Proliferative responses to estradiol, IL-1 alpha and TGF beta by cells expressing alkaline phosphatase in human osteoblast-like cell cultures

Abstract

The use of primary (nontransformed) bone cell cultures is hampered by their cellular heterogeneity. Primary cultures of osteoblast-like cells have been shown to proliferate in response to several osteotropic agents, but because mixed cell populations are present it is uncertain whether a true osteoblastic response was observed. By combining (1) localization of [3H]-thymidine incorporation into the nuclei of actively dividing cells by autoradiography with (2) subsequent induction of osteoblast differentiation by 1,25(OH)2D3 to optimize the number of cells expressing high alkaline phosphatase activity and (3) its localization by histochemical staining, it is possible to measure the proliferation of cells that are capable of expressing a more mature osteoblastic phenotype in heterogeneous human trabecular bone cell cultures. Over a 72-hour incubation period, rhIL-1 alpha (0.2-2 ng/ml) exerted a dose-dependent stimulation of proliferation of cells expressing alkaline phosphatase. Purified human TGF beta 1 produced a biphasic increase in the proliferation of these cells (0.01-1 ng/ml) but 17 beta and 17 alpha-estradiol (10(-12)-10(-8) M) failed to consistently regulate cell growth. Furthermore, 17 beta-estradiol did not reproducibly modulate proliferation induced by IL-1 alpha or TGF beta when added together in cultures. This procedure represents a more accurate method for the assessment of osteoblast proliferation in primary bone cell cultures and demonstrates that estrogen is not mitogenic for human osteoblasts and does not potentiate the actions of putative local stimulators of osteoblast replication.