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

In rat osteoblast-like cells, a time-dependent sequence of growth and diffe rentiation-dependent genes has been identified and a model of osteoblast di fferentiation in culture suggested. We investigated the expression of the b one matrix-associated proteins osteonectin and procollagen I and of the bon e cell phenotype-related proteins alkaline phosphatase and osteocalcin duri ng cell culture in primary human osteoblast like cells. Primary human expla nt cultures from nine young healthy donors were established under highly st andardized conditions. Cells in the second passage were analyzed on differe nt days from day 1 to 32, comparing cells growing under the influence of as corbate with controls. Gene expression was determined by Northern blot anal ysis or polymerase chain reaction. Osteocalcin expression was also investig ated after 1,25-(OH)(2)D-3 stimulation. On the protein level, newly synthes ized collagen I, alkaline phosphatase activity, and secretion of osteocalci n were analyzed at all time paints. On comparing our findings to the patter n 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 sta ge." 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 proce eded to the "matrix maturation stage," as defined by Owen and colleagues, i ndependent of ongoing proliferation. We were unable to observe the minerali zation period as demonstrated by the missing increase of osteocalcin expres sion and lack of nodule formation in our human osteoblast model. In contras t to the rat system, we found a proliferation stimulating influence of asco rbate, suggesting species-specific differences in response to differentiati on factors. From these data, we conclude that general considerations on phy siology and pathophysiology of bone cell differentiation have to be confirm ed in the human osteoblastic cell system. (C) 1999 Wiley-Liss, Inc.