FLOW CYTOMETRIC ANALYSIS OF RECOMBINANT HUMAN OSTEOGENIC PROTEIN-1 (BMP-7) RESPONSIVE SUBPOPULATIONS FROM FETAL-RAT CALVARIA BASED ON INTRACELLULAR OSTEOPONTIN CONTENT

The bone morphogenetic proteins (BMPs) are characterized by their abil ity to induce both chondrogenic and osteogenic differentiation of mese nchymal cells in vivo and in vitro. Primary cultures of fetal rat calv arial cells contain a broad spectrum of osteogenic cells at various st ages of differentiation, but the responsive subpopulations are incompl etely characterized. To identify responsive cells in osteogenic cell d ifferentiation, we have treated fetal rat calvarial cells with recombi nant osteogenic protein-1 and used flow cytometric analyses of intrace llular osteopontin, and of cartilage and bone nodule formation, to eva luate the effects. When administered as a single dose at confluence, o steogenic protein-1 stimulated bone nodule formation in fetal rat calv arial cultures in dose-dependently way. To determine the response of o steogenic subpopulations at two discrete stages of differentiation, fe tal rat calvaria cells were cultured for 2 days (proliferative stage) or 12 days (early mineralization stage) and treated with 100 ng/ml rec ombinant osteogenic protein-1 for 12 h before analysis by flow cytomet ry. Flow cytometry analyses of cell suspensions revealed that osteogen ic protein-1 increased the total protein content of cells, and selecti vely increased the mean expression of osteopontin and the size and gra nularity of osteopontin expressing cells, particularly at day 12, cons istent with a stimulation of osteogenic differentiation and matrix for mation. Pulse administration of 100 ng/ml osteogenic protein-1 to sort ed, osteopontin-negative subpopulations enriched for stem cells reduce d by more than four-fold the number and size of bone nodules while pro moting chondrogenesis and adipogenesis. In contrast, a pulse administr ation of osteogenic protein-1 to more differentiated, large osteoponti n-positive cells increased bone nodule formation two-fold. Continuous administration of 100 ng/ml osteogenic protein-1 to the large osteopon tin-positive and small osteopontin-negative cell populations obliterat ed bone nodule formation and promoted chondrogenesis. We conclude that pulse administration of osteogenic protein-1 promotes osteogenic diff erentiation of cells committed to the osteogenic lineage, whereas undi fferentiated periosteal cells are induced to differentiate along the c hondrogenic pathway. In contrast, continuous exposure to osteogenic pr otein-1 promotes chondrogenesis in populations of commit ted osteogeni c cells and in undifferentiated periosteal cells.