Apoptosis in the Osteonecrosis of the Femoral Head

Of the patients with steroid-induced osteonecrosis, 5 were males and 3 were females. Their mean age at surgery was 44.3 years. Four cases had received kidney transplants and the other 4 cases received high-dose steroid therapies after neurosurgical operations. For patients with steroid-induced osteonecrosis, information on the exact total cumulative dose, mean daily dose, type of steroids, and duration of steroid therapy was unavailable.

Of the patients with alcohol-induced osteonecrosis, 28 were males and 1 was female. Their mean age at surgery was 54.7 years. All patients had a history of alcohol intake of more than 5 times per week. The average amount and period of alcohol intake was approximately 99.0 g per day and 25.7 years, respectively.

Of the patients with post-traumatic cases, 3 were males and 3 were females. Their mean age at surgery was 50.3 years. Five cases underwent total hip arthroplasty due to femoral neck fractures and the remaining case was treated with 2 months of traction for a traumatic hip dislocation.

Of the patients with idiopathic osteonecrosis, 5 were males and 10 were females. Their mean age at surgery was 57.8 years. In this group, the possible risk factors for osteonecrosis could not be found.

Using TUNEL immunofluorescence staining, no or few TUNEL-positive osteocytes were observed in the tissue samples of patients with post-traumatic or idiopathic osteonecrosis, whereas many positive osteocytes were observed in patients with steroid- or alcohol-induced osteonecrosis (Fig. 1).

Fig. 1
After the terminal deoxynucleotydyl transferase mediated dUTP nick-end labeling (TUNEL) reaction, (A) condensed apoptotic osteocytes in cancellous bone from a femoral head removed from a patient with steroid-induced osteonecrosis show intense green fluorescence (× 400). (B) In a patient with alcohol-induced osteonecrosis, intermediate fluorescence is shown (× 100). Non-apoptotic cells are not visible using immunofluorescence microscopy. (C) In a patient with idiopathic osteonecrosis, no fluorescence is shown (× 100).

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DAB coupling showed that the ratio of TUNEL-positive osteocytes was high in patients with steroid- and alcohol-induced osteonecrosis and low or rare in patients with post-traumatic and idiopathic osteonecrosis (Fig. 2). The mean percentages were similar in the steroid- and alcohol-induced groups, and in the post-traumatic and idiopathic groups (p > 0.05). However, there were significant differences between the former and latter two groups (p < 0.05) (Table 2). There were no significant differences in the number of apoptotic osteocytes according to the disease stages (p > 0.05).

Fig. 2
Optical microscopy shows (A) extensive apoptosis in femoral head osteocytes from a patient with steroid-induced osteonecrosis, (B) absence of apoptotic osteocytes in a patient with idiopathic osteonecrosis and (C) moderate apoptotic osteocytes in a patient with alcohol-induced osteonecrosis. The apoptotic osteocytes were stained brown with diaminobenzidine substrates, whereas non-apoptotic osteocytes are stained blue with hematoxylin (× 400).

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Table 2
Immunoreactivity of Terminal Deoxynucleotydyl Transferase Mediated dUTP Nick-End Labeling (TUNEL) Staining

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The mechanisms of apoptosis and cell necrosis in ischemic conditions are radically different.1, 5-8) Cell necrosis is always accompanied by the surrounding inflammatory reactions, whereas apoptosis occurs without inflammation. Apoptosis exerts a homeostatic function in relation to the tissue dynamics because the steady state of continuously renewing tissues is achieved by a balance between cell replication and cell death.9)

Several techniques have been used to detect apoptosis. The TUNEL method used in this study is based on the specific binding of the terminal deoxynucleotidyl transferase (TdT) to 3-OH (3-hydroxyl) ends of fragmented DNA from apoptotic cells, yielding polydeoxylnucleotide polymers.10) The TUNEL method makes it possible for direct observations and quantification of the fraction of cells undergoing apoptosis without damaging the tissue architecture. In some cells undergoing apoptotic cell death, DNA cleavage can be absent or incomplete. Moreover, steric hindrance, which can be caused by extracellular matrix components, can prevent the access of TdT to DNA strand breaks. In either case, false negative results can be obtained. Extensive DNA fragmentation can occur during the later stages of necrosis, which can result in false positive findings.11, 12) Therefore, caution must be taken when interpreting the TUNEL results. Within each group, there was a wide range of TUNEL-positive cells, indicating the possibility of false positive and false negative errors in this study.

In this study, paraffin blocks were prepared routinely for a post-surgical histological examination. Although formalin cross-links proteins or peptides, thereby maintaining the morphological structure, long-term storage might reduce the antigenicity. Vis et al.13) reported that prostate needle biopsy specimens fixed in formalin, embedded in paraffin and mounted on glass slides showed a significant decrease in antigenicity after 5 years storage. However, the antigenicity was maintained when the tissue was archived in paraffin blocks and sectioned immediately before staining. Manne et al.14) reported similar results in breast tissues. Although these specimens had been stored for 3 to 15 months, it is likely that the antigenicity had been preserved because the glass slides were made from paraffin blocks immediately before staining.

The relationship between high dose steroid therapy and the development of osteonecrosis of the femoral head was first described in patients who underwent renal transplantation. Harrington et al.15) reported that approximately 34% of patients who ingested an average of 2,900 mg prednisolone during the first 3 weeks after transplantation developed osteonecrosis, compared to 10% of patients, who ingested an average of 1,200 mg prednisolone. In patients with systemic lupus erythematosus, a high initial corticosteroid dosage was suggested to be associated with the development of osteonecrosis.16, 17) However, others reported that the total cumulative steroid dose and duration of steroid therapy were not related to the development of osteonecrosis, hence the causal relationship is unclear.18-20)

The pathogenesis of alcohol-induced osteonecrosis has not been defined but the pathological changes occurring in patients with steroid- and alcohol-induced osteonecrosis are similar.21) It was hypothesized that steroid- and alcohol-induced osteonecrosis of the femoral head are due to intraosseous hypertension caused by fat-cell hypertrophy and proliferation, fat embolism and a disturbance of the circulation.15) However, there is no definitive histological evidence of fat emboli during human osteonecrosis.22) Based on these findings, Glimcher and Kenzora23) suggested that steroids, alcohol and/or their metabolites affect osteocytes directly and cause apoptosis. They also proposed that the toxic metabolites may accumulate and induce apoptosis, particularly in areas in which venous stasis occurs, such as a femoral head.

It was proposed that apoptosis may be related to the pathogenesis of osteonecrosis of the femoral head.3) Calder et al.2) suggested that previously mentioned toxic metabolites might activate apoptosis modulators, such as inducible nitric oxide synthase (iNOS), leading to locally toxic levels of NO in osteoblasts and osteocytes followed by cell death or apoptosis and the development of osteonecrosis of the femoral head. Weinstein et al.24) reported that mice receiving glucocorticoids for 4 weeks exhibited a 3-fold increase in osteoblast apoptosis in vertebral cancellous bone, and osteoblast and osteocyte apoptosis was higher in patients with glucocorticoid-induced osteoporosis.

The percentage of TUNEL-positive osteocytes was high in the groups of patients with steroid- and alcohol-induced osteonecrosis, but was low or rare in patients with post-traumatic and idiopathic necrosis. These findings support the hypothesis that apoptosis plays a key role in the pathogenesis of steroid- and alcohol-induced osteonecrosis of the femoral head.

To date, studies of the relationship between apoptosis and osteonecrosis of the femoral head have been limited to a small number of patients or to animal models. Large case-control studies have not been performed and the precise pathogenic mechanism by which apoptosis causes osteonecrosis has not been defined. These findings suggest a need for further research into the role of apoptosis in the development of osteonecrosis of the femoral head.