Insulin-like Growth Factor I Does Not Stimulate Bone Resorption in Cultured Neonatal Mouse Calvarial Bones

Insulin-like growth factor I (IGF-I) has documented anabolic effects on osteoblasts, whereas its influence on osteoclasts and on bone resorption is unclear. We have investigated the effects of IGF-I on osteoclast recruitment and bone resorption in vitro. IGF-I (at and above 1 nM) stimulated the formation of multinucleated tartrate-resistant acid phosphatase positive cells in murine bone marrow cultures, incubated for 9 days. The number of multinucleated cells increased to 540 ± 160% of control (mean ± SEM) in cultures treated with 10 nM IGF-I. IGF-I (0.1–100 nM) had no effect by itself on ⁴⁵Ca-release from prelabelled neonatal mouse calvarial bones. However, IGF-I (100 nM) had an inhibitory effect on bone resorption induced by prostaglandin E₂ and 1,25(OH)₂D₃. These findings indicate that IGF-I enhances the formation of osteoclasts-like cells in long-term bone marrow cultures. In bone organ cultures, however, IGF-I has an inhibitory effect on stimulated bone resorption, suggesting that IGF-I inhibits existing osteoclasts and, alternatively, that IGF-I interferes with the osteoblast-derived factor(s) that stimulate existing osteoclasts. Received: 15 August 1995 / Accepted: 1 April 1996     

Regulation of plasminogen activator and plasminogen activator inhibitor production by growth factors and cytokines in rat calvarial cells

Summary Fetal rat osteoblast-enriched calvarial cells were used to study the effects of various growth factors and cytokines on plasminogen activator (PA) and plasminogen activator inhibitor (PAI) activities and the possible relationship of these effects to bone resorption. Confluent cultures were exposed to various factors under serum-free conditions, and levels of PA and PAI activities were examined in both conditioned medium (CM) and cell layer using the¹²⁵I-fibrin plate assay, fibrin zymogram, and reverse fibrin zymogram. According to the¹²⁵I-fibrin plate assay or zymogram, incubation of cells with acidic fibroblast growth factor (aFGF), basic FGF (bFGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) elevated the PA activity in the CM as well as in the cell layer extract. Incubation with interleukin 1α (IL-1α), tumor necrosis factor α (TNFα), and insulin-like growth factor I (IGF-I) produced no change in PA activity in either CM or cell layer. Addition of transforming growth factor β (TGFβ) to calvarial cells resulted in nearly undetectable PA activity in CM with the fibrin plate assay but increased PA activity on the fibrin zymogram after PAI was separated from PA by SDS-PAGE. A reverse fibrin zymogram indicated that PAI activity was greatly enhanced in TGFβ-treated CM. TGFβ treatment also increased PA activity in the cell layer of calvarial cells. Treatment of calvarial cells with bFGF and PDGF slightly increased PAI secretion into medium. This increase, however, was not as dramatic as the increase of PA induced by these two agents. IL-1α and TNFα did not change PAI concentration in CM. No detectable PAI activity was found in the cell layer in control and treated groups. The PA found in the CM and cell layer of rat calvarial cells was the urokinase type; the PAI stimulated by TGFβ was the endothelial cell type, PAI-1. The regulation of PA activity by growth factors and cytokines did not correlate with their resorption-stimulating activities. Thus, PA secreted by osteoblasts may not be the only factor involved in the initiation of bone resorption. Delineation of the function of PA and PAI in the physiology of bone tissue awaits further studies.     

An In Vitro and In Vivo Study of Cytokines in the Acute-Phase Response Associated with Bisphosphonates

We studied the acute phase response, including specific cytokine production, [interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor α(TNFα)] following a single dose of Aredia (disodium pamidronate) in patients with increased bone turnover and, in vitro, the role played by specific cytokines in the acute-phase reaction which may follow the administration of aminobisphosphonates. An in vivo exploratory study was done on 24 in- and outpatients with increased bone turnover given a single intravenous dose of pamidronate 60 mg. Measurements were taken at baseline and at 24, 48, and 72 hours. The main outcome measures were changes from baseline in serum IL-1, IL-6, and TNFα. In addition, C-reactive protein (CRP), white blood cell count (WCC), lymphocyte count, and elastase concentration were measured. Symptomatic evaluation was made of fever, bone pain, and rigors. In vitro, whole blood from eight healthy volunteers was exposed to various concentrations of the three bisphosphonates—pamidronate, clodronate, and zoledronate. Measurements were taken immediately before and at 3, 6, and 10 hours after exposure to drugs. The main outcome measures were changes in serum IL-1, IL-6, and TNFα. In vivo, there was a statistically significant (P < 0.001) increase in median values of TNFα in all post-baseline measurements. Median values for IL-6 also showed a significant (P < 0.001) increase at 24 hours after dosing. There were no statistically significant changes in median IL-1 values. Few patients showed any change from baseline in total WCC or in lymphocyte count, but 62.5% of patients with normal range baseline values for CRP increased to above normal levels after treatment. Fourteen patients experienced fever; 2 reported rigors. There was no correlation between fever and changes in cytokines. There were no serious adverse experiences or premature discontinuations due to poor tolerability, and 91% of the patients expressed willingness to receive pamidronate again. In vitro, an increase in TNFα and a mild increase in IL-6 was seen with all bisphosphonates, with the greatest effects seen with the highest concentration of both pamidronate and zoledronate. No changes were observed in IL-1 with any agent. Significant changes in both TNFα and IL-6 were observed within 3 days of a single dose of pamidronate in patients treated for the first time confirming previous findings. However, the lack of change in IL-1 in vivo and in vitro does not support the hypothesis that this cytokine plays a major role in the acute phase reaction. The cellular mechanism of the interaction among aminobisphosphonates, Il-6, and TNFα requires further investigations. The results of the in vitro study are consistent with the in vivo findings. Received: 30 September 1996 / Accepted: 21 May 1997     

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     

Basic Fibroblast Growth Factor in the Presence of Dexamethasone Stimulates Colony Formation, Expansion, and Osteoblastic Differentiation by Rat Bone Marrow Stromal Cells

Basic fibroblast growth factor (bFGF) is known to stimulate endosteal bone formation in vivo by a mechanism possibly mediated via osteoblast precursor cells present in the bone marrow. In high density cultures of primary bone marrow cells, and in the presence of glucocorticoids, bFGF stimulates the formation of a bone-like matrix; however, due to the dense nature of these cultures, the exact mechanism of action is unclear. In an adaptation of the fibroblastic colony formation unit assay, in which the bone marrow cells are grown in the presence of dexamethasone, β-glycerophosphate, and ascorbate, mineralized colonies are formed which stem from single mesenchymal precursor cells and grow in isolation from each other. Using this system we have been able to investigate the mechanism by which bFGF stimulates the formation of bone like tissue in vitro. We have shown that bFGF increases the formation of a calcified collagenous matrix in vitro by (1) increasing the total number of fibroblastic colonies formed, (2) increasing the proportion of differentiated colonies that synthesize collagen and calcify, and (3) stimulating the proliferation and collagen accumulation of the individual colonies. A maximal increase in total and differentiated colony numbers was seen after only 5 days exposure to bFGF, however, continued exposure to bFGF continued to increase the size and collagen content of the individual colonies. Bearing in mind the endosteal location of newly formed bone seen after treatment with bFGF, these processes may well play an active role in this effect. Received: 17 January 1997 / Accepted: 30 July 1998     

A Human Homolog of the 150 kD Avian Osteoclast Membrane Antigen Related to Superoxide Dismutase and Essential for Bone Resorption is Induced by Developmental Agents and Opposed by Estrogen in FLG 29.1 Cells

Osteoclast development from hematopoietic bone marrow precursors is associated with the expression of various enzymes, receptors, adhesion molecules, and other specialized components. Among these is a novel 150 kD superoxide dismutase-related membrane glycoprotein, originally identified by its reaction with the anti-osteoclast monoclonal antibody 121F. This antigen is uniquely restricted to osteoclasts in bone, universally present on osteoclasts from multiple species, induced during osteoclast differentiation in vitro and in ovo, and required at high levels for avian osteoclastic bone pit resorption. Expression of a comparable human antigen was investigated using human leukemic FLG 29.1 cells capable of differentiating towards an osteoclast-like phenotype. Phorbol ester, 1,25 (OH)₂ vitamin D₃, and osteoblast-derived soluble factors elicited dose and time-dependent inductions of this antigen as measured by enzyme-linked immunosorbent assay (ELISA) and immunocytochemical staining, coincident with their display of multiple other osteoclastic features. Synergistic interactions of these modulators led to further elevations in the ultimate expression levels of this antigen, although not to the full extent associated with in vivo-formed avian osteoclasts. The potent antiresorptive hormone 17β-estradiol, but not its inactive α isomer, partially suppressed the phorbol ester-induced elevation of the 121F antibody-reactive antigen in FLG 29.1 cells as it does in avian osteoclast-like cells. Characterization of the human antigen isolated from FLG 29.1 cells by 121F immunoaffinity purification demonstrated that this regulated membrane component was synthesized by these human cells, more abundant following their differentiation into osteoclast-like cells, and similar biochemically and immunologically to the 150 kD integral membrane glycoprotein previously described from avian osteoclasts. Therefore, this report is the first documentation that human osteoclast-like FLG 29.1 cells express, in a developmentally regulated fashion, a homolog of the specific 150 kD avian osteoclast surface antigen that is related to superoxide dismutase, a protective free radical scavenging enzyme and is essential for osteoclastic bone resorption. Received: 1 April 1996 / Accepted: 19 July 1996     

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     

Chondroclasts and Osteoclasts in Bones of Young Rats: Comparison of Ultrastructural and Functional Features

The aim of the present study was to characterize cells involved in resorption during endochondral bone formation. We investigated whether the cells involved in cartilage breakdown at the epiphyseal/metaphyseal border, i.e., chondroclasts, share the characteristics of bone/cartilage-resorbing osteoclasts at the metaphyseal/diaphyseal border regarding ultrastructural features and functional activity. Morphometric evaluation showed that chondroclasts do not form ruffled borders and clear zones, i.e., well-known resorption characteristics, to the same extent as osteoclasts, present at the lower metaphysis. Instead, chondroclasts tend to express an undifferentiated surface adjacent to the matrix, not structurally different from the basolateral plasma membrane. Tartrate-resistant acid phosphatase (TRAP) was used as a marker for functional activity. Immunohistochemical staining by light microscopy was strong in both chondroclasts and in osteoclasts. Furthermore, in situ hybridization revealed large amounts of TRAP mRNA in chondroclasts as well as in osteoclasts. Ultrastructural immunohistochemistry suggests extensive secretion of the TRAP enzyme in the ruffled border area of both chondroclasts and osteoclasts. Intracellular accumulation was seen particularly in chondroclasts, possibly as a consequence of a relative disinclination to develop a ruffled border. Thus, semiquantitative estimation of TRAP distribution showed an inverse relationship between extracellular and intracellular TRAP in chondroclasts and osteoclasts. These results indicate that chondroclasts and osteoclasts differ, not only with respect to location but possibly also by mode of action. The observed differences may reflect the maturation sequence of these multinucleated cells when associated with different metaphyseal trabecular surfaces. Received: 22 January 1998 / Accepted 8 April 1998     

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     

Soluble Factors Secreted by Macrophage-like Cells In Vitro Cause Osteoprogenitor Cell Detachment

Explant cultures of adult rabbit bone were obtained from a variety of skeletal sites, with the intention of using the cells in a new type of bone graft. The explant cultures contained large numbers of esterase-positive, alkaline phosphatase-negative, TRAP-negative macrophage-like cells (MP), as well as osteoblast-like osteoprogenitor (OP) cells (alkaline phosphatase-positive). OP numbers were assessed by cell counts and MTT assay. The presence of the MP cells appeared to give rise to a reduction in OP numbers in culture, through cell detachment and cell lysis. In addition, after passaging, many OP were unable to reattach to the culture vessels in the presence of MP, depending upon the surface area available for reattachment. The presence of tumor necrosis factor α (TNFα) in culture medium from these cell cultures was demonstrated by a specific enzyme-linked immunosorbent assay (ELISA). A direct relationship was demonstrated between MP numbers and TNFα concentration and an inverse relationship between MP numbers and OP numbers in co-cultures. This was also found when OP cells were exposed to different concentrations of rTNFα, in place of the MP. Incubation with anti-TNFα inhibited the effect of MP and TNFα on OP. These results suggest that MP are able to cause detachment and lysis of OP cells, probably by secretion of a soluble factor in vitro which may be TNFα. Received: 3 January 1997 / Accepted: 5 March 1998     

Cytokine Production and Bone Mineral Density at the Lumbar Spine and Femoral Neck in Premenopausal Women

Cytokines such as interleukin-1 (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) can influence both bone resorption and bone formation. The objective of this cross-sectional study was to examine the relationship between cytokine production by peripheral blood mononuclear cells (PBMC) and bone mineral density (BMD); the annual rate of change in BMD was examined. Subjects participating in a randomized clinical trial entitled the Women's Healthy Lifestyle Project in Allegheny County, Pennsylvania were used. They included 50 healthy premenopausal women, aged 45–52 years, who had regular menses within the past 3 months and were not on replacement estrogens. Dual-energy X-ray absorptiometry measurements at the AP lumbar spine and femoral neck were made at baseline and at the first annual exam using a Hologic QDR 2000 densitometer. Cytokine production of IL-1β, IL-6, and TNF-α by PBMC was measured at the annual exam. The median values for stimulated cytokine production by PBMC were 3.92 ng/ml, 31.3 ng/ml, and 1.05 ng/ml, for IL-1β, IL-6, and TNF-α, respectively. There were modest correlations between cytokine production and cross-sectional BMD, ranging from r =−0.30 to r =−0.13. Trends of greater spinal bone loss were observed in women with ``high' (≥75th percentile) cytokine production of stimulated IL-1β and IL-6 (IL-1β: ``high' =−1.56% ± 0.70 versus ``low' (<75th percentile) =−0.56% ± 0.35, P= 0.21). In contrast, greater annual gains in femoral neck BMD were observed in those with high cytokine production of IL-1β and IL-6 (IL-1β: high = 3.39% ± 1.16 versus low =−0.85 ± 0.58, P= 0.002). There was no association between stimulated TNF production and annual change in BMD. In this population of healthy premenopausal women, the relationship between cytokine production by PBMC and the rate of change in BMD was significantly different for the lumbar spine and femoral neck, possibly reflecting differences in the proportion of trabecular and cortical bone at these sites. Received: 5 February 1997 / Accepted: 11 May 1998     

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     

Alendronate Reduces Adhesion of Human Osteoclast-like Cells to Bone and Bone Protein-Coated Surfaces

Bisphosphonates (BPs) are potent inhibitors of bone resorption and are therapeutically effective in disease of increased bone turnover, but their mechanism(s) of action remain to be elucidated. Using as experimental model human osteoclast-like cell lines derived from giant cell tumors of bone, extensively characterized for their osteoclast features, we investigated the adhesive properties of osteoclasts on bone slices and on different proteins of the extracellular matrix in the presence of BPs. Adhesion assays using bone slices pretreated with ALN, at the established active concentration, showed that, although the morphology of osteoclasts plated onto pretreated bone slices was not modified, the number of adherent cells was reduced by the treatment of about 50% vs. controls. The effect of ALN on the adhesion of osteoclast-like cells onto specific extracellular matrix proteins, such as bone sialoprotein-derived peptide, containing the RGD sequence, conjugated to BSA (BSP-BSA) and fibronectin (FN), was also tested. In the case of FN the treatment with ALN of protein-coated wells did not modify the percentage of cell adhesion compared with the control, whereas onto BSP-BSA the presence of ALN significantly reduced adhesion of about 40–45%, suggesting that the inhibitory effect of ALN on cell adhesion could probably be due to the interference with receptors specifically recognizing bone matrix proteins as α_Vβ₃ integrins. Furthermore, ALN induced Ca-mediated intracellular signals in osteoclasts, triggering a 2-fold increase in intracellular calcium concentration. Received: 8 August 1997 / Accepted: 27 January 1998     

Successful Detection of Active Osteoclasts In Situ by Systemic Administration of an Osteoclast-Specific Monoclonal Antibody

Cell-surface proteins preferentially expressed on osteoclasts are thought to play important roles in the functional modulation of the osteoclasts. Recently, we found a novel cell-surface antigen designated Kat1-antigen (Kat1-Ag) specifically expressed on rat osteoclasts. It would be useful to regulate the functional activity of the osteoclasts directly via an osteoclast-specific antigen expressed on the cell surface of the osteoclasts. In order to establish the basis of such an application, in the present study we established a method for the direct detection of osteoclasts in situ by a systemic administration of the anti-Kat1-Ag monoclonal antibody (mAb Kat1) to rats, and we successfully detected functional osteoclasts in situ. Prior to performing in vivo experiments, we examined the reactivity of the mAb Kat1 to the isolated rat osteoclasts. Approximately 40–80% of the osteoclasts were reactive with mAb Kat1, suggesting that this mAb recognizes osteoclasts in a specific differentiation or functional state. Calcitonin treatment of osteoclast-like cells formed in vitro from bone marrow cells resulted in a conversion of Kat1-positive osteoclast-like cells into Kat1-negative multinucleated cells, showing the positive correlation between the Kat1-Ag expression and the potential bone-resorbing activity of osteoclasts. Administration of this lineage-specific mAb to the peritoneal cavity of newborn rats resulted in a successful recruitment of mAb Kat1 to the newly formed osteoclasts and functional osteoclasts in a highly specific manner. Detailed analysis by immunoelectron microscopy revealed that this mAb specifically bound to the basolateral side of the active osteoclasts, which were identified by their typical ruffled border and clear zone, whereas the mAb did not react to postfunctional osteoclasts. These findings demonstrate a high potential utility of mAb Kat1 in osteoclast-targeted regulation of bone remodeling. Received: 9 July 1996 / Accepted: 20 February 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     

Pasteurella multocida Toxin Stimulates Bone Resorption by Osteoclasts via Interaction with Osteoblasts

In this study we used an in vitro assay system with osteoblast and osteoclast co-cultures to assess the effect of purified recombinant Pasteurella multocida toxin on bone resorption. Resorption was measured by the release of a telopeptide breakdown product of type I collagen. It was found that P. multocida did not stimulate bone resorption by osteoclasts directly and also did not stimulate bone breakdown via the release of collagenase or other proteases from osteoblasts. During co-culture of osteoblasts and osteoclasts, with cell-cell contact prevented, P. multocida toxin produced no significant effect. Osteoblast-conditioned media gave a biphasic effect; low concentrations of P. multocida toxin stimulated bone resorption, whereas 100 ng/ml inhibited resorption by osteoclasts. However, when both cell types were co-cultured with cell-cell contact permitted, P. multocida toxin induced a large concentration-dependent increase in bone resorption over a 7-day period. This suggested that P. multocida toxin causes bone breakdown via an osteoblast-dependent mechanism and that a membrane-bound receptor may be involved. Received: 8 July 1997 / Accepted: 8 April 1998     

The Effect of Alendronate on Cytokine Production, Adhesion Molecule Expression, and Transendothelial Migration of Human Peripheral Blood Mononuclear Cells

Since both osteoclasts and macrophages belong to the mononuclear phagocytic system it is conceivable that bisphosphonates not only affect bone metabolism but also inflammatory responses. The migration of mononuclear cells into perivascular tissue is a central event in inflammatory reactions. We studied the effects of the aminobisphosphonate alendronate on the transendothelial migration of human peripheral blood mononuclear cells in an in vitro model. Alendronate (at a concentration of 100 μM) significantly increased the percentage of peripheral blood mononuclear cells that migrated through endothelial cell monolayers. Similar results were obtained with another aminobisphosphonate, viz, pamidronate. An overnight treatment of the endothelial cell monolayers with alendronate did not alter the rate of peripheral blood mononuclear cells that subsequently migrated. The overnight cultivation of the peripheral blood mononuclear cells in the presence of alendronate resulted in an increased surface expression of CD54 (intercellular adhesion molecule-1, ICAM-1) in both CD14⁺ and CD3⁺ cells; in CD14⁺ cells also the expression of CD49d (α₄ subunit of late activation antigen-4, VLA-4) increased after alendronate treatment. Alendronate treatment of peripheral blood mononuclear cells also resulted in an increased production of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). We conclude that alendronate has a distinct effect on the transendothelial migration of human peripheral blood mononuclear cells in vitro. Alendronate may either directly or indirectly, e.g., by augmenting the production of proinflammatory cytokines, influence the expression of certain adhesion molecules and thereby facilitate transendothelial migration. These effects could be related to the transient leukopenia reported following intravenous administration of relatively high doses of aminobisphosphonates for the treatment of hypercalcemia of malignancy. Received: 11 September 1997 / Accepted: 20 February 1998     

Biochemical Markers of Bone Turnover Reflect Femoral Bone Loss in Elderly Women

Although over 90% of hip fractures occur in patients over age 70, few data are available on femoral bone loss in this age group. To examine the relationship between biochemical markers of bone turnover and femoral bone loss in the elderly, 36 female and 17 male, healthy, community-dwelling elderly over age 65 (mean ± SD age: women 71 ± 4 years, men 75 ± 5 years) were followed for 3 years. Annual bone mineral density measurements of the hip and lumbar spine by dual-energy x-ray absorptiometry (DXA) were obtained and biochemical markers of bone resorption (urinary N-telopeptide crosslinks, free pyridinoline, total pyridinoline, total deoxypyridinoline, and hydroxyproline) and bone formation (serum osteocalcin, bone-specific alkaline phosphatase) were obtained at the end of year 3. In elderly women, longitudinal bone loss at the total hip was negatively correlated with markers of bone resorption (r =−0.39 to −0.52, P < 0.05), bone formation (r =−0.38, P < 0.05), and age (r =−0.39, P < 0.05). Markers of bone resorption were correlated with markers of bone formation (r = 0.63 to 0.74, P < 0.01). In multiple regression analysis, urinary N-telopeptide crosslinks (marker of resorption), serum osteocalcin (marker of formation), and serum parathyroid hormone explained 43% of the variability of bone loss at the total hip in women. These parameters were not related to bone loss in men. We conclude that femoral bone loss increases with age in women over 65. Measurements of specific biochemical markers of bone turnover are correlated with longitudinal bone loss in elderly women. These markers may help identify women at greatest risk for bone loss who would benefit most from therapeutic interventions. Received: 28 January 1996 / Accepted: 3 May 1996     

2-Methoxyestradiol Inhibits Longitudinal Bone Growth in Normal Female Rats

2-Methoxyestradiol (2-MeO-E₂), a major metabolite of 17β-estradiol, may function as a physiological tumor suppressor and is being investigated for clinical applications. It has been reported to target rapidly dividing cells. We investigated the effects of 2-MeO-E₂ on the growth plate of young rats because normal longitudinal bone growth requires rapid proliferation of cartilage and endothelial cells. Sexually mature (3-month-old) normal female rats were treated with 2-MeO-E₂ (100 mg/kg/day) for 13 days and it was found to have no effect on uterine weight but reduced serum cholesterol. The estrogen metabolite had no effect on either cortical or cancellous bone. In contrast, 2-MeO-E₂ dramatically reduced longitudinal bone growth rate at the proximal tibia from 55 ± 2 to 20 ± 2 μm/day (P < 0.001) and growth plate thickness from 153 ± 14 to 70 ± 6 μm (P < 0.001). The latter decrease was due to significant reductions in the height of both the proliferative (P < 0.001) and the hypertrophic (P < 0.001) zones. These results in normal female rats demonstrate that 2-MeO-E₂ inhibited longitudinal bone growth but had no effect on either radial bone growth or cancellous bone turnover. 2-MeO-E₂ was shown by these studies to have the ability to discriminate between bone and cartilage, as well as between reproductive and nonreproductive estrogen-target tissues. Thus, 2-MeO-E₂ is a naturally produced estrogen metabolite that demonstrates unique tissue selectivity. Received: 21 July 1999 / Accepted: 12 January 2000