Autocrine signals promote osteoblast survival in culture

We have studied the survival requirements of osteoblasts to test the hypoth esis that osteoblasts undergo programmed cell death (PCD) or apoptosis unle ss they are continuously signalled by other cells not to do so. Osteoblasts survived for 6 days in culture at high cell density in the absence of othe r cell types, serum or exogenous proteins, but they died with the morpholog ical features of apoptosis in these conditions at low cell density. Osteobl ast survival was enhanced during the first 2 days of culture by the additio n of the sulphydryl compound, cysteine to the culture medium which was conv erted intracellularly to the antioxidant glutathione. Catalase, an enzyme d ecomposing hydrogen peroxide, also protected the cells, whereas superoxide dismutase had no effect. Therefore, osteoblasts in culture are sensitive to toxic compounds derived from molecular oxygen, i.e. hydroxyl radicals or h ydrogen peroxide spontaneously generated in CMRL medium containing ascorbat e and ferrous ions. Conditioned medium from high density cultures prevented osteoblast apoptosis in low density cultures, as long as antioxidants were also present. The enhancing effect of conditioned medium on osteoblast sur vival was prevented by neutralizing antibodies to insulin-like growth facto r-I (IGF-I) and IGF-II but not by antibodies to either platelet-derived gro wth factor (PDGF) or basic fibroblast growth factor (bFGF). These results s uggest that in addition to regulating cell growth and differentiation, IGF- I and IGF-II also function as survival factors for osteoblasts. Our data al so indicate that antioxidants are required for osteoblast survival and that they enhance growth factor mediated osteoblast survival.