A New fluorometric assay for determination of osteoblastic proliferation: Effects of glucocorticoids and insulin-like growth factor-I

Abstract

A novel fluorometric proliferation assay, AlamarBlue (AB), was used to study the proliferative capacity of isolated human osteoblasts (hOBs). AB is an oxidation-reduction indicator that yields a fluorescent signal in response to metabolic activity. The assay was performed by replacing the experiment media in a microtiter plate with a 10% AB solution and measuring fluorescence after a 3-8-hour incubation. The assay was optimized with respect to incubation time, cell density, and AB concentration. When the results of the AB assay were compared with cell counting in a Bürker chamber there were consistently good correlations (r > 0.9), regardless of the agonist with which the cells were treated. The mean intraassay coefficient of variance (CV) values were 9.9-11.8% in experiments where osteoblasts were treated for 12 days with insulin-like growth factor-I (IGF-I; 100 nM), or dexamethasone (1 micro;M). IGF-I dose dependently, at and above 1 nM, stimulated proliferation of hOBs. This effect was detectable after 3 days and reached 130-140% of untreated controls after 12 days in culture. The effects of dexamethasone (DEX) on the proliferation rate of hOBs were more complex. In short-term cultures, 3 days, DEX dose dependently stimulated proliferation. However, at and above 6 days, DEX exerted a biphasic effect, with stimulation seen at 1-10 nM and a marked inhibition of cell proliferation at and above 100 nM. dexamethasone, hydrocortisone, prednisolone, and deflazacort had almost identical biphasic effects on osteoblastic proliferation in 12 day cultures with a stimulation seen at 1-10 nM, and a marked inhibition down to 50-60% of untreated controls at and above 100 nM. When IGF-I (0. 1-100 nM; 12 day culture) was combined with different doses of DEX, IGF-I still dose dependently stimulated the proliferation rate in hOBs regardless of the amount of DEX added. The stimulatory effect of DEX (10 nM, 12 days culture) was additive to the effect of 100 nM IGF-I. We conclude that AB is an easy and reliable assay for osteoblastic cell proliferation, well suited for large scale studies of cell growth using small amounts of cells, and that IGF-I partly reverses the glucocorticoid-induced inhibition of osteoblastic proliferation.

Thrombin, but not bradykinin, stimulates proliferation in isolated human osteoblasts, via a mechanism not dependent on endogenous prostaglandin formation

“…The light emitted from a vertical light path on each well was read at 590 nm. The fluorescence thus obtained has been shown to be directly proportional to the number of cells in each well (Jonsson et al 1997). …”

Inflammatory cytokines regulate proliferation of cultured human osteoblasts

“…The light emitted from a vertical light path on each well in 96-well microtiter plates was read at 590 nm. The fluorescence thus obtained is directly proportional to the cell number in each well (Jonsson et al 1997). The fluorescence obtained in control wells was set to 100% and effects of agonists were compared to this value.…”

Sol–gel derived aluminosilicate coatings on alumina as substrate for osteoblasts

“…After 14 days, with the exception of T/Rmac, the cell activities showed a distinct drop, and for the sol-gel aluminosilicates there seemed to be an inverse correlation between the cell activity at 14 days and cell-specific Ca precipitation. The reductive cell activity is known to correlate with osteoblast numbers during proliferation [38], which, however, is not necessarily true for post-confluent cultures where cell metabolism has changed and the cells become embedded in mineralizing ECM [39]. Thus, in the case of T/Rmac the high cell activity at 14 days together with the lowest cell-specific Ca precipitation can be interpreted as delayed mineralization, apparently due to the macroporous surface.…”