The efficacy of statins in preventing glucocorticoid-related osteonecrosis in animal models

Article focus

• Our study aimed to determine whether statin usage reduces the risk of glucocorticoid (GC)-related osteonecrosis (ON) in animal models.

Key messages

• Our results suggest that statin usage combined with other treatments could reduce the risk of developing GC-related ON in animal models.

Strengths and limitations

• This is the first meta-analysis to review the efficacy of statins in preventing GC-induced ON in animal models.

• No distinction was made for animal models and the type or dose of statins and GCs.

Introduction

Osteonecrosis (ON) is a common, progressive and devastating disease with an insidious onset. ON presents without specific clinical symptoms and signs and most commonly affects young adults in the third and fourth decade of their life.^1-3 Management options^1,2 for ON vary from joint salvaging procedures to joint replacement, and are based on the stages described by the Association of Research Circulation Osseous (ARCO) classification. Conservative treatment, represented by core decompression, may be efficient in the early stages and in small lesions of osteonecrosis (stages I, II). However, concerns have been raised due to the potential for core decompression to weaken the cancellous bone within and adjacent to the necrotic zones.⁴ With respect to joint replacement surgeries, approximately 10 000 to 20 000 new cases of ON are diagnosed in the United States every year, and it is estimated that 5% to 12% of total hip arthroplasties are performed every year in order to treat this disease.^1,2

Glucocorticoid (GC) usage is the leading cause of non-traumatic ON of the femoral head.⁵ ON develops in 9% to 40% of patients who receive high-dose or long-term steroid therapy.⁶ Despite the strong association of GC with ON, the underlying mechanisms of ON have been unclear. Several hypotheses have been introduced that explain the mechanism of GC-induced ON. The proposed pathogenesis includes lipid metabolism disturbance, apoptosis, increased oxidative stress and disturbances of the coagulation-fibrinolysis system due to steroid hormones.^1-3,7,8 In particular, intraosseous hypertension, intravenous fat embolisms, and compression of vessels by progressive accumulation of marrow fat store, are commonly accepted theories.⁹ Based on these findings, an increasing number of studies have been initiated to explore the effects of lipid-lowering agents on preventing ON.^9-20

Statins (3-hydroxymethyl-3-glutaryl-CoA (HMG-CoA) reductase inhibitors), widely used for the treatment of hyperlipidemia as well as for preventing coronary artery diseases,^21,22 have been likewise an attractive candidate for prevention of GC-induced ON. Beneficial effects do not only result from lowering cholesterol level, but also from pleiotropic effects including improvement of endothelial dysfunction, antioxidant effects, reduction of platelet activity and decreased bone cell apoptosis.^21-23 These various effects of statins may play an important role in the prevention of GC-induced ON. Previous research studies^14-16 have shown serum lipid levels were significantly lower in the statin group than in the control group, which received steroids only.

Interestingly, Iwakirietal et al¹⁵ have shown that increased CYP3A activity owing to statins is a possible mechanism for the protective effect, given the fact that extrinsic glucocorticoids are made inactivate predominantly by hepatic CYP3A. Histological examination^14-16 revealed that rabbits treated with steroids and statins maintained more physiological bone marrow fat cell size and fraction of marrow filled by fat. In vitro and in vivo studies^9,18 demonstrated that statin acts on bone marrow mesenchymal stem cells and modulate their differentiation by enhancing osteogenesis through increasing expression of Cbfa1/Runx2 improving activity of the osteocalcin promoter, and inhibiting the adipogenesis through decreasing expression of the adipocyte-specific genes PPARγ2 (fat cell transcription factor) and 422aP (fat-specific). Statins are also associated with an elevated bone morphogenetic protein-2 gene expression, alkaline phosphatase activity, matrix mineralisation, and enhanced osteogenesis by the bone cells in vitro.⁹ Although several animal and limited clinical studies indicated that statins may have a protective role against ON a recent large-scale cohort study¹⁰ revealed that ON-free survival was similar in renal transplantation patients with and without statin exposure.

It is therefore not clear if statins could effectively prevent GC-related ON. Therefore, the primary purpose of this meta-analysis is to determine whether statin usage reduces the risk of GC-related ON in animal models.

Materials and Methods

Results

Search results and study characteristics

According to the aforementioned specific criteria, 11 eligible studies,^{13-18,23,26-29} published between 1997 and 2014 were included in our meta-analysis. Figure 1 shows the detailed process of study selection. In total, we included 554 animals consisting of 391 rabbits, 128 rats and 35 chickens. Animal model ON was induced using steroid alone in all studies except for one,²³ which combined steroid with endotoxin. The study conducted by Nozaki et al¹³ consisted of rats with a 50% incidence of spontaneous ON of the femoral head at the age of 16 to 18 weeks, while the remaining studies consisted of healthy animals. Characteristics of the included studies are summarised in Tables II to VII. The median of the reported quality score was 3 (2 to 5).

Fig. 1

Flow diagram showing the process of article selection for meta-analysis.

Table II.

Basic characteristics of included studies

Study ID	Study location	Species	n (C)	n (E)	Gender	Age (wks)	Weight
Li et al 201423	China	Rabbit	15	15	Female	24 to 28	2.5 kg to 3.1 kg
Xie et al 201328	China	Rat	30	60	Mixed	8	250 g to 280 g
Nozaki et al 201213	Japan	Rat	23	15	Male	13	NR
Li et al 201127	China	Rabbit	16	16	Female	28 to 32	2.8 kg to 3.4 kg
Kang et al 201014	China	Rabbit	24	52	Male	28 to 32	2.8 kg to 3.4 kg
Zeng et al 200926	China	Rabbit	9	20	Mixed	Adult	2.5 kg to 3.0 kg
Iwakiri et al 200815	Japan	Rabbit	30	30	Female	28 to 32	3.1 kg to 4.2 kg
Pengde et al 200816	China	Rabbit	26	25	Male	28 to32	2.8 kg to 3.4 kg
Nishida et al 200817	Japan	Rabbit	30	35	Male	28 to 32	NR
Kang et al 200729	China	Rabbit	16	32	Mixed	28 to 32	2.7 kg to 3.3 kg
Cui et al 199718	United States	Chicken	25	10	Female	Adult	NR

1. n (C), number of animals in control group; n (E), number of animals in experimental group; NR, not reported

Table III.

Characteristics of animal models of steroid-induced osteonecrosis

Study ID	Animal models
	Steroids exposure	Dose (mg/kg)	Route of delivery
Li et al 201423	MPSL (+ET)	20 (10 μg), once	i.m. (left gluteus)
Xie et al 201328	MPSL	20, daily, 4 wks	i.p.
Nozaki et al 201213	MPSL	4 mg in total	s.c. (back)
Li et al 201127	MPSL	20, once	i.m. (right gluteus)
Kang et al 201014	MPSL	20, once	i.m. (right gluteus)
Zeng et al 200926	DSP	2.5, NR	i.m.
Iwakiri et al 200815	MPSL	20, once	i.m. (right gluteus)
Pengde et al 200816	MPSL	20, once	i.m. (right gluteus)
Nishida et al 200817	MPSL	20, once	i.m. (right gluteus)
Kang et al 200729	MPSL	20, once	i.m. (right gluteus)
Cui et al 199718	MPSL	3, weekly, 12 wks	i.m.

1. MPSL, methylprednisolone; DSP, dexamethasone sodium phosphate; ET, endotoxin; i.m., intramuscular; i.v., intravenous; i.p., intraperitoneal; s.c., subcutaneous injection; NR, not reported

Table IV.

Characteristics of statin intervention of included studies

Study ID	Types	Statins administration
		Dose(mg/kg.d,)	Duration (wks)	Route of delivery	Treatment point before steroid
Li et al 201423	atorvastatin	2.5	8	Food admixture	0 wk
Xie et al 201328	lovastatin	25	14	Gavage	2 wks
Nozaki et al 201213	pravastatin	15	4	Drinking water	2 wks
Li et al 201127	pravastatin	2.5	12	Food admixture	0 wk
Kang et al 201014	lovastatin	5	14	Food admixture	2 wks
Zeng et al 200926	simvastatin	20mg, daily	NR	PO	0 wk
Iwakiri et al 200815	pravastatin/simvastatin	2/5	6/6	IV/IV	3 wks/3 wks
Pengde et al 200816	lovastatin	5	14	Food admixture	2 wks
Nishida et al 200817	pravastatin	0.7	4	IV	2 wks
Kang et al 200729	lovastatin	300	14	Food admixture	2 wks
Cui et al 199718	lovastatin	20 mg, daily	NR	PO	0 wk

1. PO, peros; IV, intravenous; NR, not reported

Table V.

The methodological quality of individual study

Study ID	(1)	(2)	(3)	(4)	(5)	(6)	(7)	Score
Li et al 201423		*	*		*			3
Xie et al 201328		*			*	*		3
Nozaki et al 201213	*	*			*	*	*	5
Li et al 201127		*			*			2
Kang et al 201014		*			*	*		3
Zeng et al 200926		*			*			2
Iwakiri et al 200815		*			*	*	*	4
Pengde et al 200816		*			*	*		3
Nishida et al 200817	*	*			*			3
Kang et al 200729		*			*			2
Cui et al 199718		*			*			2

1. *

   One score

1. Studies fulfilling the criteria of (1) sample size calculation; (2) inclusion and exclusion criteria; (3) randomisation; (4) allocation concealment; (5) reporting of animals excluded from analysis; (6) blinded assessment of ON; (7) reporting potential conflicts of interest and study funding

Table VI.

Evaluation of osteonecrosis of included studies

Study ID	Diagnosis of osteonecrosis
	Unilateral or bilateral	Sample from	Methods
Li et al 201423	Bilateral	Femoral head	Radiological examination
Xie et al 201328	NR	Femoral head	Histological examination
Nozaki et al 201213	Bilateral	Proximal femur	Histological examination
Li et al 201127	Unilateral	Femoral head	Histological examination
Kang et al 201014	Bilateral	Femur and humerus*	Histological examination
Zeng et al 200926	Bilateral	Femoral heads	Radiologicalexamination
Iwakiri et al 200815	Bilateral	Proximal femur	Histological examination
Pengde et al 200816	Bilateral	Femur and humerus*	Histological examination
Nishida et al 200817	Bilateral	Femur and humerus*	Histological examination
Kang et al 200729	Bilateral	Proximal femur	Histological examination
Cui et al 199718	Bilateral	Femoral head	Histological examination

1. *

   The whole area of the proximal one third and distal condyles of both the femur and the humerus

1. NR, not reported

Table VII.

Outcomes of each included study

Study ID	Animals treated with statin and steroid		Animals treated with steroidalone
	Osteonecrosis	Total	Osteonecrosis	Total
Li et al 201423	4	15	7	15
Xie et al 201328	19	60	17	30
Nozaki et al 201213	11	15	23	23
Li et al 201127	5	16	10	16
Kang et al 201014	13	52	16	24
Zeng et al 200926	1	20	3	9
Iwakiri et al 200815	9	30	25	30
Pengde et al 200816	9	25	18	26
Nishida et al 200817	13	35	21	30
Kang et al 200729	8	32	11	16
Cui et al 199718	0	10	14	25

Primary analysis

The overall estimate of the effect of statins was 2.06 (95% CI 1.71 to 2.50, p < 0.001), an approximately two-fold improvement in outcome (Fig. 2). Compared with the control group, the RR was 1.91 (95% CI 1.50 to 2.42) in studies using statins alone while the RR was 2.10 (95% CI 1.68 to 2.62) in studies using statins with other interventions such as bisphosphonate or anticoagulant. Then we further compared the effect of statin exposure alone to that of statins combined with other interventions using data from experiments containing both groups. Results revealed that combined usage significantly decreased the risk of ON (RR = 1.23, 95% CI, 1.02 to 1.47).

Fig. 2

Forest plot showing the overall risk estimate of osteonecrosis between groups with or without statins (M-H, Mantel–Haenszel; CI, confidence interval; df, degrees of freedom). Risk ratio (RR) on the left axis indicates statin usage decreases the risk of osteonecrosis compared with the control group, whereas RR greater than 1 indicates animals with statin usage are at increased risk of osteonecrosis. The 95% CI reveals that the result is statistically significant when “1” is not included in the interval.

Experiments with a relatively low score (< 3 points) gave more conservative estimates of effect size than those with a high score (≥ 3 points) (Fig. 3).

Fig. 3

Forest plot showing subgroup analysis based on study quality score (M-H, Mantel–Haenszel; CI, confidence interval; df, degrees of freedom). Risk ratio (RR) on the left axis indicates statin usage decreases the risk of osteonecrosis compared with the control group, whereas RR greater than 1 indicates animals with statin usage are at increased risk of osteonecrosis. The 95% CI reveals that the result is statistically significant when “1” is not included in the interval, and vice versa.

Significant protection by statins against ON was seen for all included species (Fig. 4) but not for gender (RR = 2.24, 1.62 to 3.09 versus RR = 2.25, 1.58 to 3.20, p = 0.98).

Fig. 4

Forest plot showing subgroup analysis based on species (M-H, Mantel–Haenszel; CI, confidence interval; df, degrees of freedom). Risk ratio (RR) on the left axis indicates statin usage decreases the risk of osteonecrosis compared with the control group, whereas RR greater than 1 indicates animals with statin usage are at increased risk of osteonecrosis. The 95% CI reveals that the result is statistically significant when “1” is not included in the interval, and vice versa.

Effect sizes were similar between studies where statin exposure was started at two weeks before methylprednisolone injection and simultaneously with methylprednisolone administration (Fig. 5). As to outcome measurement, experiments using radiographic examination of ON gave more conservative estimates of effect size (RR = 1.40, 0.97 to 2.02, Fig. 6) than those using histological examination (RR = 2.20, 1.77 to 2.75, Fig.6).

Fig. 5

Forest plot showing subgroup analysis based on treatment time point (M-H, Mantel–Haenszel; CI, confidence interval; df, degrees of freedom). Risk ratio (RR) on the right axis indicates statin usage decreases the risk of osteonecrosis compared with the control group, whereas RR greater than 1 indicates animals with statin usage are at increased risk of osteonecrosis. The 95% CI reveals that the result is statistically significant when “1” is not included in the interval, and vice versa.

Fig. 6

Forest plot showing subgroup analysis based on measurement (M-H, Mantel–Haenszel; CI, confidence interval; df, degrees of freedom). Risk ratio (RR) on the left axis indicates statin usage decreases the risk of osteonecrosis compared with the control group, whereas RR greater than 1 indicates animals with statin usage are at increased risk of osteonecrosis. The 95% CI reveals that the result is statistically significant when “1” is not included in the interval, and vice versa.

A funnel plot (Fig. 7) analysis showed no evident publication bias towards positive studies, further confirmed by Egger’s regression asymmetry test (p > 0.05).

Fig. 7

Funnel plots evaluating publication bias among included studies.

Discussion

This is the first meta-analysis to review the efficacy of statins in preventing GC induced ON in animal models. Our study indicated that animals with statin usage, either alone or combined with other treatment, were at decreased risk of developing GC-related ON. Moreover, subgroup analysis revealed that compared with statin alone, statin usage combined with other treatments significantly decreased the risk of ON (RR = 1.23, 95% CI 1.02 to 1.47). However, we could find no significant risk difference for different gender or for different treatment points (two weeks before or simultaneously with GC injection).

The overall risk estimate of statins in the present study supports the findings of previous work conducted by Pritchett¹¹ who found that only three patients (3/284, 1%) treated with statins developed ON, much less than the 3% to 20% incidence usually reported for patients receiving high-dose steroids, However, our findings do not correlate with those of the cohort study by Ajmal et al,¹⁰ in which 2881 renal transplantation patients were evaluated and 4.4% (15/338) of patients on statins developed ON compared with 7% (180/2543) of patients who were not on statins. The authors concluded that statin usage does not appear to lower the risk of ON. There are several possible explanations for the discrepancy in the results. Firstly, the dosage of GC administrated to patients following renal transplantation was uncertain at baseline. Higher doses, even of short duration, present greater risks.² Secondly, the indication for statin treatment was confined to patients with hypercholesterolaemia. And thirdly, the measure of ON was not reported and it is also unknown whether the measurement was taken identically for each patient.

In a randomised double-blinded placebo-controlled study, Lydonet, Schweitzer and Belmont³⁰ also found that the incidence of newly developed ON in patients with 40 mg daily of atorvastatin was not statistically different to that found in those administered hydroxychloroquine or bisphosphonates. However, it must be noted that the patients in the control group were administrated bisphosphonates, which may reduce the risk of developing ON to some degree.^4,26,28

Our results also revealed that statin usage in combination with bisphosphonate or anticoagulant significantly decreased the risk of ON, compared with statins alone, suggesting a protective role for bisphosphonates or anticoagulants against developing GC-induced ON. These findings have been supported elsewhere in the literature.^{14,20,28,29,31}

In this current study, we could find no significant difference in risk between animal gender. This differs from the published studies conducted by Ajmal et al,¹⁰ who found that male gender is associated with an increased risk (34%) of ON compared with female patients. The reason for this finding is not clear but it may be related to different mechanisms of fat metabolisms between genders.^32,33

With regard to animal species, risk estimate was lower in rabbits than in rats. To our knowledge, this has not been reported previously. A possible explanation for this might be that animal models used in Nozaki et al’s study¹³ consist of rats with a 50% incidence of spontaneous ON of the femoral head, rather than rats in healthy condition.

Both of the intervention procotols (statin exposure started at two weeks before versus simultaneously with methylprednisolone injection) demonstrated protective action against development of ON, but there was no significant difference in effect size between the two groups. To the best of our knowledge, this comparison has not been reported previously. A possible explanation for this finding might be that different statin dosage and species were used across studies. It also may be due to a possible threshold effect for development of ON.^14,29

As for assessment of ON, it was more conservatively estimated with the use of radiographic examination than with the use of histological examination. Our findings are consistent with those of Kang et al³⁴ who found no significant changes of ON examined using radiographs during two two to 12-week periods. Accordingly, the difference in risk estimate between various measurements could be explained by the lower detection rate of radiographic examination. Given the small sample size of animals examined using radiographs, however, the results should be considered with caution.

Several limitations must be considered in interpreting our findings. First, the time point at which the outcome was measured varied from study to study. However, ON was evaluated in all included studies at a time point more than two weeks after steroid injection, since two weeks is a time point that has been reported to be crucial in the development of ON.^7,16 Secondly, the sites measured for diagnosis of ON were not controlled for all included studies. The more sites per animal evaluated, the greater risk for the detection of ON. Thirdly, effect size might be weakened in animals with comorbidities Finally, the preventive effects of different statin dosage, and various types of statins on the development of ON, were not analysed.

In conclusion, the present study revealed that statin usage could reduce the risk of GC-related ON in animal models, suggesting statins are efficacious in preventing GC-induced ON to some degree in animals. However, while the overall results of this meta-analysis suggest that statins have substantial effect size, concerns should be raised that the true efficacy of statins might be substantially lower than reported here because of the possible influence of publication or other bias. Furthermore, given the limited application of experimental studies to humans, the heterogeneity among studies and lack of high quality evidence, the results should be extrapolated to the clinical setting with great caution.