| Object: Ferumoxides(superparmagnetic iron oxide, SPIO) were used to label rabbit BMSCs(bone marrow mesenchymal stem cells) as a magnetical probe. To explore the effects of various concentrations of Ferumoxide on the viability and polification of BMSCs. To observe the magnetic resonace tracing in vivo and the curative effects on femoral head necrosis with Ferumoxides-labeled BMSCs plantation, and to provide the logical proofs for the magnetic resonace tracing in vivo and the treatment of femoral head necrosis.Methods: 1. Rabbit BMSCs were isolated and trained by Ficoll density gradient centrifugation and repetitive adherence to culture plate technique. And the cells were identified by CD29+/CD34- using immunoflouresence technique. 2. BMSCs were incubated with DMEM containing 20% FBS supplanted with 5.6μg/ml,11.2μg/ml,22.4μg/ml and 44.8μg/ml of Ferumoxides, respectively. Prussian blue stain was employed for identifying intracellular irons, transmission electron microscopy was used to observe the cisterna containing irons, and the concentration of intracellular irons was measured by Atomic absorption spectroscopy, which the effective-labeled concentrations of Ferumoxides on BMSCs. 3. To analysize The effects of SPIO on polification of BMSCs by MTT assay. 4. Rabbit femoral head necrosis model was established by horse serum and hormone method or liquid nitrogen frozen method. Comparing the two methods by ordinary observation. 5. After marked with Ferumoxides the BMSCs were transplanted into the femoral head necrosis models via three ways: in situ, in Region and in the ear-vein of rabbits. Then, MRI tracing was conducted. By MRI scanning sequences including SE T2WI (TR 6000ms, TE 100ms), FSE T2WI(TR 2200ms, TE 90ms) and GRE T2*WI(TR 500ms,TE 30ms), the image changes of transplanted BMSCs marked by Ferumoxides were observed. Comparing the three ways of transplanting Ferumoxides-marked BMSCs by ordinary observation, X-ray, general femoris head sample and histology. 6. By analyzing the area percentage of newly formed bone trabecula in the defect samples through high power lens to explore the site to transplant Ferumoxides-marked BMSCs in the treatment of femoral head necrosis.Results: 1. Inverted phase contrast microscope was adopted to investigate the morphology of the isolated cells. The higher viability cells were observed and the results of immunoflouresence showed CD29 not CD34 was expressed on the surface of the cells, which proved that the isolated and cultured cells were BMSCs, not HSC(Hematopoietic stem cell,HSC. 2. The detection of the concentration of intracellular irons showed that BMSCs can be marked by incubating with various concentrations of Ferumoxides in dose-dependent manner. 3.The results of MTT assay showed no significant among the viability of cells from 5.6μg/ml,11.2μg/ml,22.4μg/ml and 44.8μg/ml groups which indicates that Ferumoxides has no effects on the growth of BMSCs. 4. Both horse serum and hormone method (A group) and liquid nitrogen frozen method (B group) lead to obvious femoral head necrosis, which suggests that Rabbit femoral head necrosis model was established successfully. However, the positive rate of bone lacuna of Group B is significantly higher than that of Group A. The model establishing time of group A is much longer than group B; the strike in group A is more intensive than group B. Two rabbits in group A died of hypersensitiveness. Although the method of liquid nitrogen frozen method is traumatic occlusion, the strike is relatively less intensive and the method is technically convenient. 5. Post the three ways of transplant, in situ, in Region and in the ear-vein, decreased-signal region was detected in MRI scanning sequences of SE T2WI, FSE T2WI and GRE T2*WI, respectively. However, the emerging and extinctive time of the decreased-signal region was different among the three scanning sequences. It was found that the decreased-signal region of the MRI scanning sequences was the target of the present experiment. MR image showed that the decrease degree was different significantly among the three scanning sequences, GRE T2*WI |
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