Human osteogenic protein-1 induces both chondroblastic and osteoblastic differentiation of osteoprogenitor cells derived from newborn rat calvaria.
Osteogenetic protein-1 (OP-1), a member of the TGF-beta superfamily, induces endochondrial bone formation at subcutaneous sites in vivo and stimulates osteoblastic phenotypic expression in vitro. Primary cultures of newborn rat calvarial cells contain a spectrum of osteogenic phenotypes ranging from undifferentiated mesenchymal osteoprogenitor cells to parathyroid hormone (PTH)-responsive osteoblasts. We examined whether treatment of this cell population with recombinant human osteogenic protein-1 could induce chondrogenesis in vitro. Markers of chondroblastic versus osteoblastic differentiation included alcian blue staining at pH 1, alkaline phosphatase-specific activity, osteocalcin radioimmunoassay, and expression of collagen mRNAs. 6 d of treatment (culture days 1-7) with 4-100 ng OP-1/ml caused dose-dependent increases in alcian blue staining intensity and alkaline phosphatase activity (4.7- and 3.4-fold, respectively, at 40 ng/ml), while osteocalcin production decreased twofold. Clusters of round, refractile, alcian blue-stained cells appeared by day 3, increased in number until day 7, and then became hypertrophic and gradually became less distinct. Histochemically, the day 7 clusters were associated with high alkaline phosphatase activity and became mineralized. mRNA transcripts for collagen types II and IX were increased by OP-1, peaking at day 4, while type X collagen mRNA was detectable only on day 7 in OP-1-treated cultures. Delay of OP-1 exposure until confluence (day 7) amplifies expression of the normal osteoblastic phenotype and accelerates its developmental maturation. In contrast, early OP-1 treatment commencing on day 1 strongly amplifies chondroblastic differentiation. In the same protocol, TGF-beta 1 alone at 0.01-40 ng/ml fails to induce any hypertrophic chondrocytes, and in combination with OP-1, TGF-beta 1 blocks OP-1-dependent chondroinduction. OP-1 is believed to act on a subpopulation of primitive osteoprogenitor cells to induce endochondrial ossification, but does not appear to reverse committed osteoblasts to the chondrocyte phenotype.
Asahina
I
I
Children's Hospital Medical Center, Boston, Massachusetts.
Sampath
T K
TK
Nishimura
I
I
Hauschka
P V
PV
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D001205:Ascorbic Acid / Q000378:metabolism; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins; D002356:Cartilage / Q000166:cytology*; D002454:Cell Differentiation; D002455:Cell Division; D005994:Glycerophosphates / Q000378:metabolism; D006801:Humans; D010006:Osteoblasts / Q000166:cytology*; D015675:Osteocalcin / Q000378:metabolism; D010641:Phenotype; D011506:Proteins / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012886:Skull / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology; D016212:Transforming Growth Factor beta / Q000378:metabolism
C525819:BMP7 protein, human; C525821:Bmp7 protein, rat; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins; D005994:Glycerophosphates; D011506:Proteins; D016212:Transforming Growth Factor beta; D015675:Osteocalcin; D000469:Alkaline Phosphatase; D001205:Ascorbic Acid; C031463:beta-glycerophosphoric acid