Practical guide to characterize biomolecule adsorption on solid surfaces (Review)

International audienc

Cell adhesion in free-standing multilayer films made of chitosan and alginate

The method for preparing multilayer ultrathin films by the consecu- tive deposition of oppositely charged polyelectrolytes has gained tre- mendous recognition due the user friendly preparation, capability of incorporating high loads of different types of biomolecules in the films, fine control over the materials’ structure, and robustness of the products under ambient and physiological conditions. However the preparation of such films needs the assembly on a substrate and, sometimes, cannot be detached from it, which has limited the appli- cation of such films in areas as tissue engineering and regenerative medicine (TERM).Thus, the production of free-standing films is of extreme importance once it allows the direct experimental determi- nation of many physical properties of fundamental significance such as ion permeation and mechanical properties that can be tuned for real-world applications. In this work, we investigated the elaboration of free-standing multilayer films made of chitosan (CHI) and alginate (ALG), by detaching a polyelectrolyte multilayer film from its under- lying substrate without any postprocessing step. The conditions for optimized film growth were investigated. The adhesion of C2C12 myoblast cells on the CHI/ALG membrane was assessed by cytoskele- tal and nuclear staining. A good cell adhesion and spreading was observed all over the surface. The results demonstrate the potential of such biocompatible free standing membranes made of CHI and ALG for applications in TERM

Surface probe measurements of the elasticity of sectioned tissue, thin gels and polyelectrolyte multilayer films : correlations between substrate stiffness and cell adhesion

Surface probe measurements of the elasticity of thin-film matrices as well as biological samples prove generally important to understanding cell attachment across such systems. To illustrate this, sectioned arteries were probed by Atomic Force Microscopy (AFM) within the smooth muscle cell (SMC)-rich medial layer, yielding an apparent Young’s modulus Emedia ~ 5-8 kPa. Polyacrylamide gels with Egel spanning several-fold above and below this range were then cast 5-70 μm thick and coated with collagen: SMC spreading shows a hyperbolic dependence in projected cell area versus Egel. The modulus that gives half-max spreading is E1/2-spread ~ 8-10 kPa, proving remarkably close to Emedia. More complex, layer-by-layer micro-films of poly(L-lysine)/hyaluronic acid were also tested and show equivalent trends of increased SMC spreading with increased stiffness. Adhesive spreading of cells thus seems to correlate broadly with the effective stiffness of synthetic materials and tissues

Magnetic-responsive hydrogels for cartilage tissue engineering

Publicado em "Journal of Tissue Engineering and Regenerative Medicine", vol. 7, supp. 1 (2013)The use of magnetic nanoparticles (MNPs) has been explored as an alternative approach to overcome current limitations of regenerative medicine strategies. Cell engineering approaches where MNPs are incorporated within three-dimensional constructs, such as scaffolds or hydrogels may constitute a novel and attractive approach towards the development of a magnetically-responsive system. These systems would enable remote controlled actions over tissue engineered constructs in vitro and in vivo. Moreover, growing evidence suggests that the application of a magnetic field may enhance biological performance over commonly used static culture conditions providing stimulation for cell proliferation, migration and differentiation. In this work we analyze the role of magnetic stimulation on the behavior of human adipose derived stem cells (hASCs) laden in k-carrageenan hydrogels aiming at cartilage tissue engineering approaches. Thermo-responsive natural-based κ-carrageenan hydrogels were used as 3D templates since previous studies(1) report the adequate environment provided by these materials to support the viability and chondrogenic differentiation of several types of cells

Improvement of stability and cell adhesion properties of polyelectrolyte multilayer films by chemical cross-linking.

Poly(L-lysine)/hyaluronan (PLL/HA) films were chemically cross-linked with a water soluble carbodiimide (EDC) in combination with a N-hydroxysulfo-succinimide (NHS) to induce amide formation. Fourier transform infrared spectroscopy confirms the conversion of carboxylate and ammonium groups into amide bonds. Quartz crystal microbalance-dissipation reveals that the cross linking reaction is accompanied by a change in the viscoelastic properties of the films leading to more rigid films. After the cross-linking reaction, both positively and negatively ending films exhibit a negative zeta potential. It is shown by fluorescence recovery after photobleaching measured by confocal laser scanning microscopy that cross-linking dramatically reduces the diffusion of the PLL chains in the network. Cross linking also renders the films highly resistant to hyaluronidase, an enzyme that naturally degrades hyaluronan. Finally, the adhesion of chondrosarcoma cells on the films terminating either with PLL or HA is also investigated. Whereas the non cross-linked films are highly resistant to cell adhesion, the cells adhere and spread well on the cross-linked films.comparative studyjournal articleresearch support, non-u.s. gov't2004 Mar-Aprimporte

Myoconductive and osteoinductive free-standing polysaccharide membranes

Free-standing (FS) membranes have increasing applications in the biomedical field as drug delivery systems for wound healing and tissue engineering. Here, we studied the potential of free-standing membranes made by the layer-by-layer assembly of chitosan and alginate to be used as a simple biomimetic system of the periosteum. The design of a periosteum-like membrane implies the elaboration of a thick membrane suitable for both muscle and bone formation. Our aim was to produce well-defined ∼50 μm thick polysaccharide membranes that could be easily manipulated, were mechanically resistant, and would enable both myogenesis and osteogenesis in vitro and in vivo. The membranes were chemically crosslinked to improve their mechanical properties. Crosslinking chemistry was followed via Fourier transform infrared spectroscopy and the mechanical properties of the membranes were assessed using dynamic mechanical analysis. The loading and release of the potent osteoinductive growth factor bone morphogenetic protein 2 (BMP-2) inside and outside of the FS membrane was followed by fluorescence spectroscopy in a physiological buffer over 1 month. The myogenic and osteogenic potentials of the membranes in vitro were assessed using BMP-2-responsive skeletal myoblasts. Finally, their osteoinductive properties in vivo were studied in a preliminary experiment using a mouse ectopic model. Our results showed that the more crosslinked FS membranes enabled a more efficient myoblast differentiation in myotubes. In addition, we showed that a tunable amount of BMP-2 can be loaded into and subsequently released from the membranes, depending on the crosslinking degree and the initial BMP-2 concentration in solution. Only the more crosslinked membranes were found to be osteoinductive in vivo. These polysaccharide-based membranes have strong potential as a periosteum-mimetic scaffold for bone tissue regeneration.This work was financially supported by the Foundation for Science and Technology (FCT) through the scholarship SFRH/BPD/96797/2013, Fundo Social Europeu (FSE), and Programa Diferencial de Potencial Human (POPH) granted to Sofia G. Caridade. C.M. is indebted to the Association Francaise contre les Myopathies for financial support via a post-doctoral fellowship (AFM project 16673). J.A. acknowledges the Whitaker International Fellows and Scholars Program for support via a post-doctoral fellowship. This work was supported by the European Commission (FP7 program) via a European Research Council starting grant (BIOMIM, GA 259370 to C.P.) and by the AFM (grant Microtiss, 16530). We thank Isabelle Paintrand for her technical help with the confocal apparatus

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.This work was financially supported by Foundation for Science and Technology (FCT) through the Scholarship SFRH/BD/64601/2009 granted to S.G.C. C.M. is indebted to Grenoble INP for financial support via a postdoctoral fellowship. This work was supported by the European Commission (FP7 Program) via a European Research Council starting grant (BIOMIM, GA 259370 to C.P.). C.P. is also grateful to Institut Universitaire de France and to Grenoble Institute of Technology for financial support. We thank Isabelle Paintrand for her technical help with the confocal apparatus and Patrick Chaudouet for his help with SEM imaging

Binding of the chemokine CXCL12α to its natural extracellular matrix ligand heparan sulfate enables myoblast adhesion and facilitates cell motility

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

Secondary structure of rhBMP-2 in a protective biopolymeric carrier material

Efficient delivery of growth factors is one of the great challenges of tissue engineering. Polyelectrolyte multilayer films (PEM) made of biopolymers have recently emerged as an interesting carrier for delivering recombinant human bone morphogenetic protein 2 (rhBMP-2 noted here BMP-2) to cells in a matrix-bound manner. We recently showed that PEM made of poly(l-lysine) and hyaluronan (PLL/HA) can retain high and tunable quantities of BMP-2 and can deliver it to cells to induce their differentiation in osteoblasts. Here, we investigate quantitatively by Fourier transform infrared spectroscopy (FTIR) the secondary structure of BMP-2 in solution as well as trapped in a biopolymeric thin film. We reveal that the major structural elements of BMP-2 in solution are intramolecular β-sheets and unordered structures as well as α-helices. Furthermore, we studied the secondary structure of rhBMP-2 trapped in hydrated films and in dry films since drying is an important step for future applications of these bioactive films onto orthopedic biomaterials. We demonstrate that the structural elements were preserved when BMP-2 was trapped in the biopolymeric film in hydrated conditions and, to a lesser extent, in dry state. Importantly, its bioactivity was maintained after drying of the film. Our results appear highly promising for future applications of these films as coatings of biomedical materials, to deliver bioactive proteins while preserving their bioactivity upon storage in dry state.This work was supported by the French Ministry of Research through an ANR-EmergenceBIO grant (ANR-09-EBIO-012-01), by the European Commission (FP7 program) via a European Research Council starting grant (BIOMIM, GA 259370), and by GRAVIT (081012_FIBIOS). C.P. is grafetul to IUF for financial support