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.

The effect of mechanical strain on hyaluronan metabolism in embryonic fibrocartilage cells.

The development of the synovial joint cavity between the cartilage anlagen of the long bones is thought to be mediated by differential matrix synthesis at the developing articular surfaces. In addition, many studies have shown that removal of movement-induced mechanical stimuli from developing diarthrodial joints prevents cavity formation or produces a secondary fusion of previously cavitated joints. Herein, we describe an inductive influence of mechanical strain on hyaluronan metabolism and the expression of hyaluronan-binding proteins in cultured cells isolated from the articular surface of the distal tibial condyles of 18-day chick embryos. The effect of 10 min of mechanical strain on hyaluronan release into culture media, intracellular uridine diphospho-glucose dehydrogenase activity (an enzyme required for hyaluronan saccharide precursor production), cell surface hyaluronan-binding protein expression and HA synthase mRNA expression were analysed up to 24 h later. Six hours after the application of strain, there was a significant increase in the accumulation of hyaluronan released into tissue culture media by strained fibrocartilage cells compared with controls, an effect still detectable after 24 h. Strained cells also showed increased activity for uridine diphospho-glucose dehydrogenase and expressed higher levels of the hyaluronan-binding protein CD44 at 24 h. In addition, at 24 h mRNA for HA synthase 2 was expressed in all samples whereas mRNA for HA synthase 3 was only expressed in strained cells. These results further highlight the role for movement-induced stimuli in differential extracellular matrix metabolism during joint development and also show that strain may facilitate differential HA synthase gene expression.

Mechanical strain stimulates nitric oxide production by rapid activation of endothelial nitric oxide synthase in osteocytes.

Previous studies have indicated that physiological levels of dynamic mechanical strain produce rapid increases in nitric oxide (NO) release from rat ulna explants and primary cultures of osteoblast-like cells and embryonic chick osteocytes derived from long bones. To establish the mechanism by which loading-induced NO production may be regulated, we have examined: nitric oxide synthase (NOS) isoform mRNA and protein expression, the effect of mechanical loading in vivo on NOS mRNA expression, and the effect of mechanical strain on NO production by bone cells in culture. Using Northern blot analyses, in situ hybridization, and immunocytochemistry we have established that the predominant NOS isoform expressed in rat long bone periosteal osteoblasts and in a distinct population of cortical bone osteocytes is the endothelial form of NOS (eNOS), with little or no expression of the inducible NOS or neuronal NOS isoforms. In contrast, in non-load-bearing calvariae there are no detectable levels of eNOS in osteocytes and little in osteoblasts. Consistent with these observations, ulnar explants release NO rapidly in response to loading in vitro, presumably through the activation of eNOS, whereas calvarial explants do not. The relative contribution of different bone cells to these rapid increases in strain-induced NO release was established by assessment of medium nitrite (stable NO metabolite) concentration, which showed that purified populations of osteocytes produce significantly greater quantities of NO per cell in response to mechanical strain than osteoblast-like cells derived from the same bones. Using Northern blot hybridization, we have also shown that neither a single nor five consecutive daily periods of in vivo mechanical loading produced any significant effect on different NOS isoform mRNA expression in rat ulnae. In conclusion, our results indicate that eNOS is the prevailing isoform expressed by cells of the osteoblast/osteocyte lineage and that strain produces increases in the activity of eNOS without apparently altering the levels of eNOS mRNA.

Enhancement by sex hormones of the osteoregulatory effects of mechanical loading and prostaglandins in explants of rat ulnae.

Explants of ulnae from 5-week-old male and female rats were cleaned of marrow and soft tissue and, in the presence and absence of 10(-8) M 17 beta-estradiol (E2) or 5 alpha-dihydrotestosterone (DHT), mechanically loaded or treated with exogenous prostanoids previously shown to be produced during loading. Over an 18-h period, mechanical loading (peak strain 1300 mu epsilon, 1 Hz, 8 minutes, maximum strain rate 25,000 mu epsilon/s), prostaglandin E2 (PGE2) and prostacyclin (PGI2) (10(-6) M), each separately produced quantitatively similar increases in cell proliferation and matrix production in bones from males and females, as indicated by incorporation of [3H]thymidine into DNA and [3H]proline into collagen. E2 and DHT both increased [3H]thymidine and [3H]proline incorporations, E2 producing greater increases in females than in males. Indomethacin abrogated the effects of loading, but had no effects on those of sex hormones. Loading, or prostanoids, together with sex hormones, produced responses generally equal to or greater than the addition of the individual influences acting independently. In females there was a synergistic response in [3H]thymidine incorporation between loading and E2, which was quantitatively similar to the interaction between E2 and PGE2 or PGI2. The interaction between loading and E2 for [3H]proline incorporation was not mimicked by these prostanoids. In males the synergism in [3H]proline incorporation seen between loading and DHT was mimicked by that between PGI2 and DHT. We conclude that loading stimulates increased bone cell proliferation and matrix production in situ through a prostanoid-dependent mechanism. This response is equal in size in males and females. Estrogen and testosterone increase proliferation and matrix production through a mechanism independent of prostanoid production. The interactions between loading and hormones are reproduced in some but not all cases by E2 and prostaglandins. E2 with loading and prostaglandins has greater effects in female bones, while DHT with loading and prostaglandins has greater effects in males.

Early responses to dynamic strain change and prostaglandins in bone-derived cells in culture.

Mechanical loading of bone explants stimulates prostaglandin E2 (PGE2) and prostacyclin (PGI2) release and increases glucose 6-phosphate dehydrogenase (G6PD) activity. This response is blocked by indomethacin and imitated by exogenous PGs. In the experiments reported here, primary cultures of rat long bone-derived osteoblast-like cells were exposed to a dynamic strain and exogenous PGs in the culture dish. Strain (3400 mu epsilon, 600 cycles, 1 Hz) caused an immediate release of PGI2 into the culture medium but had no effect on PGE2. Strain also caused an increase in G6PD activity per cell and an increase in the smallest transcript of insulin-like growth factor II (IGF-II) (IGF-II T3) but had no effect on the expression of transforming growth factor-beta1 (TGF-beta1). Indomethacin inhibited strain-induced release of PGI2 and suppressed strain-induced stimulation of IGF-II T3 transcript. PGI2 (1 microM) increased G6PD activity and mRNA levels of all three transcripts of IGF-II but had no effect on the mRNA levels of IGF-I or TGF-beta1. PGE2 (1 microM) stimulated G6PD activity and caused a marked increase in IGF-I and the largest transcript of IGF-II (IGF-II T1) but had no effect on the IGF-II transcripts T2 and T3 or on TGF-beta1 mRNA levels. These findings show similarities in response between osteoblast-like cells strained in monolayer culture and bone cells in loaded bone explants in situ. They provide support for a role for IGF-II and PGI2 in the early strain-related response of osteoblasts in loading-related bone modeling/remodeling.

Mechanical loading and sex hormone interactions in organ cultures of rat ulna.

The separate and combined effects of loading and 17 beta-estradiol (E2) or 5 alpha-dihydrotestosterone (DHT) on [3H]thymidine and [3H]proline incorporation were investigated in cultured ulna shafts from male and female rats. Ulnae were cultured and loaded to produce physiological strains in the presence or absence of 10(-8) M E2 or DHT. Loading engendered similar increases in incorporation of [3H]thymidine and [3H]proline in male and female bones. E2 engendered greater increases in incorporation in females than in males, and DHT greater increases in males than in females. In males E2 with loading produced increases in both [3H]thymidine and [3H]proline incorporation, which approximated to the arithmetic addition of the increases due to E2 and loading separately. In females E2 with loading produced increases greater than those in males, and substantially greater than the addition of the effects of E2 and loading separately. Loading with DHT in males also showed additional [3H]thymidine and [3H]proline incorporation. In females there was additional incorporation of [3H]proline, but not [3H]thymidine. The location of incorporation of [3H]thymidine and [3H] proline was consistent with their level of incorporation reflecting periosteal osteogenesis, in which case the early osteogenic effects of sex hormones are gender-specific when acting alone and in combination with loading. In males the effects of estrogen and testosterone add to, but do not enhance, the osteogenic responses to loading. In females testosterone with loading produces an additional effect on [3H]proline incorporation but no greater effect than loading alone on that of [3H]thymidine. In contrast, estrogen and loading together produce a greater effect than the sum of the two influences separately. Because premenopausal bone mass will have been achieved under the influence of loading and estrogen acting together, these findings suggest that the bone loss which follows estrogen withdrawal may result, at least in part, from reduction in the effectiveness of the loading-related stimulus on bone cell activity. This stimulus is normally responsible for maintaining bone mass and architecture.

Mechanical strain-induced NO production by bone cells: a possible role in adaptive bone (re)modeling?

The structural competence of the skeleton is maintained by an adaptive mechanism in which resident bone cells respond to load-induced strains. To investigate the possible role of the messenger molecule nitric oxide (NO) in this response, we studied NO production in well-characterized organ culture systems, rat long bone-derived osteoblast-like (LOBs) cells, and embryonic chick osteocytes (LOCYs) in monolayer culture. In superfused cancellous bone cores, loading (for 15 min) produces increases in NO2- (stable NO metabolite) release during the loading period, which paralleled those in PGI2 and PGE2. Loading of rat vertebrae and ulnae produces increases in NO2- release, and in ulnae NO synthase inhibitors diminish these responses. Transient rapid increases in NO release are stimulated by strain in both LOBs and LOCYs. Polymerase chain reaction amplification of extracted mRNA shows that rat ulnae, LOBs, and LOCYs express both the inducible and neuronal (constitutive) isoforms of NO synthase. Adaptability to mechanical strain relies on assessment of the strain environment followed by modification of bone architecture. Immediate increases in NO production induced by loading suggest the involvement of NO in strain measurement and cellular communication to establish strain distribution, as well as potentially in adaptive changes in bone cell behavior.

Calvarial and limb bone cells in organ and monolayer culture do not show the same early responses to dynamic mechanical strain.

Responses to mechanical strain in calvaria and limb bone organ cultures were compared by measuring cellular glucose 6-phosphate dehydrogenase (G6PD) activity in situ and prostaglandin release. Normal functional strains were recorded in the ulnae (1000 mu epsilon) and calvarium (30 mu epsilon) in vivo in 110 g rats. Organ cultures of ulnae and calvaria from similar animals were loaded to produce dynamic strains (600 cycles, 1 Hz) of 1000 mu epsilon in the ulna, and 100 or 1000 mu epsilon in calvaria. In ulnae, both PGE2 and PGI2 were released and resident osteocytes and osteoblasts showed increased G6PD activity. Neither response was seen in calvaria. However, exogenous PGI2 (10(-5)-10(-9) M) stimulated G6PD activity in osteocytes and osteoblasts in organ cultures of both calvaria and ulnae. In ulnar cells the response was linear, in calvarial cells it was biphasic with maximum activity at 10(-7) M. Osteoblasts derived from ulnae and cultured on plastic plates subjected to dynamic strain (600 cycles, 1 Hz, 4000 mu epsilon) showed increased G6PD activity. There was no such response in similarly treated calvarial-derived cells. Calvarial bone cells differ from those of the ulna in that they do not respond to physiological strains in their locality with increased prostanoid release or G6PD activity either in situ or when seeded onto dynamically strained plastic plates. Cells from both sites in organ culture show increased G6PD activity in response to exogenous PGI2, but their dose:responses differ in shape. These differences may reflect the extent to which functional loading influences bone architecture in these two sites.

Noninvasive loading of the rat ulna in vivo induces a strain-related modeling response uncomplicated by trauma or periostal pressure.

Adaptive changes in bone modeling in response to noninvasive, cyclic axial loading of the rat ulna were compared with those using 4-point bending of the tibia. Twenty cycles daily of 4-point bending for 10 days were applied to rat tibiae through loading points 23 and 11 mm apart. Control bones received nonbending loads through loading points 11 mm apart. As woven bone was produced in both situations, any strain-related response was confounded by the response to direct periosteal pressure. Four-point bending is not, therefore, an ideal mode of loading for the investigation of strain-related adaptive modeling. The ulna's adaptive response to daily axial loading over 9 days was investigated in 30 rats. Groups 1-3 were loaded for 1200 cycles: Group 1 at 10 Hz and 20 N, Group 2 at 10 Hz and 15 N, and Group 3 at 20 Hz and 15 N. Groups 4 and 5 received 12,000 cycles of 20 N and 15 N at 10 Hz. Groups 1 and 4 showed a similar amount of new bone formation. Group 5 showed the same pattern of response but in reduced amount. The responses in Groups 2 and 3 were either small or absent. Strains were measured with single-element, miniature strain gauges bonded around the circumference of dissected bones. The 20 N loading induced peak strains of 3500-4500 mustrain. The width of the periosteal new bone response was proportional to the longitudinal strain at each point around the bone's circumference. It appears that when a bone is loaded in a normal strain distribution, an osteogenic response occurs when peak physiological strains are exceeded. In this situation the amount of new bone formed at each location is proportional to the local surface strain. Cycle numbers between 1200 and 12,000, and cycle frequencies between 10 and 20 Hz have no effect on the bone's adaptive response.

A new light microscopy classification of spermatogenesis in the bull and the boar applicable to tissues processed for electron microscopy.

A new light microscopy classification of spermatogenesis in the bull and the boar was formulated to be used in conjunction with electron microscopy. It uses information available from all cell types and allows for very small portions of testicular epithelium at any orientation to be evaluated. The classification devised, being simple in outline, allows for the natural overlap of cell types. Furthermore, it emphasises the direct comparability of the morphological events occurring during spermatogenesis in the bull and the boar.

Cellular responses to mechanical loading in vitro.

A technique has been established in which cancellous bone biopsies may be simultaneously perfused and subjected to mechanical load bearing. Assessments of cell viability over a period of 24 h were based on the cAMP response to parathyroid hormone, intracellular lactate dehydrogenase activity, and electron micrograph morphology. Two cellular responses to mechanical loading were demonstrated similar to those that follow "osteogenic" loading in vivo, as reported previously. These were (1) a rise in intracellular G6PD in lining cells immediately after loading, and (2) an increase in RNA synthesis measured in osteocytes 6 h after loading. In vivo the osteogenic response to loading was modulated by indomethacin. In these in vitro experiments, addition of indomethacin inhibited both the loading-related G6PD and the RNA responses.

Load-induced proteoglycan orientation in bone tissue in vivo and in vitro.

Previous studies of Alcian blue-induced birefringence in adult avian cortical bone showed that a short period of intermittent loading rapidly produces an increased level of orientation of proteoglycans within the bone tissue. In the absence of further loading, this persists for over 24 hours. We have proposed that this phenomenon could provide a means for "capturing" the effects of transient strains, and so provide a persistent, constantly updated strain-related influence on osteocyte populations related to the bones' averaged recent strain history, in effect, a "strain memory" in bone tissue. In our present study, we use the Alcian blue-induced birefringence technique to demonstrate that proteoglycan orientation also occurs after intermittent loading of both cortical and cancellous mammalian bone in vivo and in vitro. We also show that the change in birefringence is proportional to the magnitude of the applied strain, and that the reorientation occurs rapidly, reaching a maximal value after only 50 loading cycles. Examination of electron micrographs of bone tissue after staining with cupromeronic blue allows direct visualization and quantification of the change in proteoglycan orientation produced by loading. This shows that intermittent loading is associated with a realignment of the proteoglycan protein cores, bringing them some 5 degrees closer to the direction of collagen fibrils in the bone matrix.

Experimental Brugia pahangi and B. malayi infections of callitrichid primates.

The callitrichid primates, Callithrix jacchus jacchus (the marmoset) and Saguinus labiatus (the tamarin) were inoculated with infective larvae of Brugia malayi and B. pahangi. Microfilaraemia at low levels developed in 3 out of 4 C.j. jacchus infected with B. malayi and living or dead adult worms found in all 4. Only one of 4 C.j. jacchus became microfilaraemic (mf + ve) when given B. pahangi and adults were found in two. Of 4 S. labiatus given B. pahangi one became very lightly mf + ve and adults were found in 3. It is concluded that these animals are not suitable hosts for chemotherapeutic experiments.

Evaluation of a sanitation programme using eggs of Ascaris lumbricoides in household yard soils as indicators.

Soil samples were analysed for the presence of Ascaris lumbricoides eggs as indicators of environmental pollution in household yards in Maputo, Mozambique, with the objective of evaluating the impact of a programme for the promotion of improved latrine construction. The locations for soil sample collection were defined by a random grid on which household activities were mapped. In addition, parasitological examinations were carried out amongst household residents. No significant difference was found between the type of latrine in use and the presence of Ascaris eggs in the soil or human Ascaris infection. Households with at least one infected person appeared more likely to have Ascaris eggs in the yard. It was notable that egg counts around the latrines were only slightly greater than in other areas of the yard and less than those immediately in front of the dwelling. This is taken to indicate that faecal pollution of the household environment is due more to promiscuous defecation than to poor construction or maintenance of the latrines. The findings highlight the need to complement sanitation 'hardware' with the necessary health education 'software'. Ascaris eggs are useful indicators but robust standardized methods are needed for their extraction from household soils.

The effect of diethylcarbamazine on the in vitro serum-mediated adherence of feline granulocytes to microfilariae of Brugia pahangi.

The effect of the antifilarial drug diethylcarbamazine (DEC) on immunologically mediated destruction of the microfilariae (mf) of Brugia pahangi in vitro is addressed by studying the drug's influence on the granulocyte adherence to the surface of mf and the subsequent death of these parasites. DEC enhanced the adherence of cat blood-derived granulocytes to the surface of mf through the meditation of cuticle direct antibodies but not that mediated by complement alone; the parasites became immobilized and degenerated subsequent to cell adherence. The rate of cell adherence in the antibody-containing cultures was enhanced by DEC. As previously reported, this drug had no direct effect on the viability of the parasites. Preincubation of the cells in DEC had a greater effect on cell adherence than did preincubating the parasites in the drug, thus supporting the contention that this antifilarial compound may act on host components. Further investigation of this possibility may contribute significantly to a more complete understanding of why DEC can cause severe side reactions in filariae infected patients.

Increased 3H-uridine levels in osteocytes following a single short period of dynamic bone loading in vivo.

Both ulnas of skeletally mature roosters (Gallus domesticus) were deprived of functional load bearing by proximal and distal submetaphyseal osteotomies. Twenty-four hours later the animals were injected with 1.5 mCi of 3H-uridine and the ulna on one side was subjected to a single period of a cyclical load engendering physiological strain levels at 1 Hz for 6 min. Twenty-four hours after loading the animals were killed. Autoradiographic examination of comparable regions of cortex in sections from the bone's midshafts showed that in the loaded bones, 72 +/- 2.7% of osteocytes were labeled compared with 12 +/- 3.5% in the corresponding areas of their contralateral nonloaded pair (P less than 0.001). The number of grains per labeled osteocyte was also higher in the loaded side (6 +/- 0.5 compared with 4 +/- 0.5, P less than 0.01). There was no obvious correlation between the longitudinal strain distribution during artificial loading and the distribution of labeled osteocytes throughout the bone cross-section. However, previous long-term experiments using a similar loading preparation had consistently shown the site of most periosteal new bone formation to also not be directly related to the local strain magnitude. Perhaps it is significant that the greatest percentage of labeled cells were found in the cortex where the long-term experiments had shown most new bone formation to subsequently occur.

Expression of cross-reactive surface antigens by microfilariae and adult worms of Brugia pahangi during infections in cats.

Microfilariae of Brugia pahangi were labelled with 125-Iodine using the reagent IODOGEN. Electron microscope autoradiographs of sections of iodinated microfilariae showed that the label was strictly confined to their sheath. Adult worms were also iodinated by the same procedure. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of detergent extracts of radio-labelled parasites revealed components of molecular weights 113, 81-71, 46 and 33 kDa in microfilariae, and of molecular weights 29, 20 and 16 kDa in adult worms. All but the 33 kDa component of microfilariae were immunoprecipitable with sera of infected cats and therefore antigenic. Antibodies to the 81-71 kDa and the 46 kDa microfilarial antigens were detected by immunoprecipitation before patency. Similarly, the 29 kDa antigen of adult worms was immunoprecipitable before the fourth moult. Therefore, during infection in cats, these antigens cross-react with epitopes present on earlier developmental stages.

Parasitological observations on Meriones unguiculatus singly or multiply infected with Brugia pahangi.

When jirds were infected with a single inoculum of 25-50 infective larvae of Brugia pahangi an overall mean recovery of adult worms of 44.5% (n = 41) was obtained. There was no difference in recoveries between male and female jirds. If jirds were repeatedly inoculated with larvae into the peritoneal cavity yields were only slightly reduced. Yields were 30.5% for 5 infections (n = 10), 26.7% for 10 infections (n = 8), 34.4% for 15 infections (n = 10) and 28.5% for 20 infections (n = 7). Twice as many worms were recovered from intraperitoneally inoculated jirds than from subcutaneously inoculated jirds.

The resistance to re-infection of cats repeatedly inoculated with infective larvae of Brugia pahangi.

Seven microfilaraemic and five amicrofilaraemic cats which had been repeatedly infected with Brugia pahangi were challenged along with normal cats 28, 14 and 1 day before autopsy. The lymphatics of the amicrofilaraemic cats contained no female adult worms originating from the repeat infections and only two adult males (both from the same cat). Only 5.2% of the worms in the control cats were recovered from the amicrofilaraemic cats. Most of the challenge worms were killed in the first 24 h. The microfilaraemic cats all contained fertile adult male and female worms derived from the repeated infection but in such low numbers as to indicate considerable resistance to infection. Compared to their controls 26.4% of the challenge worms were recovered. Analysis of the life-cycle stages recovered showed that in both groups there was attrition of all stages and that although a number of worms reached L5 these were all killed later in the amicrofilaraemic cats.

The infectivity of microfilariae of Brugia pahangi of different ages to Aedes aegypti.

By transferring microfilariae of Brugia pahangi which had been born over a 24-hour period in the peritoneal cavities of jirds (Meriones unguiculatus) to the blood circulation of other jirds, infections of known age were tested for their ability to develop into third-stage larvae (L3) in mosquitoes Aedes aegypti. Microfilariae less than three days old were not able to develop to L3. Microfilariae which had been in circulation for three days to six months were capable of developing if ingested.