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Biomaterials are widely employed across diverse biomedical applications and represent an attractive strategy to explore how extracellular matrix components influence cellular response. In this study, the previously developed streptavidin platforms is aimed to use to investigate the role of glycosaminoglycans (GAGs) in bone morphogenetic protein 2 (BMP2) signaling. However, it is observed that the interpretation of findings is skewed due to the GAG-unrelated, non-specific binding of BMP2 on components of biomaterials. Non-specific adsorption of proteins is a recurrent and challenging issue for biomaterial studies. Despite the initial incorporation of anti-fouling polyethylene glycol (PEG) chains within biomaterials, the residual non-specific BMP2 adsorption still triggered BMP2 signaling within the same range as conditions of interest. The various options are explored to prevent BMP2 non-specific adsorption and a successful blocking condition involving a combination of bovine serum albumin and trehalose are identified. Furthermore, the effect of this blocking step improved when using gold platforms instead of glass, particularly with Chinese hamster ovary (CHO) cells. With this specific example, it is suggested that non-specific adsorption of BMPs on biomaterials may be a general concern - often undetected by classical surface-sensitive techniques - that needs to be addressed to better interpret cellular responses., (© 2024 The Author(s). Macromolecular Bioscience published by Wiley‐VCH GmbH.)

The role of glycosaminoglycans (GAGs) in modulating bone morphogenetic protein (BMP) signaling represents a recent and underexplored area. Conflicting reports suggest a dual effect: some indicate a positive influence, while others demonstrate a negative impact. This duality suggests that the localization of GAGs (either at the cell surface or within the extracellular matrix) or the specific type of GAG may dictate their signaling role. The precise sulfation patterns of heparan sulfate (HS) responsible for BMP2 binding remain elusive. BMP2 exhibits a preference for binding to HS over other GAGs. Using well-characterized biomaterials mimicking the extracellular matrix, our research reveals that HS promotes BMP2 signaling in the extracellular space, contrary to chondroitin sulfate (CS), which enhances BMP2 bioactivity at the cell surface. Further observations indicate that a central IdoA (2S)-GlcNS (6S) tri-sulfated motif within HS hexasaccharides enhances binding. Nevertheless, BMP2 exhibits a degree of adaptability to various HS sulfation types and sequences. Molecular dynamic simulations attribute this adaptability to the BMP2 N-terminal end flexibility. Our findings illustrate the complex interplay between GAGs and BMP signaling, highlighting the importance of localization and specific sulfation patterns. This understanding has implications for the development of biomaterials with tailored properties for therapeutic applications targeting BMP signaling pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: Bone morphogenetic proteins (BMPs) and transforming growth factors (TGF-β) are members of the TGF-β superfamily, known for their roles in several physiological and pathological processes. These factors are known to bind in vivo to BMP and TGF-β receptors, respectively, which induces the phosphorylation of Smad (pSmad) transcription factors. This pathway is generally studied with Western blot and luciferase bioluminescence assay, which presents some limitations., Purpose: In this work, we developed and optimized a high-throughput assay to study pSmad pathways using immunofluorescence (IF) as an alternative to Western blot. We aimed to overcome the technical challenges usually faced in the classical IF assay in image acquisition, analysis, and quantification., Methods: We used C2C12 cells as a cellular model. The cells were stimulated with BMP-2 and TGF-β1 that were delivered either in solution (soluble) or via a biomaterial presenting the growth factor (GF), that is in a "matrix-bound" manner. Image acquisition parameters, analysis methods, and quantification of pSmads using IF were optimized for cells cultured on two types of supports: on bare glass and on a biomimetic coating made by self-assembly of the biopolymers hyaluronic acid and poly(l-lysine), which was crosslinked and then loaded with the GFs., Results: We performed high-content kinetic studies of pSmad expression for cells cultured in 96-well microplates in response to soluble and matrix-bound BMP-2 and TGF-β1. The detection limit of the IF-based assay was found to be similar to Western blot. Additionally, we provide a proof-of-concept for drug testing using inhibitors of BMP and TGF-β receptors, under conditions where specific signaling pathways are engaged via the ligand/receptor interactions. Altogether, our findings offer perspectives for future mechanistic studies on cell signaling and for studies at the single cell level using imaging methods., (© 2024 The Author(s). Biotechnology Journal published by Wiley‐VCH GmbH.)

Bone morphogenetic proteins (BMPs) are important targets to incorporate in biomaterial scaffolds to orchestrate tissue repair. Glycosaminoglycans (GAGs) such as heparin allow the capture of BMPs and their retention at the surface of biomaterials at safe concentrations. Although heparin has strong affinities for BMP2 and BMP4, two important types of growth factors regulating bone and tissue repair, it remains difficult to embed stably at the surface of a broad range of biomaterials and degrades rapidly in vitro and in vivo. In this report, biomimetic poly(sulfopropyl methacrylate) (PSPMA) brushes are proposed as sulfated GAG mimetic interfaces for the stable capture of BMPs. The growth of PSPMA brushes via a surface-initiated activator regenerated by electron transfer polymerization is investigated via ellipsometry, prior to characterization of swelling and surface chemistry via X-ray photoelectron spectroscopy and Fourier transform infrared. The capacity of PSPMA brushes to bind BMP2 and BMP4 is then characterized via surface plasmon resonance. BMP2 is found to anchor particularly stably and at high density at the surface of PSPMA brushes, and a strong impact of the brush architecture on binding capacity is observed. These results are further confirmed using a quartz crystal microbalance with dissipation monitoring, providing some insights into the mode of adsorption of BMPs at the surface of PSPMA brushes. Primary adsorption of BMP2, with relatively little infiltration, is observed on thick dense brushes, implying that this growth factor should be accessible for further binding of corresponding cell membrane receptors. Finally, to demonstrate the impact of PSPMA brushes for BMP2 capture, dermal fibroblasts were then cultured at the surface of functionalized PSPMA brushes. The presence of BMP2 and the architecture of the brush are found to have a significant impact on matrix deposition at the corresponding interfaces. Therefore, PSPMA brushes emerge as attractive coatings for scaffold engineering and stable capture of BMP2 for regenerative medicine applications.

Glycosaminoglycans (GAGs) play a crucial role in tissue homeostasis by regulating the activity and diffusion of bioactive molecules. Incorporating GAGs into biomaterials has emerged as a widely adopted strategy in medical applications, owing to their biocompatibility and ability to control the release of bioactive molecules. Nevertheless, immobilized GAGs on biomaterials can elicit distinct cellular responses compared to their soluble forms, underscoring the need to understand the interactions between GAG and bioactive molecules within engineered functional biomaterials. By controlling critical parameters such as GAG type, density, and sulfation, it becomes possible to precisely delineate GAG functions within a biomaterial context and to better mimic specific tissue properties, enabling tailored design of GAG-based biomaterials for specific medical applications. However, this requires access to pure and well-characterized GAG compounds, which remains challenging. This review focuses on different strategies for producing well-defined GAGs and explores high-throughput approaches employed to investigate GAG-growth factor interactions and to quantify cellular responses on GAG-based biomaterials. These automated methods hold considerable promise for improving the understanding of the diverse functions of GAGs. In perspective, the scientific community is encouraged to adopt a rational approach in designing GAG-based biomaterials, taking into account the in vivo properties of the targeted tissue for medical applications., (© 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

The automation of liquid-handling routines offers great potential for fast, reproducible, and labor-reduced biomaterial fabrication but also requires the development of special protocols. Competitive systems demand for a high degree in miniaturization and parallelization while maintaining flexibility regarding the experimental design. Today, there are only a few possibilities for automated fabrication of biomaterials inside multiwell plates. We have previously demonstrated that streptavidin-based biomimetic platforms can be employed to study cellular behaviors on biomimetic surfaces. So far, these self-assembled materials were made by stepwise assembly of the components using manual pipetting. In this work, we introduce for the first time a fully automated and adaptable workflow to functionalize glass-bottom multiwell plates with customized biomimetic platforms deposited in single wells using a liquid-handling robot. We then characterize the cell response using automated image acquisition and subsequent analysis. Furthermore, the molecular surface density of the biomimetic platforms was characterized in situ using fluorescence-based image correlation spectroscopy. These measurements were in agreement with standard ex situ spectroscopic ellipsometry measurements. Due to automation, we could do a proof of concept to study the effect of heparan sulfate on the bioactivity of bone morphogenetic proteins on myoblast cells, using four different bone morphogenetic proteins (BMPs) (2, 4, 6, and 7) in parallel, at five increasing concentrations. Using such an automated self-assembly of biomimetic materials, it may be envisioned to further investigate the role of a large variety of extracellular matrix (ECM) components and growth factors on cell signaling.

We have established a self-calibrated method, called pbFFS for photobleaching fluctuation fluorescence spectroscopy, which aims to characterize molecules or particles labeled with an unknown distribution of fluorophores. Using photobleaching as a control parameter, pbFFS provides information on the distribution of fluorescent labels and a reliable estimation of the absolute density or concentration of these molecules. We present a complete theoretical derivation of the pbFFS approach and experimentally apply it to measure the surface density of a monolayer of fluorescently tagged streptavidin molecules, which can be used as a base platform for biomimetic systems. The surface density measured by pbFFS is consistent with the results of spectroscopic ellipsometry, a standard surface technique. However, pbFFS has two main advantages: it enables in situ characterization (no dedicated substrates are required) and can be applied to low masses of adsorbed molecules, which we demonstrate here by quantifying the density of biotin-Atto molecules that bind to the streptavidin layer. In addition to molecules immobilized on a surface, we also applied pbFFS to molecules diffusing in solution, to confirm the distribution of fluorescent labels found on a surface. Hence, pbFFS provides a set of tools for investigating the molecules labeled with a variable number of fluorophores, with the aim of quantifying either the number of molecules or the distribution of fluorescent labels, the latter case being especially relevant for oligomerization studies.

While a soft film itself is not able to induce cell spreading, BMP-2 presented via such soft film (so called "matrix-bound BMP-2") was previously shown to trigger cell spreading, migration and downstream BMP-2 signaling. Here, we used thin films of controlled stiffness presenting matrix-bound BMPs to study the effect of four BMP members (BMP-2, 4, 7, 9) on cell adhesion and differentiation of skeletal progenitors. We performed automated high-content screening of cellular responses, including cell number, cell spreading area, SMAD phosphorylation and alkaline phosphatase activity. We revealed that the cell response to bBMPs is BMP-type specific, and involved certain BMP receptors and beta chain integrins. In addition, this response is stiffness-dependent for several receptors. The basolateral presentation of the BMPs allowed us to discriminate the specificity of cellular response, especiallyd the role of type I and II BMP receptors and of β integrins in a BMP-type and stiffness-dependent manner. Notably, BMP-2 and BMP-4 were found to have distinct roles, while ALK5, previously known as a TGF-β receptor was revealed to be involved in the BMP-pathway., (Copyright © 2022. Published by Elsevier Ltd.)

Bone morphogenetic proteins (BMPs) are an important family of growth factors playing a role in a large number of physiological and pathological processes, including bone homeostasis, tissue regeneration, and cancers. In vivo, BMPs bind successively to both BMP receptors (BMPRs) of type I and type II, and a promiscuity has been reported. In this study, we used biolayer interferometry to perform parallel real-time biosensing and to deduce the kinetic parameters (k a , k d ) and the equilibrium constant (K D ) for a large range of BMP/BMPR combinations in similar experimental conditions. We selected four members of the BMP family (BMP-2, 4, 7, 9) known for their physiological relevance and studied their interactions with five type-I BMP receptors (ALK1, 2, 3, 5, 6) and three type-II BMP receptors (BMPR-II, ACTR-IIA, ACTR-IIB). We reveal that BMP-2 and BMP-4 behave differently, especially regarding their kinetic interactions and affinities with the type-II BMPR. We found that BMP-7 has a higher affinity for the type-II BMPR receptor ACTR-IIA and a tenfold lower affinity with the type-I receptors. While BMP-9 has a high and similar affinity for all type-II receptors, it can interact with ALK5 and ALK2, in addition to ALK1. Interestingly, we also found that all BMPs can interact with ALK5. The interaction between BMPs and both type-I and type-II receptors in a ternary complex did not reveal further cooperativity. Our work provides a synthetic view of the interactions of these BMPs with their receptors and paves the way for future studies on their cell-type and receptor specific signaling pathways.

The procedure commonly adopted to characterize cell materials using atomic force microscopy neglects the stress state induced in the cell by the adhesion structures that anchor it to the substrate. In several studies, the cell is considered as made from a single material and no specific information is provided regarding the mechanical properties of subcellular components. Here we present an optimization algorithm to determine separately the material properties of subcellular components of mesenchymal stem cells subjected to nanoindentation measurements. We assess how these properties change if the adhesion structures at the cell-substrate interface are considered or not in the algorithm. In particular, among the adhesion structures, the focal adhesions and the stress fibers were simulated. We found that neglecting the adhesion structures leads to underestimate the cell mechanical properties thus making errors up to 15%. This result leads us to conclude that the action of adhesion structures should be taken into account in nanoindentation measurements especially for cells that include a large number of adhesions to the substrate., (© 2021 IOP Publishing Ltd.)

It is nowadays well-accepted that the extracellular matrix (ECM) is not a simple reservoir for growth factors but is an organization center of their biological activity. In this review, we focus on the ability of the ECM to regulate the biological activity of BMPs. In particular, we survey the role of the ECM components, notably the glycosaminoglycans and fibrillary ECM proteins, which can be promoters or repressors of the biological activities mediated by the BMPs. We examine how a process called mechano-transduction induced by the ECM can affect BMP signaling, including BMP internalization by the cells. We also focus on the spatio-temporal regulation of the BMPs, including their release from the ECM, which enables to modulate their spatial localization as well as their local concentration. We highlight how biomaterials can recapitulate some aspects of the BMPs/ECM interactions and help to answer fundamental questions to reveal previously unknown molecular mechanisms. Finally, the design of new biomaterials inspired by the ECM to better present BMPs is discussed, and their use for a more efficient bone regeneration in vivo is also highlighted., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The chemical and physical properties of the extracellular matrix (ECM) are known to be fundamental for regulating growth factor bioactivity. The role of heparan sulfate (HS), a glycosaminoglycan, and of cell adhesion proteins (containing the cyclic RGD (cRGD) ligands) on bone morphogenetic protein 2 (BMP2)-mediated osteogenic differentiation has not been fully explored. In particular, it is not known whether and how their effects can be potentiated when they are presented in controlled close proximity, as in the ECM. Here, we developed streptavidin platforms to mimic selective aspects of the in vivo presentation of cRGD, HS and BMP2, with a nanoscale-control of their surface density and orientation to study cell adhesion and osteogenic differentiation. We showed that whereas a controlled increase in cRGD surface concentration upregulated BMP2 signaling due to β 3 integrin recruitment, silencing either β 1 or β 3 integrins negatively affected BMP2-mediated phosphorylation of SMAD1/5/9 and alkaline phosphatase expression. Furthermore, the presence of adsorbed BMP2 promoted cellular adhesion at very low cRGD concentrations. Finally, we proved that HS co-immobilized with cRGD both sustained BMP2 signaling and enhanced osteogenic differentiation compared to BMP2 directly immobilized on streptavidin, even with a low cRGD surface concentration. Altogether, our results show that HS facilitated and sustained the synergy between BMP2 and integrin pathways and that the co-immobilization of HS and cRGD peptides optimised BMP2-mediated osteogenic differentiation. Statement of significance The growth factor BMP2 is used to treat large bone defects. Previous studies have shown that the presentation of BMP2 via extracellular matrix molecules, such as heparan sulfate (HS), can upregulate BMP2 signaling. The potential advantages of dose reduction and local specificity have stimulated interest in further investigations into biomimetic approaches. We designed a streptavidin model surface eligible for immobilizing tunable amounts of molecules from the extracellular space, such as HS, adhesion motifs (cyclic RGD) and BMP2. By studying cellular adhesion, BMP2 bioactivity and its osteogenic potential we reveal the combined effect of integrins, HS and BMP2, which contribute in answering fundamental questions regarding cell-matrix interaction., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

The control over the adsorption or grafting of biomolecules from a liquid to a solid interface is of fundamental importance in different fields, such as drug delivery, pharmaceutics, diagnostics, and tissue engineering. It is thus important to understand and characterize how biomolecules interact with surfaces and to quantitatively measure parameters such as adsorbed amount, kinetics of adsorption and desorption, conformation of the adsorbed biomolecules, orientation, and aggregation state. A better understanding of these interfacial phenomena will help optimize the engineering of biofunctional surfaces, preserving the activity of biomolecules and avoiding unwanted side effects. The characterization of molecular adsorption on a solid surface requires the use of analytical techniques, which are able to detect very low quantities of material in a liquid environment without modifying the adsorption process during acquisition. In general, the combination of different techniques will give a more complete characterization of the layers adsorbed onto a substrate. In this review, the authors will introduce the context, then the different factors influencing the adsorption of biomolecules, as well as relevant parameters that characterize their adsorption. They review surface-sensitive techniques which are able to describe different properties of proteins and polymeric films on solid two-dimensional materials and compare these techniques in terms of sensitivity, penetration depth, ease of use, and ability to perform "parallel measurements."

Over the last decade, there has been a growing interest in the development of new materials to improve bone morphogenetic protein-2 (BMP-2) delivery for tissue regeneration. This study reports the development and application of model surfaces that present BMP-2 via heparan sulfate (HS), a ubiquitous component of the extracellular matrix (ECM). On these surfaces, HS is grafted by its reducing end, to mimic the natural arrangement of HS proteoglycans in the ECM. The binding of each component on these biomimetic surfaces is highly controlled, in terms of stoichiometry of molecules and BMP-2/grafted-HS affinity, as determined by surface-sensitive techniques. For comparison, this study also uses surfaces presenting immobilized BMP-2 alone. Functional validations of the surfaces are performed using a murine myoblast cell line (C2C12) and primary human mesenchymal stromal cells. In both cell types, HS-bound BMP-2 and surface-immobilized BMP-2 significantly prolong SMAD 1/5 phosphorylation, compared to BMP-2 added to the culture media. Moreover, HS-bound BMP-2 enhances p-SMAD 1/5 levels in C2C12 cells and reduces noggin antagonistic activity. Thus, grafted HS positively affects BMP-2 cellular activity. This innovative surface design, which mimics natural interactions of growth factors with ECM components, constitutes a promising candidate for future regenerative medicine applications., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The chemokine CXCL12α is a potent chemoattractant that guides the migration of muscle precursor cells (myoblasts) during myogenesis and muscle regeneration. To study how the molecular presentation of chemokines influences myoblast adhesion and motility, we designed multifunctional biomimetic surfaces as a tuneable signalling platform that enabled the response of myoblasts to selected extracellular cues to be studied in a well-defined environment. Using this platform, we demonstrate that CXCL12α, when presented by its natural extracellular matrix ligand heparan sulfate (HS), enables the adhesion and spreading of myoblasts and facilitates their active migration. In contrast, myoblasts also adhered and spread on CXCL12α that was quasi-irreversibly surface-bound in the absence of HS, but were essentially immotile. Moreover, co-presentation of the cyclic RGD peptide as integrin ligand along with HS-bound CXCL12α led to enhanced spreading and motility, in a way that indicates cooperation between CXCR4 (the CXCL12α receptor) and integrins (the RGD receptors). Our findings reveal the critical role of HS in CXCL12α induced myoblast adhesion and migration. The biomimetic surfaces developed here hold promise for mechanistic studies of cellular responses to different presentations of biomolecules. They may be broadly applicable for dissecting the signalling pathways underlying receptor cross-talks, and thus may guide the development of novel biomaterials that promote highly specific cellular responses., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Chemokines control the migration of cells in normal physiological processes and in the context of disease such as inflammation, autoimmunity and cancer. Two major interactions are involved: (i) binding of chemokines to chemokine receptors, which activates the cellular machinery required for movement; and (ii) binding of chemokines to glycosaminoglycans (GAGs), which facilitates the organization of chemokines into haptotactic gradients that direct cell movement. Chemokines can bind and activate their receptors as monomers; however, the ability to oligomerize is critical for the function of many chemokines in vivo Chemokine oligomerization is thought to enhance their affinity for GAGs, and here we show that it significantly affects the ability of chemokines to accumulate on and be retained by heparan sulfate (HS). We also demonstrate that several chemokines differentially rigidify and cross-link HS, thereby affecting HS rigidity and mobility, and that HS cross-linking is significantly enhanced by chemokine oligomerization. These findings suggest that chemokine-GAG interactions may play more diverse biological roles than the traditional paradigms of physical immobilization and establishment of chemokine gradients; we hypothesize that they may promote receptor-independent events such as physical re-organization of the endothelial glycocalyx and extracellular matrix, as well as signalling through proteoglycans to facilitate leukocyte adhesion and transmigration., (© 2017 The Authors.)

Bone morphogenetic protein 2 (BMP-2) has been known for decades as a strong osteoinductive factor and for clinical applications is combined solely with collagen as carrier material. The growing concerns regarding side effects and the importance of BMP-2 in several developmental and physiological processes have raised the need to improve the design of materials by controlling BMP-2 presentation. Inspired by the natural cell environment, new material surfaces have been engineered and tailored to provide both physical and chemical cues that regulate BMP-2 activity. Here we describe surfaces designed to present BMP-2 to cells in a spatially and temporally controlled manner. This is achieved by trapping BMP-2 using physicochemical interactions, either covalently grafted or combined with other extracellular matrix components. In the near future, we anticipate that material science and biology will integrate and further develop tools for in vitro studies and potentially bring some of them toward in vivo applications., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The glycosaminoglycan heparan sulfate (HS), present at the surface of most cells and ubiquitous in extracellular matrix, binds many soluble extracellular signalling molecules such as chemokines and growth factors, and regulates their transport and effector functions. It is, however, unknown whether upon binding HS these proteins can affect the long-range structure of HS. To test this idea, we interrogated a supramolecular model system, in which HS chains grafted to streptavidin-functionalized oligoethylene glycol monolayers or supported lipid bilayers mimic the HS-rich pericellular or extracellular matrix, with the biophysical techniques quartz crystal microbalance (QCM-D) and fluorescence recovery after photobleaching (FRAP). We were able to control and characterize the supramolecular presentation of HS chains--their local density, orientation, conformation and lateral mobility--and their interaction with proteins. The chemokine CXCL12α (or SDF-1α) rigidified the HS film, and this effect was due to protein-mediated cross-linking of HS chains. Complementary measurements with CXCL12α mutants and the CXCL12γ isoform provided insight into the molecular mechanism underlying cross-linking. Fibroblast growth factor 2 (FGF-2), which has three HS binding sites, was also found to cross-link HS, but FGF-9, which has just one binding site, did not. Based on these data, we propose that the ability to cross-link HS is a generic feature of many cytokines and growth factors, which depends on the architecture of their HS binding sites. The ability to change matrix organization and physico-chemical properties (e.g. permeability and rigidification) implies that the functions of cytokines and growth factors may not simply be confined to the activation of cognate cellular receptors., (© 2015 The Authors.)

We demonstrate the quartz crystal microbalance as a novel method to quantify the reaction yields and stability of the terminal conjugation of chemically complex molecules. Oxime ligation is identified as a facile, broadly applicable method for the reducing-end conjugation of glycosaminoglycans that overcomes the limited stability and yield of popular hydrazone ligation.

Active cell migration and invasion is a peculiar feature of glioma that makes this tumor able to rapidly infiltrate into the surrounding brain tissue. In our recent work, we identified a novel class of glioma-associated-stem cells (defined as GASC for high-grade glioma--HG--and Gasc for low-grade glioma--LG) that, although not tumorigenic, act supporting the biological aggressiveness of glioma-initiating stem cells (defined as GSC for HG and Gsc for LG) favoring also their motility. Migrating cancer cells undergo considerable molecular and cellular changes by remodeling their cytoskeleton and cell interactions with surrounding environment. To get a better understanding about the role of the glioma-associated-stem cells in tumor progression, cell deformability and interactions between glioma-initiating stem cells and glioma-associated-stem cells were investigated. Adhesion of HG/LG-cancer cells on HG/LG-glioma-associated stem cells was studied by time-lapse microscopy, while cell deformability and cell-cell adhesion strengths were quantified by indentation measurements by atomic force microscopy and single cell force spectroscopy. Our results demonstrate that for both HG and LG glioma, cancer-initiating-stem cells are softer than glioma-associated-stem cells, in agreement with their neoplastic features. The adhesion strength of GSC on GASC appears to be significantly lower than that observed for Gsc on Gasc. Whereas, GSC spread and firmly adhere on Gasc with an adhesion strength increased as compared to that obtained on GASC. These findings highlight that the grade of glioma-associated-stem cells plays an important role in modulating cancer cell adhesion, which could affect glioma cell migration, invasion and thus cancer aggressiveness. Moreover this work provides evidence about the importance of investigating cell adhesion and elasticity for new developments in disease diagnostics and therapeutics.

Glycosaminoglycans (GAGs) are ubiquitously present at the cell surface and in extracellular matrix, and crucial for matrix assembly, cell-cell and cell-matrix interactions. The supramolecular presentation of GAG chains, along with other matrix components, is likely to be functionally important but remains challenging to control and to characterize, both in vivo and in vitro. We present a method to create well-defined biomimetic surfaces that display GAGs, either alone or together with other cell ligands, in a background that suppresses non-specific binding. Through the design of the immobilization platform - a streptavidin monolayer serves as a molecular breadboard for the attachment of various biotinylated ligands - and a set of surface-sensitive in situ analysis techniques (including quartz crystal microbalance and spectroscopic ellipsometry), the biomimetic surfaces are tailor made with tight control on biomolecular orientation, surface density and lateral mobility. Analysing the interactions between a selected GAG (heparan sulphate, HS) and the HS-binding chemokine CXCL12α (also called SDF-1α), we demonstrate that these surfaces are versatile for biomolecular and cellular interaction studies. T-lymphocytes are found to adhere specifically to surfaces presenting CXCL12α, both when reversibly bound through HS and when irreversibly immobilized on the inert surface, even in the absence of any bona fide cell adhesion ligand. Moreover, surfaces which present both HS-bound CXCL12α and the intercellular adhesion molecule 1 (ICAM-1) synergistically promote cell adhesion. Our surface biofunctionalization strategy should be broadly applicable for functional studies that require a well-defined supramolecular presentation of GAGs along with other matrix or cell-surface components., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Guidance molecules, such as Sema3A or Netrin-1, can induce growth cone (GC) repulsion or attraction in the presence of a flat surface, but very little is known of the action of guidance molecules in the presence of obstacles. Therefore we combined chemical and mechanical cues by applying a steady Netrin-1 stream to the GCs of dissociated hippocampal neurons plated on polydimethylsiloxane (PDMS) surfaces patterned with lines 2 µm wide, with 4 µm period and with a height varying from 100 to 600 nm. GC turning experiments performed 24 hours after plating showed that filopodia crawl over these lines within minutes. These filopodia do not show staining for the adhesion marker Paxillin. GCs and neurites crawl over lines 100 nm high, but less frequently and on a longer time scale over lines higher than 300 nm; neurites never crawl over lines 600 nm high. When neurons are grown for 3 days over patterned surfaces, also neurites can cross lines 300 nm and 600 nm high, grow parallel to and on top of these lines and express Paxillin. Axons - selectively stained with SMI 312 - do not differ from dendrites in their ability to cross these lines. Our results show that highly motile structures such as filopodia climb over high obstacle in response to chemical cues, but larger neuronal structures are less prompt and require hours or days to climb similar obstacles.

The ability to control the differentiation of stem cells into specific neuronal types has a tremendous potential for the treatment of neurodegenerative diseases. In vitro neuronal differentiation can be guided by the interplay of biochemical and biophysical cues. Different strategies to increase the differentiation yield have been proposed, focusing everything on substrate topography, or, alternatively on substrate stiffness. Both strategies demonstrated an improvement of the cellular response. However it was often impossible to separate the topographical and the mechanical contributions. Here we investigate the role of the mechanical properties of nanostructured substrates, aiming at understanding the ultimate parameters which govern the stem cell differentiation. To this purpose a set of different substrates with controlled stiffness and with or without nanopatterning are used for stem cell differentiation. Our results show that the neuronal differentiation yield depends mainly on the substrate mechanical properties while the geometry plays a minor role. In particular nanostructured and flat polydimethylsiloxane (PDMS) substrates with comparable stiffness show the same neuronal yield. The improvement in the differentiation yield obtained through surface nanopatterning in the submicrometer scale could be explained as a consequence of a substrate softening effect. Finally we investigate by single cell force spectroscopy the neuronal precursor adhesion on the substrate immediately after seeding, as a possible critical step governing the neuronal differentiation efficiency. We observed that neuronal precursor adhesion depends on substrate stiffness but not on surface structure, and in particular it is higher on softer substrates. Our results suggest that cell-substrate adhesion forces and mechanical response are the key parameters to be considered for substrate design in neuronal regenerative medicine., (Copyright © 2013 Wiley Periodicals, Inc.)

We used optical tweezers to analyze the effect of jasplakinolide and cyclodextrin on the force exerted by lamellipodia from developing growth cones (GCs) of isolated dorsal root ganglia (DRG) neurons. We found that 25 nM of jasplakinolide, which is known to inhibit actin filament turnover, reduced both the maximal exerted force and maximal velocity during lamellipodia leading-edge protrusion. By using atomic force microscopy, we verified that cyclodextrin, which is known to remove cholesterol from membranes, decreased the membrane stiffness of DRG neurons. Lamellipodia treated with 2.5 mM of cyclodextrin exerted a larger force, and their leading edge could advance with a higher velocity. Neither jasplakinolide nor cyclodextrin affected force or velocity during lamellipodia retraction. The amplitude and frequency of elementary jumps underlying force generation were reduced by jasplakinolide but not by cyclodextrin. The action of both drugs at the used concentration was fully reversible. These results support the notion that membrane stiffness provides a selective pressure that shapes force generation, and confirm the pivotal role of actin turnover during protrusion., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Embryonic stem (ES) cell differentiation in specific cell lineages is a major issue in cell biology particularly in regenerative medicine. Differentiation is usually achieved by using biochemical factors and it is not clear whether mechanical properties of the substrate over which cells are grown can affect proliferation and differentiation. Therefore, we produced patterns in polydimethylsiloxane (PDMS) consisting of groove and pillar arrays of sub-micrometric lateral resolution as substrates for cell cultures. We analyzed the effect of different nanostructures on differentiation of ES-derived neuronal precursors into neuronal lineage without adding biochemical factors. Neuronal precursors adhered on PDMS more effectively than on glass coverslips. We demonstrated that neuronal yield was enhanced by increasing pillars height from 35 to 400 nm. On higher pillar neuronal differentiation reaches ∼80% 96 h after plating and the largest differentiation enhancement of pillars over flat PDMS was observed during the first 6 h of culture. We conclude that PDMS nanopillars accelerate and increase neuronal differentiation., (Copyright © 2011 Wiley Periodicals, Inc.)

During early development of the central nervous system, there is an excessive outgrowth of neuronal projections, which later need to be refined to achieve precise connectivity. Axon pruning and degeneration are strategies used to remove exuberant neurites and connections in the immature nervous system to ensure the proper formation of functional circuitry. To observe morphological changes and physical mechanisms underlying this process, early differentiating embryonic stem cell-derived neurons were used combining video imaging of live growth cones (GCs) with confocal laser scanning microscopy and atomic force microscopy, both on fixed and living neurons. Using this method, we could highlight the presence of submicrometric fragments in still and in some of the retracting GCs. The observed fragmentation is not an artifact of atomic force microscopy scanning or fixation, or the result of apoptosis. Therefore, the morphology of GCs depends on their overall motility, and fragmentation seems to be the fate of GCs that have not found a correct destination.

We have analyzed the morphology of growth cones of differentiating neurons from rat dorsal root ganglia (DRG) with conventional Laser Scanning Confocal Microscopy (LSCM) and Atomic Force Microscopy (AFM). Images of immunofluorescent DRG growth cones colabeled for actin and tubulin were superimposed to images obtained with AFM at different scanning forces. In order to reduce changes of the image surface caused by the pressure of the AFM tip, we have developed a procedure to obtain 0pN AFM images. Further analysis of these images revealed topographical structures with nanoscale dimensions, referred to as "invaginations" or "holes". These holes had an area varying from 0.01 to 3.5 microm(2) with a depth varying from 2 to 178 nm. Comparative analysis with LSCM images showed that these holes correspond to regions where staining of both actin and tubulin was negligible. Filopodia height varied from 40 to 270 nm and their diameter from 113 to 887 nm. These results show that the combination of LSCM and AFM reveal structural details with a nanoscale dimension of DRG growth cones, difficult to resolve with conventional microscopy.

Atomic force microscopy (AFM) provides the possibility to map the 3D structure of viewed objects with a nanometric resolution, which cannot be achieved with other imaging methods such as conventional video imaging and confocal fluorescent microscopy. Video imaging with CCD cameras can provide an analysis of biological events with a temporal and spatial resolution not possible with AFM, while confocal imaging allows the simultaneous acquisition of immunofluorescence images. In this communication we present a simple method to combine AFM and confocal images to study differentiating embryonic stem (ES) cells-derived and dorsal root ganglia (DRG) neurons in culture. Neurons were grown on coverslips with micrometric markers that allow finding and imaging the same neuron with different microscopes. AFM and confocal images were registered using conventional methods used in Computer Science. The combination of these two techniques allows relating functional properties to morphological features of imaged neurons.