The design of advanced functional materials with nanometer- and micrometer-scale control over their properties is of considerable interest for both fundamental and applied studies because of the many potential applications for these materials in the fields of biomedical materials, tissue engineering, and regenerative medicine. The layer-by-layer deposition technique introduced in the early 1990s by Decher, Moehwald, and Lvov is a versatile technique, which has attracted an increasing number of researchers in recent years due to its wide range of advantages for biomedical applications: ease of preparation under "mild" conditions compatible with physiological media, capability of incorporating bioactive molecules, extra-cellular matrix components and biopolymers in the films, tunable mechanical properties, and spatio-temporal control over film organization. The last few years have seen a significant increase in reports exploring the possibilities offered by diffusing molecules into films to control their internal structures or design "reservoirs," as well as control their mechanical properties. Such properties, associated with the chemical properties of films, are particularly important for designing biomedical devices that contain bioactive molecules. In this review, we highlight recent work on designing and controlling film properties at the nanometer and micrometer scales with a view to developing new biomaterial coatings, tissue engineered constructs that could mimic in vivo cellular microenvironments, and stem cell "niches."

Efficient delivery of growth or survival factors to cells is one of the most important long-term challenges of current cell-based tissue engineering strategies. The extracellular matrix acts as a reservoir for a number of growth factors through interactions with its components. In the matrix, growth factors are protected against circulating proteases and locally concentrated. Thus, the localized and long-lasting delivery of a matrix-bound recombinant human bone morphogenetic protein 2 (rhBMP-2) from a biomaterial surface would mimic in vivo conditions and increase BMP-2 efficiency by limiting its degradation. Herein, it is shown that crosslinked poly(L-lysine)/hyaluronan (HA) layer-by-layer films can serve as a reservoir for rhBMP-2 delivery to myoblasts and induce their differentiation into osteoblasts in a dose-dependent manner. The amount of rhBMP-2 loaded in the films is controlled by varying the deposition conditions and the film thickness. Its local concentration in the film is increased up to approximately 500-fold when compared to its initial solution concentration. Its adsorption on the films, as well as its diffusion within the films, is evidenced by microfluorimetry and confocal microscopy observations. A direct interaction of rhBMP-2 with HA is demonstrated by size-exclusion chromatography, which could be at the origin of the rhBMP-2 "trapping" in the film and of its low release from the films. The bioactivity of rhBMP-2-loaded films is due neither to film degradation nor to rhBMP-2 release. The rhBMP-2-containing films are extremely resistant and could sustain three successive culture sequences while remaining bioactive, thus confirming the important and protective effect of rhBMP-2 immobilization. These films may find applications in the local delivery of immobilized growth factors for tissue-engineered constructs and for metallic biomaterial surfaces, as they can be deposited on a wide range of substrates with different shapes, sizes, and composition.

Beside chemical properties and topographical features, mechanical properties of gels have been recently demonstrated to play an important role in various cellular processes, including cell attachment, proliferation, and differentiation. In this work, we used multilayer films made of poly (L-lysine)/Hyaluronan (PLL/HA) of controlled stiffness to investigate the effects of mechanical properties of thin films on skeletal muscle cells (C2C12 cells) differentiation. Prior to differentiation, cells need to adhere and proliferate in growth medium. Stiff films (E 0 > 320 kPa) promoted formation of focal adhesions and organization of the cytoskeleton as well as an enhanced proliferation, whereas soft films were not favorable for cell anchoring, spreading or proliferation. Then C2C12 cells were switched to a low serum containing medium to induce cell differentiation, which was also greatly dependent on film stiffness. Although myogenin and troponin T expressions were only moderately affected by film stiffness, the morphology of the myotubes exhibited striking stiffness-dependent differences. Soft films allowed differentiation only for few days and the myotubes were very short and thick. Cell clumping followed by aggregates detachment could be observed after ~2 to 4 days. On stiffer films, significantly more elongated and thinner myotubes were observed for up to ~ 2 weeks. Myotube striation was also observed but only for the stiffer films. These results demonstrate that film stiffness modulates deeply adhesion, proliferation and differentiation, each of these processes having its own stiffness requirement.