Effects of transforming growth factor beta1 on bonelike tissue formation in three-dimensional cell culture. II: Osteoblastic differentiation

Tissue Eng. Sep-Oct 2004;10(9-10):1414-25. doi: 10.1089/ten.2004.10.1414.

Abstract

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.

MeSH terms

  • Animals
  • Bone Marrow Cells / cytology*
  • Bone Marrow Cells / drug effects
  • Bone Marrow Cells / physiology*
  • Bone Substitutes*
  • Cell Differentiation / drug effects
  • Cell Differentiation / physiology
  • Cell Proliferation / drug effects
  • Cells, Cultured
  • Dose-Response Relationship, Drug
  • Humans
  • Male
  • Osteoblasts / cytology*
  • Osteoblasts / drug effects
  • Osteoblasts / physiology*
  • Osteogenesis / drug effects
  • Osteogenesis / physiology*
  • Rats
  • Rats, Sprague-Dawley
  • Stromal Cells / cytology
  • Stromal Cells / drug effects
  • Stromal Cells / physiology
  • Tissue Engineering / methods*
  • Transforming Growth Factor beta / genetics
  • Transforming Growth Factor beta / pharmacology*
  • Transforming Growth Factor beta1

Substances

  • Bone Substitutes
  • TGFB1 protein, human
  • Tgfb1 protein, rat
  • Transforming Growth Factor beta
  • Transforming Growth Factor beta1