Purpose: Since human bone is one of the most common transplanted tissues there is a strong clinical need for a bone graft, which can replace autologous bone. Freeze-dried bone allografts are used extensively, however, their poor osteogenic potential due to the lack of live cells restrain their clinical application. Aim: to develop an in vitro protocol to seed cancellous bone allografts with bone marrow and dental pulp derived mesenchymal stem cells in order to improve biocompatibility and osteogeneicity. Materials: We coated the surface of cancellous mineralized human bone with aqueous and freeze-dried human serum albumin, collagen type I and fibronectin or their combinations. Instead of allografts we also tested hydroxi-apatite or lyophilized bovine graft scaffolds. We compared the seeding efficiency and the proliferation rate of the mesenchymal stem cells (MSCs) on the grafts in vitro. Methods: We incubated the constructs under standard cell culture conditions or under dynamic fluid movements in a rotating bioreactor. We monitored cell attachment with confocal and scanning electron microscopy and evaluated the cell number and viability with either fluorescent cell counting or the alamar-blue assay. Results: Less than the 50 percent of MSCs attached onto the surface of mineralized allografts and their quantity gradually decreased during the experiment until the 18th day. Coating of mineralized allografts with aqueous collagen type I or fibronectin did not influence appreciably the initial attachment and the survival of MSCs compared with uncoated mineralized allografts. In contrast, aqueous albumin as coating material markedly increased the quantity of retained MSCs on the surface of allografts at the outset, however, the number of detectable cells reduced to zero at 18 days. Freeze-drying of serum albumin onto the surface of allografts reversed the tendency and MSCs showed significant proliferation. Interestingly, albumin coating was beneficial only on mineralized bone allografts, and had no effect when applied on lyophilized bovine bone or hydroxy-apatite scaffolds. Conclusions: Our results indicate that allografts can be colonized in vitro with bone marrow or dental pulp derived MSCs in a rotaing bioreactor system before transplantation. Since albumin coating requires only a lyophilization step, this might be integrated into the manufacturing process of human cancellous allograft as an affordable biocompatibility improving step. Funding: This study was supported by EIT-EITECH-BRIDGE, TÁMOP 4.2.2-08/1/KMR-2008-0004, TÉT AR6-016 and CELLTHER, and the Bolyai and Öveges Fellowships.