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Experimental osteonecrosis induced by a combination of low-dose lipopolysaccharide and high-dose methylprednisolone in rabbits
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Joint Bone Spine 75 (2008) 573e578 http://france.elsevier.com/direct/BONSOI/
Original article
Experimental osteonecrosis induced by a combination of low-dose lipopolysaccharide and high-dose methylprednisolone in rabbits Xinghuo Wu, Shuhua Yang*, Deyu Duan, Yukun Zhang, Jing Wang Department of Orthopaedics, Union Hospital, Tongji Medical College, Science and Technology of Huazhong University, Wuhan 430022, China Accepted 21 November 2007 Available online 9 May 2008
Abstract Objectives: The pathogenetic mechanisms involved in steroid-induced osteonecrosis are poorly understood. Appropriate experimental models of the human disease are indispensable to the understanding of successful treatment modalities for osteonecrosis. Methods: In the present experiment we devised a novel rabbit model of steroid-induced osteonecrosis by use of two low-dose LPS and three high-dose MPS to investigate the development of osteonecrosis. Thirty eight rabbits were used and tissue assessments were performed on proximal third and distal condyles of femora and humeri obtained 6 weeks after the administration of LPS and MPS. MRI of these regions and intraosseous pressure of proximal femur were obtained at 0 and 6 weeks. Other assessments included serum plasminogen activator/inhibitor ratio, cholesterol level, LDL/HDL ratio, and triglyceride levels at various time points. Results: The study showed that with this osteonecrosis induction protocol there was low animal mortality (6.2%), high rate of osteonecrosis (90%), induction of thrombotic state, and hypercholesterol/lipidemia. Discussion: On the whole, this is a novel modified animal model of steroid associated osteonecrosis and it would be useful for elucidating the pathogenesis of steroid associated osteonecrosis and developing preventive and therapeutic strategies. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Steroid-associated osteonecrosis; Experimental; Lipopolysaccharide; Methylprednisolone
1. Introduction Osteonecrosis (ON) literally means ‘‘death of bone’’ (osteo ¼ bone, necrosis ¼ death), and it is now a commonly recognized disorder with significant morbidity. Osteonecrosis is caused by impaired blood supply to the bone, but it is not always clear what causes that impairment. Osteonecrosis is believed to be a multifactorial disease that is associated in some cases with both a genetic predilection and exposure to certain risk factors. These risk factors include corticosteroid use, alcohol intake, smoking, and various chronic diseases (renal disease, hematological disease, inflammatory bowel disease, post-organ transplantation, hypertension, and gout) [1]. One of the most common risk factors for osteonecrosis is the use
* Corresponding author. Tel.: þ86 027 85351689. E-mail address: [email protected] (S. Yang).
of corticosteroids, especially in high doses, is an independent variable. Corticosteroids are commonly used to treat inflammatory diseases such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, severe asthma, and vasculitis. Studies suggest that long-term use of oral or intravenous (IV) corticosteroids is associated with non-traumatic osteonecrosis. In order to deepen the understanding of the prevention, diagnosis and treatment of steroid-induced ON, it is important to establish an animal ON model with high reproducibility of the necrosis. Because the development of animal models is considered important to clarify the developmental mechanisms of osteonecrosis and to devise prophylactic strategies, various such models have been described, including the rabbit ON model produced by the Shwartzman reaction with two injections of 100 mg/kg body weight of lipopolysaccharide (LPS) [2] and a single IV injection of LPS of 10 mg/kg body weight [3]. Other currently used osteonecrosis models were IM weekly
1297-319X/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.jbspin.2007.11.004
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injections of 4 mg/kg of MPS [4,5] and a single IM injection of 20 mg/kg of MPS [6,7] with good reproducibility. The necrotic areas in steroid-treated rabbits with a Shwartzman reaction are often massive, extending from the epiphysis to the diaphysis of the femur. Corticosteroids reinforce the processes leading to thrombotic vascular occlusion by injuring endothelial cells and amplifying hypercoagulability. High-dose LPS combined with subsequent three injections of high-dose methylprednisolone (MPS) (H-LPS 2 þ H-MPS 3) could induce higher ON incidence (85%) but accompanied with high mortality of experimental animals (50%) [2]. However, as mentioned above, the developmental mechanisms of steroidinduced osteonecrosis are not yet clear, none of the available animals models being ideal. In testing intervention strategies for steroid-associated ON, there is a need to develop inductive protocols for establishing ON lesions with a high ON incidence yet low or even no mortality. Accordingly, we hypothesized that two injections of low-dose LPS combined with subsequent three injections of high-dose MPS (L-LPS 2 þ H-MPS 3) might induce a high incidence of ON but low or even no mortality in rabbits. For confirming the effectiveness of above purposed inductive protocol for establishment of a steroid-associated ON animal model, the present study was designed to use hematological, radiological, histological and biomechanical methods to investigate the ON incidence and mortality in a rabbit model. 2. Methods All experimental procedures adhered to the recommendations of Experimental Animal Center of Tongji medical College and the US Department of Health for the care and use of laboratory animals, and were approved by the Ethics Committee of our Tongji medical college.
at 70 C for evaluating pre-thrombotic status, including t-PA/PAI-I (ratio of tissue type plasminogen activator to plasminogen activator inhibitor) by enzyme-linked immunosorbent assay technique using corresponding mouse monoclonal anti-human antibodies (TC, Vienna, Austria). Other hematochemistry examinations were performed to estimate the plasma levels of cholesterol, triglycerides, and ratio of lowdensity lipoprotein cholesterol to high-density lipoprotein cholesterol (LDL/HDL cholesterol ratio). The total cholesterol, triglyceride, LDL and HDL were measured by enzymatic methods. 2.3. Magnetic resonance imaging (MRI) MRI was performed for bilateral proximal and distal femora and humeri before LPS injection and 6 weeks after the last injection of MPS, using a 1.5 T superconducting system (Siemens Magneton Vision). Under sedation with 10% Chloral Hydrate (3 ml/kg, i.m.), the rabbits were placed in supine with the lower limb flexed and fixed by adhesive tape. An extremity coil (transmit-receive surface coil) was used on the target site. Preliminary sagittal and oblique axial images were obtained to define the femoral longitudinal axis. Imaging parameters were as follows: T1-weighted MRI images (T1W, repetition time [TR]/echo time [TE] ¼ 490/14 ms), T2-weighted MRI images (T2W, repetition time [TR]/echo time [TE] ¼ 2632/96 ms), a section thickness of 3.0 mm, intersection gap of 1 mm, and imaging matrix of 512 512. 2.4. Measurements of intraosseous pressure
Thirty-eight 28-week-old male New-Zealand white rabbits with body weight of 3.5e4.5 kg were housed at the Experimental Animal Center of the investigators’ hospital and received a standard laboratory diet and water ad libitum. Thirty-two rabbits were assigned into the treatment group (LM group), and six rabbits were used for control group (CON group). In LM group, two injections of 10 mg/kg body weight of LPS (Sigma) were given intravenously, and then three injections of 20 mg/kg body weight of MPS (Pfizer) were given intramuscularly, at a time interval of 24 h. In addition, six rabbits of CON group were injected once with physiologic saline (PS; 1 ml/kg body weight) into the right gluteus medius muscle as a control group. Two out of 32 rabbits died of pneumonia within 4 days after last injection of MPS.
Intraosseous pressure in the proximal femur was determined in all rabbits before treatment (0 weeks) and at 6 weeks after the steroid injection. Pentobarbital sodium (30 mg/kg body weight) was given intravenously for anesthesia. A standard lateral approach to expose the proximal aspect of the right femur just distal to the greater trochanter was made under sterile conditions. A drill hole (1.0 mm in diameter) was made from the outer cortex 2.5 cm distal to the proximal end of the greater trochanter, where ON is seen in this animal model. After allowing some back-bleeding, an 18-gauge polyethylene catheter was inserted tightly 5 mm into the hole and connected to a pressure transducer by way of a polyethylene tube filled with heparinized saline. The pressure transducer was connected to a blood pressure amplifier (FY-2; ChengDu; China) for measurement of the intraosseous pressure. Because initial pressures varied slightly and several minutes were required before a steady reading was obtained, the recording at 5 min was taken as the intraosseous pressure [3]. The systemic arterial pressure was measured simultaneously via an arterial catheter in the rabbit’s ear.
2.2. Hematological examination
2.5. Tissue preparation
Blood sample was collected in a fasting state from each rabbit through the auricular arteries at 0, 1, 2, 4 and 6 weeks after the steroid injection. Half of the plasma was then stored
After measurements of MRI and intraosseous pressure, the animals were anesthetized with an intravenous injection of pentobarbital sodium (30 mg/kg of body weight), and were
2.1. Animals, grouping, and treatment
X. Wu et al. / Joint Bone Spine 75 (2008) 573e578
then killed by exsanguination via an aortectomy. For light microscopic examinations, both proximal and distal femora and humeri (a total of eight bone samples) were obtained at the time of death and were fixed for 1 week with 10% formalin0.1 mol/L phosphate buffer (pH 7.4). The bone samples were decalcified with 25% formic acid for 3 days and were then neutralized with 0.35 mol/L sodium sulfate for 3 days. The specimens were embedded in paraffin, cut into 4 mm sections, and then stained with hematoxylin and eosin. The bone samples were cut along the coronal plane in the proximal onethird and axial plane in the distal part (condyle). 2.6. Evaluation of ON Whole areas of the proximal one-thirds and distal condyles of both femora and humeri, a total of eight regions, were histopathologically examined for the presence of ON. For each group, the frequency of ON, its location, the number of necrotic foci, the size of the ON area and its histology were examined. The presence of fat emboli was also determined in each rabbit. Diagnosis of ON was blindly made by three authors on the basis of diffuse presence of empty lacunae or pyknotic nuclei of osteocytes in the bone trabeculae, accompanied by surrounding bone marrow cell necrosis [8,9]. The specimen was determined as having a necrotic focus when all three pathologists evaluated it as having necrosis. All rabbits that had at least one osteonecrotic lesion out of eight areas examined were considered to be rabbits with ON (ONþ rabbits), while those with no osteonecrotic lesions were considered to be rabbits without ON (ON rabbits). 2.7. Calculation of the size of bone marrow fat cells As described in a previous report, the fat cell size of each rabbit was calculated as the average of the Feret’s diameter of all bone marrow fat cells with clearly defined profile in 4 randomly selected fields (up-down-left-right) of each dissected part of proximal one-thirds and distal condyles of both femora and humeri (32 fields for 8 dissected parts from each rabbit). Fat cells that had undergone necrosis were excluded from imaging analysis. The histological sections were digitized into a microscope imaging system for quantification using image analysis software (HPIAS-1000) [10,11]. The corresponding morphometric data were processed automatically by the computer system. 2.8. Statistical analysis The incidence of ON was defined as the number of ONþ rabbits divided by total number of rabbits in the group. Intraosseous pressures, sizes of marrow fat cells, and hematologic data in rabbits with or without ON were compared using Student’s or Welch’s t-test. For correlations between the ratio of tPA/PAI-I and the LDL/HDL cholesterol ratio, we used the Spearman correlation coefficient. All values in the figures
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and text were expressed as mean standard deviation (SD). A P-value of <0.05 was considered statistically significant. 3. Results 3.1. MRI scanning Six weeks after last injection of MPS, MRI findings showed irregular low signal on Tl-weighted images and irregular low or high signal on T2-weighted image in 73.3% (22/30) LM group rabbits, whereas no significant change was found in CON group rabbits (Fig. S1; see the supplementary material associated with this article online). 3.2. Histopathologic features Osteonecrosis was recognized as a zonal tissue phenomenon. Histologically, ON lesions showed an accumulation of bone marrow cell debris, bone trabeculae demonstrating empty lacunae and/or ghost nuclei in the lacunae, and an increase in the fat cells of the bone marrow. Reparative appositional bone formation also presented around the necrotic bone, but not significant (Fig. S2). There was widespread lipid deposition in the osteocyte lacunae of the ONþ rabbits. Neither subchondral nor intraosseous collapse could be seen in any femoral or humeral bones in LM group rabbits. 3.3. Prevalence of osteonecrosis and calculation of fat cell size A total of 90% of the rabbits (27/30) in LM group developed ON 6 weeks after last injection of MPS, while no ON lesion was found in CON group rabbits. In a location-specific histopathological examination for the femora, 73.3% (22/30) were found having developed ON in the proximal femora while 26.7% (8/30) developed ON in the distal femora. As for the humeri, 20% (6/30) were found having developed ON in the proximal humeri while no ON lesions observed in the distal humeri. The necrotic areas of both the femoral and humeral bones were seen mainly in the metaphysis. The mortality of experimental animals was 6.2% (2/32). In CON group rabbits, the marrow fat cell diameter was (34.8 1.4 mm), and the marrow fat cells were almost homogeneous in size. In LM group rabbits, there were a larger number of enlarged marrow fat cells. The marrow fat cell diameter was (64.7 6.6 mm) in the ONþ rabbits and was (51.4 5.5 mm) in the ON rabbits. The diameter of the marrow fat cells was significantly larger in LM group rabbits than in CON group rabbits (P < 0.01) (Fig. S3). 3.4. Measurements of intraosseous pressure and systemic arterial pressures The values of intraosseous pressure in the proximal femur and the systemic arterial pressure were summarized in Tables 1 and 2. Intraosseous pressure in LM group rabbits both with and without ON increased significantly 6 weeks after administration
X. Wu et al. / Joint Bone Spine 75 (2008) 573e578
576 Table 1 Intraosseous pressure in the proximal femur (mmHg) Group
Before administration
6 weeks after administration
P-value
Rabbits with ON (N ¼ 27) Rabbits without ON (N ¼ 3) Control (N ¼ 6)
17.6 6.8 15.8 3.6 16.4 3.6
52.6 12.8* 32.2 5.4* 15.8 4.2
0.000 0.004 0.848
KEY: ON, osteonecrosis. Data are the mean standard deviation. Compared to before administration, *P < 0.01.
(P < 0.01). The pressure was significantly higher in ONþ rabbits than in ON rabbits. There were no significant differences of systemic arterial pressures between either 0 weeks or 6 weeks in LM group and CON group rabbits (P > 0.05). 3.5. Hematological findings In LM group rabbits, a significant decrease in the ratio of tPA/PAI-I was found from baseline at week 1 after MPS injection and returned to the baseline thereafter (P < 0.05). Plasma levels of cholesterol and triglyceride in LM group rabbits both with and without ON increased significantly at 1 week (P < 0.05), peaked at 2 weeks, and these high levels were maintained during the observation periods, which were consistent with the change of LDL/HDL ratio. No detectable changes were found in the hematological examinations for the control rabbits during the entire study period (P > 0.05) (Fig. S4). 4. Discussion The development of ON is not caused by a single precipitating event. There are multiple theories about the pathogenesis of steroid-induced ON, and most of these theories are mutually supportive and not exclusive. For explanation of the pathogenesis of the steroid-induced osteonecrosis, various theories have been proposed regarding the developmental mechanisms of steroid-induced osteonecrosis, including the increased size and number of fatty cells, increased intraosseous pressure, fatty degeneration of osteocytes, fat embolism, and extraosseous arterial occlusion [5,9,12,13]. In addition, coagulation abnormalities and hyperlipidaemia were among the postulated pathogenic mechanisms for ON development. Jones et al. [14] proposed a fat embolization theory whereby hyperlipidaemia and associated abnormalities in the blood coagulation system were a possible mechanism of ON. However, the precise mechanism of steroid-induced osteonecrosis Table 2 The systemic arterial pressures (mmHg) Group
Before administration
6 weeks after administration
Rabbits with ON (N ¼ 27) Rabbits without ON (N ¼ 3) Control (N ¼ 6)
84.2 9.4 82.6 8.4 82.2 6.2
90.6 11.2 88.4 8.8 87.2 7.6
KEY: ON, osteonecrosis. Data are the mean standard deviation.
remains unclear. Although, the pathogenesis of osteonecrosis is controversial and multifactorial, the glucocorticoid therapy is the most important factor contributing to the lesion. Corticosteroids are known to induce not only hyperlipidaemia but also a hypercoagulable and hypofibrinolytic state of plasma. Both hypercoagulability and hypofibrinolysis have been reported in human non-traumatic ON cases, including a high plasminogen activator inhibitor (PAI-I) and the presence of anticardiolipin antibodies [15,16]. These factors enhance the coagulability predisposing to thrombosis and may cause damage to blood vessels. In vitro study of others supported the mechanism that LPS was able to induce hypercoagulable and/or hypofibrinolytic state partially through reduction of endothelia expression of thrombomodulin (TM) [17]. In fact, endothelial TM was an important intermediary substance, which directly or indirectly participated in both inhibition of coagulation and promotion of fibrinolysis, and the ratio of t-PA/PAI-I reflected balance between anti-fibrinolysis and pro-fibrinolysis [18e20]. There were significant changes in the coagulation system in rabbit ON model, which enhanced the coagulability predisposing to thrombosis and might cause damage to blood vessels. In the present study for intravascular mechanistic investigation, tPA/PAI-I ratio (an indicator of hypofibrinolysis) were evaluated etiologically. The results showed that hypofibrinolysis was synergistically induced by a combination of LPS and MPS injection. Abnormal lipid metabolism is related to steroid-induced ON of the femoral head, and hyperlipaemia and increased free fatty acids are considered to be important risk factors [5,14,21,22]. The disturbed lipid metabolism of the rabbits is reflected in hypertrophy of bone marrow adipocytes and an elevated ratio of low-density to high-density lipoprotein cholesterol (LDL/HDL ratio). The lipid transport resulting form the higher LDL/HDL ratio might have induced intraosseous hyperlipidemic state, which eventually leads to extravascular marrow compression from lipid deposit. This lipid-induced hypertrophy of the fat cells cannot expand the marrow cavity within the inflexible osseous cage. Steroid administration increases the intraosseous pressure in spite of decreased blood flow, and the intraosseous hypertension inhibits regeneration of the blood vessels [4,23,24]. Then ischaemic events might result from vascular interruption through thrombi, lipid emboli or high intraosseous pressure associated with bone marrow fat-cell enlargement; these would subsequently lead ON development [25e27]. Moreover, steroid-induced cholesterol deposition reduces the fluidity and permeability of the cell membranes, contributing to the death of the osteocytes. Treatment of rabbits with the lipid-clearing clofibrate reduces steroid-induced hepatosteatosis, hyperlipidemia, and accumulation of lipids in osteocytes and protects the femoral head against necrosis [28,29]. In our haematological study for extravascular mechanistic investigation, serum lipid levels increased sharply 1 week after the initial steroid administration, both LDL/HDL cholesterol ratio and fat-cell size were significantly higher in LM group rabbits. These data suggested that ON was closely related to serum lipid levels (high LDL/HDL
X. Wu et al. / Joint Bone Spine 75 (2008) 573e578
cholesterol ratio), and we regarded these levels as high enough to accelerate the coagulation system. MRI can show avascular necrosis in its earliest stages and provide a picture of the area affected and the bone rebuilding process. In the present study, MRI provided a picture of the area affected and the bone rebuilding process, of 73.3% (22/ 30) rabbits with osteonecrosis. The end-point evaluations in the present study demonstrated that the proposed treatment protocol induced a higher incidence of ON of 90% and lower mortality of 6.2%. In fact, several authors using similar designs have obtained comparable results. Irisa et al. [3] documented a high incidence of osteonecrosis (77%) and low mortality (11%) in rabbits that were treated with only lowdose LPS. Miyanishi et al. [26] documented a similar incidence of osteonecrosis (76%) but no mortality in rabbits that were treated with once injection of MPS (20 mg/kg). Recently, Qin et al. [30] documented an even higher rate of osteonecrosis (93%) and no mortality in rabbits that were treated with one injection of low-dose LPS followed by three doses of MPS (L-LPS 1 þ H-MPS 3). In contrast, Yamamoto et al. [2] used two injections of high-dose lipopolysaccharide (LPS) combined with subsequent three injections of highdose methylprednisolone (MPS) (H-LPS 2 þ H-MPS 3), which induced higher ON incidence (85%) but accompanied with high mortality of experimental animals (50%). According to the etiology of steroid-associated ON, the lower ON incidence by the protocol (H-MPS 1) was due to lack of LPSinduced pre-thrombosis state before steroid treatment. On the other hand, the higher mortality by the protocol (HLPS 2 þ H-MPS 3) was due to high-dose-LPS-induced severe endotoxin shock. The present study developed a protocol by modifying the reported protocol (H-LPS 2 þ HMPS 3) by replacing the two high-dose LPS injection with two low-dose LPS injection. It (L-LPS 2 þ HMPS 3) showed a higher incidence of ON lesion (90%) and lower mortality (6.2%) in rabbits as compared with the previously published protocol (H-LPS 2 þ H-MPS 3) due to the avoidance of severe LPS-induced shock by lowering the given LPS dose. As comparison, a better understanding of the influence of LPS-MPS on the ON incidence and animal mortality is needed, and likely, differences do exist. The present experimental study showed that our experimental protocol with low-dose LPS and subsequent pulsed highdose MPS injections was an effective one to induce ON in rabbits with high incidence and low mortality. Osteonecrosis of patients and animals is akin in some aspects but diverges in others. We demonstrated many histopathological and pathogenetic features of steroid-induced ON in rabbits similar to those observed in human ON prior to subchondral bone collapse. First, ON lesions in these rabbit models present histological characteristics similar to human ON, i.e. osteocytic death surrounded by necrotic bone marrow with or without repair tissue [31,32]. Second, significantly increased intraosseous pressure was reported in patients with ON [8,13]. Third, a higher lipid deposition was noted in human osteonecrotic femoral heads [27,33]. Fourth, hypercoagulability and hypofibrinolysis have been reported to play etiologic roles in
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human ON [15,20,27]. These similarities suggested significant relevance of this animal model to human ON. In summary, this model would be useful for elucidating the pathogenesis of steroid-associated ON. Both high ON incidence and low mortality in rabbits treated with this inductive protocol suggested its effectiveness for developing preventive and therapeutic strategies. Acknowledgements The authors wish to thank Mulan Yang, Jianzhong Fan, and Qigui Zou for their very helpful technical assistance. This study was supported by: National Natural Science Foundation of China (No. 3017094). Appendix A. Supplementary data Supplementary material (Figs. S1eS4) associated with this article can be found at http://www.sciencedirect.com, at doi:10.1016/j.jbspin.2007.11.004. References [1] Mont MA, Jones LC, Hungerford DS. Nontraumatic osteonecrosis of the femoral head: ten years later. J Bone Joint Surg Am 2006;88: 1117e32. [2] Yamamoto T, Hirano K, Tsutsui H, et al. Corticosteroid enhances the experimental induction of osteonecrosis in rabbits with Shwartzman reaction. Clin Orthop Relat Res 1995;316:235e43. [3] Irisa T, Yamamoto T, Miyanishi K, et al. Osteonecrosis induced by a single administration of low-dose lipopolysaccharide in rabbits. Bone 2001;286:641e9. [4] Yamamoto T, Irisa T, Sugioka Y, et al. Effects of pulse methylprednisolone on bone and marrow tissues: corticosteroid-induced osteonecrosis in rabbits. Arthritis Rheum 1997;40:2055e64. [5] Kabata T, Kubo T, Matsumoto T, et al. Onset of steroid-induced osteonecrosis in rabbits and its relationship to hyperlipaemia and increased free fatty acids. Rheumatology 2005;44:1233e7. [6] Ichiseki T, Matsumoto T, Nishino M, et al. Oxidative stress and vascular permeability in steroid-induced osteonecrosis model. J Orthop Sci 2004;9:509e15. [7] Ichiseki T, Kaneuji A, Katsuda S, Ueda Y, Sugimori T, Matsumoto T. DNA oxidation injury in bone early after steroid administration is involved in the pathogenesis of steroid-induced osteonecrosis. Rheumatology 2005;44:456e60. [8] Yamamoto T, DiCarlo EF, Bullough PG. The prevalence and clinicopathological appearance of extension of osteonecrosis in the femoral head. J Bone Joint Surg Br 1999;81:328e32. [9] Motomura G, Yamamoto T, Miyanishi K, et al. Bone marrow fat-cell enlargement in early steroid-induced osteonecrosisda histomorphometric study of autopsy cases. Pathol Res Pract 2005;200:807e11. [10] Shaw SL, Salmon ED, Quatrano RS. Digital photography for the light microscope: results with a gated, video-rate CCD camera and NIH-image software. Biotechniques 1995;19:946e55. [11] Miyanishi K, Yamamoto T, Irisa T, et al. Bone marrow fat cell enlargement and a rise in intraosseous pressure in steroid-treated rabbits with osteonecrosis. Bone 2002;30:185e90. [12] Arlet J. Nontraumatic avascular necrosis of the femoral head. Past, present, and future. Clin Orthop Relat Res 1999;277:12e21. [13] Mankin HJ. Nontraumatic necrosis of bone (osteonecrosis). New Engl J Med 1992;326:1473e9. [14] Jones Jr JP. Fat embolism and osteonecrosis. Orthop Clin North Am 1985;16:595e633.
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[15] Glueck CJ, Freiberg R, Glueck HI, et al. Hypofibrinolysis: a common, major cause of osteonecrosis. Am J Hematol 1994;45:156e66. [16] Korompilias AV, Gilkeson GS, Ortel TL, et al. Anticardiolipin antibodies and osteonecrosis of the femoral head. Clin Orthop Relat Res 1997;345:174e80. [17] Glueck CJ, Freiberg RA, Wang P. Role of thrombosis in osteonecrosis. Curr Hematol Rep 2003;2:417e22. [18] Moore KL, Andreoli SP, Esmon NL, et al. Endotoxin enhances tissue factor and suppresses thrombomodulin expression of human vascular endothelium in vitro. J Clin Invest 1987;79:124e30. [19] Glueck CJ, Freiberg RA, Fontaine RN, et al. Hypofibrinolysis, thrombophilia, osteonecrosis. Clin Orthop Relat Res 2001;386:19e33. [20] Jones LC, Mont MA, Le TB, et al. Procoagulants and osteonecrosis. J Rheumatol 2003;30:783e91. [21] Zalavras C, Dailiana Z, Elisaf M, et al. Potential aetiological factors concerning the development of osteonecrosis of the femoral head. Eur J Clin Invest 2000;30:215e21. [22] Miyanishi K, Yamamoto T, Irisa T, et al. Effects of different corticosteroids on the development of osteonecrosis in rabbits. Rheumatology (Oxford) 2005;44:332e6. [23] Wang GJ, Cui Q, Balian G. The pathogenesis and prevention of steroid induced osteonecrosis. Clin Orthop 2000;370:295e310. [24] Boss JH, Misselevich I. Osteonecrosis of the femoral head of laboratory animals: the lessons learned from a comparative study of osteonecrosis in man and experimental animals. Vet Pathol 2003;40:345e54.
[25] Lieberman JR, Berry DJ, Mont MA, et al. Osteonecrosis of the hip: management in the 21st century. Instr Course Lect 2003;52:337e55. [26] Miyanishi K, Yamamoto T, Irisa T, et al. A high low-density lipoprotein cholesterol to high-density lipoprotein cholesterol ratio as a potential risk factor for corticosteroid-induced osteonecrosis in rabbits. Rheumatology 2001;40:196e201. [27] Jones Jr JP. Fat embolism, intravascular coagulation and osteonecrosis. Clin Orthop Relat Res 1993;292:294e308. [28] Nishimura T, Matsumoto T, Nishino M, et al. Histopathologic study of veins in steroid treated rabbits. Clin Orthop 1997;334:37e42. [29] Aldridge 3rd JM, Urbaniak JR. Avascular necrosis of the femoral head: etiology, pathophysiology, classification, and current treatment guidelines. Am J Orthop 2004;33:327e32. [30] Qin L, Zhang G, Sheng H, et al. Multiple bioimaging modalities in evaluation of an experimental osteonecrosis induced by a combination of lipopolysaccharide and methylprednisolone. Bone 2006;39:863e71. [31] Plenk Jr H, Gstettner M, Grossschmidt K, et al. Magnetic resonance imaging and histology of repair in femoral head osteonecrosis. Clin Orthop Relat Res 2001;386:42e53. [32] Chernetsky SG, Mont MA, LaPorte DM, et al. Pathologic features in steroid and nonsteroid associated osteonecrosis. Clin Orthop Relat Res 1999;368:149e61. [33] Boskey AL, Raggio CL, Bullough PG, et al. Changes in the bone tissue lipids in persons with steroid- and alcohol-induced osteonecrosis. Clin Orthop Relat Res 1983;172:289e95.
Joint Bone Spine 75 (2008) 573e578 http://france.elsevier.com/direct/BONSOI/
Original article
Experimental osteonecrosis induced by a combination of low-dose lipopolysaccharide and high-dose methylprednisolone in rabbits Xinghuo Wu, Shuhua Yang*, Deyu Duan, Yukun Zhang, Jing Wang Department of Orthopaedics, Union Hospital, Tongji Medical College, Science and Technology of Huazhong University, Wuhan 430022, China Accepted 21 November 2007 Available online 9 May 2008
Abstract Objectives: The pathogenetic mechanisms involved in steroid-induced osteonecrosis are poorly understood. Appropriate experimental models of the human disease are indispensable to the understanding of successful treatment modalities for osteonecrosis. Methods: In the present experiment we devised a novel rabbit model of steroid-induced osteonecrosis by use of two low-dose LPS and three high-dose MPS to investigate the development of osteonecrosis. Thirty eight rabbits were used and tissue assessments were performed on proximal third and distal condyles of femora and humeri obtained 6 weeks after the administration of LPS and MPS. MRI of these regions and intraosseous pressure of proximal femur were obtained at 0 and 6 weeks. Other assessments included serum plasminogen activator/inhibitor ratio, cholesterol level, LDL/HDL ratio, and triglyceride levels at various time points. Results: The study showed that with this osteonecrosis induction protocol there was low animal mortality (6.2%), high rate of osteonecrosis (90%), induction of thrombotic state, and hypercholesterol/lipidemia. Discussion: On the whole, this is a novel modified animal model of steroid associated osteonecrosis and it would be useful for elucidating the pathogenesis of steroid associated osteonecrosis and developing preventive and therapeutic strategies. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Steroid-associated osteonecrosis; Experimental; Lipopolysaccharide; Methylprednisolone
1. Introduction Osteonecrosis (ON) literally means ‘‘death of bone’’ (osteo ¼ bone, necrosis ¼ death), and it is now a commonly recognized disorder with significant morbidity. Osteonecrosis is caused by impaired blood supply to the bone, but it is not always clear what causes that impairment. Osteonecrosis is believed to be a multifactorial disease that is associated in some cases with both a genetic predilection and exposure to certain risk factors. These risk factors include corticosteroid use, alcohol intake, smoking, and various chronic diseases (renal disease, hematological disease, inflammatory bowel disease, post-organ transplantation, hypertension, and gout) [1]. One of the most common risk factors for osteonecrosis is the use
* Corresponding author. Tel.: þ86 027 85351689. E-mail address: [email protected] (S. Yang).
of corticosteroids, especially in high doses, is an independent variable. Corticosteroids are commonly used to treat inflammatory diseases such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, severe asthma, and vasculitis. Studies suggest that long-term use of oral or intravenous (IV) corticosteroids is associated with non-traumatic osteonecrosis. In order to deepen the understanding of the prevention, diagnosis and treatment of steroid-induced ON, it is important to establish an animal ON model with high reproducibility of the necrosis. Because the development of animal models is considered important to clarify the developmental mechanisms of osteonecrosis and to devise prophylactic strategies, various such models have been described, including the rabbit ON model produced by the Shwartzman reaction with two injections of 100 mg/kg body weight of lipopolysaccharide (LPS) [2] and a single IV injection of LPS of 10 mg/kg body weight [3]. Other currently used osteonecrosis models were IM weekly
1297-319X/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.jbspin.2007.11.004
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injections of 4 mg/kg of MPS [4,5] and a single IM injection of 20 mg/kg of MPS [6,7] with good reproducibility. The necrotic areas in steroid-treated rabbits with a Shwartzman reaction are often massive, extending from the epiphysis to the diaphysis of the femur. Corticosteroids reinforce the processes leading to thrombotic vascular occlusion by injuring endothelial cells and amplifying hypercoagulability. High-dose LPS combined with subsequent three injections of high-dose methylprednisolone (MPS) (H-LPS 2 þ H-MPS 3) could induce higher ON incidence (85%) but accompanied with high mortality of experimental animals (50%) [2]. However, as mentioned above, the developmental mechanisms of steroidinduced osteonecrosis are not yet clear, none of the available animals models being ideal. In testing intervention strategies for steroid-associated ON, there is a need to develop inductive protocols for establishing ON lesions with a high ON incidence yet low or even no mortality. Accordingly, we hypothesized that two injections of low-dose LPS combined with subsequent three injections of high-dose MPS (L-LPS 2 þ H-MPS 3) might induce a high incidence of ON but low or even no mortality in rabbits. For confirming the effectiveness of above purposed inductive protocol for establishment of a steroid-associated ON animal model, the present study was designed to use hematological, radiological, histological and biomechanical methods to investigate the ON incidence and mortality in a rabbit model. 2. Methods All experimental procedures adhered to the recommendations of Experimental Animal Center of Tongji medical College and the US Department of Health for the care and use of laboratory animals, and were approved by the Ethics Committee of our Tongji medical college.
at 70 C for evaluating pre-thrombotic status, including t-PA/PAI-I (ratio of tissue type plasminogen activator to plasminogen activator inhibitor) by enzyme-linked immunosorbent assay technique using corresponding mouse monoclonal anti-human antibodies (TC, Vienna, Austria). Other hematochemistry examinations were performed to estimate the plasma levels of cholesterol, triglycerides, and ratio of lowdensity lipoprotein cholesterol to high-density lipoprotein cholesterol (LDL/HDL cholesterol ratio). The total cholesterol, triglyceride, LDL and HDL were measured by enzymatic methods. 2.3. Magnetic resonance imaging (MRI) MRI was performed for bilateral proximal and distal femora and humeri before LPS injection and 6 weeks after the last injection of MPS, using a 1.5 T superconducting system (Siemens Magneton Vision). Under sedation with 10% Chloral Hydrate (3 ml/kg, i.m.), the rabbits were placed in supine with the lower limb flexed and fixed by adhesive tape. An extremity coil (transmit-receive surface coil) was used on the target site. Preliminary sagittal and oblique axial images were obtained to define the femoral longitudinal axis. Imaging parameters were as follows: T1-weighted MRI images (T1W, repetition time [TR]/echo time [TE] ¼ 490/14 ms), T2-weighted MRI images (T2W, repetition time [TR]/echo time [TE] ¼ 2632/96 ms), a section thickness of 3.0 mm, intersection gap of 1 mm, and imaging matrix of 512 512. 2.4. Measurements of intraosseous pressure
Thirty-eight 28-week-old male New-Zealand white rabbits with body weight of 3.5e4.5 kg were housed at the Experimental Animal Center of the investigators’ hospital and received a standard laboratory diet and water ad libitum. Thirty-two rabbits were assigned into the treatment group (LM group), and six rabbits were used for control group (CON group). In LM group, two injections of 10 mg/kg body weight of LPS (Sigma) were given intravenously, and then three injections of 20 mg/kg body weight of MPS (Pfizer) were given intramuscularly, at a time interval of 24 h. In addition, six rabbits of CON group were injected once with physiologic saline (PS; 1 ml/kg body weight) into the right gluteus medius muscle as a control group. Two out of 32 rabbits died of pneumonia within 4 days after last injection of MPS.
Intraosseous pressure in the proximal femur was determined in all rabbits before treatment (0 weeks) and at 6 weeks after the steroid injection. Pentobarbital sodium (30 mg/kg body weight) was given intravenously for anesthesia. A standard lateral approach to expose the proximal aspect of the right femur just distal to the greater trochanter was made under sterile conditions. A drill hole (1.0 mm in diameter) was made from the outer cortex 2.5 cm distal to the proximal end of the greater trochanter, where ON is seen in this animal model. After allowing some back-bleeding, an 18-gauge polyethylene catheter was inserted tightly 5 mm into the hole and connected to a pressure transducer by way of a polyethylene tube filled with heparinized saline. The pressure transducer was connected to a blood pressure amplifier (FY-2; ChengDu; China) for measurement of the intraosseous pressure. Because initial pressures varied slightly and several minutes were required before a steady reading was obtained, the recording at 5 min was taken as the intraosseous pressure [3]. The systemic arterial pressure was measured simultaneously via an arterial catheter in the rabbit’s ear.
2.2. Hematological examination
2.5. Tissue preparation
Blood sample was collected in a fasting state from each rabbit through the auricular arteries at 0, 1, 2, 4 and 6 weeks after the steroid injection. Half of the plasma was then stored
After measurements of MRI and intraosseous pressure, the animals were anesthetized with an intravenous injection of pentobarbital sodium (30 mg/kg of body weight), and were
2.1. Animals, grouping, and treatment
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then killed by exsanguination via an aortectomy. For light microscopic examinations, both proximal and distal femora and humeri (a total of eight bone samples) were obtained at the time of death and were fixed for 1 week with 10% formalin0.1 mol/L phosphate buffer (pH 7.4). The bone samples were decalcified with 25% formic acid for 3 days and were then neutralized with 0.35 mol/L sodium sulfate for 3 days. The specimens were embedded in paraffin, cut into 4 mm sections, and then stained with hematoxylin and eosin. The bone samples were cut along the coronal plane in the proximal onethird and axial plane in the distal part (condyle). 2.6. Evaluation of ON Whole areas of the proximal one-thirds and distal condyles of both femora and humeri, a total of eight regions, were histopathologically examined for the presence of ON. For each group, the frequency of ON, its location, the number of necrotic foci, the size of the ON area and its histology were examined. The presence of fat emboli was also determined in each rabbit. Diagnosis of ON was blindly made by three authors on the basis of diffuse presence of empty lacunae or pyknotic nuclei of osteocytes in the bone trabeculae, accompanied by surrounding bone marrow cell necrosis [8,9]. The specimen was determined as having a necrotic focus when all three pathologists evaluated it as having necrosis. All rabbits that had at least one osteonecrotic lesion out of eight areas examined were considered to be rabbits with ON (ONþ rabbits), while those with no osteonecrotic lesions were considered to be rabbits without ON (ON rabbits). 2.7. Calculation of the size of bone marrow fat cells As described in a previous report, the fat cell size of each rabbit was calculated as the average of the Feret’s diameter of all bone marrow fat cells with clearly defined profile in 4 randomly selected fields (up-down-left-right) of each dissected part of proximal one-thirds and distal condyles of both femora and humeri (32 fields for 8 dissected parts from each rabbit). Fat cells that had undergone necrosis were excluded from imaging analysis. The histological sections were digitized into a microscope imaging system for quantification using image analysis software (HPIAS-1000) [10,11]. The corresponding morphometric data were processed automatically by the computer system. 2.8. Statistical analysis The incidence of ON was defined as the number of ONþ rabbits divided by total number of rabbits in the group. Intraosseous pressures, sizes of marrow fat cells, and hematologic data in rabbits with or without ON were compared using Student’s or Welch’s t-test. For correlations between the ratio of tPA/PAI-I and the LDL/HDL cholesterol ratio, we used the Spearman correlation coefficient. All values in the figures
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and text were expressed as mean standard deviation (SD). A P-value of <0.05 was considered statistically significant. 3. Results 3.1. MRI scanning Six weeks after last injection of MPS, MRI findings showed irregular low signal on Tl-weighted images and irregular low or high signal on T2-weighted image in 73.3% (22/30) LM group rabbits, whereas no significant change was found in CON group rabbits (Fig. S1; see the supplementary material associated with this article online). 3.2. Histopathologic features Osteonecrosis was recognized as a zonal tissue phenomenon. Histologically, ON lesions showed an accumulation of bone marrow cell debris, bone trabeculae demonstrating empty lacunae and/or ghost nuclei in the lacunae, and an increase in the fat cells of the bone marrow. Reparative appositional bone formation also presented around the necrotic bone, but not significant (Fig. S2). There was widespread lipid deposition in the osteocyte lacunae of the ONþ rabbits. Neither subchondral nor intraosseous collapse could be seen in any femoral or humeral bones in LM group rabbits. 3.3. Prevalence of osteonecrosis and calculation of fat cell size A total of 90% of the rabbits (27/30) in LM group developed ON 6 weeks after last injection of MPS, while no ON lesion was found in CON group rabbits. In a location-specific histopathological examination for the femora, 73.3% (22/30) were found having developed ON in the proximal femora while 26.7% (8/30) developed ON in the distal femora. As for the humeri, 20% (6/30) were found having developed ON in the proximal humeri while no ON lesions observed in the distal humeri. The necrotic areas of both the femoral and humeral bones were seen mainly in the metaphysis. The mortality of experimental animals was 6.2% (2/32). In CON group rabbits, the marrow fat cell diameter was (34.8 1.4 mm), and the marrow fat cells were almost homogeneous in size. In LM group rabbits, there were a larger number of enlarged marrow fat cells. The marrow fat cell diameter was (64.7 6.6 mm) in the ONþ rabbits and was (51.4 5.5 mm) in the ON rabbits. The diameter of the marrow fat cells was significantly larger in LM group rabbits than in CON group rabbits (P < 0.01) (Fig. S3). 3.4. Measurements of intraosseous pressure and systemic arterial pressures The values of intraosseous pressure in the proximal femur and the systemic arterial pressure were summarized in Tables 1 and 2. Intraosseous pressure in LM group rabbits both with and without ON increased significantly 6 weeks after administration
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576 Table 1 Intraosseous pressure in the proximal femur (mmHg) Group
Before administration
6 weeks after administration
P-value
Rabbits with ON (N ¼ 27) Rabbits without ON (N ¼ 3) Control (N ¼ 6)
17.6 6.8 15.8 3.6 16.4 3.6
52.6 12.8* 32.2 5.4* 15.8 4.2
0.000 0.004 0.848
KEY: ON, osteonecrosis. Data are the mean standard deviation. Compared to before administration, *P < 0.01.
(P < 0.01). The pressure was significantly higher in ONþ rabbits than in ON rabbits. There were no significant differences of systemic arterial pressures between either 0 weeks or 6 weeks in LM group and CON group rabbits (P > 0.05). 3.5. Hematological findings In LM group rabbits, a significant decrease in the ratio of tPA/PAI-I was found from baseline at week 1 after MPS injection and returned to the baseline thereafter (P < 0.05). Plasma levels of cholesterol and triglyceride in LM group rabbits both with and without ON increased significantly at 1 week (P < 0.05), peaked at 2 weeks, and these high levels were maintained during the observation periods, which were consistent with the change of LDL/HDL ratio. No detectable changes were found in the hematological examinations for the control rabbits during the entire study period (P > 0.05) (Fig. S4). 4. Discussion The development of ON is not caused by a single precipitating event. There are multiple theories about the pathogenesis of steroid-induced ON, and most of these theories are mutually supportive and not exclusive. For explanation of the pathogenesis of the steroid-induced osteonecrosis, various theories have been proposed regarding the developmental mechanisms of steroid-induced osteonecrosis, including the increased size and number of fatty cells, increased intraosseous pressure, fatty degeneration of osteocytes, fat embolism, and extraosseous arterial occlusion [5,9,12,13]. In addition, coagulation abnormalities and hyperlipidaemia were among the postulated pathogenic mechanisms for ON development. Jones et al. [14] proposed a fat embolization theory whereby hyperlipidaemia and associated abnormalities in the blood coagulation system were a possible mechanism of ON. However, the precise mechanism of steroid-induced osteonecrosis Table 2 The systemic arterial pressures (mmHg) Group
Before administration
6 weeks after administration
Rabbits with ON (N ¼ 27) Rabbits without ON (N ¼ 3) Control (N ¼ 6)
84.2 9.4 82.6 8.4 82.2 6.2
90.6 11.2 88.4 8.8 87.2 7.6
KEY: ON, osteonecrosis. Data are the mean standard deviation.
remains unclear. Although, the pathogenesis of osteonecrosis is controversial and multifactorial, the glucocorticoid therapy is the most important factor contributing to the lesion. Corticosteroids are known to induce not only hyperlipidaemia but also a hypercoagulable and hypofibrinolytic state of plasma. Both hypercoagulability and hypofibrinolysis have been reported in human non-traumatic ON cases, including a high plasminogen activator inhibitor (PAI-I) and the presence of anticardiolipin antibodies [15,16]. These factors enhance the coagulability predisposing to thrombosis and may cause damage to blood vessels. In vitro study of others supported the mechanism that LPS was able to induce hypercoagulable and/or hypofibrinolytic state partially through reduction of endothelia expression of thrombomodulin (TM) [17]. In fact, endothelial TM was an important intermediary substance, which directly or indirectly participated in both inhibition of coagulation and promotion of fibrinolysis, and the ratio of t-PA/PAI-I reflected balance between anti-fibrinolysis and pro-fibrinolysis [18e20]. There were significant changes in the coagulation system in rabbit ON model, which enhanced the coagulability predisposing to thrombosis and might cause damage to blood vessels. In the present study for intravascular mechanistic investigation, tPA/PAI-I ratio (an indicator of hypofibrinolysis) were evaluated etiologically. The results showed that hypofibrinolysis was synergistically induced by a combination of LPS and MPS injection. Abnormal lipid metabolism is related to steroid-induced ON of the femoral head, and hyperlipaemia and increased free fatty acids are considered to be important risk factors [5,14,21,22]. The disturbed lipid metabolism of the rabbits is reflected in hypertrophy of bone marrow adipocytes and an elevated ratio of low-density to high-density lipoprotein cholesterol (LDL/HDL ratio). The lipid transport resulting form the higher LDL/HDL ratio might have induced intraosseous hyperlipidemic state, which eventually leads to extravascular marrow compression from lipid deposit. This lipid-induced hypertrophy of the fat cells cannot expand the marrow cavity within the inflexible osseous cage. Steroid administration increases the intraosseous pressure in spite of decreased blood flow, and the intraosseous hypertension inhibits regeneration of the blood vessels [4,23,24]. Then ischaemic events might result from vascular interruption through thrombi, lipid emboli or high intraosseous pressure associated with bone marrow fat-cell enlargement; these would subsequently lead ON development [25e27]. Moreover, steroid-induced cholesterol deposition reduces the fluidity and permeability of the cell membranes, contributing to the death of the osteocytes. Treatment of rabbits with the lipid-clearing clofibrate reduces steroid-induced hepatosteatosis, hyperlipidemia, and accumulation of lipids in osteocytes and protects the femoral head against necrosis [28,29]. In our haematological study for extravascular mechanistic investigation, serum lipid levels increased sharply 1 week after the initial steroid administration, both LDL/HDL cholesterol ratio and fat-cell size were significantly higher in LM group rabbits. These data suggested that ON was closely related to serum lipid levels (high LDL/HDL
X. Wu et al. / Joint Bone Spine 75 (2008) 573e578
cholesterol ratio), and we regarded these levels as high enough to accelerate the coagulation system. MRI can show avascular necrosis in its earliest stages and provide a picture of the area affected and the bone rebuilding process. In the present study, MRI provided a picture of the area affected and the bone rebuilding process, of 73.3% (22/ 30) rabbits with osteonecrosis. The end-point evaluations in the present study demonstrated that the proposed treatment protocol induced a higher incidence of ON of 90% and lower mortality of 6.2%. In fact, several authors using similar designs have obtained comparable results. Irisa et al. [3] documented a high incidence of osteonecrosis (77%) and low mortality (11%) in rabbits that were treated with only lowdose LPS. Miyanishi et al. [26] documented a similar incidence of osteonecrosis (76%) but no mortality in rabbits that were treated with once injection of MPS (20 mg/kg). Recently, Qin et al. [30] documented an even higher rate of osteonecrosis (93%) and no mortality in rabbits that were treated with one injection of low-dose LPS followed by three doses of MPS (L-LPS 1 þ H-MPS 3). In contrast, Yamamoto et al. [2] used two injections of high-dose lipopolysaccharide (LPS) combined with subsequent three injections of highdose methylprednisolone (MPS) (H-LPS 2 þ H-MPS 3), which induced higher ON incidence (85%) but accompanied with high mortality of experimental animals (50%). According to the etiology of steroid-associated ON, the lower ON incidence by the protocol (H-MPS 1) was due to lack of LPSinduced pre-thrombosis state before steroid treatment. On the other hand, the higher mortality by the protocol (HLPS 2 þ H-MPS 3) was due to high-dose-LPS-induced severe endotoxin shock. The present study developed a protocol by modifying the reported protocol (H-LPS 2 þ HMPS 3) by replacing the two high-dose LPS injection with two low-dose LPS injection. It (L-LPS 2 þ HMPS 3) showed a higher incidence of ON lesion (90%) and lower mortality (6.2%) in rabbits as compared with the previously published protocol (H-LPS 2 þ H-MPS 3) due to the avoidance of severe LPS-induced shock by lowering the given LPS dose. As comparison, a better understanding of the influence of LPS-MPS on the ON incidence and animal mortality is needed, and likely, differences do exist. The present experimental study showed that our experimental protocol with low-dose LPS and subsequent pulsed highdose MPS injections was an effective one to induce ON in rabbits with high incidence and low mortality. Osteonecrosis of patients and animals is akin in some aspects but diverges in others. We demonstrated many histopathological and pathogenetic features of steroid-induced ON in rabbits similar to those observed in human ON prior to subchondral bone collapse. First, ON lesions in these rabbit models present histological characteristics similar to human ON, i.e. osteocytic death surrounded by necrotic bone marrow with or without repair tissue [31,32]. Second, significantly increased intraosseous pressure was reported in patients with ON [8,13]. Third, a higher lipid deposition was noted in human osteonecrotic femoral heads [27,33]. Fourth, hypercoagulability and hypofibrinolysis have been reported to play etiologic roles in
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human ON [15,20,27]. These similarities suggested significant relevance of this animal model to human ON. In summary, this model would be useful for elucidating the pathogenesis of steroid-associated ON. Both high ON incidence and low mortality in rabbits treated with this inductive protocol suggested its effectiveness for developing preventive and therapeutic strategies. Acknowledgements The authors wish to thank Mulan Yang, Jianzhong Fan, and Qigui Zou for their very helpful technical assistance. This study was supported by: National Natural Science Foundation of China (No. 3017094). Appendix A. Supplementary data Supplementary material (Figs. S1eS4) associated with this article can be found at http://www.sciencedirect.com, at doi:10.1016/j.jbspin.2007.11.004. References [1] Mont MA, Jones LC, Hungerford DS. Nontraumatic osteonecrosis of the femoral head: ten years later. J Bone Joint Surg Am 2006;88: 1117e32. [2] Yamamoto T, Hirano K, Tsutsui H, et al. Corticosteroid enhances the experimental induction of osteonecrosis in rabbits with Shwartzman reaction. Clin Orthop Relat Res 1995;316:235e43. [3] Irisa T, Yamamoto T, Miyanishi K, et al. Osteonecrosis induced by a single administration of low-dose lipopolysaccharide in rabbits. Bone 2001;286:641e9. [4] Yamamoto T, Irisa T, Sugioka Y, et al. Effects of pulse methylprednisolone on bone and marrow tissues: corticosteroid-induced osteonecrosis in rabbits. Arthritis Rheum 1997;40:2055e64. [5] Kabata T, Kubo T, Matsumoto T, et al. Onset of steroid-induced osteonecrosis in rabbits and its relationship to hyperlipaemia and increased free fatty acids. Rheumatology 2005;44:1233e7. [6] Ichiseki T, Matsumoto T, Nishino M, et al. Oxidative stress and vascular permeability in steroid-induced osteonecrosis model. J Orthop Sci 2004;9:509e15. [7] Ichiseki T, Kaneuji A, Katsuda S, Ueda Y, Sugimori T, Matsumoto T. DNA oxidation injury in bone early after steroid administration is involved in the pathogenesis of steroid-induced osteonecrosis. Rheumatology 2005;44:456e60. [8] Yamamoto T, DiCarlo EF, Bullough PG. The prevalence and clinicopathological appearance of extension of osteonecrosis in the femoral head. J Bone Joint Surg Br 1999;81:328e32. [9] Motomura G, Yamamoto T, Miyanishi K, et al. Bone marrow fat-cell enlargement in early steroid-induced osteonecrosisda histomorphometric study of autopsy cases. Pathol Res Pract 2005;200:807e11. [10] Shaw SL, Salmon ED, Quatrano RS. Digital photography for the light microscope: results with a gated, video-rate CCD camera and NIH-image software. Biotechniques 1995;19:946e55. [11] Miyanishi K, Yamamoto T, Irisa T, et al. Bone marrow fat cell enlargement and a rise in intraosseous pressure in steroid-treated rabbits with osteonecrosis. Bone 2002;30:185e90. [12] Arlet J. Nontraumatic avascular necrosis of the femoral head. Past, present, and future. Clin Orthop Relat Res 1999;277:12e21. [13] Mankin HJ. Nontraumatic necrosis of bone (osteonecrosis). New Engl J Med 1992;326:1473e9. [14] Jones Jr JP. Fat embolism and osteonecrosis. Orthop Clin North Am 1985;16:595e633.
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