Johan N. M. Heersche

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.

Glucocorticoid‐Induced Osteoporosis: Both In Vivo and In Vitro Concentrations of Glucocorticoids Higher Than Physiological Levels Attenuate Osteoblast Differentiation

SINCE HENCH ET AL. (1) first reported the use of cortisone for treatment of patients with rheumatoid arthritis, glucocorticoids (GCs) have been widely used for anti-inflammatory and immunosuppressive therapy. However, GCs have major effects on several organ systems, including the cardiovascular, endocrine, gastrointestinal, ophthalmic, and musculoskeletal systems, and these often lead to major side effects. Among the most dramatic side effects of in vivo long-term GC excess is the development of GC-induced osteoporosis. Several lines of evidence indicate that the major mechanisms whereby GCs induce osteoporosis are decreased bone matrix deposition by osteoblasts, stimulation of osteoclastic bone resorption, and decreased intestinal calcium resorption with resulting mild secondary hyperparathyroidism (for reviews see Refs. 3, 5–8). However, the diverse and complex effects of GCs on bone metabolism are still incompletely understood, mainly due to differences in experimental outcomes in different systems and culture conditions, which could be a result of species differences, age of the experimental animals used, maturation stage of the cells studied, heterogeneity of osteoblast populations, differences between transformed cells and normal cells, or length and dose of GC treatment used. In this brief review, we will focus on the effects of GCs on bone formation. With regard to this aspect of GC effects, it has been reported in a variety of in vitro systems that GCs induce cells of the osteoblast lineage to differentiate into mature cells expressing the osteoblastic phenotype, e.g., parathyroid hormone–stimulated cAMP synthesis, osteopontin, bone sialoprotein, alkaline phosphatase (AP), and mineralized bone nodule formation. However, in several in vitro systems, GCs have also been shown to decrease type I collagen synthesis and mRNA levels, osteocalcin production and mRNA levels, and the expression of insulin-like growth factor I (IGF-I) and selected IGF-binding proteins (IGFBPs) which enhance IGF activity (e.g., IGFBP-3 and IGFBP-5). The reason for the observed differences is not clear, but it is known that the GC receptor can mediate not only transcriptional activation but also repression of transcriptional expression by interfering with the binding or activity of other transcription factors or by interacting directly with negative glucocorticoid response elements (nGREs). For example, the GC-induced inhibition of 1,25-dihydroxyvitamin D3– mediated osteocalcin transcription is likely to occur by binding of the GC receptor to a nGRE overlapping the TATA box in the proximal promoter region of osteocalcin gene. In addition, mRNA levels can also be controlled by GCs through selective changes in mRNA stability. Indeed, Delany et al. have shown that GC-mediated down-regulation of a1(I) collagen mRNA occurs by a decrease in the stability of a1(I) collagen mRNA in bone cell populations derived from fetal rat calvariae. Although a number of such mechanisms for negative-transcriptional or post-transcriptional gene regulation by GCs have been described, it is still unclear in many systems how positive and negative regulation interact to result in the observed effects (for review see Ref. 38). Furthermore, the observed differences may well be related to the fact that the effects of GCs are often dependent on the timing of GC treatment and the stage of differentiation of the osteoblast-like cells used. For example, when cultures obtained from chick embryo calvariae were continuously exposed to dexamethasone (Dex) from the onset of culture, Dex increased AP activity, but when Dex was added late in the culture period after bone had formed, AP activity decreased. In addition, Dietrich et al. have shown that in fetal rat calvariae, 30–100 nM

Characteristics of in vitro osteoblastic cell loading models.

Normal loading strains of 200-2000 (mu)epsilon to bone result in bending forces, generating mechanical stretch and pressure gradients in canaliculi that drive extracellular fluid flow, resulting in stress on the membranes of osteocytes, lining cells, and osteoblasts. Under excess loading, as well as during unloading (e.g., microgravity, bed rest), the fluid shift and resultant change in interstitial fluid flow may play a larger role in bone remodeling than mechanical stretch. The in vitro model systems used to investigate mechanical loading of bone generate either fluid shear, hydrostatic compression, biaxial stretch, uniaxial stretch, or a combination of two or more of these forces. The results of in vitro experiments suggest that fluid shear is a major factor affecting bone cell metabolism. Both the flow-loop apparatus (which produces pulsatile flow and uses fluid shear as its principal stimulus) and the uniaxial silicone plate stretching apparatus (which generates cyclic stretch) create a reproducible and consistent stimulus. Endpoints measured in flow experiments, however, are short term and usually short lived, and it is unknown whether these changes impact the function of differentiated osteoblasts. Endpoints measured in uniaxial stretch experiments are generally long-term-sustained effects of mechanical perturbation and more easily relatable to changes in osteoblastic activity. Biaxial stretch devices create both bending and compressive forces, resulting in different types of force on the cells, with the relative amount of each depending on the position of the cell in the device. Therefore, systems that incorporate pulsatile fluid flow or uniaxial stretch as the principal stimulus should be further developed and implemented in the study of the relationship between mechanical loading and bone response.

Resorptive state and cell size influence intracellular pH regulation in rabbit osteoclasts cultured on collagen-hydroxyapatite films

Diseases exhibiting excessive bone loss are often characterized by an increase in the size and number of osteoclasts in affected areas, suggesting that osteoclast size is associated with increased resorptive activity or efficiency. Because osteoclastic bone resorption depends on proton extrusion via a bafilomycin A1-sensitive vacuolar type H+ ATPase (V-ATPase), we investigated the relationship between osteoclast size and state of activity on the one hand, and proton-extruding mechanisms (bafilomycin A1-sensitive V-ATPase and amiloride-sensitive Na+/H+ exchange) on the other. In determining resorptive activities of individual osteoclasts, osteoclast-containing cell suspensions obtained from newborn rabbit long bones were cultured on apatite-collagen complex (ACC)-coated coverslips. Large osteoclasts resorbed 2.5 times more per cell than small osteoclasts, but the amount resorbed per nucleus was the same for the two categories. However, a much larger percentage of large osteoclasts was resorbing compared with small osteoclasts. To study pH regulatory mechanisms in individual large and small osteoclasts, the cells were loaded with the pH-sensitive indicator BCECF and analyzed by single-cell fluorescence. Small and large resorbing osteoclasts had significantly higher basal pH(i) than their nonresorbing counterparts. Also, small nonresorbing osteoclasts were insensitive to bafilomycin A1 addition or Na+ removal from the medium, large nonresorbing osteoclasts responded slightly, and all resorbing osteoclasts (small and large) responded strongly. Differences were also seen in the recovery from an acid load: both small and large nonresorbing osteoclasts were more sensitive to amiloride inhibition, while large resorbing cells were more sensitive to bafilomycin A1 inhibition. Small resorbing cells were inhibited equally by bafilomycin A1 and amiloride. These results clearly show that a greater proportion of large osteoclasts are active in resorption and that pH(i) regulation is associated with enhanced proton pump activity in actively resorbing osteoclasts. Thus, large and small osteoclasts differ in the proportion of cells that are resorbing, while pH regulatory mechanisms differ mainly between resorbing and nonresorbing cells.

Differential chemotactic responses of different populations of fetal rat calvaria cells to platelet-derived growth factor and transforming growth factor β

We tested the chemotactic response to platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF beta) of cells released enzymatically from fetal rat calvaria (RC). Both factors were chemotactic for RC cells, but the magnitude of the chemotactic response differed markedly between different populations and varied with time in culture of the cell populations. Cells released earlier from the calvaria showed a greater response than osteoblast-enriched populations released later. The optimal concentration of PDGF was the same for both alkaline phosphatase (AP)-positive and AP-negative cells within the populations. However AP-positive cells showed two peaks of response to TGF beta; one peak coincided with the TGF beta concentration also maximally affecting AP-negative cells, while the other occurred at a concentration 50-100 times higher. The results indicate that PDGF and TGF beta are chemotactic for both AP-positive and AP-negative cells in populations of cells derived from fetal calvariae, that chemotactic response declined with longer periods of time in culture, and that AP-positive osteoblast-like cells respond to a concentration of TGF beta that does not affect the AP-negative cells in the population.

Progesterone Stimulates Proliferation and Differentiation of Osteoprogenitor Cells in Bone Cell Populations Derived From Adult Female but not From Adult Male Rats

We examined the effects of the synthetic glucocorticoid dexamethasone (Dex) and of the sex steroids progesterone (Prog) and testosterone (Testo) on proliferation and differentiation of progenitors for osteoblasts, adipocytes, and macrophages in cell populations derived from lumbar vertebrae of adult male and female rats. To assay for these progenitors, we used a previously described colony assay, where progenitors are identified by the appearance of colonies of the differentiated phenotype in long term cultures of cell populations containing these progenitors. In cell populations derived from both males and females, Dex (10(-9)-10(-6) mol/L) induced a similar dose-dependent increase in the number of osteoblast colonies (bone nodules), colonies of alkaline phosphatase (AP)-positive cells, adipocyte colonies and macrophage colonies (ED2-positive cells). Prog (10(-8)-10(-5) mol/L), on the other hand, increased bone nodule formation in female-derived populations but not in male-derived populations. Maximal stimulation was seen at 10(-5) mol/L. 17 beta-Estradiol (E2) enhanced the Prog-induced increase in the number of bone nodules in a dose-related fashion. Maximal stimulation was seen at 10(-8) mol/L E2. E2 (10(-9)-10(-6) mol/L) had no effect on Dex-induced bone nodule formation, indicating that the effect is specific for Prog-induced stimulation of bone nodule formation. Prog also caused a dose-dependent increase in the number of colonies of AP-positive cells. Interestingly, the effect of Prog on the number of AP-positive colonies was the same in populations derived from both sexes. Prog-induced stimulation of adipocyte and macrophage development in female-derived populations was significantly greater than that in male-derived populations. Testo (10(-9)-10(-5) mol/L) had no effect on any of the parameters evaluated in populations derived from either males or females. These observations demonstrate that the effect of Prog on proliferation and differentiation of progenitors for osteoblasts, adipocytes, and macrophages in cell populations derived from lumbar vertebrae of adult male or female rats is sex-dependent and is seen either only (bone nodule formation) or more pronounced (adipocyte and macrophage development) in female-derived populations.

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.

Macrophage Colony Stimulating Factor Increases Bone Resorption in Dispersed Osteoclast Cultures by Increasing Osteoclast Size

Several reports indicate that macrophage colony stimulating factor (MCSF) is one of the major factors required for osteoclast proliferation and differentiation. Paradoxically, it has also been reported that MCSF inhibits osteoclastic activity. We therefore decided to investigate in detail the effects of MCSF on resorption and osteoclast formation to try and clarify this issue. Osteoclast‐containing cultures were obtained from rabbit long bones and cultured on plastic culture dishes or devitalized bovine bone slices. MCSF (4–400 ng/ml) stimulated osteoclastic bone resorption in a time‐dependent manner and at all doses examined. After 48 h of culture in the presence of MCSF, we observed a 2‐fold increase in the total area of bone resorbed, as well as a significant increase in the area of bone resorbed per osteoclast and the number of resorption pits per osteoclast. This effect was paralleled by an increase in the number of larger osteoclasts (as determined by the number of nuclei per cell) and an increase in the size and depth of the resorption pits. Since the total number of osteoclasts remained the same, the MCSF‐induced increase in resorptive activity appeared to be related to an increase in the average size of the osteoclasts. When resorption was expressed as the amount of bone resorbed per osteoclast nucleus, larger osteoclasts resorbed more per nucleus, suggesting that large osteoclasts, as a population, are more effective resorbers than small osteoclasts. Interestingly, when osteoclasts were plated at one‐fifth the standard density, the amount of bone resorbed per osteoclast decreased considerably, indicating that resorptive activity is also affected by cell density of osteoclasts and/or of other cells present. However, at this lower density MCSF still increased osteoclast size and resorption by the same fold increase over control, suggesting that the effect of MCSF was independent of factors related to cell density.