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.

Material properties and in vitro biocompatibility of a newly developed bone cement

“…Except for some small donor-dependent variations, no cytotoxic effects on pHOB cell density, morphology and viability could be observed during incubation with COOL formulation A independent on the setting time. In accordance to Siggelkow et al 30 , pHOB cells from both donors showed a typical long-stretched fibroblast-like appearance during early proliferation, which switched to polygonal during continuing proliferation. In contrast, pHOB cells incubated with Refobacin ® R added to the cultures directly after preparation showed extremely high death rates, probably due to chemical necroses caused by the release of free, not polymerized, toxic MMA monomers.…”

“…Another component of the ceramic mixture is CZP, which is biostable 2,30 and increases the radiopacity of COOL. It accounts for less than 10 wt.…”

Sustained In Vitro Expansion of Bone Progenitors Is Cell Density Dependent

“…Consistent with the findings of an earlier study on the proliferation and differentiation of osteoprogenitor cells in the presence or absence of dex [32], CFU-O were not detectable beyond 18-20 population doublings. It is also of interest that in the hematopoietic system dex in combination with erythropoietin and stem cell factor was able to induce erythroid progenitors to undergo a similar number of cell divisions (15)(16)(17)(18)(19)(20)(21)(22), corresponding to a 10 5 -10 6 -fold amplification before a loss in proliferation and differentiation capacity was measured [43]. Dex acts directly on human erythroid progenitors, maintaining their colony-forming capacity and delaying their differentiation, with the glucocorticoid receptor acting as a key regulator for the decision between self-renewal and differentiation [43,44].…”

“…Identifying a relationship between population growth, nodule formation, and expression of surface or other osteoblast markers is the object of much study [15,16,28,[49][50][51][52]. To this end, separation of the input RC population into four purified fractions (ALP showed that nodules are able to form within each fraction [53].…”

Effect of advanced oxidation protein products on the proliferation and osteogenic differentiation of rat mesenchymal stem cells

“…During proliferation, the production of collagen I initially occurs, followed by an increased in the expression of ALP and calcium deposition (28). Collagen I comprises approximately 90% of the organic material as the most abundant protein component of the bone matrix.…”