Layer‐By‐Layer Films as a Biomimetic Reservoir for rhBMP‐2 Delivery: Controlled Differentiation of Myoblasts to Osteoblasts

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 ≈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. Kept in reserve: Controlled differentiation of myoblast cells in myotubes (see images, top) or osteoblasts (bottom) occurs via poly(L‐lysine)/hyaluronan (PLL/HA) multilayer films loaded with increasing amounts of recombinant human bone morphogenetic protein 2 (rhBMP‐2). Myogenic (troponin T, green) and osteogenic (alkaline phosphatase, blue) markers are used.