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Rheumatology Advance Access originally published online on March 7, 2008
Rheumatology 2008 47(5):591-596; doi:10.1093/rheumatology/ken037
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Prevention of corticosteroid-induced osteonecrosis in rabbits by intra-bone marrow injection of autologous bone marrow cells

T. Asada1, T. Kushida1, M. Umeda1, K. Oe1, H. Matsuya1, T. Wada1, K. Sasai1, S. Ikehara2 and H. Iida1

1Department of Orthopaedic Surgery and 2First Department of Pathology, Kansai Medical University, Osaka, Japan.

Correspondence to: T. Kushida, Department of Orthopaedic Surgery, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan. E-mail: kushidat{at}takii.kmu.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. Femoral head osteonecrosis (ON) is a serious complication of steroid administration. We evaluated bone marrow transplantation (BMT) for preventing corticosteroid-induced ON.

Methods. Rabbits, injected with methylprednisolone (MPSL; 20 mg/kg), were divided into four groups: (i) MPSL alone; MPSL injection only, (ii) MPSL+needling; 2 days after MPSL injection, a hole (1.2 mm diameter) was drilled from the outer cortex 2.5 cm distal to the proximal end of the greater trochanter, (iii) MPSL+saline; 2 days after MPSL injection, 2 ml saline was injected directly into the bone marrow cavity, and (iv) MPSL+BMT; 2 days after MPSL injection, 1 x 107/2 ml bone marrow cells (BMCs) were injected directly into the bone marrow cavity. Platelets, fibrinogen, prothrombin time and total cholesterol in peripheral blood were measured before and after treatment. Tissues were stained with haematoxylin and eosion and terminal deoxynucleotidyl-mediated deoxyuridine triphosphate nick-end labelling stain and immunostained for VEGF, while cell proliferation and viability of whole BMCs in the femur were analysed by cell cycle analysis and [3H]-thymidine uptake.

Results. The ON incidence in rabbits treated with MPSL alone, MPSL+needling and MPSL+saline was 72.7, 70.0 and 66.7%, respectively, while in the MPSL+BMT group, the incidence was 0%. Serological findings in the MPSL+BMT group were almost normalized. VEGF and TUNEL staining were reduced in the MPSL+BMT group compared with all other groups. There were significantly fewer BMCs in G1 phase from the MPSL+BMT group than the other groups, while uptake of [3H]-thymidine was significantly increased.

Conclusion. Direct injection of autologous BMCs into femurs prevents corticosteroid-induced ON following treatment with high-dose, short-term steroids.

KEY WORDS: Corticosteroid, Osteonecrosis, Animal model, Bone marrow transplantation, Bone marrow cells


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Corticosteroid-induced osteonecrosis (ON) of the femoral head is one of the most serious complications when steroids are administered to treat spinal cord injury [1, 2], for immunosuppression after bone marrow transplantation (BMT) or transplantation of organs such as the kidney or liver [3–5], or as treatment for autoimmune diseases such as RA and SLE [3, 6]. The incidences of corticosteroid-induced ON in patients administered steroids for spinal cord injury, kidney transplantation and BMT are 5, 21 and 19%, respectively [2, 4, 7]. In addition, the incidence of ON in patients administered steroids for SLE is 22–44% [6, 8]. It has been reported that many factors such as thromboembolism, fat embolism, thrombophilia, hypofibrinolysis, intramedullary haemorrhage, vasculitis and increased bone marrow pressure are related to corticosteroid-induced ON [3, 9–14]. Therefore, several therapies such as treatment with anti-lipid agents or anti-coagulant agents have been carried out to prevent corticosteroid-induced ON [15–19]. However, as the relationship between these various factors and corticosteroid-induced ON is not yet clear, there is to date no established method for its prevention.

Recently, there have been reports that, in rabbits, a single injection of high-dose MPSL (20 mg/kg) or low-dose MPSL (4 mg/kg) was able to cause corticosteroid-induced ON lesions of the metaphysis and diaphysis but not the epiphysis of the long bones [20, 21]. Furthermore, there have also been reports that, using this rabbit model, several methods for the prevention of corticosteroid-induced ON incidence were attempted [21–23].

Bone marrow cells (BMCs) contain not only haematopoietic stem cells (HSCs) but also mesenchymal stem cells (MSCs). Osteoblasts, which differentiate from MSCs, play a crucial role in the activation of osteoclasts. Therefore, the balance between osteoclasts and osteoblasts is important in the regulation of osteogenesis. Very recently, a new method for harvesting BMCs (‘perfusion method’), which is able to prevent contamination with peripheral lymphocytes, has been developed [24, 25]. In this study, we injected autologous BMCs harvested by the ‘perfusion method’ into the femurs of steroid-administered rabbits and examined the effects of BMT on the prevention of corticosteroid-induced ON.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Rabbits
Adult female Japanese white rabbits ranging in age from 28 to 32 weeks and weighing 3–4 kg (defined as animals with closed growth plates) were used. The rabbits were kept in single cages and fed a standard diet. The left hip was used for the experiment in all rabbits. All rabbits were obtained from Shimizu Laboratory Supplies (Kyoto, Japan). The study protocol was approved by the Animal Experimentation Committee, Kansai Medical University (Osaka, Japan).

Preparation of BMCs and injection into the bone marrow cavity
Rabbits were anaesthetized using Halothane (Takeda Pharmaceutical Company Limited, Osaka, Japan), and bone marrow fluid was collected by the perfusion method [25]. In brief, as shown in Fig. 1A, the bone marrow fluid was collected from the right femur. One needle (18-gauge needle; Terumo Company Limited, Tokyo, Japan) was inserted into the proximal side of the femur and a second needle was inserted into the distal side. The proximal needle was connected to a syringe (30 ml, Code No. SS-30ES; Terumo Company Limited) containing heparin (10 U/ml), and the distal needle was connected to a syringe containing 30 ml of saline (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan). The saline was pushed gently from the syringe into the medullary cavity to flush out the bone marrow of the right femur into the second syringe. After perfusion, the harvested whole BMCs were cultured in DMEM (Invitrogen, Grand Island, NY, USA) with 10% fetal bovine serum (FBS) (Invitrogen) supplemented with antibiotics in a humidified atmosphere containing 5% CO2 at 37°C for 2 days.


Figure 1
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  		FIG. 1. Experimental protocol. Adult female Japanese white rabbits ranging in age from 28 to 32 weeks and weighing 3–4 kg were used. The bone marrow fluid was collected by the perfusion method (A). The rabbits were injected once with 20 mg/kg of MPSL into the gluteus medius muscle. After the injection of MPSL, we prepared four groups as follows: (i) MPSL alone, (ii) MPSL+needling, (iii) MPSL+saline and (iv) MPSL+BMT. The diagnosis of ON was determined 2 weeks after the injection of MPSL. The whole BMCs were injected directly into the bone marrow cavity according to the method described previously (B).

 
The BMCs were injected directly into the bone marrow cavity according to the method described previously [26]. In brief, an 18-gauge needle was inserted into the proximal side of the femur and the donor BMCs (1 x 107/2 ml) were injected into the bone marrow cavity of the left femur, as shown in Fig. 1B.

Protocol
Rabbits were injected once with 20 mg/kg of methylprednisolone (MPSL) (Pfizer Japan Inc., Tokyo, Japan) [20]. After injection, the rabbits were divided into four groups as follows: (i) MPSL alone; injection of MPSL only, (ii) MPSL+needling; 2 days after injection of MPSL, a drill hole (1.2 mm in diameter) was made from the outer cortex 2.5 cm distal to the proximal end of the greater trochanter under sterile condition, (iii) MPSL+saline; 2 days after injection of MPSL, 2 ml of saline were injected directly into the bone marrow cavity, and (iv) MPSL+BMT; 2 days after injection of MPSL, 1 x 107/2 ml of BMCs were injected directly into the bone marrow cavity.

Serological findings
Peripheral blood samples were obtained from the rabbits prior to injection of MPSL and from the rabbits treated with MPSL alone, MPSL+needling, MPSL+saline and MPSL+BMT, 2 weeks after the injection of MPSL. Levels of platelets (PLT), prothrombin time (PT), fibrinogen (Fib) and total cholesterol (T-cho) were measured.

Tissue preparation
The treated rabbits were sacrificed and the femurs were removed. The femurs were fixed for 1 week in 10% formalin. Bone samples were decalcified with EDTA (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 2 weeks. The specimens were embedded in paraffin, cut into 4-µm sections, and stained with haematoxylin and eosin (HE). In addition, sections were immunostained for VEGF, using an anti-VEGF monoclonal antibody, clone JH121 (Upstate Biotechnology, Lake Placid, NY, USA). In brief, sections were incubated with anti-VEGF monoclonal antibody overnight. After washing with 0.05 M phosphate buffer (pH 7.6), sections were labelled with peroxidases and goat anti-mouse Ig antibody (Histofine Mousestain Kit: Nichirei Biosciences Inc., Tokyo, Japan). Following a wash with 0.05 M phosphate buffer (pH 7.6), the coloured reaction product was developed using 3-3'-diaminobenzidine (DAB). Sections were then counter-stained with Mayer's haematoxylin to show the nuclei. Terminal deoxynucleotidyl-mediated deoxyuridine triphosphate nick-end labelling staining (TUNEL) was also carried out to detect apoptosis as previously described [27].

Pathological examination and evaluation of corticosteroid-induced ON
Corticosteroid-induced ON was determined at 2 weeks after MPSL injection by HE stain. As described by Yamamoto et al. corticosteroid-induced ON was determined on the basis of the diffuse presence of empty lacunae or pyknotic nuclei of osteocytes in the bone trabeculae, accompanied by surrounding bone marrow cell necrosis [20]. Only bone marrow cell necrosis composed of both haematopoietic cell necrosis and fat cell necrosis in which no bone trabecula was included was counted as corticosteroid-induced ON; lesions composed of only a few empty lacunae within the normal bone trabecula and/or fat cell necrosis alone were excluded from the diagnosis of corticosteroid-induced ON [20–22].

Cell cycle analysis
To evaluate the cell proliferation of the whole BMCs from the femurs in each group, 2 weeks after MPSL injection, we analysed the cell cycle using flow cytometry. In brief, cells were treated with propidium iodide (PI; Sigma-Aldrich Co., St Louis, MO, USA) and ribonuclease A (RNase; Sigma-Aldrich Co.) and the stained cells were analysed using a FACScan (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

Analysis of viability of the BMCs
To evaluate the viability of the whole BMCs from the femur in each group, 2 weeks after MPSL injection, we analysed the uptake of [3H]-thymidine (Perkin Elmer, Inc., Waltham, MA, USA). The whole BMCs were set up in a 96-well culture plate. Each well contained 5 x 104 BMCs. The BMCs were labelled with 18.5 kBq of [3H]-thymidine per well for 3 and 9 days in DMEM with 10% FBS supplemented with antibiotics and the radioactivity was counted.

Statistical analyses
The number of femurs with ON lesions was compared using the {chi}2 test with Bonferroni correction for multiple comparisons. Analyses of serological data, cell cycle and [3H]-thymidine were compared using Student's t-test. Statistical differences were considered significant when the P-value was <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Histological findings in the femurs 14 days after MPSL
In this study, to examine the effects of BMT into the bone marrow cavity on corticosteroid-induced ON, we analysed the histology of the proximal and distal sides of femurs by HE staining. In the femurs of the rabbits treated with MPSL alone, with MPSL+needling or with MPSL+saline, empty lacunae and pyknotic nuclei of osteocytes within the bone trabeculae were observed and BMC necrosis was also seen (Fig. 2B–D). In contrast, the femurs from the rabbits treated with MPSL+BMT did not show ON findings, except for a few empty lacunae within the normal bone trabeculae or fat cell necrosis (Fig. 2E), and otherwise appeared histologically normal (Fig. 2A). These findings indicate that BMT is able to prevent corticosteroid-induced ON in the femur.


Figure 2
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  		FIG. 2. Histological features 2 weeks after MPSL. The histology of the femurs in normal rabbits is shown (A). The rabbits administered MPSL alone showed the diffuse presence of empty lacunae, pyknotic nuclei of osteocytes within the bone trabeculae and bone marrow necrosis (B). The rabbits administered MPSL+needling (C) and MPSL+saline (D) also showed necrosis. The rabbits administered MPSL+BMT showed absence of necrosis (E). (HE; original magnification x400).

 
Prevalence of ON
As shown in Table 1, the incidence of corticosteroid-induced ON in the left femurs of rabbits treated with MPSL alone, MPSL+needling and MPSL+saline was 72.7% (8 of 11), 70.0% (7 of 10) and 66.7% (10 of 15), respectively. Furthermore, the incidence in the right (donor side) and left (recipient side) femurs of rabbits treated with MPSL+BMT was 50.0% (5 of 10) and 0% (0 of 15), respectively. These findings suggest that BMT confers significant protection from corticosteroid-induced ON not on the indirectly injected side (donor side) but on the directly injected side (recipient side) in comparison with those treated with MPSL alone, MPSL+needling and MPSL+saline.


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  		TABLE 1. Prevalence of ON

 
Serological findings
To examine the effects of MPSL on fat metabolism (fibrinogenolysis and coagulation) and to investigate the effects of BMT on metabolism, we analysed the levels of PT, Fib, PLT and T-cho. As shown in Fig. 3A and D, PT and T-cho levels significantly decreased in the rabbits treated with MPSL+BMT in comparison with those treated with MPSL alone, MPSL+needling and MPSL+saline and were returned to normal levels. In addition, as shown in Fig. 3B and C, Fib and PLT significantly increased in the rabbits treated with MPSL+BMT in comparison with MPSL alone, MPSL+needling and MPSL+saline, and approached normal levels. These findings indicate that BMT can improve fat metabolism, fibrinogenolysis and coagulation in the rabbits after injection of MPSL.


Figure 3
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  		FIG. 3. Serological findings. Blood samples were obtained from all rabbits prior to injection of MPSL and from the rabbits treated with MPSL alone, MPSL+needling, MPSL+saline and MPSL+BMT 2 weeks after the injection of MPSL. We examined the blood levels of PT (A), Fib (B), PLT (C) and T-cho (D). The results are expressed as the mean ± S.D. of six rabbits. *P < 0.05; normal vs MPSL alone, MPSL+needling, MPSL+saline or MPSL+BMT.

 
Ischaemic and apoptotic changes in the femurs 14 days after MPSL injection
VEGF, an angiogenic promoter, is rapidly induced in response to local hypoxia. To investigate the relationship between the development of corticosteroid-induced ON and local ischaemia, we analysed the expression of VEGF in the bone marrow of the femurs. We found that the femurs of the rabbits treated with MPSL alone, MPSL+needling and MPSL+saline contained many VEGF-positive regions (Fig. 4A–C). On the other hand, the femurs of the rabbits treated with MPSL+BMT showed only a few positive regions (Fig. 4D).


Figure 4
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  		FIG. 4. Ischaemic changes in the femurs 2 weeks after MPSL. We examined local hypoxia of the femurs using VEGF staining. In the rabbits administered MPSL alone, many VEGF-positive cells were observed (A). The rabbits administered MPSL+needling (B) and MPSL+saline (C) also showed many VEGF-positive cells. In the rabbits administered MPSL+BMT there were few VEGF-positive cells (D). (Original magnification x400).

 
Furthermore, to investigate the relationship between the development of corticosteroid-induced ON and the level of apoptosis, we compared the number of apoptotic cells using TUNEL staining in the bone marrow of the femurs. The result showed that the femurs of rabbits treated with MPSL alone, MPSL+needling and MPSL+saline contained many positive regions (Fig. 5A–C). In contrast, in those treated with MPSL+BMT, only a few positive regions were observed (Fig. 5D). These findings indicate that corticosteroid treatment induces ischaemic changes in the femurs and consequently causes apoptotic changes. Thus, BMT prevents such corticosteroid-induced ischaemic changes and the resulting apoptotic changes in the femurs.


Figure 5
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  		FIG. 5. Apoptotic changes in the femurs 2 weeks after MPSL. We investigated the presence of apoptotic cells in the femoral neck by TUNEL staining. In the rabbits administered MPSL alone, there were many cells in which nuclei showed chromatin condensation and nuclear fragmentation (A). The rabbits administered MPSL+needling (B) and MPSL+saline (C) also showed many TUNEL-positive cells. In the rabbits administered MPSL+BMT, a few cells containing chromatin condensation were observed (D). (Original magnification x400).

 
Cell cycle analysis
To examine cell proliferation of the whole BMCs in the femurs, we analysed the cell cycle using flow cytometry. As shown in Fig. 6, the percentage of cells in the G1 phase of the cell cycle in the femurs of rabbits treated with MPSL+BMT was significantly reduced compared with MPSL alone or MPSL+saline and approached the normal level. Based on these findings, we suggest that steroid treatment induces cell cycle arrest of whole BMCs in the femurs of MPSL-treated rabbits and that transplanting whole BMCs suppresses steroid-induced cell cycle arrest and normalizes the cell cycle of BMCs in the femurs.


Figure 6
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  		FIG. 6. Cell cycle analyses. Cell proliferation of the whole BMCs from the femurs in each group 2 weeks after MPSL by cell cycle analysis was examined. The percentages of cells in the G1 phase in each group are shown. The results are expressed as the mean ± S.D. of six rabbits. *P < 0.05; MPSL+BMT vs MPSL alone or MPSL+saline.

 
Analyses of BMC viability
To examine the viability of the whole BMCs, we analysed [3H]-thymidine incorporation. As shown in Fig. 7, uptake of [3H]-thymidine in BMCs at 3 days after MPSL+BMT (recipient side) was significantly increased compared with normal, MPSL alone and MPSL+saline. Furthermore, uptake of [3H]-thymidine in BMCs at 9 days after MPSL+BMT (recipient side) was significantly increased compared with all other groups including MPSL+BMT (donor side). On the basis of these findings, we suggest that the viability of whole BMCs in the femurs into which the BMCs are directly injected, continues to increase and plays an important role on the treatment of ON.


Figure 7
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  		FIG. 7. Analyses of viability of the BMCs. We examined the viability of the whole BMCs from the femurs in each group 2 weeks after MPSL injection by [3H]-thymidine incorporation. The results are expressed as the mean ± S.D. of six rabbits. *P < 0.05; MPSL+BMT (recipient side) vs MPSL alone, MPSL+saline or MPSL+BMT (donor side).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
Steroids are indispensable for the treatment of spinal cord injury, organ transplantation and autoimmune diseases such as SLE [1–8]. However, when steroids are administered, corticosteroid-induced ON of the femoral head is one of the most serious complications. Corticosteroid-induced ON of the femoral head is characterized by variable areas of dead trabecular bone and bone marrow, extending to but excluding the subchondral plate. Sakamoto et al. [28] reported that when a prospective study using MRI to look for corticosteroid-induced ON of the femoral head was performed, the band which indicates corticosteroid-induced ON was detected 2 months after administration of steroids.

To date, the mechanisms underlying corticosteroid-induced ON remain unclear. Previous studies have reported that abnormal lipid metabolism such as hyperlipaemia and hyper free-fatty acidaemia is related to corticosteroid-induced ON of the femoral head [3, 13, 14]. Other studies have reported that thrombophilia and hypofibrinolysis are also related to the incidence of corticosteroid-induced ON [3, 12, 14]. Miyanishi et al. [29] found that larger doses of steroids increased fat-cell size and reported that drilling a hole (1.0 mm diameter) in the femurs of rabbits given steroids could decrease intraosseous pressure. In this study, the incidence of ON in the femurs treated with MPSL alone and MPSL+saline were 72.7 and 66.7%, respectively. These findings suggest that the injection procedure itself may help to relieve intraosseous pressure and may help to reduce the incidence of ON and that injection of whole BMCs into the femurs is effective not only on the directly injected side but also on the indirectly injected side due to systemic action. In this group, the levels of T-cho significantly increased and the levels of PLT, PT and Fib significantly decreased in comparison with normal rabbits. These abnormalities of lipid metabolism, thrombophilia and hypofibrinolysis were associated with fat embolism and thromboembolism in the intramedullary cavity of the femurs, leading to corticosteroid-induced ON. In contrast, in rabbits treated with MPSL+BMT, these haematological data were restored to normal levels. There have been reports that steroids and hypoxia-inducible factor-1{alpha} induce cell cycle arrest of endothelial cells, which regulate the blood coagulating system and fibrinolytic system [30, 31]. Therefore, we suggest that the damaged endothelial cells cause deregulation of the blood coagulating system and fibrinolytic system. Recently, it has been shown that BMCs include many kinds of immature cells that are able to differentiate into haematopoietic cells and endothelial progenitor cells [32] and that BMCs induce angiogenesis due to a variety of factors [33]. Furthermore, MSCs have been reported to differentiate into endothelial cells and to regulate angiogenesis according to their mechanical environment [34]. Therefore, we suggest that BMT can restore the damaged endothelial cells leading to prevention of thrombophilia and hypofibrinolysis caused by corticosteroids.

Weinstein et al. [35] reported the inhibition of osteoblastogenesis and the promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Furthermore, Manolagas et al. [36] reported that the lifespan of osteoblasts and osteocytes decreased, and that osteoclast numbers transiently increased during the early stages of steroid therapy, but decreased subsequently. In this study, many TUNEL-positive areas, indicating apoptotic regions, could be detected in the femurs of rabbits treated with MPSL alone. Moreover, VEGF is rapidly induced in response to local hypoxia and these rabbits showed many VEGF-positive regions in the femurs. Based on these findings, we suggest that corticosteroid-induced ischaemic changes are associated with apoptotic cell death within the medullary space, resulting in induction of ON.

In the G1 phase of the cell cycle, the percentage of cells decreases as cell proliferation progresses, while the rates increase when the cell cycle is arrested. In this study, the percentage of cells in the G1 phase in the whole BMCs treated with MPSL+BMT significantly decreased in comparison with MPSL alone and MPSL+saline, and was restored to normal levels (Fig. 6). Furthermore, the viability of the whole BMCs at 9 days in the MPSL+BMT (recipient side) group, as shown by uptake of [3H]-thymidine, was significantly increased compared with all other groups including MPSL+BMT (donor side) (Fig. 7). These findings suggest that the cell proliferation and viability of the whole BMCs in the femurs treated with MPSL+BMT continued to increase, leading to regeneration after corticosteroid-induced ON.

On the basis of these findings, we hypothesize that whole BMCs that are not treated with corticosteroids can reduce thromboembolism and fat embolism, because the transplanted whole BMCs can regulate fibrinogenolysis and coagulation in the peripheral blood. As a result, the increase of bone marrow pressure is avoided and thus hypoxia and ischaemic changes in the femurs are prevented. Furthermore, accelerating osteogenesis in the femurs by transplanting whole BMCs prevents the development of corticosteroid-induced ON (Fig. 8). Based on these findings, we strongly suggest that whole BMCs directly injected into the femurs can prevent corticosteroid-induced ON in patients treated with high-dose and short term steroid medication as a therapy for acute spinal cord injury or organ transplantation.


Figure 8
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  		FIG. 8. Mechanisms underlying prevention of ON by BMT. BMT exerts its effect on the stem cells, leading to the acceleration of osteogenesis. Whole BMCs, which are not treated by corticosteroid, reduce thromboembolism and fat embolism, because normal BMCs can regulate fibrinogenolysis and coagulation in the peripheral blood. As a result, bone marrow pressure decreases, and hypoxia and ischaemic changes in the femurs are normalized. Thus, the acceleration of osteogenesis in the femurs by transplanted whole BMCs leads to the prevention of development of corticosteroid-induced ON. Dotted arrows: cascade of events, leading to ON induced by high-dose corticosteroids. Solid arrows: effect of BMT.

 
Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
Funding: This study was supported by a grant from the ‘Science Frontier’ programme of the Ministry of Education, Culture, Sports, Science and Technology, a grant from the ‘The 21st Century Center of Excellence (COE)’ programme of the Ministry of Education, Culture, Sports, Science and Technology and by a grant from ‘The Osaka Medical Research Foundation For Incurable Diseases’.

Disclosure statement: The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Bracken MB. Methylprednisolone and acute spinal cord injury: an update of the randomized evidence. Spine (2001) 26:S47–54.[CrossRef][Web of Science][Medline]
  2. Wing PC, Nance P, Connell DG, Gagnon F. Risk of avascular necrosis following short term megadose methylprednisolone treatment. Spinal Cord (1998) 36:633–6.[CrossRef][Web of Science][Medline]
  3. Mont MA, Hungerford DS. Non-traumatic avascular necrosis of the femoral head. J Bone Joint Surg (1995) 77A:459–74.[Free Full Text]
  4. Torii Y, Hasegawa Y, Kubo T, et al. Osteonecrosis of the femoral head after allogeneic bone marrow transplantation. Clin Orthop Relat Res (2001) 382:124–32.[CrossRef][Medline]
  5. Lieberman JR, Scaduto AA, Wellmeyer E. Symptomatic osteonecrosis of the hip after orthotopic liver transplantation. J Arthroplasty (2000) 15:767–71.[CrossRef][Web of Science][Medline]
  6. Oinuma K, Harada Y, Nawata Y, et al. Osteonecrosis in patients with systemic lupus erythematosus develops very early after starting high dose corticosteroid treatment. Ann Rheum Dis (2001) 60:1145–8.[Abstract/Free Full Text]
  7. Kubo T, Yamazoe S, Sugano N, et al. Initial MRI findings of non-traumatic osteonecrosis of the femoral head in renal allograft recipients. Magn Reson Imaging (1997) 15:1017–23.[CrossRef][Web of Science][Medline]
  8. Nagasawa K, Ishii Y, Mayumi T, et al. Avascular necrosis of bone in systemic lupus erythematosus: possible role of haemostatic abnormalities. Ann Rheum Dis (1989) 48:672–6.[Abstract/Free Full Text]
  9. Atsumi T, Kuroki Y. Role of impairment of blood supply of the femoral head in the pathogenesis of idiopathic osteonecrosis. Clin Orthop Relat Res (1992) 277:22–30.[Medline]
  10. Fisher DE. The role of fat embolism in the etiology of corticosteroid-induced avascular necrosis: clinical and experimental results. Clin Orthop Relat Res (1978) 130:68–80.[Medline]
  11. Jones JP Jr. Fat embolism, intravascular coagulation, and osteonecrosis. Clin Orthop Relat Res (1993) 292:294–308.[Medline]
  12. Glueck CJ, Freiberg R, Tracy T, Stroop D, Wang P. Thrombophilia and hypofibrinolysis: pathophysiologies of osteonecrosis. Clin Orthop Relat Res (1997) 334:43–56.[Medline]
  13. Wang TY, Avlonitis EG, Relkin R. Systemic necrotizing vasculitis causing bone necrosis. Am J Med (1988) 84:1085–6.[CrossRef][Web of Science][Medline]
  14. Saito S, Inoue A, Ono K. Intramedullary haemorrhage as a possible cause of avascular necrosis of the femoral head. The histology of 16 femoral heads at the silent stage. J Bone Joint Surg Br (1987) 69:346–51.[Web of Science][Medline]
  15. Meroni PL, Raschi E, Testoni C, et al. Statins prevent endothelial cell activation induced by antiphospholipid (anti-beta2-glycoprotein I) antibodies: effect on the proadhesive and proinflammatory phenotype. Arthritis Rheum (2001) 44:2870–8.[CrossRef][Web of Science][Medline]
  16. Cui Q, Wang GJ, Su CC, Balian G. The Otto Aufranc Award. Lovastatin prevents steroid induced adipogenesis and osteonecrosis. Clin Orthop Relat Res (1997) 344:8–19.[Medline]
  17. Nagasawa K, Tada Y, Koarada S, et al. Prevention of steroid-induced osteonecrosis of femoral head in systemic lupus erythematosus by anti-coagulant. Lupus (2006) 15:354–7.[Abstract/Free Full Text]
  18. Pritchett JW. Statin therapy decreases the risk of osteonecrosis in patients receiving steroids. Clin Orthop Relat Res (2001) 386:173–8.[CrossRef][Medline]
  19. Hayashi T, Saito N, Shoji T, Togawa M, Okada N, Tsubakihara Y. Cyclosporin A mono-therapy in nephrotic syndrome with contra-indication of steroid therapy. Intern Med (1999) 38:272–5.[CrossRef][Web of Science][Medline]
  20. Yamamoto T, Irisa T, Sugioka Y, Sueishi K. Effects of pulse methylprednisolone on bone and marrow tissues: corticosteroid-induced osteonecrosis in rabbits. Arthritis Rheum (1997) 40:2055–64.[Web of Science][Medline]
  21. 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) 44:1233–7.[Abstract/Free Full Text]
  22. Miyanishi K, Yamamoto T, Irisa T, et al. Effects of cyclosporin A on the development of osteonecrosis in rabbits. Acta Orthop (2006) 77:813–9.[CrossRef][Web of Science][Medline]
  23. Motomura G, Yamamoto T, Miyanishi K, Jingushi S, Iwamoto Y. Combined effects of an anticoagulant and a lipid-lowering agent on the prevention of steroid-induced osteonecrosis in rabbits. Arthritis Rheum (2004) 50:3387–91.[CrossRef][Web of Science][Medline]
  24. Kushida T, Inaba M, Ikebukuro K, et al. Comparison of bone marrow cells harvested from various bones of cynomolgus monkeys at various ages by perfusion or aspiration methods: a preclinical study for human BMT. Stem Cells (2002) 20:155–62.[CrossRef][Web of Science][Medline]
  25. Kushida T, Inaba M, Ikebukuro K, et al. A new method for bone marrow cell harvesting. Stem Cells (2000) 18:453–6.[CrossRef][Web of Science][Medline]
  26. Kushida T, Inaba M, Hisha H, et al. Intra-bone marrow injection of allogeneic bone marrow cells: a powerful new strategy for treatment of intractable autoimmune diseases in MRL/lpr mice. Blood (2001) 97:3292–9.[Abstract/Free Full Text]
  27. Kabata T, Kubo T, Matsumoto T, et al. Apoptotic cell death in steroid induced osteonecrosis: an experimental study in rabbits. J Rheumatol (2000) 27:2166–71.[Web of Science][Medline]
  28. Sakamoto M, Shimizu K, Iida S, Akita T, Moriya H, Nawata Y. Osteonecrosis of the femoral head: a prospective study with MRI. J Bone Joint Surg Br (1997) 79:213–9.[CrossRef][Medline]
  29. 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) 30:185–90.[Medline]
  30. Iida T, Mine S, Fujimoto H, Suzuki K, Minami Y, Tanaka Y. Hypoxia-inducible factor-1alpha induces cell cycle arrest of endothelial cells. Genes Cells (2002) 7:143–9.[Abstract]
  31. Okada Y, Tanikawa T, Iida T, Tanaka Y. Vascular injury by glucocorticoid; involvement of apoptosis of endothelial cells. Clin Calcium (2007) 17:872–7.[Medline]
  32. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science (1997) 275:964–7.[Abstract/Free Full Text]
  33. Hamano K, Li TS, Kobayashi T, et al. The induction of angiogenesis by the implantation of autologous bone marrow cells: a novel and simple therapeutic method. Surgery (2001) 130:44–54.[CrossRef][Web of Science][Medline]
  34. Kasper G, Dankert N, Tuischer J, et al. Mesenchymal stem cells regulate angiogenesis according to their mechanical environment. Stem Cells (2007) 25:903–10.[CrossRef][Web of Science][Medline]
  35. Weinstein RS, Robert LJ, Michael Parfitt A, Stavros CM. Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone. J Clin Invest (1998) 102:274–82.[Web of Science][Medline]
  36. Manolagas SC, Weinstein RS. New developments in the pathogenesis and treatment of steroid-induced osteoporosis. J Bone Miner Res (1999) 14:1061–6.[CrossRef][Web of Science][Medline]
Submitted 10 June 2007; revised version accepted 15 January 2008.
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