Common herbs, essential oils, and monoterpenes potently modulate bone metabolism.

During our survey of herbs looking for activity on bone metabolism, we found that the dried leaves of sage strongly inhibit bone resorption. Therefore, we investigated several common herbs rich in essential oils (sage, rosemary, and thyme) and essential oils extracted from these herbs and other plants (oils of sage, rosemary, juniper, pine, dwarf pine, turpentine, and eucalyptus) as well as their monoterpene components (thujone, eucalyptol, camphor, borneol, thymol, alpha-pinene, beta-pinene, bornylacetate as well as menthol) and found that they inhibit bone resorption when added to the food of rats. Pine oil, used as a representative essential oil, protects an osteoporosis model, the aged ovariectomized rat, from bone loss. The monoterpenes borneol, thymol, and camphor are directly inhibitory in the osteoclast resorption pit assay. Nonpolar monoterpenes may require metabolism to be active in vitro, for example, cis-verbenol, a metabolite of alpha-pinene occurring in human urine, inhibits osteoclast activity in contrast to the parent compound. Within 30 min borneol inhibits the formation of actin rings, a characteristic of resorbing osteoclasts indicating cell polarization. Both the in vitro and the in vivo effects of borneol are reversible. Our study demonstrates for the first time that essential oils and monoterpenes are efficient inhibitors of bone resorption in the rat.

Functional adaptation of human joints to mechanical stimuli.

OBJECTIVE: This study tests the hypothesis that functional adaptation occurs in human joints, and that substantial differences in joint 'loading history' explain the phenotypic variability observed in human cartilage morphology. METHOD: We examined 18 triathletes (nine men and nine women) who had been physically active throughout life (training for >10 h per week for the last 3 years), and 18 volunteers that had never been physically active on a regular basis. The right knee joints were imaged with a previously validated fat-suppressed gradient-echo MR sequence. Cartilage volume, thickness, joint surface areas, and normalized cartilage signal intensity were determined with post-processing software, specifically designed for these applications. RESULTS: The knee joint cartilage thickness, and signal intensity were not significantly different between athletes and inactive volunteers, but male athletes displayed significantly larger knee joint surfaces (P< 0.01; +8.8%). Female athletes displayed a significantly larger medial tibia (P< 0.05; +18.9%), the difference in the total knee surface area reaching borderline significance (P=0.08; +7.0%). CONCLUSIONS: The results suggest that joint size can be modulated during growth, but that (opposite to muscle and bone) the thickness of the cartilage does not adapt to mechanical stimulation. This finding may reveal a general principle in the development and functional adaptation of diarthrodial joints, elucidating an important mechanism for reducing mechanical stress in biphasic cartilage layers.

Gender differences in knee joint cartilage thickness, volume and articular surface areas: assessment with quantitative three-dimensional MR imaging.

OBJECTIVE: To compare the cartilage thickness, volume, and articular surface areas of the knee joint between young healthy, non-athletic female and male individuals. SUBJECTS AND DESIGN: MR imaging was performed in 18 healthy subjects without local or systemic joint disease (9 female, age 22.3 +/- 2.4 years, and 9 male, age 22.2 +/- 1.9 years.), using a fat-suppressed FLASH 3D pulse sequence (TR = 41 ms, TE = 11 ms, FA = 30 degrees) with sagittal orientation and a spatial resolution of 2 x 0.31 x 0.31 mm3. After three-dimensional reconstruction and triangulation of the knee joint cartilage plates, the cartilage thickness (mean and maximal), volume, and size of the articular surface area were quantified, independent of the original section orientation. RESULTS AND CONCLUSIONS: Women displayed smaller cartilage volumes than men, the percentage difference ranging from 19.9% in the patella, to 46.6% in the medial tibia. The gender differences of the cartilage thickness were smaller, ranging from 2.0% in the femoral trochlea to 13.3% in the medial tibia for the mean thickness, and from 4.3% in the medial femoral condyle to 18.3% in the medial tibia for the maximal cartilage thickness. The differences between the cartilage surface areas were similar to those of the volumes, with values ranging from 21.0% in the femur to 33.4% in the lateral tibia. Gender differences could be reduced for cartilage volume and surface area when normalized to body weight and body weight x body height. The study demonstrates significant gender differences in cartilage volume and surface area of men and women, which need to be taken into account when retrospectively estimating articular cartilage loss in patients with symptoms of degenerative joint disease. Differences in cartilage volume are primarily due to differences in joint surface areas (epiphyseal bone size), not to differences in cartilage thickness.

Empirical model for the autotrophic biodegradation of thiocyanate in an activated sludge reactor.

AIMS: The aim of this investigation was to develop an empirical model for the autotrophic biodegradation of thiocyanate using an activated sludge reactor. METHODS AND RESULTS: The methods used for this purpose included the use of a laboratory scale activated sludge reactor unit using thiocyante feed concentrations from 200 to 550 mg x l(-1). Reactor effluent concentrations of <1 mg x l(-1) thiocyanate were consistently achieved for the entire duration of the investigation at a hydraulic retention time of 8 h, solids (biomass) retention of 18 h and biomass (dry weight) concentrations ranging from 2 to 4 g x l(-1). A biomass specific degradation rate factor was used to relate thiocyanate degradation in the reactor to the prevailing biomass and thiocyanate feed concentrations. A maximum biomass specific degradation rate of 16 mg(-1) x g(-1) x h(-1) (mg thiocyanate consumed per gram biomass per hour) was achieved at a thiocyanate feed concentration of 550 mg x l(-1). The overall yield coefficient was found to be 0.086 (biomass dry weight produced per mass of thiocyanate consumed). CONCLUSION: Using the results generated by this investigation, an empirical model was developed, based on thiocyanate feed concentration and reactor biomass concentration, to calculate the required absolute hydraulic retention time at which a single-stage continuously stirred tank activated sludge reactor could be operated in order to achieve an effluent concentration of <1 mg x l(-1). The use of an empirical model rather than a mechanistic-based kinetic model was proposed due to the low prevailing thiocyanate concentrations in the reactor. SIGNIFICANCE AND IMPACT OF THE STUDY: These results represent the first empirical model, based on a comprehensive data set, that could be used for the design of thiocyanate-degrading activated sludge systems.

Comparison of knee joint cartilage thickness in triathletes and physically inactive volunteers based on magnetic resonance imaging and three-dimensional analysis.

The objective of this study was to employ quantitative magnetic resonance imaging for the analysis of knee joint cartilage thickness in triathletes and physically inactive volunteers. The right knee joints of nine male triathletes (10 hours training per week for at least 3 years) and nine inactive male volunteers (<1 hour of physical activity per week throughout life) were imaged with a previously validated fat-suppressed gradient echo sequence. The cartilage plates were reconstructed three-dimensionally, and the cartilage thickness was computed independently of the original section orientation with a three-dimensional Euclidian distance transformation. There was a high interindividual variability of the mean and the maximal cartilage thickness values in all surfaces, both in the triathletes and in the inactive volunteers. In the patella, the femoral trochlea, and the lateral femoral condyle, the mean and maximal cartilage thickness values were slightly higher in the triathletes, but they were somewhat lower in the medial femoral condyle, and in the medial and lateral tibial plateau. However, the differences did not attain statistical significance. These results are unexpected in view of the functional adaptation observed in other musculoskeletal tissues, such as muscle and bone, in which a more obvious relationship with the magnitude of the applied mechanical stress has been observed.

Some vegetables (commonly consumed by humans) efficiently modulate bone metabolism.

We have hypothesized that some vegetables which are part of the regular human diet may contain modulators of bone metabolism. To mimic a typical Western diet with large proportions of refined components, rats were pair-fed a semi-purified diet to which, in the treated animals, the dried material under investigation was added. Effects are expressed as % of untreated control. Bone parameters in rats were assessed in the proximal tibia by pQCT. Bone resorption (BR) was assessed by the urinary excretion of [3H]-tetracycline from prelabeled rats. Daily administration of 1 g of onion during 4 weeks increased total bone mineral content by 17.4% (p<0.05), trabecular bone mineral density by 13.6% (p<0.05). One g of onion/day administered to male rats blunted BR by 23-/+5% (p<0.05). Daily administration of onion to ovariectomized rats inhibited BR in a dose-dependent manner. At the highest dose (1.5 g of onion) BR was inhibited by 26-/+4% (p<0.01) as compared to 24-/+3% (p<0.001) for estradiol (27microg/kg/day). An additional 13 vegetables displayed significant effects on BR at the dose of 1g/day. Interestingly, 1g/day of soy did not inhibit BR in this model. Also, skimmed milk, meat and egg (all 1 g/day) were ineffective. Thus, common vegetables consumed by humans potently modulate bone metabolism in the rat. This opens the possibility to develop the basis for a low-cost, safe and effective nutritional approach to osteoporosis.

Food fractionation is a powerful tool to increase bone mass in growing rats and to decrease bone loss in aged rats: modulation of the effect by dietary phosphate.

The incidence of osteoporotic fractures has been associated with low bone mass. To reduce this incidence, it is therefore important to try to prevent the development of low bone mass by either increasing bone mass built up during adolescence and/or preventing bone loss in later life. It has been shown that food fractionation, a procedure that prevents the diurnal rhythm of bone resorption, increases bone mass in growing rats fed a high calcium (Ca), high phosphate (Pi) diet. In this paper, data are presented that show that providing growing rats with the same daily amount of a high Ca, low Pi diet (a Pi content similar to that of a human diet) in portions every 6 h instead of one meal increases total bone mineral content, trabecular bone mineral density, and cortical thickness, and markedly reduces the decrease in these parameters in aged rats. The effect is smaller when a high Ca, high Pi diet is fractionated. This could be the consequence of the transient postprandial increase in parathyroid hormone (PTH) following one large meal of a high Ca, high Pi diet, since the effect is similar to that reported after PTH injections which are anabolic for bone. Thus, a high dietary Pi must be considered as a confounding factor when treatments affecting bone mass are investigated in rats. The present data show that feeding habits have a profound effect on bone mass in the rat, independent of age. Whether bone mass in humans is also under the control of dietary habits is not yet clear. If so, frequent small meals of appropriate composition may help to prevent osteoporosis.

Apoptosis during castration-induced regression of the prostate is Fos dependent.

Apoptotic cell death was shown to be accompanied or preceded by an elevated expression of the c-fos protooncogene and DNA binding activity of transcription factor AP-1. We used Fos-deficient mice to study the role of c-Fos during programmed cell death in the prostate. In normal mice apoptosis is induced in the prostate within 2-4 days after castration. Histological features of reduced secretory activity and morphological signs of programmed cell death become obvious. No apparent decrease in secretory activity and no epithelial cell death were observed in Fos-deficient animals after castration. Fragmentation of nuclear DNA was measured by in situ terminal transferase reaction. DNA fragmentation was observed in the prostate epithelium of control mice after castration whereas no similar fragmentation was found in Fos-deficient animals. After castration an AP-1 complex accumulated in the prostate of Fos deficient mice which mainly consists of FosB, Fra-2 and JunD whereas in control animals the AP-1 complex in addition contained c-Fos. Our data strongly suggest that c-Fos is required for programmed cell death of prostate epithelial cells.

[Sex-specific analysis of cartilage volume in the knee joint--a quantitative MRI-based study]. / Geschlechtsspezifische Analyse der Knorpelvolumina des Kniegelenks--eine quantitative MRT-basierte Studie.

The objective of the present study was to determine differences in the normal knee joint cartilage volume of males and females, the analysis of the percentage distribution of the cartilage tissue onto the various joint surfaces, and the determination of the relationship between the cartilage volume, the body weight, and the tibial head diameter. We examined the knee joints of nine healthy men and nine women with a low level of physical activity. The cartilage volume was assessed with magnetic resonance imaging, applying a fat-suppressed gradient-echo sequence with a resolution of 2 x 0.31 x 0.31 mm3 and 3D image reconstruction. In the men, the absolute volumes of the femur and tibia, but not those of the patella, were significantly higher than in the women. The differences between the sexes were considerably lower after normalisation to the body weight and the tibial head diameter and were no more statistically significant. The interindividual variability was reduced after normalisation to these two parameters, the body weight being more effective. We did not observe sex-specific differences in the percentage of the total cartilage volume taken up by the various joint surfaces. Our results suggest that, in young individuals without cartilage lesions, there exist sex-specific differences of the cartilage volume in the knee joint. However, these can be explained in terms of general differences in body constitution (body weight and bone size), without further significant influences of the sex. The knowledge of the normal, sex-specific cartilage volume is relevant when attempting to estimate the amount of tissue loss at the time at which symptoms occur in a patient with degenerative joint disease.

Morphometric analysis of gastrectomy-evoked osteopenia.

Gastrectomy leads to osteopenia in the rat. The present study describes the effects of gastrectomy on bone morphology. Rats were subjected to gastrectomy or sham operation. Four weeks after the operation the rats were killed and both tibiae were removed. Bone morphology of the left tibia was analyzed with quantitative computer tomography, the right tibia with histomorphometry. Bone length, bone mineral content, as well as indices of bone resorption and formation were measured in the metaphysis and the diaphysis. Gastrectomy had no effect on longitudinal bone growth but it led to a low bone mineral content at both sites. Bone resorption was increased by gastrectomy, as shown by an increase in the medullary cavity area in the diaphysis. Gastrectomy also reduced bone formation, as shown by a decreased periosteal circumference and a decrease in the mean periosteal bone apposition in the diaphysis. In conclusion, gastrectomy-evoked osteopenia reflects impaired formation and increased resorption of bone.

Phorbol myristate acetate downregulates the binding sites for colony-stimulating factor-1 on osteoclasts isolated from rats.

Colony-stimulating factor-1 (CSF-1) is the growth factor for the cells of the mononuclear phagocytic system and for osteoclasts. We tested whether phorbol myristate acetate (PMA), a phorbol ester activating protein kinase C, modulates the number of binding sites for CSF-1 on isolated rat osteoclasts. PMA decreased binding of CSF-1 to osteoclasts within 60 minutes. The effect of PMA was dose dependent at concentrations between 10(-9) M and 10(-6) M. The inactive phorbol ester, 4alpha-phorbol 12,13-didecanoate, had only a slight effect. Since the osteoclast preparation was contaminated with other cells, the action of PMA on the osteoclasts might have been either direct or indirect. In pure osteoclasts harvested by a micropipette, the downregulation of CSF-1 binding by PMA reached only about three-quarters of that in nonpurified preparations. Addition of osteoblastic osteosarcoma UMR106 cells increased the effect of PMA. Antiserum against CSF-1, which was added to osteoclasts contaminated with other cells, mainly osteoblasts, partially inhibited the effect of PMA, but the antiserum had no effect in pure osteoclasts. These data suggest that the effect mediated by osteoblasts or other contaminating cells is due to the release of CSF-1, which is known to downregulate its binding sites on osteoclasts. The direct action of PMA on osteoclasts decreased the binding only to about 40%, in contrast to CSF-1 which completely downregulated the binding. The data also differ from the published results about macrophages. In these cells, PMA downregulates the binding of CSF-1 completely. The CSF-1 binding sites on osteoclasts recovered within 4 hours after removal of PMA, and cycloheximide, an inhibitor of protein synthesis, inhibited the recovery.

Frequency of food intake and natural dietary components are potent modulators of bone resorption and bone mass in rats.

Prevention of low bone mass is important to reduce the incidence of osteoporotic fractures. In this report evidence is provided that feeding habits per se, that is, increased frequency of food intake as well as a diet containing soy and other raw components, decrease bone resorption and increase bone mass in growing rats. Interim results after 6 weeks indicate that food fractionation and natural dietary components are both capable of inhibiting trabecular bone loss in aged rats. These interim results indicate that the effect of both dietary interventions are additive and together are capable of nearly completely blunting the age-dependent loss of trabecular bone mineral density. These dietary manipulations are, however, only partially effective in inhibiting the strongly increased loss of trabecular bone mineral density induced by estrogen deprivation. The fact that the natural dietary components are not more effective in ovariectomized rats as compared to intact females confirms our contention that these components may not operate by mimicking the effect of estradiol. Whether bone mass in humans is also under the control of dietary habits is not known. If so, an increased frequency of meals of appropriate composition may be used to prevent osteoporosis.

Effect of bisphosphonates on the increase in bone resorption induced by a low calcium diet.

This study investigates whether bisphosphonate-treated rats are still able to adapt to low calcium supply through an increase in bone resorption assessed by measuring the urinary excretion of [3H]-tetracycline from chronically prelabeled rats. First it was shown that in this model, parathyroid hormone was responsible for the increase in bone resorption on the low calcium diet. In the second part, animals were treated with the three bisphosphonates-clodronate, alendronate, and ibandronate-given in two doses. Animals receiving a dose that already strongly inhibits bone resorption were still able to respond to a low calcium diet by increasing bone resorption, showing the potency of the latter as a stimulator of bone resorption. Higher doses were, however, able to blunt this response. As soon as the treatment was discontinued, this increase in bone resorption resumed with clodronate but not with alendronate or ibandronate.

The food-induced stimulation of bone resorption in the rat, assessed by the urinary [3H]-tetracycline excretion, is mediated by parathyroid hormone.

Using the urinary excretion of [3H]-tetracycline from prelabeled rats to monitor bone resorption, we have previously shown that food intake is associated with a rapid and large increase in bone resorption. This increase is blunted when the daily intake is fractionated into 4 portions instead of being given at once. Food fractionation also leads to a large increase in bone mass. In order to establish whether the thyroparathyroid gland is involved in this effect, thyroparathyroidectomized (TPTX) rats were studied. The food-induced increase in bone resorption was absent in TPTX rats. Therefore, the pattern of parathyroid hormone (PTH) was investigated during the development of the food-induced bone resorption, and under food fractionation. Rats were trained to eat their daily high Ca food in less than two hours. Thereafter they were given food portions of 5 or 20 grams, respectively. PTH in serum was measured at 0, 1, 2, 3, and 4 hours after food intake. In rats given the large food portions, a conspicuous increase of PTH was found. In contrast, serum PTH of rats fed the small food portion did only change to an insignificant extent. This study in rats shows that the ingestion of a large meal induces a transient increase of PTH. The present results can therefore explain the formerly observed acute increase in bone resorption following food intake and its blunting by food fractionation. It is not known, whether such a mechanism occurs also in humans. If so, these results would provide the rational basis to decrease bone resorption by food fractionation.

The diurnal rhythm of bone resorption in the rat. Effect of feeding habits and pharmacological inhibitors.

Prevention of low bone mass is important to reducing the incidence of osteoporotic fractures. This paper shows that, in rats, bone mass can be increased by feeding habits per se. Using six-hourly urinary excretion of [3H]tetracycline from prelabeled rats to monitor bone resorption, we previously found a peak of bone resorption following food administration. We now demonstrate that dividing the solid and liquid intake into portions blunts this peak and leads to a decrease in 24-h bone resorption to the level observed in thyroparathyroidectomized animals. Calcium balance increases and, when such feeding schedules are imposed for 30 d, bone mass increases. Dividing the intake is not effective in thyroparathyroidectomized animals, indicating the importance of PTH and/or calcitonin. Administration of calcitonin inhibits practically only the peak of bone resorption, suggesting that it is osteoclast mediated. In contrast, treatment with a bisphosphonate reduces basal bone resorption without a specific effect on the peak, indicating a fundamentally different mechanism of action. This is also supported by the finding that their combined effects are additive. Whether bone mass in humans is also under the control of dietary habits is not known. If so, an increased meal frequency may be used to prevent osteoporosis.

Evaluation of urinary pyridinium crosslink excretion as a marker of bone resorption in the rat.

The aim of this study was to evaluate the value of the urinary excretion of the pyridinium crosslinks, pyridinoline (Pyr) and deoxypyridinoline (D-Pyr), as markers of bone resorption in the rat. The excretion of the crosslinks was compared with that of urinary [3H]tetracycline ([3H]TC) excretion from chronically [3H]TC-prelabeled animals, a technique established to monitor bone resorption in the rat. Bone resorption was modulated by Ca restriction, infusion of PTH, thyroparathyroidectomy, and administration of different bisphosphonates. Furthermore, the urinary crosslinks were assessed in three different osteopetrotic mutations in the rat. We found a delayed response of Pyr and D-Pyr excretion to acute changes in bone resorption compared with [3H]TC excretion. This delay was 1 day after Ca restriction and longer after other treatments, such as PTH administration or bisphosphonate treatment, with which it was more than 3 weeks. In contrast, chronic states with stimulation or inhibition of bone resorption showed similar changes in excretion of the urinary crosslinks and [3H]TC, except after PTH administration. The excretion of the crosslinks was greatly reduced in osteopetrotic rats (op/op, tl/tl, and ia/ia) and increased to normal levels in tl/tl rats after stimulation of bone resorption by M-CSF administration. These results suggest that, in rats, urinary excretion of the pyridinium crosslinks reflects bone resorption in chronic but not always in acute conditions. The cause of this discrepancy is still unclear.

Sodium EDTA enhances intestinal absorption of two bisphosphonates.

Bisphosphonates are poorly absorbed when given orally and their absorption is subject to a large inter- and intraindividual variability. This poor absorbability is thought to result, at least in part, from formation of unabsorbable complexes with calcium. It was therefore investigated whether the calcium chelator EDTA could improve intestinal absorption of two bisphosphonates, 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (AHBuBP), and dichloromethylenebisphosphonate (Cl2MBP). Absorption was assessed indirectly by measuring the suppression of hypercalcemia induced in thyroparathyroidectomized rats by a retinoid. The absorption of AHBuBP was in the range of 1-3%. EDTA increased absorption about tenfold at a AHBuBP dose of 0.6 mg P/kg and about twofold at lower doses, with the minimal effective dose of EDTA being 10 mg/kg. The absorption of Cl2MBP was also increased by EDTA, although to a smaller extent, the lowest effective dose being 100 mg/kg EDTA. Thus, EDTA can, in certain circumstances, increase the intestinal absorption of bisphosphonates. The mechanism might involve an increase in available bisphosphonate and a change in mucosal permeability. The amount of EDTA required is, however, too high for use clinically.