Flow cytometric analysis of recombinant human osteogenic protein-1 (BMP-7) responsive subpopulations from fetal rat calvaria based on intracellular osteopontin content.

Abstract

The bone morphogenetic proteins (BMPs) are characterized by their ability to induce both chondrogenic and osteogenic differentiation of mesenchymal cells in vivo and in vitro. Primary cultures of fetal rat calvarial cells contain a broad spectrum of osteogenic cells at various stages of differentiation, but the responsive subpopulations are incompletely characterized. To identify responsive cells in osteogenic cell differentiation, we have treated fetal rat calvarial cells with recombinant osteogenic protein-1 and used flow cytometric analyses of intracellular osteopontin, and of cartilage and bone nodule formation, to evaluate the effects. When administered as a single dose at confluence, osteogenic protein-1 stimulated bone nodule formation in fetal rat calvarial cultures in dose-dependently way. To determine the response of osteogenic subpopulations at two discrete stages of differentiation, fetal rat calvaria cells were cultured for 2 days (proliferative stage) or 12 days (early mineralization stage) and treated with 100 ng/ml recombinant osteogenic protein-1 for 12 h before analysis by flow cytometry. Flow cytometry analyses of cell suspensions revealed that osteogenic protein-1 increased the total protein content of cells, and selectively increased the mean expression of osteopontin and the size and granularity of osteopontin expressing cells, particularly at day 12, consistent with a stimulation of osteogenic differentiation and matrix formation. Pulse administration of 100 ng/ml osteogenic protein-1 to sorted, osteopontin-negative subpopulations enriched for stem cells reduced by more than four-fold the number and size of bone nodules while promoting chondrogenesis and adipogenesis. In contrast, a pulse administration of osteogenic protein-1 to more differentiated, large osteopontin-positive cells increased bone nodule formation two-fold. Continuous administration of 100 ng/ml osteogenic protein-1 to the large osteopontin-positive and small osteopontin-negative cell populations obliterated bone nodule formation and promoted chondrogenesis. We conclude that pulse administration of osteogenic protein-1 promotes osteogenic differentiation of cells committed to the osteogenic lineage, whereas undifferentiated periosteal cells are induced to differentiate along the chondrogenic pathway. In contrast, continuous exposure to osteogenic protein-1 promotes chondrogenesis in populations of committed osteogenic cells and in undifferentiated periosteal cells.