MRI活体示踪超顺磁性氧化铁标记兔骨髓间充质干细胞的自体皮下移植

doi:10.3969/j.issn.2095-4344.2012.49.015

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (49): 9201-9208.doi: 10.3969/j.issn.2095-4344.2012.49.015

• 干细胞移植 stem cell transplantation • 上一篇下一篇

MRI活体示踪超顺磁性氧化铁标记兔骨髓间充质干细胞的自体皮下移植

金旭红¹，张寿¹，杨柳²，文亚明²，段小军²

¹中南大学湘雅医学院附属海口市中心医院骨科，海南省海口市 570208；²解放军第三军医大学西南医院关节外科中心，重庆市 400038

收稿日期:2011-02-14 修回日期:2012-03-27 出版日期:2012-12-02 发布日期:2013-01-16
通讯作者: 张寿，主任医师，教授，硕士生导师，中南大学湘雅医学院附属海口市中心医院骨科，海南省海口市 570208
作者简介:金旭红，副主任医师，硕士生导师，主要从事骨科方面的研究。
基金资助:
海口市重点科技计划项目(2010-168)，应用自体骨髓间充质干细胞微创修复关节软骨缺损；(2009-049-11)利用脐血间充质干细胞构建生物工程软骨；海南省自然科学基金项目(309107)，应用同种异体脐血干细胞构建生物工程软骨修复软骨缺损的实验研究。

In vivo magnetic resonance imaging tracking of superparamagnetic iron oxide labeled rabbit bone marrow mesenchymal stem cells after subcutaneoustransplantation

Jin Xu-hong¹, Zhang Shou¹, Yang Liu², Wen Ya-ming², Duan Xiao-jun²

¹Department of Orthopedics, Haikou Hospital Affiliated to Xiangya School of Medicine, Central South University, Haikou 570208, Hainan Province, China; ²Center of Joint Surgery, Southwest Hospital, Third Military Medical University, Chongqing 400038, China

Received:2011-02-14 Revised:2012-03-27 Online:2012-12-02 Published:2013-01-16
About author:Jin Xu-hong, Associate chief physician, Master’s supervisor, Department of Orthopedics, Haikou Hospital Affiliated to Xiangya School of Medicine, Central South University, Haikou 570208, Hainan Province, China jxh53@yahoo.cn
Supported by:
Key Science and Technology Planning Program of Haikou, No. 2010-168*, 2009-049-11*; Natural Science Foundation of Haikou, No. 309107*

摘要/Abstract

摘要：

背景：高磁场MRI已被成功应用于示踪移植超顺磁性氧化铁标记骨髓基质干细胞的研究，但目前还有应用低磁场MRI示踪移植干细胞的报道。
目的：探讨用0.2 T MRI活体示踪自体皮下移植磁化标记骨髓基质干细胞的分布和迁移的可行性。
方法：从兔骨髓中分离培养骨髓基质干细胞，采用超顺磁性氧化铁和BrdU双重标记后，与壳聚糖复合植入兔自体大腿皮下。自体皮下移植未标记骨髓基质干细胞和皮下单纯注射超顺磁性氧化铁为对照。
结果与结论：超顺磁性氧化铁标记骨髓基质干细胞经普鲁士蓝染色和电镜检查证实细胞胞浆含致密铁颗粒。超顺磁性氧化铁标记后自体皮下移植的兔骨髓基质干细胞在GRET2*WI序列成像时产生特征性的低信号改变至少维持8周，且信号逐渐从移植部位进入组织深处。但普鲁士蓝染色和BrdU免疫组化显示大部分的移植细胞仍停留在原移植部位。提示体外超顺磁性氧化铁能有效地标记骨髓基质干细胞，利用0.2 T MRI活体示踪自体皮下移植的超顺磁性氧化铁标记兔骨髓基质干细胞分布和迁移是可行的。

关键词: 超顺磁性氧化铁, 骨髓基质干细胞, 移植, 磁共振成像, 组织工程

Abstract:

BACKGROUND: Magnetic resonance imaging with high field units has been successfully used in tracking the superparamagnetic iron oxide labeled bone marrow mesenchymal stem cells. However, the validity of a low-field magnetic resonance imaging unit for detecting labeled cells has not been thoroughly investigated.
OBJECTIVE: To explore the feasibility of 0.2-T magnetic resonance imaging for in vivo tracking the distribution and migration of magnetically labeled bone marrow mesenchymal stem cells after subcutaneous transplantation.
METHODS: Bone mesenchymal stem cells were isolated from rabbit bone marrow. Before implantation, bone marrow mesenchymal stem cells were double labeled in vitro with superparamagnetic iron oxide and BrdU, then the cells were composited with chitosan and subcutaneously implanted in the thigh. Thighs subcutaneously implanted with unlabeled bone marrow mesenchymal stem cells and simply superparamagnetic iron oxides were used as control.
RESULTS AND CONCLUSION: The dense iron particles could be seen in the cytoplasm of the superparamagnetic iron oxide labeled bone marrow mesenchymal stem cells through Prussian blue staining and electron microscopy detection. Subcutaneously transplanted superparamagnetic iron oxide labeled autologous rabbit bone marrow mesenchymal stem cells showed low signal changes and maintained at least 8 weeks during T2*-weighted gradient-echo sequence imaging, and the signal gradually entered into the tissue from the graft site. Prussian blue staining and BrdU immunohistochemistry showed that most of the transplanted cells remained in the original transplantation site. It indicated that bone marrow stromal stem cells could be effectively labeled with superparamagnetic iron oxide in vitro, and it was possible to in vivo track the subcutaneously transplanted superparamagnetic iron oxide labeled autologous rabbit bone marrow mesenchymal stem cells with 0.2-T magnetic resonance imaging.

中图分类号:

R394.2

金旭红，张寿，杨柳，文亚明，段小军. MRI活体示踪超顺磁性氧化铁标记兔骨髓间充质干细胞的自体皮下移植[J]. 中国组织工程研究, 2012, 16(49): 9201-9208.

Jin Xu-hong, Zhang Shou, Yang Liu,Wen Ya-ming, Duan Xiao-jun. In vivo magnetic resonance imaging tracking of superparamagnetic iron oxide labeled rabbit bone marrow mesenchymal stem cells after subcutaneoustransplantation[J]. Chinese Journal of Tissue Engineering Research, 2012, 16(49): 9201-9208.

图/表（结果） 5

Quantitative analysis of experimental animals

All the 14 rabbit models entered the final analysis. There was no dead animal in each group.

MSCs labeled with SPIO

Bone marrow MSCs isolated from the bone marrow blood were cultured and proliferated in vitro, and the cells shaped as fusiform and grew in vortex (Figure 1a). After 12 hours of incubation with SPIO nanoparticles, more than 90% of the cells turned positive upon Prussian blue staining. Prussian blue staining demonstrated iron-containing sites as blue spots in the cytoplasm (Figures 1b-c). Trypan blue staining did not suggested a decreased viability of the labeled cells when compared with unlabeled cells. Transmission electron microscopy confirmed the presence of iron oxide nanoparticles inside the cytoplasm and vesicles (Figure 1d). BrdU immunohistochemistry showed the BrdU-positive MSCs (Figure 1e). Figure 1 Morphological changes of bone marrow mesenchymal stem cells (MSCs) labeled with superparamagnetic iron oxide (SPIO)

Clear hypointense signals in the labeled MSCs and increasing numbers of hypointense regions with increasing concentrations of labeled MSCs are shown in Figure 2. GRET2*WI sequence demonstrated significant decreases in the signal intensity for 1×10⁶ and 5×10⁵ labeled cells compared with unlabeled cells, but 1×10⁶ labeled cells had the greater loss of signal

intensity than that of the 5×10⁵ labeled cells. Figure 2 In vitro cells magnetic resonance imaging (T2*-weighted gradient-echo of 5 Eppendorf tubes)

At 5 days post-grafting in group A animals, a lonely significant hypointense signal void located in the subcutaneous site can be identified, which was detected at a distance of 0.5 cm from implantation site on axial MR images (Figure 3c), the maximum diameter of hypointense signal void with irregular
borders dispersing to 1.7 cm can be identified on sagittal MR images, and there was no decreases in the signal intensity. The hypointense signal void was disappeared after 5 days post-grafting in group C animals, and no contrast could be observed on MR images of unlabeled cells in B group animals. At 2 weeks post-grafting in group A, the lonely significant hypointense signal void was observed connecting with marked hypointense signal caused by the injection site, and appeared as a dark elongated line long as 0.6 cm on axial MR images (Figure 3d), the maximum diameter of hypointense signal area dispersing to 2.0 cm on sagittal MR images, and it had involved in the muscle layer. Over the following weeks, we observed the hypointense signal line was intensified and elongated, and the size of hypointense signal area was increased. At 8 weeks post-grafting, the hypointense signal line reached its maximum of 1.1 cm (Figure 3e), and the maximum diameter of hypointense signal area which encroached in the muscle deep layer was observed spreading to 2.6 cm. But a decreasing in hypointense signal intensity was seen (Figure 3f). Figure 3 Transverse and saggital sections T2*-weighted gradient echo images from rabbit hind limb after autologous implantation of labeled mesenchymal stem cells in subcutaneous tissue

Histological and immunohistochemical evaluation of the implanted MSCs

Both Prussian blue and anti-BrdU staining results coincided regarding the areas of contrast enhancement as seen on MR images. At 5 days post-injection, histology showed that a large number of Prussian blue-positive cells remained within the implant constructs, and some Prussian blue-positive cells were found at the interface between C-GP implant and host subcutaneous tissue (Figure 4a), a finding consistent with that of BrdU staining (Figure 4b). The presence of iron-oxide particles was discerned inside the cells, no iron-oxide particles appeared in the extracellular space. Both Prussian blue and anti-BrdU staining revealed a relative increase of visibly stained MSCs in the adjacent surrounding subcutaneous tissue over time. At 8 weeks post-injection, Prussian blue-positive cells could still be strongly detected but the number of it was decreased in the implantation constructs (Figure 5a), and a large number of Prussian blue-positive cells were also detected entering the surrounding subcutaneous and muscle layer, suggesting a migration from the cell implantation constructs toward the adjacent surrounding spaces over time, which coincided regarding the results of anti-BrdU staining (Figure 5b). Inflammatory response was evident in both groups at day 5 and week 2, and then inflammatory cells were reduced gradually. The vast majority of scaffolds were absorbed and degraded. BrdU immunohistochemistry was used to identify the implanted MSCs, but showed no positive Prussian blue staining cells at group B. No Prussian-staining or BrdU-positive cells were observed in group C.

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引言

材料方法

Design

A randomized controlled animal experiment.

Time and setting

This experiment was performed at the Center of Joint Surgery, Southwest Hospital, Third Military Medical University from December 2010 to September 2011.

Materials

A total of 14 male Japanese White rabbits, aged 8-week-old and weighting 2-2.5 kg, were provided by the Experimental Animal Center of the Third Military Medical University in China. The protocols employed were in direct accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China^[16].

Methods

Isolation and culture of MSCs

The rabbit MSCs were isolated as described previously^[17]. All Japanese White rabbits were anesthetized by intramuscular injection of ketamine (50 mg/kg). The hind legs were shaved and draped in a sterile fashion. Briefly, rabbit bone marrow was harvested from the proximal tibias via an 18-gauge needle, and 1.0-1.5 mL of bone marrow was collected into a test tube containing 500 units of heparin. Monocytes layer was obtained by density centrifugation and then washed twice with PBS. The cells were resuspended in DMEM containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin, and the cell concentration was adjusted to 2×10⁵ cells/cm². Then, the cells were plated into 37.5 cm² flasks. The medium was firstly changed 48 hours after seeding to remove nonadherent cells. Later, the medium was replaced every 3 days as the cells grew into confluence. On day 14, the cells were harvested by incubation with 0.25% trypsin and re-seeded in 37.5 cm² flasks at 1×10⁴ cells/cm² and grew to confluence. For both in vitro and in vivo assays, only the MSCs after two passages were used.

SPIO labeling of cells

As described previously^[18], a commercially available ferumoxides suspension, Feridex Ⅳ was diluted with culture medium to 50 mg/L upon use. Protamine sulfate used as the transfection agent was prepared as a fresh stock solution of 10 mg/mL in distilled water. Then 6 mg/L Protamine (Pro) sulfate diluted from stock solutions was mixed on a rotating shaker with ferumoxides for 60 minutes in cell culture medium at room temperature, Then, an equal volume of the solution containing Fe-Pro complexes was added into the existing medium in rabbit MSCs culture to a final concentration of SPIO of 25 μg/L and Pro sulfate of 3 mg/L. The cells were incubated for 12 hours at 37 ℃ in air containing 5% CO₂.

After incubation with the Fe-Pro complex, the cells were washed three times with PBS to remove excess ferumoxides. Rat MSCs were co-labeled with 30 μmol/L BrdU at 2 hours prior to implantation.

Iron detection

For Prussian blue staining of intracellular iron particles, the cells were trypsinized and adhered to the slides for 24 hours, fixed with 4% glutaraldehyde, washed and incubated for 30 minutes with 2% potassium ferrocyanide in 6% hydrochloric acid, and then counterstained with nuclear fast red. Cells were considered positive on Prussian blue staining if intracytoplasmic blue granules were detected. The percentage of labeled cells was calculated with the mean of cell numbers in 10 high-power fields.

Before electron microscopy examination, the MSCs were fixed in 2.5% buffered glutaraldehyde for 30 minutes at 4 ℃, followed by treated with 1% osmium tetroxide for 30 minutes. The cells were dehydrated in a concentration gradient of ethanol, immersed in propylene-oxide, and embedded with Epon 812. The samples were sliced into ultrathin sections (60 nm). These sections were examined under a Tecnai-10 transmission electron microscope.

In vitro proliferation of MSCs

The viability of labeled MSCs was assessed by trypan blue exclusion and in vitro proliferation assays. Briefly, the labeled MSCs were seeded into a 96-well plate (5 000 cells/well) and cell growth curves were plotted. The samples were assayed at each time point over 9 days. The absorbance at 490 nm (with 570 nm as reference wavelength) was measured by a Thermomaxmicroplate reader.

In vitro cellular MRI

To determine the sensitivity of the lower field dedicated extremity MR system for the detection of labeled cells, labeled and unlabeled MSCs were trypsinized, centrifuged and resuspended in 0.5 mL of 1% agarose (1×10⁶, 5×10⁵ labeled cells, 1×10⁶ unlabeled cells). Two Eppendorf tubes containing 1% agarose, and distilled water, were also used respectively. Then the five tubes were placed into a box filled with water. In vitro cellular imaging was performed on a low-field dedicated extremity MR imager using a T2*-weighted gradient-echo (GRET2*WI) sequence. Sequence parameters were as follows: repetition time/echo time (TR/TE)=500 ms/18 ms, field of view=180 mm×180 mm, matrix size =286×192, slice thickness=4 mm, gap=0.4 mm, flip angle=40°.

Experimental protocol

Fourteen Japanese White rabbits were divided into three groups. In group A, SPIO and BrdU co-labeled, autologous MSCs that seeded in chitosan and glycerophosphate (C-GP) gel were injected subcutaneously into rabbits (n=6). In group B, BrdU-labeled autologous MSCs that seeded in C-GP gel were subcutaneously injected (n=6). In group C, single 5 μL of 25 mg/L SPIO particles were subcutaneously injected into rabbits (n=2).

In vivo experiments and in vivo MRI

All Japanese white rabbits were anesthetized by 30 g/L sodium pentobarbital (1 mL/kg). The hind legs were shaved and draped in a sterile fashion. Using a Hamilton syringe with a 25-gauge needle, engineered MSCs (5×10⁷) that seeded in 1 mL C-GP gel were injected into medial subcutaneous tissue of the left thigh, 3 cm proximal to the superior pole of the patellar in rabbits. Following injection, the rabbits were raised separately and observed everyday.

The rabbits were imaged under general anesthesia at 1 and 5 days and 2, 4 and 8 weeks post-injection, and MR images of the rabbit joints were obtained on a clinical lower field dedicated extremity MR imager. Sagittal and axial images were obtained by a GRET2*WI sequence. Imaging parameters were as follows: TR/TE=500 ms/18 ms, field of view=180 mm× 180 mm, matrix size =286×192, slice thickness=4 mm, gap=0.4 mm, flip angle=40°.

Histology and immunohistochemistry

The rabbits were sacrificed at 5 days, 4 and 8 weeks after surgery, the whole graft was removed from the thigh subcutaneous tissue. Macroscopic observations on the graft and surrounding graft tissues were made at necropsy. For histological evaluations, all samples were carefully fixed in 4% paraformaldehyde overnight, followed by paraffin embedment and sectioning (5 μm thick). The sections were stained with hematoxylin and eosin before histological examination.

Injected MSCs were detected by Prussian blue staining for iron to produce ferric ferrocyanide and by anti-BrdU staining using a fitc-conjugated goat anti-mouse IgG as a secondary antibody. To detect BrdU-labeled MSCs in the samples, Methanol-H₂O₂ treatment was applied to eliminate autogenous peroxidase activity and 10% normal goat serum was used to block non-specific immunoreaction. Sections were labeled overnight at 4 ℃ with anti-BrdU monoclonal antibody diluted 1:100 in PBS. The samples were washed three times with PBS and reacted with fitc-conjugated goat anti-mouse IgG as a secondary antibody in a dilution of 1:100 in PBS for 30 minutes at 4 ℃.

Main outcome measures

A low-field dedicated extremity MRI unit (0.2 T) was applied to detect the SPIO-labeled MSCs following subcutaneous implantation in vivo at different time points in rabbit models.

讨论

The most important results from the present study was that magnetically labeled MSCs with SPIO can be visualized and traced both in vitro and in vivo using a 0.2 T field MRI equipment. As low-field MR systems traditionally suffered from hardware and technical drawbacks, the majority of published reports regarding MRI of tracking labeled cells using superconducting magnets at high field strength field or at intermediate field^[19-21]. Imaging of the magnetically labeled cells after implantation in the host organism with a low-field MRI is great challenging, the quality of the images would be

severely degraded. In recent years, great improvements have been made in the development of low-field MRI. Currently, a low field dedicated extremity MRI unit (0.2-T) is exclusively used as a sensitive and specific tools for both diagnosis and monitoring the peripheral joints disease activity and joint damage^{[15, 22-23]}. However, none have directly investigated whether it could provide information on tracking magnetically labeled MSCs in vivo as that of a high field MRI unit, a critical appraisal of the diagnostic value of MRI of the implant cells is therefore in order.

Thus, we carried out the first experiment of low-field dedicated extremity MRI units tracing autologous SPIO-labeled cells after subcutaneous implantation into rabbits, and observed the fate of the labeled implanted cells and if it caused immunological rejection in the recipient subcutaneous tissue. In order to avoid the interfered noise caused by animals breathing, we chose the thigh of hindlimb as the implant site.

To be visualized on MR images, cells need to be magnetically labeled. Many studies have demonstrated a successful labeling of MSCs with SPIO nanoparticles^[24-29]. Using a similar approach as previously described^[17], we labeled MSCs with a clinically approved MR contrast agent and that such labeled MSCs could be detected in the subcutaneous tissue of a living animal using standard MRI methods^[18]. Most studies have reported that standard MR protocols such as GRET2*WI can best assess the magnetically labeled cells from the surrounding tissues after implantation in the host organism, owing to significant shortening of the T2 and T2* relaxation times by iron oxide nanoparticles incorporated in the endosomes^{[18, 25, 30-31]}. MR detection sensitivity of magnetic labeled cells with magnetic field strength, coil specifications, imaging parameters and the concentration of SPIO marker, is closely related to particle size^[32]. Our results showed that low-field dedicated extremity MRI can detect limitation of 5×10⁴ labeled cells in vitro. And in accordance with our study, it was shown that the presence and localization of implanted MSCs in the subcutaneous tissue may be easily visualized on GRET2*WI sequences, as labeled MSCs creates large artifacts on MR images. The hypointensity of the cell implant remained visible for the entire 8 weeks period, and the labeled MSCs display a widespread migration from the implantation site location to the surrounding host spaces. Both Prussian blue and anti-BrdU staining results analysis show that most labeled cells remain in the site of implantation constructs. However, more cells were found in the host tissue after 8 weeks compared with 2 weeks, and the iron-oxide particles were still present inside the cells. These findings of the histological analysis were in agreement with those of signal intensity changes on in vivo images. This indicates that the injected MSCs have a strong ability of migration in vivo, but the factors and mechanisms involved in MSCs migration remain unclear. At present, several studies have demonstrated that the migration distance were different^[33-34], we think that the inconsistent observations are most likely due to different cell types lead to different migration patterns. Afterwards, the intensity of hypointense signals appeared to decrease throughout the later time points, which may mainly relate to the migration of labeled cells, dilution of iron oxide particles after cell proliferation, and biodegradation of iron oxide particles.

An unexpected finding was that the presence of separate significant hypointense signal void distance from implantation site far as 0.5 cm on axial MR images at 5 days post-grafting in group A animals, we believe this phenomenon is caused by cell migration but not the injection pressure driven artifact which appeared to be connected with the injection site. Furthermore, Tissur hemorrhage or iron-containing proteins such as hemoglobin, ferritin and hemosiderin, is not sufficient to cause the dark region effects in MR image^[35]. We observed that the hypointense signal void was disappeared after 5 days post-grafting in group C animals, this indicates that the iron particles which did not incorporate into cells can be metabolized or eliminated by the host tissue. However, inflammatory response expressed by an increase in cell proliferation and vascularization was obvious in group A at day 5, and then inflammatory cells were decreased gradually. In addition, no animal developed complication, such as red swelling and effusion. These findings suggest that the labeled implanted cells did not cause the evident immunological rejection in the subcutaneous tissue. A limitation in this study was lack of a limb holder make the image position inconsistent. When interpreting the results of this comparative without standing these limitations, our study shows that magnetically labeled MSCs with SPIO nanoparticles may be visualized in vitro and in vivo using a 0.2-T field MR unit, it raises promising prospects for future explorations of MSCs for cell therapy in regenerative medicine. Further studies are needed to clarify the sensitivity and accuracy of the 0.2-T field MR unit for detection of magnetically labeled MSCs.

MRI活体示踪超顺磁性氧化铁标记兔骨髓间充质干细胞的自体皮下移植

In vivo magnetic resonance imaging tracking of superparamagnetic iron oxide labeled rabbit bone marrow mesenchymal stem cells after subcutaneoustransplantation

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