Experimental Study On The Effects And Mechanisms Of Low-frequency Pulsed Electromagnetic Fields Therapy For Steroid-induced Avascular Necrosis Of Femoral Head

Background-Steroid induced avascular necrosis of the femoral head (SANFH) is the most common and intractable type of Nontraumatic ANFH. If the necrotic area is too large, spontaneous healing is impossible, and surgery is imperative when subchondral bone collapses. Though more and more cases of steroid-associated ANFH were recently reported, the detailed pathogenesis is not well understood. Proposed mechanisms include apoptosis of osteocyte, hyperlipidemia, fat emboli, hypercoagulable condition, osteoporosis, oxidative stress and etc. But there is no unanimous system to explain all the pathological process. A reasonable pathomechanism some scholars believe is that excessive use of glucocorticoids can disturb the lipid metabolism homeostasis and suppress the bone formation by directly destroying the balance between adipogenesis and osteogenesis of bone marrow mesenchymal stem cells (BMSCs).At present, pulsed electromagnetic fields (LF-PEMF) have been widely used for the treatment of orthopedic disorders, such as arthritis, bone nonunion and osteoporosis. LF-PEMF also has been proven to be an effective non-invasive physiotherapy in the prevention and treatment of osteonecrosis of the femoral head. Though LF-PEMF is known to have beneficial effects on ANFH, the exact mechanism and the optimum program of the treatment of LF-PEMF on steroid-associated ANFH are still unknown. A previous study has shown that LF-PEMF can prevent steroid-associated osteonecrosis in rats by increasing the expression of TGF-β1and decreasing serum lipid levels. In present study, we want to study the pathological mechanism of SANFH and explore the effects of LF-PEMF by animal and cell experimentation.Part I:Experimental study on the establishment of animal model and the pathogenesis of SANFH in ratsObjective:To successfully established the animal models of SANFH and to explore the possible pathogenesis of the disease.Methods:Thirty-six Wistar rats (255±13g) were randomly divided into experimental and control groups,18in each group:(1) model group:Each rat received10μg/kg Lipopolysaccharide (LPS) intravenously; then received20mg/kg methylprednisolone by intramuscular injection, once every24hours for three consecutive days.(2) Control group:animals had received the same dose sterile saline by using the same injection at the same time. At the time point of24hours (24h),2weeks (2w), and4weeks (4w) after hormone injection,6rats were respectively sacrificed. Then we examined the related index after obtaining the blood and femoral head specimens:Lipid changes by automatic biochemical analyzer; Histopathological changes by HE and Masson staining; the expression of PPARRy-2and Runx2mRNA by PCR test.Results:24h,2w,4w after modeling, in normal control group, pathological changes of the femoral head in HE staining are consistent. In the model group, the pathological changes is consistent with the normal control on24h; On2w, there are large amounts of empty bone lacunae in trabecular bone, the number of the hematopoietic cells in bone marrow had decreased, the amount and volume of fat cells had increased; On4w, typical osteonecrosis occurred, with an incidence of83%(5/6). The Structural of subchondral bone were damaged, showing a wide range of the bone empty lacunae and large necrotic cell debris in the bone marrow. In the model group, lipids,24h after modeling, began to rise, with a peak on2w, and dropped slightly on4w. Masson staining shows:Enormous amount of mature bone tissue in the normal femoral head and more immature new bone in model group. PCR results showed:On2w and on4w, compared with the normal group, the level of PPAR-y2mRNA expression in model group was higher. On the other hand, the level of Runx2mRNA expression was lower than which in control group.Conclusions:(1)The method, using steroid combined with a single dose of lipopolysaccharide, can successfully establish an animal model of SANFH in the early stage;(2) The application of large doses of steroid can increase the expression of PPAR-y2mRNA and decrease the expression of Runx2mRNA, then led to the balance between the osteogenic and adipogenic in femoral head tissue broken, further cause lipid metabolism disorders and the inhibition of bone formation, finally resulting in osteonecrosis of the femoral head.Part II:Experimental Study on the effects and mechanisms of LF-PEMF therapy for SANFH in the early stage in ratsObjective:This study was designed to investigate the effects of low-frequency pulsed electromagnetic fields (LF-PEMF) on SANFH based on the animal model and explore the therapy mechanism.Methods:Forty-two rats were divided into three groups:(1) Steroid group (S, n=16);(2) Steroid+LF-PEMF group (S+P, n=16);(3) Control group (C, n=10). In the case of (1) and (2), all rats were first intravenously given10μg/kg lipopolysaccharide on day1, and then intramuscularly injected with20mg/kg methylprednisolone acetate with an interval24h on day2,3and4. After4weeks, group S+P was treated with LF-PEMF (4.5ms square pulse, repeated at15Hz, with a peak of12mT) for4h a day for the next8weeks. Group S had not been exposed to LF-PEMF. Group C was chosen as control group, without steroid use and exposure to LF-PEMF. After8-week treatment, the osteonecrosis incidence, the histological changes, and the mRNA and protein expression of PPAR-y2and Runx2were measured and analyzed.Results:Compared with S group, there’re a lower osteonecrosis incidence (31%vs.69%, P<0.05) and empty osteocyte lacuna rate (36.16±15.34vs.59.55±21.70, P<0.01) in group S+P. Furthermore, the treatment of LF-PEMF could suppress the expressions of PPAR-y2and improve the expressions of Runx2in the femoral head (P<0.05).Conclusions:All data suggest that LF-PEMF is an effective physiotherapy in the treatment of steroid-induced SANFH, and the possible underlying mechanisms include protecting the balance between adipogenesis and osteogenesis in femoral head.Part HI:Experimental Study on the effects and mechanisms of LF-PEMF therapy on steroid-induced adipogenic differentiation of BMSCsObjective:To study the effects and mechanisms of LF-PEMF therapy on steroid-induced adipogenic differentiation of BMSCs.Methods:BMSCs were isolated and purified from Wistar rats’bone marrow by using the repeated adherent method. The growth curve of PI, P3and P6BMSCs were drawn by MTT method. The differentiation ability was evaluated through osteogenic and adipogenic differentiation experiment. The surface antigens were detected by flow cytometry. Then we chose P3BMSCs for further experiment. BMSCs randomly divided into four groups. The control group (C):Cells were cultured by using complete culture medium, without steroid use and exposure to LF-PEMF; LF-PEMF therapy group (P):complete culture medium with exposure to LF-PEMF,2h/day for7days; Steroid group (S):high glucose DMEM medium with dexamethasone (1x10-6mol/L), without exposure to LF-PEMF; Steroid and LF-PEMF group (S+P):high glucose DMEM medium with dexamethasone (1x10-6mol/L), with exposure to LF-PEMF,2h /day for7days. Seven days later, cells were collected to detect intracellular triglyceride and ALP content. The PPAR-y2and Runx2mRNA expression were detected by RT-PCR.Results:(1) The results showed the cells grown well with eumorphism and high adherence ability. The cell growth curve had "S" shape and showed a gradually decay trend with cells algebra. Red calcified nodules were observed by Alizarin red staining. Visible lipid droplets were observed by oil red "O" staining. Flow cytometry result showed the high expression of CD44and CD90and the low expression of CD34.(2)Seven days after the intervention of various factors, the experiment results showed, compared with other groups, the levels of TG content and PPAR-y2mRNA expression were more significantly higher in the S group, while the levels of ALP content and Runx2mRNA expression were more significantly lower in the S group.Conclusions:(1) We can get BMSCs with good performance and high purity from the bone marrow of rats by using the repeated adherent method.(2)The usage of high doses of steroid can induce BMSCs to differentiate into fat cells in vitro and destroy the balance of the normal differentiation of BMSCs, then resulting in increased lipogenesis, decreased bone formation. This theory may be one of the pathogenesis of SANFH.(3) In these further experiments, the results showed LF-PEMF therapy can protect the balance of the differentiation of BMSCs from high doses of steroid and reduce fat formation and increase bone formation and repair, so as to achieve the therapeutic purposes for SANFH in the early stage.