Site-specific differences in osteoblast phenotype, mechanical loading response and estrogen receptor-related gene expression

Borzo Gharibi, Mandeep S Ghuman, Giuseppe Cama, Simon C F Rawlinson, Agamemnon E Grigoriadis, Francis J Hughes

The osteoporosis-resistant nature of skull bones implies inherent differences exist between their cellular responses and those of other osteoporosis-susceptible skeletal sites. Phenotypic differences in calvarial and femoral osteoblastic responses to induction of osteogenesis, mechanical loading, estrogen, growth factor and cytokine stimulation were investigated. Primary rat calvarial and femoral adult male osteoblasts were cultured and osteoblastic mineralisation and maturation determined using Alizarin Red staining and expression of osteogenic marker genes assessed. Expression of known mechanically-responsive genes was compared between sites following loading of scaffold-seeded cells in a bioreactor. Cell proliferation and differentiation following growth factor and estrogen stimulation were also compared. Finally expression of estrogen receptors and associated genes during osteogenic differentiation were investigated. Calvarial osteoblasts exhibited delayed maturation (45d. vs 21d.) and produced less mineralised matrix than femoral osteoblasts when osteogenically induced. PDGF-BB and FGF2 both caused a selective increase in proliferation and decrease in osteoblastic differentiation of femoral osteoblasts. Mechanical stimulation resulted in the induction of the expression of Ccl2 and Anx2a selectively in femoral osteoblasts, but remained unchanged in calvarial cells. Estrogen receptor beta expression was selectively upregulated 2-fold in calvarial osteoblasts. Most interestingly, the estrogen responsive transcriptional repressor RERG was constitutively expressed at 1000-fold greater levels in calvarial compared with femoral osteoblasts. RERG expression in calvarial osteoblasts was down regulated during osteogenic induction whereas upregulation occurred in femoral osteoblasts. Bone cells of the skull are inherently different to those of the femur, and respond differentially to a range of stimuli. These site-specific differences may have important relevance in the development of strategies to tackle metabolic bone disorders.