In vitro effects of combined and sequential bone morphogenetic protein administration.

OBJECTIVE To assess the effects of combined and sequential administration of bone morphogenetic protein 2 (BMP-2) and BMP-7 on osteoblastic differentiation compared with administration of single growth factors. DESIGN In vitro study of osseous differentiation in murine pluripotent cells using assays of extracellular matrix calcification, alkaline phosphatase activity, and expression of osseous markers. Mesenchymal cells were cultured with BMP-2, BMP-7, or a combination of these growth factors or were sequentially exposed to the growth factors. RESULTS Sequential administration of BMP-2 and BMP-7 resulted in increased extracellular matrix calcification and expression of osteocalcin, whereas all groups treated with BMP up-regulated expression of the osteoblastic transcription factor Runx2/cbfa1, type I collagen, and the inhibitory BMP second messenger Smad6. None of the experimental groups demonstrated increased expression of osteopontin or Smad1, and only cells treated with concurrent administration of BMP-2 and BMP-7 increased Smad5 expression. Alkaline phosphatase activity was increased from baseline only in cells treated with BMP-2 alone. CONCLUSIONS Culture with BMP-2 and BMP-7, their sequential administration, and their coadministration had variable effects on osseous differentiation in mesenchymal cells. These results demonstrate the need for increased understanding of the role of growth factors and their combinations in bone development and have important implications for the ongoing development of osteoinductive therapies.