A novel therapeutic approach for the treatment of bone defects is gene therapy assisted bone tissue engineering using bone marrow stromal cells (hBMSC). The aim of this study was to investigate the influence of human epidermal growth factor (hEGF) on proliferation and alkaline phosphatase (AP) activity of primary hBMSC in vitro. hBMSC cultures were achieved by explantation culture of bone chips. Following exposure to 0-10 ng recombinant hEGF (rhEGF)/ml cell numbers were determined by automated cell counting and cell bound AP activity was measured spectrophotometrically. hBMSC were transfected with hEGF plasmids and the proliferative effect was studied by cocultivation of transfected and untreated cells using porous cell culture inserts. The persistence of hEGF expression even after cell transfer was studied by the generation of possibly osteogenic constructs introducing transfected hBMSC in fibrin glue and bovine cancellous bone. The maximum increase in proliferation (156 +/- 7%) and AP activity (220 +/- 34%) was detected after exposition to 10 ng rhEGF/ml. In the separation chamber assay transfected cells produced hEGF concentrations up to 3.6 ng/ml, which induced a mean proliferation increase of 93% which could be significantly inhibited by a neutralizing hEGF antibody. Further, EGFsecretion of transfected hBMSC in 3D-culture was verified. Recombinant and transgenic hEGF stimulate proliferation of primary hBMSC in vitro. Lipotransfection of hBMSC with hEGF plasmids allows the transient and site directed delivery of biologically active transgenic hEGF. The introduction of mitogenic, angiogenic and chemoattractive factors in gene therapy assisted bone tissue engineering is discussed by the example of EGF.