Jin Xu-hong, Yang Liu, Duan Xiao-jun, Xie Bing, Li Zhong, Tan Hong-bo
Center of Joint Surgery, Southwest Hospital, Third Military Medical University, Chongqing, China.
Zhonghua Yi Xue Za Zhi. 2007 Dec 4;87(45):3213-8.
To evaluate the feasibility of in vivo magnetic resonance imaging (MRI) with 1.5T system tracking of the survival, migration and differentiation of magnetically labeled seed cells-bone marrow-derived mesenchymal stem cells (MSCs) injected into the articular cavity.
Rabbit MSCs were isolated, purified, expanded, and then coincubated in vitro with supermagnetic iron oxide particles (SPIO) and 5-bromo-2-deoxyuridine (BrdU). Prussian blue staining and transmission electron microscopy were performed to observe the intracellular iron. Some labeled MSCs were subjected to chondrogenic differentiation and the phenotype was examined to assess their chondrogenic differentiation capacity. MSCs colabeled with SPIO nanoparticles and (BrdU were suspended in chitosan and glycerophosphate (C-GP) gel. Eighteen rabbits underwent damage to the femoral trochlea to create cartilage defect models, and randomly divided into 3 groups 1 week later: Group A (n=6) undergoing injection of the MSC suspension in C-GP gel into the intra-articular space of knee joints, Group B (n=6), injected with un-labeled MSC suspension in C-GP gel, and Group C (n=6), without injection. MRI of the knee was performed 1, 4, 8, and 12 weeks after the injection respectively on a certain numbers of rabbits. and then the rabbits were killed with their knee joints taken out to undergo immunohistochemistry. The MR imaging findings were compared with the histological findings.
Prussian blue staining and transmission electron microscopy showed intracytoplasmic nanoparticles in the SPIO-labeled cells. Safranin-O staining showed deposition of proteoglycan and type II collagen outside both the labeled and unlabeled MSCs, showing chondrogenesis. GRE T2-weighted MR image showed marked hypointense signal void areas, representing the implanted MSCs, in the intra-articular space after the MSC injection in Group A that persisted for 12 weeks at least; 2 week after the MSC injection hypointense signal could be seen in the defect, which peaked in the signal intensity about 4 weeks later, and then gradually decreased in the signal intensity; and 12 weeks after the injection no recognizable hypointense signal in the defect was detected. Immunohistochemical staining demonstrated the presence of Prussian blue-positive cells and BrdU-positive cells in the tissue sections in the areas corresponding well to the signal intensity loss regions in the MRI images. Group B and Group C showed no signal intensity loss in the intra-articular spaces by GRE T2-weighted MR imaging. Histological observation showed that the defects were repaired with fibrocartilage in Groups A and B, and with fiber tissue in Group C.
Labeled with SPIO, the MSCs remains their ability of chondrogenic differentiation. It is feasible to track the fate and dynamic redistribution of magnetically labeled MSCs, the seed cells, injected into the articular cavity by 1.5T MRI, an efficient noninvasive technique.
评估采用1.5T系统进行体内磁共振成像（MRI）追踪注入关节腔的磁性标记种子细胞——骨髓间充质干细胞（MSCs）存活、迁移及分化情况的可行性。
分离、纯化、扩增兔MSCs，然后在体外与超顺磁性氧化铁颗粒（SPIO）和5-溴-2-脱氧尿苷（BrdU）共同孵育。进行普鲁士蓝染色和透射电子显微镜检查以观察细胞内铁。对部分标记的MSCs进行成软骨分化并检测其表型以评估成软骨分化能力。将用SPIO纳米颗粒和BrdU共标记的MSCs悬浮于壳聚糖和甘油磷酸（C-GP）凝胶中。18只兔造成股骨滑车损伤以建立软骨缺损模型，1周后随机分为3组：A组（n=6）将C-GP凝胶中的MSCs悬液注入膝关节腔；B组（n=6）注入C-GP凝胶中的未标记MSCs悬液；C组（n=6）不进行注射。分别在注射后1、4、8和12周对一定数量的兔进行膝关节MRI检查，然后处死兔并取出膝关节进行免疫组织化学检查。将MRI检查结果与组织学检查结果进行比较。
普鲁士蓝染色和透射电子显微镜检查显示SPIO标记的细胞内有胞质纳米颗粒。番红O染色显示标记和未标记的MSCs周围均有蛋白聚糖和Ⅱ型胶原沉积，表明有成软骨形成。梯度回波T2加权MR图像显示A组注射MSCs后关节腔内有明显的低信号空洞区，代表植入的MSCs，至少持续12周；注射MSCs后2周在缺损处可见低信号，信号强度约在4周后达到峰值，然后逐渐降低；注射后12周缺损处未检测到可识别的低信号。免疫组织化学染色显示组织切片中普鲁士蓝阳性细胞和BrdU阳性细胞的存在，其区域与MRI图像中信号强度降低区域对应良好。B组和C组梯度回波T2加权MR成像显示关节腔内无信号强度降低。组织学观察显示A组和B组缺损由纤维软骨修复，C组由纤维组织修复。
用SPIO标记后，MSCs仍保留其成软骨分化能力。采用1.5T MRI追踪注入关节腔的磁性标记种子细胞MSCs的命运和动态再分布是可行的，这是一种有效的非侵入性技术。
