【作者】 金旭红；

【导师】 杨柳；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 背景多种原因所致的关节软骨缺损在医学临床较为常见,目前所用的保守治疗和手术治疗方法均存在明显缺陷。关节软骨组织工程技术可为其再生修复提供新的治疗手段。近年来骨髓间充质干细胞(BMSCs)因具有良好的体外扩增能力、且具有软骨分化潜能,已成为体外构建组织工程软骨的重要种子细胞来源,许多实验表明移植入宿主体内的BMSCs能促进宿主体内缺损功能的修复,展示了光明的前景。然而目前困扰关节组织工程技术临床应用的一个重要难题――对体内原位种子细胞的研究缺乏有效的识别和追踪监测手段,因而难以明确外源性种子细胞在软骨缺损修复中的作用和转归,体内新生软骨组织的细胞来源,细胞移植术的疗效。因此迫切需要探索一种对体内原位移植细胞进行追踪和监测的安全、有效、无创的手段,以促进组织工程软骨修复关节软骨缺损技术研究的进一步深入。目的本项目拟在课题组既往关节软骨组织工程研究获得重要进展的基础上,借鉴国内外最新研究成果,研究体外SPIO标记种子细胞BMSCs的适宜方法、磁标记物对种子细胞生物学特性的影响、MRI监测体外磁标记细胞的灵敏度、准确度及MRI活体示踪自体皮下移植磁化标记BMSCs的可行性,最后通过不同时相点1.5T MRI在体示踪种子细胞在活体内的存活、迁徙及分布,以及结合BrdU细胞示踪技术作为阳性对照,并判定其磁标记细胞的分化转归过程,完成其自体移植修复关节软骨缺损的动物实验应用研究,为关节软骨组织工程种子细胞的在体示踪提供一种安全无创、动态直观的新技术和新方法。方法1、体外纳米磁标记BMSCs的体外细胞生物学特性及其MR成像从兔骨髓中分离培养BMSCs,不同浓度SPIO(50μg/ml、25μg/ml、12.5μg/ml)联合硫酸鱼精蛋白转染剂与BMSCs孵育12h,未标记细胞设为对照组。普鲁士染色和电镜检查鉴定细胞内是否含铁颗粒;胎盼蓝染色检测细胞存活和MTT法测定生长曲线的变化;磁标记BMSCs转入各定向培养基中进行诱导培养2w后;鉴定磁标记BMSCs的多向分化潜能:对成骨定向诱导组进行钙结节茜素红染色和碱性磷酸酶(ALP)组化染色,对成脂肪定向诱导组观察细胞形态学变化或油红-O染色,对成软骨定向诱导组进行番红-O染色和II型胶原免疫组化染色检测胞外基质的分泌和表达;应用1.5T MR梯度回波T2加权(GRET2*WI)扫描序列和自旋回波T2加权(SET2WI)扫描序列对磁标记细胞成像示踪。2、MR成像示踪磁标记兔BMSCs自体皮下移植BMSCs经体外采用SPIO和BrdU双重标记后,与壳聚糖-甘油磷酸钠(C-GP)支架复合植入兔自体大腿皮下,在术后1h、第5d及2w、4w、8w应用0.2T MR GRET2*WI序列对磁标记细胞成像行连续示踪,扫描后即处死动物并取材行组织切片普鲁士染色及免疫组化BrdU检查。实验组为自体皮下移植SPIO标记BMSCs(n=6),设立自体皮下移植未标记BMSCs(n=6)和皮下单纯注射SPIO组(n=2)为两组对照。3、MR在体成像示踪磁标记的BMSCs修复兔关节软骨缺损建立兔膝直径4mm深约3mm的股骨髁软骨缺损模型,1周后将经SPIO和BrdU双重标记的BMSCs与C-GP支架1ml复合,然后注射到自体软骨损伤关节腔中,术后1h、4w、8w及12w应用1.5T MR GRET2*WI序列对膝关节腔内注入的磁标记BMSCs进行扫描示踪,并与组织切片普鲁士染色及免疫组化BrdU对照。实验组为损伤侧膝关节注入1ml含1×10~8个磁标记BMSCs与C-GP支架混悬液;设立注入1ml含1×10~8个未标记BMSCs与C-GP支架混悬液、损伤侧膝关节不做任何处理为两组对照(n=6)。结果1、体外纳米磁标记BMSCs细胞生物学特性及其MR成像磁标记细胞普鲁士染色和电镜检查显示细胞胞浆内含致密铁颗粒;胎盼蓝染色和MTT分析测定生长曲线证实磁标记对BMSCs活性和增殖无影响(P>0.05);纳米磁标记BMSCs在体外具有向成骨细胞、软骨细胞和脂肪细胞表型诱到的潜能。1.5TMR扫描GRET2*WI序列和SET2WI序列提示与未标记细胞SI相比,1×10~6个标记细胞、5×10~5个标记细胞信号强度均有不同程度显著性下降(P<0.05)其中GRET2*WI的信号强度衰减率显著性高于T2WI序列(P<0.05)。在两个序列中1×10~6(标记细胞)信号强度衰减率均高于5×10~5(标记细胞)信号强度衰减率,但不具有有显著性差异。2、MR成像示踪磁标记兔BMSCs自体皮下移植自体皮下移植的磁标记兔BMSCs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。术后1h兔后肢0.2T MR GRET2*WI序列成像示磁标记细胞在皮下注入部位形成直径约1.5cm的特异性类圆形低信号影。术后5d观察到距注射部位后侧0.5cm处皮下出现孤立的特异性低信号影,原皮下注射部位特异性低信号影直径扩大至1.7cm,低信号强度未见减弱。术后2w见皮下孤立的特异性低信号影已与原皮下注射部位特异性低信号影融合,并呈线性延伸为0.6cm,低信号区域直径扩大为2.0cm,侵及肌层。术后4w见低信号区域进一步扩大。术后8w见皮下注射部位向周围发出的低信号线显著长达1.1cm,低信号区域直径扩大为2.6cm,侵及肌肉深层,低信号强度减弱。MRI信号改变区域与组织学切片普鲁士染色及免疫组化BrdU显示植入细胞结果相对应。术后第5d移植部位见植入细胞密集,植入物与宿主组织界面周围散在出现植入细胞。术后2w至4w见移植部位与宿主组织界面周围出现的植入细胞较前增加,但植入细胞主要聚集在移植部位内,术后8w移植部位植入细胞减少,宿主组织内出现的植入细胞较前显著增加。HE染色观察到术后初期在植入区域出现炎性反应,但术后1周炎性反应消失,所有动物的移植部位均未出现切口红肿和分泌物。3、MR在体成像示踪磁标记的BMSCs修复兔关节软骨缺损体外磁标记的BMSCs与C-GP复合注射入关节腔后1.5T MR GRET2*WI序列成像显示关节腔内磁标记BMSCs产生弥漫性颗粒状低信号影改变至少12w,术后1h可见关节腔内出现弥漫性颗粒状异常低信号改变,主要分布于和腘窝部位,术后4w观察到软骨缺损部位、软骨下骨处特异性低信号影改变。但随着移植时间延长,低信号强度逐渐减弱,术后12w软骨缺损处特异性低信号影不明显,而关节腔内髌上囊、腘窝处结节状低信号改变仍清晰存在。MRI信号改变区域与组织学切片普鲁士染色及免疫组化BrdU显示植入细胞结果相对应。术后4w见软骨修复区有少量植入细胞存在,大量植入细胞主要分布于髌上囊、腘窝处滑膜和软骨下骨,术后8w软骨修复区植入细胞消失,滑膜中植入细胞数目亦减少,术后12w软骨修复区亦未见植入细胞,而髌上囊滑膜和软骨下骨部位仍较多存在植入细胞。结论1、SPIO联合硫酸鱼精蛋白转染剂能成功标记BMSCs,磁标记对细胞存活、增殖及潜在多向分化能力无影响,磁标记细胞在MR上产生特征性的低信号改变,临床1.5TMR成像示踪标记细胞可行,以GRET2*WI序列成像最为敏感。2、自体皮下移植的磁标记兔BMSCs在0.2T GRET2*WI序列产生特征性的低信号改变至少8w,术后1h、第5d、2w、4w、8w不同时相MR连续成像观察到植入细胞从皮下移植部位向远处迁移并逐渐进入宿主组织。术后2w、4w、8w时植入细胞的组织学改变与MRI结果基本一致。移植的磁标记细胞在具有免疫功能的皮下未诱发明显的免疫反应。利用0.2T MR连续示踪自体皮下移植的磁标记BMSCs活体内的分布和迁移是可行的。3、兔BMSCs经SPIO标记后仍然具有成软骨细胞诱导能力;磁标记后植入关节腔内的BMSCs可以在临床1.5T MR上产生明显的低信号改变至少12w,术后1h、4w、8w、12w时不同时相MR连续成像观察到关节腔内部分植入细胞向软骨缺损迁移聚集随后又逐渐减少,至术后12w时软骨缺损部位植入细胞消失,此时植入细胞主要分布于关节腔内髌上囊、腘窝、软骨下骨。术后4w、8w、12w时植入细胞的组织学改变与MRI结果基本一致,关节腔内注入体外扩增培养的磁标记BMSCs,不能促进软骨缺损修复。应用MRI在体示踪磁标记细胞技术可以连续示踪组织工程软骨种子细胞BMSCs在活体关节腔内的分布和迁移,可望为组织工程种子细胞的示踪提供一种无创动态、直观简便的方法。更多还原

【Abstract】 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.更多还原