| Objective:To determine the feasibility of labeling rabbit bone marrow mesenchymal stem sells (BMSC) with home-made superparamagnetic iron oxide nanoparticles (SPION), the optimal labeling duration and concentration and its influence on the viability of cells. Materials and Methods:Rabbit BMSCs were primarily cultured to the 5th passage, and CD44, CD34 and CD45 were detected. Then BMSCs were tagged by home-made SPION at 0.03mg 0.015mg 0.007mg and 0.0035mg per milliliter respectively for 24 hours. The labeling efficiency was evaluated through Prussian blue staining and the optimal labeling concentration was thus established. With this optimal concentration, BMSCs were labeled for 24,48, 72 and 96 hours respectively. The labeling percentage was assessed also by Prussian blue staining to find the optimal labeling duration. The viability of BMSCs was detected via the typan blue staining after they were labeled at the optimal concentration for optimal duration. Results:After 5 passages, BMSCs were positive for CD44, and negative for CD34 and CD45. As for the labeling percentage after labeling for 24 hours, the 0.007mg/ml (80.6% ±1.7%) group was significantly higher than 0.0035mg/ml group (55.8% ± 1.4%,P<0.01), the 0.015mg/ml group (90.4%±1.2%) was significantly higher than the 0.007mg/ml group (P<0.01), and there was no significant difference between the 0.015mg/ml group and the 0.03mg/ml group (92.3% ±1.1%, P>0.05). As for the labeling percentage at the labeling concentration of 0.015mg/ml, the 48-hour group (91.3% ±0.8%) was significantly higher than the 24-hour group (89.4% ±0.8%) P <0.05, the 72-hour (98.7%±0.9%) group was significantly higher than the 48-hour group (P<0.01), and there was no significant difference between the 72-hour group and the 96-hour group (98.4%±0.1%, P>0.05). The typan blue staining show that the survival rate of BMSCs labeled at 0.015mg/ml for 72 hours was 95%.Conclusion:Labeling the BMSCs of rabbits with the home-made SPION was feasible. The optimal labeling efficiency could be achieved at 0.015mg/ml for 72 hours, and the viability of labeled BMSCs was not damaged.Objective:To evalaute the feasibility and effect of auto-transplantation of bone marrow mesenchymal stem sells (BMSC) in treatment of the hind limb ischemia in rabbits and the feasibility of in vivo tracking transplanted BMSCs preoperatively labeled by home-made superparamagnetic iron oxide nanoparticles (SPION) by magnetic resonance imaging (MRI). Materials and Methods:Fourteen 1.5-month-old New Zealand rabbits were divided into group A and group B (7 each). In group A, bone marrow was extracted from the femora in each rabbit and BMSCs were cultured. Then the hind ischemia model was constructed in all rabbits of both groups. BMSCs were labeled by SPION for 72 hours and by 5-bromodeoxyuridine (Brdu) for 48 hours after culture for 4 passages. Ten days after ischemia model procedure, labeled BMSCs were transplanted back to the corresponding donor rabbits in group A and PBS containing SPION was injected intramuscularly in group B. Calf blood ratio (defined as the systolic blood pressure of the intact hind limb to that of the ischemic one) and MRI were performed once a week for 3 consecutive weeks. At completion of the transplantation and in the 3rd postoperative week, magnetic resonance angiography was added at the time of MRI detection. Then, rabbits were sacrificed, and HE and Prussian blue staining, immunohistochemical staining for CD34 and Brdu, scanning electron microscopy were performed, and the capillary density and capillary/muscle ratio were calculated.Results:In culture, BMSCs were positive for CD44 and negative for CD34 and CD45. One rabbit in group B died of incision infection 15 days after the ischemia model procedure. The calf blood ratio was 0 in both groups at the completion of ischemia model procedure, increased in both groups (no significant difference, A 0.38±0.02, B 组 0.36±0.04, P>0.05) 1 week later, further improved in both groups (A 0.54±0.04 significantly higher than B 0.35±0.02, P<0.01), and still further rised in both groups(A 0.73±0.04 significantly higher than B 0.42±0.04, P<0.01). The capillary density of group A (198.9±2.3) was significantly higher than that of group B (80.5±5.1, P<0.01). The capillary/muscle ratio of group A (0.84±0.03) was also significantly higher than that of group B (0.35±0.04, P<0.01). In group A, the scanning electron microscopy identified SPION in the endothelial cell of vessels, and vascular endothelial cells were simultaneously positive for Prussian blue staining and Brdu and CD34 immunohistological staining, and MRI depicted that the hypointense signal was localized at the injection position immediately after the transplantation and then gradually separated into small particles and dispersed around the injection site during the following 3 weeks, and MRA showed abundant collateral vessels in the ischemic limbs, and all these findings on MRI corresponded to those on histological examinations. In group B, the Prussian blue staining-positive particles were only detected extracellularly in the tissue, and MRI demonstrated that the hypointense signal was localized at the injection position both shortly after the transplantation and during the next 3 weeks, and MRA revealed scarce collateral vessels in the ischemic limbs.Conclusion:Auto-transplantation of BMSCs may precipitate the formation of collateral vessels and ameliorate ischemia in the hind limb of rabbits, which implicates its applicability to patients with critical limb ischemia. Home-made SPION can be employed as a marker for non-invasive magnetic resonance tracking of implanted BMSCs...
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