

Cardiovascular disease is a leading cause of morbidity and mortality. Smooth muscle cells (SMC) comprising the vascular wall can switch phenotypes from contractile to synthetic, which can promote the development of aberrant remodelling and intimal hyperplasia (IH). MicroRNA-21 (miR-21) is a short, non-coding RNA that has been implicated in cardiovascular diseases including proliferative vascular disease and ischaemic heart disease. However, its involvement in the complex development of atherosclerosis has yet to be ascertained. Smooth muscle cells (SMC) were isolated from human saphenous veins (SV). miR-21 was over-expressed and the impact of this on morphology, proliferation, gene and protein expression related to synthetic SMC phenotypes monitored. Over-expression of miR-21 increased the spread cell area and proliferative capacity of SV-SMC and expression of MMP-1, whilst reducing RECK protein, indicating a switch to the synthetic phenotype. Furthermore, platelet-derived growth factor BB (PDGF-BB; a growth factor implicated in vasculoproliferative conditions) was able to induce miR-21 expression via the PI3K and ERK signalling pathways. This study has revealed a mechanism whereby PDGF-BB induces expression of miR-21 in SV-SMC, subsequently driving conversion to a synthetic SMC phenotype, propagating the development of IH. Thus, these signaling pathways may be attractive therapeutic targets to minimise progression of the disease. © 2018 IUBMB Life, 70(7):649-657, 2018.

An RNAi screen of Rho signalling networks identifies RhoH as a regulator of Rac1 in prostate cancer cell migration

BackgroundCell migration is essential for development and tissue repair, but it also contributes to disease. Rho GTPases regulate cell migration, but a comprehensive analysis of how each Rho signalling component affects migration has not been carried out.ResultsThrough an RNA interference screen, and using a prostate cancer cell line, we find that approximately 25% of Rho network components alter migration. Some genes enhance migration while others decrease basal and/or hepatocyte growth factor-stimulated migration. Surprisingly, we identify RhoH as a screen hit. RhoH expression is normally restricted to haematopoietic cells, but we find it is expressed in multiple epithelial cancer cell lines. High RhoH expression in samples from prostate cancer patients correlates with earlier relapse. RhoH depletion reduces cell speed and persistence and decreases migratory polarity. Rac1 activity normally localizes to the front of migrating cells at areas of dynamic membrane movement, but in RhoH-depleted cells active Rac1 is localised around the whole cell periphery and associated with membrane regions that are not extending or retracting. RhoH interacts with Rac1 and with several p21-activated kinases (PAKs), which are Rac effectors. Similar to RhoH depletion, PAK2 depletion increases cell spread area and reduces cell migration. In addition, RhoH depletion reduces lamellipodium extension induced by PAK2 overexpression.ConclusionsWe describe a novel role for RhoH in prostate cancer cell migration. We propose that RhoH promotes cell migration by coupling Rac1 activity and PAK2 to membrane protrusion. Our results also suggest that RhoH expression levels correlate with prostate cancer progression.

Eigenstrain as a mechanical set-point of cells.

Cell contraction regulates how cells sense their mechanical environment. We sought to identify the set-point of cell contraction, also referred to as tensional homeostasis. In this work, bovine aortic endothelial cells (BAECs), cultured on substrates with different stiffness, were characterized using traction force microscopy (TFM). Numerical models were developed to provide insights into the mechanics of cell-substrate interactions. Cell contraction was modeled as eigenstrain which could induce isometric cell contraction without external forces. The predicted traction stresses matched well with TFM measurements. Furthermore, our numerical model provided cell stress and displacement maps for inspecting the fundamental regulating mechanism of cell mechanosensing. We showed that cell spread area, traction force on a substrate, as well as the average stress of a cell were increased in response to a stiffer substrate. However, the cell average strain, which is cell type-specific, was kept at the same level regardless of the substrate stiffness. This indicated that the cell average strain is the tensional homeostasis that each type of cell tries to maintain. Furthermore, cell contraction in terms of eigenstrain was found to be the same for both BAECs and fibroblast cells in different mechanical environments. This implied a potential mechanical set-point across different cell types. Our results suggest that additional measurements of contractility might be useful for monitoring cell mechanosensing as well as dynamic remodeling of the extracellular matrix (ECM). This work could help to advance the understanding of the cell-ECM relationship, leading to better regenerative strategies.

A Breakdown of Cellular Mechanisms Required for Cell and Focal Adhesion Area Sensitivity to Substrate Stiffness

Cell spread area and focal adhesion (FA) sizes are known to increase with substrate stiffness (Yeung et al., Cell Motil. Cytoskeleton., 2005). Different models have been developed, some of which can predict increases in cell spread area with substrate stiffness while others can predict increases in only the FA area. Here, we systematically add one mechanism at a time to start recapitulating both these behaviors together. We start with an adapted model (Walcott et. al. Biophysical J., 2011) of FA growth implemented in a 2D cell that spreads at a constant rate. This model demonstrates that while the total FA area increases with substrate stiffness the cell spread area is not sensitive to the stiffness. In order for the cell spread area to increase with substrate stiffness the spreading model must include a coupling between the spreading rate, the FA complex evolution, and intracellular stresses (Vernerey & Farsad, J. Math. Bio., 2013). When the spreading rate is coupled with cellular stresses and FA evolution, we demonstrate the ability of our model to qualitatively reproduce the increase in cell spread area and FA area with substrate stiffness. Further we try to generate biological insight on the role of the model parameters in their ability to reproduce cellular behavior for various experimentally studied cellular systems such as 3T3 fibroblasts and human mesenchymal stem cells. Based on this we also articulate the gaps in the current model and make a case for potentially bringing in additional mechanisms that can account for the role of stress fiber orientation (Deshpande et al, PNAS, 2006) which can enhance our ability to understand the interplay between the different mechanisms in predicting cellular responses across a broader range of cell types.

Integrin Molecular Tensions in Live Cells are Altered by Substrate Rigidity

Mounting evidence suggests that mammalian cells are capable of sensing the rigidity of their local environment and responding to it. However, it remains unclear whether cells sense cellular force alteration caused by matrix compliance (stress sensing) or sense cytoskeleton remodeling caused by matrix deformation (strain sensing). Because integrins are the major mechano-sensitive receptors that transmit cellular force at the cell-matrix interface, the tension transmitted by integrin molecules (integrin tension) may serve as fundamental mechanical signal in cell mechanotransduction and rigidity sensing. Therefore, measuring and mapping integrin molecular tension in live cells on elastic substrates will shed light to the mechanism of cell rigidity sensing. In this work, we applied integrative tension sensor (ITS), a sensor converting molecular tension above a threshold to fluorescent signal, to calibrate and map integrin tension directly by fluorescence imaging. In experiments, ITS with 54 pN tension threshold was grafted on PDMS (Polydimethylsiloxane) substrates with Young's modulus ranging from 1kPa to 1.8 MPa. Integrin tension above 54 pN in live cells was mapped with high resolution (0.4 µm) and sensitivity on these substrates. The results show that cell spreading area and integrin tension activity both increased with increasing PDMS gel stiffness for CHO-K1, NIH-3T3 and skin fibroblast cell lines. Also, keratocytes migrated faster on softer PDMS substrates and integrin tension activity was the highest on the stiffest PDMS sample. These experiments for the first time monitored integrin tension in live cells on elastic substrates, and clearly demonstrated that the substrate rigidity affects integrin tension activity, providing evidence in favor of stress sensing hypothesis.

Increased Substrate Stiffness Elicits a Myofibroblastic Phenotype in Human Lamina Cribrosa Cells.

Alteration in the extracellular matrix (ECM) of the optic nerve head (ONH) causes lamina cribrosa (LC) fibrosis and affects the mechanical integrity of the ONH. Increased ECM tissue stiffness drives myofibroblast activation leading to tissue fibrosis throughout the body. Here using primary human LC cells, we investigate the effect of substrate stiffness on profibrotic changes, which might be a key molecular mechanism driving ECM remodeling of the LC in primary open-angle glaucoma (POAG) glaucoma. Primary human LC cells from normal and age-matched POAG glaucoma donors were cultured on substrates with defined mechanical properties of 5 and 100 kPa to replicate the range of mechanical microenvironments that cells may experience in vivo. Cell morphology, spread area, actin stress fibers, vinculin-focal adhesion formation, and α-smooth muscle actin (α-SMA) signal were examined using immunofluorescence staining. The elastic modulus of cells was measured using atomic force microscopy (AFM). Significantly greater cell spread area along with increased actin filament development, and vinculin-focal adhesion formation (number and size) were found in both normal and glaucoma LC cells cultured on stiff substrates. These changes were positively associated with elevated cell stiffness measured by AFM. Changes in spreading and cytoskeleton organization of glaucoma LC cells were significantly more pronounced than those in normal cells. The transformation to a myofibroblast-like cell phenotype was identified in both LC cells exposed to stiffer substrates, as indicated by an increased α-SMA signal and its colocalization with the actin stress fibers. These findings demonstrated that a stiffer cell microenvironment activates a myofibroblastic transformation in human LC cells, and therefore contributes to LC remodelling and fibrosis in glaucoma.

Stiffness-dependent motility and\xa0proliferation uncoupled by deletion of CD44

Information in the microenvironment guides complex cellular decisions such as whether or not to proliferate and migrate. The effects of soluble extracellular signals on these cellular functions are fairly well understood, but relatively little is known about how the extracellular matrix (ECM), and particularly the mechanical information in the ECM, guides these cellular decisions. Here, we show that CD44, a major receptor for the glycosaminoglycan ECM component hyaluronan, coordinates the motility and proliferative responses to ECM stiffening. We analyzed these cellular responses on fibronectin-coated polyacrylamide hydrogels prepared at a physiologic range of ECM stiffness and found that stiffening of the ECM leads to both cell cycling and cell motility in serum-stimulated primary mouse dermal fibroblasts. Remarkably, deletion of CD44 impaired stiffness-stimulated motility of the primary cells without affecting other hallmark cellular responses to ECM stiffening including cell spread area, stress fiber formation, focal adhesion maturation, and intracellular stiffening. Even stiffness-mediated cell proliferation was unaffected by deletion of CD44. Our results reveal a novel effect of CD44, which is imposed downstream of ECM-mechanosensing and determines if cells couple or uncouple their proliferative and motility responses to ECM stiffness.

Plasma of Argon Increases Cell Attachment and Bacterial Decontamination on Different Implant Surfaces.

This in vitro study tested the effects of argon atmospheric pressure dielectric barrier discharge (APDBD) on different implant surfaces with regard to physical changes, bacterial decontamination, and osteoblast adhesion. Seven hundred twenty disks with three different surface topographies-machined (MAC), titanium plasma-sprayed (TPS), and zirconia-blasted and acid-etched (ZRT)-were tested in this experiment. Bacterial adhesion tests were performed repeatedly on a simplified biofilm of Streptococcus mitis. Bacteria were incubated in the presence of the samples, which were subsequently either left untreated as controls or treated with APDBD for 30, 60, and 120 seconds. Samples were then metalized, prior to the recurring acquisition of images using a scanning electronic microscope (SEM). Protein adsorption, surface wettability, and early biologic response were determined for both treated (120 seconds) and untreated implant surfaces. For depicting the eukaryotic cell behavior, preosteoblastic murine cells were used. Cells were conveniently stained, and nuclei were counted. Cell viability was assessed by a chemiluminescent assay at 1, 2, and 3 days. On all treated samples, values of the contact angle measurements were lower than 10 degrees. The untreated samples showed values of contact angle of 80, 100, and 110 degrees, respectively, for MAC, TPS, and ZRT. The protein adsorption on TPS and ZRT was significantly increased after the plasma of argon treatment. However, no significant effect was noted on the MAC disks. The number and the cell spreading area of adherent osteoblasts significantly increased in all treated surfaces. Nonetheless, argon treatment did not influence the osteoblast proliferation and viability at different time points. Bacteria adhesion was significantly reduced, even after 60 seconds of argon treatment. Preliminary data showed that argon atmospheric pressure dielectric barrier discharge disinfected the implant surface, with potential to promote osteoblast attachment and spreading, suggesting this may be a possible approach to clean a peri-implantitis-contaminated implant surface.

Structure and biocompatibility improvement mechanism of highly oriented poly(lactic acid) produced by solid die drawing

Highly oriented long-chain-branched poly(lactic acid) (LCB-PLA) was fabricated through solid die drawing technology for the purpose of further improving the mechanical properties and biocompatibility of PLA as bone fixation materials. Compared with neat PLA, higher draw ratio can be obtained for LCB-PLA during the drawing process, which led to the sharp increase of the mechanical properties of PLA samples. For LCB-PLA sample with 13.2 of draw ratio, the tensile strength and modulus reached up to 235.4MPa and 8.2GPa, respectively, which satisfied the requirement of the weight-bearing implant fixation. The MTT and ALP results suggested that the proliferation, differentiation and ALP activity of osteoblasts on the surface of PLA samples increased with draw ratio. In addition, cells with flat and extension morphology adhered and grew better, and the spreading area of cells on LCB-PLA samples was larger than that on PLA samples, which suggested that long chain branching and uniaxial orientation exhibited optimal role in promoting cell growth and maintaining their morphology. After orientation, due to the increase of crystallinity and CH3 content on the sample surface, the hydrophobicity increased, promoting the protein adsorption and thus the cell adhesion on it; moreover, cell adhered more stably on the CO enriched and grooved surfaces of the oriented samples, further improving the cell reproduction and aggregation.

Enhanced antiadhesive properties of chitosan/hyaluronic acid polyelectrolyte multilayers driven by thermal annealing: Low adherence for mammalian cells and selective decrease in adhesion for Gram-positive bacteria

The development of antifouling coatings with restricted cell and bacteria adherence is fundamental for many biomedical applications. A strategy for the fabrication of antifouling coatings based on the layer-by-layer assembly and thermal annealing is presented. Polyelectrolyte multilayers (PEMs) assembled from chitosan and hyaluronic acid were thermally annealed in an oven at 37°C for 72h. The effect of annealing on the PEM properties and topography was studied by atomic force microscopy, ζ-potential, circular dichroism and contact angle measurements. Cell adherence on PEMs before and after annealing was evaluated by measuring the cell spreading area and aspect ratio for the A549 epithelial, BHK kidney fibroblast, C2C12 myoblast and MC-3T3-E1 osteoblast cell lines. Chitosan/hyaluronic acid PEMs show a low cell adherence that decreases with the thermal annealing, as observed from the reduction in the average cell spreading area and more rounded cell morphology. The adhesion of S. aureus (Gram-positive) and E. coli (Gram-negative) bacteria strains was quantified by optical microscopy, counting the number of colony-forming units and measuring the light scattering of bacteria suspension after detachment from the PEM surface. A 20% decrease in bacteria adhesion was selectively observed in the S. aureus strain after annealing. The changes in mammalian cell and bacteria adhesion correlate with the changes in topography of the chitosan/hyaluronic PEMs from a rough fibrillar 3D structure to a smoother and planar surface after thermal annealing.

Effects of Functional Groups of Materials on Nonspecific Adhesion and Chondrogenic Induction of Mesenchymal Stem Cells on Free and Micropatterned Surfaces.

Functional groups of materials are known to affect cell behaviors, yet the corresponding effect on stem cell differentiation is always coupled with that of cell spreading; it is thus unclear whether the chemical groups influence cell differentiation directly or via cell spreading indirectly. Herein we used a unique surface patterning technique to decouple the corresponding effects. Mesenchymal stem cells (MSCs) derived from bone marrow were seeded on surfaces coated with alkanethiols with one of four functional end groups (-CH3, -OH, -COOH, and -NH2) and underwent 9 days of chondrogenic induction. The measurements of quartz crystal microbalance with dissipation confirmed less proteins adsorbed from the cell culture media on the neutral -CH3 and -OH surfaces than on the charged -COOH and -NH2 surfaces. The neutral surfaces exhibited less cell spreading and higher extents of chondrogenic differentiation than the charged surfaces, according to the characterizations of immunofluorescence staining and quantitative real-time polymerase chain reaction. We further used a transfer lithography technique to prepare patterned surfaces on nonfouling poly(ethylene glycol) hydrogels to localize single MSCs on microislands with self-assembly monolayers of different alkanethiols, under given microisland areas and thus well-defined spreading areas of cells. While small microislands were always beneficial for chondrogenic induction, we found that the type of functional groups had no significant effect on chondrogenic induction under the given cell spreading areas, implying that the chemical groups influence cell differentiation only indirectly. Our results hence illustrate that functional groups regulate stem cell differentiation via tuning protein adsorption and then nonspecific cell adhesion and thus cell spreading.

Free energy analysis of cell spreading

In this study we present a steady-state adaptation of the thermodynamically motivated stress fiber (SF) model of Vigliotti et al. (2015). We implement this steady-state formulation in a non-local finite element setting where we also consider global conservation of the total number of cytoskeletal proteins within the cell, global conservation of the number of binding integrins on the cell membrane, and adhesion limiting ligand density on the substrate surface. We present a number of simulations of cell spreading in which we consider a limited subset of the possible deformed spread-states assumed by the cell in order to examine the hypothesis that free energy minimization drives the process of cell spreading. Simulations suggest that cell spreading can be viewed as a competition between (i) decreasing cytoskeletal free energy due to strain induced assembly of cytoskeletal proteins into contractile SFs, and (ii) increasing elastic free energy due to stretching of the mechanically passive components of the cell. The computed minimum free energy spread area is shown to be lower for a cell on a compliant substrate than on a rigid substrate. Furthermore, a low substrate ligand density is found to limit cell spreading. The predicted dependence of cell spread area on substrate stiffness and ligand density is in agreement with the experiments of Engler et al. (2003). We also simulate the experiments of Théry et al. (2006), whereby initially circular cells deform and adhere to “V-shaped” and “Y-shaped” ligand patches. Analysis of a number of different spread states reveals that deformed configurations with the lowest free energy exhibit a SF distribution that corresponds to experimental observations, i.e. a high concentration of highly aligned SFs occurs along free edges, with lower SF concentrations in the interior of the cell. In summary, the results of this study suggest that cell spreading is driven by free energy minimization based on a competition between decreasing cytoskeletal free energy and increasing passive elastic free energy.

Cytotoxic effects of docetaxel as a candidate drug of drug-eluting stent on human umbilical vein endothelial cells and the signaling pathway of cell migration inhibition, adhesion delay and shape change.

Docetaxel (DTX), a paclitaxel analogue, can efficiently inhibit proliferation of vascular smooth muscle cells and has broadly been used as an antiangiogenesis drug. However, as a candidate drug of drug-eluting stent, the effects of DTX on human umbilical vein endothelial cells (HUVECs) are still not well understood. Herein, we investigated the effects of DTX on proliferation, apoptosis, adhesion, migration and morphology of HUVECs in vitro. We found that DTX had the cytostatic and cytotoxic effects at low and high concentrations, respectively. DTX could inhibit the proliferation and migration of HUVECs, induce HUVECs apoptosis, delay HUVECs adhesion and decrease spreading area and aspect ratio of individual cells. The signaling pathway that DTX led to the migration inhibition, adhesion delay and shape change of HUVECs is the VE-cadherin mediated integrin β1/FAK/ROCK signaling pathway. The study will provide a theoretical basis for the clinical application of DTX.

Innovative surface modification of Ti6Al4V alloy by electron beam technique for biomedical application

The low elastic modulus, high corrosion resistance and excellent biological response allow titanium alloys to be used for permanent orthopaedic devices. Furthermore, the design of specific multi scale surface topographies on titanium alloys can provide a fast osseointegration. This work highlights the use of electron beam as a promising technique to produce a designed surface topography and improve the tribological behaviour of Ti6Al4V alloy. The produced surface topography due to the transport of molten material is influenced by the deflection figure, the physical properties of the material and the energy input. The analysis of the surface roughness shows an increment of the area up to 26% and a canal shape in a range from 1.3μm up to 9μm depth and from 68.6μm up to 119.7μm width. The high solidification rate reached during the process affects the microstructure, provoking the formation of martensite and thus the improvement of hardness. In vitro studies with pre-osteoblastic MC3T3-E1 cells performed for several cultivation times show the cells with a polygonal shape and built connections through elongated filopodia. A notable increase of cell spreading area on surface structure with a finer canal shape is found after 48h cultivation time.