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Original Studies |
Bone Research Group (Medical Research Council) (B.S.N., A.T., J.R.), Cambridge University Department of Medicine, Addenbrooke’s Hospital, Cambridge, United Kingdom CB2 2QQ; and Department of Obstetrics and Gynecology (R.W.S.), University of Wales Medical College, Cardiff, Wales, United Kingdom
Address all correspondence and requests for reprints to: B. S. Noble, Bone Research Group (Medical Research Council), Cambridge University Department of Medicine, Addenbrooke’s Hospital, Cambridge, United Kingdom CB2 2QQ.
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Abstract |
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Transiliac biopsies were obtained from six premenopausal women, between 30–45 yr old, diagnosed as having endometriosis. Biopsies were taken before and after 24 weeks of GnRH-a therapy. Biopsies were snap-frozen and cryostat sectioned. Osteocyte viability, determined by the presence of lactate dehydrogenase (LDH) activity, was reduced in all but one subject after treatment. Furthermore, in every subject, the proportion of osteocytes showing evidence of DNA fragmentation typical of apoptosis increased, as demonstrated using in situ DNA nick translation (P = 0.008). Gel electrophoresis of extracted DNA and morphological studies of chromatin condensation and nuclear fragmentation confirmed that changes typical of apoptosis were affecting the osteocytes.
It was concluded that GnRH-a therapy caused a higher prevalence of dead osteocytes in iliac bone, probably caused by the increase in the observed proportion of osteocytes showing apoptotic changes. The capacity of bone to repair microdamage and to modulate the effects of mechanical strain is currently believed to be dependent on osteocyte viability. Our findings have therefore revealed a possible mechanism whereby estrogen deficiency could lead to increased bone fragility with or without an accompanying net bone loss.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Recent work has suggested a permissive role for estrogen in the normal response of bone cells to loading (6), and reports of a reduction in the response of bone to loading during estrogen deficiency (7) support this concept. Although osteocytes have been shown to have estrogen receptors (8), the effect of estrogen on osteocyte function is unknown. If osteocytes are indeed mechanosensors/transducers in the loading response, any reduction in the response to loading may, in principle, be caused by a reduction in individual osteocyte function or a reduction in the number of osteocytes available to undertake these functions. Osteocyte death has been shown to occur in bone and may, in part, be a function of age (9, 10) and disease (11). The mechanism by which osteocytes die is unknown, but we recently have reported that in some circumstances, human osteocytes die via a mechanism that is similar, if not identical, to apoptosis (12, 13). In addition, we have suggested that signals released by these apoptotic cells might play a role in the local control of bone resorption (13). Because of their inaccessibility associated with residence in a mineralized matrix, it seems likely that phagocytosis of osteocytes after apoptosis may be substantially delayed. If this were true, it would lead to persistence of apoptotic material in lacunae, resulting in a relatively high index of apoptosis in osteocytes, similar to that seen in atherosclerotic plaques (14).
Estrogen withdrawal results in bone loss, which is thought to be the product of an imbalance in the processes of bone formation and resorption whereby the relative activity of resorption is functionally higher than normal. In part, this may be caused by increased osteoclast survival, because estrogen promotes osteoclast apoptosis (15). Because osteocytes might play an important role in increasing local repair mechanisms, act as mechanosensors, and also contribute to the maintenance of the balance between bone formation and resorption, we have investigated the impact of estrogen loss on the viability of osteocytes in human bone. During the treatment of endometriosis, using GnRH analogues (GnRH-a), a hypoestrogenic state is produced that decreases the extent of the endometriosis but has previously been demonstrated to result in increased bone turnover and bone loss (16).
In this study, we have found substantial changes in osteocyte viability and the numbers of osteocytes with fragmented DNA, after treatment of subjects with GnRH-a, using an in situ nick translation assay. To ascertain the mechanism of osteocyte death, additional criteria that previously have been used to assign cell death to necrosis or apoptosis were studied. Agarose gel electrophoresis was used to identify DNA fragmentation into oligonucleosomal sized increments. Nuclear fragmentation and chromatin condensation are other morphological criteria for apoptosis, and these were investigated using light microscopy. Overall, our data suggest that estrogen withdrawal jeopardizes osteocyte viability by promoting apoptosis in osteocytes in human cancellous and cortical bone.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Transiliac biopsies were obtained from 6 premenopausal women, 30–45 yr old, with a diagnosis of endometriosis, attending the Obstetrics and Gynecology out-patients department at the Royal Free Hospital. These patients formed a subset of 13 subjects taking part in a study of the effect of estrogen withdrawal on bone turnover and structure who had biopsies before and after 6 months of treatment (17). The reduced numbers of subjects used in our study was the result of the use of substantial proportions of the original biopsies in other studies (18, 19), to the extent that in some cases, insufficient bone remained available for us to study. Each subject gave informed written consent to the study as directed by the hospital’s ethics committee. The patients had biopsies both before and after treatment with 24 weeks of GnRH-a therapy, comprising either Goserelin (3.6 mg sc every 4 weeks) or Triptorelin (3.75 mg im every 4 weeks). Three of the patients were treated with Tibolone (Livial, St. Louis, MO). Biopsies were snap-frozen in a hexane chilling bath before being stored at -70 C, were split, and half were fixed and later embedded in LR White medium resin (London Resin Co., London, UK) (17). Cryostat sections of 10-µm thickness were cut from the chilled material and were transferred to 3'-aminopropyltriethoxy-silane (APES) (Sigma)-coated slides (Poole, Dorset, UK). The two sections used per biopsy for each of the measurement criteria were spaced at least three sections apart to avoid sampling the same nucleus twice. Consecutive sections were used to relate two or more measurement criteria to each other.
In situ analysis of osteocyte DNA fragmentation using nick translation
The percentage of osteocytes demonstrating significant numbers of DNA breaks was determined using a DNA nick translation technique that was deliberately designed to be of moderate sensitivity, to increase specificity (13). Fragmented DNA was detected in cell nuclei in situ by this adaption of previously described methods (20, 21). Briefly, 10 µm-thick cryostat sections of bone were transferred to APES slides, fixed in 4% formaldehyde for 10 min, and air dried before demineralization in 0.25 mol/L ethylenediamine tetra-acetate (Sigma) in 50 mmol/L Tris HCl, pH 7.4, for 10 min. One group of sections was treated with deoxyribonuclease I (Sigma) (0.2 mg/mL PBS, Sigma) for 1 h (positive control) to produce breaks in the DNA. Sections were then treated with nick translation mixture, which consisted of 3 µmol/L digoxigenin (DIG)-labelled deoxyuridine 5'-triphosphate (DIG-11-dUTP); 3 µmol/L each of dGTP, dATP, and dCTP; 50 mmol/L Tris HCl, pH 7.5; 5 mmol/L MgCl2; and 0.1 mmol/L dithiothreitol [either with or without (negative control) 0.5 µL/100 µL Kornberg polymerase for 1 h at 37 C] (all reagents obtained from Boehringer Mannheim, Germany). Sections were washed in phosphate buffered saline (PBS) (Sigma) and incubated with FITC-labelled anti-DIG antibody, 5% normal sheep serum in PBS for 1 h at room temperature. After washing, sections were counterstained for nuclear DNA with propidium iodide (Sigma) for 3 min, washed thoroughly in water before being mounted in Citifluor mounting medium (Agar Scientific, Essex, UK), and visualized by fluorescence photomicrography.
Cell viability assessment in situ
Cells that were viable at the time of sampling were identified
in cryostat sections by means of their LDH activity. The number of
LDH-positive osteocytes (considered to be live osteocytes) were
expressed as a percentage of total osteocyte number. Histochemical
staining was undertaken using a modification of the methods of
Farquharson (22). Briefly, 10-µm tissue sections were reacted in 1.75
mg/mL disodium salt (
-nicotinamide adenine dinucleotide) (Boehringer
Mannheim), 60 mmol/L lactic acid, 3 mg/mL Nitroblue tetrazolium
(Sigma), and 40% Polypep (Sigma) to stabilize, pH 8.0, for 3 h at
37 C in a humidified chamber. After reaction, sections were rinsed in
warm water, fixed in 4% formaldehyde in PBS for 10 min, and then
mounted in Farrant’s mounting medium (Nustain, Nottingham, UK).
DNA gel electrophoresis
DNA was extracted from the bone component of 15 undecalcified cryostat sections (20 µm thick) of each iliac biopsy using a Nucleon DNA extraction kit (Scotlab, Glasgow, UK) (13). After extraction, DNA was precipitated in ethanol and pelleted. The ethanol supernatant was treated with 0.2 mol/L sodium acetate at -20 C overnight and then pelleted. Both pellets were solubilized in TBE and loading buffer before being run on a 1.5% agarose gel at 20 volts overnight (approximately 10 µg DNA was loaded per sample). A 100-bp standard was included as a reference. The gel was incubated in ethidium bromide before visualization with ultraviolet light and image capture (Eagle eye transilluminator, Stratagene, Cambridge, UK).
Nuclear morphology
Evidence of osteocyte chromatin condensation and nuclear fragmentation (which, when present, are suggestive of apoptosis) was investigated using both 0.1% methylene blue and 1% propidium iodide (Sigma).
Polycut LR White-embedded sections (7 µm thick) were stained in 0.5% methylene blue, 0.5% Azure II (Gurr, Poole, UK), and 1% borax (Sigma) for 5 sec before being thoroughly washed in distilled water. Sections were then air dried, mounted in DPX (Merck, Poole, UK), and visualized using light microscopy.
Polycut LR White (plastic)-embedded sections were decalcified in 0.25 mol/L ethylenediamine tetra-acetate (Sigma) in 50 mmol/L Tris HCl, pH 7.4, for 10 min before being stained in 1% propidium iodide for 30 sec, washed thoroughly in distilled water, air dried, and mounted in Citifluor. Nuclear DNA was then visualized under fluorescence microscopy to permit identification of nuclear fragmentation.
Quantification of viability and apoptotic criteria
In principle, each biopsy was assessed in its entirety. About 30% of sections showed regions of up to 10% by area that were affected by artifacts, particularly marrow smearing, and close proximity to ragged section edges, and these areas were avoided. Two nonadjacent sections from each biopsy were scored for the number of osteocyte lacunae, the number of live osteocytes (in situ LDH viability assay), and the number of apoptotic osteocytes (nick translation assay). In all cases, at least 500 osteocytes were assessed per section. These criteria were used to calculate the percentage of apoptotic osteocytes, the percentage of LDH negative osteocytes, lacuna density, and the percentage of lacunae-containing cells.
Biochemical markers of bone formation and resorption
Biochemical markers of bone formation and resorption were measured using a variety of techniques (as summarized in Refs. 17, 18, 23, and 24).
Statistical analysis
Student’s paired two-tailed t test was employed in all statistical analysis of the six subjects with paired biopsies. Results are reported as means ± SD; P < 0.05 was considered to be statistically significant. Chi-squared tests were used for individual subject results.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In this study, GnRH-a treatment rapidly reduced estrogen levels from premenopausal to postmenopausal values, after which time, the suppression was maintained for the period of treatment (17).
The data in this study are a subset of a larger study that included the effect on biochemical markers, and which also indicated that an increase in bone remodeling had occurred after estrogen withdrawal. The biochemical indicators of bone resorption, the ratios hydroxyproline:creatinine and fasting calcium:creatinine, seemed to increase after GnRH-a treatment (18). The indicators of bone formation (alkaline phosphatase and osteocalcin) also seemed to increase (18). There was no significant difference in any of these biochemical criteria, between subjects receiving GnRH-a alone or GnRH-a and Tibolone.
Osteocyte lacunar density
There was no significant difference in the overall density of
osteocyte lacunae (P = 0.34) between the pre- and
posttreatment biopsies (Fig. 1
). There
was no significant difference in these measurements between
Tibolone-plus-GnRH patients and GnRH-alone patients in either
pretreatments (P = 0.58) or posttreatment biopsies
(P = 0.93). When bone was categorized as either
cancellous or cortical, regardless of treatment, no significant
difference was found in lacunar density between the bone types
(P = 0.31).
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In paired samples, the percentage of viable osteocytes
(i.e. demonstrating LDH activity) was reduced after
treatment, relative to their pretreatment biopsies, in all but one
subject (mean reduction, including all subjects 8.6%; mean, excluding
uncharacteristic subject, 13%). (Fig. 2). The inclusion of data from the
individual in which osteocyte viability had increased negated the
attainment of statistical significance. On an individual basis, the
change in osteocyte viability was significant (P <
0.05) in four of the five subjects showing a reduction (Fig. 2). By
preliminary visual assessment, nonviable osteocytes did not appear in
large homogeneous areas but could be found in close proximity to viable
cells (Fig. 3). In addition, viable and
nonviable osteocytes were not separately grouped or separated by any
demarcation or cement line, as is typically seen in necrotic bone (9).
The addition of Tibolone treatment did not make a significant
difference in the proportion of nonviable osteocytes (P
= 0.32).
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The percentage of osteocytes (propidium iodide-positive lacunae)
displaying DNA breaks, using the in situ nick translation
technique, was significantly increased after GnRH treatment (Fig. 4a), with a mean increase of 374.9%
(P = 0.008). This increase was seen in all individuals
with paired biopsies (Fig. 4b). This increase persisted when the
subjects were grouped according to Tibolone treatment, and there was no
significant difference in the proportion of osteocytes with DNA breaks
between treatments, within either pre- (P = 0.78) or
posttreatment groups (P = 0.54). Also, there was no
significant difference in the percentage of osteocytes displaying DNA
breaks between cortical and cancellous bone (P = 0.8).
The osteocytes displaying DNA breaks were not seen to be clustered,
were not preferentially found in osteoid, and were typically seen in
close proximity to viable osteocytes negative for DNA breaks (Fig. 5).
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Samples of DNA, extracted from cryostat sections of bone obtained
from two different biopsies taken after treatment, displayed
oligonucleosomal sized DNA fragments characteristic of apoptotic cells
(lanes 2 and 3), whereas DNA from a pretreatment sample was intact
(lane 1) (Fig. 6). DNA smears
characteristic of necrosis were seen in no sample examined in this
study.
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Osteocyte nuclear chromatin condensation and nuclear blebbing were
observed in a number of cells in each posttreatment biopsy, when the
methylene blue-stained samples were examined (Fig. 7). In addition, when nuclear DNA was
stained, using propidium iodide, a number of osteocytes in the post
treatment samples showed evidence of nuclear fragmentation, forming
DNA-containing blebs in the cytoplasm, which were found also at the
periphery of the cell and in the osteocyte lacunae itself (Fig. 8, a and b). This morphology was far less
common in the pretreatment samples. The majority of osteocytes in the
samples displayed normal morphology with a smooth nuclear profile (Fig. 8c).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In the present study, the proportion of viable osteocytes in iliac bone has been studied. By comparison with published data on the femoral head, osteocyte viability in our pretreatment group was normal for the age group studied. However, after GnRH agonist treatment, this proportion was reduced and seemed more similar to previous estimates of the proportion of nonviable osteocytes seen in elderly women (9, 25). In the present study, the observed reduction in viable osteocyte numbers was noted after only 6 months of estrogen withdrawal. The clinical significance of nonviability of osteocytes is currently unknown. However, it has been argued that it may increase bone brittleness or vulnerability of microfractures propagating into large-scale fractures. Thus, our findings might have important implications for the clinical management of women with hypoestrogenic states. It also could contribute to our understanding of why some women become vulnerable to insufficiency fractures quite soon after a natural menopause, before they have had much opportunity to lose large amounts of bone.
The nonviable osteocytes seen in this study, which have been newly acquired in association with estrogen suppression, are the result of either necrosis or apoptosis, and several criteria point to the apoptotic route. If the distribution of empty lacunae is considered, it is apparent that they are not clustered in large homogeneous areas reported to be typical in cases of avascular necrosis and osteoarthritis, in which dead bone often is separated from live bone by cement lines (11). Using DNA ladders as a marker of apoptosis (28, 29, 30), we saw evidence for apoptosis in bone from post-GnRH-a-treated patients, whereas in pretreatment samples, there was none. The presence of DNA ladders indicates that a significant amount of apoptosis is occurring but does not identify the cells responsible. To clarify which cells were affected, we used in situ nick translation. When this was done, the osteocytes were the only cell type showing significantly increased numbers with DNA fragmentation in situ post treatment, suggesting that they may be contributing substantially to the appearance of the characteristic DNA ladders in posttreatment samples.
Nick translation methods do not identify specifically oligonucleosomal fragmentation, but the larger number of DNA breaks associated with apoptosis, relative to necrosis, provides the method with some specificity (31, 32). In our system, we deliberately reduced the polymerase concentration used in previous studies, to decrease the sensitivity of the technique, such that it only stained known apoptotic cells in the hypertrophic region of the human growth plate (13, 33). Cells showing in situ evidence of DNA fragmentation were found intermingled with cells showing no fragmentation, and this distribution was reminiscent of the pattern of distribution of empty lacunae observed in the same samples. This pattern is dissimilar to that seen for dead osteocytes in avascular necrosis (11). Because we saw no evidence of osteocytes in osteoid being preferentially labelled for DNA fragmentation, it would seem unlikely that the apoptotic osteocytes seen were originally apoptotic osteoblasts that have become enclosed in matrix.
The morphological changes associated with apoptosis are variable but include cell shrinkage, chromatin condensation, nuclear fragmentation, and cell blebbing with production of apoptotic bodies (29). One technical limitation of our study was that it was not possible to observe osteocyte morphology at the EM level because the original biopsy material was either frozen or plastic embedded. However, using light microscopy, we did observe chromatin condensation and nuclear fragmentation in some cells in the posttreatment samples consistent with apoptosis.
The group of patients we studied did not seem to be atypical, compared with women studied previously pre- and post GnRH-a treatment, with respect to bone loss and bone turnover (16, 17). Despite the small numbers of individuals included in the present study, caused by ethical and technical constraints, some biochemical markers of both bone formation and resorption increased after treatment. The simultaneous treatment with GnRH-a plus Tibolone showed a small, but statistically insignificant, reduction in bone turnover, compared with the group treated with GnRH-a alone. The addition of Tibolone, along with GnRH-a, did not affect the reduction in osteocyte viability and the increase in the percentage of osteocytes with fragmented DNA. Tibolone is a weakly estrogenic and progestrogenic molecule. It interacts poorly with the estrogen receptor (1.3% relative to estrogen) and slightly better with the progesterone receptor, although some of its metabolites are a little better at estrogen receptor interaction (3–4% of estrogen) (34). Its lack of protective effect against estrogen loss in this study could be related to its poor estrogen receptor interaction or might imply that estrogen’s role in osteocyte viability involves a nongenomic pathway incompatible with the structure of the synthetic molecule. These possibilities would seem to be substantiated by our preliminary investigations using an ovariectomized rat model, in which we have found that estrogen add-back therapy prevented the apoptotic changes in osteocytes (35).
Histomorphometric analysis, carried out by Compston (17) on the larger group of subjects recruited to this study, showed raised indices of bone turnover in the cancellous bone of the ileum associated with treatment. A consequence of increased remodeling is that the accumulation of nonviable osteocytes in bone is likely to be partly offset by replacement of old bone by new. In pediatric calvarial bone, which undergoes high levels of modeling activity associated with skull expansion, empty lacunae rarely are seen, despite the presence of significant numbers of apoptotic osteocytes, presumably because the bone is replaced every few days (13). In the present study, the empty lacunae found in adult bone might have resulted from a number of different processes. Osteocytes packaged as apoptotic bodies, in principle, might have been available for ingestion by neighboring osteocytes via the canaliculi. Alternatively, apoptotic cells that have not been phagocytosed during bone resorption might undergo a secondary necrosis, with resulting dissolution of cellular material. It also is possible that osteocytes that undergo apoptosis and fragment into apoptotic bodies might remain in their lacunae in the fragmented state for extended periods of time. This latter possibility might account for the relatively high percentage of cells staining positive for DNA fragmentation after GnRH treatment. Consistent with this hypothesis are previous studies reporting dead osteocytes persisting for 16 weeks in lacunae (36) and the occurrence of so-called pearls of mineralization morphologically similar to fragmented osteocytes thought eventually to lead to micropetrosis (37). In addition, recent work on atherosclerotic lesions has proposed a persistence of apoptotic cellular material as an explanation of the high apoptotic index in this tissue (14). Thus, the time course of apoptosis in osteocytes is unknown but might be considerably longer than the 1–3 days taken for complete removal of apoptotic cells in other nonmatrix-bound cell types in vivo (38, 20).
The present study is not capable of determining whether the reduction in estrogen levels associated with GnRH-a therapy directly or indirectly causes the increase in indices of osteocyte apoptosis and death. When the increased bone loss, brought about by either GnRH-a treatment or ovariectomy, have been compared in man (39), the response was shown to be quantitatively the same; and the GnRH-a changes were considered to be caused by the estrogen loss, rather than the changes in pituitary hormones (such as FSH and LH) that accompany GnRH-a therapy. It has been shown that a proportion of osteocytes, quantitatively similar to the proportion of apoptotic cells seen in this study, have estrogen receptors (8). Estrogen status seems to modify the adaptive loading response thought to be controlled by the osteocyte (6) and, therefore, estrogens may play a physiological role in altering osteocyte function in some way. Nevertheless, the present study cannot rule out a response in the osteocytes related to some other aspect of GnRH-a treatment.
In conclusion, we have shown that suppression of endogenous estrogen by GnRH-a increases the proportion of nonviable osteocytes in human iliac bone after 24 weeks of therapy. The mechanism by which the cells die is similar, in many respects, to classical apoptosis and results in ratios of dead-to-live osteocytes similar to those reported previously in studies of much older postmenopausal women. The clinical implications of the reduction in live osteocytes is unclear, but current concepts of osteocyte function suggest that it might lead to bone fragility and impairment of the adaptive response to loading. Additionally, the possibility that bone tissue containing a high proportion of apoptotic osteocytes is preferentially resorbed has been suggested in other work on pediatric calvaria (12, 13). In the present study, the observation that increased bone remodeling is associated with an increase in the proportion of apoptotic osteocytes is consistent with this hypothesis and has led us to consider the question of whether apoptosis lies on the causal pathway that increases bone resorption. However, based on current data, the association of apoptosis with resorption could be explained equally by the act of resorption killing osteocytes (13). Future studies should address this question and clarify the potential signaling role of apoptotic osteocytes.
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Acknowledgments |
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Footnotes |
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2 Recipient of an MRC studentship.
Received December 9, 1996.
Revised May 15, 1997.
Accepted May 28, 1997.
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References |
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 J. E. Compston Sex Steroids and Bone Physiol Rev, January 1, 2001; 81(1): 419 - 447. [Abstract] [Full Text] [PDF]
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S. C. Manolagas Birth and Death of Bone Cells: Basic Regulatory Mechanisms and Implications for the Pathogenesis and Treatment of Osteoporosis Endocr. Rev., April 1, 2000; 21(2): 115 - 137. [Abstract] [Full Text]
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J. S. Dodd, J. A. Raleigh, and T. S. Gross Osteocyte hypoxia: a novel mechanotransduction pathway Am J Physiol Cell Physiol, September 1, 1999; 277(3): C598 - C602. [Abstract] [Full Text] [PDF]
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 S. B. Wade, P. Oommen, W. C. Conner, D. J. Earnest, and R. C. Miranda Overlapping and Divergent Actions of Estrogen and the Neurotrophins on Cell Fate and p53-Dependent Signal Transduction in Conditionally Immortalized Cerebral Cortical Neuroblasts J. Neurosci., August 15, 1999; 19(16): 6994 - 7006. [Abstract] [Full Text] [PDF]
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 S. R. Cummings, W. S. Browner, D. Bauer, K. Stone, K. Ensrud, S. Jamal, B. Ettinger, and The Study of Osteoporotic Fractures Research Group Endogenous Hormones and the Risk of Hip and Vertebral Fractures among Older Women N. Engl. J. Med., September 10, 1998; 339(11): 733 - 738. [Abstract] [Full Text] [PDF]
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R. Marcus M.D. Editorial: New Perspectives on the Skeletal Role of Estrogen J. Clin. Endocrinol. Metab., July 1, 1998; 83(7): 2236 - 2238. [Full Text]
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