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.

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.

Control of bone architecture by functional load bearing.

The continuing ability of the skeleton to withstand functional loads without damage requires that bone mass and architecture are adjusted according to the loads experienced. Load bearing is the only functional influence that requires a particular bone architecture, and functionally engendered strains within the bone tissue provide the only feedback containing the necessary information on the relationship between current architecture and prevailing load history. The specific strain-related objectives of the adaptive modeling and remodeling response to load bearing have not been adequately defined. They appear to be different for cortical and cancellous bone and vary according to cortical location. Experiments suggest that adaptive modeling and remodeling is sensitive to dynamic but not static strain change and that the osteogenic response to a period of dynamic strain is quickly saturated but is higher when the rate of change in strain is high and the distribution of strain unusual. Presumably it is the cumulative effect of this osteogenic response to load bearing that normally maintains bone mass above that seen in disuse situations. Through their independent effects on bone cell behavior, nutritional and hormonal factors can enable, enhance, limit, or frustrate full expression of the osteogenic response to strain change. However, such systemic factors do not appear to be able to engender or successfully imitate the sustained cumulative local response to load bearing that normally maintains functionally appropriate bone mass and architecture. Experiments in vivo and in vitro suggest that in osteocytes and surface osteoblasts the almost immediate response to strain change is increased production of prostacyclin. Surface osteoblasts also produce prostaglandin E.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Estrogen enhances the stimulation of bone collagen synthesis by loading and exogenous prostacyclin, but not prostaglandin E2, in organ cultures of rat ulnae.

The shafts of ulnae from 110 g male rats were cultured, and after a period of 5 h preincubation one of each pair of bones was either loaded cyclically (500 g, 1 Hz, 8 minutes) to produce physiologic strains (-1300 mu epsilon) or treated with exogenous prostacyclin (PGI2) or prostaglandin E2 (10(-6) M, 8 minutes) in the presence or absence of 17 beta-estradiol (10(-8) M). PGI2, PGE2, and loading stimulated almost immediate increases in glucose 6-phosphate dehydrogenase (G6PD) activity in osteocytes and osteoblasts. This increase was uniform throughout the section with exogenous PGs in the medium but was related to local strain magnitude in loading. Elevated G6PD levels in response to loading and PGI2 persisted for 18 h, by which time, ALP activity in surface osteoblasts was elevated and [3H]proline incorporation into collagen increased. PGE2 produced similar immediate and sustained increases in G6PD activity and [3H]proline incorporation after 18 h but no change in ALP activity. Bones cultured for 18 h with 17 beta-estradiol increased their [3H]proline incorporation, as did those loaded, and treated with PGI2 and PGE2. Loading and PGI2 but not PGE2 produced similar proportional increases in [3H]proline incorporation above the increased baseline of estradiol alone. These results suggest that estrogen and loading together produce a greater osteogenic response than either separately. If so, estrogen withdrawal would result in a rapid fall in bone mass to establish a new equilibrium appropriate to the reduced effectiveness of the loading-related stimulus. Such a fall in bone mass is a characteristic feature of estrogen withdrawal at the menopause.

The estrogen receptor's involvement in osteoblasts' adaptive response to mechanical strain.

The estrogen receptor's role in bone cells' response to mechanical strain was investigated by studying the effect of the estrogen receptor modulators ICI 182, 780 and tamoxifen on the proliferation of primary cultures of rat long bone-derived osteoblasts stimulated by the independent and combined effects of 17beta-estradiol, mechanical strain, and the mitogens basic fibroblast growth factor (bFGF), truncated insulin-like growth factor (tIGF)-I and tIGF-II, and epidermal growth factor (EGF). 17Beta-estradiol (10(-10) M to 10(-8) M) increased [3H]thymidine incorporation equally in cells from males and females, as did a single period of cyclical strain in the plastic strips onto which the cells had been seeded (peak strain 3,400 microepsilon, 600 cycles, 1 Hz). At 10(-8) M, neither ICI 182,780 nor tamoxifen had any effect on basal [3H]thymidine incorporation in these cells, but both compounds prevented their proliferative responses to 10(-8) M 17beta-estradiol. Tamoxifen eliminated and ICI 182,780 substantially reduced the proliferation stimulated by strain. 17Beta-estradiol partially rescued the strain-related response from the effect of tamoxifen but not that of ICI 182,780. Both tamoxifen and ICI 182,780 reduced proliferation stimulated by 10(-8) M EGF but had no effect on that by 10(-7) M bFGF or tIGF-I and tIGF-II. That both ICI 182,780 and tamoxifen, which in other tissues act as estrogen antagonists, should reduce osteoblast proliferation stimulated by 17beta-estradiol and EGF, but not that by FGF or the IGFs, was expected since the mitogenic effects of estrogen and EGF involve the estrogen receptor, whereas those of FGF and the IGFs do not. That these compounds should prevent osteoblasts' proliferative response to strain suggests that strain also stimulates mitogenesis by a mechanism involving the estrogen receptor. If this is so, bones' reduced ability to maintain their structural strength after the menopause could be explained by less effective strain-related (re)modeling when estrogen is absent and, among other changes, the estrogen receptor could be down-regulated.

Early loading-related changes in the activity of glucose 6-phosphate dehydrogenase and alkaline phosphatase in osteocytes and periosteal osteoblasts in rat fibulae in vivo.

The tibiae and fibulae of 14-week-old rats were subjected to a single 5 minutes period of cyclic longitudinal loading at 1 Hz. The activity of the enzymes glucose 6-phosphate dehydrogenase (G6PD) and alkaline phosphatase (ALP) in osteocytes and periosteal osteoblasts was measured immediately and 24 h after loading. In osteocytes G6PD activity was increased immediately after loading but returned to control values 24 h later. There was no detectable ALP activity in these cells regardless of loading history. In periosteal osteoblasts G6PD activity was raised immediately after loading and remained higher than controls 24 h later. ALP activity in periosteal cells was unaffected immediately after loading but 24 h later was substantially increased. These findings are consistent with osteocytes and periosteal cells both being immediately responsive to periods of intermittent loading in their adjacent matrices. In both cell types an early feature of this response is an increase in G6PD activity. In osteocytes this response is short-lived, suggesting that it is an early biochemical change associated with strain perception that does not progress to matrix synthesis. The increase in G6PD activity with unaffected ALP levels in periosteal cells immediately after loading is consistent with a similar response. In these cells the increase in G6PD accompanied by increased ALP levels 24 h after loading suggests that the loading-related response progresses to new bone formation.

Direct transformation from quiescence to bone formation in the adult periosteum following a single brief period of bone loading.

The concept of resorption preceding formation in a coupled response is well established as the normal sequence of remodeling in adult bone. So prevalent is this concept, however, that the idea of the direct activation of osteogenic modeling in normal adult bone is often ignored. This experiment documents the direct transformation of the normal, quiescent, adult periosteum to active bone formation. The osteogenic stimulus was provided by a single short period of dynamic loading. Periosteal activation and the production of new bone within 5 days of loading was unaccompanied by resorption or the presence of osteoclasts. We therefore conclude that an adult resting periosteum can become directly converted to formation as a physiologic response to an appropriate osteogenic stimulus without the need for resorption. To distinguish this process from remodeling we suggest it be called renewed modeling. It is notable that a single short exposure to an "osteogenic" loading regime can influence the full cascade of cellular events between quiescence and active bone formation.

Early strain-related changes in cultured embryonic chick tibiotarsi parallel those associated with adaptive modeling in vivo.

A model was developed for the application of cyclic mechanical loads to 17 day embryonic chick tibiotarsi in culture. A single 20 minute period of intermittent loading at 0.4 Hz, producing physiologic peak strains and strain rates, resulted in two peak strain magnitude-related responses that were previously reported in vivo: (1) a rapid increase in glucose 6-phosphate dehydrogenase activity in osteoblasts and osteocytes and (2) increased RNA synthesis, as shown by increased incorporation of [3H]uridine into extracted RNA. The RNA response was detectable 8 h following loading but was more pronounced by 24 h. Both responses were blocked by indomethacin (10(-6) M). These results demonstrate that embryonic chick bones in organ culture exhibit cellular responses to loading similar to those previously identified in adult canine cancellous bone cultures in vitro and adult avian cortical bone in vivo. These findings are consistent with a sequence of events between loading and new bone formation that includes an immediate strain magnitude-related, prostanoid-dependent increase in activity of the pentose monophosphate shunt in osteoblasts and osteocytes, followed by a similarly strain magnitude-related increase in RNA synthesis over the subsequent 24 h.

Early strain-related changes in enzyme activity in osteocytes following bone loading in vivo.

The skeleton's architecture is matched to the changing loads to which it is subjected because mechanical loading directly or indirectly influences the activity of cell populations to deposit, maintain, or remove bone tissue as appropriate. This responsiveness to load bearing presupposes that certain cells are sensitive to load itself or to its consequences within the tissue. The nature of this effect and the cells concerned have not yet been determined. In this series of experiments, bones were exposed in vivo to a single short period of dynamic loading, which if repeated daily had been shown to result in increased new bone formation. There was an increase in the activity of glucose 6-phosphate dehydrogenase (G6PD) in the periosteal cells adjacent to the bone surface 6 min after this single period of loading. This increase was proportional to the strain magnitude in the bone tissue in the immediate vicinity of the cells. In osteocytes, although the G6PD activity in each individual cell was unchanged by loading, the number of cells displaying activity was increased. This increase was also directly proportional to the applied strain in that area of the cortex (52% compared with 26% active osteocytes at a strain of 0.002). Activation of G6PD was unaccompanied by any equivalent changes in the activities of either glyceraldehyde 3-phosphate dehydrogenase (GA3PD) or lactate dehydrogenase (LDH). This finding is consistent with loading increasing the activity of the oxidative part of the pentose monophosphate shunt pathway. It is also consistent with stimulation of a synthetic process, such as the production of RNA from ribose 5-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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 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.

Bone's early responses to mechanical loading differ in distinct genetic strains of chick: selection for enhanced growth reduces skeletal adaptability.

Bone's functional competence is established and maintained, at least partly, by mechanisms involving appropriate adaptation to mechanical loading. These appear to fail in chickens selectively bred either for maximum egg (Egg-type) or meat (Meat-type) production, which show high rates of fracture and skeletal abnormality, respectively. By measuring several early strain-induced responses in cultured embryonic tibiotarsi from commercially bred (Egg-type and Meat-type) and wild-type (Wild-type) chicks, we have investigated the possibility that these skeletal failures are the product of a compromised ability to respond appropriately to loading-induced mechanical strain. Axial loads engendering peak dynamic (1 Hz) longitudinal strains of between -1300 microepsilon and -1500 microepsilon (for 10 minutes) in vitro in tibiotarsi from the three types of 18-day-old chicks increased periosteal osteoblast glucose 6-phosphate dehydrogenase (G6PD) activity in both Wild-type (26%, p < 0.01) and Egg-type (49%, p < 0.001) chicks in situ, while Meat-type chicks did not show any significant changes (11%). Load-induced increases in medium nitrite accumulation (stable nitric oxide [NO] metabolite) were produced in Egg-type and Wild-type tibiotarsi (82 +/- 12%, p < 0.01; 39 +/- 8%, p < 0.01), respectively. In contrast, loading produced no change in NO release from Meat-type chick tibiotarsi. These changes in NO release correlated with load-related increases in G6PD activity (R2 = 0.98, p < 0.05) in the different chick types. Wild-type and Meat-type tibiotarsal periosteal osteoblasts responded in a biphasic manner to exogenous prostacyclin (PGI2), with maximal stimulation of G6PD activity at 10(-7) M and 10(-6) M PGI2. However, Egg-type chick osteoblasts showed smaller, progressive increases up to 10(-5) M PGI2. These results indicate that early phases of the adaptive response to loading differ in different genetic strains of embryonic chick; that skeletal abnormalities which develop in genetically selected, high growth rate chicks may reflect a compromised ability to respond to load; and that load-induced increases in osteoblastic G6PD activity appear to be closely associated with increased rates of NO release. It is probable that similar genetically related differences in bones' responsiveness to mechanical loading occur in other species.

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.

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.

Loading-related increases in prostaglandin production in cores of adult canine cancellous bone in vitro: a role for prostacyclin in adaptive bone remodeling?

Cyclic mechanical loading sufficient to engender strains of physiologic magnitude applied to recently excised canine cancellous bone cores in vitro increased the release of prostaglandin E (PGE) and prostacyclin (PGI2, measured as its breakdown product 6-keto-PGF1 alpha), during a 15 minute loading period in which PG levels were measured in perfusing medium at 5 minute intervals. Peak production occurred in the 0-5 minute sample. Mean levels preload compared to during load were PGE, 2.66 and 3.67 ng/ml (p less than 0.002); and 6-keto-PGF1 alpha, 543 and 868 pg/ml (p less than 0.007). The elevated levels then declined to preload levels during the loading period. However, the 5-10 minute but not the 10-15 minute samples still contained levels greater than preload values. A second 15 minute period of load, 1 h following the end of the first, produced smaller increases in the levels of release that were statistically significant only for the first 0-5 minute sample during load (preload compared to load mean values, PGE, 1.09-1.66 ng/ml, p less than 0.02; 6-keto-PGF1 alpha, 401-558 pg/ml, p less than 0.04). Immunolocalization revealed PGE and 6-keto-PGF1 alpha in lining cells and 6-keto-PGF1 alpha but not PGE in osteocytes. Addition to the medium of 1 microM PGE2, approximating the concentration produced by loading, had no significant effect on the specific activity of the extractable RNA fraction labeled with [3H]uridine, whereas 1 microM PGI2 produced an increase similar to that seen previously with loading.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of tenascin-C in bones responding to mechanical load.

A number of early biochemical responses of bone cells to mechanical loading have been identified, but the full sequence of events from the sensing of strain to the formation of new bone is poorly characterized. Extracellular matrix proteins can modulate cell behavior and would be ideal molecules to amplify the early response to loading. The extracellular matrix protein, tenascin-C, supports differentiation of cultured osteoblast-like cells. The current study was carried out to investigate whether expression patterns of tenascin-C in loaded bones support a role for this protein as a mediator of the osteoregulatory response to loading. Tenascin-C expression was investigated by Northern blot analysis in rat ulnae subjected to an established noninvasive loading regimen engendering physiological strain levels. RNA extracted from loaded compared with contralateral control bones 6 h after loading showed a significant increase in tenascin-C transcript expression. The presence of tenascin-C was investigated by immunohistochemistry in bones of animals killed 3, 5, or 15 days after the initiation of daily loading. In animals killed at 3 or 5 days, periosteal surfaces undergoing load-induced reversal from resorption to formation showed enhanced tenascin-C staining. In animals killed at 15 days, the bone formed in response to loading was clearly demarcated from old bone by strong tenascin-C staining of reversal lines. Within this new bone, tenascin-C staining was seen in the lacunae of older but not more recently embedded osteocytes. The results presented here indicate that tenascin-C expression by bone cells is enhanced in the early osteogenic response to loading. This may indicate that tenascin-C acts as a mediator of the mechanically adaptive response.

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.