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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 8 2907-2912
Copyright © 2000 by The Endocrine Society

Original Studies

Apoptosis of Osteocytes in Glucocorticoid-Induced Osteonecrosis of the Hip1

Robert S. Weinstein, Richard W. Nicholas and Stavros C. Manolagas

Division of Endocrinology and Metabolism, Center for Osteoporosis and Metabolic Bone Diseases (R.S.W., S.C.M.), Departments of Internal Medicine and Orthopedics (R.W.N.), and Central Arkansas Veterans Healthcare System, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205

Address all correspondence and requests for reprints to: Robert S. Weinstein, M.D., Division of Endocrinology and Metabolism, University of Arkansas for Medical Sciences, Slot 587, 4301 West Markham Street, Little Rock, Arkansas 72205-7199. E-mail: weinsteinroberts{at}exchange.uams.edu


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
An increase in osteoblast and osteocyte apoptosis has been demonstrated in mice and humans receiving glucocorticoids and may be involved in the pathogenesis of the associated osteonecrosis. To examine the spatial relationship between osteocyte apoptosis and glucocorticoid-induced osteonecrosis, we determined the prevalence of osteocyte apoptosis in whole femoral heads obtained from patients who underwent prosthetic hip replacement because of osteonecrosis due to chronic glucocorticoid treatment (n = 5), alcoholism (n = 3), and trauma (n = 1) as well as in femoral neck cores from patients with sickle cell disease (n = 5). Abundant apoptotic osteocytes and cells lining cancellous bone were found juxtaposed to the subchondral fracture crescent in femurs from the patients with glucocorticoid excess. In contrast, apoptotic bone cells were absent from the specimens taken from patients with trauma or sickle cell disease and were rare with alcohol abuse. These results indicate that glucocorticoid-induced osteonecrosis is a misnomer. The bone is not necrotic; instead, it shows prominent apoptosis of cancellous lining cells and osteocytes. Glucocorticoid-induced osteocyte apoptosis, a cumulative and irreparable defect, could uniquely disrupt the mechanosensory function of the osteocyte network and thus start the inexorable sequence of events leading to collapse of the femoral head.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
OSTEONECROSIS, the in situ death of a segment of bone, is estimated to develop in over 25% of patients receiving glucocorticoid therapy (1). Although collapse of the femoral head and severe osteoporosis were observed early after the advent of glucocorticoid therapy (2), subsequent efforts to reduce the dose or discontinue administration of the drugs have generally been unsuccessful because of the necessity for glucocorticoids in transplantation (3) and in treatment of pulmonary, rheumatological, autoimmune, hemopoietic, and gastrointestinal diseases. Consequently, an increasing number of patients receive glucocorticoids for months, years, or a lifetime. The risk of osteonecrosis increases with the both the dose and duration of treatment (1, 4, 5). Most commonly affected is the femoral head, often showing bilateral involvement. Despite the increased awareness of this devastating complication, the mechanism of the in situ death of portions of bone and the impending collapse of the joint remains obscure. Currently, efforts to treat the disorder have little effect, and the clinical course is usually progressive, eventually requiring total hip replacement.

The name osteonecrosis (also known as aseptic, avascular, or ischemic necrosis) may be misleading, as it has not been demonstrated that the bone cells die by necrosis. Indeed, the cell swelling and inflammatory responses that characterize necrosis in soft tissues usually do not occur (1, 6). Glucocorticoid-induced osteonecrosis has been attributed to fat emboli, microvascular tamponade of the blood vessels of the femoral head by marrow fat or fluid retention, and poorly mending fatigue fractures (1).

Another possibility, however, is that programmed cell death or apoptosis may be part of the mechanism of the osteonecrosis. We have recently reported that mice receiving glucocorticoids for 4 weeks exhibit a 3-fold increase in the prevalence of osteoblast apoptosis in murine vertebral cancellous bone and show apoptosis in 28% of the osteocytes in metaphyseal cortical bone (7). An increase in osteoblast and osteocyte apoptosis was also documented in patients with glucocorticoid-induced osteoporosis (7).

Here we report the identification of abundant apoptotic osteocytes in sections of whole femoral heads obtained during total hip replacement for glucocorticoid-induced osteonecrosis, whereas apoptotic bone cells were absent from femoral specimens removed because of traumatic or sickle cell osteonecrosis, suggesting that the so-called glucocorticoid-induced osteonecrosis may actually be osteocyte apoptosis.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Patients

Femoral heads were obtained from the Departments of Surgical Pathology and Orthopedics at the University of Arkansas for Medical Sciences in compliance with the guidelines of the human research assurance review committee. The specimens were removed during total hip replacement for osteonecrosis of the femoral head in 1 patient with femoral neck fracture and rupture of ligamentum teres, 5 patients receiving high dose glucocorticoid treatment, and 3 with chronic alcoholism. Femoral neck specimens (3–4 mm diameter, 4- to 5-cm long biopsies) were also obtained from 5 patients with sickle cell disease who underwent core decompression for osteonecrosis at the Medical College of Georgia (Augusta, GA). Informed consent was obtained from patients in compliance with the human research committee of Medical College of Georgia. Three of these patients had prior collapse of the opposite femoral head, and 1 had collapse of the contralateral femoral head at annual follow-up. Osteonecrosis was confirmed by radiography, bone scanning, computed tomography, and magnetic resonance imaging in all cases (8). A band saw was used to cut the fresh whole femoral heads into 5-mm slices, which were then fixed in 10% formaldehyde for 4–7 days before gentle decalcification in 5% formic acid for 2–3 weeks (9). Formic acid is used in antigen retrieval procedures and may enhance staining characteristics (10). Decalcification was monitored with daily chemical testing using ammonium oxalate to avoid under- or overdecalcification (overdecalcification can remove proteins from the bone matrix) (9). Specimens were cleared in methyl salicylate, rather than in xylene, to reduce brittleness and further enhance staining. The decalcified femoral head specimens were then embedded in polymer-augmented paraffin at 65–67 C at 15–18 psi vacuum (9). Sections were taken from multiple levels throughout the block and stained with hematoxylin and eosin. Normal iliac crest bone was obtained from 25 volunteers (11). Transiliac bone biopsy specimens taken from 2 patients with glucocorticoid-induced osteoporosis (22 and 36 yr old, receiving 15–25 mg/day prednisone for 3–6 yr) were used as positive controls (7). The specimens from the normal volunteers and the patients with glucocorticoid-induced osteoporosis or sickle cells disease were taken after they had received 2 courses of oral tetracycline 23, 22, 21, 6, 5, and 4 days before biopsy. Iliac crest biopsies and femoral head core specimens were fixed for 24 h in 4 C Millonig’s phosphate-buffered 10% formalin, pH 7.4, embedded undecalcified in methyl methacrylate, and stained as previously described (11). For each specimen, sections were examined at x400 magnification in a minimum of 20 fields, selected from the area adjacent to the subchondral fracture crescent and moving distally. An apoptotic index was made by marking the presence of predominantly stained cells as 3+, abundant staining as 2+, rare staining as 1+, and the absence of staining as 0. Each slide was encoded so that the histomorphometric technician was blinded to the identity and diagnosis of the specimens.

DNA nick end labeling of bone sections

Sections were mounted on silane-coated glass slides (Scientific Device Laboratory, Inc., Des Plains, IL) and incubated in 10 mmol/L citrate buffer, pH 6.0, in a microwave oven at 98 C for 5 min. Slides were then placed in 0.5% pepsin in 0.1 N HCl for 20 min at 37 C, rinsed with Tris-buffered saline, reincubated in 30% H2O2 in methanol for 5 min, and rinsed again. DNA fragmentation was detected by the TUNEL reaction (transferase-mediated digoxigenin-deoxy-UTP nick end labeling) using Klenow terminal deoxynucleotidyl transferase (Oncogene Research Products, Cambridge, MA) in sections counterstained with 3% methyl green. This system allows for sensitive and specific staining of the high concentrations of 3'-OH ends that accumulate with DNA fragmentation due to apoptosis (7, 12). To further improve the sensitivity of the reaction, sections were subsequently incubated for 1–2 min with 0.15% CuSO4 in 0.9% NaCl (12). The TUNEL reaction was noted within cell nuclei, and the cells whose nuclei were clearly dark brown from the peroxidase-antidigoxigenin antibody conjugate instead of blue-green from the methyl green were interpreted as positive. With every set of TUNEL slides, sections of rat mammary tissue, taken 4–6 days after weaning, and sections from two patients with glucocorticoid-induced osteoporosis were used as positive controls. Cancellous bone samples from normal volunteers were used as negative controls (11). In these transcortical iliac biopsies, occasional marrow cell apoptosis was noted, but apoptotic bone cells were absent (7). Additional negative controls were made by omitting the transferase. Morphological changes characteristic of apoptosis were examined carefully to minimize ambiguity regarding the interpretation of results. With these precautions, TUNEL has been unequivocally associated with apoptosis (7, 12). In addition, TUNEL has been used with DNA fragmentation and immunohistochemical studies to demonstrate apoptosis of osteoblastic cells and osteoblasts both in vitro and in vivo (7, 12, 13).

Hoechst staining

To highlight the characteristic morphological changes of apoptosis in the nucleus of bone cells in histological sections of femoral biopsy specimens (14), sections were deplasticized, hydrated in distilled water, and placed in Tris-buffered saline, pH 7.6, for 5 min. Excess buffer was wiped from the sections, and 100 µL of a 50 ng/mL solution of the DNA-specific bisbenzimide dye Hoechst 33258 (Molecular Probes, Inc., Eugene, OR) was placed on the 25 x 25-mm sections and incubated in a dark humidifying chamber at room temperature for 2 min. The sections were then washed twice in distilled water for 3 min each time, dried, and mounted with Crystal Mount (Fisher Scientific, Pittsburgh, PA). The paraffin-embedded femoral heads were deparaffinized and then treated identically to the plastic sections, except that 200–300 µL of a 50 ng/mL solution of Hoechst 33258 was applied to these 50 x 55-mm sections.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
The cases of osteonecrosis of the femoral head were seen between 1991 and 1998. Of these, six were men and eight were women; their ages at diagnosis ranged from 20–70 yr (mean, 40 yr). Prednisone was the most common glucocorticoid used, and dosage ranged from 15–80 mg/day administered for 1–8 yr. The total dosage administered and the time interval between glucocorticoid therapy and the onset of hip pain were impossible to determine with any degree of accuracy. The patients were seen and specimens taken before complete collapse and fragmentation of the joint. Clinical characteristics and findings are given in (Table 1Go).


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  		Table 1. Clinical characteristics and findings in the patients with osteonecrosis

 
Using the TUNEL procedure, apoptotic osteocytes and cells lining cancellous bone were plentiful in the proximal femoral heads resected from patients with glucocorticoid-induced osteonecrosis, whereas apoptotic bone cells were absent from specimens removed because of trauma or sickle cell disease and were rare in alcohol-induced femoral necrosis (Fig. 1Go). Apoptotic osteocytes were also easily recognized by the intense blue fluorescence of the Hoechst dye in condensed nuclei (Figs. 2Go and 3Go). The apoptotic cells were more frequently recorded adjacent to the subchondral fracture crescent (Fig. 4Go), whereas empty osteocytic lacunae, the cardinal sign of bone necrosis (1, 15, 16), were infrequent. Moreover, the presence of apoptotic cells was associated with reduced cancellous bone area, increased marrow adipocytes, and decreased hemopoietic marrow in the patients with glucocorticoid-induced osteonecrosis. In these patients, signs of inflammation and necrosis, such as hyperemia, round cell infiltration, or lipid cyst formation, were absent.



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  		Figure 1. Evidence of osteocyte apoptosis in glucocorticoid-induced osteonecrosis. Sections of whole femoral heads obtained during total hip replacement. A–F are stained with hematoxylin and eosin. G–I are stained by TUNEL. Magnification: A–C, x1; D–F, x2.5; G–I, x400; inset, x630. Apoptotic osteocytes (arrow) were rare in osteonecrosis due to alcohol abuse (B, E, and H) and were absent in traumatic osteonecrosis (C, F, and I). Extensive apoptosis (arrows) was seen in femoral head osteocytes from a patient with glucocorticoid-induced osteonecrosis (A, D, and G). Apoptosis usually occurred focally, with groups of apoptotic osteocytes in close proximity to normal cells. Apoptotic osteocytes had condensed nuclei and fragmented chromatin (inset, arrowheads).

 


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  		Figure 2. After staining with Hoechst dye, condensed apoptotic osteocytes in cancellous bone from a femoral head removed from a patient with glucocorticoid-induced osteonecrosis show intense blue fluorescence (A; excitor, 365 nm; beam splitter, 395 nm; emission, 397 nm), whereas uniformly separated unstained osteocytes are barely visible (B is same section as in A viewed with Nomarski differential interference contrast microscopy). Original magnification, x200.

 


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  		Figure 3. The devastating skeletal impact of glucocorticoids is shown by the intensely luminescent, apoptotic osteocytes (arrows) recently buried in a new packet of bone (outlined by the yellow tetracycline markers). This section was taken from a transcortical iliac biopsy from a patient with glucocorticoid-induced osteoporosis and is stained with Hoechst dye. Original magnification, x400.

 


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  		Figure 4. Schematic diagram of the femoral head illustrated in Fig. 1AGo.

 
In the core biopsies taken from the patients with sickle cell disease and femoral osteonecrosis, hemopoietic tissue was replaced by granular reticular necrosis, oil cysts, and foam cells. Marrow fibrosis was noted in two specimens. In cancellous bone tissue, extensive crush damage, trabecular fragmentation, and hemorrhage were present. Osteocytic lacunae were often empty. Four of the five specimens had lamellar osteoid (mean ± SD, 11.8 ± 4.4 µm; normal, 7.6 ± 1.9) and widely separated double tetracycline labels. The mean rate of mineral apposition was accelerated (0.98 ± 0.39 µm/day; normal, 0.63 ± 0.21). TUNEL-positive cells were absent.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In the current study we have demonstrated that glucocorticoid-induced osteonecrosis is characterized by osteocyte and lining cell apoptosis, a defect that could interrupt the mechanosensory network of the femoral head and thus promote joint collapse. Therefore, we suggest that glucocorticoid-induced osteonecrosis is a misnomer; the disease is glucocorticoid-induced osteocyte apoptosis.

Osteonecrosis can result from a variety of causes, including femoral neck fracture, high dose glucocorticoid treatment, alcohol abuse, gout,2 sickle cell disease, Gaucher’s disease, Caisson disease, osteochondritis, dislocation of the hip, systemic lupus erythematosus and other systemic vasculitides (see Footnote 1), Hodgkin’s disease, dyskeratosis congenita, irradiation, and myxedema (1, 18, 19, 20, 21, 22). However, the cellular mechanisms responsible for the relentless progressive deterioration and eventual collapse of the joint remain unknown. Our previous observations of apoptotic osteoblasts and osteocytes in patients with osteoporosis due to long-term glucocorticoid treatment (7) prompted us to search for evidence of apoptosis as a possible mechanism underlying the progressive destruction of the femoral head in glucocorticoid-induced osteonecrosis. In contrast to osteonecrosis due to femoral neck fracture, ethanol abuse, and sickle cell disease, whole femoral head sections taken from patients with glucocorticoid-induced osteonecrosis showed that the cancellous osteocytes and cells lining cancellous bone were predominantly apoptotic.

By analogy with the hemopoietic system, glucocorticoid-induced apoptosis of the cells of the osteoblast lineage could be expected. Both the plasma cell and osteoblast have highly prominent perinuclear clear zones representing their hypertrophied Golgi apparatus, and both cells are exquisitely sensitive to glucocorticoid excess. The role of apoptosis in pathological conditions is increasingly being recognized. Recently, apoptosis was reported as a mechanism in heart failure, polycythemia vera, and polycystic kidney disease (14, 23, 24).

Apoptosis is quite different from necrotic cell death. As the functional opposite of mitosis, apoptosis is required to regulate cell numbers in the maintenance of adult tissues such as corneal epithelium, intestinal mucosa, epidermis, blood, and bone (25). Cells dying by apoptosis display marked nuclear condensation, chromatin contraction, volume shrinkage, and activation of an endonuclease that cleaves DNA into oligonucleosomes of 180–200 bp or multiples thereof. A crucial characteristic of apoptosis is that it can be prevented in a tissue-specific way (26). Therefore, defining the events that regulate this death program presents a major challenge. There has been concern about the pitfalls of TUNEL labeling and the need to optimize staining protocols for each tissue and fixation technique (27, 28). In this study, TUNEL was optimized for bone sections, and positive labeling was verified with the presence of typical signs of apoptosis: marginated masses of chromatin, nuclear condensation, and shrinkage, as seen with the Hoechst DNA-specific bisbenzimide dye. Moreover, the absence of an inflammatory infiltrate in glucocorticoid-induced osteonecrosis argues against necrosis and for apoptosis. Furthermore, TUNEL staining was absent in osteonecrosis due to trauma or sickle cell disease.

Most of the previous work on the histopathology of osteonecrosis of the proximal femur has been focused on advanced cases in which collapse and severe deformity of the femoral head had already occurred (6, 15, 16, 17, 29, 30). To complicate matters further, prior work often combined all of the causes of osteonecrosis together in one group, used only core biopsies with their limited volume or archival specimens stored for years in formalin, and was plagued by tissue damage due to the biopsy or surgical procedures and artifacts from specimen preparation (16, 30). In some studies, 1-cm-thick slabs of femoral head tissue were cut and fixed for several weeks before unmonitored demineralization with strong nitric acid or ethylenediamine tetraacetate and clearing in xylene (17, 29, 30), procedures that generate tissue artifacts and promote brittleness (9). With this material, empty osteocytic lacunae were thought to be the cardinal sign of bone necrosis (1, 16, 30), but a far more likely explanation is that the pyknotic, apoptotic osteocytes were lost during tissue processing (15). Histological evidence of early osteonecrosis has been reported less than 3 months after the administration of high dose glucocorticoid treatment (29). Rapid removal of the damaged osteocytes is, however, unlikely because of their anatomical isolation from scavenger cells and unique unavailability for phagocytosis and the need for extensive degradation to small molecules to dispose of the cells through the narrow canalicular system. Therefore, the process would be prolonged and affected osteocytes would accumulate, a set of conditions that may contribute to osteonecrosis.

Although the osteocyte is the most abundant bone cell, its function in bone metabolism remains unclear. Osteocytes descend from osteoblasts and remain connected by gap junctions after they are incorporated into the bone matrix. Glucocorticoid-induced osteocyte apoptosis could disrupt the proposed mechanosensory role of these cells and thus prevent functional adaptation of bone (Fig. 5Go) (31). Microdamage would then be unrepaired because of lack of detection by the osteocytes, an idea first proposed by Frost in 1964 (17).



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  		Figure 5. Chronic glucocorticoid therapy caused the accumulation of markedly pyknotic, apoptotic osteocytes and lining cells (dark brown). Note that the osteocyte-canaliculi-lining cell network (purple) now links dead cells. Two intact osteocytes are shown (blue). TUNEL and a toluidine blue counterstain were used. Original magnification, x250.

 
In conclusion, glucocorticoid-induced osteonecrosis may actually be osteocyte apoptosis, a cumulative and irreparable defect that would uniquely disrupt the mechanosensory role of the osteocyte-canalicular network and thus lead to the inexorable sequence of events resulting in collapse of the femoral head. Glucocorticoid-induced osteocyte apoptosis would explain the correlation between total steroid dose and the incidence of osteonecrosis and its occurrence after glucocorticoid administration has ceased (1, 4, 5).


   Acknowledgments
 
We are indebted to Drs. Aubrey J. Hough, Jr., Carl L. Nelson, and A. Michael Parfitt for helpful discussions; and to Frances Swain, Julie Crawford, Tony Chambers, and Robert Skinner for excellent technical assistance.


   Footnotes
 
1 This work was supported by the NIH (PO1-AG13918 and RO1-AR46191) and a Research Enhancement Award Program (REAP) Grant and Merit Review Grants (to R.S.W. and S.C.M.) from the V.A. Back

2 In these conditions, osteonecrosis appears to occur only after a period of excessive glucocorticoid administration (17 ). Back

Received November 10, 1999.

Revised February 25, 2000.

Accepted March 6, 2000.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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  10. Kitamoto T, Ogomori K, Tateishi J, Prusiner SB. 1987 Formic acid pretreatment enhances immunostaining of cerebral and systemic amyloids. Lab Invest. 57:230–236.[Medline]
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  14. Woo D. 1995 Apoptosis and loss of renal tissue in polycystic kidney diseases. N Engl J Med. 333:18–25.[Abstract/Free Full Text]
  15. Wong SYP, Evans RA, Needs C, Dunstan CR, Hills E, Garvan J. 1987 The pathogenesis of osteoarthritis of the hip. Clin Orthop Rel Res. 214:305–312.
  16. Sweet DE, Madewell JE. 1988 Pathogenesis of osteonecrosis. In: Resnick D, Niwayama G, eds. Diagnosis of bone and joint disorders, 2nd Ed. Philadelphia: Saunders, Harcourt Brace; 3188–3237.
  17. Glimcher MJ, Kenzora JE. 1979 The biology of osteonecrosis of the human femoral head and its clinical implications. I. Tissue biology; II. The pathological changes in the femoral head as an organ and in the hip joint. III. Discussion of the etiology and genetics of the pathological sequelae: comments on treatment. Clin Orthop Rel Res. 138:284–309; 139:283–312; 140:273–312.
  18. Klippel JH, Gerber LH, Pollak L, Decker JL. 1979 Avascular necrosis in systemic lupus erythematosus: silent symmetric osteonecroses. Am J Med. 67:83–87.[CrossRef][Medline]
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  23. Narula J, Haider N, Virmani R, et al. 1996 Apoptosis in myocytes in end-stage heart failure. N Engl J Med. 335:1182–1189.[Abstract/Free Full Text]
  24. Silva M, Richard C, Benito A, Sanz C, Olalla I, Fernandez-Luna JL. 1998 Expression of Bcl-x in erythroid precursors from patients with polycythemia vera. N Engl J Med. 338:564–571.[Abstract/Free Full Text]
  25. Kerr JFR, Wyllie AH, Currie AR. 1972 Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 26:239–257.[Medline]
  26. Thompson CB. 1995 Apoptosis in the pathogenesis and treatment of disease. Science. 267:1456–1462.[Abstract/Free Full Text]
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