Force propagation between epithelial cells depends on active coupling and mechano-structural polarization.

Cell-generated forces play a major role in coordinating the large-scale behavior of cell assemblies, in particular during development, wound healing, and cancer. Mechanical signals propagate faster than biochemical signals, but can have similar effects, especially in epithelial tissues with strong cell-cell adhesion. However, a quantitative description of the transmission chain from force generation in a sender cell, force propagation across cell-cell boundaries, and the concomitant response of receiver cells is missing. For a quantitative analysis of this important situation, here we propose a minimal model system of two epithelial cells on an H-pattern ('cell doublet'). After optogenetically activating RhoA, a major regulator of cell contractility, in the sender cell, we measure the mechanical response of the receiver cell by traction force and monolayer stress microscopies. In general, we find that the receiver cells show an active response so that the cell doublet forms a coherent unit. However, force propagation and response of the receiver cell also strongly depend on the mechano-structural polarization in the cell assembly, which is controlled by cell-matrix adhesion to the adhesive micropattern. We find that the response of the receiver cell is stronger when the mechano-structural polarization axis is oriented perpendicular to the direction of force propagation, reminiscent of the Poisson effect in passive materials. We finally show that the same effects are at work in small tissues. Our work demonstrates that cellular organization and active mechanical response of a tissue are key to maintain signal strength and lead to the emergence of elasticity, which means that signals are not dissipated like in a viscous system, but can propagate over large distances.

Light-driven biological actuators to probe the rheology of 3D microtissues.

The mechanical properties of biological tissues are key to their physical integrity and function. Although external loading or biochemical treatments allow the estimation of these properties globally, it remains difficult to assess how such external stimuli compare with cell-generated contractions. Here we engineer microtissues composed of optogenetically-modified fibroblasts encapsulated within collagen. Using light to control the activity of RhoA, a major regulator of cellular contractility, we induce local contractions within microtissues, while monitoring microtissue stress and strain. We investigate the regulation of these local contractions and their spatio-temporal distribution. We demonstrate the potential of our technique for quantifying tissue elasticity and strain propagation, before examining the possibility of using light to create and map local anisotropies in mechanically heterogeneous microtissues. Altogether, our results open an avenue to guide the formation of tissues while non-destructively charting their rheology in real time, using their own constituting cells as internal actuators.

Dynamic Functional Connectivity between order and randomness and its evolution across the human adult lifespan.

Functional Connectivity (FC) during resting-state or task conditions is not static but inherently dynamic. Yet, there is no consensus on whether fluctuations in FC may resemble isolated transitions between discrete FC states rather than continuous changes. This quarrel hampers advancing the study of dynamic FC. This is unfortunate as the structure of fluctuations in FC can certainly provide more information about developmental changes, aging, and progression of pathologies. We merge the two perspectives and consider dynamic FC as an ongoing network reconfiguration, including a stochastic exploration of the space of possible steady FC states. The statistical properties of this random walk deviate both from a purely "order-driven" dynamics, in which the mean FC is preserved, and from a purely "randomness-driven" scenario, in which fluctuations of FC remain uncorrelated over time. Instead, dynamic FC has a complex structure endowed with long-range sequential correlations that give rise to transient slowing and acceleration epochs in the continuous flow of reconfiguration. Our analysis for fMRI data in healthy elderly revealed that dynamic FC tends to slow down and becomes less complex as well as more random with increasing age. These effects appear to be strongly associated with age-related changes in behavioural and cognitive performance.

Confinement-Induced Transition between Wavelike Collective Cell Migration Modes.

The structural and functional organization of biological tissues relies on the intricate interplay between chemical and mechanical signaling. Whereas the role of constant and transient mechanical perturbations is generally accepted, several studies recently highlighted the existence of long-range mechanical excitations (i.e., waves) at the supracellular level. Here, we confine epithelial cell monolayers to quasi-one-dimensional geometries, to force the establishment of tissue-level waves of well-defined wavelength and period. Numerical simulations based on a self-propelled Voronoi model reproduce the observed waves and exhibit a phase transition between a global and a multinodal wave, controlled by the confinement size. We confirm experimentally the existence of such a phase transition, and show that wavelength and period are independent of the confinement length. Together, these results demonstrate the intrinsic origin of tissue oscillations, which could provide cells with a mechanism to accurately measure distances at the supracellular level.

On the spatiotemporal regulation of cell tensional state.

Since the emergence of mechanobiology, mechanical signals have been shown to influence almost every process in biology. Cells transduce mechanical signals into biochemical signaling pathways, adjust their behavior and/or phenotype before transmitting these signals to neighboring cells. Mechanical signals thus appear as information, which can be "written" by cells in the surrounding extracellular matrix, "transmitted" through it and "read" by other cells. This brief review summarizes our current understanding of the mechanisms regulating the tensional state of cells and tissues subjected to mechanical perturbations, prior to examining existing or potential experimental approaches to study these mechanisms.

Magneto-active substrates for local mechanical stimulation of living cells.

Cells are able to sense and react to their physical environment by translating a mechanical cue into an intracellular biochemical signal that triggers biological and mechanical responses. This process, called mechanotransduction, controls essential cellular functions such as proliferation and migration. The cellular response to an external mechanical stimulation has been investigated with various static and dynamic systems, so far limited to global deformations or to local stimulation through discrete substrates. To apply local and dynamic mechanical constraints at the single cell scale through a continuous surface, we have developed and modelled magneto-active substrates made of magnetic micro-pillars embedded in an elastomer. Constrained and unconstrained substrates are analysed to map surface stress resulting from the magnetic actuation of the micro-pillars and the adherent cells. These substrates have a rigidity in the range of cell matrices, and the magnetic micro-pillars generate local forces in the range of cellular forces, both in traction and compression. As an application, we followed the protrusive activity of cells subjected to dynamic stimulations. Our magneto-active substrates thus represent a new tool to study mechanotransduction in single cells, and complement existing techniques by exerting a local and dynamic stimulation, traction and compression, through a continuous soft substrate.

Quiescence of human muscle stem cells is favored by culture on natural biopolymeric films.

BACKGROUND: Satellite cells are quiescent resident muscle stem cells that present an important potential to regenerate damaged tissue. However, this potential is diminished once they are removed from their niche environment in vivo, prohibiting the long-term study and genetic investigation of these cells. This study therefore aimed to provide a novel biomaterial platform for the in-vitro culture of human satellite cells that maintains their stem-like quiescent state, an important step for cell therapeutic studies. METHODS: Human muscle satellite cells were isolated from two donors and cultured on soft biopolymeric films of controlled stiffness. Cell adhesive phenotype, maintenance of satellite cell quiescence and capacity for gene manipulation were investigated using FACS, western blotting, fluorescence microscopy and electron microscopy. RESULTS: About 85% of satellite cells cultured in vitro on soft biopolymer films for 3 days maintained expression of the quiescence marker Pax7, as compared with 60% on stiffer films and 50% on tissue culture plastic. The soft biopolymeric films allowed satellite cell culture for up to 6 days without renewing the media. These cells retained their stem-like properties, as evidenced by the expression of stem cell markers and reduced expression of differentiated markers. In addition, 95% of cells grown on these soft biopolymeric films were in the G0/G1 stage of the cell cycle, as opposed to those grown on plastic that became activated and began to proliferate and differentiate. CONCLUSIONS: Our study identifies a new biomaterial made of a biopolymer thin film for the maintenance of the quiescence state of muscle satellite cells. These cells could be activated at any point simply by replating them onto a plastic culture dish. Furthermore, these cells could be genetically manipulated by viral transduction, showing that this biomaterial may be further used for therapeutic strategies.

Signal mingle: Micropatterns of BMP-2 and fibronectin on soft biopolymeric films regulate myoblast shape and SMAD signaling.

In vivo, bone morphogenetic protein 2 (BMP-2) exists both in solution and bound to the extracellular matrix (ECM). While these two modes of presentation are known to influence cell behavior distinctly, their role in the niche microenvironment and their functional relevance in the genesis of a biological response has sparsely been investigated at a cellular level. Here we used the natural affinity of BMP-2 for fibronectin (FN) to engineer cell-sized micropatterns of BMP-2. This technique allowed the simultaneous control of the spatial presentation of fibronectin-bound BMP-2 and cell spreading. These micropatterns induced a specific actin and adhesion organization around the nucleus, and triggered the phosphorylation and nuclear translocation of SMAD1/5/8 in C2C12 myoblasts and mesenchymal stem cells, an early indicator of their osteoblastic trans-differentiation. We found that cell spreading itself potentiated a BMP-2-dependent phosphorylation of SMAD1/5/8. Finally, we demonstrated that FN/BMP-2-mediated early SMAD signaling depended on LIM kinase 2 and ROCK, rather than myosin II activation. Altogether, our results show that FN/BMP-2 micropatterns are a useful tool to study the mechanisms underlying BMP-2-mediated mechanotransduction. More broadly, our approach could be adapted to other combinations of ECM proteins and growth factors, opening an exciting avenue to recreate tissue-specific niches in vitro.

Amyloid-like aggregates formation by blood plasma fibronectin.

Fibronectin (FN) is a multifunctional glycoprotein of the extracellular matrix (ECM) playing critical roles in physiological and pathological cell processes like adhesion, migration, growth, and differentiation. These various functions of FN are modulated by its supramolecular state. Indeed, FN can polymerize into different types of assemblies like fibrils and aggregates. However, the mechanism of polymerization and the effects of such assemblies on cell behaviors still remain to be elucidated. Here we show that upon irreversible thermal denaturation, human blood plasma fibronectin forms high molecular weight aggregates. These compact and globular aggregates show amyloid features: they are stabilized by intermolecular b-sheets, they bind Thioflavin T and they are resistant to reducing and denaturing agents. Their characterization by electrospray ionization charge detection mass spectrometry shows that two populations can be distinguished according to the mass and charge density. Despite their amyloid features and the presence of hydrophobic patches on their surface, these aggregates are not toxic for cells. However, their binding abilities to gelatin and RGD are drastically decreased compare to native FN, suggesting possible effects on ECM-cell interactions.

Multiscale Porosity Directs Bone Regeneration in Biphasic Calcium Phosphate Scaffolds.

Large and load-bearing bone defects are challenging to treat and cause pain and disfigurement. The design of efficacious bone scaffolds for the repair of such defects involves a range of length scales from the centimeter down to the micrometer-scale. Here, we assess the influence on bone regeneration of scaffold rod spacing (>300 µm) and microporosity (<50 µm), as well as the combination of different structures and materials in the same scaffold, i.e., at the millimeter scale. We use four single-domain scaffolds, microporous (MP) or nonmicroporous (NMP) and with either a "small" or "large" rod spacing. Multidomain scaffolds combine four regions corresponding to the macro- and microarchitectures of the single-domain scaffolds. The scaffolds are implanted in pig mandibles for 3 weeks and bone regeneration is assessed by measuring the average bone volume fraction, BVF̅, the bone distribution and the trabecular thickness from micro-CT data. For the single-domain scaffolds, BVF̅ was 45 ± 3% for MP-small, 39 ± 2% for MP-large, 25 ± 2% for NMP-small, and 25 ± 2% for NMP-large. MP scaffolds have significantly higher BVF̅ and a more uniform bone distribution compared to NMP, regardless of rod spacing. The average trabecular thickness is significantly larger in MP compared to NMP, and in "large" compared to "small" scaffolds. Microporosity affects trabecular thickness throughout the scaffold, while rod spacing affects it only at the scaffold periphery. In multidomain scaffolds, MP-large and NMP-large domains have similar BVF̅ as compared to their respective single-domain counterparts. These results suggest that combining different architectures into one scaffold conserves the properties of each domain. Hence, bone growth and morphology can be tailored by controlling scaffold architecture from the millimeter down to the micrometer level. This will allow the customization of scaffold designs for the treatment of large and load-bearing bone defects.

Beyond mice: Emerging and transdisciplinary models for the study of early-onset myopathies.

The use of the adapted models to decipher patho-physiological mechanisms of human diseases is always a great challenge. This is of particular importance for early-onset myopathies, in which pathological mutations often impact not only on muscle structure and function but also on developmental processes. Mice are currently the main animal model used to study neuromuscular disorders including the early-onset myopathies. However strategies based on simple animal models and on transdisciplinary approaches exploring mechanical muscle cell properties emerge as attractive, non-exclusive alternatives. These new ways provide valuable opportunities to improve our knowledge on how mechanical, biochemical, and genetic/epigenetic cues modulate the formation, organization and function of muscle tissues. Here we provide an overview of how single cell and micro-tissue engineering in parallel to non-mammalian, Drosophila and zebrafish models could contribute to filling gaps in our understanding of pathogenic mechanisms underlying early-onset myopathies. We also discuss their potential impact on designing new diagnostic and therapeutic strategies.

Stiffness-dependent cellular internalization of matrix-bound BMP-2 and its relation to Smad and non-Smad signaling.

Surface coatings delivering BMP are a promising approach to render biomaterials osteoinductive. In contrast to soluble BMPs which can interact with their receptors at the dorsal side of the cell, BMPs presented as an insoluble cue physically bound to a biomimetic matrix, called here matrix-bound (bBMP-2), are presented to cells by their ventral side. To date, BMP-2 internalization and signaling studies in cell biology have always been performed by adding soluble (sBMP-2) to cells adhered on cell culture plates or glass slides, which will be considered here as a "reference" condition. However, whether and how matrix-bound BMP-2 can be internalized by cells and its relation to canonical (SMAD) and non-canonical signaling (ALP) remain open questions. In this study, we investigated the uptake and processing of BMP-2 by C2C12 myoblasts. This BMP-2 was presented either embedded in polyelectrolyte multilayer films (matrix-bound presentation) or as soluble form. Using fluorescently labeled BMP-2, we showed that the amount of matrix-bound BMP-2 internalized is dependent on the level of crosslinking of the polyelectrolyte films. Cav-1-mediated internalization is related to both SMAD and ALP signaling, while clathrin-mediated is only related to ALP signaling. BMP-2 internalization was independent of the presentation mode (sBMP-2 versus bBMP-2) for low crosslinked films (soft, EDC10) in striking contrast with high crosslinked (stiff, EDC70) films where internalization was much lower and slower for bBMP-2. As anticipated, internalization of sBMP-2 barely depended on the underlying matrix. Taken together, these results indicate that BMP-2 internalization can be tuned by the underlying matrix and activates downstream BMP-2 signaling, which is key for the effective formation of bone tissue. STATEMENT OF SIGNIFICANCE: The presentation of growth factors from material surfaces currently presents significant challenges in academic research, clinics and industry. Being able to deliver efficiently these growth factors by a biomaterial will open new perspectives for regenerative medicine. However, to date, very little is known about how matrix-bound growth factors are delivered to cells, especially whether they are internalized and how they are signaling to drive key differentiation events. These initial steps are crucial as they will guide the subsequent processes leading to tissue regeneration. In this work, we investigate the uptake and processing by cells of BMP-2 ligands embedded in polyelectrolyte multilayer films in comparison to soluble BMP-2. We show that BMP-2 responsive cells can internalize matrix-bound BMP-2 and that internalization is dependent on the cross-linking level of the polyelectrolyte films. In addition, we show that internalization is mediated by both clathrin- and caveolin-dependent pathways. While inhibiting clathrin-dependent endocytosis affects only non-canonical signaling, blocking caveolin-1-dependent endocytosis reduces both canonical and non-canonical BMP signaling. The signaling pathways found for matrix-bound BMP-2 are similar to those found for soluble BMP-2. These results highlight that BMP-2 presented by a biomaterial at the ventral side of the cell can trigger major endocytic and associated signaling pathways leading to bone regeneration.

Micropore-induced capillarity enhances bone distribution in vivo in biphasic calcium phosphate scaffolds.

UNLABELLED: The increasing demand for bone repair solutions calls for the development of efficacious bone scaffolds. Biphasic calcium phosphate (BCP) scaffolds with both macropores and micropores (MP) have improved healing compared to those with macropores and no micropores (NMP), but the role of micropores is unclear. Here, we evaluate capillarity induced by micropores as a mechanism that can affect bone growth in vivo. Three groups of cylindrical scaffolds were implanted in pig mandibles for three weeks: MP were implanted either dry (MP-Dry), or after submersion in phosphate buffered saline, which fills pores with fluid and therefore suppresses micropore-induced capillarity (MP-Wet); NMP were implanted dry. The amount and distribution of bone in the scaffolds were quantified using micro-computed tomography. MP-Dry had a more homogeneous bone distribution than MP-Wet, although the average bone volume fraction, BVF‾, was not significantly different for these two groups (0.45±0.03 and 0.37±0.03, respectively). There was no significant difference in the radial bone distribution of NMP and MP-Wet, but the BVF‾, of NMP was significantly lower among the three groups (0.25±0.02). These results suggest that micropore-induced capillarity enhances bone regeneration by improving the homogeneity of bone distribution in BCP scaffolds. The explicit design and use of capillarity in bone scaffolds may lead to more effective treatments of large and complex bone defects. STATEMENT OF SIGNIFICANCE: The increasing demand for bone repair calls for more efficacious bone scaffolds and calcium phosphate-based materials are considered suitable for this application. Macropores (>100µm) are necessary for bone ingrowth and vascularization. However, studies have shown that microporosity (<20µm) also enhances growth, but there is no consensus on the controlling mechanisms. In previous in vitro work, we suggested that micropore-induced capillarity had the potential to enhance bone growth in vivo. This work illustrates the positive effects of capillarity on bone regeneration in vivo; it demonstrates that micropore-induced capillarity significantly enhances the bone distribution in the scaffold. The results will impact the design of scaffolds to better exploit capillarity and improve treatments for large and load-bearing bone defects.

Substrate Stiffness Combined with Hepatocyte Growth Factor Modulates Endothelial Cell Behavior.

Endothelial cells (ECs) play a crucial role in regulating various physiological and pathological processes. The behavior of ECs is modulated by physical (e.g., substrate stiffness) and biochemical cues (e.g., growth factors). However, the synergistic influence of these cues on EC behavior has rarely been investigated. In this study, we constructed poly(l-lysine)/hyaluronan (PLL/HA) multilayer films with different stiffness and exposed ECs to these substrates with and without hepatocyte growth factor (HGF)-supplemented culture medium. We demonstrated that EC adhesion, migration, and proliferation were positively correlated with substrate stiffness and that these behaviors were further promoted by HGF. Interestingly, ECs on the lower stiffness substrates showed stronger responses to HGF in terms of migration and proliferation, suggesting that HGF can profoundly influence stiffness-dependent EC behavior correlated with EC growth. After the formation of an EC monolayer, EC behaviors correlated with endothelial function were evaluated by characterizing monolayer integrity, nitric oxide production, and gene expression of endothelial nitric oxide synthase. For the first time, we demonstrated that endothelial function displayed a negative correlation with substrate stiffness. Although HGF improved endothelial function, HGF was not able to change the stiffness-dependent manner of endothelial functions. Taken together, this study provides insights into the synergetic influence of physical and biochemical cues on EC behavior and offers great potential in the development of optimized biomaterials for EC-based regenerative medicine.

Quick and easy microfabrication of T-shaped cantilevers to generate arrays of microtissues.

Over the past decade, a major effort was made to miniaturize engineered tissues, as to further improve the throughput of such approach. Most existing methods for generating microtissues thus rely on T-shaped cantilevers made by soft lithography and based on the use of negative SU-8 photoresist. However, photopatterning T-shaped microstructures with these negative photoresists is fastidious and time-consuming. Here we introduce a novel method to quickly generate T-shaped cantilevers dedicated to generation of cellular microtissues, based on the use of positive photoresist. With only two layers of photoresist and one photomask, we were able to fabricate arrays of microwells in less than 3 h, each containing two T-shaped cantilevers presenting either a rectangular or a circular geometry. As a proof of concept, these arrays were then replicated in poly(dimethylsiloxane) and microtissues composed of NIH 3T3 fibroblasts encapsulated in collagen I were generated, while the two cantilevers simultaneously constrain and report forces generated by the microtissues. Immunostainings showed longitudinally aligned and elongated fibroblasts over the whole microtissue after 8 days of culture. The method described here opens the potential to quick prototyping platforms for high-throughput, low-volume screening applications.

Construction and myogenic differentiation of 3D myoblast tissues fabricated by fibronectin-gelatin nanofilm coating.

In this study, we used a recently developed approach of coating the cells with fibronectin-gelatin nanofilms to build 3D skeletal muscle tissue models. We constructed the microtissues from C2C12 myoblasts and subsequently differentiated them to form muscle-like tissue. The thickness of the constructs could be successfully controlled by altering the number of seeded cells. We were able to build up to ∼76 µm thick 3D constructs that formed multinucleated myotubes. We also found that Rho-kinase inhibitor Y27632 improved myotube formation in thick constructs. Our approach makes it possible to rapidly form 3D muscle tissues and is promising for the in vitro construction of physiologically relevant human skeletal muscle tissue models.

Differences in Morphology and Traction Generation of Cell Lines Representing Different Stages of Osteogenesis.

Osteogenesis is the process by which mesenchymal stem cells differentiate to osteoblasts and form bone. The morphology and root mean squared (RMS) traction of four cell types representing different stages of osteogenesis were quantified. Undifferentiated D1, differentiated D1, MC3T3-E1, and MLO-A5 cell types were evaluated using both automated image analysis of cells stained for F-actin and by traction force microscopy (TFM). Undifferentiated mesenchymal stem cell lines were small, spindly, and exerted low traction, while differentiated osteoblasts were large, had multiple processes, and exerted higher traction. Size, shape, and traction all correlated with the differentiation stage. Thus, cell morphology evolved and RMS traction increased with differentiation. The results provide a foundation for further work with these cell lines to study the mechanobiology of bone formation.

Spatio-Temporal Control of LbL Films for Biomedical Applications: From 2D to 3D.

Introduced in the '90s by Prof. Moehwald, Lvov, and Decher, the layer-by-layer (LbL) assembly of polyelectrolytes has become a popular technique to engineer various types of objects such as films, capsules and free standing membranes, with an unprecedented control at the nanometer and micrometer scales. The LbL technique allows to engineer biofunctional surface coatings, which may be dedicated to biomedical applications in vivo but also to fundamental studies and diagnosis in vitro. Initially mostly developed as 2D coatings and hollow capsules, the range of complex objects created by the LbL technique has greatly expanded in the past 10 years. In this Review, the aim is to highlight the recent progress in the field of LbL films for biomedical applications and to discuss the various ways to spatially and temporally control the biochemical and mechanical properties of multilayers. In particular, three major developments of LbL films are discussed: 1) the new methods and templates to engineer LbL films and control cellular processes from adhesion to differentiation, 2) the major ways to achieve temporal control by chemical, biological and physical triggers and, 3) the combinations of LbL technique, cells and scaffolds for repairing 3D tissues, including cardio-vascular devices, bone implants and neuro-prosthetic devices.

Magnetic approaches to study collective three-dimensional cell mechanics in long-term cultures (invited).

Contractile forces generated by cells and the stiffness of the surrounding extracellular matrix are two central mechanical factors that regulate cell function. To characterize the dynamic evolution of these two mechanical parameters during tissue morphogenesis, we developed a magnetically actuated micro-mechanical testing system in which fibroblast-populated collagen microtissues formed spontaneously in arrays of microwells that each contains a pair of elastomeric microcantilevers. We characterized the magnetic actuation performance of this system and evaluated its capacity to support long-term cell culture. We showed that cells in the microtissues remained viable during prolonged culture periods of up to 15 days, and that the mechanical properties of the microtissues reached and maintained at a stable state after a fast initial increase stage. Together, these findings demonstrate the utility of this microfabricated bio-magneto-mechanical system in extended mechanobiological studies in a physiologically relevant 3D environment.

Microfabrication of a platform to measure and manipulate the mechanics of engineered microtissues.

Engineered tissues can be used to understand fundamental features of biology, develop organotypic in vitro model systems, and as engineered tissue constructs for replacing damaged tissue in vivo. However, a key limitation is an inability to test the wide range of parameters that might impact the engineered tissue in a high-throughput manner and in an environment that mimics the three-dimensional (3D) native architecture. We developed a microfabricated platform to generate arrays of microtissues embedded within 3D micropatterned matrices. Microcantilevers simultaneously constrain microtissue formation and report forces generated by the microtissues in real time, opening the possibility to use high-throughput, low-volume screening for studies on engineered tissues. Thanks to the micrometer scale of the microtissues, this platform is also suitable for high-throughput monitoring of drug-induced effect on architecture and contractility in engineered tissues. Moreover, independent variations of the mechanical stiffness of the cantilevers and collagen matrix allow the measurement and manipulation of the mechanics of the microtissues. Thus, our approach will likely provide valuable opportunities to elucidate how biomechanical, electrical, biochemical, and genetic/epigenetic cues modulate the formation and maturation of 3D engineered tissues. In this chapter, we describe the microfabrication, preparation, and experimental use of such microfabricated tissue gauges.