title: Recombinant human bone morphogenetic protein-2 stimulates differentiation in primary cultures of fetal rat calvarial osteoblasts.
abstract: Recombinant human bone morphogenetic protein (rhBMP-2) was examined for its in vitro effects on biochemical markers representing osteoblast phenotype. Primary cultures of fetal rat calvarial osteoblasts were used in this study. The results indicated that rhBMP-2 stimulated alkaline phosphatase activity, parathyroid hormone (PTH)-induced cyclic AMP production, and collagen biosynthesis in a dose-dependent manner in confluent cultures. The percent collagen synthesis also increased in a dose-dependent manner. Alkaline phosphatase activity was stimulated in a time-dependent manner by rhBMP-2 that reached its maximum 5 days after initiation. Cycloheximide (2 micrograms/ml) inhibited rhBMP-2-stimulated alkaline phosphatase indicating de novo protein synthesis of the enzyme. Transforming growth factor-beta 1 (TGF-beta 1)-induced inhibition of alkaline phosphatase activity observed in confluent primary cultures was completely abolished by rhBMP-2 at a concentration that was 43 times greater than the TGF-beta 1 concentration. Also, rhBMP-2 produced a small stimulation of alkaline phosphatase activity in cells grown in the absence of ascorbic acid; however, the effect was greatly enhanced in cells cultivated in the presence of ascorbic acid (50 micrograms/ml). In view of the potentiating effect of ascorbic acid on rhBMP-2-induced stimulation of alkaline phosphatase, we speculate that ascorbic acid could amplify the osteoinductive effects of rhBMP-2 and thereby augment the efficacy of the BMP when used as bone repair material in vivo. rhBMP-2 (4.3-86 ng/ml) did not exhibit mitogenic effects on cultured osteoblasts. These data suggest that rhBMP-2 has the ability to induce expression of various markers associated with the osteoblast phenotype in primary cultures of fetal rat calvarial osteoblasts. In addition, we speculate that TGF-beta 1 may play a regulatory role in BMP-induced bone formation and ascorbic acid may potentiate the effects of rhBMP-2 in vivo.
authors.affiliation: Biochemistry Section, US Army Dental Research Detachment, Walter Reed Army Medical Center, Washington, DC, USA.
authors.forename: A
authors.initials: A
authors.lastname: Chaudhari
authors.forename: E
authors.initials: E
authors.lastname: Ron
authors.forename: M P
authors.initials: MP
authors.lastname: Rethman
mesh_terms: D000469:Alkaline Phosphatase / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000235:genetics* / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D003094:Collagen / Q000096:biosynthesis / Q000187:drug effects; D000242:Cyclic AMP / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D010006:Osteoblasts / Q000187:drug effects*; D010281:Parathyroid Hormone / Q000494:pharmacology; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D012886:Skull / Q000166:cytology* / Q000187:drug effects / Q000196:embryology*; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000494:pharmacology
chemical_list: C525718:BMP2 protein, human; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D010281:Parathyroid Hormone; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2; D003094:Collagen; D000242:Cyclic AMP; D000469:Alkaline Phosphatase; D001205:Ascorbic Acid