The impairement of bone formation and mineralization in BSP−/− mouse calvaria cell cultures is partly rescued by increasing cell density

International audienceBone sialoprotein regulates osteoblast activity and bone formation. In knockout (BSP-/-) mouse bone marrow (BM) stromal cell cultures, the pool of osteoprogenitor (OP) cells (CFU-F number) is not different from wild type (+/+), nor is their early differentiation (same numbers of alkaline phosphatase positive colonies =CFU-ALP, although these are smaller), while the number of osteoblast, mineralized colonies (CFU-OB) is dramatically reduced. Because ossification of newborn BSP-/- mouse calvaria is delayed, we analysed the impact of the mutation on in vitro osteogenesis in cultures of mouse calvaria cells (MCC), isolated from 6 days old mice by collagenase digestion. In contrast to BM, CFU-F, CFU-ALP and CFU-OB numbers were lower in BSP-/- MCC cultures. Consistent with less OP, BSP-/- cultures displayed lower proliferation and delayed growth. In MCC cultures seeded at 5000cell/cm2 osteoblast marker expression did not differ between genotypes until D6. By D14 (=first CFU-OBs) ALP, Coll1, OSX, Runx2 as well as terminal differentiation markers, OCN, PHEX, DMP1 and MEPE increased strongly in BSP+/+ cultures but was low/absent in BSP-/-, with no mineralization. In contrast, osteopontin (OPN) was over-expressed in BSP-/- dishes. At high density (≥25000cell/cm2), marker levels were similar for both genotypes, and BSP-/- cultures mineralized. OPN is a potent inhibitor of mineralization, and was reported to be a substrate for PHEX. Very low PHEX expression in low density BSP-/- cultures suggests that OPN is less degraded and might inhibit mineralization. Increased PHEX expression at higher density would permit OPN degradation and mineralization. Lack of BSP thus reduces MCC culture clonogenicity, differentiation and activity, consistent with lower bone formation in vivo. A BSP-/- bone microenvironment may alter proliferation/cell fate in early OP, explaining the smaller size of CFU-PAL observed in BM cultures