Protein-coated Surfaces Research Articles

Effects of ECM protein-coated surfaces on the generation of retinal pigment epithelium cells differentiated from human pluripotent stem cells.

Retinal degeneration diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), initially manifest as dysfunction or death of the retinal pigment epithelium (RPE). Subretinal transplantation of human pluripotent stem cell (hPSC)-derived RPE cells has emerged as a potential therapy for retinal degeneration. However, RPE cells differentiated from hPSCs using current protocols are xeno-containing and are rarely applied in clinical trials. The development of hPSC-derived RPE cell differentiation protocols using xeno-free biomaterials is urgently needed for clinical applications. In this study, two protocols (the activin A and NIC84 protocols) were selected for modification and use in the differentiation of hiPSCs into RPE cells; the chetomin concentration was gradually increased to achieve high differentiation efficiency of RPE cells. The xeno-free extracellular matrix (ECM) proteins, laminin-511, laminin-521 and recombinant vitronectin, were selected as plate-coating substrates, and a Matrigel (xeno-containing ECM)-coated surface was used as a positive control. Healthy, mature hPSC-derived RPE cells were transplanted into 21-day-old Royal College of Surgeons (RCS) rats, a model of retinal degeneration disease. The visual function of RCS rats was evaluated by optomotor response (qOMR) and electroretinography after transplantation of hPSC-derived RPE cells. Our study demonstrated that hPSCs can be efficiently differentiated into RPE cells on LN521-coated dishes using the NIC84 protocol, and that subretinal transplantation of the cell suspensions can delay the progression of vision loss in RCS rats.

Photoactivatable substrates show diverse phenotypes of leader cells in collective migration when moving along different extracellular matrix proteins.

In cancer metastasis, collectively migrating clusters are discriminated into leader and follower cells that move through extracellular matrices (ECMs) with different characteristics. The impact of changes in ECM protein types on leader cells and migrating clusters is unknown. To address this, we investigated the response of leader cells and migrating clusters upon moving from one ECM protein to another using a photoactivatable substrate bearing photocleavable PEG (PCP), whose surface changes from protein-repellent to protein-adhesive in response to light. We chose laminin and collagen I for our study since they are abundant in two distinct regions in living tissues, namely basement membrane and connective tissue. Using the photoactivatable substrates, the precise deposition of the first ECM protein in the irradiated areas was achieved, followed by creating well-defined cellular confinements. Secondary irradiation enabled the deposition of the second ECM protein in the new irradiated regions, resulting in region-selective heterogeneous and homogenous ECM protein-coated surfaces. Different tendencies in leader cell formation from laminin into laminin compared to those migrating from laminin into collagen were observed. The formation of focal adhesion and actin structures for cells within the same cluster in the ECM proteins responded according to the underlying ECM protein type. Finally, integrin β1 was crucial for the appearance of leader cells for clusters migrating from laminin into collagen. However, when it came to laminin into laminin, integrin β1 was not responsible. This highlights the correlation between leader cells in collective migration and the biochemical signals that arise from underlying extracellular matrix proteins.

Frictional behaviour of plant proteins in soft contacts: unveiling nanoscale mechanisms.

Despite the significance of nanotribology in the design of functional biomaterials, little is known about nanoscale friction in the presence of protein-coated soft contact surfaces. Here, we report a detailed investigation of frictional behaviour of sustainable plant proteins at the nanoscale for the first time, using deformable bio-relevant surfaces that achieve biologically relevant contact pressures. A combination of atomic force microscopy, quartz crystal microbalance with dissipation monitoring, and friction force microscopy with soft polydimethylsiloxane (PDMS, 150 kPa) surfaces was employed to elucidate the frictional properties of model plant proteins, i.e. lupine, pea, and potato proteins at the nanoscale while systematically varying the pH and ionic strength. Interactions of these plant proteins with purified mucins were also probed. We provide the much-needed direct experimental evidence that the main factor dictating the frictional properties of plant proteins is their affinity towards the surface, followed by the degree of protein film hydration. Proteins with high surface affinity, such as pea and potato protein, have better lubricating performance than lupine at the nanoscale. Other minor factors that drive lubrication are surface interactions between sliding bodies, especially at low load, whilst jamming of the contact area caused by larger protein aggregates increases friction. Novel findings reveal that interactions between plant proteins and mucins lead to superior lubricating properties, by combining high surface affinity from the plant proteins and high hydration by mucins. We anticipate that fundamental understanding gained from this work will set the stage for the design of a plethora of sustainable biomaterials and food with optimum nanolubrication performance.

Indirect Immobilised Jagged-1 Enhances Matrisome Proteins Associated with Osteogenic Differentiation of Human Dental Pulp Stem Cells: A Proteomic Study.

The indirect immobilisation of Jagged-1 (Jagged-1) promoted osteogenic differentiation of human dental pulp cells (hDPs). Furthermore, the analysis of the Reactome pathway of RNA sequencing data indicates the upregulated genes involved with the extracellular matrix (ECM). Hence, our objective was to investigate the effects of Jagged-1 on proteomic profiles of human dental pulp stem cells (hDPSC). hDPSCs were cultured on the surface coated with human IgG Fc fragment (hFc) and the surface coated with rhJagged1/Fc recombinant protein-coated surface. Cells were differentiated to the osteogenic lineage using an osteogenic differentiation medium (OM) for 14 days, and cells cultured in a growth medium were used as a control. The protein component of the cultured cells was extracted into the cytosol, membrane, nucleus, and cytoskeletal compartment. Subsequently, the proteomic analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS). Metascape gene list analysis reported that Jagged-1 stimulated the expression of the membrane trafficking protein (DOP1B), which can indirectly improve osteogenic differentiation. hDPSCs cultured on Jagged-1 surface under OM condition expressed COL27A1, MXRA5, COL7A1, and MMP16, which played an important role in osteogenic differentiation. Furthermore, common matrisome proteins of all cellular components were related to osteogenesis/osteogenic differentiation. Additionally, the gene ontology categorised by the biological process of cytosol, membrane, and cytoskeleton compartments was associated with the biomineralisation process. The gene ontology of different culture conditions in each cellular component showed several unique gene ontologies. Remarkably, the Jagged-1_OM culture condition showed the biological process related to odontogenesis in the membrane compartment. In conclusion, the Jagged-1 induces osteogenic differentiation could, mainly through the regulation of protein in the membrane compartment.

A model for bubble dynamics in a protein solution

This paper presents a model for bubble dynamics in a protein solution, including varying surface tension and dynamic adsorption/desorption of protein onto the bubble surface. We model the protein-coated bubble surface as a linear viscoelastic interface. Two cases are studied to understand the importance of the surface excess stress: (1) bubble dissolution in a protein solution due to gas diffusion; and (2) bubble response to an imposed fluctuating pressure field. In the first case study, the surface excess stress stabilizes the bubble against dissolution. Initially, the surface excess stress is negligible, and the dissolution rate is governed by the Weber number, which compares the gas inertial force and the surface tension force. As the bubble shrinks, the surface excess stress grows and eventually balances the surface tension. After that, the dissolution rate is governed by the protein desorption rate and the elasto-capillary number, which compares the surface tension and the surface dilatational elasticity. Our model predictions for the dissolution process agree with experiments before the bubble buckles. In the second case study, the surface dilatational viscosity and dilatational elasticity add resistance and stiffness to the system, respectively. Including the surface excess stress increases (or reduces) the amplitude of the bubble radius if the frequency of the imposed fluctuating pressure is greater (or less) than a critical value. These results highlight the importance of the surface rheology on the protein-coated bubble dynamics, which has applications in drug delivery and ultrasound contrast agents.

Protein-coated nanostructured surfaces affect the adhesion of Escherichia coli.

Developing new implant surfaces with anti-adhesion bacterial properties used for medical devices remains a challenge. Here we describe a novel study investigating nanotopography influences on bacterial adhesion on surfaces with controlled interspatial nanopillar distances. The surfaces were coated with proteins (fibrinogen, collagen, serum and saliva) prior to E. coli-WT adhesion under flow conditions. PiFM provided chemical mapping and showed that proteins adsorbed both between and onto the nanopillars with a preference for areas between the nanopillars. E. coli-WT adhered least to protein-coated areas with low surface nanopillar coverage, most to surfaces coated with saliva, while human serum led to the lowest adhesion. Protein-coated nanostructured surfaces affected the adhesion of E. coli-WT.

Stress-Related Regulation Is Abnormal in the Psoriatic Uninvolved Skin.

Keratinocyte stress-response of the uninvolved psoriatic epidermis is known to be altered compared to healthy cells. Therefore, we aimed to reveal potential mechanisms underlying this alteration. We compared the expression of annotated cell-stress-related proteins between uninvolved psoriatic and healthy skin using the protein array method. Data were analyzed by the Reactome over-representation test. We found that p27/CDKN1B and cytochrome C showed at least a two-fold increase, while cyclooxygenase-2, indolamine-2,3-dioxygenase-1, serum paraoxonase 1, serum paraoxonase 3, serine-46-phosphorylated tumor protein p53, and superoxide-dismutase-2 showed a two-fold decrease in expression in the uninvolved skin. Over-representation analysis suggested the Forkhead-box protein O (FOXO)-mediated transcription as the most significant pathway affected by the differently expressed cell-stress-related proteins (DECSRPs). DECSRPs indicate increased FOXO-mediated transcription of cell-cycle genes and reduced interleukin-signaling in the psoriatic uninvolved skin. Nuclear positivity of the FOXO-signaling-related p27/CDKN1B and FOXO1 are negatively correlated with the disease severity and showed increased expression in the uninvolved epidermis and also in healthy primary keratinocytes, which were grown on cartilage oligomeric matrix protein-coated surfaces. Our results indicate a cell-cycle inhibitory process, as a stress-related compensatory mechanism in the uninvolved epidermis, that could be responsible for blocking keratinocyte hyperproliferation in the psoriatic uninvolved skin, thus maintaining the symptomless skin phenotype.

Effect of upstream priming on transient downstream platelet-substrate interactions

Upstream exposure of platelets to activating proteins ‘primes’ platelets for increased downstream adhesion, though the mechanics of platelet translocation before permanently arresting are not well understood. To investigate platelet translocation on platelet-binding proteins, primed platelets’ transient contacts with immobilized proteins were recorded and analyzed. Using a microfluidic channel, representative of a vascular graft, platelet-activating proteins were covalently attached to the upstream priming, center, and downstream capture positions. Image sequences of platelet interactions with the center protein were captured as platelet-rich plasma (PRP) was perfused through the channel. There was an increase in both platelet pause events and net platelet adhesion on von Willebrand factor, collagen, or fibrinogen following upstream exposure to the same protein. Upstream priming also caused a decrease in average platelet velocity. The duration of transient platelet arrests on the protein-coated surface and the distance that platelets travel between pause events depended on the protein with which they were interacting. The most significant increase in platelet pause events frequency and decrease in average velocity occurred on immobilized von Willebrand factor, compared to the control with no upstream priming. These results demonstrate that platelet priming increases downstream platelet-protein interactions prior to permanent adhesion.

Friction between soft contacts at nanoscale on uncoated and protein-coated surfaces.

The understanding of friction on soft sliding biological surfaces at the nanoscale is poorly understood as hard interfaces are frequently used as model systems. Herein, we studied the influence of elastic modulus on the frictional properties of model surfaces at the nanoscale for the first time. We prepared model silicone-based elastomer surfaces with tuneable modulus ranging from hundreds of kPa to a few MPa, similar to those found in real biological surfaces, and employed atomic force microscopy to characterize their modulus, adhesion, and surface morphology. Consequently, we used friction force microscopy to investigate nanoscale friction in hard-soft and soft-soft contacts using spherical colloidal probes covered by adsorbed protein films. Unprecedented results from this study reveal that modulus of a surface can have a significant impact on the frictional properties of protein-coated surfaces with higher deformability leading to lower contact pressure and, consequently, decreased friction. These important results pave the way forward for designing new functional surfaces for serving as models of appropriate deformability to replicate the mechanical properties of the biological structures and processes for accurate friction measurements at nanoscale.

Surfactant Interactions with Protein-Coated Surfaces: Comparison between Colloidal and Macroscopically Flat Surfaces.

Surface interactions with polymers or proteins are extensively studied in a range of industrial and biomedical applications to control surface modification, cleaning, or biofilm formation. In this study we compare surfactant interactions with protein-coated silica surfaces differing in the degree of curvature (macroscopically flat and colloidal nanometric spheres). The interaction with a flat surface was probed by means of surface plasmon resonance (SPR) while dynamic light scattering (DLS) was used to study the interaction with colloidal SiO2 (radius 15 nm). First, the adsorption of bovine serum albumin (BSA) with both SiO2 surfaces to create a monolayer of coating protein was studied. Subsequently, the interaction of these BSA-coated surfaces with a non-ionic surfactant (a decanol ethoxylated with an average number of eight ethoxy groups) was investigated. A fair comparison between the results obtained by these two techniques on different geometries required the correction of SPR data for bound water and DLS results for particle curvature. Thus, the treated data have excellent quantitative agreement independently of the geometry of the surface suggesting the formation of multilayers of C10PEG over the protein coating. The results also show a marked different affinity of the surfactant towards BSA when the protein is deposited on a flat surface or individually dissolved in solution.

Kinetics of Platelet Adhesion to Protein-Coated Surface in Whole Blood Samples at High Flow Rates.

We studied platelet adhesion to fibrinogen-coated surface in whole blood samples under conditions of high flow rates. The degree of platelet adhesion was evaluated by the intensity of laser light scattered from protein-coated optical surface with adhered platelets. The intensity of adhesion in whole blood samples at high flow rates was by 2.7 (2.4; 4.1) times higher than in platelet-rich plasma samples. Among the factors intensifying platelet adhesion in the whole blood at high flow rates, von Willebrand factor is of utmost importance. At low flow rates, platelet adhesion almost totally depends on platelet-fibrinogen interaction. At high flow rates, the interactions of platelets with both fibrinogen and von Willebrand factor become equally important.

A comparative analysis of detachment forces and energies in initial and mature cell-material interaction

Single cell force spectroscopy (SCFS) enables data on interaction forces to be acquired during the very early adhesion phase. However, SCFS detachment forces and energies have not been compared so far with the forces and energies after maturation of the cell-material contact on a single cell level and with comparable time resolution. We used FluidFM® to physically attach single cells to the cantilever by aspiration through a microfluidic channel, in order to achieve the higher forces required for detaching maturely adhering cells. Combining these two approaches allowed us to compare cell adhesion in the initial and maturation phases of adhesion for two exemplary cell-substrate combinations – L929 fibroblasts on fibronectin and MC3T3 osteoblasts on collagen type I. Uncoated glass substrates were used as a reference. For both cell lines, SCFS measurements after contact times of 5, 15 and 30 s revealed significantly higher maximum detachment forces (MDFs) and energies on glass compared to the protein-coated surfaces in the 0.5–4 nN (1–40 fJ) range. FluidFM® measurements after 1, 2 and 3 days of culture revealed a significant absolute increase in the MDFs and detachment energies for both cell lines on protein-coated substrates to values of about 600 nN and 10 pJ. On glass, the MDFs were similar for MC3T3 cells, while they were significantly lower for L929 cells. For both cell types, the differences in detachment energy were significant. These differences underline the importance of investigating early and mature adhesion states to obtain a holistic assessment of the cell-material interactions.

Fluorescence emission and interaction mechanism of protein-coated gold and copper nanoclusters as ion sensors in different ionic environments

Lysozyme protein-induced luminescent gold and copper nanoclusters (AuNCs and CuNCs) are prepared and their sensing capability is studied in the presence of different valent ions (Na+, Ca2+, Hg2+, Pb2+, Fe3+ and La3+ ions) in aqueous solutions of up to 100 µM salt concentration. Both types of nanocluster show good stability in aqueous conditions and, among all the chosen valent ions, it is found that maximum fluorescence quenching is observed in the presence of Hg2+ and Pb2+ ions for AuNCs and CuNCs, respectively. From steady-state fluorescence study and by using Stern–Volmer plots, the Stern–Volmer constant and other important thermodynamical parameters, i.e. changes in enthalphy, entropy and Gibbs free energy, are calculated. We propose a two step interaction process between the nanoclusters and the dissolved ions: first the interaction occurs between the protein-coated nanocluster surface and the ions, followed by the interaction of the metallic nanocluster core with the ions. Although thermodynamic investigation implies that both types of interaction are electrostatic in nature, selective fluorescence quenching of AuNCs and CuNCs in the presence of Hg2+ and Pb2+ ions occurs due to noncovalent metallophilic interactions between the cluster core and the dissolved ions.

Long-range interactions between protein-coated particles and POEGMA brush layers in a serum environment

Hydrophilic poly[oligo(ethylene glycol) methyl methacrylate] (POEGMA) brush layers with different thickness and graft densities were prepared by surface-initiated atom transfer radical polymerization (SI-ATRP) to construct a model surface to examine protein-surface interactions in a serum environment. The thickness of the POEGMA brush layers could be well controlled by the polymerization time and density of the immobilized initiators. The interactions between these brush-modified surfaces and the protein-coated polystyrene (PS) particles in newborn calf serum (NBCS) environment were then measured by total internal reflection microscopy (TIRM). In addition, protein adsorption properties onto the polymer brush surface layers were examined by atomic force microscopy (AFM). Relatively large amounts of protein adsorbed to short (4nm and 9nm-thick) POEGMA-coated surfaces or surfaces grafted with a low density of polymer chains. It was considered that shorter polymer chains or chains with low grafted density cannot fully cover the surfaces, proteins in serum could directly interact with the material surface and then deposited to form an adsorbed layer. The TIRM measurements showed that such adsorbed protein layer could mediate the interactions between the two surfaces by generating steric or bridging forces, resulting in different interaction potentials. Some particles were freely diffusing, some experienced intermittent diffusion and more than 50% of particles were irreversibly deposited to the surfaces covered by short polymer brushes. However, for longer (17 and 30nm-thick) POEGMA brush layer surfaces, material surface would be sufficiently covered by the dense coating and the first step of protein adsorption on surface was avoided. TIRM measurements showed that around 95% of the protein-coated particles could freely move in the serum and no attractive force between two surfaces was detected. The steric repulsion generated from the long POEGMA brush layer in the swollen state was long-range and strong so that the protein adsorption is very unlikely. These results concluded that the adsorbed protein layer on POEGMA surfaces plays an important role in regulating the interaction between protein-coated particles and POEGMA surfaces which are highly repellent toward protein adsorption.

Interaction of plasma-generated water cluster ions with chemically-modified Si surfaces investigated by infrared absorption spectroscopy

We have investigated the interaction of water cluster ions generated by discharge plasma, with chemically modified Si surfaces using infrared absorption spectroscopy in the multiple internal reflection geometry. We observe that water cluster ions readily adsorb on SiO2-covered Si surfaces to form water droplets. We demonstrate that positively- and negatively-charged cluster ions adsorb on the SiO2-covered Si surface in different manners, indicating ionic interaction of the water droplets with the negatively-charged SiO2 surface. Water droplets formed on the protein-coated surface rupture the amide bond of the proteins, suggesting the function of protein decomposition of water cluster ions.

Effects of protein species and surface physicochemical features on the deposition of nanoparticles onto protein-coated planar surfaces

Proteins are often an important component of many bulk surfaces in biological and environmental systems that are coated with complex organic compounds that may also interact with nanoparticles.

How cells respond to environmental cues - insights from bio-functionalized substrates.

Biomimetic materials have long been the (he)art of bioengineering. They usually aim at mimicking in vivo conditions to allow in vitro culture, differentiation and expansion of cells. The past decade has witnessed a considerable amount of progress in soft lithography, bio-inspired micro-fabrication and biochemistry, allowing the design of sophisticated and physiologically relevant micro- and nano-environments. These systems now provide an exquisite toolbox with which we can control a large set of physicochemical environmental parameters that determine cell behavior. Bio-functionalized surfaces have evolved from simple protein-coated solid surfaces or cellular extracts into nano-textured 3D surfaces with controlled rheological and topographical properties. The mechanobiological molecular processes by which cells interact and sense their environment can now be unambiguously understood down to the single-molecule level. This Commentary highlights recent successful examples where bio-functionalized substrates have contributed in raising and answering new questions in the area of extracellular matrix sensing by cells, cell-cell adhesion and cell migration. The use, the availability, the impact and the challenges of such approaches in the field of biology are discussed.

The interaction of protein-coated bionanoparticles and surface receptors reevaluated: how important is the number of bonds?

Specifically designed bionanoparticles with a function-oriented protein-coating layer interact with self-prepared receptor surfaces as the counterpart. Based on surface plasmon resonance biosensing experiments, a model framework is validated to estimate the number of bonds formed between these bionanoparticles and the receptor surface based on multivalent interactions. Our multi-site kinetic model is able to analyze the adsorption rate constants and the number of bonds from experimental data of natural and synthetic bionanoparticles. The influence of the mass transport on the adsorption kinetics is modeled including a diffusional boundary layer where a helpful analytical solution has been derived. Our model framework extends previous studies to include a higher number of bonds, ranging from 1 up to 1000. An almost linear relationship between the number of bonds and the adsorption amount of bionanoparticles makes the model framework suitable to predict, for example, ligand density and to further assess coating performance. The proposed model framework can serve as a design tool for multivalent interaction experiments under variable process conditions.

Scanning Resonator Microscopy: Integrating Whispering Gallery Mode Sensing with Atomic Force Microscopy

Scanning resonator microscopy (SRM) is developed to integrate whispering gallery mode (WGM) sensing with atomic force microscopy (AFM). The hybrid technique combines the exquisite refractive index sensing of whispering gallery mode resonators with the topography mapping capabilities of AFM. A 45 μm diameter barium titanate microsphere is attached to the end of a conventional AFM cantilever and acts as both a WGM resonator and stylus for mapping surface topography. Calibration plots, taken in contact-mode feedback, show that the WGM spectrum responds to changes in both solution and substrate refractive index. SRM imaging of a glass substrate reveals changes in surface refractive index that correspond to a small, 36 nm high feature measured simultaneously in the contact-mode topography image. Spectral measurements confirm that the contrast arises from refractive index changes and not coupling with sample topography, thus validating the approach. Additional measurements on thin polymer films and protein-coated surfaces are presented and discussed in terms of possible areas of application for SRM.

Thrombotic Responses of Endothelial Outgrowth Cells to Protein-Coated Surfaces

There is significant clinical need for viable small-diameter vascular grafts. While there are many graft biomaterials in development, few have been clinically successful. Evaluation of grafts with a clinically relevant model is needed to drive development. This work examined extracellular matrix coatings on the thrombotic phenotype of endothelial outgrowth cells (EOCs). EOCs were tested on flat plates and tubular grafts. Flat plate studies examined collagen I, collagen IV, fibronectin and α-elastin coatings. EOCs attached or proliferated more readily on collagen I and fibronectin surfaces as determined by total DNA. The production of activated protein C (APC) by EOCs was also dependent on the surface coating, with collagen I and fibronectin displaying a higher activity than both collagen IV and α-elastin on flat plate studies. Based on these results, only collagen I and fibronectin coatings were tested on expanded polytetrafluoroethylene (ePTFE) in the ex vivo model. Tubular samples showed significantly greater tissue factor pathway inhibitor gene expression on collagen I than on fibronectin. Platelet adhesion was not significantly different, but EOCs on collagen I produced significantly lower APC than on fibronectin, suggesting that differences exist between the flat plate and tubular cultures. Overall, while the hemostatic phenotype of EOCs displayed some differences, cell responses were largely independent of the matrix coating. EOCs adhered strongly to both fibronectin- and collagen-I-coated ePTFE grafts under ex vivo (100 ml/min) flow conditions suggesting the usefulness of this clinically relevant cell source, testing modality, and shunt model for future work examining biomaterials and cell conditioning before implantation.