## 4 Discussion  
Spinal cord injury continues to be a devastating injury to affected individuals and their families and creates enormous financial, psychological, and emotional costs [26]. Damaged nerve cells cannot regenerate or show limited regeneration, and thus, initial treatment can have lifelong effects. Despite years of research, current treatment is limited to early administration of high-dose steroids and acute surgical intervention to minimize cord edema and the subsequent cascade of secondary delayed injury [1; 27]. Paralysis remains incurable. MSCs can also differentiate into nerve cells. These morphological and phenotypic changes make MSCs good candidates for clinical applications for tissue regeneration and neurological treatment. To effectively regenerate tissues and functions after SCI, stem cells capable of differentiating into nerve cells or glial cells and that can be directly injected into the lesion or administered by an intravascular transplant method are needed. However, direct injection may cause secondary neural injury of the spinal cord, and the poor environment into which stem cells are transplanted may decrease the cell survival, migration rate, and preventing tissue regeneration. To overcome these limitations, SPIONs could be used to label MSCs to increase stem cell migration under magnetic induction. Studies have shown that this system promotes the induction of tissue regeneration [28]. SPIONs have been applied as contrast agents in magnetic resonance imaging [16] and have been suggested to be useful in orthopedic clinical application of stem-cell-based therapies for the tagging, tracking, and activation of stem cells [29; 30]. After targeting SPIONs to MSCs, through inducing the magnetic field, it will stimulate the differentiation of neurons and release more neurotrophic factors to increase the therapeutic effect [31]. In recent animal studies of SCI regeneration using SPIONs, stem cells were injected into the spinal canal of the L5 lumbar spine and were induced using a magnetic field. Analysis of the stem cell distribution revealed that the stem cells aggregated effectively in the target area. However, no studies have examined the stability of SPIONs and differentiation ability of SPION-internalized stem cells [32]. Therefore, we confirmed cell stability by producing SPION-internalized MSCs using fluorescence-labeled markers and analyzed stem cell migration induced by a magnetic field at the cellular level. D1 mouse MSCs could undergo osteogenic, chondrogenic, and neurogenic differentiation, which were labeled with dopamine-coated SPIONs [33]. The existence of the carbon layer in the polydopamine-coated SPION surface could effectively increase the stability and bioactivity of the sample. It also makes easy conjugation with the fluorescence probe, as well as, viable detection both in vitro and in vivo. Similarly, through the 675 nm-NHS coating the surface gets further modified to become a theranostic agent and is diagnosed roentgenology with CT or MRI [34]. Hence, both dopamine and 675-nm NHS act on the surface of the SPION and do not influence any phase transfer through the SPIONs. MTT assays were used to analyze the cytotoxicity of various concentrations of SPIONs, which was highest at 100 ug/mL. Additionally, 50 ug/mL SPIONs showed the highest migration efficiency without causing toxicity. SPIONs in the cytoplasm of cells were detected by Bio-TEM. Nearly all the SPIONs in the cells were in vesicles in the cytoplasm and nuclear membrane, indicating that absorption occurred through endocytosis. Some SPIONs particles were detected around the nucleus. This may be because, during nuclear collapses, SPIONs in the cytoplasm were near the nucleus. Some cells contained numerous SPIONs in the endosome, but this likely resulted from the aggregation between SPIONs because of the dopamine coating. To determine whether SPIONs internalized by the cells affected cell stability, we evaluated the optimal concentration of 50 ug/mL SPION-treated D1 stem cells from days 1 to 7.