JOURNAL OF BONE AND MINERAL RESEARCH Volume 5, Supplement 1, 1990 Mary Ann Liebert, Inc., Publishers
The Effects of Fluoride on Osteoblast Progenitors In Vitro c.o. BELLOWS, J.N.M. HEERSCHE, and lE. AUBIN ABSTRACT The number of discrete, three-dimensional bone nodules formed in vitro from a class of osteoprogenitor cells present in fetal rat calvaria cell populations (RC cells) is linearly related to the number of cells plated, implying that this system functions as a colony assay for the expression of osteoprogenitor cells. To determine the effect of fluoride on the expression of these cells, primary RC cells were grown for periods of up to 21 days in a-MEM (minimal essential medium) containing 5-15 % heat-inactivated fetal bovine serum (FBS), 50 ug/ml ascorbic acid, 10mM Na 13-glycerophosphate, and NaF at concentrations from 10 !lM to 5 mM. The continuous presence of NaF resulted in an increase in the number of bone nodules with maximal response occurring at 500 !lM (p < 0.001). A similar response at 500 !lM NaF was observed also with regard to alkaline phosphatase activity. NaF levels up to 500 !lM did not affect the growth of the mixed RC cell population, however, higher concentrations (1 mM) significantly reduced cell numbers (p < 0.001) suggestive of cytotoxicity. Plating efficiency tests for colony formation in the presence of 0.5 to 2 mM NaF showed that the decreases in nodule formation observed at concentrations above 500 !lM correlated with cytotoxicity. NaC1 at 1mM had no effect on nodule formation, alkaline phosphatase activity, or cell growth. The results show that NaF stimulates osteoprogenitor cell number in vitro and that the maximal effect occurs at concentrations close to toxic levels.
INTRODUCTION LUORIDE (F) APPEARS TO BE THE ONLY THERAPEUTIC AGENT
that results
F in increased bone mass; possibly by inducing a positive uncoupling of bone remodeling, that is, a selective increase in bone formation over bone resorption.v'50 fells) were counted. A dose-dependent inhibition of colony formation is evident over the range of 0.6 to 1.2 mM. Points represent the mean ± 95% CL of 12 dishes from two experiments. (b) Nodule formation. RC cells were plated at 2x10 4 cells/35-mm dish and maintained for 15 days in vitro. The decrease in nodule number parallels the decrease in colony formation caused by NaF cytotoxicity. Points represent the mean ± 95% CL of 4--6 dishes.
multilayered and nodule formation was first beginning (Day 9; Fig. 4). Maximal stimulation occurred at 0.5 mM (p < .001 at both time points) which paralleled the most effective dose for nodule formation. Whereas the cytotoxic effects of NaF at high concentrations were evident in each experiment, the extent of stimulation in the number of bone nodules formed at lower concentration varied from one experiment to another. Table 1 summarizes the effects of 0.5 mM NaF (the peak stimulatory concentration) on bone formation in 10 independent isolates of RC cells over approximately 1.5 years. The maximum stimulation in number of bone nodules formed was approximately 1.5-fold over control in these experiments (the number formed in supplemented medium without NaF) and this effect fell to values as low as 1.1-fold (not significantly different from control). The mean fold increase was 1.32 with a 95% confidence interval of 0.08. The possibility that factors in the serum as well as differences between cell isolates might modulate NaF effects on nodule formation was tested by analyzing the effect of NaF in the presence of different serum concentrations. The number of nodules decreased progressively with decreasing FBS concentrations in control cultures, while nodule number decreased to a much less extent with 0.5 mM NaF, resulting in a greater fold increase at lower FBS concentrations (Fig. 5). In lower concentrations of NaF (10 and 75 lAM), nodule numbers did not differ significantly from those in control cultures.
DISCUSSION Fluoride administration in vivo typically results in increased bone mass, however, very few in vitro systems have been able to duplicate these results. Osteoprogenitor cells present in mixed fetal RC cell populations proliferate and differentiate in longterm culture to form discrete bone nodules. By limiting dilution analysis we have shown that the number of these osteoprogen-
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BELLOWS ET AL. TABLE I. FLUORIDE EFFECTS ON BONE NODULE FORMATION IN DIFFERENT CELL ISOLATES
Date
Control 45.6 273.0 77.0 28.2 44.4 213.3 119.8 164.8 54.0 77.6
26/11/87 3/2/88 6/8/86 28/7/87 16/7/87 11/2/88 2/2/88 23/3/88 12/3/86 21/1/88
0.5 mMF
± 4.2 ± 25.9 ± 8.6 ± 3.9 ± 7.8 ± 13.6 ± 7.6 ± 11.1 ± 3.3 ± 15.3
50.3 309.0 97.3 35.5 59.4 274.8 166.3 233.0 76.5 119.0
± 4.1 ± 34.7 ± 13.7 ± 2.5 ± 7.0 ± 19.4 ± 13.6 ± 27.2 ± 7.4 ± 12.9
p
Fold stimulation
NS NS NS
The Effects of Fluoride on Osteoblast Progenitors In Vitro c.o. BELLOWS, J.N.M. HEERSCHE, and lE. AUBIN ABSTRACT The number of discrete, three-dimensional bone nodules formed in vitro from a class of osteoprogenitor cells present in fetal rat calvaria cell populations (RC cells) is linearly related to the number of cells plated, implying that this system functions as a colony assay for the expression of osteoprogenitor cells. To determine the effect of fluoride on the expression of these cells, primary RC cells were grown for periods of up to 21 days in a-MEM (minimal essential medium) containing 5-15 % heat-inactivated fetal bovine serum (FBS), 50 ug/ml ascorbic acid, 10mM Na 13-glycerophosphate, and NaF at concentrations from 10 !lM to 5 mM. The continuous presence of NaF resulted in an increase in the number of bone nodules with maximal response occurring at 500 !lM (p < 0.001). A similar response at 500 !lM NaF was observed also with regard to alkaline phosphatase activity. NaF levels up to 500 !lM did not affect the growth of the mixed RC cell population, however, higher concentrations (1 mM) significantly reduced cell numbers (p < 0.001) suggestive of cytotoxicity. Plating efficiency tests for colony formation in the presence of 0.5 to 2 mM NaF showed that the decreases in nodule formation observed at concentrations above 500 !lM correlated with cytotoxicity. NaC1 at 1mM had no effect on nodule formation, alkaline phosphatase activity, or cell growth. The results show that NaF stimulates osteoprogenitor cell number in vitro and that the maximal effect occurs at concentrations close to toxic levels.
INTRODUCTION LUORIDE (F) APPEARS TO BE THE ONLY THERAPEUTIC AGENT
that results
F in increased bone mass; possibly by inducing a positive uncoupling of bone remodeling, that is, a selective increase in bone formation over bone resorption.v'50 fells) were counted. A dose-dependent inhibition of colony formation is evident over the range of 0.6 to 1.2 mM. Points represent the mean ± 95% CL of 12 dishes from two experiments. (b) Nodule formation. RC cells were plated at 2x10 4 cells/35-mm dish and maintained for 15 days in vitro. The decrease in nodule number parallels the decrease in colony formation caused by NaF cytotoxicity. Points represent the mean ± 95% CL of 4--6 dishes.
multilayered and nodule formation was first beginning (Day 9; Fig. 4). Maximal stimulation occurred at 0.5 mM (p < .001 at both time points) which paralleled the most effective dose for nodule formation. Whereas the cytotoxic effects of NaF at high concentrations were evident in each experiment, the extent of stimulation in the number of bone nodules formed at lower concentration varied from one experiment to another. Table 1 summarizes the effects of 0.5 mM NaF (the peak stimulatory concentration) on bone formation in 10 independent isolates of RC cells over approximately 1.5 years. The maximum stimulation in number of bone nodules formed was approximately 1.5-fold over control in these experiments (the number formed in supplemented medium without NaF) and this effect fell to values as low as 1.1-fold (not significantly different from control). The mean fold increase was 1.32 with a 95% confidence interval of 0.08. The possibility that factors in the serum as well as differences between cell isolates might modulate NaF effects on nodule formation was tested by analyzing the effect of NaF in the presence of different serum concentrations. The number of nodules decreased progressively with decreasing FBS concentrations in control cultures, while nodule number decreased to a much less extent with 0.5 mM NaF, resulting in a greater fold increase at lower FBS concentrations (Fig. 5). In lower concentrations of NaF (10 and 75 lAM), nodule numbers did not differ significantly from those in control cultures.
DISCUSSION Fluoride administration in vivo typically results in increased bone mass, however, very few in vitro systems have been able to duplicate these results. Osteoprogenitor cells present in mixed fetal RC cell populations proliferate and differentiate in longterm culture to form discrete bone nodules. By limiting dilution analysis we have shown that the number of these osteoprogen-
S104
BELLOWS ET AL. TABLE I. FLUORIDE EFFECTS ON BONE NODULE FORMATION IN DIFFERENT CELL ISOLATES
Date
Control 45.6 273.0 77.0 28.2 44.4 213.3 119.8 164.8 54.0 77.6
26/11/87 3/2/88 6/8/86 28/7/87 16/7/87 11/2/88 2/2/88 23/3/88 12/3/86 21/1/88
0.5 mMF
± 4.2 ± 25.9 ± 8.6 ± 3.9 ± 7.8 ± 13.6 ± 7.6 ± 11.1 ± 3.3 ± 15.3
50.3 309.0 97.3 35.5 59.4 274.8 166.3 233.0 76.5 119.0
± 4.1 ± 34.7 ± 13.7 ± 2.5 ± 7.0 ± 19.4 ± 13.6 ± 27.2 ± 7.4 ± 12.9
p
Fold stimulation
NS NS NS
