1,25 dihydroxyvitamin-D3 attenuates the confluence-dependent differences in the osteoblast characteristic proteins alkaline phosphatase, procollagen I peptide, and osteocalcin.

In the present study a cell culture model of primary human osteoblasts based on degrees of confluence was investigated by measuring basal and 1,25(OH)2D3stimulated levels of the osteoblast characteristic proteins alkaline phosphatase (AP), procollagen I-peptide (PICP), and osteocalcin (OC), as well as the corresponding gene expression. Primary osteoblast-like cell cultures from seven donors were treated in the second passage with 1,25(OH)2D3 (5 x 10(-8) M for 48 hours) and investigated at four stages of confluence (stage I 50%, stage II 75%, stage III 100%, and stage IV 7 days postconfluence). In untreated cultures passing through the different stages of confluence, we saw a 1.8-fold increase of AP activity, a 2. 3-fold increase of OC secretion, but a decrease of PICP levels to 0. 36-fold. Gene expression showed only minor variation between the different confluence stages. 1,25(OH)2D3 did not significantly affect PICP production. Alkaline phosphatase protein was stimulated during proliferation until confluence, with no effect thereafter. Surprisingly, OC secretion and mRNA expression were stimulated in all four stages to the same absolute level independent of basal values. We conclude that our results correspond to other studies showing differentiation-stage dependent changes of basal levels of osteoblast-specific proteins. However, 1,25(OH)2D3 stimulation decreased the confluence-dependent difference for AP and abolished it for osteocalcin, thus leading to a more differentiated phenotype of the osteoblast. Therefore, 1,25(OH)2D3 stimulation might improve the reproducibility of results obtained at different confluence stages from cultures of clinical samples.

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.