The MRI Tracer Study Of SPIO-labeled Rabbit Bone Marrow Mesenchymal Stem Cell Transplantation For Treatment Of Femoral Head Avascular Necrosis

Object: SPIO(superparmagnetic iron oxide) were used to label rabbit BMSCs(bone marrow mesenchymal stem cells) as a magnetical probe. To observe the magnetic resonace tracing in vivo and the curative effects on femoral head necrosis with SPIO-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 morphology and flow cytometry technique.2. BMSCs were incubated with DMEM/F12containing10%FBS supplanted with25ug/ml SPIO jointed0.75ug/ml PLL.To identify the osteogenic differentiation capacity by alkaline phosphatase assay and alizarin red vital staining and to identify the adipogenic differentiation capacity by oil red O dyeing.3. Rabbit femoral head necrosis model was established by liquid nitrogen frozen method and confirmed by morphologica,histological and MRI detection.4.After the SPIO-labeled rabbit BMSCs transplanted into the rabbit femoral head necrosis has been established via in situ way.Then,MRI tracing was conducted,the image changes of transplanted BMSCs marked by SPIO were observed among the three scanning sequences of SE T2WI,FSE T2WI and GRE T2*WI.Compared with control group by histology,to calculate the area percentage of newly formed bone trabecula in the defect samples through high power lens and make statistical analysis.Results:1.We can detect a great quantity of adherent cells with great activity and generation capacity by inverted phase contrast microscope.Flow cytometric analysis results show that the cultured cells witn high level expression of CD29, CD44, CD90and low level expression of CD34,CD45. To confirm the cultured cells is rabbit BMSCs.2.BMSCs were incubated with DMEM/F12containing10%FBS supplanted with25ug/ml SPIO jointed0.75ug/ml PLL,safely and effectively.The SPIO-labeled BMSCs and unlabeled BMSCs have the same osteogenic and adipogenic differentiation capacity.3.Liquid nitrogen frozen method lead to obvious femoral head necrosis with empty lacunae rate of35%±2.2%in the4week,which suggests that Rabbit femoral head necrosis model was established successfully.4. In situ cell transplantation group.the emerging and extinctive time of the decreased-signal region was different among the three scanning sequences of SE T2WI,FSE T2WI and GRE T2*WI.It was found that the decreased-signal region of the MRI scanning sequences was the target of the present experiment.No obvious signal change in the control side.The results of calculating the area percentage of newly formed bone trabecula in the defect samples through high power lens and statistical analysis showed that group A*(19.31±2.81)%has no significant difference with group B*(20.87±1.55)%(P>0.05),has significant differences compared with the negative control group C*(2.96±1.25)%(P＜0.01).Conclusion:The SPIO-labeled BMSCs and unlabeled BMSCs have the same effect in the treatment of femoral head necrosis.The SPIO-labeled BMSCs can observe obviously by MRI detection in vitro and it lasted for six weeks.