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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2010 Jun;91(3):235–243. doi: 10.1111/j.1365-2613.2010.00705.x

Effects of an anticoagulant and a lipid-lowering agent on the prevention of steroid-induced osteonecrosis in rabbits

Pengde Kang *, Hong Gao †, Fuxing Pei *, Bin Shen *, Jing Yang *, Zongke Zhou *
PMCID: PMC2884091  PMID: 20353425

Abstract

This study was designed to evaluate the effects of the combined treatment with an anti-coagulant (enoxaparin) agent and a lipid-lowering agent (lovastatin) on prevention or decrease in the occurrence of steroid-induced osteonecrosis in rabbits. A total of 112 rabbits, which were injected intramuscularly with 20 mg/kg of methylprednisolone acetate were divided into four groups and treated as follows: one group received enoxaparin combined with lovastatin (EL; n= 30), another received enoxaparin alone (EA; n= 28), another received lovastatin alone (LA; n= 28) and the last received no treatment (non-prophylactic; NP, n= 26). Haematological examination for serum lipid levels and prothrombin time was carried out and both femora and humeri were examined histopathologically for the presence of osteonecrosis (ON) before injection and at 2, 4, 8 and 12 weeks after the injection. The incidence of ON in the EL group (15%) was significantly lower than that observed in the NP group (68%). The incidence in the EA and LA groups was also significantly lower than that in the NP group (31%, 35%vs. 68%). The fat cell sizes of the bone marrow in both EL (46.49 ± 1.27 μm) and LA (50.8 ± 2.31 μm) groups were lower than in the NP group (59.89 ± 6.33 μm). The prothrombin time was prolonged and plasma lipid levels were reduced in the EL group during the study. Combination treatment with an anti-coagulant agent and a lipid-lowering agent can reduce the incidence of steroid-induced ON in rabbits. Future evaluation in clinical practice is necessary.

Keywords: corticosteroid, enoxaparin, lovastatin, osteonecrosis, rabbits

The association between the steroid use and the development of osteonecrosis of the femoral head (ONFH) has been well established since 1957 (Lemoine 1957). Increased use of steroids for immunosuppression in patients with organ transplants and for conditions such systemic lupus erythematosus and rheumatoid arthritis has resulted in an increased incidence of ONFH. However, the pathophysiology of ONFH has not been completely elucidated. Contemporary studies of the pathophysiology of ONFH have focused on the microcirculation of the femoral head and the ischaemia consequences of microvascular occlusion (Lafforgue 2006; Mont et al. 2006). The microcirculation of the femoral head is vulnerable to the occlusion from both intravascular thrombosis and extravascular compression. Thrombotic and fat emboli have been found in both arterioles and venules in specimens of osteonecrosis (ON) tissue and have been associated with osteocyte necrosis in some animal models (Hernigou & Beaujean 1997; Hirano et al. 1997; Yamamoto et al. 1997; Chemetsky et al. 1999; Koo et al. 1999). In an animal model study of osteonecrosis, hyperlipidaemia associated with abnormal thrombophilic coagulatory and bone marrow fat-cell packing was linked to the development of ONFH (Jones 1993; Miyanishi et al. 2002, 2005; Kang et al. 2008; Masada et al. 2008). Many authors have suggested that interference with the blood supply, by various means, plays a major role in the pathogenesis of ON. Intra-osseous hypertension, intravascular fat emboli and coagulation and compression of vessels by progressive accumulation of marrow fat stores are the commonly accepted theories (Lemoine 1957; Wang et al. 1978, 2000; Hungford & Lennox 1985; Mont et al. 2006; Pengde et al. 2008).

Although total hip arthroplasty is a definitive treatment for the advanced ONFH, the procedure historically has had a higher rate of failure in the young active patient (Lemoine 1957; Xenakis et al. 1997; Ortiguera et al. 1999; Hungerford 2007). Therefore, prevention of the onset of ONFH would be a better strategy for such disorders. The use of pharmacological agents for the treatment and prevention of ONFH has received considerable attention in recent years. The aim of using these agents, which include lipid-lowing drugs, anti-coagulants, is to address specific physiological risk factors for ONFH, such as lipid emboli, adipocyte hypertrophy, venous thrombosis and increased intra-osseous pressure.

Lovastatin can not only decrease the level of cholesterols but also counteract the effects of steroids on the differentiation of the precursor cells in the bone marrow into the adipocytes. Lovastatin can decrease the expression of the fat-cell transcription factor PPARγ2 and increase the expression of the osteoblast gene Runx2/Cbfa1 and shunt the uncommitted osteoprogenitor cells in the bone marrow from the adipocytic to the osteoblastic differentiation pathway (Li et al. 2003). Lovastatin has been associated with the increased bone morphogenetic proteins-2 (BMP-2) gene expression (Cui et al. 1997; Wang et al. 2000), alkaline phosphatase activity, matrix mineralization and enhanced in vitro osteogenesis (Cui et al. 1997; Hirano et al. 1997). Statins increase bone volume in rats and bone formation in mice (Mundy et al. 1999; Emmanucle et al. 2003). Cholesterol reducing drugs lower the intra-osseous pressure in femoral heads of the steroid-treated rabbits (Mundy et al. 1999).

Enoxaparin, a low molecular weight heparin, is one of the anti-coagulants, which has a less ability to inactivate thrombin. It has also been used to treat patients with thrombophilic or hypofibrinolytic disorders and the early-stages ONFH. The results showed that enoxaparin can slow down the progression of ONFH and stabilize or even reverse the changes of ONFH while providing significant pain relief (Roy & Glueck 1999; Glueck et al. 2001).

The effects of either anti-coagulants or lipid-lowing agents have been assessed. However, the combination of anti-coagulants and lipid-lowing agents for preventing and decreasing the steroid-induced ONFH has not been studied. Provided that anti-coagulant (enoxaparin) therapy combined with the lipid-lowing agent (lovastatin) is started during the use of corticosteroids, ONFH may be prevented or decreased. As several rabbit models for steroid-induced ON have been reported in literature (Miyanishi et al. 2005; Kang et al. 2008; Masada et al. 2008), in our study, this model was used to address the question whether anti-coagulant (enoxaparin) combined with a lipid-lowering agent (lovastatin) could prevent or could decrease the incidence of steroid-induced ON.

Materials and methods

We used a rabbit model of steroid-induced ON that has also been described previously (Miyanishi et al. 2005; Kang et al. 2008; Masada et al. 2008). The animal protocol was reviewed and approved by the Animal Care and Committees at the West China Medical School of Sichuan University.

Animals

Adult male Japanese white rabbits weighting 2.8–3.4 kg were housed at Animal Center of Sichuan University and maintained on a standard laboratory diet and water. We randomly chose 20 rabbits to receive an X-ray examination to confirm that the epiphyses were closed before the experiment started. The age of the rabbits ranged from 28 to 32 weeks. The body weights of the rabbits were measured prior to each experiment and at 1, 2 weeks, and before the rabbits were sacrificed after the steroid injection.

Treatment

In all, 112 rabbits were injected once with 20 mg/kg of body weight of methylpednisolone acetate (MPSL; Pfizer Pharmaceutical, Hangzhou, China) intramuscularly into the right gluteus medius muscle. The rabbits were divided into four groups. In the group wherein enoxaparin was combined with lovastatin (EL), rabbits received lovastatin (MerckSharp & Dohme, Hangzhou, China) orally at a dosage of 5 mg/kg/day as a 10% food admixture for 14 weeks, beginning 2 weeks before the MPSL injection. At the time of the methylpednisolone acetate injection, enoxaparin was given subcutaneously, 1 mg/kg/day for 4 weeks (EL; n= 30). In the LA group (lovastatin alone), the rabbits received lovastatin alone orally at a dosage of 5 mg/kg/day as a 10% food admixture for 14 weeks, beginning 2 weeks before the methylpednisolone acetate injection (LA; n= 28). In the enoxaparin alone (EA) group, the rabbits were administered enoxaparin subcutaneously alone at a dosage of 1 mg/kg/day for 4 weeks, beginning at the same time of the methylpednisolone acetate injection (EA; n= 28). In the non-prophylactic (NP) group, the rabbits received no treatment (NP) (n= 26). The animals were fed a standard diet and allowed free activity. Precise administration of each daily dose was achieved by feeding rabbits the standard diet, after confirming that they completely consumed a small volume (5–10 g) of food admixture of lovastatin. Six rabbits were killed respectively in each group at 2, 4, 8 and 12 weeks and then the tissue samples were prepared.

Tissue sample preparation

Six rabbits from each group were killed respectively at 2, 4, 8 and 12 weeks after the methylpednisolone injection in each group. The rabbits were anaesthetized with an intravenous injection of pentobarbital sodium (25 mg/kg) and were then killed by exsanguination via an aortectomy.

For the light microscopic examinations, both femora and humeri were obtained at the time of the rabbit death and were fixed for 1 week in 10% formalin, 0.1 mol/l phosphate buffer (pH 7.4). The bone samples were decalcified with 25% formic acid for 3 days. Samples were sectioned along the coronal plane for the proximal one-third and cut along the axial plane in the distal part (condyle). Finally, the specimens were embedded in paraffin, cut into 5 μm sections and stained with haematoxylin and eosin (Kang et al. 2008).

Evaluation of ON

The diagnosis of ON was performed histologically at 2, 4, 8 and 12 weeks after the methylpednisolone injection. Two weeks was a time point that had been reported to be crucial in the development of ON. The whole areas of the proximal one-third and the distal condyles of both femora and humeri (totaling eight regions) were examined histopathologically for ON. The slides were evaluated in a blinded fashion by three independent authors (KPD, PFX and SB). Diagnosis of ON was based on the fact that the slides either had empty lacunae near bone marrow necrosis or had pyknotic nuclei and bone marrow necrosis (Matsui et al. 1992). If the diagnoses differed among the three examiners, consensus was reached by discussing the histological findings without knowledge of the group from which the sample was obtained. The rabbit that has at least one osteonecrotic lesion in the eight examined areas was considered to have ON. We determined both the numbers of the rabbits with ON and the numbers of the osteonecrotic lesions per affected rabbit (maximum eight regions).

Measurement of the bone marrow fat cell size and fat area

At the 12-week point, bone marrow fat cell size was calculated as the average of the greatest diameters of 100 fat cells in four randomly selected fields (one field = 25 × 10−8 m2) from the proximal one-third of the femur using NIH Image software (Shaw et al. 1995). Briefly, an image of the section was taken using a camera and was directed electronically to an image processor. The greatest diameter of fat cells displayed on the video monitor was measured using an interactive mousepad-tracing instrument. The corresponding morphometric data were processed automatically by the computer system. The metaphyseal and/or diaphyseal regions were examined because ON has been seen in these areas in this rabbit model (Yamamoto et al. 1997). Fat cells that had undergone necrosis were excluded from the evaluation of fat cell size.

At the same time, six sections from each animal (three from each femoral head) were scanned using a Nikon slide scanner and analysed morphometrically. The areas of fat cells (including necrosis areas of marrow fat cells) and trabecular bone within the zone of the femoral head proximal to a straight line connecting the edges of the articular cartilage quantified histomorphometrically (Wang et al. 2000) with Mocha program according to the method suggested by the American Society of Bone and Mineral Research Histomorphometry Nomenclature Committee (Parfitt et al. 1987). The mean of the six sections from each animal was taken as the value for that rabbit.

Haematological examination

The blood samples were obtained from the auricular arteries while the animals were in fasting state prior to experimentation (0 week) and at 1, 2 and 4 weeks after the methylpednisolone injection and the blood samples were sent to the hospital clinical lab for analysis. The levels of cholesterol, triglycerides and free fatty acids were carried out to evaluate the effect of lovastatin. At the same time, the prothrombin time, evaluating the anti-coagulant activity of enoxaparin, was determined and is expressed as the international normalized ratio (INR).

Statistical analysis

Data were expressed as the mean ± SD. Fat cell sizes and fat cell areas were compared using one-way anova. The haematoxylin data in ON-positive and ON-negative were compared using the chi-square test. Haematological data were analysed by repeated-measures anova with Scheffe’s post hoc test. Statistical differences were considered significant when the P-value was less than 0.05. Statistical analyses were performed using spss 12.0 software (SPSS Inc., Chicago, IL, USA).

Results

Ten of the 112 rabbits died. Two of the rabbits in the EL group, two in the LA group, one in the EA group and two in the NP group died of pneumonia at 1 week after methylprednisolone injection. Two of the rabbits in the EL group and 1 in the EA group died of bleeding from the site of blood sampling at 2 weeks after methylprednisolone injection because the haemorrhage from the blood-sampling site could not stop. After exclusion of the 10 dead rabbits from the study, there were 26 rabbits in the EL group, 26 rabbits in the LA group, 26 rabbits in the EA group and 24 rabbits in the NP group. The amount of body weight loss during the experimental period, i.e. from the time before the experiment started to the time the animals were terminated, did not differ among the groups. The mean ± SD amount of weight loss was 353 ± 152 g in the EL group, 325 ± 126 g in the LA group, 346 ± 127 g in the EA group and 339 ± 115 g in the NP group.

Macroscopic and histopathological features

Macroscopically, the ON areas appeared as yellow patches in the metaphysis and the diaphysis. Histologically, ON lesions demonstrated an accumulation of bone marrow cell debris and the bone trabeculae had empty lacunae at the time point of 2 weeks (Figure 1a). The fat cell size and fat cell area in the bone marrow were increased with time at 8 and 12 weeks, the number of the empty lacunae was also increased (Figure 1b). These findings were consistent in all the ON-positive rabbits. Neither the subchondral bone nor the intra-osseous collapse was observed in the four groups. The granulation tissue and appositional bone formation were also not observed in the four groups.

Figure 1.

Figure 1

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(a) Osteonecrotic lesions in a rabbit model of the steroid-induced osteonecrosis (ON) in the non-prophylactic (NP) group that received no treatment. The tissues exhibited an accumulation of the bone-marrow cell debris and the bone trabeculae had empty lacunae at the time point of 2 weeks (HE, ×200). (b) Osteonecrotic lesions in a rabbit model of the steroid-induced ON in the NP group. The fat-cell size and the fat-cell area in the bone marrow were increased with time at 12 weeks after the methylpednisolone injection and the number of the empty lacunae was also increased. These findings were consistent for all the ON-positive rabbits (HE, ×200).

Incidence of ON

Osteonecrosis appeared 2 weeks after the steroid administration; its frequency increased until the fourth week and then reached a plateau. The incidence of ON was 4/26 in the EL group, 9/26 in the LA group, 8/26 in the EA group and 16/24 in the NP group. The incidence of ON in the four groups is shown in Figure 2. The incidence of ON in the EL the group, the LA group and the EA group was significantly lower than in the NP group (P<0.001, P<0.01, P<0.01 respectively). The incidence of ON in the LA group and the EA group was significantly higher than in the EL group (P<0.05, P <0.05 respectively). No significant difference in the number of ON lesions in each affected rabbit was seen among the groups.

Figure 2.

Figure 2

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The incidence of osteonecrosis (ON) in the four groups. The EL group received enoxaparin combined with lovastatin; the LA group received only lovastatin; the EA group received only enoxaparin; the non-prophylactic (NP) group received no treatment. The incidence of ON was significantly lower in the former three groups than in the NP group (P<0.001, P <0.01, P <0.01). The incidence of ON was significantly higher in the LA and EA groups than in the EL group (P <0.05, P <0.05).

Calculation of the sizes of bone marrow fat cells

At 12 weeks, the average size of bone marrow fat cells was significantly smaller in the EL group than in the NP group (46.49 ± 1.27 μm vs. 59.89 ± 6.33 μm, P<0.01). The size of bone marrow fat cells in the LA group was also significantly smaller than in either the EA group (50.8 ± 2.31 μm vs. 57.37 ± 1.52 μm, P<0.01) or the NP group (50.8 ± 2.31 μm vs. 59.89 ± 6.33 μm, P<0.01). There were no significant differences in the size of bone marrow fat cells between the EL group and the LA group.

In the LA group, the average fat cell size of bone marrow did not differ significantly between the ON-positive rabbits (2/6) and the ON-negative rabbits (4/6) (51.4 ± 3.71 μm vs. 45.9 ± 4.63 μm, P=0.165). In the NP group, the size of bone marrow fat cells in the ON-positive (4/6) rabbits was significantly larger than in the ON-negative rabbits (2/6) (60.78 ± 3.32 μm vs. 55.36 ± 2.92 μm, P<0.01). In the ON-positive rabbits, the number of the enlarged marrow fat cell was increased and some of the fat cells were necrotic. The space normally occupied by the bone marrow haematopoietic cells and the vascular channels was replaced by the enlarged marrow fat cells, and the bone trabeculae thinning also existed. In the ON-negative rabbits in the EL group (4/6) and the LA group (4/6), the sizes of some marrow fat cells were increased; however, the space for the bone marrow haematopoietic cells was relatively preserved though the bone trabeculae were relatively thinning. There was no significant difference in the size of bone marrow fat cells of the ON-negative rabbits between the EL & LA groups and the EA & NP groups.

Bone marrow fat cell areas

The bone-marrow fat-cell areas and the whole bone-marrow area were quantified histomorphometrically (×10−8 m2). The ratio of the fat-cell area to the whole bone-marrow area on the cross sections was significantly smaller in the EL group than in the EA group (0.25 ± 0.02 vs. 0.51 ± 0.02, P<0.01) and the NP group (0.25 ± 0.02 vs. 0.53 ± 0.05, P<0.01). In the LA group, the fat-cell area ratio in the bone marrow was not significantly different between the ON-positive rabbits and the ON-negative rabbits (0.23 ± 0.13 vs.0.29 ± 0.12, P=0.264). In the NP group, the fat-cell area ratio in the bone marrow was significantly larger in the ON-positive rabbits than in the ON-negative rabbits (0.56 ± 0.13 vs. 0.38 ± 0.17, P<0.01). The ratio of the trabecular bone area to the whole bone marrow area on the cross sections was significantly decreased in the NP group (P<0.01), which accounted for 32% of the total areas of the femoral head compared with 65% in the LA group. In the LA group, the ratio of the trabecular bone area was maintained at 52%.

The number of the necrotic foci

The number of the necrotic foci was 1.14 ± 0.38 in the EL group, 1.59 ± 0.43 in the EA group, 1.67 ± 0.42 in the LA group and 2.23 ± 0.47 in the NP group. The number of the necrotic foci was significantly smaller in the EL group than in the EA group, the LA group and the NP group (P<0.05, P<0.01, P<0.01). There was no significant difference between the EA group and the LA group. The number of the necrotic foci was significantly smaller in the EA and LA groups than in the NP group (P<0.05, P<0.05).

Haematological findings

In the lipid system, the total cholesterol (Figure 3a), triglycerides and free fatty acids in the EA group and the NP group were increased (P<0.01, P<0.01 respectively) between 1 and 2 weeks after the methylprednisolone injection and these high levels were maintained during the observation time (1 week to 4 weeks). The lipid levels in the EA group and the NP group were significantly higher than in either the EL group (P<0.01, P<0.01 respectively) or the LA group (P<0.01, P<0.01 respectively) at all the time points. There was no significant difference in any of the factors examined between the ON-positive rabbits and ON-negative rabbits.

Figure 3.

Figure 3

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(a) The sequential changes in the lipid levels of the cholesterol in the rabbits of the four groups. The cholesterol levels in the enoxaparin alone (EA) and non-prophylactic (NP) groups were significantly increased (P <0.01, P<0.01) between 1 and 2 weeks after the methylprednisolone injection and those high levels were maintained during the observation time (1–4 weeks). The cholesterol levels were significantly higher in the EA and NP groups than in the enoxaparin combined with lovastatin (EL) group (P<0.01, P<0.01) and in the lovastatin alone (LA) group (P<0.01, P<0.01) at all the time points. (b) The prothrombin time in the four groups. The prothrombin time was at significantly higher levels in the EL and EA groups than in the LA and NP groups during the experimental period (P<0.01, P<0.01).

The prothrombin time was significantly higher in the EL group and the EA group than in the LA group and the NP group during the experimental period (P<0.01, P<0.01) (Figure 3b).

Discussion

Corticosteroid treatment is considered an important risk factor for ON (Cui et al. 1997; Yamamoto et al. 1997; Lieberman et al. 2002; Miyanishi et al. 2005). Several factors in the pathogenesis on ON have been suggested based on both human and animal studies, including coagulation abnormalities and hyperlipidaemia (Wang et al. 1978; Jones 1993; Yamamoto et al. 1997; Miyanishi et al. 2005; Kang et al. 2008; Masada et al. 2008). The rabbit model of ON studies have demonstrated that the development of ON is closely linked to the hyperlipidaemia associated with abnormal thrombophilic coagulopathy and bone marrow fat-cell packing, which results in microvascular occlusion, high intra-osseous pressure and produces an intra-osseous compartment syndrome within the hard cortex, all of which contributed to the development ON in rabbits (Wang et al. 1978, 2000; Miyanishi et al. 2005). Studies in humans have also demonstrated that vascular occlusion may occur as a result of mechanical interruption by thrombi or lipid emboli in nutrient vessels (Yamamoto et al. 1997; Miyanishi et al. 2005). Therefore, we may postulate that the combination treatment with both an anti-coagulant agent and a lipid-lowering agent can prevent the conditions associated with the development of osteonecrosis. To answer the question, the effect of anti-coagulant, lovastatin and lipid-lowering agent, enoxaparin, on the prevention of ON in vivo was evaluated in the present study. The results demonstrated that the incidence of ON in rabbits was significantly decreased by combined treatment with both enoxaparin and lovastatin when compared with either enoxaparin or lovastatin alone or without treatment.

Enoxaparin was chosen as the an anti-coagulant for this study based on previous studies, which have demonstrated that enoxaparin could treat thrombophilic or hypofibinolitic disorders and prevent the progression of stage I and stage II ONFH (Glueck et al. 2005), stabilize or even reverse the changes of ON while providing significant pain relief (Roy & Glueck 1999; Glueck et al. 2001, 2005). In this study, enoxaparin ameliorated the thrombophilic state of plasma, as evidenced by a significantly higher prothrombin time (INR) when compared with that in the other two groups that did not receive enoxaparin. The incidence of ON was also significantly decreased. These findings have indicated that there is a close association between hypercoagulability and ONFH and that hypercoagulability plays an important role in the development of the steroid-induced ON.

Lovastatin, an inhibitor of hydroxymethylglutaryl coenzyme A reductase, can not only decrease the levels of cholesterols but also counteract the effects of the steroids on the differentiation of the precursor cells in the bone marrow adipocyte (Cui et al. 1997; Wang et al. 2000; Kang et al. 2008). The studies showed that lovastatin improves bone formation and prevents dexamethasone-mediated adipogenesis by the mesenchymal cells in vitro (Cui et al. 1997; Wang et al. 2000). Lovastatin acts on the precursor cells in bone marrow stroma to modulate their differentiation by enhancing osteoblast differentiation, acting on the level of the commitment through increased expression of the Cbfa1/Runx2 gene, also by increasing activity of the osteocalcin promoter (Li et al. 2003). Lovastatin may shunt uncommitted osteogenitor cells in marrow from adipocytic to the osteoblastic differentiation pathway by acting on the expression of adipocyte-specific genes, PPARγ2 and 422aP (Li et al. 2003). Lovastatin is also associated with increased BMP-2 gene expression (Mundy et al. 1999; Li et al. 2003), alkaline phosphatase activity and matrix mineralization, and enhances osteogenesis by the bone cells in vitro (Hirano et al. 1997; Lee et al. 2002). The results of this study have indicated that lovastatin not only decreases the serum lipid levels but also preserves the haematopoietic cells and decreases the bone fat volume in the steroid-treated rabbits, in which lovastatin can counteract the effects of the steroid on the differentiation of the precursor cells in the bone marrow into the adipocytes (Wang et al. 2000; Kang et al. 2008) and reduce the incidence of steroid-induced ON.

In this study, the ON lesions in the rabbit models present histological characteristics of empty lacunae accompanied by surrounding marrow cell necrosis. These features are similar to very early stage human ON. However, they are also similar to the processing artefacts sometimes observed in the human samples (Chemetsky et al. 1999). It is important to note that the rabbits are different from human ON in that their ON regions are not on the epiphysis, but on the medial side of the metaphysis and diaphysis, and their ON lesions develop very often, but do not collapse (Matsui et al. 1992; Yamamoto et al. 1997). These differences may be caused by metabolic differences between humans and rabbits, or by mechanical differences between quadrupeds and bipeds. We recognise that it is difficult to draw a definite conclusion for human ON from this rabbit experiment because of the interspecies differences. Although the mechanism of development of ON may not be completely the same as in humans, there are several similarities between human and rabbit ON, such as increased lipid deposition and a rise in intra-osseous pressure (Zizic et al. 1997; Miyanishi et al. 2005; Kang et al. 2008), histological features of empty lacunae accompanied by surrounding marrow cell necrosis (Arlet et al. 1984; Yamamoto et al. 1997; Kang et al. 2008) and the multifocal ON (Mankin 1992; Yamamoto et al. 1997). Thus investigating the denominators of ON in both humans and rabbits may help us understand the pathogenetic mechanism and explore the methods to prevent steroid-induced ON. We believe that the present animal model study is useful to investigate this mechanism and preventing methods of steroid-induced ON in human beings.

Although the combined use of enoxaparin and lovastatin significantly decreased the incidence of the steroid-induced ON, 15% of the rabbits still developed ON in this study, which indicated that there were multiple factors for the steroid-induced ON, such as the genetic predisposition and the direct cell toxicity associated with corticosteroids (Wang 1992; Asano et al. 2003; Chao et al. 2003; Motomura et al. 2004; Liu et al. 2005; Mont et al. 2006). Besides, the individual variability in the response to the steroids and the lipid-lowering drugs may also affect the incidence of the steroid-induced ON and the effects of the lipid-lowering drugs. Furthermore, there may be a threshold in the development of the steroid-induced ON. Once the threshold was reached, the development of ON was initiated and finally ON was formed despite the prophylactic treatment (Lieberman et al. 2002; Motomura et al. 2004). Finally, not only do some of the animals with treatment get osteonecrosis, but many of the animals without treatment (but with steroid) do not get osteonecrosis. This may be a limitation in the use of this kind of models.

In summary, the combined treatment with both enoxaparin and lovastatin can significantly decrease the incidence of osteonecrosis in the steroid-treated rabbits. Coagulation abnormalities and hyperlipidaemia, which result in the vascular occlusion and ischaemia by the formation of the thrombi or lipid emboli in the nutrient vessels, may play an important role in the development of steroid-induced ON in rabbits. The precise mechanism needs to be further explored in vivo. Studies focusing on the effect of anti-coagulant and lipid-lowering agents in humans are needed to determine their clinical impacts on ON.

Acknowledgments

This work was supported by National Natural Science Fund of China (30772202). The authors thank Hong-sheng Dang, MD and Wei Liu, MD, PhD, for their technical support. We also acknowledge the following individuals for their contributions to this study: Zhang Hui, MD, Yun-yong Lian, MD.

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