It is shown that blend multilayers of hyaluronan (HA) and heparin (HEP) as polyanions and poly(L-lysine) (PLL) as a polycation can be used to prepare films with different thicknesses and chemical compositions. The amounts of recombinant human BMP-2 (rhBMP-2) loaded and the fraction initially released from the films depend on the film's chemical composition. The amounts of rhBMP-2 loaded in the films are much higher for HA mass fractions of more than 0.4. The bioactivity of the rhBMP-2-loaded films is investigated on C2C12 myoblasts, which differentiates into osteoblasts in contact with the films. The alkaline phosphatase expression for cells grown on nanoblend films of various compositions falls over a unique curve. This suggests that the cells “sensing” the rhBMP-2 are not influenced by the film's chemistry. The rhBMP-2 can sustain at least three successive culture sequences while remaining bioactive, thus confirming the important and protective effect of rhBMP-2. Altogether, these results indicate that crosslinked PLL/HA films have superior properties for the incorporation of rhBMP-2 and on its long-lasting bioactivity.