A Novel Human Bone Marrow Stroma-Derived Cell Line TF274 Is Highly Osteogenic In Vitro and In Vivo

A novel, immortalized, human bone marrow stroma-derived cell line TF274 is described which has the ability to form bone both in vitro and in vivo. Under basal conditions these cells expressed alkaline phosphatase (ALP) and type I collagen genes which are characteristic of the osteoblast phenotype. ALP levels were upregulated in the presence of osteotropic agents such as parathyroid hormone (PTH), transforming growth factor beta (TGF-β), and BMP-2. In addition, PTH also increased cAMP levels in these cells. The capacity of these cells to form bone in vitro was evaluated by culturing them in the presence of L-ascorbic acid and β-glycerophosphate. Matrix mineralization in these cultures was assessed by Alizarin Red staining and increased ⁴⁵Ca uptake. Under these conditions mineralized nodule formation was observed in less than 2 weeks. Northern analysis of TF274 cells at various times during the mineralization process indicated a temporal expression of the osteocalcin gene that is typically associated with differentiating osteoblasts. The osteogenic nature of TF274 cells was confirmed in vivo using the severe combined immunodeficient (SCID) mouse model. Antibodies to human leukocyte antigens (HLA), class I antigens, and human OK^a blood group antigen were used to demonstrate that the lesions formed were of human origin. By 21 days, the lesion consisted of a homogeneous focus of ALP-positive cells containing areas of mineralized bone lined with tartarate-resistant acid phosphatase (TRAP) positive osteoclasts. Thus, the TF274 cells exhibit osteogenic potential both in vitro and in vivo. This immortalized cell line represents a consistent source of cells that can be used to study human osteoblast differentiation both in vitro and in vivo. Received: 30 July 1997 / Accepted: 23 January 1998     

Endothelin-1 and Paget's Bone Disease: Is There a Link?

The abundance of endothelial cells in bone marrow and the proximity of these cells to osteoclasts and osteoblasts suggest a role for endothelin-1 (ET-1) on bone metabolism. In vitro, the direct contact with bone endothelial cells induces osteoclastic progenitors to differentiate into mature elements. Recently it has been reported that ET-1 inhibits osteoclastic bone resorption and cell mobility through a specific receptor on osteoclasts; other authors demonstrated that ET-1 exerts a mitogenic activity on osteoblast-like cells (MC3T3) by stimulating tyrosin phosphorylation. We measured ET-1 circulating levels in patients with active Paget's bone disease, a condition with accelerated bone turnover. For the study we recruited 11 patients with Paget's bone disease (5F, 6M; mean age 68.2 ± 3.6) in the acute stage of the disease; 10 healthy subjects (7F, 3M; mean age 66.5 ± 3.9) were also enrolled as controls. Plasma ET-1 levels were measured with RIA kits provided by Nichols Institute. Patients showed significantly (P < 0.01) higher ET-1 circulating levels than controls (6.35 ± 1.9 versus 3.4 ± 1.2 pg/ml) with a positive correlation (r = 0.63; P= 0.038) with serum alkaline phosphatase (ALP), but not with urinary hydroxyproline. The higher levels of ET-1 in our patients suggest a physiopathological role for this peptide in the disease and, could perhaps represent a new useful marker of Paget's bone disease activity. Received: 29 April 1997 / Accepted: 20 February 1998     

Response of Human Osteoblasts to Polymethylmetacrylate In Vitro

The effects of a polymethylmetacrylate (PMMA) powder with a diameter between 0.5 and 25 μm have been studied in vitro on several human osteoblast populations obtained from different sources. Parameters of cell activity such as cell growth, collagen synthesis, osteocalcin, and interleukin-6 (IL-6) production have been evaluated. Cell proliferation and collagen synthesis were inhibited after exposure to bone cement, whereas osteocalcin and IL-6 production were stimulated. These results suggest that PMMA particles could affect osteoblast activity in a way that could contribute, together with other factors, to periprosthetic osteolysis through two different pathways: a reduced periprosthetic bone formation due to the reduced osteoblast proliferation and collagen synthesis, and an osteoblast-mediated activation of osteoclastic bone resorption as suggested by the increased osteocalcin and IL-6 synthesis. In fact, osteocalcin has been demonstrated to have a role in osteoclast recruitment to bone surfaces, and IL-6 is known to induce osteoclastogenesis and to directly stimulate bone resorption. Received: 20 December 1996 / Accepted: 2 July 1997     

Calcium Absorption and Bone Loss in Ovariectomized Rats Fed Varying Levels of Dietary Calcium

The following studies were undertaken to examine whether estrogen deficiency impairs calcium absorption in aged rats, and to determine whether impaired calcium absorption and the level of dietary calcium are related to the degree of bone loss due to estrogen deficiency. Sixty rats were sham operated (Sham) or ovariectomized (Ovx) to make them estrogen deficient and divided into three dietary groups of 10 rats per group: Group 1 (Sham) and Group 2 (Ovx) were maintained on a diet containing 0.5% calcium; Group 3 (Sham) and Group 4 (Ovx) were maintained on a diet containing 0.1% calcium; Group 5 (Sham) and Group 6 (Ovx) were maintained on a diet containing 0.02% calcium. Calcium absorption was measured in all animals at the beginning of the study and 2 weeks, 1 month, 2 months, and 3 months following surgery, then the animals were sacrificed. In Ovx rats fed 0.5% Ca diet, calcium absorption decreased progressively and the decrease became statistically significant 8 and 12 weeks following ovariectomy (P < 0.05). A similar ovariectomy-related impairment of calcium absorption was not observed in animals fed diets with lower calcium content, making the Ovx rat a tenuous model of intestinal calcium malabsorption. Low dietary calcium decreased cancellous bone mineral content and density at the proximal tibial metaphysis and the decrease was augmented by ovariectomy. The degree of osteopenia due to ovariectomy was not related to the level of dietary calcium or the efficiency of calcium absorption. Received: 7 July 1998 / Accepted: 23 December 1998     

Bone Mass and Markers of Bone and Calcium Metabolism in Postmenopausal Women Treated with 1,25-Dihydroxyvitamin D (Calcitriol) for Four Years

To evaluate the long-term effect of calcitriol treatment on bone mineral density (BMD) of the femoral neck and lumbar spine and the parameters of calcium and bone metabolism in elderly women, 55 healthy, postmenopausal women, all aged 66 years, were enrolled in the study. Eighteen started a 4-year supplementation with 0.5 μg of calcitriol daily and 37 served as controls. Calcium intake of all the subjects was adjusted to 800 mg daily. In 4 years femoral neck BMD increased by 3.0% in the calcitriol group, but decreased by 1.6% in the control group (P= 0.009). The respective changes in lumbar spine BMD were +2.3% and +0.9% (P= 0.067). Two years' treatment with calcitriol increased the intestinal absorption of strontium by 57% (P < 0.001), doubled the urinary excretion of calcium (P < 0.001), and decreased the mean parathyroid hormone (PTH) level by 32% (P < 0.01). In the calcitriol group the marker of bone formation, serum osteocalcin, decreased by 27% (P < 0.01), and the marker of bone resorption, serum C-telopeptide of type I collagen (CTx), by 33% (P= 0.05) after 2 years. In two subjects the calcitriol dose had to be reduced because of hypercalciuria. We conclude that calcitriol treatment increases bone mass at the femoral neck and lumbar spine, the increases being maintained for up to 4 years. The gain in bone mass results from reduced bone turnover which is partly a consequence of the enhanced intestinal absorption of calcium and suppressed serum PTH levels. Received: 8 January 1999 / Accepted: 29 February 2000     

Bone Sialoprotein (BSP) is a Crucial Factor for the Expression of Osteoblastic Phenotypes of Bone Marrow Cells Cultured on Type I Collagen Matrix

In this study, we demonstrated that type I collagen matrix induced the expression of osteoblastic phenotypes of bone marrow cells, and that antibone sialoprotein (BSP) monoclonal antibody suppressed the expression of these phenotypes. On the other hand, BSP accelerated the expression of osteoblastic phenotypes of bone marrow cells. The adherent bone marrow cells were harvested from rat femur and cultured on type I collagen matrix gels in medium containing 15% fetal calf serum, neither β-glycerophosphate nor glucocorticoid. Cells showed osteoblastic phenotypes (high alkaline phosphatase activity, osteocalcin synthesis, and responsiveness against parathyroid hormone) on collagen matrix gels at week 3 after the inoculation, and simultaneously, BSP was detected in the conditioned medium by Western blotting using an anti-BSP monoclonal antibody. However, cells in the conventional culture dishes did not show osteoblastic phenotypes during the experimental period. To investigate the physiological function of BSP in osteoblastic differentiation, bone marrow cells were cultured on collagen matrix with an anti-BSP monoclonal antibody for 3 weeks. This treatment suppressed the expression of the osteoblastic phenotypes, and the effect of the antibody was abolished by the addition of bovine bone BSP. Furthermore, bovine bone BSP stimulated the expression of osteoblastic phenotypes of bone marrow cells. Our results indicate that BSP plays a crucial role in the expression of osteoblastic phenotypes of bone marrow cells. Received: 17 February 1999 / Accepted: 14 December 1999     

Effects of Human Tumor Cell Lines on Local New Bone Formation In Vivo

Although some tumors cause osteolytic lesions, there are some that stimulate new bone formation. This is an important phenomenon because the responsible mechanisms probably represent an aberration of normal physiological bone formation, and identifying the factors involved in the process may lead to new therapies for various bone diseases. To clarify our understanding of the potential mechanism responsible, we compared and quantitated the extent of new bone formation stimulated by human tumors (HeLa, Hep-2, AV-3, FL, WISH and KB), some of which have osteogenic activity in vivo [2]. Tumor cells were injected over the calvaria of nude mice to examine formation of new bone. The tumor cells produced three histologically distinct patterns of new bone growth: (1) WISH and KB stimulated appositional bone growth adjacent to periosteal bone surfaces; (2) HeLa and Hep2 induced new bone growth over calvarial surface even when distant from the tumor mass; (3) FL stimulated bone formation adjacent to periosteum as well as ectopic bone formation in sites distant from bone. All tumors except AV3 induced mean new bone thickness >100 μm, and Hep-2 cells produced bone 330 μm thick. PCR and Northern blot analysis of mRNA isolated from cultured tumor cells revealed that all cell lines expressed mRNA for TGFβ, (fibroblast growth factor) FGF-1, FGF-2, and IGF-I, and most cell lines produced mRNA for PDGF. Only FL expressed large amounts of mRNA for BMP2. In serum-free conditioned media from Hep2 and HeLa cells purified by heparin affinity chromatography, we have identified FGF-1, FGF-2, and PDGF by immunodetection with specific antibodies. Our results show that new bone growth caused by these tumors is likely due to the production of bone growth factors by the tumor cells, and that the overall effects on bone may be due to several factors working in concert. Received: 15 January 1996 / Accepted: 3 May 1996     

Different Behavior of Human Osteoblast-Like Cells Isolated from Normal and Heterotopic Bone In Vitro

In this study, a characterization of human bone-forming cells responsible for heterotopic ossification was carried out in vitro. The biological and biochemical cell characteristics of the heterotopic osteoblast-like (HOB) cells were compared with those of orthotopic osteoblast-like (OB) cells from normal bone and stromal bone marrow cells believed to contain a subpopulation of osteogenic precursor cells. We found that HOB's from the spongiosa of heterotopic ossification required less time until the beginning of migration and the achievement of confluence in vitro compared with OBs from femoral shaft spongiosa. The fraction of mitotically active cells assessed by a clonogenic assay was higher as well in HOB cells. The in vitro studies of mitogenesis and the efficiency of colony formation of osteogenic cells indicate that with increasing differentiation and relative age they become more dependent on growth factors in the medium, otherwise the morphology of osteoblast-like cells changes and they pass irreversibly into the postmitotic stage of the cell cycle. The activity of the alkaline phosphatase is distinctly higher in the HOB than in the OB cells, HOB cells exhibit a lower level of osteocalcin expression compared with OB cells. No significant difference was found between OB and HOB cells in the amount of procollagen of type I sequestered by the cells. After 30 days, HOB and OB cells formed a mineralized matrix on exposure to 2 mM β-glycerophosphate. Since HOBs were isolated from heterotopic bone that had developed within 3–6 months after hip surgery, the differences in cellular behavior compared with OBs may be attributed to the relatively young age of HOB cells. Received: 29 March 1996 / Accepted: 21 May 1997     

The Resorption of Vital and Devitalized Bone In Vitro: Significance for Bone Grafts

Several studies have suggested that devitalized bone is less satisfactory than live tissue for surgical grafting purposes because an initial resorption step, prior to new formation, is lacking. We have compared the osteoclastic resorption of cultured bone containing living osteocytes with that of similar bone in which the osteocytes were dead. In experiment I, transverse slices cut from freshly harvested adult rabbit femora were either placed in phosphate buffered saline (Set 1) or subjected to freezing and thawing (Set 2). In experiment II, a heated set (Set 3) was prepared in addition. All slices were cultured with osteoclasts for 24 hours, eight slices per set being seeded with bone cells in experiment I and three per set in experiment II. The areas and volumes of resorption pits formed during the culture period were measured using reflection confocal microscopy. In both experiments, the mean values for the areas of the pits were smaller in the bone containing live osteocytes (P < 0.03, Mann Whitney test), and in experiment II the volumes of the pits in Set 1 were smaller than those in Set 3 (P < 0.0001, Mann Whitney test). However, in neither experiment was there a significant difference between the Sets in the volume:area ratios (mean depths) of the pits. The findings show that devitalized bone is resorbed by osteoclasts at least as readily as bone containing vital osteocytes in vitro, and indicate that if grafted devitalized bone resorbs less well in vivo it is not because the bone tissue is intrinsically resistant to osteoclastic resorption. Received: 25 November 1997 / Accepted: 24 June 1998     

Inhibitory Effects of 1,25(OH)2D3 on Membrane-Associated and Cytosolic Casein Kinase Activity in MC3T3-E1 Osteoblast-like Cells

The secretion of phosphorylated matrix proteins is high in osteoblasts. Phosphorylation of these proteins may be catalyzed by casein kinases (CK), and CK may play an important role in the site of bone mineralization. In this study, we examined the effects of 1,25(OH)₂D₃ on CK activities in MC3T3-E1 osteoblast-like cells. Different concentrations (ranging from 10⁻⁷ to 10⁻¹¹M) of 1,25(OH)₂D₃ were included in a culture medium. After incubation for various lengths of time, MC3T3-E1 cells were homogenized and segregated into cytosolic (c) and microsomal (m) fractions. To measure CK activity, each fraction was used as an enzyme source to phosphorylate casein. MC3T3-E1 cells showed the highest cCK activity after incubation for 21 days, and showed the highest mCK activity after incubation for 14 days. 1,25(OH)₂D₃ inhibited mCK activity at the early stage of culture, but inhibited cCK activity at the late stage of culture. In contrast, 1,25(OH)₂D₃ had a slight stimulatory effect on CK activity in the culture medium of MC3T3-E1 cells. Our data suggest that cCK and mCK may play different roles in the function of osteoblasts, and 1,25(OH)₂D₃ regulates intracellular and extracellular casein kinase activities related to the function of osteoblasts. Received: 26 June 1997 / Accepted: 23 March 1998     

In Vivo Treatment with Calcitriol (1,25(OH)2D3) Reverses Age-Dependent Alterations of Intestinal Calcium Uptake in Rat Enterocytes

The vitamin D endocrine system has been involved in the impairment of intestinal calcium absorption during aging. Alterations in the nongenomic mechanism of calcitriol (1,25-dihydroxy-vitamin D₃; [1,25(OH)₂D₃] have been recently evidenced. In enterocytes isolated from aged rats, 1,25(OH)₂D₃ stimulation of Ca²⁺ channels through the cAMP/PKA pathway is blunted. We have now investigated whether in vivo administration of calcitriol to senescent rats reverses the absence of hormonal effects in isolated intestinal cells. In enterocytes from 20–24-month-old rats given 1,25(OH)₂D₃ for 3 days (30 ng/100 g bw/day), calcitriol (10⁻¹⁰ M, 3–5 minutes) stimulated Ca²⁺ uptake and intracellular cAMP to the same degree and protein quinase A (PKA) activity to a lesser degree than in enterocytes from young animals. Significantly higher basal levels of cAMP and PKA detected in enterocytes from old rats were not affected by prior injection of animals with 1,25(OH)₂D₃. When the aged rats were injected with 25(OH)D₃, similar Ca²⁺ influx, cAMP, and PKA responses to in vitro stimulation with calcitriol were obtained. 1,25(OH)₂D₃-dependent changes in Ca²⁺ uptake by enterocytes from both young and old rats treated with calcitriol were totally suppressed by the cAMP antagonist Rp-cAMPS, whereas the response to the agonist Sp-cAMPS was markedly depressed in aged animals. These results suggest that intestinal resistance to nongenomic 1,25(OH)₂D₃ stimulation of duodenal cell Ca²⁺ uptake develops in rats upon aging and show that in vivo administration of 1,25(OH)₂D₃ or its precursor to senescent rats restores the ability of the hormone to stimulate duodenal cell calcium influx through the cAMP messenger system. Received: 26 December 1997 / Accepted: 12 May 1998     

Parathyroid Hormone,Prostaglandin E2, and 1,25-Dihydroxyvitamin D3 Decrease the Level of Na+-Ca2+ Exchange Protein in Osteoblastic Cells

We previously described Na⁺-Ca²⁺ exchange in osteoblastic rat osteosarcoma cells (UMR-106) and demonstrated that Na⁺-dependent Ca²⁺ transport was inhibited by 24-hour treatment of cells with parathyroid hormone (PTH), prostaglandin E₂ (PGE₂), or 1,25(OH)₂D₃. To determine whether this inhibition of Na⁺-Ca²⁺ exchange is at the level of exchanger protein synthesis we have examined exchanger protein levels using immunoblot analysis. UMR-106 cells were treated for 24 hours with or without PTH, PGE₂, or 1,25(OH)₂D₃. Plasma membrane fractions (7500 g) were obtained and proteins were separated by SDS-PAGE, transferred to nylon membranes, and immunoblotted with a polyclonal antibody to the canine cardiac Na⁺-Ca²⁺ exchanger. In rat cardiac membranes, we detected 125 and 75 kD bands, similar to findings for the canine exchanger. In the osteoblastic UMR cell membranes, a specific band was detected at 90 kD that decreased 65% after treatment of cells with PTH. Inhibition by PTH was dose dependent, was maximal with 10⁻⁷ M PTH, and required 16–24 hour treatment time. Similar inhibition was observed after a 24 hour treatment with 10⁻⁶ M PGE₂ or 10⁻⁸ M 1,25(OH)₂D₃. These results demonstrate the presence of a specific protein in UMR cells that cross-reacts with antibody directed against the cardiac Na⁺-Ca²⁺ exchanger. Thus, the previously reported inhibition of Na⁺-Ca²⁺ exchange activity by calcemic agents in osteoblasts appears to be due to regulation of exchanger protein levels in these osteoblastic cells. Received: 5 February 1996 / Accepted: 18 October 1996     

Avian Osteoclast Cells are Stimulated to Resorb Calcified Matrices by and Possess Receptors for Leukotriene B4

Leukotriene B₄ (LTB₄) is elevated in inflammatory conditions and appears to be a potential mediator of inflammation. We have recently shown that this 5-lipoxygenase metabolite of arachidonic acid stimulates bone resorption in vitro and in vivo. In order to determine the mechanism whereby LTB₄ causes bone resorption, avian osteoclasts were examined for the effects of LTB₄ and for the presence of LTB₄ receptors. Isolated avian osteoclast mononuclear precursor cells, which fuse in culture to form multinucleated cells, were chosen for receptor binding studies because this population is a morphologically similar source of osteoclasts, and large numbers of these cells can be obtained from egg-laying hens. Binding of LTB₄ and activation would support the hypothesis of a direct effect of this compound on osteoclasts. LTB₄ stimulated isolated avian osteoclasts to form resorption lacunae on calcified matrices and to increase their content of tartrate-resistant acid phosphatase (TRAP), a marker of activated osteoclasts. Receptor binding studies were performed at day 1, when the cells were mononuclear, at day 4, when mononuclear precursors were actively fusing, and at day 7, when fusion has slowed. Scatchard analysis of saturation binding data showed two classes of binding sites, a high- and low-affinity binding site with dissociation constants (K_D) of 0.2–0.4 nM and 5.6–24 nM. Association studies showed rapid binding of LTB₄ to the cells within 10 minutes. These data show that LTB₄ accelerates fusion and activates highly enriched populations of avian osteoclasts and that LTB₄ receptors are present in this cell population. Received: 30 November 1997 / Accepted: 12 May 1998     

Increase in the Potential of Osteoblasts to Support Bone Resorption by Osteoclasts In Vitro in Response to Roughness of Bone Surface

The development of the potential of osteoblasts to support bone resorption by osteoclasts in response to roughness on bone slices was examined in the co-incubation cell system of immature osteoclasts and osteoblastic cells. The immature osteoclasts, which need alkaline phospatase (ALP)-positive osteoblastic cells for bone resorption, were generated in mouse spleen cultures with 1, 25-dihydroxyvitamin D₃ and prostaglandin E₂. ALP-negative osteoblastic cells from mouse calvaria were incubated on rough surfaced bone slices for 3 days. The number of ALP-positive cells increased greatly on the rough surface, but little on the smooth surface. When immature osteoclasts were added and incubated for 1 more day, the resorption pit number and the total pit areas on the smooth surface were not much different from those before incubation but were approximately four times higher on the rough surface. Received: 21 July 1998 / Accepted: 12 March 1999     

Up-Regulation of Inducible Nitric Oxide (NO) Synthase and NO Production in HL-60 Cells Stimulated to Differentiate by Phorbol 12-Myristate 13-Acetate Plus 1,25-Dihydroxyvitamin D3 Is Not Obtained With Dimethylsulfoxide Plus 1,25-Dihydroxyvitamin D3

In previous studies we found that the calciotropic hormone 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] augments the action of either prostaglandin E₁ (PGE₁) or NaF to induce differentiation of human promyelocytic HL-60 cells, a process that features increased generation of nitric oxide (NO) via up-regulation of inducible nitric oxide synthase (iNOS). We have now examined the short-term interaction of 1,25(OH)₂D₃ with phorbol 12-myristate 13-acetate (PMA) and dimethylsulfoxide (DMSO) in these cells. PMA (100 nM) alone generally up-regulated several classical indices of macrophagic differentiation and stimulated cellular production of interleukin (IL)-1α, IL-6, tumor-necrosis factor (TNF)-α, PGE₂, and NO. Increased generation of NO primarily resulted from increased expression of cellular iNOS. When 1,25(OH)₂D₃ (10 nM) was added to PMA treatments, most PMA-induced changes, particularly its effects to up-regulate iNOS-dependent NO production and change cell morphology, were multiplicatively augmented. In contrast, DMSO (1.3%) alone, an inducer of granulocytic differentiation, increased cytokine production, but failed to stimulate NO production or induce iNOS. In contrast to its striking interaction with PMA, 1,25(OH)₂D₃ could not augment DMSO's differentiative effects. Changes in cellular cytokine production were eliminated as the driving force in HL-60 differentiation when specific neutralizing antibodies failed to produce any attenuation of iNOS up-regulation or of the shifts in cell morphology. However, indomethacin (30 μM) blocked the synergistic interaction between 1,25(OH)₂D₃+ PMA to shift cell morphology and stimulate NO production. Subsequently adding PGE₂ (1 ng/ml) to indomethacin-treated cells restored the ability of 1,25(OH)₂D₃+ PMA to interactively increase cellular NO production, but failed to fully replicate the strong shift in cell morphology typical of PMA + 1,25(OH)₂D₃ treatments. Our findings suggest that interaction between 1,25(OH)₂D₃ and PMA to induce macrophagic differentiation increases iNOS-dependent NO production by a mechanism involving a cyclooxygenase product(s), possibly PGE₂. Received: 13 March 1997 / Accepted: 14 November 1997     

Bone Mineral Density Is a Predictor of Survival

The purpose of this study was to examine the relationship between bone mineral density (BMD) and survival in both sexes and to compare BMD with other established risk factors such as blood pressure and cholesterol. A population-based prospective study of 1924 individuals (850 men, 1074 women) was performed in G?teborg from 1980 to 1983. Measurements of BMD were obtained in 1468 (76%) of the participants (653 men, 815 women). This selection of individuals generated 10,965 person years, and death was registered for 289 men and 197 women in the 7-year period (2661 days) after bone mineral measurement. Later information on date of death was obtained from the official population register. This information covers 7 years from the time of survey of the last examined participant (in Dec. 1983). At the beginning of the study, BMD was measured in the calcaneus by dual photon absorptiometry (DPA), and blood pressure, serum cholesterol, serum triglycerides, and body mass index (BMI) were also recorded. The study was coordinated with the National Register of Causes of Death and the National Cancer Register. A modified version of the Cox proportional hazards model was used to calculate and determine the age-adjusted relations between nontrauma mortality and BMD. When the various quartiles of BMD were compared prospectively from 70, 75, and 79 years of age with survival figures during the 2661-day follow-up period, the first and the second quartiles with the lowest BMD at entry showed the lowest survival rate in both men (P= 0.01) and women (P= 0.01). A decrease of 1 SD of BMD in a univariate analysis was associated with a 1.39-fold increase in mortality in both men (95% confidence interval 1.25–1.56, P < 0.001) and women (95% confidence interval 1.22–1.58, P < 0.001), and a multivariate analysis demonstrated a relative risk of 1.23 (95% confidence interval 1.10–1.41, P < 0.001) in men and 1.19 (95% confidence interval 1.02 to 1.39, P= 0.019) in women. All relations were adjusted for sex, age, and follow-up. This study indicates that BMD is a predictor of survival, especially for subjects over 70. Bone mineral density was found to be a better predictor of death than blood pressure and cholesterol. This study indicates that, after adjustments have been made for diseases, low bone mass is an independent predictor of mortality and might be a marker of general health or functional aging. Its measurement might therefore be a valuable tool in general health investigations. Received: 26 December 1996 / Accepted: 27 January 1998     

Hormonal Regulation of the Osteoblastic Phenotype Expression in Neonatal Murine Calvarial Cells

Osteoblastic cell cultures from fetal rat calvariae have provided a popular model for studying the effects of dexamethasone (DEX) and 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D₃] on gene expression but data from murine calvarial cells are scarce. Species-specific responses of rat and mouse osteoblastic cells to these hormones have been reported previously. In the present study, we investigated the effects of DEX and 1,25(OH)₂D₃ on expression of the osteoblastic phenotype by mouse calvarial cells. These murine osteoblast-like (MOB) cells expressed alkaline phosphatase (ALP) activity and osteocalcin and formed calcified nodules. Unlike the rat calvarial cells, ALP activities and nodule formation in MOB were inhibited by DEX. 1,25(OH)₂D₃ enhanced and DEX lowered the amount of osteocalcin synthesized by MOB. 1,25(OH)₂D₃ did not affect the number of nodules, but increased their sizes. Treating the cells for 2 days with only DEX at the beginning of the culture enhanced the effect of 1,25(OH)₂D₃ on ALP. We found that in murine calvarial cells, DEX inhibits and 1,25(OH)₂D₃ enhances ALP activity, osteocalcin synthesis, and calcified nodule formation. This is in contrast to previous reports of rat calvarial cells where DEX is a positive and 1,25(OH)₂D₃ can be a negative regulator of the osteoblastic phenotype. These results suggest that profound species-specific differences exist between mice and rats in the regulation of the osteoblastic phenotype. Received: 15 October 1997 / Accepted: 16 June 1998     

Skeletal Site-Dependent Expression of the Androgen Receptor in Human Osteoblastic Cell Populations

There are obvious sexual differences in adult skeletal morphology which for the most part are related to differences in size. Higher androgen serum levels in males exert potent osteoanabolic effects and therefore may contribute to this sexual dimorphism of the skeleton. The presence of androgen receptors (AR) in bone cells is a prerequisite for a direct osteoanabolic action of androgens. To investigate the possibility that, in addition to gender-related differences in androgen serum levels, there are also gender-related differences in the osteoblastic expression pattern of the androgen receptor, we examined AR mRNA expression, androgen binding sites, and mitogenic responses to the androgen dihydrotestosterone (DHT) in human osteoblastic cell (HOC) populations. HOCs were isolated from bone biopsy specimens derived from different skeletal sites of healthy adult males and females (2–69 years old). We found that male and female HOCs of all examined ages express similar AR mRNA levels and similar numbers of androgen binding sites. Using whole-cell-binding assays, we observed 3129–8417 androgen binding sites per femoral HOC with apparent KDs of 1.45–2.83 nM depending on the age of the investigated HOC population. Mandibular and cortical HOC of both sexes expressed higher AR mRNA levels, significantly more androgen binding sites per cell, and exhibited significantly greater mitogenic responses to DHT than iliac crest-derived and trabecular HOC of the same skeletal system and the same skeletal-site, respectively. In early adulthood, HOCs of both sexes appear to express somewhat higher AR mRNA levels and to possess more androgen binding sites than prepubertal and senescent HOC. Because sex hormone serum levels rise in puberty, we investigated the regulation of the AR mRNA expression by various steroids. We found that dexamethasone (dexa) and in some experiments also 17β-estradiol (E2) and 1,25-dihydroxyvitamin D3 (D3) increased AR mRNA levels and androgen binding in HOC cultures. A pretreatment with dexa, E2, and D3 significantly increased the mitogenic response of HOCs to DHT. We conclude that (1) higher androgen serum levels in males together with a higher AR expression at certain skeletal sites may contribute to the development of sex-related differences in skeletal morphology, (2) glucocorticoids induce AR gene expression in HOC cultures, and (3) glucocorticoids, E2, and D3 enhance the mitogenic action of DHT. Received: 3 June 1996 / Accepted: 30 April 1997     

Biochemical Responses of Bone Metabolism to 1,25-Dihydroxyvitamin D Administration in Black and White Women

The basis for the racial difference in bone mass between black and white women is not known. Lower bone turnover, better renal calcium conservation, and decreased sensitivity to parathyroid hormone (PTH) have been proposed as explanations. A dynamic comparison of osteoblast function, utilizing stimulation by 1,25-dihydroxyvitamin D [1,25(OH)₂D], has not been tested between these two ethnic groups. We compared well-matched black (n= 15) and white (n= 15) premenopausal women, before and during 5 days of 1,25(OH)₂D administration (1.0 μg/day) in order to assess dynamic indices of bone metabolism. As expected, at baseline, black women had lower levels of serum 25-hydroxyvitamin D and biochemical markers of bone turnover with slightly higher levels of PTH. Black women also had superior renal calcium conservation than white women at baseline. In response to 1,25(OH)₂D administration, black women had a slightly greater increase in serum calcium and greater decrement in PTH. Moreover, black women showed a lesser increment in urinary calcium than white women and a more robust increase in two markers of bone formation – osteocalcin and carboxyterminal propeptide of type 1 procollagen – than white women. There were no changes in bone resorption indices in either race upon 1,25(OH)₂D administration. These data provide preliminary evidence that black women conserve calcium more efficiently under both static and dynamic conditions, and also appear to have better osteoblastic functional reserve than white women. Received: 22 June 1999 / Accepted: 6 September 1999