The use of confluence stages does not decrease the overall variability in primary human osteoblasts but can give additional information on differentiation in vitro.

Primary cultures of human osteoblast-like cells are frequently used to study osteoblast function. Due to inhomogeneous growth of primary osteoblasts in culture, it is of interest if the use of confluence stages for analysis would reduce the overall variability. Consequently, we have tested the influence of degree of confluence and passage number on the growth and differentiation of human primary osteoblast-like cells. Phenotypic features of primary human osteoblast-like cells were compared at four successive cell densities defined as stage of confluence I (50%), stage II (75%), stage III (100%) and stage IV (5 days post confluence). The stability of the system was also tested by comparing these observations obtained using cells from the 2nd and 4th passages. As a sign for further differentiation, the number of AP-positive cells increased with a decrease in proliferation. The secretion of procollagen-I decreased to 50% during culture while procollagen I mRNA doubled from proliferation to confluence. A constant activity of alkaline phosphatase, procollagen-I secretion and procollagen I gene expression over passages was seen together with a decrease in growth. The paper introduces a potential model of osteoblastic differentiation in vitro for human primary osteoblast-like cells. We were able to show an increasing differentiation with a decrease in proliferation and the stability of this differentiation behaviour over cell passages in this model, but we were not able to reduce the overall variability.