| [Objective] To establish a stable animal model for glucocorticoid-induced avascular necrosis of femoral head in rabbits, provide experimental data for the clinical supervising of glucocorticoid-induced avascular necrosis of femoral head by blood biochemical analysis and histological examination and imaging changes in the rabbit model, using light microscopy from the morphological point of view. And explore the mechanism of osteonecrosis femoral head. to discuss the good or bad aspects of the ONFH in different ways, which provides the animal experimental models for the study of clinical pathogenesis.Separation the bone marrow mesenchymal stem cells of healthy group and the model of rabbit, Comparison the proliferation and osteogenic differentiation of two cells, Provide thee clinical basis about bone marrow mesenchymal stem cell transplantation for treatment of early ischemic necrosis of the femoral head.[Methods] healthy adult New Zealand white rabbits were 48, male and female open, body weight (3.0±0.3) kg, were randomly divided into 4 groups. A group (control group),12:intramuscular injection of normal saline every day as D group; B group (a group injection of horse serum and three times injections hormone),12:by ear vein injection of lOmL/kg Horse serum,3 weeks after injection of 6mL/kg Horse serum by ear vein again.2 weeks after the hip muscular injection of 40mg/kg MPS sodium succinate (methylprednisolone), a total of 3 times, each time interval 24 h. C group(a group of Single injection of endotoxin and three times injections hormone),12:by ear vein injection of 10μg/kg LPS (E. coli endotoxin),24h after the hip muscular injection of 40mg/kg MPS sodium succinate (methylprednisolone), a total of 3 times, each time interval 24 h. D group(a group of wice injection of endotoxin and three times injections hormone),12:by ear vein injection of 10μg/kg LPS (E. coli endotoxin), a total of 2 times, each time interval 24h. after 24h the hip muscular injection of 40mg/kg MPS sodium succinate (methylprednisolone), a total of 3 times, each time interval 24 h. After injection, the generally circs were observed the animals; 4,6,8,10,12 in the first week after administration line serum cholesterol, triglycerides, blood rheology, coagulation function tests, and the application of statistical software version SPSS11.5 using the T test to analyzed for two sets of data, The single-factor analysis of variance were used to compare multiple sets of data for q text, compared to P<0.05 as a statistically significant difference with the standard analysis of the data. Executed in the first week of 6,8,10,12 rabbits with bilateral femoral head line from the whole specimens, histopathological sections of conventional light microscopy and vascular endothelial growth factor (VGEF) Immunohistochemical staining of microvascular thrombosis of the femoral head (MSB) staining, according to diffusion in the air or bone lacuna bone karyopyknosis whether the cells around the bone tissue necrosis of femoral head avascular necrosis as a histopathologic feature of the criteria for each group of animals the compare of femoral head necrosis statistics. X-ray and MRI imaging were performed in the rabbits before the first injection of lipopolysaccharide or methylprednisolone, and at 4, 8, and 12 weeks after the last injection of lipopolysaccharide or methylprednisolone.Extracted the bone marrow in bilateral femur of rabbit at sterile conditions, By the adherence, we isolate the bone marrow mesenchymal stem cells of the healthy group and the model group. To compare the characteristics of the two cells, including the morphos of the cells, the rate of adherence, cell kinetics, cell surface marker, ALP solution and Von kossa staining, quantity detection of ALP and the caicium in induced cells. To analyse the data.[Results] At 4 weeks after the final injection, the body weight of groups A was increased.At 3 weeks after the final injection, the body weight of groups B,C and D was decreased. Listlessness, less activity and reduced diet were found in the rabbits of groups C and D after injected with lipopolysaccharide. C have an animal die after the final injection at the end of 2d and 10d. D have an animal die after the final injection at the end of 1d and 8d. Compared with A, the subsequent modeling B,C and D groups in of serum cholesterol (cholesterol, CHOL), triglyceride (triglyceride, TG), the whole blood viscosity low shear rate, viscosity of plasma, erythrocyte aggregation index were significantly increased (P<0.05), But more significant of D group. Since the beginning of 10 weeks after the modeling, The comparison between the two groups were statistically significant indicators (P<0.05). Showed by imaging and pathology, with the same time of A, B, C group, the size of D group osteonecrosis of femoral head increased significantly, the bone of trabecular sparse, thinning and fracture more obvious, the number of fat cells in intramedullary femoral head were significantly increased, the number of empty lacunae increased.It can be seen by the cell culture, (1)The morphous of two cells are chord shape,like fibroblast cells. (2) By quantity detection of adherence rate and analysis of variance with repeated measurement data, have significant difference exist between-two groups of the adherence rate(P<0.05). Shows the adhesion rate of model group were lower than the healthy group. (3) The ability of proliferation of model group were lower than the healthy group, the postdigestive 2nd and 5th generation cells have 6-9 days log phase growth after the latent period about 24～48 hours,then transit to the platform period. (4)By immunocytochemistry, adipose mesenchymal stem cells and bone marrow mesenchymal stem cells express CD44 positive.(5)The ALP and Von kossa staining of the two cells are both positive. (6)By quantity detection of alkaline phosphatase and analysis of variance with repeated measurement data, have significant difference exist between two groups of the quantity of ALP(P<0.05). The quantity of ALP of the two cells have diversity at different time. (7) By quantity detection of accumulation of calcium and analysis of variance with repeated measurement data, have significant difference exist between two groups of the quantity of accumulation of calcium (P<0.05). The accumulation of calcium of the two cells have diversity at different time. [Conclusion] (1) The model application of hormone and double injection of E. coli endotoxin, the resulting dyslipidemia, serum cholesterol, triglyceride content increased, while the formation of hyperlipidemia fatty degeneration of bone marrow cells, leading to slow blood flow, stasis, whole blood viscosity, plasma viscosity and erythrocyte aggregation index increased, micro-thrombosis and hence the occurrence of avascular necrosis of femoral head there is a close relationship.coagulation dysfunction, prothrombin time, activated partial prothrombin time, blood was hypercoagulable state, microcirculation, and promote micro-thrombosis and progress to increase the incidence of avascular necrosis of femoral head.(2) Under identical experimental condition, the group of wice injection of endotoxin and three times injections hormone show up Hormonal Osteonecrosis of the Femoral Head which is the most early and the most obviously. The rate of the empty lacunaes increased with the time of experiment.(3) Under the same conditions, compared with the healthy group, The bone marrow mesenchymal stem cells of model group in vitro proliferation and the capacity of bone induction were reduced in varying degrees. |
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