The Experimental Study Of High-Energy Shock Waves On Treatment Early Femoral Head Necrosis

Osteonecrosis of femoral head is induced by various reasons which cause disturbance of blood circulation. Osteonecrosis of femoral head is an increasingly common cause of musculoskeletal disability as well as a major diagnostic and therapeutic challenge. Trauma, treatment with corticosteroids, alcoholism and coagulation abnormality are the most common conditions associated with it. It is a burning question needed to be resolved. The course of disease can be reversed for early femoral head necrosis. It usually takes 3 to 5 years for femoral head necrosis progresses to degenerative changes of the hip joint. Untreated osteonecrosis of femoral head usually results in collapse of the femoral head. There are many methods can be used to treat femoral head necrosis. However, no therapy has been developed that preserves the activity of the femoral head. Osteonecrosis of femoral head usually progresses to joint destruction, requiring total hip replacement (THR) in individuals. Heavy economic burden and suffering of the patients are caused by the disease. Accordingly, the development of an effective and noninvasive treatment would be extremely valuable.Early femoral head necrosis in rabbit was induced by methylprednisolone and lipopolysaccharide. The necrotic femoral head was subjected to high energy shock wave (HESW) treatment at different energy flux densities and impulses. The effect of HESW on microvessel density, bone remodeling and cellular ultrastructure of necrotic femoral head was observed. Optimum parameters of HESW in energy intensity and impulse were analyzed. The bone morphogenetic protein 2(BMP-2) and vascular endothelial growth factor (VEGF) expression was assessed by histochemistry, immunohistochemistry, quantitative real-time PCR and Western blot analysis. The mechanism of femoral head necrosis treated with HESW was explored. The theoretical basis for clinical application of HESW was provided.Firstly, animal model of early femoral head necrosis was induced. Early femoral head necrosis in rabbit was induced by methylprednisolone and lipopolysaccharide. Healthy adult New Zealand rabbits were injected twice with lipopolysaccharide, and then were injected three times with methylprednisolone; the interval between injections was 24 hours. At the end of six weeks after the injection the animals were put to death by intravenous injection of air from the vein of aural edge. X-ray, CT, MRI, ultrastructural observations and histomorphology studies were performed to evaluate the animal model. Methylprednisolone combined with lipopolysaccharide can induce typical rabbit model for early necrosis of femoral head.Secondly, the effect of HESW on microvessel density, bone remodeling and cellular ultrastructure of necrotic femoral head was observed after the necrotic femoral head was treated with HESW. Animal model of early femoral head necrosis was induced and a single HESW treatment (0.26 mJ/mm2 energy flux density, 1 Hz, 2000 impulses; Orthospec?, Israel) was applied to the necrotic femoral head. Each rabbit with necrotic femoral head was anesthetized and placed in a prone position with all four limbs stabilized in brace. The ultrasound transmission gel was used as contact medium between the HESW apparatus and skin. The left hind limbs (treatment side) of all rabbits received shock wave treatment to the femoral heads and the right limbs (control side) of all rabbits received no shock wave treatment. X-ray, CT, MRI, ultrastructural observations and histomorphology studies were performed and the effect of HESW on microvessel density, bone remodeling and cellular ultrastructure of necrotic femoral head was observed. No changes could be detected on X-ray, CT and MR images in rabbits of all groups 4 weeks after the treatment. By 12 weeks after shock wave treatment, low density patchy shadows in X-ray at treatment side of femoral head decreased or disappeared and no obvious changes were seen at control side of femoral head. Radiolucent cystic area and low density patchy shadows on CT scans at treatment side of femoral head decreased or disappeared and no obvious changes were seen at control side of femoral head. Irregular low signal on T1-weighted images and irregular low or high signal on T2-weighted images on MR images at treatment side of femoral head decreased or disappeared. Irregular low signal on T1-weighted images and irregular low or high signal on T2-weighted images on MR images at control side of femoral head were still obvious. At 4 and 12 weeks after shock wave treatment, microvessel density of the femoral heads in the treatment side was significantly higher than that in the control side. We have performed a histomorphometric analysis of subchondral bone of the femoral heads for both groups. Necrotic femoral heads treated with shock waves, when compared to controls, were characterized by higher percent trabecular area (Tb.Ar %), tabecular thichness (Tb.Th), trabecular number (Tb.N), nodes/termini ratio (NTR), percent labeled primeter (%L.Pm), inter label width (IrL. W i) and bone formation rate (BFR/BV). However, tabecular separation (Tb.Sp) was lower in necrotic femoral heads treated with shock waves than in controls. Under epifluorescent light, the tetracycline labels in shock wave treated femoral head are intense and the distances between two lines are wide, the tetracycline labels in untreated femoral head are weak and the distances between two lines are narrow. Osteoblasts of treatment group were round or oval, had plenty of rough endo-plasmic reticulum and mitochondria in the cytoplasm, nuclear staining is weak. This suggests that the function of the osteoblasts in treatment group is active. HESW may have effect in promoting angiogenesis of necrotic femoral head, improving the function of osteoblasts and vascular endothelia in the femoral heads, and accelerating the repair process of femoral head necrosis.Thirdly, the VEGF and BMP-2 expression was assessed by histochemistry, immunohistochemistry, quantitative real-time PCR and Western blot analysis after the necrotic femoral head was treated with HESW. Animal model of early femoral head necrosis was induced and a single HESW treatment (0.26 mJ/mm2 energy flux density, 1 Hz, 2000 impulses; Orthospec?, Israel) was applied to the necrotic femoral head. Each rabbit with necrotic femoral head was anesthetized and placed in a prone position with all four limbs stabilized in brace. The ultrasound transmission gel was used as contact medium between the HESW apparatus and skin. The left hind limbs (treatment side) of all rabbits received shock wave treatment to the femoral heads and the right limbs (control side) of all rabbits received no shock wave treatment. The animals were killed with an overdose of pentobarbital at 24h (n=6) and 1 (n=6), 2 (n=6), 4 (n=6), 8 (n=6) and 12 (n=6) weeks after shock wave therapy. The femoral heads were harvested and freed of soft tissue and cartilage. They were assigned for histomorphometry, immunohistochemistry, Western blot analysis, and quantitative real-time reverse transcriptase PCR. In the subchondral bone of the necrotic femoral heads untreated with shock waves, a slight VEGF immunoreactivity (VEGF-IR) was found in the osteoblasts and the endothelial cells during experiments and a slight BMP-2 immunoreactivity (BMP-2-IR) was found in the osteoblasts and the fibroblastic cells during experiments. During the 2 and 4 weeks after shock wave therapy, the osteoblasts and the endothelial cells showed an increased level of VEGF-IR, the osteoblasts and the fibroblastic cells showed an increased level of BMP-2-IR. At 8 weeks, VEGF-IR was increased in the fibroblastic and endothelial cells of the vascular granulation tissue invading the necrotic bone. Bone matrix and osteocytes also showed VEGF-IR, BMP-2-IR was increased in the fibroblastic cells of the vascular granulation tissue invading the necrotic bone. Bone matrix also showed BMP-2-IR. At 12 weeks, VEGF-IR and BMP-2-IR were observed in the trabecular bone and the marrow. Osteoblasts adjacent to the newly formed woven bone showed intensive expression of the VEGF and BMP-2. The gene expression of VEGF and BMP-2 were markedly higher in shock wave treated femoral heads compared to untreated femoral heads throughout the time points. Statistically significant differences were observed at 1, 2, and 4 weeks after shock wave treatment. An increase in VEGF and BMP-2 proteins was consistently observed in the subchondral bone samples obtained from the shock wave treated femoral heads at 2, 4 and 8 weeks after the shock wave treatment in comparison with the samples from the untreated femoral heads. These data clearly show that extracorporeal shock waves can significantly up-regulate the expression of VEGF and BMP-2. The up-regulation of VEGF and BMP-2 may play a role to induce the ingrowth of neovascularization and accelerate the repair process of femoral head necrosis.Finally, the necrotic femoral head was treated with HESW at different energy flux densities and impulses. Optimum parameters of HESW in energy intensity and impulse for the treatment of femoral head necrosis were analyzed. Animal model of early femoral head necrosis was induced and a single HESW treatment (0.16mJ/mm2, 2000 impulses ),(0.21mJ/mm2, 2000 impulses),(0.26mJ/mm2, 2000 impulses),(0.32 mJ/mm2, 2000 impulses),(0.26 mJ/mm2, 1000 impulses) or (0.26 mJ/mm2, 4000 impulses) was applied to the left necrotic femoral heads of Group A(n=8),B(n=8),C(n=8),D(n=8),E(n=8),F(n=8). Rabbits of Group G (n=8) received no shock wave treatment. Each rabbit with necrotic femoral head was anesthetized and placed in a prone position with all four limbs stabilized in brace. The ultrasound transmission gel was used as contact medium between the HESW apparatus and skin. Ultrastructural observations and histomorphology studies were performed and the effect of high energy shock wave on microvessel density, bone remodeling and cellular ultrastructure of necrotic femoral head was observed. At 12 weeks after shock wave treatment, microvessel density of the femoral heads in Group of B,C,D,F was significantly higher than that in Group of G. At 12 weeks after shock wave treatment, percent trabecular area in Group of B,C,D,F was significantly higher than that in Group of G. Osteoblasts of B,C,D and F group were round or oval, had plenty of rough endo-plasmic reticulum and mitochondria in the cytoplasm, nuclear staining is weak. This suggests that necrotic femoral heads treated with shock waves at 0.21-0.32mJ/mm2 for more than 2000 impulses can repair more quickly and angiogenesis of the necrotic femoral head is promoted.