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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

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 17β-estradiol (E2) or 5α-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 μϵ, 1 Hz, 8 minutes, maximum strain rate 25,000 μϵ/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.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

FRACTURE RESISTANCE is ensured in weight-bearing bones by adapting their mass and architecture in response to their mechanical environment.1 When this adaptive mechanism fails, bone mass and architecture become inadequate for the loads experienced and the risk of fracture increases. In women the bone fragility of postmenopausal osteoporosis is obviously associated with estrogen withdrawal.2–7 In males lack of estrogen is also associated with fragile bones8 and osteoporosis is an increasing problem.9 It is important therefore to understand the potential role of sex hormones in the process of bone's adaptive response to load bearing. Frost has hypothesized that estrogen's potential role is that of altering the strain-related setting on the mechanostat, the conceptual equivalent of a thermostat.10,11

In our previous studies using explants of rat ulnae12,13 we have investigated the effects of mechanical loading and estrogen on the early stages of osteogenesis, notably osteoblast recruitment (as indicated by [3H]thymidine incorporation) and matrix production by osteoblasts (as indicated by [3H]proline incorporation). In bones from females, but not males, loading and estrogen in combination produced greater increases in [3H]thymidine and [3H]proline incorporation than the arithmetic addition of the two effects acting separately. We interpreted this as circumstantial evidence that one of estrogen's actions is to amplify the load-related stimulus which we suppose to be used by bone cells as the functional input for their mechanically adaptive responses.13 Change from an amplified to an unamplified load-related stimulus would be interpreted by the bone cells as relative disuse, the appropriate response to which is reduction in bone mass. If this interpretation is correct, it would explain why loss of any direct osteoregulatory effect of estrogen at the menopause is not compensated for by the homeostatic mechanism normally responsible for strain regulation.

In the experiments reported here, we extend our previous studies in male and female rat ulna explants to determine whether the interactions between loading and sex hormones can be accounted for by the interaction of sex hormones with those prostanoids previously shown to be produced by loading. Specifically we investigated: (1) the effects of indomethacin on the response to loading and the interaction of loading and sex hormones; (2) whether the prostaglandins that we have demonstrated to be produced by physiological loads in bone explants (prostaglandin E2 [PGE2] and prostacyclin [PGI2]),14 can substitute for mechanical loading in their effect on cell proliferation and matrix production; and (3) whether these prostaglandins also show similar interactions with the sex hormones, estrogen and testosterone (E2 and DHT), as those produced by loading.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Preparation of bones for culture, loading, and treatments with exogenous agents

Ulnae from 5-week-old (110 ± 5 g of body weight) male and female Sprague-Dawley rats (Charles River, Margate, Kent, U.K.) were prepared as described previously.12,13 Rats were sacrificed by barbiturate overdose, a 12 mm section of the ulna shaft taken and marrow removed. After incubation for 5 h, each pair of ulnae was washed three times in calcium and magnesium-free phosphate buffered saline (PBS) (GIBCO, Paisley, Scotland, U.K.). In those experiments where the bones were treated with exogenous substances, appropriate media previously equilibrated to 37°C containing either vehicle, indomethacin (10−6 M), PGE2 (10−6 M), PGI2 (10−6 M), and/or E2 (10−6 M) or 5α-dihydrotestosterone (DHT) (10−6 M) (all from Sigma Chemical Co., St. Louis, MO, U.S.A.) were dispensed and the cultures incubated for 8 minutes. Those bones subjected to loading were loaded for 8 minutes at this stage. After this the bones were washed in PBS and incubated in fresh media for a further 18 h without exogenous PGE2 or PGI2 but where appropriate with indomethacin, E2, or DHT. For experiments involving [3H]thymidine incorporation the medium was supplemented with 10 μM thymidine and the post-treatment incubation medium with 1.0 μCi/ml L[5′-3H]thymidine (specific activity of 16.4 Ci/mmol; Amersham, Buckinghamshire, U.K.), and where appropriate E2 or DHT. For experiments involving [3H]proline incorporation, the medium was supplemented with 1000 μm L-proline and the post-treatment incubation medium with 10 μCi/ml L[5-3H] proline (specific activity of 25 Ci/mmol; Amersham), and where appropriate E2 or DHT.

Bones were loaded as described previously.12,13 Each bone was cyclically loaded with a weight of 500 g at a frequency of 1 Hz for a period of 8 minutes. The longitudinal peak strain12,13 generated on the bone surface was −1300 μϵ on the medial side and 700 μϵ on the lateral with a maximum strain rate of 25,000 μϵ/s. Following loading, the bones were washed three times in PBS and then cultured for a further 18 h as described above.

[3H]thymidine incorporation

The method used to quantify [3H]thymidine incorporation into the cultured rat ulna was a modification of that by Osborne et al.15 and Dietrich and Paddock.16 Cultured bones were washed three times in ice-cold PBS and placed in 5% TCA (Sigma) for 2 h to remove unincorporated isotope. The bones were then washed again in PBS, minced, and homogenized in 1.0 ml PBS. One hundred microliters of carrier DNA solution (1.0 μg of salmon sperm DNA [Sigma]/1.0 μl PBS) and 1.0 ml 10% TCA were then added to the homogenates. The samples were thoroughly mixed and left at 4°C for 16 h. The TCA insoluble fractions were then recovered by centrifuging at 4°C at 1500 g for 20 minutes. The samples were then washed twice with 5% TCA and 90% ethanol. The resultant pellets were dissolved in 8 ml of ACSII scintillant (Amersham) and the radioactivity was read with a 1214 Rackbeta liquid scintillation counter (LKB Wallac, London, U.K.).

[3H]proline incorporation

The pepsin sensitivity method was used to determine [3H]proline incorporation as described by Webster and Harvey17 and Meghji et al.18 with some modifications.12 In brief, ulnae were placed in 5% ice-cold TCA for 2 h to remove unbound isotope and small peptides and then rinsed in PBS three times and blotted. They were then homogenized and digested with 2 ml of pepsin solution (0.5 mg/ml in 0.5 M acetic acid) for 16 h, and the collagen was precipitated by adding 100 μl of carrier collagen solution (Sigma type V, 1.0 mg/ml in 0.5 M acetic acid) and 0.5 ml of 25% (w/v) sodium chloride/0.5 M acetic acid solution.

After 3 h the precipitate was collected by centrifugation at 3000 g for 30 minutes. The resultant pellet was then dissolved in 1.0 ml of 0.5 M acetic acid, reprecipitated with sodium chloride/acetic acid solution, and recovered again as described above. The final collagen pellets were dissolved in 1.0 ml of 0.5 M acetic acid, mixed with 10 ml of ACSII, and radioactivity was determined by using a 1214 Rackbeta liquid scintillation counter (LKB Wallac).

Statistical analysis

The results presented are from paired ulnae of male and female rats, with one ulna of each pair being treated and the other acting as its control. Comparison between treatments outside the pairs was possible since there was no significant difference between the controls in each group.

The comparisons between treated and control bones were made using the paired t-test on the original numerical data, not the percentage figures illustrated and quoted in the text. All results are reported as means ± SEM. Comparisons of differences between treated and control bones of different groups were made using analysis of variance detailed by Snedecor and Cochran.19 Least significant difference was determined and p < 0.05 was considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Effects of prostaglandins

PGE2 (10−6 M) increased [3H]thymidine incorporation in male bones by 38% and female bones by 29% (Fig. 1A) and increased [3H]proline incorporation in males by 37% and females by 56% (Fig. 1B). These differences in the increases between males and females are not significant.

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Figure FIG. 1. Effects of prostaglandin E2 (PGE2) and prostacyclin (PGI2) on (A) [3H]thymidine and (B) [3H]proline incorporations in male and female rat ulnae in culture. Results are from six pairs of ulnae and expressed as percentage increases of treated bones compared with controls. Their were no significant differences between the control of different groups. All values shown are significantly greater than their paired controls (p < 0.01).

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PGI2 (10−6 M) increased [3H]thymidine incorporation in male bones by 60% and female bones by 49% (Fig. 1A) and increased [3H]proline incorporation in males by 42% and females by 53% (Fig. 1B). There is no significant difference in these increases between male and female bones.

Effects of indomethacin on loading and sex hormone-related incorporations of [3H]thymidine and [3H]proline

Indomethacin (10−6 M) acting alone had no effect on basal [3H]thymidine or [3H]proline incorporation (data not shown). 17β-estradiol (10−8 M) increased [3H]thymidine and [3H]proline incorporation in both sexes but more in females than in males (Figs. 2A and 2B). Similarly 5α-dihydrotestosterone (10−8 M) increased [3H]thymidine and [3H]proline incorporations in both sexes but significantly more in males than in females (Figs. 2A and 2B). Indomethacin had no effect on the elevation of [3H]thymidine (Fig. 2A) or [3H]proline (Fig. 2B) incorporation due to E2 or DHT.

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Figure FIG. 2. Effects of mechanical loading (Load), 17β-estradiol (E2), 5β-dihydrotestosterone (DHT), and Indomethacin (Indo) on (A) [3H]thymidine and (B) [3H]proline incorporations. Results are from six pairs of ulnae and expressed as percentage increases of treated bones compared with controls. Their were no significant differences between the control of different groups. All values shown are significantly greater than their paired controls (p < 0.01) except loading in the presence of indomethacin (Load + Indo) (p > 0.5). Indo (10−6 M) does not influence the effects of E2 or DHT but abrogates the effects of mechanical loading and the additional or synergistic effects of loading in the presence of E2 or DHT. *p < 0.05 and **p < 0.01 indicate significant differences between male and female bones with same treatments.

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Loading increased [3H]thymidine (Fig. 2A) and [3H]proline (Fig. 2B) incorporation in both male and female bones to an equal extent. These increases, however, were abrogated by indomethacin when it was present during the loading and postloading periods.

In male bones, loading in the presence of E2 or DHT produced increases in [3H]thymidine (Fig. 2A) and [3H]proline (Fig. 2B) incorporations that approximated the arithmetic addition of loading and hormone acting individually in the absence of indomethacin. However, in female bones, loading in the presence of E2 produced increases in both [3H]thymidine (Fig. 2A) and [3H]proline (Fig. 2B) incorporations that were not only twice the level of those in males (p < 0.01), but also approximately 50% more than the addition of the two influences acting independently. DHT had no effect on loading response in females, producing an increase in [3H]thymidine incorporation only approximately equal to loading alone (Fig. 2A) and an increase in [3H]proline incorporation approximately equal to the addition of the two influences acting independently (Fig. 2B).

Indomethacin, when present during the loading and post-loading periods, eliminated not only the effect of loading, but also the enhancing effects of loading on both [3H]thymidine (Fig. 2B) and [3H]proline (Fig. 2B) incorporations in the presence of sex hormones.

Effects of sex hormones and prostaglandins in combination

Prostaglandin E2 and 17β-estradiol:

In males, [3H]thymidine incorporation (Fig. 3A) was increased by PGE2 in the presence of E2 by 65%, which was approximately the same as the arithmetic sum of the two agents acting independently. However, in females the increase was substantially higher than the arithmetic sum of the two influences separately (105%) and significantly (p < 0.01) higher than the increase in males (65%).

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Figure FIG. 3. Effects of 17β-estradiol (E2), 5α-dihydrotestosterone (DHT), prostaglandin E2 (PGE2), and prostacyclin (PGI2) in combination on (A) [3H]thymidine and (B) [3H]proline incorporations in male and female rat ulnae in culture. Results are from six pairs of ulnae and expressed as percentage increases of treated bones compared with controls. There were no significant differences between the control of different groups. All values shown are significantly greater than their paired controls (p < 0.01). *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences between male and female bones with the same treatment.

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For [3H]proline incorporation (Fig. 3B), the level of increases in both male and female bones by PGE2 in the presence of E2 were similar to the level of increase by either E2 or PGE2 alone and thus substantially less than the addition of the increases produced by them independently.

Prostacyclin and 17β-estradiol:

The increase in [3H]thymidine incorporation (Fig. 3A) in males, caused by PGI2 in the presence of E2, was 56%. This was approximately the same as PGI2 acting independently. In contrast, in females the increase with PGI2 and E2 together was 108%, which was higher than the arithmetic sum of PGI2 and E2 acting alone (85%) and significantly (p < 0.001) higher than the 56% increase in males.

The increases in [3H]proline incorporation (Fig. 3B), caused in both male and female bones by PGI2 in the presence of E2, were similar to the arithmetic sum of the two agents acting independently.

Prostaglandin E2 and 5α-dihydrotestosterone

Increases in [3H]thymidine incorporation (Fig. 3A) in both male and females by PGE2 in the presence of DHT were similar to the arithmetic sum of the two agents acting independently.

The increase in [3H]proline incorporation (Fig. 3B) in male bones caused by PGE2 in the presence of DHT was 105%. This was approximately the same as the arithmetic sum of the two agents acting independently and significantly (p < 0.01) higher than the increase in females, which at 52% was approximately the same level as that produced by PGE2 alone.

Prostacyclin and 5α-dihydrotestosterone

For [3H]thymidine incorporation (Fig. 3A) in both male and females, the increases by PGI2 in the presence of DHT were not substantially different from the arithmetic sum of the two agents acting independently.

For [3H]proline incorporation (Fig. 3B), the increase in male bones by PGI2 in the presence of DHT was 160%. This was substantially greater than the arithmetic sum of the two agents acting independently. It was also significantly (p < 0.001) higher than the increase in females, which was only 73% and similar to the arithmetic sum of the two agents acting independently.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

We have previously reported synergistic interaction13 between loading and estrogen in female rat bones. The results presented here extend those observations to the effects of loading and testosterone in males and to investigating the extent to which the effects of loading can be mimicked by the prostanoids shown to be produced by loading.

The results from straining bone explants in the absence of sex hormones shows that mechanical stimulation increases both [3H]thymidine and [3H]proline incorporation and that these early response to dynamic strain are similar in males and females. The effect of indomethacin confirms the findings of previous studies that this early response contains a prostanoid-dependent step.12,14,20 Previous work suggested that loading stimulates the production of at least two prostanoids (PGE2 and PGI2)14 and that when these are applied exogenously to bone explants at a concentration of 10−6 M that they increase G6PD activity to a similar extent as recent exposure to physiological strain.12 At this same concentration in this study, these prostanoids independently increase [3H]thymidine and [3H]proline incorporation to the same extent as loading, producing similar physiological levels of strain. These prostanoid-dependent responses are also quantitatively similar in males and females.

Estrogen and testosterone (10−8 M) also increased [3H]thymidine and [3H]proline incorporation in ulna explants. This response was unaffected by indomethacin and so appears not to require the production of prostanoids.

Although the effects of loading at the level used in this study, and exogenous prostanoids at 10−6 M, are similar in both sexes, their interaction with sex hormones is different. When the ulnar explants were exposed to loading, PGE2 or PGI2 in combination with sex hormones, the resulting incorporation was always greater than the effect of a single influence alone, except for the increases in [3H]thymidine incorporation stimulated by PGI2 in combination with E2 in males. Although in some cases the effects of prostanoid and hormone acting in combination were less than their arithmetic sum when acting individually, in others their effect in combination exceeded the sum of their independent effects.

In males E2 together with either PGE2 or PGI2 had an additive effect on [3H]thymidine incorporation. In females there was synergistic interaction with E2 and loading, E2 and PGE2, and E2 and PGI2. In both sexes, therefore, the effects of PGE2 or PGI2 with E2 were similar to those of loading with E2.

For [3H]proline incorporation only loading produced synergism with E2 in females, and PGI2 with DHT in males. PGE2 and E2 together had only additive effects in females but not males, whereas PGI2 and E2 together had additive effects in both sexes. That prostanoids do not mimic the effects of loading on matrix production may mean that in the estrogen-replete state, PGE2 and PGI2 are only partially responsible for bone's adaptive responses to mechanical loading.

PGE2 or PGI2 in the presence of DHT had only additive effects on cell proliferation in both male and female bones. This is similar to the effect of loading with DHT. By contrast, both PGE2 and PGI2 in the presence of DHT showed synergistic effects on matrix production in male bones which are greater than loading.

Synergism in bone mass and density induced by estrogen and mechanical loading has also been reported by Yeh et al.21 in ovariectomized rats in vivo. The involvement of prostaglandins in the cascade between mechanical loading and bone remodeling has also been suggested by a number of studies demonstrating both in vivo and in vitro that prostaglandins and their related enzymes, such as prostaglandin G/H synthases and cyclo-oxygenases, are stimulated by mechanical loading.22–27 The effects of prostaglandins on bone modeling and remodeling have also been investigated but these studies have usually involved PGE2 rather than PGI2. Early reports suggested that PGE2 was a powerful stimulator of bone resorption,28,29 but more recent studies support its role as a strong stimulator of bone formation in growing rats in vivo and in cultured bone cells in vitro.30–35 The effects of PGE2 on bone formation may be mediated by IGF-I,35–37 the transcription of which may be up-regulated by estrogen.37 Our current results are not entirely consistent with our previous reports14,22 that exogenous PGI2 not PGE2 stimulated IGF-II production. However, this report was from experiments in another preparation of another species and the IGF produced was not related to proliferation and matrix production which are the variables measured here.

Forwood38 recently reported that a specific cyclo-oxygenase inhibitor, NS 398, and indomethacin at various concentrations, could abrogate bone formation induced by four-point bending in rat tibiae in vivo. Pilbeam et al.39 have demonstrated that exogenous prostaglandins at micromolar concentrations can induce the enzyme prostaglandin G/H synthase which in turn may increase prostaglandin production in bone cells. These workers suggested that small and intermittent signals such as those that might arise from mechanical loading might act on bone formation through such a mechanism. Klein-Nulend et al.40 have also reported strain-related prostaglandin up-regulation in association with induction of prostaglandin G/H synthase mRNA at various time points following pulsating fluid flow in bone cells.

The distinction in our studies between additional and synergistic responses to loading, prostaglandins, and sex hormones should not be taken as absolute since we have not investigated the separate and combined effects of these influences at the full range of concentrations necessary to draw such distinctions. However, the fact that synergies between loading, prostaglandins, and sex hormones are demonstrated at any physiologically relevant concentration suggests that such interactions may exist in vivo.

This is consistent with the hypothesis that particularly in females estrogen not only has a direct effect on bone cells but also may play an important role in bones' early adaptive responses to load bearing.10,13,41,42 If the early responses in rat organ culture accurately reflect the long-term situation in humans in vivo, then premenopausal bone mass is the consequence of an adaptive response to load bearing in which estrogen amplifies the early stages of the bone's osteogenic response to loading. Under these circumstances, estrogen withdrawal could be expected to result in bone loss and a reduced ability to match bone mass and architecture appropriately to bone loading. We suggest that this may be the underlying lesion of postmenopausal osteoporosis.

In conclusion, loading explants of long bones from growing rats produces two prostanoids:prostaglandin E2 and prostacyclin. Loading that produces physiological strain levels and exogenous application of these prostanoids at a concentration of 10−6 both stimulate increased [3H] thymidine and [3H] proline incorporation to an equal extent in bones from males and females. The stimulatory effects of loading are abrogated by indomethacin, suggesting a prostanoid-dependent process. The sex hormones E2 and DHT have quantitatively similar effects on proliferation and matrix production. These effects were unaffected by indomethacin, suggesting that they act in a prostanoid-independent manner. The positive effects on [3H]proline and [3H]thymidine incorporation of loading or prostanoids, and the appropriate sex hormone, generally add to one another in males, but in some cases in females the combined responses are greater than the addition of the individual influences acting independently. The basis for the synergistic response between loading and estrogen in females in relation to [3H]thymidine incorporation may be accounted for quantitatively by the interaction between estrogen and the prostanoids PGE2 and PGI2 at the concentrations used in this study. The synergistic interaction between loading and estrogen in relation to [3H]proline incorporation appears to require factors other than PGE2 and PGI2.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

This work was supported by grants from the Medical Research Council, the Biotechnology and Biological Sciences Research Council, and the Wellcome Trust.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References
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