Thomas Boudou

A hitchhiker's guide to mechanobiology.

More than a century ago, it was proposed that mechanical forces could drive tissue formation. However, only recently with the advent of enabling biophysical and molecular technologies are we beginning to understand how individual cells transduce mechanical force into biochemical signals. In turn, this knowledge of mechanotransduction at the cellular level is beginning to clarify the role of mechanics in patterning processes during embryonic development. In this perspective, we will discuss current mechanotransduction paradigms, along with the technologies that have shaped the field of mechanobiology.

Free-Standing Polyelectrolyte Membranes Made of Chitosan and Alginate

Free-standing films have increasing applications in the biomedical field as drug delivery systems for wound healing and tissue engineering. Here, we prepared free-standing membranes by the layer-by-layer assembly of chitosan and alginate, two widely used biomaterials. Our aim was to produce a thick membrane and to study the permeation of model drugs and the adhesion of muscle cells. We first defined the optimal growth conditions in terms of pH and alginate concentration. The membranes could be easily detached from polystyrene or polypropylene substrate without any postprocessing step. The dry thickness was varied over a large range from 4 to 35 μm. A 2-fold swelling was observed by confocal microscopy when they were immersed in PBS. In addition, we quantified the permeation of model drugs (fluorescent dextrans) through the free-standing membrane, which depended on the dextran molecular weight. Finally, we showed that myoblast cells exhibited a preferential adhesion on the alginate-ending membrane as compared to the chitosan-ending membrane or to the substrate side.

Variation of Polyelectrolyte Film Stiffness by Photo-Cross-Linking: A New Way To Control Cell Adhesion

We report on the preparation of polyelectrolyte films based on biopolymers whose nanomechanical properties can be tuned by photo-cross-linking. Cationic poly(L-lysine) was layer-by-layer assembled with anionic hyaluronan (HA) derivatives modified by photoreactive vinylbenzyl (VB) groups. The study of the multilayer buildup by quartz crystal microbalance with dissipation monitoring showed that the presence of VB groups does not influence significantly the multilayer growth. Then the VB-modified HA incorporated into the films was cross-linked upon UV irradiation. UV spectroscopy measurements showed that the cross-linking rate of the multilayers increases with the amount of VB groups grafted onto HA chains. Force measurements performed by atomic force microscopy with a colloidal probe proved that the rigidity of the cross-linked films increases with the grafting degree of HA chains and consequently the number of cross-links. Cell culture assays performed on non-cross-linked and photo-cross-linked films with myoblast cells demonstrated that cell adhesion and proliferation are considerably improved with increasing film rigidity.

Internal Composition versus the Mechanical Properties of Polyelectrolyte Multilayer Films: The Influence of Chemical Cross-Linking

Different types of polyelectrolyte multilayer films composed of poly(L-lysine)/hyaluronan (PLL/HA), chitosan/hyaluronan (CHI/HA) and poly(allylamine hydrochloride)/poly(L-glutamic acid) (PAH/PGA) have been investigated for their internal composition, including water content, ion pairing, and ability to be covalently cross-linked, as well as for their mechanical properties. Film buildup under physiological conditions was monitored by the quartz crystal microbalance with dissipation monitoring (QCM-D) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), which allows unambiguous quantification of the different groups present in the polyelectrolytes. (PAH/PGA) films emerged as the most dense films with the lowest hydration (29%) and the highest COO(-) molar density. In addition, PAH is greatly in excess in these films (3 PAH monomers per PGA monomer). The formation of amide bonds during film cross-linking using the water-soluble carbodiimide EDC was also investigated. All of the films could be cross-linked in a tunable manner, but PAH/PGA exhibited the highest absolute number of amide bonds created, approximately 7 times more than for (PLL/HA) and (CHI/HA) films. The Youngs modulus E of the films measured by AFM nanoindentation was shown to vary over 1 to 2 orders of magnitude for the different systems. Interestingly, a linear relationship between E and the density of the covalent cross-links created was observed for (PLL/HA) and (CHI/HA) films whereas (PGA/PAH) films exhibited biphasic behavior. The mean distance between covalent cross-links was estimated to be approximately 11 nm for (PLL/HA) and (CHI/HA) films and only approximately 6 nm for (PAH/PGA) films for the maximum EDC concentration tested (100 mg/mL).

Surface Functionalization of Hyaluronic Acid Hydrogels by Polyelectrolyte Multilayer Films

Hyaluronic acid (HA), an anionic polysaccharide, is one of the major components of the natural extracellular matrix (ECM). Although HA has been widely used for tissue engineering applications, it does not support cell attachment and spreading and needs chemical modification to support cellular adhesion. Here, we present a simple approach to functionalize photocrosslinked HA hydrogels by deposition of poly(l-lysine) (PLL) and HA multilayer films made by the layer-by-layer (LbL) technique. PLL/HA multilayer film formation was assessed by using fluorescence microscopy, contact angle measurements, cationic dye loading and confocal microscopy. The effect of polyelectrolyte multilayer film (PEM) formation on the physicochemical and mechanical properties of hydrogels revealed polyelectrolyte diffusion inside the hydrogel pores, increased hydrophobicity of the surface, reduced equilibrium swelling, and reduced compressive moduli of the modified hydrogels. Furthermore, NIH-3T3 fibroblasts seeded on the surface showed improved cell attachment and spreading on the multilayer functionalized hydrogels. Thus, modification of HA hydrogel surfaces with multilayer films affected their physicochemical properties and improved cell adhesion and spreading on these surfaces. This new hydrogel/PEM composite system may offer possibilities for various biomedical and tissue engineering applications, including growth factor delivery and co-culture systems.

Polyelectrolyte Multilayer Nanofilms Used as Thin Materials for Cell Mechano‐Sensitivity Studies

Three types of multilayer films made from poly(L-lysine)/hyaluronan, chitosan/hyaluronan, and poly(allylamine hydrochloride)/poly(L-glutamic acid), were used to investigate the interplay between film mechano-chemical properties and cell adhesion. We showed that C2C12 myoblast adhesion and proliferation depended on the extent of film cross-linking for all films whatever their internal chemistry. Cell spreading areas were found to correlate with the films stiffness and to be distributed over a unique curve. Immuno-staining of the cytoskeletal components revealed the formation of F-actin stress fibers and vinculin plaques only on stiff films. Finally, we compared our results with previous studies performed on polyacrylamide and PDMS gels, two recognized materials for mechano-sensitivity studies. We found that the effect of substrate stiffness on cell spreading is material-dependent.

Decoupling Cell and Matrix Mechanics in Engineered Microtissues Using Magnetically Actuated Microcantilevers

A novel bio-magnetomechanical microtissue system is described for magnetic actuation of arrays of 3D microtissues using microcantilevers. This system enables both in situ measurements of fundamental mechanical properties of engineered tissue, such as contractility and stiffness, as well as dynamic stimulation of the microtissues. Using this system, cell and extracellular matrix contributions to the tissue mechanical properties are decoupled for the first time under both static and dynamic loading conditions.

Gradients of physical and biochemical cues on polyelectrolyte multilayer films generated via microfluidics

The cell microenvironment is a complex and anisotropic matrix composed of a number of physical and biochemical cues that control cellular processes. A current challenge in biomaterials is the engineering of biomimetic materials which present spatially controlled physical and biochemical cues. The layer-by-layer assembly of polyelectrolyte multilayers (PEM) has been demonstrated to be a promising candidate for a biomaterial mimicking the native extracellular matrix. In this work, gradients of biochemical and physical cues were generated on PEM films composed of hyaluronan (HA) and poly(l-lysine) (PLL) using a microfluidic device. As a proof of concept, four different types of surface concentration gradients adsorbed onto the films were generated. These included surface concentration gradients of fluorescent PLL, fluorescent microbeads, a cross-linker, and one consisting of a polyelectrolyte grafted with a cell adhesive peptide. In all cases, reproducible centimeter-long linear gradients were obtained. Fluorescence microscopy, Fourier transform infrared spectroscopy and atomic force microscopy were used to characterize these gradients. Cell responses to the stiffness gradient and to the peptide gradient were studied. Pre-osteoblastic cells were found to adhere and spread more along the stiffness gradient, which varied linearly from 200 kPa-600 kPa. Myoblast cell spreading also increased throughout the length of the increasing RGD-peptide gradient. This work demonstrates a simple method to modify PEM films with concentration gradients of non-covalently bound biomolecules and with gradients in stiffness. These results highlight the potential of this technique to efficiently and quickly determine the optimal biochemical and mechanical cues necessary for specific cellular processes.

Polyelectrolyte multilayer nanoshells with hydrophobic nanodomains for delivery of Paclitaxel.

Efficient and effective delivery of poorly water-soluble drug molecules, which constitute a large part of commercially available drugs, is a major challenge in the field of drug delivery. Several drugs including paclitaxel (PTX) which are used for cancer treatment are hydrophobic, exhibit poor aqueous solubility and need to be delivered using an appropriate carrier. In the present work, we engineered PTX-loaded polyelectrolyte films and microcapsules by pre-complexing PTX with chemically modified derivative of hyaluronic acid (alkylamino hydrazide) containing hydrophobic nanocavities, and subsequent assembly with either poly(l-lysine) (PLL) or quaternized chitosan (QCHI) as polycations. The PTX loading capacity of the films was found to be dependent on number of layers in the films as well as on the initial concentration of PTX pre-complexed to hydrophobic HA, with a loading capacity up to 5000-fold the initial PTX concentration. The films were stable in physiological medium and were degraded in the presence of hyaluronidase. The PTX-loaded microcapsules were found to decrease the viability and proliferation of MDA MB 231 breast cancer cells, while unloaded microcapsules did not impact cell viability. All together, our results highlight the potential of hyaluronan-based assemblies containing hydrophobic nanodomains for hydrophobic drug delivery.

Nonlinear elastic properties of polyacrylamide gels: Implications for quantification of cellular forces

Because of their tunable mechanical properties, polyacrylamide gels (PAG) are frequently used for studying cell adhesion and migratory responses to extracellular substrate stiffness. Since these responses are known to heavily depend on the tensional balance between cell contractility and substrate mechanical resistance, a precise knowledge of PAGs mechanical properties becomes quite crucial. Using the micropipette aspiration technique, we first exhibited the nonlinear elastic behavior of PAG and then successfully modeled it by an original strain-energy function. This function depends on the Poissons ratio and on two material parameters, which have been explicitly related to acrylamide and bis-acrylamide concentrations. Implications of these results have been highlighted with regard to traction force microscopy experiments where cellular force quantification is derived from displacements of beads embedded in PAG. We found that considering PAG as a linear elastic medium tends to significantly underestimate traction forces for substrate displacements larger than 2 mum. Interestingly, we also showed that in the range of cellular force amplitude and PAG stiffness currently used in cell traction force experiments, finite size effects become critical for PAG substrate thickness below 60 mum. Thus, our improved characterization of PAG nonlinear mechanical properties through a new constitutive law could have significant impact onto biological experimentations where such extracellular substrates experience large strains.