Ovariectomy of 12-month-old rats: effects on osteoprogenitor numbers in bone cell populations isolated from femur and on histomorphometric parameters of bone turnover in corresponding tibia.

Ovariectomy (OVX) in rats results in increased bone turnover and decreased bone volume and bone mineral density when measured in the metaphyses of long bones. We have investigated the effects of OVX on changes in the number of progenitors in cell populations derived from the metaphyseal bone of femurs of ovariectomized rats at 12 months of age, by using colony assays, bone nodule assays, and limiting dilution analysis at 1.5 and 9 months post-OVX. We have also measured histomorphometric parameters of bone formation and resorption in the corresponding tibia at the same time-points. A significant increase, as shown by bone nodule assays and limiting dilution analysis, occurs in the number of progesterone- and dexamethasone-responsive osteoprogenitors in cell populations isolated from ovariectomized rats at the 9-month post-OVX time-point. Progesterone-responsive osteoprogenitors are also increased at 1.5 months post-OVX. The number of fibroblast colony-forming units does not change. Histomorphometry has shown that OVX causes an increase in osteoblast surfaces, mineralizing surfaces, and bone formation rate at both 1.5 and 9 months post-OVX. The mineral apposition rate is increased at 1.5 months post-OVX. OVX also increases parameters of bone resorption at both time-points, the net result being a decrease in bone mineral density and cancellous bone volume at 9 months post-OVX. Thus, OVX in rats at 12 months of age is associated with an increase in the number of both progesterone- and dexamethasone-responsive osteoprogenitors 9 months post-OVX; this corresponds with increases in the histomorphometric parameters of bone formation.

Different effects of insulin and insulin-like growth factors I and II on osteoprogenitors and adipocyte progenitors in fetal rat bone cell populations.

We investigated the effects of insulin (1-1,000 nM), insulin-like growth factor (IGF)-I, and IGF-II (3-100 nM each) alone or together with 10 nM dexamethasone (DEX) or 10 nM 1,25-dihydroxyvitamin D(3) (1,25[OH](2)D(3)) on proliferation and differentiation of adipocyte and osteoblast progenitors in bone cell populations derived from fetal rat calvaria. The effects on differentiation were evaluated by counting the number of bone or osteoid nodules and adipocyte colonies and the effects on proliferation, by measuring their size by image analysis. The types of cells studied were 1,25(OH)(2)D(3)- and DEX-responsive adipocyte progenitors and DEX-dependent and independent osteoprogenitors. Both IGF-I and IGF-II stimulated osteoprogenitor differentiation both alone and in the presence of DEX, while insulin stimulated osteoprogenitor differentiation only in the absence of DEX. Neither IGF-I/-II nor insulin affected proliferation of osteoprogenitors. Insulin had little effect on adipocyte differentiation by itself but strongly stimulated differentiation in the presence of either 1,25(OH)(2)D(3) or DEX, while IGF-II stimulated adipocyte differentiation in both the absence and presence of 1,25(OH)(2)D(3) or DEX. IGF-I by itself or in the presence of DEX strongly stimulated adipocyte cell differentiation but had little effect in the presence of 1,25(OH)(2)D(3). Our results demonstrate that insulin, IGF-II, and IGF-I have specific and different effects on the differentiation and proliferation of different groups of progenitor cells.

Effect of simulated weightlessness on osteoprogenitor cell number and proliferation in young and adult rats.

Experiments with rats flown in space or hind limb unloaded (HU) indicate that bone loss in both conditions is associated with a decrease in bone volume and osteoblast surface in cancellous and cortical bone. We hypothesize that the decrease in osteoblastic bone formation and osteoblast surface is related to a decrease in the number of osteoprogenitors and/or decreased proliferation of their progeny. We tested this hypothesis by evaluating the effect of 14 days of HU on the number of osteoprogenitors (osteoblast colony forming units; CFU-O), fibroblastic colony forming units (CFU-F), and alkaline phosphatase-positive CFU (CFU-AP) in cell populations derived from the proximal femur (unloaded) and the proximal humerus (normally loaded) in 6-week-old and 6-month-old rats. To confirm the effect of unloading on bone volume and structure, static histomorphometric parameters were measured in the proximal tibial metaphysis. Effects of HU on proliferation of osteoprogenitors were evaluated by measuring the size of CFU-O. HU did not affect the total number of progenitors (CFU-F) in young or adult rats in any of the cell populations. In femoral populations of young rats, HU decreased CFU-O by 71.0% and mean colony size was reduced by 20%. HU decreased CFU-AP by 31.3%. As expected, no changes in CFU-O or CFU-AP were seen in cell populations from the humerus. In femoral cell populations of adult rats, HU decreased CFU-O and CFU-AP by 16.6% and 36.6%, respectively. Again, no effects were seen in cell populations from the humerus. In 6-week-old rats, there was a greater decrease in bone volume, osteoblast number, and osteoblast surface in the proximal tibial metaphysis than that observed in adult rats. Both trabecular thickness and trabecular number were decreased in young rats but remained unaffected in adults. Neither osteoclast number nor surface was affected by unloading. Our results show that the HU-induced decrease in the number of osteoprogenitors observed in vitro parallels the effects of HU on bone volume and osteoblast number in young and old rats in vivo, suggesting that the two may be interdependent. HU also reduced CFU-O colony size in femoral populations indicating a diminished proliferative capacity of osteoblastic colonies.

Proliferation, differentiation and self-renewal of osteoprogenitors in vertebral cell populations from aged and young female rats.

A significant contribution to the bone loss associated with aging is likely to be a decline in bone formation. We have characterized and compared the number, capacity for proliferation and differentiation and the self-renewal ability of osteoprogenitors of aged (17-26-month-old) and young (1.5-month-old) female Wistar rats using limiting dilution analyses and continuous subculture experiments. Cells were obtained from outgrowths of explants of lumbar vertebrae (L1-L6) and grown in alpha-minimal essential medium (alpha-MEM), 10% FBS and 50 microg/ml ascorbic acid with or without dexamethasone (Dex; 0.3-300 nM) or progesterone (Prog; 0.01-10 microM). Growth curves for cell populations of both age groups were similar with population doubling times of 27.1 and 26.7 h for the aged and young animals, respectively. Osteoprogenitors from both age groups formed bone nodules when cultured in the presence of either Dex or Prog. Limiting dilution analysis in the presence of 10 nM Dex showed no difference between the aged and young rats in the number of colony forming units-fibroblast (CFU-F), alkaline phosphatase-positive colony forming units-fibroblast (AP+ CFU-F) or colony forming units-osteoblast (CFU-O). No differences were also found for any progenitor within the aged group. Limiting dilution analysis in the presence of 3 microM Prog showed no differences in the numbers of CFU-F, AP+ CFU-F or CFU-O between the aged and young groups or within the aged group. Continuous subculture of cells in the presence of 10 nM Dex revealed that the number of nodules per 10(4) plated cells increased in second subculture over first subculture cells in the young group but decreased in the aged group. Also, in third to fifth subculture cells, the number of nodules was lower in the aged group than in the young group. A similar pattern was observed in the presence of 3 microM Prog. Results indicate that the cell population doubling times, growth characteristics, and the number of CFU-F and osteoprogenitors in vertebral bone cell populations from aged rats and young rats are similar. This suggests that the bone loss associated with aging is not caused by a decrease in osteoprogenitor cell number. However, cell populations from the aged rats showed a reduced capacity for self-renewal in vitro, which would ultimately translate into a reduced number of osteoblasts and might be partly responsible for a decrease in bone formation in aged animals.

The frequency of common progenitors for adipocytes and osteoblasts and of committed and restricted adipocyte and osteoblast progenitors in fetal rat calvaria cell populations.

In fetal rat calvaria (RC) cell populations, adipocyte differentiation is stimulated by both dexamethasone (Dex) and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], whereas osteoblast differentiation is stimulated by Dex but inhibited by 1,25(OH)2D3. We examined whether the osteoblastic and adipocytic colonies were derived from a common progenitor, from committed and restricted adipocyte and osteoblast progenitors, or from both and whether the adipocyte progenitors stimulated by 1,25(OH)2D3 constitute a population of progenitors that is different from that stimulated by Dex. RC cells were isolated by sequential enzyme digestion yielding five populations designated I-V. In population I the effect of Dex on adipocyte formation was greater than that of 1,25(OH)2D3, whereas the effect of 1,25(OH)2D3 was greater than that of Dex in populations III-V. We next applied replica plating techniques to further investigate the response characteristics of individual osteoprogenitors and adipocyte progenitors by looking at the fate of duplicate colonies derived from the same progenitor under different culture conditions. RC cells were plated at 1,000-1,500 cells/100 mm culture dish and a 17-microm mesh polyester membrane overlaid onto master dishes on day 4 or day 5 and removed on day 11 or day 12. Then, replicas and master dishes were cultured separately in medium containing either Dex, 1,25(OH)2D3, or Dex plus 1,25(OH)2D3 for a further 17-21 days and then fixed and stained with both Sudan IV and the von Kossa technique. Nine hundred twenty-seven matched colonies present on both master dishes and replica membranes were screened and colonies were classified as either adipocytic, osteoblastic (bone or osteoid), or fibroblastic. Results show convincingly that most of the osteoprogenitors present in fetal RC cells are committed and restricted to the osteoblastic cell lineage (95.29%); that the 1,25(OH)2D3-responsive adipocyte progenitors are different from the Dex-responsive adipocyte progenitors, but both are restricted to form adipocytes and finally; and that a common osteoblastladipocyte progenitor is present in a low frequency (4.71% of osteoprogenitors).

Expression of mRNAs for type-I collagen, bone sialoprotein, osteocalcin, and osteopontin at different stages of osteoblastic differentiation and their regulation by 1,25 dihydroxyvitamin D3.

We have used in situ hybridization to evaluate the effects of 1,25 dihydroxyvitamin D3 (1,25 (OH)2 D3) on the expression of mRNA for bone-matrix proteins and to determine whether mature osteoblasts respond differently to 1,25 (OH)2 D3 than younger, newly differentiated osteoblasts. Rat calvaria cells were cultured for 7, 12, 15, and 19 days to obtain a range of nodules from very young to very mature. At each time point, some cultures were treated with 10 nM 1,25 (OH)2 D3 for 24 h prior to fixation. In control cultures, type-I collagen mRNA was detectable in osteoblastic cells in very young nodules and increased with increasing maturity of the nodules and the osteoblasts lining them. The bone sialoprotein mRNA signal was weak in young osteoblasts, increased in older osteoblasts, and decreased in mature osteoblasts. Weak osteocalcin and osteopontin signals were seen only in osteoblasts of intermediate and mature nodules. 1,25 (OH)2 D3 treatment markedly upregulated osteocalcin and osteopontin mRNAs and downregulated mRNA levels of bone sialoprotein and, to a lesser extent, type-I collagen in both young and mature osteoblasts. However, a marked diversity of signal levels for bone sialoprotein, osteocalcin, and osteopontin existed between neighboring mature osteoblasts, particularly after 1,25 (OH)2 D3 treatment, which may therefore selectively affect mature osteoblasts, depending on their differentiation status or functional stage of activity.

Aluminum accelerates osteoblastic differentiation but is cytotoxic in long-term rat calvaria cell cultures.

We have examined the effects of aluminum (Al) on osteoprogenitor proliferation and differentiation, cell survival, and bone formation in long-term rat calvaria (RC) cell cultures. RC cells were grown in alpha minimal essential medium containing 10% fetal bovine serum, 50 microg/ml ascorbic acid, and 10 mM beta-glycerophosphate with or without Al added to final concentrations of 1 microM-1 mM. Al caused a dose-dependent increase in the number of bone nodules present at early times (day 11) but had no significant effect on nodule numbers at later times (day 17). Time course experiments showed that Al increased nodule number beginning from day 7. Alkaline phosphatase activity, assessed at four stages during the differentiation sequence of RC cell cultures (from 4 to 13 days) was stimulated by Al at all times. However, Al decreased colony formation, inhibited cell growth in late log phase, and decreased saturation density of the treated cultures. Al concentrations of 30 microM and above resulted in degeneration of the cell layer and an increasing fibrillar appearance of the matrix present in between or adjacent to nodules when cultures were maintained for more than 15 days. The presence of Al significantly decreased the viability of cells obtained from 13-17 days cultures, as determined by plating efficiency and trypan blue exclusion. We frequently observed cellular toxicity (in 8 of 10 experiments) in cultures containing 300 microM Al, and by days 17-19, cells, nodules, and matrix were disintegrating in these cultures. We conclude that Al accelerates the rate of osteoprogenitor cell differentiation and the formation of bone nodules while concomitantly inhibiting nodule mineralization. However, concentrations that accelerate differentiation appear to be cytotoxic in long-term cultures.

Osteoprogenitor cells in cell populations derived from mouse and rat calvaria differ in their response to corticosterone, cortisol, and cortisone.

Osteoprogenitors present in cell populations derived from fetal or newborn rat and mouse calvaria differentiate in long term culture and form osteoblastic bone-forming colonies (bone nodules). Previous reports have indicated considerable differences between bone cell populations derived from these two species with regard to their proliferation in response to glucocorticoids. In the present investigation, we have focused on proliferation and differentiation of osteoprogenitor cells in these bone cell populations and evaluated the effect of corticosterone, the principal glucocorticoid of both mouse and rat. Cells were isolated by sequential collagenase digestion from calvaria of newborn (2-5 days) CD-1 mice [mouse calvariae (MC) cells] and term fetal Wistar rats [rat calvaria (RC) cells] and cultured for up to 25 days in alpha-minimal essential medium containing 10% fetal bovine serum (FBS), antibiotics, 50 microg/mL ascorbic acid, and 8-10 mmol/L beta-glycerophosphate. In agreement with previous observations by us and others, corticosterone increased cell growth in RC cell cultures, but inhibited cell growth in MC cultures. In RC cell cultures, corticosterone (1-1000 nmol/L) increased the nodule number in a dose-dependent manner (p < 0.001 for all concentrations above 3 nmol/L) with a maximal effect at 300 and 1000 nmol/L (threefold increase over control). In MC cells, on the other hand, corticosterone (0.3-1000 nmol/L) increased the nodule number only at 30 nmol/L (50%, p < 0.01) but inhibited nodule formation by 33% (p < 0.001) at 1000 nmol/L. In both RC and MC cultures a linear relationship was found between the number of cells plated and number of nodules formed. When cultures were treated with cortisol (30-300 nmol/L), similar effects were observed; the number of nodules dose dependently increased in RC cell cultures and dose dependently decreased in MC cell cultures. Significantly, however, the inactive glucocorticoid cortisone also increased bone nodule formation in RC cell cultures and decreased bone nodule formation in MC cell cultures. Carbenoxolone, which blocks 11 beta hydroxysteroid dehydrogenase and thus prevents conversion of cortisone to cortisol, partially inhibited the cortisone-induced effects on bone nodule formation in both RC and MC cell cultures, indicating that both RC and MC cells can convert inactive glucocorticoids to active metabolites. In conclusion, our results show that the glucocorticoids corticosterone and cortisol inhibit proliferation and differentiation of osteoprogenitors in MC cell cultures but stimulate these processes in rat-derived osteoprogenitors.

The decrease in bone mass associated with aging and menopause.

The human skeleton accumulates bone up to approximately age 30, after which bone is gradually lost. Although estrogen replacement therapy prevents postmenopausal bone loss, it is not certain that estrogen deficiency alone is responsible for the decrease in bone mass. Progesterone deficiency could also be a factor, and progesterone replacement therapy has been shown to prevent postmenopausal bone loss associated with ovarian dysfunction. This article reviews what is known about bone remodeling and bone loss as a function of age and gender, discusses evidence from studies in rats that progesterone plays an important role in regulating bone formation, and suggests directions for future studies in predicting the success or failure of implant therapy based on the number and kinds of osteoprogenitor cells present.

Progesterone-mediated stimulation of osteoprogenitor proliferation and differentiation in cell populations derived from adult or fetal rat bone tissue depends on the serum component of the culture media.

We have shown previously that progesterone (Prog) and dexamethasone (Dex) stimulate osteoprogenitor proliferation and differentiation in cell populations derived from adult rat vertebrae and in primary cultures of fetal rat calvariae. In these two in vitro systems, osteoprogenitors can be identified by the appearance of colonies of differentiated osteoblasts producing bone (bone nodule formation). Culture conditions supporting proliferation and differentiation of osteoprogenitors include a requirement for the presence of serum in the culture media. Our major interest in the present study was to investigate whether Prog- and Dex-mediated osteoprogenitor proliferation and differentiation was observed to the same degree in different lots of fetal bovine serum (FBS). In addition, we wanted to investigate whether osteoprogenitors present in cell populations derived from fetal calvarial bone and those present in populations derived from adult vertebral bone would respond similarly under the different culture conditions. We found that, in populations derived from adult rat vertebrae, the effects of the serum component of the culture medium on the number of bone nodules induced by Prog and on the dose-dependency of the Prog effect were striking: in culture media containing the most effective serum the number of bone nodules was 22-fold higher than that in the least effective serum. In addition, Prog responses were detectable at 10(-5) M only in some sera but were significant at 10(-7) M in others. The effect of Dex in the adult rat vertebrae-derived populations was much less dependent on the serum used: the number of bone nodules in culture media containing the most effective serum was only 1.3 times greater than that in media containing the least effective serum. In cell populations derived from fetal calvariae, the serum dependence of the Prog response was less pronounced: a 4.3-fold increase over control was observed in the most effective serum, and a 2.4-fold increase in the least effective serum. No effects of the serum component of the culture medium on the Dex response were detectable. Thus, Prog-induced bone nodule formation appears to be strongly dependent on the particular type of FBS used for osteoprogenitors present in bone cell populations derived from adult rat vertebrae but much less so in populations obtained from fetal rat calvariae. Preliminary experiments suggest that the estrogen content of the culture media may be one of the determinants regulating Prog responsiveness of the osteoprogenitors. Dex-induced proliferation and differentiation of osteoprogenitors in bone cell populations derived from both adult rat vertebrae and fetal rat calvariae, on the other hand, did not appear to be strongly dependent on factor(s) present in the FBS component of the culture medium.

Aluminum inhibits both initiation and progression of mineralization of osteoid nodules formed in differentiating rat calvaria cell cultures.

Osteoid nodules form in cultures of fetal rat calvaria (RC) cells grown in medium containing 10% fetal bovine serum (FBS) and 50 microns/ml of ascorbic acid. When 10 mM beta-glycerophosphate (beta-GP) is added, the nodules mineralize in two phases: an initiation phase that is dependent upon alkaline phosphatase activity for cleavage of beta-GP to inorganic phosphate (P(i)) and a progression phase that proceeds independently of the activity of alkaline phosphatase and does not require exogenous phosphate. We have used this system to investigate the effects of aluminum (Al3+)on mineralization. When AlCl3 was added to culture medium at concentrations of 0, 3, 10, 30, 100, and 300 muM, the total concentrations of aluminum were 0.98, 6.07, 16.82, 40.19, 88.45, and 284.52 muM, respectively. The corresponding free Al3+ concentrations, assessed after ultrafiltration, were found to be 1.11, 1.75, 3.40, 6.22, 5.38, and 12.11 muM. In cultures in which osteoid was formed and mineralization initiated in the presence of added Al+ (3-300 muM), a dose-dependent inhibition of mineralization occurred. Osteoid formed in the presence of added Al3+ mineralized normally when Al3+ was removed from cultures at the time of initiation of mineralization with beta-GP. In osteoid nodules grown in the absence of Al3+, addition of Al3+ (3-300 muM) at the start of the initiation phase of mineralization resulted in a dose-dependent inhibition of mineralization. Addition of Al3+ to cultures after mineralization had been initiated in the absence of Al3+ inhibited progression of mineralization at added Al3+ concentrations of 10 muM and above. Al3+ did not decrease the conversion of beta-GP to P(i) and caused a small but significant increase in alkaline phosphatase activity at added concentrations of 100 muM or greater. The data show that Al3+ inhibits both the initiation and progression phases of mineralization starting at added concentrations of 3-10 muM (approximately 1.7-3.4 muM free Al3+) and that mineralization of osteoid formed in the presence of Al3+ is unaffected if Al3+ is removed prior to the initiation of mineralization.

Tri-iodothyronine (T3) and dexamethasone interact to modulate osteoprogenitor cell differentiation in fetal rat calvaria cell cultures.

We investigated the role of 3,5,3'-tri-iodothyronine (T3) in regulating differentiation of osteoprogenitor cells and also studied the effects of the glucocorticoid hormone dexamethasone (Dex) on the T3-induced effects on osteoprogenitor populations. This was done by determining the effects of either hormone alone, or of combinations of the two hormones, on the number of bone nodules formed in long-term cultures of rat calvaria cells. In this system, Dex has been shown to increase bone nodule formation, the maximal effective dose being 10 nM (Bellows et al. Endocrinology 121: 1985-1992; 1987). In standard culture medium containing 15% fetal bovine serum FBS), low concentrations of T3 (0.001-0.1 nM) had no effect on the number of bone nodules, while higher concentrations of 1-100 nM inhibited. However, in culture medium containing 10 nM Dex, the lower concentrations of T3 markedly increased the number of nodules. Short term pulse experiments with these low concentrations of T3 in the presence of Dex indicated that stimulation of nodule formation occurred only when T3 was present prior to confluency. Higher concentrations of T3 (1-100 nM) decreased nodule number whether or not Dex was added. We then cultured cells in medium containing FBS from which T3 and T4 were removed by treatment with AG-1 chi-10 resin. In both + or - Dex conditions, bone nodule formulation was increased 1.5 to 2-fold in T3, T4-depleted medium when compared with cultures maintained in standard culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

1,25-dihydroxyvitamin D3 stimulates adipocyte differentiation in cultures of fetal rat calvaria cells: comparison with the effects of dexamethasone.

Progenitor cells for several mesenchymally derived cell types exist within freshly isolated fetal rat calvaria (RC) cell populations. We have characterized the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on the differentiation of adipocytes from primary RC cells and compared these effects with those of dexamethasone (Dex). RC cells were plated at 3 x 10(4)/35-mm dish, and cultures were maintained for 14-19 days in alpha-Minimum Essential Medium containing 10% fetal bovine serum, 50 micrograms/ml ascorbic acid, 10 mM Na beta-glycerophosphate, and 0.1-100 nM 1,25-(OH)2D3 or 1-1000 nM Dex. Morphological (quantitation of adipocyte foci number and area after staining cultures with Sudan IV) and biochemical (glycerol-3-phosphate dehydrogenase activity) methods of assessing adipogenesis were used. In the presence of 1,25-(OH)2D3, adipocyte foci developed about 3 days after confluency as clusters of rounded or stellate cells. Stimulation of adipocyte foci development was dose dependent from 0.1-100 nM and was maximal with 10 nM 1,25-(OH)2D3; half-maximal stimulation occurred at about 1 nM. The presence of ascorbic acid and beta-glycerophosphate was not required for 1,25-(OH)2D3-induced stimulation of adipocytes, but both significantly increased the number of adipocyte foci in the presence of 1,25-(OH)2D3. The critical period for initiation of adipocyte differentiation with 1,25-(OH)2D3 was between 1-9 days, and once committed along the adipogenic pathway, adipocytes maintained their differentiated state in the absence of 1,25-(OH)2D3. Short term (48-h) pulses of 1,25-(OH)2D3 resulted in slight, but significant, increases in adipocyte formation. Other vitamin D3 metabolites were less effective than 1,25-(OH)2D3 in stimulating adipocyte differentiation. Dex (1-100 nM) also caused a dose-dependent increase in the differentiation of adipocyte foci in RC cell cultures. The adipocyte foci that developed in the presence of Dex frequently appeared earlier in culture, i.e. when cells reached confluency on days 6-7, and were more diffuse than those forming with 1,25-(OH)2D3. The stimulation of adipocyte differentiation by 1,25-(OH)2D3, however, was greater than that by Dex in mixed RC II-V cells. The combined effects of 1,25-(OH)2D3 and Dex were additive at low concentrations and synergistic at higher concentrations of either 1,25-(OH)2D3 or Dex. The data show that bone cell populations isolated from fetal RC contain adipocyte progenitors and that 1,25-(OH)2D3 as well as Dex are potent regulators of adipocyte differentiation within these bone cell populations.

Differential effects of fluoride during initiation and progression of mineralization of osteoid nodules formed in vitro.

Osteoid nodules form in cultures of fetal rat calvarial (RC) cells grown in medium containing 10% FBS and 50 micrograms/ml of ascorbic acid. When 10 mM beta-glycerophosphate (beta-GP) is added, osteoid nodules mineralize in two phases: an initiation phase, which is dependent upon alkaline phosphatase activity for conversion of beta-GP to P(i), and a progression phase that proceeds independently of alkaline phosphatase activity and does not require exogenous phosphate. We have now used this system to investigate the effects of fluoride (F-) on mineralization. In cultures in which osteoid was formed and mineralization initiated in the presence of F-, a dose-dependent inhibition of the initiation of mineralization occurred over a concentration range of 25-500 microM F- (p < 0.001 in all cases). The initiation of mineralization was not inhibited if F- was removed from the cultures at the time when mineralization was initiated with beta-GP. In osteoid nodules grown in the absence of F-, addition of F- resulted in a dose-dependent inhibition of the initiation of mineralization, with significant decreases in 45Ca uptake occurring at F- concentrations of 3 microM (p < 0.01) and higher. However, if F- was added to cultures after mineralization was initiated in the absence of F-, a stimulation of 45Ca uptake was observed at F- concentrations of 250 microM and above (p < 0.001). F- (1-1000 microM) did not affect the conversion of beta-GP to P(i) or alkaline phosphatase activity in the cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the 1,25-(OH)2D3-induced inhibition of bone nodule formation in long-term cultures of fetal rat calvaria cells.

We investigated the effects of 1,25-dihydroxyvitamin D3[1,25-(OH)2D3], on osteoprogenitor cell differentiation and bone nodule formation at various stages of differentiation by evaluating the effects on long term cultures of fetal rat calvaria (RC) cells. RC cells were plated at 3 x 10(4) cells/35-mm dish in alpha-minimal essential medium containing 15% fetal bovine serum, ascorbic acid, and beta-glycerophosphate (beta-GP), conditions under which bone nodules form. 1,25-(OH)2D3 inhibited bone nodule formation in a dose-dependent manner with total inhibition occurring at 1-10 nM and half-maximal inhibition occurring at approximately 0.06 nM. 1,25-(OH)2D3 also significantly stimulated RC cell growth in a dose-dependent manner in both the presence and absence of ascorbic acid. Addition of 1 nM 1,25-(OH)2D3 at different times after the start of culture inhibited nodule formation when added before and up to the early multilayering stage (up to day 11 of culture), but had no effect on nodule number when added later. When 1,25-(OH)2D3 was added at the start of the culture period and removed at the early multilayering stage, nodule formation was also inhibited. Pulses of 48-h duration also inhibited nodule formation, with maximal effect occurring between days 3 and 11. Thus, 1,25-(OH)2D3 inhibited osteoprogenitor cell differentiation during the earlier stages of culture before visible bone nodule formation occurred and the effect was not reversible upon removal of 1,25-(OH)2D3. In cultures grown to the multilayering stage in medium without ascorbic acid and beta-GP and then changed to medium with ascorbic acid and beta-GP, 1,25-(OH)2D3 inhibited when present before, but not after, the addition of ascorbic acid and beta-GP. Two other vitamin D3 metabolites, 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] and 1,24,25-trihydroxyvitamin D3 [1,24,25-(OH)3D3] had inhibitory effects similar to 1,25-(OH)2D3. The effects were dose dependent for each metabolite tested and correlated with the biological effectiveness of these metabolites in other systems: i.e. 1,25-(OH)2D3 was more effective than 1,24,25-(OH)3D3 which in turn was more effective than 24,25-(OH)2D3. The data show that 1,25-(OH)2D3 inhibits osteoprogenitor cell differentiation at an early stage and at a time during which cell growth is stimulated.

Inorganic phosphate added exogenously or released from beta-glycerophosphate initiates mineralization of osteoid nodules in vitro.

Rat calvaria (RC) cells grown in medium containing ascorbic acid form nodules of osteoid and cells. When 10 mM beta-Glycerophosphate (beta-GP) is added, the osteoid mineralizes in two phases: an initiation phase that is dependent upon alkaline phosphatase activity and a progression phase that proceeds independently of the activity of alkaline phosphatase and does not require added beta-GP (Bellows et al., Bone Miner 1991;14:27-40). The present experiments were performed to determine whether beta-GP is converted to inorganic phosphate (Pi) during the initiation phase of the mineralization process and whether increased Pi can replace beta-GP in the initiation phase. Measurements of Pi concentrations in the culture medium showed that during the first 8 h of the initiation phase of mineralization, 10 mM beta-GP was rapidly degraded resulting in Pi concentrations of 9-10 mM. The production rate of Pi from beta-GP was linear (r = 0.996) and the alkaline phosphatase activity in the same cultures indicated a potential for conversion of beta-GP to Pi that was greater than the actual conversion rate. The addition of 2-5 mM Pi in the absence of beta-GP also initiated mineralization. Mineralization initiated by either beta-GP or Pi progressed in the absence of added beta-GP or Pi. 100 microM Levamisole inhibited the initiation of beta-GP-induced mineralization and the conversion of beta-GP to Pi, but did not affect Pi-induced initiation of mineralization. The addition of 1-5 mM Pi to cultures in which mineralization had been initiated by 10 mM beta-GP had no significant effect on the progression phase of mineralization. Neither beta-BP nor Pi initiated 45Ca uptake in cultures without nodules (RC population I) and the histological appearance of the mineralized tissue in either phosphate source appeared identical. The present experiments show that beta-GP is rapidly and virtually completely degraded to Pi during the initiation phase of mineralization and that the addition of increased concentrations of Pi can replace beta-GP in the initiation phase of mineralization in the absence of non-specific 45Ca uptake or apparent cellular toxicity.

Initiation and progression of mineralization of bone nodules formed in vitro: the role of alkaline phosphatase and organic phosphate.

Osteoid nodules form but do not mineralize in fetal rat calvaria cell cultures grown in alpha-minimal essential medium with 10% fetal bovine serum in the absence of Na beta-glycerophosphate (beta-GP). To study factors involved in the initiation and progression of mineralization, cultures were treated with beta-GP and radiolabelled with 0.1-0.2 microCi/ml 45Ca after nodules had formed (17-19 days in medium without beta-GP). Concentrations of beta-GP from 1 to 14 mM induced a dose-dependent increase in 45Ca uptake. 45Ca uptake was restricted to nodule-containing cultures and did not occur in cultures without nodules. Continuous labelling over 72 h compared with 2 h pulses over the same time period showed that little mineralization occurred over the first 8-12 h and that the rate of mineralization was maximal and constant after 24 h exposure to beta-GP. Calcium uptake from medium was slow during the first 12 h of beta-GP exposure but increased rapidly thereafter until the medium calcium concentration reached a steady state of between 0.5 and 0.6 mM. Measurement of calcium concentration in the medium after mineralization had been initiated (24 h after beta-GP exposure) showed a linear calcium uptake into nodules (r = 0.990) over a 7 h period at a rate of 9.2 micrograms calcium/h/culture. Initiation of mineralization was prevented by 100 microM levamisole, but not by 100 microM dexamisole. When 100 microM levamisole was added 24 h after mineralization had been initiated by the addition of beta-GP, the progression of mineralization was unaffected. Similarly, after mineralization had been initiated for 24 h by 10 mM beta-GP, mineralization continued independent of the presence of beta-GP. The data show that the initiation and progression of mineralization are separate phenomena and that organic phosphate and alkaline phosphatase play a crucial role in the initiation of mineralization but are not required for the continuation of mineralization of bone nodules.

Modulation of plasminogen activators and plasminogen activator inhibitors by TGF-beta, IL-1 alpha and EGF in fetal rat calvaria cells at different times of culture.

Fetal rat calvaria cells (RC cells) grown in long term culture in the presence of ascorbic acid and organic phosphate proliferate and differentiate to form mineralized nodules of bone. Since transforming growth factor beta (TGF-beta), interleukin 1-alpha (IL-1 alpha) and epidermal growth factor (EGF) affect both bone resorption and bone formation, we have studied the ability of these growth factors to affect plasminogen activators and plasminogen activator inhibitors release by RC cells at different times throughout this proliferation/differentiation sequence. Cultures in log phase growth (day 4), when first multilayering (day 7) and when bone nodules were forming (day 13) were exposed to either TGF-beta, IL-1 alpha, EGF or vehicle. Conditioned medium was collected after 6 and 24 h and plasminogen activators and plasminogen activator inhibitors were analysed by fibrin autography and reverse fibrin autography. TGF-beta-mediated changes in plasminogen activator were apparent at day 4. By day 7 two molecular weight species of plasminogen activator were noted; a 65 kDa species, prominent at 24 h exposure was blocked by anti-tPA antibody, and a 38 kDa plasminogen activator, prominent after 6 h of stimulation was not blocked by anti-tPA antibody. Plasminogen activator-plasminogen activator inhibitor complexes are also increased. IL-1 alpha caused similar increases in plasminogen activator and plasminogen activator inhibitor with maximal activity measured at day 13, coincident with the time when bone nodules were forming. EGF-mediated changes were less by comparison. TGF-beta significantly decreased bone nodule formation after both a 6 and 24 h serum-free exposure, whereas IL-1 alpha and EGF decreased nodule number only after the 24 h exposure. The data suggest that the three factors influence the expression of plasminogen activator and plasminogen activator inhibitor by RC cells and their effect is different at different times of culture.

Biosynthesis of bone proteins [SPP-1 (secreted phosphoprotein-1, osteopontin), BSP (bone sialoprotein) and SPARC (osteonectin)] in association with mineralized-tissue formation by fetal-rat calvarial cells in culture.

To determine the relationship between the expression of bone proteins and the formation of mineralized-tissue matrix, the biosynthesis of non-collagenous bone proteins was studied in cultures of fetal-rat calvarial cells, which form mineralized nodules of bone-like tissue in the presence of beta-glycerophosphate. The temporal pattern of protein synthesis in both mineralizing and non-mineralizing cultures was studied by metabolic labelling with [35S]methionine, 35SO4(2-) or 32PO4(3-) over a 5-day period. After a 24 h labelling period, the culture media were harvested and the cell layers extracted sequentially with aq. 0.5 M-NH3, followed by 4 M-guanidinium chloride (GdmCl), 0.5 M-EDTA and a second extraction with 4 M-GdmCl. Protein associated with collagenous bone matrix was analysed after digestion with bacterial collagenase. On the basis of [35S]methionine labelling, the major proteins extracted from the mineralizing matrix were secreted phosphoprotein-1 (SPP-1; osteopontin), bone sialoprotein (BSP) and a 14 kDa phosphoprotein. The presence of SPP-1 and BSP in the conditioned media of both mineralizing and non-mineralizing cultures and their incorporation into the mineralizing nodules indicated that these proteins associate with preformed mineral crystals. However, some BSP was also present in GdmCl extracts and, together with a 35 kDa sulphated protein, was released from a bacterial-collagenase digestion of the tissue residue in both non-mineralizing and mineralizing cultures. Two forms of sulphated SPP-1 were identified, a highly phosphorylated 44 kDa species being the predominant form in the mineralized matrix. The BSP was more highly sulphated but less phosphorylated than SPP-1. Bone SPARC (secreted protein, acid and rich in cysteine) protein (osteonectin) was present almost entirely in the conditioned media and did not incorporate 32PO4(3-) or 35SO4(2-). The SPP-1 and the 14 kDa protein were susceptible to thrombin digestion, the 44 kDa SPP-1 being specifically cleaved into 28 and 26 kDa fragments. The fragments were labelled uniformly with [35S]methionine, but the 28 kDa fragment incorporated more 35SO4(2-), but less 32PO4(3-), than the 26 kDa fragment. These studies demonstrate that SPP-1 and BSP are the major osteoblast-derived bone proteins to bind to the bone mineral. That BSP also binds to the collagenous bone matrix indicates a potential role for this protein in linking the hydroxyapatite with collagen.

Parathyroid hormone reversibly suppresses the differentiation of osteoprogenitor cells into functional osteoblasts.

The effects of PTH on osteoprogenitor cell differentiation have been analyzed by quantifying its effects on bone nodule formation in an in vitro assay. Fetal rat calvaria cells were plated at 3 x 10(4) cells/35-mm dish, and cultures were maintained for 17-23 days in alpha-Minimal Essential Medium containing ascorbic acid, Na beta-glycerophosphate, and 10% fetal bovine serum. Continuous exposure to PTH at concentrations from 1 pM to 1 nM (2 x 10(-5) to 2 x 10(-2) IU/ml) caused a dose-dependent inhibition of bone nodule formation. Half-maximal inhibition occurred at 0.05 nM, and total inhibition at 1 nM, concentrations much lower than those required to elicit a significant cAMP response in rat calvaria cells. PTH at the concentrations used did not affect cell growth or saturation density. While continuous exposure to 1 nM PTH eliminated bone nodule formation, a single 48-h pulse administered at any time during the 17-day culture period had no effect. When 1 nM PTH was added on day 1 and removed at different times during the culture period, a time-related release from inhibition was observed. Cultures exposed to 1 nM PTH until nodules had developed in the corresponding control cultures and then switched to medium without added PTH rapidly formed clusters of differentiated osteoblasts and nodules within 3 days. PTH added at different times during the culture period and present continuously there-after suppressed formation of new nodules, the magnitude of the effect being a function of the duration of exposure. The results show that PTH at physiological concentrations is a potent suppressor of osteoblast differentiation and that its effect occurs at a late stage in the differentiation of osteoprogenitor cells, probably preventing differentiation of preosteoblasts into osteoblasts.