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     

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     

Complex Shape Changes in Isolated Rat Osteoclasts: Involvement of Protein Kinase C in the Response to Calcitonin

The cytoplasmic spreading of osteoclasts has been used to assess responsiveness to agents such as calcitonin and associated signal transduction mechanisms. Although cyclic AMP and intracellular calcium are known mediators of calcitonin effects in osteoclasts, the role of protein kinase C (PKC) is less clear. We have used time-lapse videomicroscopy of isolated rat osteoclasts to characterize shape changes induced by calcitonin, forskolin, and phorbol 12-myristate-13-acetate (PMA) in the absence and presence of PKC blockers. Treatment with calcitonin reduced cytoplasmic plan area but increased perimeter length, resulting in a characteristic ``stellate' appearance, whereas forskolin produced ``nonstellate' contraction. The response of osteoclasts to PMA was dose dependent. High concentrations (10⁻⁷–10⁻⁶ M) produced biphasic responses with transitory, calcitonin-like ``stellate' contraction followed by sustained expansion, whereas low concentrations (10⁻¹¹–10⁻⁹ M) produced expansion only. The effects of low-concentration PMA could be prevented by pretreatment with a PKC blocker, whereas the effects of high concentrations were only partially inhibited. The effects of forskolin were unchanged by pretreatment with the PKC blocker. Treatment with calcitonin in the presence of various PKC blockers resulted in paradoxical transient expansion followed by contraction. These results indicate that calcitonin-induced shape change in osteoclasts is a complex process involving protein kinase C in addition to cyclic AMP-dependent mechanisms and possibly other factors. Received: 31 October 1996 / Accepted: 26 April 1997     

Calcitonin,Etidronate, and Calcidiol Treatment in Bone Loss after Cardiac Transplantation

Cardiac transplantation is associated with severe bone loss caused by glucocorticoids, immunosuppressive treatment, and other factors. Treatment protocols for the prevention of bone loss is being studied. Forty patients who underwent cardiac transplantation were randomly given calcitonin (n= 13; 100 UI/d, nasal route), etidronate (n= 14; cyclical treatment 400 mg p.o./d/2 weeks/3 months), or calcidiol (n= 13; 32,000 IU/weekly) therapy for at least 18 months. Serum parameters (Ca, P, alkaline phosphatase, osteocalcin, intact PTH), urinary calcium, and vertebral mineral density (VMD; L2–L4, DXA Hologic QDR 1000) were measured immediately before treatment and after 6, 12, and 18 months of therapy after cardiac transplantation. Patients with cardiac transplantation had a VMD significantly lower than age and sex-matched Spanish controls. Prevalence of osteoporosis (Z-score below −2 SD) was 30%. Osteocalcin levels increased at 6, 12, and 18 months of treatment in the three groups. After 18 months of treatment, VMD increased significantly in the calcidiol 4.9%, vs. −1.19% and −0.19% in the calcitonin and etidronate groups, respectively. A lower incidence of fracture was found in patients treated with calcidiol during the study. In summary, we have found in this open randomized study that calcidiol was the most effective drug in the prevention and treatment of bone loss in patients after cardiac transplantation. Received: 2 February 1996 / Accepted: 25 June 1996     

Prostaglandin E2 Directly Inhibits Bone-Resorbing Activity of Isolated Mature Osteoclasts Mainly Through the EP4 Receptor

Prostaglandins (PGs) are well known to be important local factors in regulating bone formation and resorption. PGE₂ is a potent stimulator of bone resorption because of enhancing osteoclast formation by its indirect action through stromal cells. However, the direct action of PGE₂ on functionally mature osteoclasts is still controversial. In this study using highly purified rabbit mature osteoclasts, we examined the direct effect of PGE₂ on osteoclastic bone-resorbing activity and its mechanism. PGE₂ inhibited resorption pit formation on a dentine slice by the purified osteoclasts in a dose- and time-dependent manner. The inhibitory effect appeared as early as 4 hours after the PGE₂ addition. Forskolin and 12-0-tetradecanoyl phorbol-13-acetate (TPA), respective activators of adenylate cyclase and protein kinase C, also decreased the osteoclastic bone-resorbing activity. PGE₂ increased the content of intracellular cAMP in a dose range effective for the inhibition of bone resorption, whereas the prostanoid did not alter the intracellular level of inositol triphosphate. The inhibition of osteoclastic bone resorption by PGE₂ was amplified and diminished by a cAMP phosphodiesterase inhibitor (isobutyl methylxanthine) and a protein kinase A inhibitor (Rp-cAMP), respectively. Of four different subtypes of PGE₂ receptors (EPs), EP4 mRNA was predominantly expressed in isolated osteoclasts, whereas the other types of EP mRNA were detected in only small amounts. These results suggest that the PGE₂ inhibitory effect was mediated by an adenylate cyclase system coupled with EP4. This possible association of PGE₂ with EP4 in mature osteoclasts was supported by the finding that a specific agonist of EP4 (AE-604) inhibited the bone-resorbing activity and elevated the intracellular cAMP content. However, butaprost, a selective EP2 agonist, also mimicked the PGE₂ effects on isolated osteoclasts although EP2 mRNA expression was minimal. In conclusion, PGE₂ directly inhibits bone-resorbing activity of functionally mature osteoclasts by activation of the adenylate cyclase system, perhaps mainly through EP4. Received: 21 July 1999 / Accepted: 31 January 2000     

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     

Cortical Bone Resorption in Osteoporosis

A study was made of 110 women: 35 healthy premenopausal, 40 healthy postmenopausal, and 35 women diagnosed as having postmenopausal osteoporosis. The postmenopausal women had similar ages and years since menopause (YSM). In all of the women, total bone mass was evaluated by dual-energy X-ray absorptiometry and metacarpal morphometry was evaluated by radiogrammetry on the second metacarpal of the nondominant hand, performed by computed radiography. An external metacarpal diameter of ≥7.4 mm was required as proof of having developed an adequate peak bone mass. The endosteal diameter, which is indicative of bone resorption in both groups of postmenopausal women, obtained in the postmenopausal groups was subtracted from the endosteal diameter obtained in the premenopausal group and the resulting figure was divided by the years since menopause to calculate the rate of cortical bone resorption/year for each group. The endosteal diameters values differed in the three groups studied (P < 0.0001): 3.2 ± 0.7 mm in the healthy premenopausal women; 3.9 ± 0.6 mm in the healthy postmenopausal women; and 4.7 ± 0.5 mm in the osteoporotic postmenopausal women. The rate of cortical bone resorption was 0.068 ± 0.002 mm/YSM (years since menopause) in the osteoporotic postmenopausal women and 0.033 ± 0.003 mm/YSM in the healthy postmenopausal women (P < 0.0001). These figures reflect the importance of bone resorption, as opposed to deficient bone formation, as a cause of osteoporosis. Received: 27 January 1995 / Accepted: 21 August 1996     

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     

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     

Loss of the Differentiated Phenotype Precedes Apoptosis of ROS 17/2.8 Osteoblast-Like Cells

Osteoblast cells, recruited from mesenchymal precursors, initiate the final phase of bone remodeling by secreting the protein components of the bone matrix. Upon completion of remodeling, some of these osteoblasts may further differentiate, giving rise to matrix-embedded osteocytes and bone lining cells. The fate of the remaining osteoblasts is unknown, although by analogy with other cell systems, apoptotic cell death may be involved. We induced and characterized the apoptotic process in ROS 17/2.8 osteosarcoma cells by growing and maintaining confluent cultures in low serum medium. At confluence, but prior to apoptosis, the levels of collagen type I, alkaline phosphatase, and osteocalcin mRNAs declined abruptly. Expression of two housekeeping genes (ribosomal protein RPS6 and GAPDH) remained unchanged. Some 72 hours later cells began to show morphological and biochemical features of apoptosis, namely, chromatin condensation, membrane budding, and internucleosomal degradation of genomic DNA. We conclude that serum starvation-induced apoptosis of ROS 17/2.8 cells can serve as a model for investigating the mechanisms of osteoblastic apoptosis. Received: 20 November 1996 / Accepted: 8 January 1998     

Molecular Chaperones Stimulate Bone Resorption

Molecular chaperones, also known as heat shock proteins (hsp), are intracellular proteins found in all cells that catalyze protein folding. We have discovered that one class of bacterial molecular chaperone, the chaperonins, are potent inducers of bone resorption. To address the question of whether the osteolytic activity of the chaperonins was unique to this protein class, or was a common attribute of molecular chaperones generally, we have examined a number of bacterial and mammalian molecular chaperones for activity in the murine calvarial bone resorption assay. All the Escherichia coli molecular chaperones (groEL, groES, and dnaK) were active. The osteolytic activity of groEL was inhibited by indomethacin and the natural antagonist of interleukin-1 receptor antagonist (IL-1ra) but was unaffected by neutralization of tumor necrosis factor (TNF) or inhibition of 5-lipoxygenase. Mammalian molecular chaperones of molecular mass 27, 47, 70, and 90 kDa were also tested and, with the exception of the 47 kDa protein, all showed activity in the murine calvarial assay. Molecular chaperones appear, therefore, to have the capacity to modulate the cellular processes in bone explant cultures, resulting in resorption of the calcified matrix. The possibility that these proteins could play a role in the normal or pathological remodeling of bone is discussed. Received: 15 October 1997 / Accepted: 24 June 1998     

Calcium Supplementation Suppresses Bone Resorption in Early Postmenopausal Women

In order to establish whether calcium supplementation suppresses bone resorption in early postmenopausal women and whether any response is related to calcium absorption status, we studied 22 healthy women (median age 52 years) all within 5 years of the menopause. Urine was collected between 9.00 p.m. and 9.00 a.m., and 9.00 a.m. and 9.00 p.m., (2 days) and a fasting blood and spot urine sample was obtained at 9 a.m. On the first day, 5 μCi of ⁴⁵Ca in 250 ml water with 20 mg calcium carrier as the chloride was given at 9.00 a.m. and a further blood sample was obtained at 10.00 a.m. to measure calcium absorption. A 1 g calcium load was given at 9.00 p.m., immediately before the second 24-hour urine collection. There was a rise in plasma ionized calcium (1.18 ± 0.010 mmol/liter versus 1.21 ± 0.011 mmol/liter, P < 0.01) and a fall in plasma PTH (4.2 ± 0.34 pmol/liter versus 3.5 ± 0.31 pmol/liter, P < 0.01) from baseline after the calcium load, and a trend for the magnitude of the change in PTH to be inversely related to calcium absorption (r =−0.33, P= 0.13). In the fasting spot urine samples, there were falls in hydroxyproline (OHPr/Cr; 14.6 ± 0.71 versus 12.6 ± 0.83, P < 0.001), pyridinoline (Pyr/Cr; 75 ± 2.8 versus 70 ± 3.5, P < 0.05), and deoxypyridinoline (Dpd/Cr; 22.7 ± 1.2 versus 19.5 ± 1.1, P < 0.005) after the calcium load. The calcium load suppressed urinary Dpd/Cr between 9.00 p.m. and 9.00 a.m. (P < 0.005), but not between 9.00 a.m. and 9.00 p.m. We conclude that acute administration of a 1 g calcium load suppresses bone resorption in early postmenopausal women, probably by decreasing PTH secretion. Received: 2 December 1996 / Accepted: 21 May 1997     

Human Osteoclast Formation from Blood Monocytes, Peritoneal Macrophages, and Bone Marrow Cells

Mononuclear precursors of the human osteoclast have been identified in both bone marrow and the circulation in man, but osteoclast membership of the mononuclear phagocyte system (MPS) and its precise cellular ontogeny remain controversial. We isolated human hematopoietic marrow cells, blood monocytes, and peritoneal macrophages and incubated each of these cell populations with UMR106 osteoblast-like cells on glass coverslips and dentine slices in both the presence and absence of 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃), macrophage-colony stimulating factor (M-CSF), and dexamethasone. Cells isolated from peripheral blood and peritoneal dialysis fluid were positive only for monocyte/macrophage markers (CD11a, CD11b, CD14, and HLA-DR) and negative for osteoclast markers [tartrate-resistant acid phosphatase (TRAP), vitronectin reception (VNR), and calcitonin (CT) receptors and did not form resorption pits on dentine slices after 24 hours in culture. Similarly marrow cells did not form resorption pits on dentine slices after 24 hours in culture. However, after 14 days in co-culture with UMR106 cells, in the presence of 1,25(OH)₂D₃ and M-CSF, numerous TRAP, CT receptor, and VNR-positive multinucleated cells capable of extensive lacunar resorption were formed in co-cultures of all these preparations. The presence of 1,25 (OH)₂D₃, M-CSF, and UMR106 were absolute requirements for osteoclast differentiation. It is concluded that precursor cells capable of osteoclast differentiation are present in the marrow compartment, the monocyte fraction of peripheral blood, and in the macrophage compartment of extraskeletal tissues and that these cells are capable of differentiating into mature functional osteoclasts. These findings argue in favor of osteoclast membership of the human MPS. Received: 3 January 1997 / Accepted: 14 November 1997     

Acute Alteration in Bone Mineral Density and Biochemical Markers for Bone Metabolism in Nephrotic Patients Receiving High-Dose Glucocorticoid and One-Cycle Etidronate Therapy

It is widely known that glucocorticoids induce and accelerate osteoporosis. High-dose glucocorticoids are administrated daily to patients in the acute phase of nephrotic syndrome. It could be inferred that high-dose glucocorticoids rapidly decrease patients' basal bone mineral density (BMD) and this accelerates the natural progress of osteoporosis associated with aging or menopause. Nine nephrotic patients (male/female: 5/4) without previous prednisolone administration were chosen to measure BMD and the level of the markers for bone turnover before and after treatment for 3 months (total prednisolone administration: 4.5 ± 0.0 g). Twenty-three patients under remission with prednisolone administration (male/female: 14/9) were included in the long-term treatment group. Patients in this group whose %YAM in the lateral lumbar spine was less than 89% were classified into a low BMD group (n = 10, male/female: 3/7). They were administered etidronate disodium at 200 mg/day for 14 days. BMD and % of young adult mean (YAM) in the lumbar spine (L2-L4 in lateral objection) and other regions were measured by dual-energy X-ray absorptiometry. As markers of bone metabolism, the urinary level of deoxypyridinoline (Dpd) was determined to evaluate osteogenesis, and serum osteocalcin was measured to evaluate bone resorption. BMD of the lumbar spine significantly decreased in the 3-month treatment group (752 ± 96 mg/cm², 7 ± 4% reduction) compared with the pretreatment group (810 ± 85 mg/cm²). BMD in the long-term treatment group decreased continuously (683 ± 135 mg/cm²). No significant differences were noted in other measurement sites. BMD in the etidronate treatment group increased significantly (597 ± 55 mg/cm²) compared with the pretreatment group (549 ± 76 mg/cm²). Etidronate did not change BMD at the sites with a normal BMD. Among the biochemical markers (BM) examined, the urinary level of Dpd (nMol/liter · Cr) significantly increased in the 3-month treatment group (8.6 ± 5.1 nMol/liter·Cr) compared with the pretreatment group (5.8 ± 2.0 nMol/liter · Cr). No significant differences were seen in the BMs measured in the long-term treatment group. The urinary Dpd level of the etidronate treatment group decreased (3.9 ± 1.4 nMol/liter · Cr) compared with the pretreatment group. These data indicate that etidronate could improve the accelerated bone resorption. In conclusion, high-dose glucocorticoid therapy causes rapid bone resorption and accelerates the natural progress of osteoporosis associated with aging or menopause. Etidronate administration prevents the progress of osteoporosis in nephrotic patients. Preventive treatment should be performed when the estimated BMD in 3 months falls below the baseline by more than 7 ± 4%, reaching the therapeutic range. Received: 31 March 1999 / Accepted: 29 September 1999     

Plasma Leptin Values in Relation to Bone Mass and Density and to Dynamic Biochemical Markers of Bone Resorption and Formation in Postmenopausal Women

After the menopause it has been noted that heavier women conserve bone better than those with lower body weight. The protective effect of obesity on bone mass has been ascribed to a high body fat content. The present study of 54 postmenopausal women was undertaken to determine whether circulating plasma levels of leptin, the newly described hormone produced in adipocytes, were correlated with age-adjusted total body bone mineral content (BMC) or bone mineral density (BMD), or with dynamic biochemical markers of bone resorption or of bone formation. Leptin values were strongly correlated with all measures of adiposity (P < 0.001). Age-adjusted values for BMC and BMD, respectively, were also positively correlated (P < 0.001) with body weight (r = 0.643, r = 0.502), total fat mass (r = 0.557, r = 0.510) and with plasma leptin concentrations (r = 0.480, r = 0.551), confirming a positive relationship between fat mass and bone mass. By contrast, no significant correlations were observed between plasma leptin and dynamic markers of bone resorption (urinary deoxypyridinoline/creatinine r =−0.105, hydroxyproline/creatinine r =−0.193) or formation (plasma osteocalcin r = 0.103). Because there was no evidence for an association between ciculating plasma levels of leptin and biochemical markers of either osteoclastic or osteoblastic activity we conclude it is unlikely that circulating leptin plays any significant direct role in controlling bone cell activity. Our results do not support the hypothesis that leptin mediates the bone-sparing effects of obesity. Received: 23 September 1997 / Accepted: 11 May 1998     

The Effect of Weight Change on DXA Scans in a 2-Year Trial of Etidronate Therapy

Variation in soft tissue composition is a potential cause of error in dual X-ray absorptiometry (DXA) measurements of bone mineral density (BMD). We investigated the effect of patients' change of weight on DXA scans in 152 women enrolled in a 2-year trial of cyclical etidronate therapy. Scans of the spine, hip, and total body were performed at baseline, 1 and 2 years on a Hologic QDR-2000. The study was completed by 135 subjects (64 on etidronate, 71 on placebo). Results were expressed as the percentage change in BMD (spine, femoral neck, total body) or bone mineral content (BMC) (total body only) at 2 years. Total body scans were analyzed using the manufacturer's `standard' and `enhanced' algorithms. Analysis was performed using multivariate regression with percentage change in BMD or BMC as the dependent variable, and treatment group and percentage change in weight as the independent variables. Weight change varied between −14.4% and +16.7%. All DXA variables showed a statistically significant treatment effect. Standard total body BMD and BMC and enhanced total body BMC all showed a significant dependence on weight change (P < 0.01, P < 0.001 and P < 0.01, respectively). No effect of weight change was seen on spine, femoral neck, or enhanced total body BMD. In order to investigate the effects of weight on long-term precision, patients were allocated to two groups according to baseline body mass index (BMI <25 and >25 kg/m², respectively). For femoral neck BMD the root mean square (RMS) residual percentage change was statistically significantly larger in the high BMI group (P < 0.05) but all other bone density variables showed no significant difference. With patients allocated to two groups according to their absolute percentage change in weight (<5% and >5%, respectively) the RMS residual percentage changes in the bone density variables were statistically significantly larger in the large weight change group for femoral neck BMD (P < 0.05) and for standard and enhanced total body BMC (P < 0.01 and P < 0.05, respectively). With the exception of the standard total body algorithm, weight change in a longitudinal study of postmenopausal women was not found to cause systematic errors in the results of DXA studies but may adversely affect precision. Received: 22 November 1996 / Accepted: 30 April 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     

Comparison of Calcitriol Treatment with Etidronate–Calcitriol and Calcitonin–Calcitriol Combinations in Turkish Women with Postmenopausal Osteoporosis: A Prospective Study

Calcitriol has been widely used in the management of osteoporosis, but its efficiency is a matter of controversy. It is not known whether combinations of calcitriol and antiresorptive agents such as etidronate and calcitonin are superior to calcitriol alone in the treatment of postmenopausal osteoporosis. To make this determination, 30 Turkish women with postmenopausal osteoporosis between 45 and 68 years of age were randomized to receive either intermittent cyclical etidronate (400 mg/day, for 14 days) followed by 60 days of cyclical calcitriol therapy 0.25 μg twice daily (group 1; n= 10), or calcitriol 0.25 μg twice daily (group 2; n= 10), or calcitriol 0.25 μg/day in combination with 100 IU intranasal salmon calcitonin taken every other day (group 3; n= 10) through a 1-year period. Bone mineral density (BMD) of lumbar spine (L2 to L4) was determined for each patient by dual-photon absorptiometry (¹⁵³Gd) at baseline, after 6 months, and at the end of the study. There was no significant difference among groups with respect to mean spinal BMD at baseline, after 6, and after 12 months. No significant spinal BMD changes occurred in any group from baseline, after 6 months, and after 12 months. Four patients in groups 1 and 2 and five patients in group 3 developed hypercalcemia at least once during therapy. Hypercalciuria occurred at least once in 9, 10, and 7 patients in groups 1, 2, and 3, respectively. One patient in group 2 developed a renal stone at the end of the study. Mean urine hydroxyproline levels did not change significantly in any group with respect to baseline. The data suggest that one-year treatment with calcitriol, given either alone or in combination with antiresorptive agents, does not improve spinal BMD in Turkish women with postmenopausal osteoporosis, and is associated with a high rate of adverse events. Received: 4 October 1996 / Accepted: 31 December 1996     

Expression of Cathepsins B, H, K, L, and S and Matrix Metalloproteinases 9 and 13 During Chondrocyte Hypertrophy and Endochondral Ossification in Mouse Fracture Callus

Fracture repair provides an interesting model for chondrogenesis and osteogenesis as it recapitulates in an adult organism the same steps encountered during embryonic skeletal development and growth. The fracture callus is not only a site of rapid production of cartilage and bone, but also a site of extensive degradation of their extracellular matrices. The present study was initiated to increase our understanding of the roles of different proteolytic enzymes, cysteine cathepsins B, H, K, L, and S, and matrix metalloproteinases (MMPs) 9 and 13, during fracture repair, as this aspect of bone repair has previously received little attention. Northern analysis revealed marked upregulation of cathepsin K, MMP-9, and MMP-13 mRNAs during the first and second weeks of healing. The expression profiles of these mRNAs were similar with that of osteoclastic marker enzyme tartrate-resistant alkaline phosphatate (TRAP). The changes in the mRNA levels of cathepsins B, H, L, and S were smaller when compared with those of the other enzymes studied. Immunohistochemistry and in situ hybridization confirmed the predominant localization of cathepsin K and MMP-9 and their mRNA in osteoclasts and chondroclasts at the osteochondral junction. MMP-13 was present in osteoblasts and individual hypertrophic chondrocytes near the cartilage-bone interphase. In cartilaginous callus, the expression of cathepsins B, H, L, and S was mainly related to chondrocyte hypertrophy. During bone remodeling both osteoblasts and osteoclasts contained these cathepsins. The present data demonstrate that degradation and remodeling of extracellular matrices during fracture healing involves activation of MMP-13 production in hypertrophic chondrocytes and osteoblasts, and cathepsin K and MMP-9 production in osteoclasts and chondroclasts. Received: 2 February 2000 / Accepted: 25 May 2000 / Online publication: 2 November 2000