Development of the osteoblast phenotype in primary human osteoblasts in culture: Comparison with rat calvarial cells in osteoblast differentiation

Abstract

In rat osteoblast-like cells, a time-dependent sequence of growth and differentiation-dependent genes has been identified and a model of osteoblast differentiation in culture suggested. We investigated the expression of the bone matrix-associated proteins osteonectin and procollagen I and of the bone cell phenotype-related proteins alkaline phosphatase and osteocalcin during cell culture in primary human osteoblast like cells. Primary human explant cultures from nine young healthy donors were established under highly standardized conditions. Cells in the second passage were analyzed on different days from day 1 to 32, comparing cells growing under the influence of ascorbate with controls. Gene expression was determined by Northern blot analysis or polymerase chain reaction. Osteocalcin expression was also investigated after 1,25-(OH)(2)D(3) stimulation. On the protein level, newly synthesized collagen I, alkaline phosphatase activity, and secretion of osteocalcin were analyzed at all time points. On comparing our findings to the pattern of gene expression suggested for the rat calvarial osteoblast system, we found a similar developmental sequence for the so-called "proliferation" as well as a similar, but lengthened, sequence for the "matrix maturation stage." During "matrix maturation," we found an ongoing proliferation despite increased alkaline phosphatase and decreased procollagen I gene expression. Our study, therefore, shows that in pHOB the gene expression profile proceeded to the "matrix maturation stage," as defined by Owen and colleagues, independent of ongoing proliferation. We were unable to observe the mineralization period as demonstrated by the missing increase of osteocalcin expression and lack of nodule formation in our human osteoblast model. In contrast to the rat system, we found a proliferation stimulating influence of ascorbate, suggesting species-specific differences in response to differentiation factors. From these data, we conclude that general considerations on physiology and pathophysiology of bone cell differentiation have to be confirmed in the human osteoblastic cell system.

Lysosome Dispersion in Osteoblasts Accommodates Enhanced Collagen Production during Differentiation

“…Lysosomes Colocalize with Procollagen-containing Vesicles in AA-treated Osteoblasts-Enhanced collagen synthesis is a hallmark feature of differentiated osteoblasts (29,39). We investigated extracellular and intracellular collagen production in control and AA-differentiated osteoblasts.…”

The influence on human osteoblasts in vitro of non-steroidal anti-inflammatory drugs which act on different cyclooxygenase enzymes

“…The effect on cell differentiation was studied by examination of collagen synthesis and alkaline phosphatase (ALP) production. 30,31 After completion of the original study, NO-donating NSAID became available and a preliminary study of their effect on cell number was added as a further comparison.…”

Choice of osteoblast model critical for studying the effects of electromagnetic stimulation on osteogenesisin vitro

“…A study showed that isolated rat osteoblasts expressed far less ALP, pro-collagen type I, transforming growth factor-β1 and interleukin 6 (IL-6) when compared to isolated human osteoblasts (Torricelli et al, 2003). There is also evidence that isolated rat osteoblasts respond species-specifically to differentiation factors (Siggelkow et al, 1999), including glucocorticoids in particular (Prince et al, 2001). These differences should be acknowledged when selecting animal cells for studying the interaction of electromagnetic energy and bone.…”