In Vivo MR Imaging Tracking Of Magnetic Iron Oxide Nanoparticles Labeled, Engineered, Autologous Bone Marrow Mesenchymal Stem Cells On Repair Of Articular Cartilage Defects

BackgroundClinically, articular cartilage defects occur commonly in association with different pathological situations. Clinical treatments for cartilage defects elicit incomplete repair, e.g. fibrocartilage. Recently, tissue-engineering procedures hold promise for the treatment of articular cartilage defects to achieve the re-generation to hyaline cartilage. However, there is still a lack of understanding regarding the characteristics of the seed cells in repairing defects. The development of tissue-engineering therapies requires an efficient and noninvasive technique to monitor the in vivo behavior of implanted cells in host tissue and thus help understand the characteristics of the seed cells.PurposeThe aim of this study was to label BMSCs with SPIO(superparamagnetic iron oxide nanoparticles, SPIO) and study the effects of magnetic labeling on the proliferation and differentiation of BMSCs, to study the feasibility of magnetic resonance imaging tracking of transplanted SPIO-labeled BMSCs in vivo after implantation into rabbit subcutaneous tissue, and to evaluate in vivo magnetic resonance imaging with 1.5T system tracking for the surviva1, migration and differentiation of magnetically labeled BMSCs injected in articular cavity in rabbit cartilage defect model.Method1. Biological characteristics and in vitro MRI of SPIO labeled BMSCs from rabbits.rabbit BMSCs were isolated, purified, expanded ,then coincubated with various doses of SPIO(50μg/ml,25μg/ml,12.5μg/ml) complexed to protamine sulfate(Pro) transfection agents overnight. Prussian blue stain and transmission electron microscopy were performed to show intracellular iron, Tetrazolium salt (MTT) assay was applied to evaluate toxicity and proliferation of magnetic labeled BMSCs. Cell differentiation capacity were assessed in vitro using appropriate functional assays.Vials containing cells underwent 1.5T MR imaging (MRI) with GRE T2*WI weighted and SET2WI sequence. Data were expressed as the mean±SD, and one-way analysis of variance and the Independent-Samples T test were used to test for significant differences.2. In vivo Magnetic Resonance Imaging Tracking of SPIO-labeled BMSCs after Autologous Transplantation In Subcutaneous Tissue of Rabbits.rabbit BMSCs were in vitro coincubated with SPIO(25μg/ml) complexed to protamine sulfate transfection agents for 12h, subsequently BMSCs were grown in medium containing BrdU for 2h.After colabeled, BMSCs were encapsulated in chitosan and g1ycero- phosphate(C-GP) gel. Autologous co-labeled BMSCs encapsulated in C-GP gel constructs were injected into thigh subcutaneous tissue of rabbits.All rabbits were performed on a clinical 0.2-T MR imager using a T2*-weighted gradient- echo(GRE T2*WI) sequence at 1 day , 5 days, 2 weeks, 4 weeks, 8 weeks after implantation. Animals were divided into 3 experimental groups: 1) rabbits injected with SPIO and BrdU colabeled BMSCs seeded inC-GP gel into autologus thigh subcutaneous tissue(n=6); 2) rabbits injected with BrdU labeled BMSCs seeded in chitosan and glycerophosphate (C-GP ) gel into autologus thigh subcutaneous tissue(n=6); 3) rabbits injected with SPIO lonely into autologus thigh subcutaneous tissue(n=2);3. In vivo MR Imaging Tracking of Magnetic Iron-oxide Nanoparticles Labeled Bone Marrow Mesenchymal Stem Cells injected Into the Intra-articular Space of Knee Joints In RabbitBMSCs colabeled with SPIO(25μg/ml) and BrdU were suspended in 1ml of C-GP gel and injected into the intra-articular space of knee joints in rabbit cartilage defect model. 18 Japanese White rabbits were equally divided into 3 groups. In group A, SPIO and BrdU co-labeled, autologous BMSCs that were seeded in C-GP gel were injected into the knee joint cavity of the rabbit models of articular cartilage defects. In group B, BrdU-labeled, autologous BMSCs that were seeded in C-GP gel were injected. In group C, no treatment was applied to the rabbit models for cartilage defects.All rabbits were imaged at 1 day, 4 weeks, 8 weeks,12 weeks post-injection. 1.5-T MR imaging findings were compared with histology.Result1. Intracytoplasmic nanoparticles were stained with Prussian blue and observed by transmission electron microscopy clearly except the unlabeled control. As compared with the nonlabeled cells, MTT values of light absorption had no statistically significant difference. It showed no significant difference in effects on the viability, growth rate and differentiation of the labeled BMSCs. And the differentiation of the labeled cells were unaffected by the endosomal incorporation of SPIO after the labeled BMSCs were incubated in appropriate inducers for 3 weeks. The lipid drop emergence and some specimens were stained with Oil-red-O. The calcium nodu1es were stained with alizarin red. For Chondrogenesis in labeled and unlabeled BMSCs, Safranin-O staining shows deposition of proteoglycan and immunohis- tochemical staining shows production of collagen type II expressed equally. GRET2*WI and T2*WI demonstrated significant decrease of signal intensity (SI) in vials containing 1×106 and 5×105 labeled cells, in comparison with unlabeled cells (P<0.05). The percentage change of SI(△SI) was significantly higher in 1×106 labeled cells than that in 5×105 labeled cells, particularly on GRET2*WI (P<0.05). Among pulse sequences, GRE T2*WI demonstrated the highest△SI(P<0.05).2. At injection sites low signal intensity could be observed on MRI examination with the scanning sequences of GRET2*WI. Low signal intensity lines could be observed targeting to the host tissue areas in experimental group.3. Marked hypointense signal void areas representing the implanted BMSCs could be observed in intra-articular space after cell injection on GRE T2-weighted MR image in group A, and persisted for 12 weeks at least. Two week after injection, we observed a hypointense signal in the defect, which reached its maximum in signal intensity at about 4 weeks and decreased for the next weeks.12 weeks after injection, no recognizable hypointense signal in the defect was detected. Histochemical staining demonstrated the presence of Prussian blue-positive cells and BrdU-positive cells in tissue sections in areas that corresponded well to the signal intensity loss regions in the MRI images. Group B and group C showed no signal intensity loss in intra-articular space on GRE T2-weighted MR image. The histological observation showed that the defects were repaired with fibrocartilage in group A and group B, fiber tissue in group C.Conclusion1. BMSCs can be labeled with Fe-Pro efficiency without significant change in cell viability and differentiation capacity.The suspension of labeled BMSCs can be imaged with standard 1.5-T MR equipment,Low signal intensity could be observed and GRET2*WI was the most sensitive sequence for detecting SPIO-labeled BMSCs.2. SPIO can be used to label BMSCs in vitro efficiently. 0.2-T MRI in vivo tracking of the transplanted SPIO-labeled BMSCs in subcutaneous tissue is effective.3. 1.5-T MRI tracking for the surviva1, migration and differentiation of magnetically labeled BMSCs injected in articular cavity in rabbit articular cartilage defect model is feasible and efficient. BMSCs cultured in vitro and injected into intra-articular space can not improve the treatment results of the articular cartilage defect. MRI would be an efficient noninvasive technique to monitor the fate and dynamic redistribution of seed cells labeled with SPIO in future articular cartilage tissue engineering applications.