Effects of transforming growth factor beta1 on bonelike tissue formation in three-dimensional cell culture. II: Osteoblastic differentiation.
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.
Lieb
E
E
Department of Pharmaceutical Technology, University of Regensburg, Regensburg, Germany.
Vogel
T
T
Milz
S
S
Dauner
M
M
Schulz
M B
MB
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D018786:Bone Substitutes; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008297:Male; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D010012:Osteogenesis / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D023822:Tissue Engineering / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1
D018786:Bone Substitutes; C508960:TGFB1 protein, human; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1