Disuse rescues the age-impaired adaptive response to external loading in mice.

UNLABELLED: We aimed to determine whether aged bone's diminished response to mechanical loading could be rescued by modulating habitual activity. By reducing background loading, aged bone's response to loading increased to a level no different to young mice. This suggests, given the right stimulus, that ageing bone can respond to mechanical loading. INTRODUCTION: Age-related decline in bone mass has been suggested to represent an impaired ability of bone to adapt to its mechanical environment. In young mice, the tibia's response to external mechanical loading has been shown to increase when habitual activity is reduced by sciatic neurectomy. Here we investigate if neurectomy can rescue bone's response to loading in old mice. METHODS: The effect of tibial disuse, induced by unilateral sciatic neurectomy (SN), on the adaptive response to a single peak magnitude of dynamic load-engendered mechanical strain was assessed in 19-month-old (aged) mice. In a second experiment, a range of peak loads was used to assess the load magnitude-related effects of loading on a background of disuse in young adult and aged mice. Bone architecture was analysed using micro-computed tomography (µCT) and dynamic histomorphometry. RESULTS: In the first experiment, SN in aged mice was associated with a significant periosteal osteogenic response to loading not observed in sham-operated mice (7.98 ± 1.7 vs 1.02 ± 2.2 % increase in periosteally enclosed area, p < 0.05). In the second experiment, SN abrogated the expected age-related difference in the bones' osteogenic response to peak strain magnitude (p > 0.05). CONCLUSIONS: These data suggest that bones' age-related decline in osteogenic responsiveness to loading does not originate in bone cells to either assess, or appropriately respond to strain, but rather is likely to be due to inhibitory "averaging" effects derived from the habitual strains to which the bone is already adapted. If such "strain averaging" is applicable to humans, it suggests that gentle exercise may degrade the beneficially osteogenic effects of short periods of more vigorous activity.

The cyclooxygenase-2 selective inhibitor NS-398 does not influence trabecular or cortical bone gain resulting from repeated mechanical loading in female mice.

UNLABELLED: A single injection of the cyclooxygenase-2 (COX-2) selective inhibitor NS-398 reduces bone's osteogenic response to a single period of mechanical loading in female rats, while women taking COX-2 selective inhibitors do not have lower bone mass. We show that daily NS-398 injection does not influence bone gain from repeated loading in female mice. INTRODUCTION: Prostaglandins are mediators of bone cells' early response to mechanical stimulation. COX-2 expression is up-regulated by exposure of these cells to mechanical strain or fluid flow, and the osteogenic response to a single loading period is reduced by COX-2 inhibition. This study determined, in female mice in vivo, the effect of longer term COX-2 inhibition on adaptive (re)modelling of cortical and trabecular bone in response to repeated loading. METHODS: Nineteen-week-old female C57BL/6 mice were injected with vehicle or NS-398 (5 mg/kg/day) 5 days a week for 2 weeks. On three alternate days each week, the right tibiae/fibulae were axially loaded [40 cycles (7 min)/day] three hours after injection. Left limbs acted as internal controls. Changes in three-dimensional bone architecture were analysed by high-resolution micro-computed tomography. RESULTS: In control limbs NS-398 was associated with reduced trabecular number but had no influence on cortical bone. In loaded limbs trabecular thickness and cortical periosteally enclosed volume increased. NS-398 showed no effect on this response. CONCLUSION: Pharmacological inhibition of COX-2 by NS-398 does not affect trabecular or cortical bone's response to repeated mechanical loading in female mice and thus would not be expected to impair the functional adaptation of bone to physical activity in women.

Who needs more veterinary schools?

Does more mean better, different, or worse, asks Lance Lanyon.

Estrogen receptors α and ß have different gender-dependent effects on the adaptive responses to load bearing in cancellous and cortical bone.

To determine the effect of estrogen receptors (ER) α and ß on bones' adaptive response to loading, we subjected the right tibiae of mice lacking ERα or ERß activity to either axial loading or to disuse. Adaptive changes in architecture were assessed by comparing differences between the right (treated) and left (control) tibiae in these genotypes as assessed by microcomputed tomography. In female ERα(-/-) mice, the net-osteogenic response to loading was lower in cortical bone compared with their wild-type littermates (11.2 vs. 20.9% in ERα(+/+)), but it was higher in both cortical and cancellous bone of male ERα(-/-) mice (cortical 20.0 vs. 4.6% in ERα(+/+); cancellous 30.0 vs. 5.3% in ERα(+/+), P < 0.05). In ERß(-/-) male and female mice, the net-osteogenic response to loading was higher in cortical bone (males 10.9 vs. 3.9% in ERß(+/+); females 18.5 vs. 15.8% in ERß(+/+), P < 0.05) but no different from controls in cancellous bone. The bone loss in response to disuse was less in cancellous bone of ERα(-/-) mice than in controls (-15.9 vs. -21.3%, respectively, P < 0.05) but no different at any other site or between any other groups. Our conclusion is that functional ERα enhances the net-osteogenic response to loading in cortical but not cancellous bone in female mice but reduces it in males. ERß decreases the response to loading in cortical bone of males and females but has no effect in cancellous bone. Bone loss due to disuse in cortical bone is unaffected by ER status, but in cancellous bone, functional ERα contributes to greater disuse-related bone loss.

Mechanical loading-related changes in osteocyte sclerostin expression in mice are more closely associated with the subsequent osteogenic response than the peak strains engendered.

UNLABELLED: Osteocyte sclerostin is regulated by loading and disuse in mouse tibiae but is more closely related to subsequent local osteogenesis than the peak strains engendered. INTRODUCTION: The purpose of this study was to assess the relationship between loading-related change in osteocyte sclerostin expression, local strain magnitude, and local bone modeling/remodeling. METHODS: The right tibiae of 19-week-old female C57BL/6 mice were subjected to non-invasive, dynamic axial loading and/or to sciatic neurectomy-induced disuse. The sclerostin status of osteocytes was evaluated immunohistochemically, changes in bone mass by micro-computed tomography, new bone formation by histomorphometry, and loading-induced strain by strain gauges and finite element analysis. RESULTS: In cortical bone of the tibial shaft, loading engendered strains of similar peak magnitude proximally and distally. Proximally, sclerostin-positive osteocytes decreased and new bone formation increased. Distally, there was neither decrease in sclerostin-positive osteocytes nor increased osteogenesis. In trabecular bone of the proximal secondary spongiosa, loading decreased sclerostin-positive osteocytes and increased bone volume. Neither occurred in the primary spongiosa. Disuse increased sclerostin-positive osteocytes and decreased bone volume at all four sites. Loading reversed this sclerostin upregulation to a level below baseline in the proximal cortex and secondary spongiosa. CONCLUSION: Loading-related sclerostin downregulation in osteocytes of the mouse tibia is associated preferentially with regions where new bone formation is stimulated rather than where high peak strains are engendered. The mechanisms involved remain unclear, but could relate to peak surface strains not accurately reflecting the strain-related osteogenic stimulus or that sclerostin regulation occurs after sufficient signal processing to distinguish between local osteogenic and non-osteogenic responses.

The adaptive response of bone to mechanical loading in female transgenic mice is deficient in the absence of oestrogen receptor-alpha and -beta.

Postmenopausal osteoporosis represents a failure of the response by which bone cells adapt bone mass and architecture to be sufficiently strong to withstand loading without fracture. To address why this failure should be associated with oestrogen withdrawal, we investigated the ulna's adaptive response to mechanical loading in adult female mice lacking oestrogen receptor-alpha (ERalpha(-/-)), those lacking oestrogen receptor-beta (ERbeta(-/-)) and their wild-type littermates. In wild-type mice, short periods of physiologic cyclic compressive loading of the ulna in vivo over a 2-week period stimulates new bone formation. In ERalpha(-/-) and ERbeta(-/-) mice this osteogenic response was respectively threefold and twofold less (P<0.05). In vitro, primary cultures of osteoblast-like cells derived from these mice were subjected to a single short period of mechanical strain. Twenty-four hours after strain the number of wild-type cells was 61+/-25% higher than in unstrained controls (P<0.05), whereas in ERalpha(-/-) cells there was no strain-related increase in cell number. However, the strain-related response of ERalpha(-/-) cells could be partially rescued by transfection with functional human ERalpha (P<0.05). ERbeta(-/-) cells showed a 125+/-40% increase in cell number following strain. This was significantly greater than in wild types (P<0.05).These data support previous findings that functional ERalpha is required for the full osteogenic response to mechanical loading and particularly the stage of this response, which involves an increase in osteoblast number. ERbeta appears to depress the ERalpha-mediated strain-related increase in osteoblast number in vitro, but in female transgenic mice in vivo the constitutive absence of either ERalpha or ERbeta appears to diminish the osteogenic response to loading.

Managing material transfer and nutrient flow in an agricultural watershed.

Place-based resource management, such as watershed or ecosystem management, is being promoted to replace the media-focused approach for achieving water quality protection. We monitored the agricultural area of a 740-ha watershed to determine the nature and scale of farm material transfers, N and P balances, and farmer decisions that influenced them. Using field data and farmer interviews we found that 3 of 15 farms, emphasizing hog, dairy, or cash crops with poultry production, accounted for more than 80% of the inputs and outputs of N and P for the 362-ha agricultural area (332 ha of managed cropland and animal facilities). Feed for hogs (38% each of total N and P) and manure applied to fields as part of the cash crop and poultry operation (28 and 38% of total N and P, respectively) were the dominant inputs. No crops grown in the watershed were fed to animals in the watershed and more manure nutrients were applied from animals outside than from those in the watershed. A strategic decision by the hog farmer to begin marketing finished hogs changed the material transfers and nutrient balances more than tactical decisions by other farmers in allocating manure to cropland. Since the components of agricultural production were not all interconnected, the fundamental assumption of place-based management programs is not well-suited to this situation. Alternative approaches to managing the effect of agriculture on water quality should consider the organization of agricultural production and the role of strategic decisions in controlling farm nutrient balances.

The effect of in vivo mechanical loading on estrogen receptor alpha expression in rat ulnar osteocytes.

The presence of estrogen receptor alpha (ER alpha) in osteocytes was identified immunocytochemically in transverse sections from 560 to 860 microm distal to the midshaft of normal neonatal and adult male and female rat ulnas (n = 3 of each) and from adult male rat ulnas that had been exposed to 10 days of in vivo daily 10-minute periods of cyclic loading producing peak strains of either -3000 (n = 3) or -4000 microstrain (n = 5). Each animal ambulated normally between loading periods, and its contralateral ulna was used as a control. In animals in which limbs were subject to normal locomotor loading alone, 14 +/- 1.2% SEM of all osteocytes in each bone section were ER alpha positive. There was no influence of either gender (p = 0.725) or age (p = 0.577) and no interaction between them (p = 0.658). In bones in which normal locomotion was supplemented by short periods of artificial loading, fewer osteocytes expressed ER alpha (7.5 +/- 0.91% SEM) than in contralateral control limbs, which received locomotor loading alone (14 +/- 1.68% SEM; p = 0.01; median difference, 6.43; 95% CI, 2.60, 10.25). The distribution of osteocytes expressing ER alpha was uniform across all sections and thus did not reflect local peak strain magnitude. This suggests that osteocytes respond to strain as a population, rather than as individual strain-responsive cells. These data are consistent with the hypothesis that ER alpha is involved in bone cells' responses to mechanical strain. High strains appear to decrease ER alpha expression. In osteoporotic bone, the high strains assumed to accompany postmenopausal bone loss may reduce ER alpha levels and therefore impair the capacity for appropriate adaptive remodeling.

Validation of a technique for studying functional adaptation of the mouse ulna in response to mechanical loading.

Functional adaptation of the mouse ulna in response to artificial loading in vivo was assessed using a technique previously developed in the rat. Strain gauge recordings from the mouse ulnar midshaft during locomotion showed peak strains of 1680 muepsilon and maximum strain rates of 0.03 sec(-1). During falls from 20 cm these reached 2620 muepsilon and 0.10 sec(-1). Axial loads of 3.0 N and 4.3 N, applied through the olecranon and flexed carpus, engendered peak strains at the lateral ulnar midshaft of 2000 muepsilon and 3000 muepsilon, respectively. The left ulnae of 17, 17-week-old female CD1 mice were loaded for 10 min with a 4 Hz trapezoidal wave engendering a strain rate of 0.1 sec(-1) for 5 days/week for 2 weeks. The mice were killed 3 days later. The response of the cortical bone of the diaphysis was assessed histomorphometrically using double calcein labels administered on days 3 and 12 of the loading period. Loading to peak strains of 2000 muepsilon stimulated lamellar periosteal bone formation, but no response endosteally. The greatest increase in cortical bone area was 4 mm distal to the midshaft (5 +/- 0.4% compared with 0.1 +/- 0.1% in controls [p < 0.01]). Periosteal bone formation rate (BFR) at this site was 0.73 +/- 0.06 microm(2)/microm per day, compared with 0.03 +/- 0.02 microm(2)/microm per day in controls (p < 0.01). Loading to peak strains of 3000 muepsilon induced a mixed woven/lamellar periosteal response and lamellar endosteal bone formation. Both of these were greatest 3-4 mm distal to the ulnar midshaft. At this level, the loading-induced periosteal response increased cortical bone area by 21 +/- 4% compared with 0.03 +/- 0.02% in controls, and resulted in a BFR of 2.84 +/- 0.42 microm(2)/microm per day, compared with 0.01 +/- 0.01 microm(2)/microm per day in controls (p < 0.05). Endosteal new bone formation resulted in a 2 +/- 0.4% increase in cortical bone area, compared with 0.4 +/- 0.3% in controls, and a BFR of 1.05 +/- 0.23 microm(2)/microm per day, compared with 0.22 +/- 0.15 microm(2)/microm per day in controls (p < 0.05). These data show that the axial ulna loading technique developed in the rat can be used successfully in the mouse. As in the rat, a short daily period of loading results in an osteogenic response related to peak strain magnitude. One important advantage in using mice over rats involves the potential for assessing the effects of loading in transgenics.

Mechanical strain and fluid movement both activate extracellular regulated kinase (ERK) in osteoblast-like cells but via different signaling pathways.

Extracellular regulated kinases (ERKs)-1 and -2 are members of the MAPK family of protein kinases involved in the proliferation, differentiation, and apoptosis of bone cells. We have shown previously that ROS 17/2.8 cells show increased activation of ERK-1 or -2, which is sustained for 24 h, when the strips onto which they are seeded are subjected to a 10 min period of cyclic four point bending that produces physiological levels of mechanical strain along with associated fluid movement of the medium. Movement of the strips through the medium without bending causes fluid movement without strain. This also increases ERK-1/2 activation, but in a biphasic manner over the same time period. Our present study investigates the role of components of signaling pathways in the activation of ERK-1/2 in ROS 17/2.8 cells in response to these stimuli. Using a range of inhibitors we show specific differences by which ERK-1 and ERK-2 are activated in response to fluid movement alone, compared with those induced in response to strain plus its associated fluid movement. ERK-1 activation induced by fluid movement was markedly reduced by nifedipine, and therefore appears to involve L-type calcium channels, but was unaffected by either L-NAME or indomethacin. This suggests independence from prostacyclin (PGI(2)) and nitric oxide (NO) production. In contrast, ERK-1 activation induced by application of strain (and its associated fluid disturbance) was abrogated by TMB-8 hydrochloride, L-NAME, and indomethacin. This suggests that strain-induced ERK-1 activation is dependent upon calcium mobilization from intracellular stores and production of NO and PGI(2). ERK-2 activation appears to be mediated by a separate mechanism in these cells. Its activation by fluid movement alone involved both PGI(2) and NO production, but its activation by strain was not affected by any of the inhibitors used. The G protein inhibitor, pertussis toxin, did not cause a reduction in the activation of ERK-1 or -2 in response to either stimulus. These results are consistent with earlier observations of ERK activation in bone cells in response to both strain (with fluid movement) and fluid movement alone, and further demonstrate that these phenomena stimulate distinct signaling pathways.

Growth rate rather than gender determines the size of the adaptive response of the growing skeleton to mechanical strain.

To determine whether male and female skeletons are equally responsive to mechanical load, the left ulnae in a group of juvenile male (n = 7), and age-matched female (n = 9) rats received a short daily period of controlled dynamic loading in vivo (1200 cycles at 2 Hz each day for 10 days) in addition to their normal exercise. Axial loads for each group were adjusted to engender a peak dynamic strain of -4000 microstrain at the medial face of the ulna midshaft, applied and released at a rate of +/-30,000 microstrain/sec. Fluorescent labels were administered at the start and finish of the loading period. Over the course of daily loading, the body mass of the male rats increased 2.5 times faster than that of the females (6.3 g/day vs. 2.5 g/day). The increase in periosteal interlabel bone area due to growth and normal exercise was also 2.5 times greater in the males than in the females. Both genders showed statistically significant (p < 0.05) increases in periosteal new bone deposition in the ulna of their loaded compared with their control limb. The pattern of osteogenic response was similar in males and females and featured increased mineral apposition rate on the lateral surface of the ulna, and arrest of modeling-drift-related resorption with its reversal to bone formation on the medial surface. In males, the absolute loading-related increase in bone area was six times greater than that in females. However, when the absolute size of the loading-related change in periosteal interlabel new bone deposition was expressed relative to that due to growth, there was no difference between males and females (Mean +/- SEM: 37 +/- 12% for males, 34 +/- 12% for females). These data confirm that the ulna of young actively growing rats of both genders responds to a short daily period of loading with an altered modeling response that involves increased bone formation and decreased resorption. Although the absolute amount of new bone formation stimulated by loading is greater in males than in females there is no difference between genders following correction for the higher rate of bone deposition seen in the males in association with their faster rate of growth.

Mechanical strain and estrogen activate estrogen receptor alpha in bone cells.

Bone cells' early responses to estrogen and mechanical strain were investigated in the ROS 17/2.8 cell line. Immunoblotting with antiphosphorylated estrogen receptor a (ER-alpha) antibody showed that when these cells were exposed for 10 minutes to estrogen (10(-8) M) or a single period of cyclic dynamic strain (peak 3400 microepsilon, 1 Hz, 600 cycles), there was an increase in the intensity of a 66-kDa band, indicating phosphorylation of ser122 in the amino terminus of ER-alpha. Increased phosphorylation was detected within 5 minutes of exposure to estrogen and 5 minutes after the end of the period of strain. Estrogen and strain also activated the mitogen-activated protein kinase (MAPK) family member extracellular regulated kinase-1 (ERK-1). Increases in ERK activation coincided with increased ER-alpha phosphorylation. Activation of ERK-1 and the phosphorylation of ER-alpha, by both estrogen and strain, were prevented by the MAP kinase kinase (MEK) inhibitor U0126 and the protein kinase A (PKA) inhibitor (PKI). These data support previous suggestions that resident bone cells' early responses to strain and estrogen share a common pathway, which involves ER-alpha. This pathway also appears to involve PKA and ERK-mediated phosphorylation of ser122 within the amino terminus of ER-alpha. Reduced availability of this pathway when estrogen levels are reduced could explain diminished effectiveness of mechanically related control of bone architecture after the menopause.

Mechanical strain stimulates osteoblast proliferation through the estrogen receptor in males as well as females.

Mechanical strain, testosterone, and estrogen all stimulate proliferation of primary cultures of male rat long bone (LOB)-derived osteoblast-like cells as determined by [3H]thymidine incorporation. The maximum proliferative effect of a single period of mechanical strain (3400 microepsilon, 1 Hz, and 600 cycles) is additional to that of testosterone (10(-8) M) or estrogen (10(-8) M). The cells' proliferative response to strain is abolished both by concentrations of tamoxifen that cause proliferation (10(-8) M) and by those that have no effect (10(-6) M). Strain-related proliferation also is reduced by the estrogen antagonist ICI 182,780 (10(-8) M) but is unaffected by the androgen receptor antagonist hydroxyflutamide (10(-7) M). Tamoxifen, ICI 182,780, and the aromatase inhibitor 4-dihydroandrostenedione, at concentrations that have no effect on basal proliferation, significantly reduce the proliferative effect of the aromatizable androgen testosterone but not that of the nonaromatizable androgen 5alpha-dihydrotestosterone. Hydroxyflutamide, at a concentration that has no effect on basal proliferation (10(-7) M), eliminates the proliferative effect of 5alpha-dihydro-testosterone but had no significant effect on that caused by testosterone. Proliferation associated with strain is blocked by neutralizing antibody to insulin-like growth factor II (IGF-II) but not by antibody to IGF-I. Proliferation associated with testosterone is blocked by neutralizing antibody to IGF-I but is unaffected by antibody to IGF-II. These data suggest that in rat osteoblast-like cells from males, as from females, strain-related proliferation is mediated through the estrogen receptor (ER) in a manner that does not compete with estrogen but that can be blocked by ER modulators. Proliferation associated with testosterone appears to follow its aromatization to estrogen and is mediated through the ER, whereas proliferation associated with 5alpha-dihydrotestosterone is mediated by the androgen receptor. Strain-related proliferation in males, as in females, is mediated by IGF-II, whereas proliferation associated with estrogen and testosterone is mediated by IGF-I.

Mechanical strain activates estrogen response elements in bone cells.

The involvement of the estrogen receptor in the early responses of bone cells to mechanical strain was investigated by subjecting subconfluent monolayer cultures of ROS.SMER #14 cells (ROS 17/2.8 cells stably transfected with additional ER alpha) to 17 beta-estradiol or a single short period of dynamic mechanical strain (600 cycles, 1 Hz). The basal proliferation rate of ROS.SMER #14 cells was similar to ROS 17/2.8 cells, whose proliferative responsiveness to strain and estrogen is similar to that of primary cultures of rat long bone-derived osteoblasts. At peak strains of 3400 mu epsilon, strain-related proliferation in ROS.SMER #14 cells was 1.4 times that of ROS 17/2.8 cells. At 10(-8) mol/L, 17 beta-estradiol-related proliferation was nearly twice greater. The ROS.SMER #14 cells were transiently transfected with an estrogen-responsive reporter, 2ERE-pS2-CAT, containing two consensus estrogen response elements (ERE) linked to a chloroamphenicol acetyl transferase gene. Strain increased normalized ERE-CAT activity threefold and estradiol (10(-8) mol/L) sixfold. Both strain-related and estradiol-related increases in proliferation and ERE-CAT activity were blocked by the estrogen antagonist ICI 182,780 (10(-6) mol/L). These data show that strain as well as estrogen stimulates increased proliferation in ROS 17/2.8 cells and increased ER alpha-related ERE activity in ROS cells transfected with ER alpha. Proliferation is greater in the cells with more estrogen receptors. Both strain- and estrogen-related proliferation and ERE activity are blocked by the estrogen antagonist ICI 182,780. This indicates that ROS cells' early responses to mechanical strain involve ER alpha and estrogen-responsive genes.

Arachidonic acid for loading induced prostacyclin and prostaglandin E(2) release from osteoblasts and osteocytes is derived from the activities of different forms of phospholipase A(2).

Mechanical loading of bone stimulates resident bone cells to produce prostacyclin (PGI(2)) and prostaglandin (PG)E(2) by a mechanism that can be differentially regulated by ion channel blockers. We have investigated differences in the loading-related PG production mechanisms in rat ulnae explants loaded ex vivo. Loading and aluminium fluoride (AlF(3), a nonselective activator of G-proteins) both increased PGI(2) and PGE(2) release into culture medium. Pertussis toxin (PTX) blocked loading-related release of PGE(2), but not PGI(2), while isotetrandrine, an inhibitor of G-protein-mediated activation of phospholipase (PL)A(2), abolished the loading-related release of both PGs. This suggests both PTX-sensitive and -insensitive G-protein-dependent, PLA(2)-mediated mechanisms for loading-related PG production. Blockade of secretory (s)PLA(2) activity prevented loading-related release of PGE(2) and PGI(2), whereas inhibition of cytosolic (c)PLA(2) activity blocked loading-related release of PGE(2) alone. cPLA(2) was localized immuno-cytochemically to osteoblasts, but not to osteocytes. sPLA(2) was localized to osteocytes and osteoblasts. Exogenous type-IA sPLA(2) and type-IB sPLA(2) stimulated significant increases in PGE(2) and PGI(2) release. PTX reduced the release of both PGs stimulated by type IA PLA(2), but not type IB. Furthermore, inhibition of protein kinase C (PKC) activity blocked loading-related release of PGE(2), but not that of PGI(2). These data suggest that loading-related release of PGI(2) and PGE(2) utilizes arachidonic acid derived from the activity of different PLA(2)s. In osteocytes and osteoblasts, arachidonic acid for PGI(2) synthesis is liberated by PTX-insensitive G-protein-dependent sPLA(2) alone. In osteoblasts, arachidonic acid for PGE(2) synthesis is released by PTX-sensitive, G-protein-dependent, cPLA(2)-mediated activity, which also requires upstream sPLA(2) and PKC activities.

Cryptosporidia on dairy farms and the role these farms may have in contaminating surface water supplies in the northeastern United States.

The prevalence and risk factors for shedding of cryptosporidia by dairy cattle and calves and the prevalence and risk factors for cryptosporidia in surface waters associated with dairy farms were determined for a well-defined watershed in the northeastern United States. Eleven dairy farms were enrolled in the study and subjected to monthly sampling over a 6-month period. Animal-, water-, and manure-management practices were determined by survey and fecal, on-farm water, and stream samples were obtained monthly and evaluated for the presence of cryptosporidia. Ninety-one percent of the dairy farms in our study had Cryptosporidium on their premises. Fifteen percent of the sampled calves 0-3 weeks of age were shedding cryptosporidia. The risk factors for calves shedding cryptosporidia were contact between calves and frequent bedding changes. The probability of shedding cryptosporidia decreased with age. Nine percent of farm-associated stream samples were cryptosporidia-positive. The single risk factor for detecting cryptosporidia in surface water was increasing frequency of spreading of manure on fields. The probability of detecting cryptosporidia in streams decreased as 5-day cumulative precipitation increased. There were no animal-associated or barnyard-management features associated with detecting cryptosporidia in farm-impacted streams.

Alpha-particle doses to cells of the bone remodeling cycle from alpha-particle-emitting bone-seekers: indications of an antiresorptive effect of actinides.

There are indications that alpha-particle-emitting bone-seekers such as plutonium or americium could enhance bone mass by suppressing bone resorption. To assess this possibility, this study calculates doses from alpha-particle emitters to the cells involved in trabecular bone turnover. Alpha-particle energy deposition in tissue from a bone surface source was calculated by Monte Carlo modeling. This was combined with bone surface cellular geometry to yield dose rates to cells during the remodeling cycle. Bone-resorbing osteoclasts receive on average 50 times the dose rate that bone-forming osteoblasts receive. Newly formed bone shields osteoblasts from alpha particles emitted by the buried deposit of alpha-particle emitters. However, at alpha-particle bone-seeking radionuclide intakes known to cause changes in remodeling (about 3700 Bq/kg body weight), the alpha-particle dose to osteoclasts corresponds to an extremely low rate of cell traversals (0.07% per cycle). It is therefore unlikely that perturbation of bone remodeling by alpha-particle bone-seeking radionuclides is directly caused by alpha-particle traversals of remodeling cells; some other indirect mechanism might be involved.

Mechanical strain stimulates ROS cell proliferation through IGF-II and estrogen through IGF-I.

The mechanism by which mechanical strain stimulates bone cell proliferation was investigated and compared with that of estrogen in ROS 17/2.8 cells. Similarity of strain-related responses between ROS cells and osteoblasts was established by demonstrating that ROS cells respond to a short single period of strain in their substrate (1000-3500 microepsilon, 600 cycles, 1 Hz) by a similar strain magnitude-related increase in glucose 6-phosphate dehydrogenase activity as rat osteoblasts and osteocytes in explants in situ. ROS17/2.8 cells also showed similar proliferative responses to strain and 17beta-estradiol, as assessed by [3H]thymidine incorporation and cell counting, as primary cultures of long bone-derived osteoblast-like cells. Strain-related increase in proliferation in ROS cells was accompanied by a 4-fold increase in levels of insulin-like growth factor-II (IGF-II) in conditioned medium. Neither strain nor estrogen had an effect on the conditioned medium levels of IGF-I. Exogenous truncated IGFs tIGF-I and tIGF-II both increased proliferation in a dose-dependent manner. The neutralizing monoclonal antibody (nMAb) to IGF-I blocked proliferation stimulated by tIGF-I but not that due to tIGF-II and vice versa. IGF-I receptor blocking antibody (IGF-IRBAb) blocked the proliferative effect of tIGF-I but not that to tIGF-II. The proliferative effect of estrogen was abolished by IGF-I nMAb and IGF-IRBAb, but these antibodies had no effect on the proliferative response to strain. In contrast IGF-II nMAb abolished the proliferative effect of strain but had no effect on that of estrogen. These data show that ROS17/2.8 cells have similar responses to strain and estrogen qualitatively and quantitatively as rat osteoblasts in situ and rat long bone-derived osteoblast-like cells in primary culture. Estrogen-related proliferation in ROS17/2.8 cells appears to be mediated by IGF-I acting through the IGF-I receptor and does not involve IGF-II. In contrast, strain-related proliferation appears to be mediated by IGF-II and does not involve either IGF-I or the IGF-I receptor.