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, differen tiate and migrate to generate specific tissues in a defined temporo-sp atial sequence. We have used antibodies to osteopontin (OPN) and fluor escence-activated cell sorting (FAGS) to determine the relationship be tween OPN expression and cell differentiation in cultures of fetal rat calvarial cells. At different stages of osteogenic differentiation, O PN 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 differentiat ion markers; high proliferative potential, capacity for self renewal a nd 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 disti nct phenotypes; a perinuclear distribution, characteristic of secreted OPN, and an intracellular perimembranous distribution co-localising w ith the CD44 receptor, characteristic of migrating cells in which OPN was increased > 10-fold as measured by immunoblotting. These studies s how that OPN is expressed early in mesenchymal cell differentiation an d is related to cell migration as well as osteogenesis.