C. G. Bellows

Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations.

SummarySingle-cell suspensions obtained from sequential enzymatic digestions of fetal rat calvaria were grown in long-term culture in the presence of ascorbic acid, Na β-glycerophosphate, and dexamethasone to determine the capacity of these populations to form mineralized bone. In cultures of osteoblastlike cells grown in the presence of ascorbic acid and β-glycerophosphate or ascorbic acid alone, three-dimensional nodules (∼75 μm thick) covered by polygonal cells resembling osteoblasts could be detected 3 days after confluency. The nodules became macroscopic (up to 3 mm in diameter) after a further 3–4 days. Only in the presence of organic phosphate did they mineralize. Nodules did not develop without ascorbic acid in the medium. Dexamethasone caused a significant increase in the number of nodules. Histologically, nodules resembled woven bone and the cells covering the nodules stained strongly for alkaline phosphatase. Immunolabeling with specific antibodies demonstrated intense staining for type I collagen that was mineral-associated, a weaker staining for type III collagen and osteonectin, and undetectable staining for type II collagen. Nodules did not develop from population I and the number of nodules formed by populations II–V bore a linear relationship to the number of cells plated (r=.99). The results indicated that enzymatically released calvaria cells can form mineralized bone nodulesin vitro in the presence of ascorbic acid and organic phosphate.

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.

Inorganic phosphate added exogenously or released from β-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.

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.

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.

Aluminum Accelerates Osteoblastic Differentiation But is Cytotoxic in Long-Term Rat Calvaria Cell Cultures

Abstract. 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 α minimal essential medium containing 10% fetal bovine serum, 50 μg/ml ascorbic acid, and 10 mM β-glycerophosphate with or without Al added to final concentrations of 1 μM—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 μM 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 μM 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.

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 dependant on factor(s) present in the FBS component of the culture medium.

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 D3 (1,25[OH]2D3) 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)2D3- 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)2D3 or DEX, while IGF-II stimulated adipocyte differentiation in both the absence and presence of 1,25(OH)2D3 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)2D3. 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.

The Role of Cells in the Calcification Process

Although the vertebrate body contains an abundance of potential sites for mineralization, not every tissue calcifies, which indicates that regulatory processes are operative. The mechanisms responsible for stimulating and inhibiting the initiation and progression of mineralization are likely to be cell-mediated, and various experimental systems have been used to study the involvement of cells, the function of which may be regulated systemically or locally. In this chapter, we will discuss experiments in which we studied the role of vitamin D metabolites as regulators of bone matrix calcification in rats in vivo and experiments designed to analyse the factors involved in regulation of de novo mineralization of bone formed in vitro. First, however, we present a brief overview of the matrix constituents associated with calcification.