Taxol inhibits osteoclastic bone resorption

Summary We have examined the effect of the anti-tumor compound taxol, on osteoclastic bone resorption. In the bone slice assay, taxol (0.1–0.001 M) dose-dependently inhibited bone resorption with an IC₅₀ of 0.08 M. Osteoclast survival on bone slices was unaffected by 0.01–1 M taxol, but 10 M was cytotoxic. Taxol (1 M) also ihibited osteoclast spreading (45%) on fibronectin-coated slides. The antiproliterative effects of taxol are due to its unique ability to stabilize microtubules. Primary osteoclasts are nonproliferating end cells, so taxol probably inhibits bone resorption by intertering with other microtubule-dependent functions such as cell polarization, motility or vesicle exocytosis. Since these inhibitory effects on osteoclasts in vitro are seen with therapeutically relevant concentrations, taxol therapy may have beneficial side-effects e.g. inhibition of hyperealcemia and bone metastases.     

Effects of vitamin D binding protein-macrophage activating factor (DBP-MAF) infusion on bone resorption in two osteopetrotic mutations

Osteopetrosis is a heterogeneous group of bone diseases characterized by an excess accumulation of bone and a variety of immune defects. Osteopetrosis (op) and incisors absent (ia) are two nonallelic mutations in the rat which demonstrated these skeletal defects as a result of reduced bone resorption. Osteopetrotic (op) rats have severe sclerosis as a result of reduced numbers of osteoclasts which are structurally abnormal. The sclerosis in ia rats is not as severe as in op mutants; they have elevated numbers of osteoclasts, but they are also morphologically abnormal, lacking a ruffled border. Both of these mutations have defects in the inflammation-primed activation of macrophages. They demonstrate independent defects in the cascade involved in the conversion of vitamin D binding protein (DBP) to a potent macrophage activating factor (DBP-MAF). Because this factor may also play a role in the pathogenesis of osteoclastic dysfunction, the effects of ex vivo-generated DBP-MAF were evaluated on the skeletal system of these two mutations. Newborn ia and op rats and normal littermate controls were injected with DBP-MAF or vehicle once every 4 days from birth until 2 weeks of age, at which time bone samples were collected to evaluate a number of skeletal parameters. DBP-MAF treated op rats had an increased number of osteoclasts and the majority of them exhibited normal structure. There was also reduced bone volume in the treated op animals and an associated increased cellularity of the marrow spaces. The skeletal sclerosis was also corrected in the ia rats; the bone marrow cavity size was significantly enlarged and the majority of the osteoclasts appeared normal with extensive ruffled borders. Superoxide production was enhanced to normal levels in the osteoclasts from the treated ia animals as well. These studies demonstrate that the skeletal defects in both mutations can be improved with exogenous DBP-MAF and that this protein is involved in the pathogenesis of the disease.     

Effect of diacylglycerols on osteoclastic bone resorption

We studied the effect of various synthetic diacylglycerols (DAGs) on bone resorption by rat and chick osteoclasts. l-stearoyl-2-arachidonoyl-sn-glycerol (DAG IV), at a concentration of 100 ΜM, caused a significant reduction in resorption pit number in both species at 6 and 24 hours without any toxic effect. Over a 6-hour incubation period, a significant inhibition was seen at 10 and 100 ΜM in both species. 1,2-dioctanoyl-sn-glycerol (DAG I) and 1, 2-dihexanoyl-sn-glycerol (DAG III) caused a marked inhibition of resorption by rat osteoclasts at 6 hours, but there was recovery of bone-resorptive ability over a 24-hour incubation period. DAGs with the -rac conformation failed to have any effect on bone resorption. In time-lapse video studies, osteoclast motility was not influenced by any of the DAGs at any of the concentrations used. Our results indicate that DAGs with the -sn conformation inhibit bone resorption, and DAGs with the -rac conformation do not. The finding that DAGs, the physiological activators of protein kinase C (PKC), inhibit bone resorption provides further evidence for an important role of the PKC pathway in the regulation of osteoclast activity.     

谷康泰灵对骨质疏松模型大鼠骨形成和骨吸收功能的影响

目的　观察药物谷康泰灵对骨质疏松 (OP)模型大鼠骨形成和骨吸收功能的影响 ,为其临床治疗骨质疏松提供实验依据。方法　SD大鼠 4 4只 ,随机分为正常组 (13只 )和骨质疏松模型组 (31只 )。以维甲酸 80mg·kg- 1 ·d- 1 灌胃 15d,诱导OP模型。模型复制成功后 ,各组处死 5只 ,正常组 (8只 )继续观察 ,模型组又随机分为无措施对照组 (8只 )、谷康泰灵治疗组 (10只 ,0 0 8mg·kg- 1 ·d- 1 谷康泰灵腹腔注射 )和雌二醇治疗组 (8只 ,每只 0 0 5mg,每周 3次苯甲酸雌二醇腹腔注射 )。治疗期 30d。观察股骨松质骨区成骨细胞及破骨细胞数量和血清中AKP、TRAP活性变化。结果　与正常组相比 ,维甲酸诱导的OP模型大鼠股骨松质骨区成骨细胞功能活跃 ,数量变化不大 ;破骨细胞数量、活跃程度显著增加 ;血清中AKP和TRAP明显增高。谷康泰灵治疗后 ,OP大鼠活跃成骨细胞数量明显增加 ,破骨细胞数量显著减少 ,血清AKP活性明显增高 ,TRAP活性下降。结论　谷康泰灵对OP大鼠骨形成和骨吸收功能有显著影响 ,可刺激成骨细胞的产生增加成骨细胞的活性 ,减少破骨细胞的数量抑制破骨细胞的活性。     

淫羊藿苷对小鼠骨髓源性破骨细胞诱导生成及骨吸收功能的影响

目的探讨淫羊藿苷对破骨细胞诱导产生及骨吸收功能的影响。方法用终浓度分别为25ng·mL^-1、30ng·mL^-1、10^-8mol·L^-1的M—CSF、RANKL、1，25（OH）2VitD3体外诱导培养小鼠骨髓源性破骨细胞，在此过程中加入终浓度分别为0、10^-7mol·L^-1、10^-6mol·L^-1、10^-5mol·L^-1的淫羊藿苷。倒置相差显微镜下观察活体细胞、HE染色、TRAP染色及降钙素受体染色鉴定破骨细胞，计数骨片上骨吸收陷窝数及面积，玻片上TRAP阳性多核细胞数。结果加药组随淫羊藿苷浓度的增加，骨片上形成的骨吸收陷窝数及面积，玻片上的TRAP阳性多核细胞数呈量的依赖性的减少，与非加药组比较，10^-5mol·L^-1、10^-5mol·L^-1浓度的淫羊藿苷组，差异有显著性（P〈0．05）。结论淫羊藿苷具有抑制破骨细胞诱导产生及骨吸收功能的作用，并随浓度增加抑制作用增强。     

Rodent peritoneal macrophages as bone resorbing cells

Summary Because of the difficulty in obtaining large, relatively pure populations of osteoclasts, most studies of bone resorption are performed on intact animals or in cultures of embryonic bone rudiments. These experimental systems, however, do not permit detailed analysis of the cellular mechanisms of matrix degradation or of the means whereby resorbing cells attach to the bone surface. Mononuclear phagocytes, which are probably ontogenetically related to the osteoclast, will resorb bone matrix in tissue culture. Consequently, we have developed an in vitro system whereby the ability of these cells to bind and resorb skeletal matrix can be precisely and individually measured using radioisotopically labeled, devitalized rat bone particles. We have found that when derived from mice, peritoneal macrophages bind approximately 80% of bone particles within the first 40 min of incubation. Significant (P<0.025) net matrix degradation, as defined by the percentage of isotope released from bone cultured with macrophages as compared to that released in the absence of cells, occurs within the first 3 h of culture and proceeds rapidly for at least the first 2 days of incubation. By this time 40%–50% of isotope usually has been released into the medium. Resident peritoneal macrophages appear to mobilize matrix as actively as those which are thioglycollate induced. By comparison, lymphocytes elicit little isotope mobilization from bone, and rat peritoneal exudate macrophages are markedly less efficient (P<0.001) at resorbing rat bone than are macrophages obtained from mice. Isotope release by peritoneal macrophages represents true cell-mediated resorption and not merely nonspecific mineral mobilization as evidenced by the facts that: (a) the magnitudes of release of isotopes representing the inorganic (⁴⁵CaCl) and organic (³H-proline) phases of bone are the same, (b) daily buffering of the cultures to pH 7.4 has little effect on⁴⁵Ca release, and (c) cell-matrix contact is required for optimal mobilization of⁴⁵Ca or³H.     

1,25-Dihydroxycholecalciferol increases bone resorption in thyroparathyroidectomised mice

Mice, 1 week old, prelabelled with⁴⁵Ca, were either thyroparathyroidectomised or sham-operated; 1 day later half of the mice of each group were injected with 1,25-dihydroxycholecalciferol (5 ng/g), and 20 h later all the mice were killed. Bone resorption in explants was then measured by an in vivo/in vitro technique previously published; compared with untreated mice it was found that 1,25-dihydroxycholecalciferol had increased resorption irrespective of whether the mice had been thyroparathyroidectomised or not. These data suggest that 1,25-dihydroxycholecalciferol is able to increase bone resorption independently of parathyroid hormone.     

Mechanisms of bone resorption in calcium-deficient rats

Summary A study of surface remodeling activity and osteocyte lacunar area was made in young and adult rats maintained on a low-calcium diet, to explore cellular mechanisms of bone resorption. The diet produced active remodeling of the endosteal part of the femoral cortex, with a decrease in the amount of bone present. Surface resorption, with numerous osteoclasts, was evident, but there was no evidence of osteocytic osteolysis in bone which, by tetracycline labeling, could be identified as existing at the commencement of the experimental period. Osteocyte lacunae in bone formed during the period of calcium deprivation were somewhat larger than lacunae in control animals, apparently because of interference with the formation or maturation of the perilacunar tissue.     

Hydrochlorothiazide inhibits osteoclastic bone resorption In vitro

Long-term thiazide diuretic use is associated with higher bone mineral density and reduced hip fracture rates, which are attributed to increased serum calcium levels and decreased parathyroid activity that lead to decreased bone resorption. The present study shows that 1–100 M hydro-chlorothiazide (HCTZ) dose dependently inhibits bone resorption by isolated rat osteoclasts in the bone slice assay with an IC₅₀ of 20 M. At these concentrations, HCTZ did not affect osteoclast survival on bone slices and had no effect on the proliferation of UMR-106 rat osteoblasts, indicating that the compound is not cytotoxic. However, such concentrations of HCTZ are unlikely to be achieved in man where therapeutic doses are usually 12.5–100 mg/day. That the in vitro effect of HCTZ on bone resorption may be due to inhibition of osteoclast carbonic anhydrase is discussed.     

Genistein对破骨细胞分化和骨吸收功能的影响及其机制探讨

目的 探讨三羟异黄酮类药Genistein对破骨细胞性骨吸收的作用及其机制。方法 1，25(OH)2D3诱导小鼠骨髓细胞形成破骨样细胞，分别加入0moL／L、10^-9moL／L、10～moL／L、10^-7moL／L、10～moL／L、10^-5moL／LGenistein，于培养3，5和8d分别计数抗酒石酸酸性磷酸酶阳性细胞个数；于培养12d利用图像分析系统对骨吸收陷窝的面积进行测量分析。另从小鼠颅盖骨分离培养获得原代成骨细胞，分别加入0，10^-8，10^-7，10^-6和10^-5moL／LGenistein并以10^-9moL／L^-7 β雌二醇为对照，采用RT-PCR的方法检测Genistein对骨保护因子(osteoprotegerin，OPG)及破骨细胞分化因子(receptor activator of NF-κB ligand，RANKL)mRNA表达的影响。结果 利用1，25(OH)2D3成功诱导出破骨样细胞；随Genistein浓度增加，抗酒石酸酸性磷酸酶阳性细胞(单核、双核和多核)个数和骨吸收陷窝面积呈剂量依赖性减少。同时，应用Genistein后原代成骨细胞中OPG和RANKL的mRAN表达都有增强，但其最终效应表现为剂量依赖性和时间依赖性地增大OPG／RANKL的浓度比。结论 Genistein通过抑制破骨前体细胞分化来抑制其体外骨吸收功能，其分子机制与OPG／RANKL mRNA表达比值的升高有关。     

Monocytes mediate osteoclastic bone resorption by prostaglandin production

Summary Monocytes are frequently found adjacent to active bone resorption surfaces in both physiological and pathological situations and may play a key role in bone resorption. There is strong circumstantial evidence that monocytes are precursors for osteoclasts in vivo, and recently they have been shown to resorb devitalized bone directly. The present study shows that monocytes can also resorb bone by stimulation of osteoclasts. Live fetal rodent bones prelabeled with⁴⁵Ca and cultured for 48–96 h in the presence of human monocytes or monocyte-conditioned medium released 80% more mineral than bones cultured in control medium. Bone matrix sustained comparable resorption as demonstrated by a 2-fold decrement in the extracted dry weights of the bones cultured in monocyte-conditioned medium. Histological examination of the bones cultured with monocytes or monocyte-conditioned medium showed increased osteoclast number and activity when compared with bones cultured in control medium. Known inhibitors of osteoclastic activity (phosphate 6 × 10⁻³M, cortisol 10⁻⁶M, and calcitonin 50 mU/ml) inhibited monocyte-conditioned medium-mediated bone resorption. The monocyte-conditioned medium contained sufficient prostaglandin E to account for the bone resorption. Indomethacin 10⁻⁵M added to the monocyte cultures blocked monocyte-conditioned media-induced bone resorption and prostaglandin release. These experiments suggest that monocytes stimulate osteoclastic bone resorption by prostaglandin production. Monocyte-induced bone resorption is partly reversed by inhibitors of osteoclast function. Monocyte-induced osteoclastic bone resorption may play an important role in physiologic bone remodeling and in bone destruction that occurs in chronic inflammatory diseases such as rheumatoid arthritis and periodontal disease.     

Effect of ACTH on PTH-stimulated bone resorption in vitro

Summary In vitro demonstration of PTH effects requires hormone concentrations greater than the “physiological” concentrations reported by radio-immunoassay or cytochemical assays. This discrepancy could be the result of binding or destruction of PTH at nonbiologically active sites. In the present study, ACTH was found to have no effect by itself on bone resorption, but addition of ACTH to bone cultures together with low concentrations of PTH resulted in a specific enhancement of PTH-stimulated bone resorption. This effect was not observed when bone resorption was stimulated by PGE₂ and 1,25(OH)₂D₃, and it was blocked by human serum. The effect of ACTH is similar to the enhancement in PTH-stimulated bone resorption by poly-l-lysine [7]. We suggest that the amplification of PTH stimulation was the result of displacement of PTH from nonbiologically active sites, making more PTH available for binding to its biologically active receptor. An alternative explanation for our results was that ACTH prevented degradation of PTH by bone-derived proteolytic enzymes. Thus the sensitivity of bioassays for PTH could be improved by adding ACTH.     

The in vitro effect of gold complexes on bone resorption

Mouse calvaria were maintained in organ culture without serum additives. The effects of three gold complexes--aurothioglucose, aurothiomalate, and auranofin--on active bone resorption (45Ca release) and hydroxyproline synthesis were determined. The influence of these compounds on DNA and protein synthesis and lysosomal enzyme release from calvaria was also assessed. All gold complexes reduced bone resorption to some extent, with auranofin being the most potent within a narrow concentration range (10(-6) M). This concentration of auranofin also significantly inhibited collagen synthesis, although DNA and protein synthesis were unaffected. None of the compounds tested appeared to mediate their action via significant inhibition of lysosomal enzyme release.     

Flurbiprofen-induced stimulation of periosteal bone formation and inhibition of bone resorption in older rats

The skeletal effects of flurbiprofen (Fb), a nonsteroidal antiinflammatory drug, was studied by histomorphometry in 9-month-old retired female breeder, Sprague-Dawley rats. Flurbiprofen was given subcutaneously at 0, 0.2, 0.1, 0.5, 2.5, or 5 mg/kg/d for 21 days. Flurbiprofen had no effect on longitudinal growth, but stimulated radial growth (+200%) over controls. In the tibial shaft, Fb stimulated the mineral apposition rate (+25%), mineral bone formation rate (+100%), and periosteal labeling length (+64%) at the 2.5 and 5.0 mg Fb/kg dose levels, and had no effect on marrow cavity size compared to controls. However, these changes were insufficient to increase cortical bone mass. In the proximal tibial metaphysis, Fb suppressed osteoclasts/mm² of metaphyseal tissue (-47%), osteoclasts/mm of bone surface (-46%), and the osteoclast/osteoblast ratio (-50%), increased the calcified cartilage core population (+100%), and had no effect on osteoblast numbers at all dose levels. There was an insignificant increase in metaphyseal cancellous bone mass. The current study leads to the conclusion that flurbiprofen-stimulated periosteal bone growth was due to direct stimulation of osteoblast recruitment and activity independent of longitudinal bone growth. Further, it confirms early findings in young rats that flurbiprofen induced depressed bone resorption without lowering bone formation. However, because of insufficient treatment time, the older rat did not accumulate bone as the young rats did.     

Promethazine inhibits osteoclastic bone resorption In vitro

Summary Several studies have shown that promethazine can reduce age-related osteopenia in mice. Furthermore, prolonged treatment with promethazine (50 mg/day) increases bone mineral content in the lumbar spine in post-menopausal women with osteopenia. However, the mechanism of action of promethazine has not been elucidated. The present study shows that promethazine HCl (0.01 – 10 M) dose-dependently inhibits bone resorption by isolated rat osteoclasts in the bone slice assay with an IC₅₀ of 1 M. Since these concentrations are likely to be achieved in vivo, it is suggested that the beneficial effect of promethazine on osteopenia is at least partly due to a direct inhibitory effect on osteoclast activity.     

Cancellous bone resorption in the proximal ilium of the ovariectomized rat

Summary Bone histomorphometry was performed on the proximal ilium of mature Sprague-Dawley rats following ovariectomy, and these rats were compared with sham-operated controls. Bone volume per unit tissue volume (BV/TV), osteoid surface, and the depth and extent of eroded cavities were measured in animals killed at intervals after operation. The rate of bone loss and the mean osteoid surface in the proximal ilium of the ovariectomized rats was significantly greater than that of the control rats over a 210-day postoperative period. The eroded surface and mean trabecular thickness in the proximal ilium of the ovariectomized rats were not significantly different from that of the control rats, and therefore failed to explain the difference in the rate of bone loss. The distribution of the depths of trabecular eroded cavities in the ilium of ovariectomized rats was different from that in the control rats. The loss of trabecular bone mass in the proximal ilium of ovariectomized, mature rats appeared due to increased activity of individual osteoclasts, rather than to increased osteoclast numbers and thinning of trabeculae.     

Protamine: A powerfulin vivo inhibitor of bone resorption

Summary Protamine is shown to be a powerful disrupter of calcium homeostasis, acutely inducing a severe hypocalcemia in both rabbits and rats. The magnitude of its effect correlates with bone turnover. Protamine does not significantly alter the renal excretion of calcium, and is effective whether or not there is calcium present in the gut. Protamine causes a significant fall in the specific activity of⁴⁵Ca in the blood in animals whose bone has been prelabeled with⁴⁵Ca. These data suggest that protamine induces hypocalcemia by blocking calcium efflux from bone. Further work seems indicated to define the biochemical mechanism of this action.     

Transient bone resorption following finger replantation: a report of 3 cases

Radiographic changes consisting of alterations in mineral content, osteopaenia or destructive neuropathy that occur following successful finger replantation have already been described. We report our experience about four fingers in three individuals in whom bone changes developed in the first three months postoperatively with complete “restitution ad integrum”.Three patients, 21-49 years old (average 36 years) sustained a clean-cut amputation of four fingers. The first patient had an amputation at the base of the middle phalanx of the index finger and the second patient at the base of the proximal phalanx of the ring finger. The third had an amputation at the base of the first metacarpal bone and the proximal phalanx of the small finger in a five finger amputation. In the first case, two dorsal veins and two palmar digital arteries and nerves were repaired. In the second case, one palmar artery and one dorsal vein were reanastomosed. In the third case at the thumb, two dorsal veins and two palmar digital arteries and nerves were reconstructed. At the small finger, one dorsal vein, one palmar digital artery and two digital nerves were reconstructed. Bone fixation was achieved with two and three K-wires or tension-band wiring. Replantation was successful in all cases. Three weeks after replantation, the X-rays showed rapid development of osteopaenia in the juxtaarticular region and metaphyses of the bone. These changes were followed by subperiosteal, intracortical and endosteal bone resorption. No further surgical procedures or splintage were needed and hand therapy was not discontinued. At 10-13 weeks (average 12 weeks) postoperatively, the X-rays showed a complete recovery with new periosteal bone formation.We suggest that the radiographic changes after finger replantation are transient, first evident subperiosteally and progressing centrally. They may reflect small-vessel compromise and microinfarction and transient hyperemia secondary to neurovascular damage or to sympathetic progressive recovery.     

Longitudinal evaluation of a bone resorption marker in elderly subjects

The variability over time in the excretion of a hone resorption metabolite (collagen type I N-telopeptide crosslink, NTx) was evaluated in a cohort of community-dwelling elderly men and women (mean age 73 years). Three annual 24-h urine samples were collected. NTx concentration was measured using an established ELISA. Total (24-h) NTx excretion as well as NTx/creatinine concentration were compared. Men had a significantly lower excretion of NTx/creatinine than women who were not on hormone replacement therapy. Overall, the within-subject long-term coefficient of variability for NTx/creatinine was 26%. The correlation coefficient between the samples taken a year apart was higher for the 24-h NTx excretion (r=0.66) than for the 24-h creatinine excretion (r=0.51). The consistency of NTx excretion over time was also evaluated in all 93 subjects with three yearly samples using Kendall’s rank correlation method; the resulting coefficient of concordance was 0.78 (significant at the 0.01 level). These results indicate that while NTx excretion varies in subject samples collected over a period of 2 years, this variability is not much greater than the daily variation reported for NTx and other bone metabolism markers. The relative reproducibility of NTx excretion over time in this age group was also evident in the coefficient of concordance. The results provide support for stratifying subjects according to level of bone resorption and identifying those subjects with high turnover who may be at greater risk of osteoporotic fracture.     

Urinary gamma-glutamyltransferase (GGT) as a potential marker of bone resorption

We recently identified γ-glutamyltransferase (GGT) as a novel bone-resorbing factor. The present study was undertaken to determine whether GGT is a marker of bone resorption in two genetic models of hyper- and hypo-function of osteoclasts, as well as in postmenopausal women with accelerated bone resorption, using type I collagen N-telopeptide (NTX) and deoxypyridinoline (DPD) as established biochemical markers. Urinary excretion of GGT, corrected for creatinine, was found to be increased in osteoprotegerin (OPG)-deficient osteoporotic mice as well as in patients with postmenopausal osteoporosis (67–83 years of age); in both cases the urinary level decreased after treatment of patients or mice with alendronate, a selective inhibitor of bone resorption, concomitantly with a reduction in DPD and NTX. Conversely, in osteopetrotic op/op mice, urinary GGT increased in parallel with DPD after induction of osteoclasts with M-CSF injection. Constant infusion of parathyroid hormone (PTH) also increased urinary GGT along with DPD. In a survey of 551 postmenopausal women (50–89 years of age) at their regular health checkup, urinary GGT excretion exhibited a high correlation with DPD (ρ = 0.49, p < 0.0001). The calculated sensitivity and specificity for diagnosing elevated bone resorption, as determined by a DPD value higher than 7.6 nM/mM Cr, were 61% and 92%, respectively, when a cut-off value of 40 IU/g Cr was assigned for urinary GGT. Since GGT activity can be measured inexpensively in large numbers in a very short time, the measurement of urinary level may provide a convenient and useful method for mass screening to identify those with increased bone turnover and hence at increased risk for bone fracture.