Entropic repulsion of cholesterol-containing layers counteracts bioadhesion.

Control of adhesion is a striking feature of living matter that is of particular interest regarding technological translation1-3. We discovered that entropic repulsion caused by interfacial orientational fluctuations of cholesterol layers restricts protein adsorption and bacterial adhesion. Moreover, we found that intrinsically adhesive wax ester layers become similarly antibioadhesive when containing small quantities (under 10 wt%) of cholesterol. Wetting, adsorption and adhesion experiments, as well as atomistic simulations, showed that repulsive characteristics depend on the specific molecular structure of cholesterol that encodes a finely balanced fluctuating reorientation at the interface of unconstrained supramolecular assemblies: layers of cholesterol analogues differing only in minute molecular variations showed markedly different interfacial mobility and no antiadhesive effects. Also, orientationally fixed cholesterol layers did not resist bioadhesion. Our insights provide a conceptually new physicochemical perspective on biointerfaces and may guide future material design in regulation of adhesion.

Remodeling of the Cardiac Extracellular Matrix Proteome During Chronological and Pathological Aging.

Impaired extracellular matrix (ECM) remodeling is a hallmark of many chronic inflammatory disorders that can lead to cellular dysfunction, aging, and disease progression. The ECM of the aged heart and its effects on cardiac cells during chronological and pathological aging are poorly understood across species. For this purpose, we first used mass spectrometry-based proteomics to quantitatively characterize age-related remodeling of the left ventricle (LV) of mice and humans during chronological and pathological (Hutchinson-Gilford progeria syndrome (HGPS)) aging. Of the approximately 300 ECM and ECM-associated proteins quantified (named as Matrisome), we identified 13 proteins that were increased during aging, including lactadherin (MFGE8), collagen VI α6 (COL6A6), vitronectin (VTN) and immunoglobulin heavy constant mu (IGHM), whereas fibulin-5 (FBLN5) was decreased in most of the data sets analyzed. We show that lactadherin accumulates with age in large cardiac blood vessels and when immobilized, triggers phosphorylation of several phosphosites of GSK3B, MAPK isoforms 1, 3, and 14, and MTOR kinases in aortic endothelial cells (ECs). In addition, immobilized lactadherin increased the expression of pro-inflammatory markers associated with an aging phenotype. These results extend our knowledge of the LV proteome remodeling induced by chronological and pathological aging in different species (mouse and human). The lactadherin-triggered changes in the proteome and phosphoproteome of ECs suggest a straight link between ECM component remodeling and the aging process of ECs, which may provide an additional layer to prevent cardiac aging.

Construction of a 3D brain extracellular matrix model to study the interaction between microglia and T cells in co-culture.

Neurodegenerative disorders are characterised by the activation of brain-resident microglia cells and by the infiltration of peripheral T cells. However, their interplay in disease has not been clarified yet. It is difficult to investigate complex cellular dynamics in living animals, and simple two-dimensional (2D) cell culture models do not resemble the soft 3D structure of brain tissue. Therefore, we developed a biomimetic 3D in vitro culture system for co-cultivation of microglia and T cells. As the activation and/or migration of immune cells in the brain might be affected by components of the extracellular matrix, defined 3D fibrillar collagen I-based matrices were constructed and modified with hyaluronan and/or chondroitin sulphate, resembling aspects of brain extracellular matrix. Murine microglia and spleen-derived T cells were cultured alone or in co-culture on the constructed matrices. Microglia exhibited in vivo-like morphology and T cells showed enhanced survival when co-cultured with microglia or to a minor degree in the presence of glia-conditioned medium. The open and porous fibrillar structure of the matrix allowed for cell invasion and direct cell-cell interaction, with stronger invasion of T cells. Both cell types showed no dependence on the matrix modifications. Microglia could be activated on the matrices by lipopolysaccharide resulting in interleukin-6 and tumour necrosis factor-α secretion. The findings herein indicate that biomimetic 3D matrices allow for co-cultivation and activation of primary microglia and T cells and provide useful tools to study their interaction in vitro.

Fibrillar biopolymer-based scaffolds to study macrophage-fibroblast crosstalk in wound repair.

Controlled wound healing requires a temporal and spatial coordination of cellular activities within the surrounding extracellular matrix (ECM). Disruption of cell-cell and cell-matrix communication results in defective repair, like chronic or fibrotic wounds. Activities of macrophages and fibroblasts crucially contribute to the fate of closing wounds. To investigate the influence of the ECM as an active part controlling cellular behavior, coculture models based on fibrillar 3D biopolymers such as collagen have already been successfully used. With well-defined biochemical and biophysical properties such 3D scaffolds enable in vitro studies on cellular processes including infiltration and differentiation in an in vivo like microenvironment. Further, paracrine and autocrine signaling as well as modulation of soluble mediator transport inside the ECM can be modeled using fibrillar 3D scaffolds. Herein, we review the usage of these scaffolds in in vitro coculture models allowing in-depth studies on the crosstalk between macrophages and fibroblasts during different stages of cutaneous wound healing. A more accurate mimicry of the various processes of cellular crosstalk at the different stages of wound healing will contribute to a better understanding of the impact of biochemical and biophysical environmental parameters and help to develop further strategies against diseases such as fibrosis.

Impact of binding mode of low-sulfated hyaluronan to 3D collagen matrices on its osteoinductive effect for human bone marrow stromal cells.

Synthetically sulfated hyaluronan derivatives were shown to facilitate osteogenic differentiation of human bone marrow stromal cells (hBMSC) by application in solution or incorporated in thin collagen-based coatings. In the presented study, using a biomimetic three-dimensional (3D) cell culture model based on fibrillary collagen I (3D Col matrix), we asked on the impact of binding mode of low sulfated hyaluronan (sHA) in terms of adsorptive and covalent binding on osteogenic differentiation of hBMSC. Both binding modes of sHA induced osteogenic differentiation. Although for adsorptive binding of sHA a strong intracellular uptake of sHA was observed, implicating an intracellular mode of action, covalent binding of sHA to the 3D matrix induced also intense osteoinductive effects pointing towards an extracellular mode of action of sHA in osteogenic differentiation. In summary, the results emphasize the relevance of fibrillary 3D Col matrices as a model to study hBMSC differentiation in vitro in a physiological-like environment and that sHA can display dose-dependent osteoinductive effects in dependence on presentation mode in cell culture scaffolds.

Distinct impacts of substrate elasticity and ligand affinity on traction force evolution.

Cell adhesion is regulated by the mechanical characteristics of the cell environment. The influences of different parameters of the adhesive substrates are convoluted in the cell response leading to questions on the underlying mechanisms, like biochemical signaling on the level of adhesion molecules, or viscoelastic properties of substrates and cell. By a time-resolved analysis of traction force generation during early cell adhesion, we wanted to elucidate the contributions of substrate mechanics to the adhesion process, in particular the impact of substrate elasticity and the molecular friction of adhesion ligands on the substrate surface. Both parameters were independently adjusted by (i) an elastic polyacrylamide hydrogel of variable crosslinking degree and (ii) a thin polymer coating of the hydrogel surface controlling the affinity (and the correlated substrate-ligand friction) of the adhesion ligand fibronectin. Our analysis showed two sequential regimes of considerable force generation, whose occurrence was found to be independent of substrate properties. The first regime is characterized by spreading of the cell and a succeeding force increase. After spreading cells enter the second regime with saturated forces. Substrate elasticity and viscosity, namely hydrogel elasticity and ligand affinity, were both found to affect the kinetics and absolute levels of traction force quantities. A faster increase and a higher saturation level of traction forces were observed for a higher substrate stiffness and a higher ligand affinity. The results complement recent modeling approaches on the evolution of forces in cell spreading and contribute to a better understanding of the dynamics of cell adhesion on viscoelastic substrates.

Quantification of protein-materials interaction by soft colloidal probe spectroscopy.

We present a robust and fast method to quantify the adhesion energy of surface anchored proteins on material surfaces using soft colloidal particles as sensors. The results obtained from studying the adhesion of fibronectin on surfaces with different hydrophobicity were in good agreement with theoretical considerations demonstrating the feasibility of the method.

Specific adhesion of carbohydrate hydrogel particles in competition with multivalent inhibitors evaluated by AFM.

Synthetic glycooligomers have emerged as valuable analogues for multivalent glycan structures in nature. These multivalent carbohydrates bind to specific receptors and play a key role in biological processes. In this work, we investigate the specific interaction between mannose ligand presenting soft colloidal probes (SCPs) attached to an atomic force microscope (AFM) cantilever and a Concanavalin A (ConA) receptor surface in the presence of competing glycooligomer ligands. We studied the SCP-ConA adhesion energy via the JKR approach and AFM pull-off experiments in combination with optical microscopy allowing for simultaneous determination of the contact area between SCP and ConA surface. We varied the contact time, loading rate and loading force and measured the resulting mannose/ConA interaction. The average adhesion energy per mannose ligand on the probe was 5 kJ/mol, suggesting that a fraction of mannose ligands presented on the SCP bound to the receptor surface. Adhesion measurements via competitive binding of the SCP in the presence of multivalent glycooligomer ligands did not indicate an influence of their multivalency on the glycooligomer displacement from the ConA surface. The absence of this "multivalency effect" indicates that glycooligomers and ConA do not associate via chelate complexes and shows that steric shielding by the glycooligomers does not slow their displacement upon competitive binding of a ligand presenting surface. These results highlight the high reversibility of carbohydrate-surface interactions, which could be an essential feature of recognition processes on the cell surface.

Cytoskeletal transition in patterned cells correlates with interfacial energy model.

A cell's morphology is intricately regulated by microenvironmental cues and intracellular feedback signals. Besides biochemical factors, cell fate can be influenced by the mechanics and geometry of the surrounding matrix. The latter point was addressed herein, by studying cell adhesion on two-dimensional micropatterns. Endothelial cells were grown on maleic acid copolymer surfaces structured with stripes of fibronectin by microcontact printing. Experiments showed a biphasic behaviour of actin stress fibre spacing in dependence on the stripe width with a critical size of approx. 15 µm. In a concurrent modelling effort, cells on stripes were simulated as droplet-like structures, including variations of interfacial energy, total volume and dimensions of the nucleus. A biphasic behaviour with regard to cell morphology and area was found, triggered by the minimum of interfacial energy, with the phase transition occurring at a critical stripe width close to the critical stripe width found in the cell experiment. The correlation of experiment and simulation suggests a possible mechanism of the cytoskeletal rearrangements based on interfacial energy arguments.

Differential Smad2/3 linker phosphorylation is a crosstalk mechanism of Rho/ROCK and canonical TGF-ß3 signaling in tenogenic differentiation.

The transforming growth factor (TGF)-ß3 is a well-known inducer for tenogenic differentiation, signaling via the Smad2/3 pathway. Furthermore, other factors like extracellular matrix or mechanical force can induce tenogenic differentiation and possibly alter the response to TGF-ß3 by signaling via the Rho/ROCK pathway. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-ß3/Smad signaling in tenogenic differentiation, with the Smad2/3 molecule hypothesized as a possible interface. Cultured as monolayers or on collagen I matrices, mesenchymal stromal cells (MSC) were treated with the ROCK inhibitor Y-27632 (10 µM), TGF-ß3 (10 ng/ml) or both combined. Control cells were cultured accordingly, without Y-27632 and/or without TGF-ß3. At different time points, MSC were analyzed by real-time RT-PCR, immunofluorescence, and Western blot. Cultivation of MSC on collagen matrices and ROCK inhibition supported tenogenic differentiation and fostered the effect of TGF-ß3. The phosphorylation of the linker region of Smad2 was reduced by cultivation on collagen matrices, but not by ROCK inhibition. The latter, however, led to increased phosphorylation of the linker region of Smad3. In conclusion, collagen matrices and the Rho/ROCK signaling pathway influence the TGF-ß3/Smad2/3 pathway by regulating different phosphorylation sites of the Smad linker region.

Biomaterial approaches for engineering and analyzing structure and metabolic states of microbial consortia within biofilms.

Microbial consortia within biofilms are frequently found in structured organization in nature and are thought to bear great potential for productive biotechnological applications, such as the degradation of complex substrates, biosensing, or the production of chemical compounds. However, in-depth understanding of their organizational principles, as well as comprehensive design criteria of structured microbial consortia for industrial applications are still limited. It is hypothesized that biomaterial engineering of such consortia within scaffolds can advance the field by providing defined in vitro mimics of naturally occurring and industrially applicable biofilms. Such systems will allow for adjustment of important microenvironmental parameters and in-depth analysis with high temporal and spatial resolution. In this review, we provide the background of biomaterial engineering of structured biofilm consortia, show approaches for their design, and demonstrate tools to analyze their metabolic state.

Correlation between Fibrin Fibrillation Kinetics and the Resulting Fibrin Network Microstructure.

Fibrin, the prominent extracellular matrix in early wound tissue, is discussed to influence immune cells and healing. The nature of fibrinogen/fibrin to form fibrillary networks is frequently exploited to engineer microenvironments for cellular analysis. This study focuses on revealing the correlation of fibril formation kinetic and the resulting network microstructure of engineered 3D fibrin networks. Different concentrations of fibrinogen (1-3 mg mL-1 ), thrombin (0.01-0.15 U mL-1 ), sodium chloride (40-120 mm), and calcium chloride (1-10 mm) are applied to assess the impact on the fibril growth kinetics by turbidity analysis and on the resulting fibril and pore diameter by laser scanning microscopy. The results highlight a direct influence of the sodium chloride concentration on fibrillation kinetics and reveal a strong correlation between fibrillation kinetics and network microstructure. With the assumption of a first-order growth kinetic, an increase of the growth constant k (0.015-0.04 min-1 ) is found to correlate to a decrease in fibril diameter (1-0.65 µm) and pore diameter (11-5 µm). The new findings enable an easy prediction of 3D fibrin network microstructure by the fibril formation kinetic and contribute to an improved engineering of defined scaffolds for tissue engineering and cell culture applications.

Phenotype Switching of Breast Cancer Cells upon Matrix Interface Crossing.

Tumor cell growth, invasion, and metastasis are dependent on the tumor microenvironment. Many studies emphasize a correlation between the material characteristics of the tumor extracellular matrix (ECM) and the invasive properties of tumor cells and even a trigger of tumor aggressiveness. Herein, we report that the previously observed trigger of migration characteristics of MDA-MB-231 breast cancer cells during transmigration across interfaces of two differently porous matrices is strongly correlated with a persistent change in cell invasiveness and aggressiveness. Using an in vitro 3D model of fibrillar collagen-I matrices, we found an increase in migration directionality, strongly elongated morphology, higher proliferation, and an increase in aggressive markers in the genetic profile after cells crossed the interface from dense to open porous matrix microstructure. Moreover, our results indicate strong nuclear deformation and increased DNA damage during transmigration of the matrix interface as a possible trigger of the more aggressive phenotype. These findings suggest that distinct tissue interfaces or altered ECM conditions with differences in microstructure may instruct or even reprogram tumor cells toward more aggressive phenotypes in vivo. The biomedical relevance of our results is corroborated by additional findings that the transmigrated cells exhibit an increased resistance against a common breast cancer therapeutic.

Development of a synthesis strategy for sulfamethoxazole derivatives and their coupling with hydrogel microparticles.

Sulfonamides were the first synthetic antibiotics broadly applied in veterinary and human medicine. Their increased use over the last few decades and limited technology to degrade them after entering the sewage system have led to their accumulation in the environment. A new hydrogel microparticle based biosensing application for sulfonamides is developed to overcome existing labour-intensive, and expensive detection methods to analyse and quantify their environmental distribution. This biosensing assay is based on the soft colloidal probe principle and requires microparticle functionalization strategies with target molecules. In this study, we developed a step-wise synthesis approach for sulfamethoxazole (SMX) derivatives in high yield, with SMX being one of the most ubiquitous sulfonamide antibiotics. After de novo synthesis of the SMX derivative, two coupling schemes to poly(ethylene glycol) (PEG) hydrogel microparticles bearing maleimide and thiol groups were investigated. In one approach, we coupled a cysteamine linker to a carboxyl group at the SMX derivative allowing for subsequent binding via the thiol-functionality to the maleimide groups of the microparticles in a mild, high-yielding thiol-ene "click" reaction. In a second approach, an additional 1,11-bis(maleimido)-3,6,9-trioxaundecane linker was coupled to the cysteamine to target the hydrolytically more stable thiol-groups of the microparticles. Successful PEG microparticle functionalization with the SMX derivatives was proven by IR spectroscopy and fluorescence microscopy. SMX-functionalized microparticles will be used in future applications for sulfonamide detection as well as for pull-down assays and screenings for new sulfomethoxazole binding targets.

Polymeric biomaterials for stem cell bioengineering.

This review covers the application of polymeric materials in stem cell bioengineering. Main emphasis is directed towards current material design concepts that mimic distinct exogenous signals of the stem cell microenvironment. Progress within the field of stem cell-specific biomaterials will be discussed, focusing on pluripotent, hematopoietic, mesenchymal and neural stem cells. The future role of biomaterials will be outlined with possible applications for cell reprogramming and engineering cancer cell microenvironments.

Microfluidics-assisted synthesis and functionalization of monodisperse colloidal hydrogel particles for optomechanical biosensors.

The soft colloidal probe (SCP) assay is a highly versatile sensing principle employing micrometer-sized hydrogel particles as optomechanical transducer elements. We report the synthesis, optimization, and conjugation of SCPs with defined narrow size distribution and specifically tailored mechanical properties and functionalities for integration into a microinterferometric optomechanical biosensor platform. Droplet-based microfluidics was used to crosslink polyethylene glycol (PEG) macromonomers by photocrosslinking and thiol-Michael addition. The effect of several synthesis parameters, i.e. PEG and radical initiator solid content, molecular weight and architecture of macromonomers, as well as UV exposure time and energy, were examined. SCPs were characterized with regard to the conversion of contained functional groups, morphology and mechanical properties by bright-field, confocal laser scanning and reflection interference contrast microscopy, as well as force spectroscopy. Functional groups were introduced during SCP synthesis and by several post-synthesis procedures, based on photoradical grafting and thiol-Michael addition. Preparation of SCPs by thiol-Michael addition and subsequent coupling of maleimide derivatives to unreacted thiols proved to be the superior strategy, while other approaches were associated with changes in the properties of the SCP. The newly developed SCPs were tested for their sensing capabilities employing the biotin-streptavidin-system. Biotin detection in the range of 10-7 to 10-10 M verified the concept of the synthesis strategy and the advantage of using monodisperse SCPs for easier and faster sensing applications of the SCP assay.

Friction-controlled traction force in cell adhesion.

The force balance between the extracellular microenvironment and the intracellular cytoskeleton controls the cell fate. We report a new (to our knowledge) mechanism of receptor force control in cell adhesion originating from friction between cell adhesion ligands and the supporting substrate. Adherent human endothelial cells have been studied experimentally on polymer substrates noncovalently coated with fluorescent-labeled fibronectin (FN). The cellular traction force correlated with the mobility of FN during cell-driven FN fibrillogenesis. The experimental findings have been explained within a mechanistic two-dimensional model of the load transfer at focal adhesion sites. Myosin motor activity in conjunction with sliding of FN ligands noncovalently coupled to the surface of the polymer substrates is shown to result in a controlled traction force of adherent cells. We conclude that the friction of adhesion ligands on the supporting substrate is important for mechanotransduction and cell development of adherent cells in vitro and in vivo.

Functional immobilization of signaling proteins enables control of stem cell fate.

The mode of ligand presentation has a fundamental role in organizing cell fate throughout development. We report a rapid and simple approach for immobilizing signaling ligands to maleic anhydride copolymer thin-film coatings, enabling stable signaling ligand presentation at interfaces at defined concentrations. We demonstrate the utility of this platform technology using leukemia inhibitory factor (LIF) and stem cell factor (SCF). Immobilized LIF supported mouse embryonic stem cell (mESC) pluripotency for at least 2 weeks in the absence of added diffusible LIF. Immobilized LIF activated signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK) signaling in a dose-dependent manner. The introduced method allows for the robust investigation of cell fate responses from interface-immobilized ligands.

Weakly coupled lipid bilayer membranes on multistimuli-responsive poly(N-isopropylacrylamide) copolymer cushions.

Polymer-cushioned lipid bilayers are frequently used to mimic the native environment of cellular membranes in respect to the extracellular matrix and intracellular structures. With the aim to actively tune lipid membrane characteristics, we pursue the approach to use temperature and pH responsive polymer thin films of poly(N-isopropylacrylamide-co-carboxyacrylamide) (PNIPAAm-co-carboxyAAM) as cushions for supported lipid bilayers. A cationic lipid bilayer composed of dioleoylphosphatidylcholine (DOPC) and dioleoyltrimethylammoniumpropane (DOTAP) (9:1) was formed on top of the polymer thin film in a drying/rehydration process. Fluorescence recovery after photobleaching (FRAP) yielded higher lipid diffusion coefficients (6.3-9.6 µm(2) s(-1)) on polymer cushions in comparison to solid glass supports (3.0-5.9 µm(2) s(-1)). No correlation of the lipid mobility was found with the swelling state of (PNIPAAm-co-carboxyAAM), which is ascribed to restrained interfacial electrostatic interactions and dispersion forces. The results revealed a minimal coupling of the lipid bilayer with the polymer cushions, and thus, bilayers supported by (PNIPAAm-co-carboxyAAM) provide interesting opportunities for unperturbed lipid diffusion combined with control of transmembrane protein mobility due to the impact of a tunable frictional drag.

Functional label-free assessment of fibroblast differentiation in 3D collagen-I-matrices using particle image velocimetry.

Fibroblasts are a diverse population of connective tissue cells that are a key component in physiological wound healing. Myofibroblasts are differentiated fibroblasts occurring in various physiological and pathological conditions, like in the healing of wounds or in the tumour microenvironment. They exhibit important functions compared to fibroblasts in terms of proliferation, protein secretion, and contractility. The gold standard to distinguish myofibroblasts is alpha-smooth muscle actin (αSMA) expression and its incorporation in stress fibres, which is only revealed by gene expression analysis and immunostaining. Here, we introduce an approach to functionally determine the myofibroblast status of live fibroblasts directly in in vitro cell culture by analysing their ability to contract the extracellular matrix around them without the need for labelling. It is based on particle image velocimetry algorithms applied to dynamic deformations of the extracellular matrix network structure imaged by phase contrast microscopy. Advanced image analysis allows us to distinguish between various differentiation stages of fibroblasts including the dynamic change over several days. We further apply machine learning classification to automatically evaluate different cell culture conditions. With this new method, we provide a versatile tool to functionally evaluate the dynamic process of fibroblast differentiation. It can be applied for in vitro screening studies in biomimetic 3D cell cultures with options to extend it to other cell systems with contractile phenotypes.