In order to recreate a periosteum like membrane, we used the layer-by-layer technique to generate thick biomimetic membranes. Chitosan and alginate films were built up on a low energy substrate by tuning pH and polyelectrolyte concentration. Free-standing (FS) membranes obtained were then simply obtained by removing the substrate in mild conditions. The obtained FS were easily manipulated in dry and wet conditions. Their crosslinking degree of the FS was modulated as well as their loading with an osteoinductive protein, the bone morphogenetic protein-2 (BMP-2), enabling myogenic or osteogenic differentiation.