We supplemented rat marrow stromal cells (rMSCs) seeded on poly(L-lactic-co-glycolic acid) fiber meshes with transforming growth factor beta1 (TGF-beta1) to improve bone tissue formation for tissue engineering. Whereas our first study (Lieb, E., et al. Tissue Eng. 10, 1399-1413, 2004) investigated the effects of TGF-beta1 on matrix formation and mineralization, this second study focused on the differentiation of rMSCs to the osteoblastic phenotype in dynamic cell culture (orbital shaker). We assessed a series of bone markers to determine a dosing regimen for TGF-beta1 that enhances collagenous matrix formation and preserves or increases osteoblastic differentiation. Bone sialoprotein and osteonectin formation were investigated immunohistochemically and by RT-PCR. For alkaline phosphatase activity (ALP), we employed an enzyme assay. Osteocalcin was examined by RT-PCR as well as by an immunoassay. Whereas bone sialoprotein appeared to be dose-dependently increased in the immunochemistical stainings after supplementation with TGF-beta1, osteonectin remained unchanged. Both ALP activity and osteocalcin were suppressed by high doses of TGF-beta1, such as single doses of 10 ng/mL or four doses of 1 ng/mL added once a week. Considering the effects of TGF-beta1 both on differentiation and on matrix formation and mineralization, TGF-beta1 at 1 ng/mL, added once a week in the first 1 to 2 weeks, was selected as an effective dose to improve bonelike tissue formation in vitro.