Tissue engineering has merged with stem cell biotechnology with development of new sources of transplantable biomaterials for the treatment of bone tissue diseases. Bone defects are expected to benefit from this new biotechnology because of the low self-regenerating capacity of bone matrix secreting cells. The differentiation of stem cells to bone cells using bi-functionalized multilayered particles is presented. The functionalized particles are composed of poly-glutamic acid (PGA) and poly-L-lysine (PLL) with two bone growth factors (BMP-2 and TGFbeta1) embedded into the multilayered film. The induction of bone from these bioactive particles incubated with embryonic stem cells was demonstrated in vitro. We report the demonstration of a multilayered particle-based delivery system for inducing bone formation in vivo. This new strategy is an alternative approach for in vivo bone formation.