| The avascular necrosis of the femoral head (AvNFH), among which the most cases were induced by steroid hormone, is a common disease. It often occurs and causes disabilities in clinic. With the development of modern medical imaging technologies,the imaging technology of morphology and function will become a new way during disease research. It is important that we explore the microcirculatin and blood perfusin about femoral head. MR perfusion can evaluate the blood perfusin of bone tissue,and get the blood flow and velocity and so evaluate the viability of bone tissue. We evaluated femoral perfusion in the steroid-induced AvNFH animal model, using dynamic contrast-enhanced magnetic resonance imaging combined with hemorrheology, to explore the value of dynamic contrast-enhanced MRI. Otherwise,to explore the influence of VEGF through the observation of the expression of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2(BMP-2) in bone tissue during the progress of steroid induced osteonecrosis with immunohistochemistry. By the methods of cell and morphology we investigate further the pathogenetic mechanism of steroid-induced AvNFH with the cultured bone marrow mesenchymal stem cell in vitro.This study is divided into two parts:Firstly,twenty-eight white New Zealand rabbits were used and randomly divided into two groups. Group A: model group (horse serum and prednisone);Group B: control group. All rabbits were examined by non-enhanced, dynamic contrast-enhanced and conventional enhancement MRI scan in the same method and in the second week, fourth week, sixth week and eighth week after steroid treatment. Vein blood was attained for hemorrheology before treatment and in the fourth week, eighth week. After rabbits were killed, all femoral heads were made paraffin section and HE stained specimen for pathological investigation. Their specimens of femoral heads were taken for the expression and alteration of VEGF and BMP-2 in the second week, fourth week, sixth week and eighth week after steroid treatment by means of immunohistochemical staining and examined under microscope.These results were showed as follows: (1) By light microscopy, there were osteocytes pyknosis or necrosis,empty bone lacunae and fat tissue with much more adipocytes increased but hematopoietic tissue decreased with treatment of prednison. (2) The steepest slope (SSmax) of time-signal intensity curve gradually declined in the early stage decreased gradually (p<0.05). Correspondingly, the number of empty lacuna increased gradually (p<0.01). (3) Compared with control group, the index of model group include of whole blood (high and low slice), viscosity of plasma and packed cell volume elevated, but only whole blood (low slice), packed cell volume were significant (p<0.05). (4) The parameter SS of dynamic contrast-enhanced MRI correlated negatively with whole blood(low slice)(r=-0.65,P<0.05). (5) The expression of VEGF protein in the osteoblasts,vascular endothelial cells and bone matrix became lower in the femoral head of model group in the second,fourth week,but increased slightly in the sixth and eighth week. The expression of BMP-2 protein in cartilage cell,osteoblasts,matix between bone marrow cells descended during treatment with prednison.These results suggested that (1) Dynamic contrast-enhanced MRI can evaluate the state of blood perfusion of femoral head and show the pathologic changes during the progress of osteonecrosis. (2) The expression of VEGF and BMP-2 protein in bone tissue are inhibited by glucocorticoid during the steroid-induced necrosis of the femoral head,then angiogenesis and new bone tissue are restrained and so the degree of ischemia, anoxia of the local environment in bone tissue become serious,which explains the decrease in femoral blood perfusion.(3) It is indicated that VEGF and BMP-2 play an important role during the progress of osteonecrosis.Secondly,MSCs were separated and cultured in 6-well plates according to the different biological characteristics between MSCs and hematopoietic stem cells. And then the third-MSCs were planted in 6-well plates, which were divided into four groups by random: empty control, group treated with 10-8 mol/L Dex, group treated with 10-7 mol/L Dex, group treated with 10-6 mol/L Dex. The cells morphology was detected by using inverted phase-contrast microscope, whose proliferation was observed by using MTT assay. After four groups of MSCs were cultured for 21 days, they were stained with oil red O. Adipocytes was investigated and counted under a light microscope. These results were showed as follows: (1) Investigation of morphology of MSC:MSCs were planted and attached to the plates. Most of the cells exhibited fibroblast-like spindle shape. Compared with that of empty control, proliferation of cells treated with dexamethasone became slow. Under the inverted phase-contrast microscope, cytoplasmic lipid droplets, i.e. triglyceride-containing vesicles, were observed in the cells treated with dexamethasone. The cells became larger and transferred to round or polygonal shape. (2) Inhibition of the proliferation of MSCs by Dexamethasone: compared with that of control group,the OD value of group treated with 10-8 mol/L Dex, groups treated with 10-7 mol/L Dex, groups treated with 10-6 mol/L Dex group was respectively 0.289±0.023,0.221±0.015,0.154±0.013, compared with the control group(0.352±0.012),the difference was significant (P<0.05). (3) Dexamethasone can directly stimulate the differentiation of MSCs into adipocytes: the positive rates of adipocytes in four groups (empty control, group treated with 10-8 mol/L Dex, groups treated with 10-7 mol/L Dex, groups treated with 10-6 mol/L Dex) was respectively 1.8±0.7,46.2±3.2,77.1±3.4,83.6±2.8(%). Compared with that of control group,the difference between them was significant (P<0.05).These results suggested that dexamethasone could not only inhibit the proliferation of bone marrow mesenchymal stem cells directly, but also induce the differentiation of MSCs into adipocytes, which was similar to the pathologic changes of steroid-induced osteonecrosis, and helpful for clarifying the pathobiological mechanism of osteonecrosis induced by steroid.To sum up, changes of these indexes in steroid-induced avascular necrosis of femoral head animal model and bone marrow mesenchymal stem cells were observed from the whole level of animals and cellular level of in vitro in this study. They were both independent and interrelated with each other by the glucocorticoid. We observed that blood perfusion decreased gradually during the progress of osteonecrosis with dynamic contrast-anhanced MRI. The expression of VEGF and BMP-2 protein in bone tissue is inhibited by glucocorticoid during the steroid-induced necrosis of the femoral head,then angiogenesis and new bone tissue are restrained and so the degree of ischemia of the local environment in bone tissue become serious,which explains the decrease in femoral blood perfusion. Otherwise,it was observed that dexamethasone can directly induce bone marrow mesenchymal stem cells differentiation into a large number of adipocytes by the ways of cell and morphology. This might lead to an increase in marrow fat volume of the femoral head following administration of high dose of steroids; eventually an increase of intraosseous pressure occured, with a decrease of vascular perfusion. Without sufficient repair of the necrotic bone the final result is avascular necrosis of the femoral head.
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