Differentiation of osteoid-producing cells in vitro: possible evidence for the requirement of a microenvironment

Periostea were dissected from 17-day-old chicken embryo calvariae, placed on millipore filters, and cultured on fluid media containing serum or on serum and plasma containing "plasma clots," in three ways: 1) with the osteogenic layer facing the filter, 2) with the osteogenic layer away from the filter, 3) folded such that the osteogenic layer was in apposition with itself within the fold. The cultures were studied histologically as well as biochemically. Periostea that were cultured folded showed differentiation of osteoblastlike cells after 2 days, and production of osteoid at day 4. Tissues cultured with the osteogenic layer away from the filter demonstrated similar osteoblastic differentiation and osteoid production. Both types of cultures exhibited an increase in histochemically detectable alkaline phosphatase activity over the 4 day culture period that was associated with osteoblasts and the osteogenic area. Periostea cultured with the osteogenic layer facing the filter produced no osteoid. In these cultures, histochemically detectable alkaline phosphatase activity decreased and virtually disappeared over the 4 day culture period. The possibility that the creation of a suitable micro-environment is required for osteodifferentiation in this culture system is discussed.