Analysis of intracellular osteopontin as a marker of osteoblastic cell differentiation and mesenchymal cell migration

Formation and repair of the hard and soft connective tissues of teeth and their supporting structures require stem cells to divide, differentiate and migrate to generate specific tissues in a defined temporo-spatial sequence. We have used antibodies to osteopontin (OPN) and fluorescence-activated cell sorting (FACS) to determine the relationship between OPN expression and cell differentiation in cultures of fetal rat calvarial cells. At different stages of osteogenic differentiation, OPN was expressed by 60-98% of cells. Populations of small OPN-negative cells with low cellular granularity (S cells) were isolated and shown to be enriched in stem cells, characterised by a lack of differentiation markers, high proliferative potential, capacity for self renewal and multipotentiality. Within 24 h of plating, S cells attached, spread and started expressing OPN, CD44, and collagens types I, II and III. Confocal microscopy of OPN in differentiating cells revealed two distinct phenotypes; a perinuclear distribution, characteristic of secreted OPN, and an intracellular perimembranous distribution co-localising with the CD44 receptor, characteristic of migrating cells in which OPN was increased > 10-fold as measured by immunoblotting. These studies show that OPN is expressed early in mesenchymal cell differentiation and is related to cell migration as well as osteogenesis.