Effect of functionalization of multilayered polyelectrolyte films on motoneuron growth
Introduction
In the field of tissue engineering, tissue regeneration and wound healing, a major challenge is the development of cellular microdevices assembling various molecular species into micron-scaled structures. While the selection of the appropriate biomolecules and the development of methods for their incorporation into such complex structures are important, other pertinent issues include the cell viability on the prepared surfaces, and the long-term stability and retention of biological activity of incorporated biomolecules. Surface topography and chemistry have been shown to direct cell–substrate interactions and cellular properties such as cell adhesion, cell–cell reactions and cytoskeletal organization. Neurons are highly sensitive to the nature of the substrate whereas other cells such as glial cells more easily adhere on any kind of untreated surfaces. Polypeptides like polylysine or polyornithine have often been used [1], [2], together with laminin and fibronectin to optimize neurons cultures [3], [4].
Decher [5], developed a technique based on the alternate adsorption of positively and negatively charged polyelectrolytes to build thin films. This approach allows the build-up of films with variable thickness or roughness. The interactions of such multilayered architectures with living cells have been recently investigated [6], [7], [8], [9], [10], [11], [12]. Proteins can be embedded in the architecture of thin films [13], [14], [15], [16], [17], [18], and keep a secondary structure close to their native form as well as their bioactivity [18], [19].
In the present study, we used multilayered polyelectrolyte films built-up by layer-by-layer deposition, and investigated the reaction of motoneurons with these nanofilms with respect to the nature of the ending polyelectrolyte type and to the film functionalization either with a growth factor, Brain Derived Neurotrophic Factor (BDNF) [20], [21], or a chemorepulsive protein, Semaphorin 3A (Sema3A) [22], [23]. The films were characterized by means of Optical Waveguide Lightmode Spectroscopy (OWLS) and Atomic Force Microscopy (AFM). The viability of the motoneurons was estimated by the acid phosphatase method and measurements were completed by morphometrical analyses.
Section snippets
Polyelectrolyte films preparation
The following polyelectrolytes were purchased from Sigma-Aldrich, Saint Quentin Fallavier, France and have been used for the build-up of multilayer films: poly(ethylene-imine) (PEI; MW 750×103), poly(sodium-4-styrenesulfonate) (PSS; MW 70×103), poly(allylamine hydrochloride) (PAH; MW 70×103), poly(l-glutamic acid) (PGA; MW 54.8×103) and the poly(l-lysine) (PLL; MW 23.4×103). Solutions of PEI, PSS, and PAH have been prepared to a concentration of 5 mg/ml in 0.15 mol/l NaCl, pH 6.5, while solutions
Multilayered polyelectrolyte films build-up
The build-up of multilayered films arises from electrostatic interactions between alternately deposited polyanions and polycations. In each step, either a polyanion or a polycation layer is added to the film. The number of cycles, the type of polyelectrolytes and the physico-chemical characteristics (salt concentration, pH,…) modulate the thickness and roughness of the multilayered film [33], [34].
In our experiments, the solid surface was glass coverslips made of negatively charged silica. We
Conclusion
Our data indicate that the response of motoneurons cultured on polyelectrolyte films can be tuned by protein embedding and are strictly influenced by the ending polyelectrolyte. The immobilization of proteins directly into the growing substrate where they remain still functional and available, even embedded under two layers of polyelectrolytes, is particularly advantageous since it allows the direct presentation of growth factors in the injury environment. In comparison to conventional coating
Acknowledgements
The authors thank Catherine Picart, Philippe Lavalle, Géraldine Koenig and all the members of INSERM U-595 for assistance and advice. This work was in part financed by the program “Ingenierie tissulaire” by INSERM-CNRS. C.V. and C.E. thanks the Faculty of Odontology of Strasbourg for financial support. F.B. was supported by a doctoral fellowship of the Ministère de l’Enseignement Supérieur et de la Recherche.
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