Recent tissue engineering efforts have been focused on the use of natural or synthetic matrices which combine the characteristics of biodegradable properties with those of protein delivery vehicles, allowing for implanted cell actions and enhanced tissue regeneration. The principal objective of this study was to assess the feasibility of ectopic bone formation in a three-dimensional fibrin construct mixed with bone morphogenic protein-2 (BMP-2) loaded in nano-carriers for the osteogenic differentiation of human mesenchymal stem cells (hMSCs). The results of our evaluation showed that the osteogenic differentiation of hMSCs embedded in the fibrin construct was affected significantly by the stimulation of growth factors loaded in nanoparticles. When the osteoinduction activity of hMSCs in fibrin construct was evaluated in an in vitro test followed by RT-PCR, real time-QPCR, Western blotting, histological and immunohistochemical examinations, significant homogeneous bone formation was observed histologically throughout the fibrin construct containing the growth factor (BMP-2) loaded into the nanoparticles. With the above detection techniques, the BMP-2-loaded nanoparticles encapsulated in fibrin constructs evidenced more potent effects of hMSCs on bone regeneration as compared to the control or BMP-2 loaded fibrin constructs without nanoparticles. In the current study, we conclude that fibrin constructs containing BMP-2 loaded nanoparticles will be a promising method by which bone regeneration can be enhanced. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.