Fig. 6. Spatial and temporal expression of BSP and osteocalcin in BMSC pellet cultures. Haematoxylin and eosin stained section (a-d), BSP immunolabelling (e,f) and osteocalcin immunolabelling (g-l) at 7, 14 and 28 days of culture (chondrogenic conditions), and at 35 days of culture (28 days under chondrogenic conditions and 7 days under mineralization-permissive conditions). Concurrent with a morphological change in the outer layer of tissue (a-d), BSP production is restricted to a progressively thicker rind of tissue during the chondrogenic phase (e-g). Deposition of extracellular BSP (h, arrows) and osteocalcin (l) is only observed when mineralization has occurred (h, arrows). Notice the similar patterns of production of BSP and type I procollagen (Fig. 5), including the apparent centripetal shift of the cellular production of either protein between day 14 and 28 (f,g, double arrows for BSP). Bar, 50 µm.