Mature primary human osteocytes in mini organotypic cultures secrete FGF23 and PTH1-34-regulated sclerostin

Osteoblasts from 4 donors were seeded onto 20 constructs. Constructs were constructed as described: Calcium phosphate (brushite / β-TCP) posts were pinned into a Sylgard 184 silicone elastomer (base and curing agent, Dow Corning Corporation) base layer in 6-well dishes. Cells were seeded in fibrinogen onto a thrombin gel and subsequently cultured in 3ml of proliferation media (osteoblast media, 50 μg/ml ascorbate-2-phosphate, 40 μg/ml proline) per well until cells had fully contracted the gel between the support posts. Week 0 constructs were harvested and snap-frozen at this point with the remainder transferred to osteogenic media (proliferation media, 10 mM β-glycerophosphate, 10 nM dexamethasone) and harvested at 2, 4, 6 and 8 weeks. RNA expression profiling during differentiation of human primary osteoblasts to osteocytes in mini organotypic cultures. RNA extracted from the onset of contruction (Time 0) and at the onset of ossification (8 weeks) and at 2 week intervals in between, from four different donors.